US20150013033A1

US20150013033A1 - Lignification reduction in plants

Info

Publication number: US20150013033A1
Application number: US14/232,018
Authority: US
Inventors: Dominique Loqué; Amyerick Guillaume Eudes
Original assignee: University of California San Diego UCSD
Current assignee: University of California San Diego UCSD
Priority date: 2011-07-13
Filing date: 2012-07-13
Publication date: 2015-01-08
Also published as: US20190300895A1; CN103827299A; US11085048B2; BR112014000570A2; WO2013010124A1; CN108130344A

Abstract

The present invention provides an expression cassette containing a polynucleotide coding sequence for a hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which is operably linked to a heterologous promoter. Also provided are methods of engineering plants that have reduced lignification, as well as cells, plant parts, and plant tissues from such engineered plants.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No. 61/507,484, filed Jul. 13, 2011, which application is herein incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Lignocellulosic plant biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic and hydrophobic branched polymer incrusted within biomass that negatively affects extraction and hydrolysis of polysaccharides during industrial processes. Engineering the monomer composition of lignin offers attractive potential for reducing its recalcitrance. The present invention offers a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers, which incorporate as end-groups in the polymer and reduce lignin chain extension. Biosynthesis of these lignification stoppers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes. Stems from plants that express HCHL accumulate higher amount of hydroxybenzaldehyde and hydroxybenzoate derivates compared to wild type plants. Part of these C₆C₁phenolics are alcohol-extractable from plant tissues and are released from extract-free cell walls upon mild alkaline hydrolysis. Engineered plants with intermediate HCHL activity level show no reduction of total lignin, sugar content and biomass yield compared to wild type plants. However, cell wall characterization by 2D-NMR reveals the presence of new molecules in the aromatic region and the analysis of lignin isolated from these plants revealed an increased amount of C₆C₁phenolic end-groups and a reduced molecular mass distribution. In addition, these engineered lines show saccharification improvement of pretreated cell wall biomass. Enhancing the incorporation of C₆C₁phenolic end-groups in lignin represents a promising strategy to alter lignin structure and reduce cell wall recalcitrance to enzymatic hydrolysis.

BRIEF SUMMARY OF THE INVENTION

In the first aspect, the present invention provides an isolated expression cassette comprising a polynucleotide sequence encoding a hydroxycinnamoyl-CoA hydratase-lyase (HCHL) and a heterologous promoter, and the promoter is operably linked to the polynucleotide sequence. In some embodiments, the HCHL is Pseudomonas fluorescens HCHL, which has the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the promoter used in this expression cassette is a secondary cell wall specific promoter, such as pIRX5, which is within the polynucleotide sequence set forth in SEQ ID NO:3.
In a second aspect, the present invention provides a method for engineering a plant having reduced lignification. The method includes these steps: (1) introducing the expression cassette described herein into the plant; and (2) culturing the plant under conditions under which the HCHL is expressed, thereby reducing lignification in the plant. In some embodiments, the plant is selected from the group consisting of Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.
In a third aspect, the present invention provides a plant that is engineered by the methods described herein, and a plant cell from such a plant, a seed, flower, leaf, or fruit from such a plant, a plant cell that contains the expression cassette described herein, and biomass comprising plant tissue from the plant or part of the plant described herein. Thus, the invention provides an engineered plant comprising a heterologous hydroxycinnamoyl-CoA hydratase-lyase (HCHL) operably linked to a promoter. In some embodiments, the polynucleotide encoding the heterologous HCHL is integrated into a plant genome. In some embodiments, the promoter is heterologous to the plant. In some embodiments, the promoter is an endogenous promoter. In some embodiment, the promoter is a secondary cell wall-specific promoter, such as an IRX5 promoter. In some embodiments, the HCHL is Pseudomonas fluorescens HCHL. The plant may be a monocot or a dicot. In some embodiments, the plant is selected from the group consisting of Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.
In further aspects, the invention provide methods of using an engineered plant of the invention, or parts of the plant, or plant biomass comprising material from the plant. In some embodiments, plant material is used in a saccharificatoni reaction, e.g., enzymatic saccharification, to generate soluble sugars at an increased level of efficiency as compared to wild-type plants that have not been modified to express HCHL. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase biomass yield or simplify downstream processing for wood industries (such as paper, pulping, and construction) as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase the quality of wood for construction purposes. In some embodiments the plants, parts of plants, or plant biomass material can be used in a combustion reaction, gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic or chemical). In some embodiments, the plants, plant parts, or plant biomass material are used as forage that is more readily digested compared to wild-type plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HCHL-mediated conversion of hydroxycinnamoyl-CoAs into hydroxybenzaldehydes. HCHL performs the hydration and cleavage of hydroxycinnamoyl-CoAs (R=H, coumaroyl-CoA; R=OH, caffeoyl-CoA; R=OCH₃, feruloyl-CoA) to produce hydroxybenzaldehydes (R=H, 4-hydroxybenzaldehyde; R=OH, 3,4-dihydroxybenzaldehyde; R=OCH₃, 4-hydroxy-3-methoxybenzaldehyde) and acetyl-CoA via the formation of the corresponding reaction intermediates 4-hydroxyphenyl-β-hydroxypropionyl-CoAs.

FIG. 2. Analysis of HCHL expression in IRX5:HCHL lines. (A) Detection by RT-PCR of HCHL transcripts using mRNA isolated from secondary stems of five independent five-week-old transformants in the T1 generation. cDNA synthesized from mRNA purified from wild type (WT) stems were used as a negative control. Tub8-specific primers were used to assess cDNA quality for each sample. (B) Detection by western blot of HCHL tagged with the AttB2 peptide (approximate size 32 kDa) using the universal antibody and 5 μg of total protein extracted from the primary stem of five independent five-week-old IRX5:HCHL transformants in the T2 generation. A protein extract from wild type stems (WT) was used as a negative control.

FIG. 3. Histochemical staining of stem sections from five-week-old Arabidopsis plants. (A) Mäule staining. (B) Phloroglucinol-HCl staining. (C) Toluidine blue O staining. i, interfascicular fibers; x, xylem. Bars represent 50 μm for (A) and (B), and 20 μm for (C). Note the collapsed xylem vessels (yellow arrows) observed for line IRX5:HCHL (4).

FIG. 4. Spectral analysis of IRX5:HCHL and wild type plants. (A) Lignin and polysaccharide content in CWR of mature senesced stems from wild type (WT) and line IRX5:HCHL (2) using FT-Raman spectroscopy. Values represent integrated intensities over the range of 1555-1690 cm⁻¹and 1010-1178 cm⁻¹for lignin and polysaccharides (cellulose/hemicellulose) quantification, respectively. Values are means of three biological replicates±SE. (B) Comparison of FT-IR spectra obtained from xylem (black line) and interfascicular fibers (grey line) in basal stem sections of wild type and line IRX5:HCHL (2). A Student's t-test was performed on absorbance values of wild type versus transgenic and plotted against wave numbers. At each wavelength, the zone between −2 and +2 corresponds to non-significant differences (p-value<0.05) between the two genotypes tested. Significant positive t-values indicated a higher absorbance value in wild type than in IRX5:HCHL plants.

FIG. 5. 2D-HSQC NMR spectra analysis of line IRX5:HCHL plants. 2D-HSQC NMR spectra of lignin from wild type (WT) stems (A) and from IRX5:HCHL (FCA1) stems (B); Difference spectrum (IRX5:HCHL (2)—wild type) showing the presence of new components in the aromatic region (C).

FIG. 6. Polydispersity profiles of CEL lignin purified from stems of wild type and line IRX:HCHL (2) plants. SEC chromatograms were obtained using (A) UV-A₃₀₀absorbance and (B) UV-F_ex250/em450fluorescence.

FIG. 7. Saccharification of biomass from mature senesced stems of IRX5:HCHL and wild type plants. Amount of reducing sugars released from 10 mg of biomass after hot water, dilute alkaline, or dilute acid pretreatment followed by 72-h enzymatic hydrolysis were measured using the DNS assay. Values are means of four biological replicates±SE.

FIG. 8. Alignment of amino acid sequences of Pseudomonas fluorescens HCHL (SEQ ID NO:1) and other homologous proteins.

FIG. 9. Organ and tissue-specific activity of the IRX5 promoter in Arabidopsis. Line CS70758, which contains a pIRX5:GUS expression cassette, was used to localize the activity of the IRX5 promoter. Young seedlings (A and B), rosettes leaves (C and D), siliques (E and F), cauline leaves (G and H), flowers (I and J), and inflorescence stems (K and L) were incubated in the GUS assay buffer for 1 h and 8 h at 37° C. Gus activity was essentially detected in the stem xylem vessels after a 1-h incubation (K). For longer incubations (8 h), GUS staining was also observed in interfascicular fibers of the stem (L), the vascular system of young seedlings (A), siliques (F) rosette (D) and cauline leaves (H), as well as in the style and anthers (J). x: xylem vessels, if: interfascicular fibers. Scale bars: 2 mm (A-B, E-F), 4 mm (C-D, G-H), 500 μm (I-J), 100 μm (K-L).

FIG. 10. Growth and development of IRX5:HCHL and wild type (WT) plants at different stages. (A) Three-week-old rosette (B) Five-week-old flowering stage. (C) Eight-week-old senescing stage.

FIG. 11. Synthesis of C₆C₁phenolics production upon HCHL activity and probable associated enzymes. The phenylpropanoid pathway (center box) and monolignol pathway (left box) are represented. HCHL converts hydroxycinnamoyl-CoAs into their corresponding hydroxybenzaldehydes. Metabolomic data showed occurrence of hydroxycinnamic acids and alcohols, suggesting involvement of aldehyde dehydrogenases (DH) and reductases (left box). UDP-glucosyltransferases (UGT) are responsible for the formation of C6-C1 phenolic glucose conjugates. Syringaldehyde is possibly derived from vanillin and 5OH-vanillin after successive monooxyenase (Monox) and O-methyltransferase activities (OMT). Asterisks indicate compounds found in higher amount in lignin of Arabidopsis expressing HCHL. Abbreviations for enzymes are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CCR, hydroxycinnamoyl-CoA reductase; CAD, coniferyl alcohol dehydrogenase; HCT, p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyl-CoA transferase; C3H, p-coumarate 3-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; FSH, ferulate 5-hydroxylase (coniferaldehyde 5-hydroxylase); COMT, caffeic acid/5-hydroxyferulic acid O-methyltransferase.

FIG. 12. Transgenic rice lines that express pAtIRX5::HCHL.

FIG. 13. Expression analysis of HCHL in the engineered rice lines. Results of an RT-PCR using RNA extracted from rice plants and HCHL-specific primers.

FIG. 14. Detection of pHBA in stems from the engineered rice lines.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “hydroxycinnamoyl-CoA hydratase-lyase” or “HCHL” refers to an enzyme that catalyzes the hydration of the double bond of lignin precursor p-coumaroy-CoA, caffeoyl-CoA, or feruloyl-CoA thioester, which is followed by a retro aldol cleavage reaction to produce a corresponding C₆C₁hydroxylbenzaldehyde and acetyl-CoA. A typical HCHL within the meaning of this invention is an HCHL from bacterium Pseudomonas fluorescens (EC 4.2.1.101-trans-feruloyl-CoA hydratase), which has the amino acid sequence set forth in FIG. 11 as SEQ ID NO:1 (GenBank Accession No. CAA73502), encoded by cDNA sequence set forth in GenBank Accession No. Y13067.1 or by a codon-optimized polynucleotide sequence set forth in SEQ ID NO:2 (synthesized by GenScript, Piscatway, N.J.). In this application, the term HCHL includes polymorphic variants, alleles, mutants, and interspecies homologs to the Pseudomonas fluorescens HCHL, some examples of which are provided in FIG. 8. A nucleic acid that encodes an HCHL refers to a gene, pre-mRNA, mRNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants, and interspecies homologs of the particular sequences described herein. Thus, an HCHL nucleic acid (1) has a polynucleotide sequence that has greater than about 50% nucleotide sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher nucleotide sequence identity, preferably over a region of at least about 10, 15, 20, 25, 50, 100, 200, 500 or more nucleotides or over the length of the entire polynucleotide, to a polynucleotide sequence encoding SEQ ID NO:1 (e.g., SEQ ID NO:2 or the polynucleotide sequence set forth in Y13067.1); or (2) encodes a polypeptide having an amino acid sequence that has greater than about 50% amino acid sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200 or more amino acids or over the length of the entire polypeptide, to a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or to any one of the amino acid sequences shown in FIG. 8 (SEQ ID NOs:4-34). The enzymatic activity of an HCHL within the meaning of this application can be verified by functional assays known in the art or described in the example section of this application, for its ability to convert any one of lignin precursors p-coumaroy-CoA, caffeoyl-CoA, and feruloyl-CoA thioester to a corresponding C₆C₁hydroxylbenzaldehyde and acetyl-CoA.
The term “substantially localized,” when used in the context of describing a plant expressing an exogenous HCHL that is substantially localized to a particular tissue, refers to the enzymatic activity and modified monolignols produced therefore in substantially higher amounts in the particular cell or tissue type of interest as compared to other cell or tissue types. In some embodiments, the presence of HCHL and modified monolignols is substantially localized to the secondary cell wall of a plant cell and in the stem of a plant.
The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
The term “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. For example, an HCHL may have an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, the amino acid sequence of Pseudomonas fluorescens HCHL.
Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10⁵, and most preferably less than about 10.
Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

(see, e.g., Creighton, Proteins (1984)).
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C. For example, stringent conditions for hybridization, such as RNA-DNA hybridizations in a blotting technique are those which include at least one wash in 0.2×SSC at 55° C. for 20 minutes, or equivalent conditions.
The term “promoter,” refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements, translational control elements (5′UTR: untranslated region) and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic or exonic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5′ to the transcribed gene, and as used herein, include the sequence 5′ from the translation start codon (i.e., including the 5′ untranslated region of the mRNA, typically comprising 50-200 bp). Most often the core promoter sequences lie within 1-3 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls.
A “constitutive promoter” is one that is capable of initiating transcription in nearly all cell types, whereas a “cell type-specific promoter” initiates transcription only in one or a few particular cell types or groups of cells forming a tissue. In some embodiments, the promoter is secondary cell wall specific. Secondary cell wall is mainly composed of cellulose, hemicellulose, and lignin and is deposited in some, but not all, tissues of a plant, such as woody tissue. As used herein, a “secondary cell wall specific” promoter refers to a promoter that initiates higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk. In some embodiments, a promoter is secondary cell wall specific if the transcription levels initiated by the promoter in secondary cell walls are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess secondary cell wall, e.g., the stem of a plant. Non-limiting examples of secondary cell wall specific promoters include the promoters directing expression of genes IRX1, IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB103, PAL1, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc, and CADd. See, e.g., Turner et al 1997; Meyer et al 1998; Jones et al 2001; Franke et al 2002; Ha et al 2002; Rohde et al 2004; Chen et al 2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong et al 2006; Mitsuda et al 2007; Zhong et al 2007a, 2007b; Zhou et al 2009; Brown et al 2009; McCarthy et al 2009; Ko et al 2009; Wu et al 2010; Berthet et al 2011. In some embodiments, the promoter is substantially identical to the native promoter sequence directing expression of the IRX5 gene (see, e.g., the promoter and transcriptional regulatory elements for IRX5 are contained in SEQ ID NO:3). Some of the above mentioned secondary cell wall promoter sequences can be found within the polynucleotide sequences provided herein as SEQ ID NOs:36-61. A promoter originated from one plant species may be used to direct gene expression in another plant species.
A polynucleotide is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).
The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
The term “expression cassette” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, RNAi, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. One example of an expression cassette is a polynucleotide construct that comprises a polynucleotide sequence encoding a HCHL protein operably linked to a promoter that is heterologous to the plant cell into which the expression cassette may be introduced. In some embodiments, an expression cassette comprises a polynucleotide sequence encoding a HCHL protein that is targeted to a position in the genome of a plant such that expression of the HCHL polynucleotide sequence is driven by a promoter that is present in the plant.
The term “plant,” as used herein, refers to whole plants and includes plants of a variety of a ploidy levels, including aneuploid, polyploid, diploid and haploid. The term “plant part,” as used herein, refers to shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), branches, roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, and plant tissue (e.g., vascular tissue, ground tissue, and the like), as well as individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, and seeds. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae.
The term “biomass,” as used herein, refers to plant material that is processed to provide a product, e.g., a biofuel such as ethanol, or livestock feed, or a cellulose for paper and pulp industry products. Such plant material can include whole plants, or parts of plants, e.g., stems, leaves, branches, shoots, roots, tubers, and the like.
The term “reduced lignification” encompasses both reduced size of a lignin polymer, e.g., a shorter lignin polymer chain due to a smaller number of monolignols being incorporated into the polymer, a reduced degree of branching of the lignin polymer or a reduced space filling (also called a reduced pervaded volume). Typically, a reduced lignin polymer can be shown by detecting a decrease in it molecular weight or a decrease in the number of monolignols by at least 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, or more, when compared to the average lignin molecule in a control plant. Methods for detecting reduced lignification are described in detail in the example section of this application.
As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “plant cell” includes a plurality of such plant cells.

II. Introduction

Plant cell walls are constituted by a polysaccharidic network of cellulose microfibrils and hemicellulose embedded in an aromatic polymer known as lignin. This ramified polymer is mainly composed of three phenylpropanoid-derived phenolics (i.e., monolignols) named p-coumaryl, coniferyl, and sinapyl alcohols which represent the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units (Boerjan et al., 2003). Monolignols have a C₆C₃carbon skeleton which consists of a phenyl ring (C₆) and a propane (C3) side chain. Lignin is crucial for the development of terrestrial plants as it confers recalcitrance to plant cell walls. It also provides mechanical strength for upright growth, confers hydrophobicity to vessels that transport water, and acts as a physical barrier against pathogens that degrade cell walls (Boudet, 2007). Notably, lignin content and composition are finely regulated in response to environmental biotic and abiotic stresses (Moura et al., 2010).
Economically, lignocellulosic biomass from plant cell walls is widely used as raw material for the production of pulp in paper industry and as ruminant livestock feed. Plant feedstocks also represent a source of fermentable sugars for the production of synthetic molecules such as pharmaceuticals and transportation fuels using engineered microorganisms (Keasling, 2010). However, negative correlations exist between lignin content in plant biomass and pulp yield, forage digestibility, or polysaccharides enzymatic hydrolysis (de Vrije et al., 2002; Reddy et al., 2005; Dien et al., 2006; Chen and Dixon, 2007; Dien et al., 2009; Taboada et al., 2010; Elissetche et al., 2011; Studer et al., 2011). Consequently, reducing lignin recalcitrance in plant feedstocks is a major focus of interest, especially in the lignocellulosic biofuels field for which efficient enzymatic conversion of polysaccharides into monosaccharides is crucial to achieve economically and environmentally sustainable production (Carroll and Somerville, 2009).
Lignin biosynthesis is well characterized and well conserved across land plants (Weng and Chapple 2010). Genetic modifications such as silencing of genes involved in particular steps of this pathway or its regulation have been employed to reduce lignin content (Simmons et al., 2010; Umezawa, 2010) but this approach often results in undesired phenotypes such as dwarfism, sterility, reduction of plant biomass, and increased susceptibly to environmental stress and pathogens (Bonawitz and Chapple, 2010). These pleiotropic effects are generally the consequences of a loss of secondary cell wall integrity, accumulation of toxic intermediates, constitutive activation of defense responses, or depletion of other phenylpropanoid-derived metabolites which are essential for plant development and defense (Li et al., 2008; Naoumkina et al., 2010, Gallego-Giraldo et al., 2011). Alternatively, changing the recalcitrant structure and physico-chemical properties of lignin can be achieved by modifying its monomer composition. For example, incorporation of coniferyl ferulate into lignin improves enzymatic degradation of cell wall polysaccharides (Grabber et al., 2008). Recently, it has been demonstrated that enrichment in 5-hydroxy-G units and reduction in S units in lignin contribute to enhanced saccharification efficiencies without affecting drastically biomass yields and lignin content (Weng et al., 2010; Dien et al., 2011; Fu et al., 2011).
In this study, as an alternative strategy to reduce lignin recalcitrance, the inventors developed a dominant approach that uses precursors derived from the lignin biosynthetic pathways to enhance production of non-conventional monolignols, namely C₆C₁phenolics. These phenol units lack propane side chain and thus have different polymerization properties compared to classic C₆C₃monolignols. Such C₆C₁phenolics are usually found in trace amount in some lignins and form the so-called ‘benzyl end-groups’ (Kim et al., 2000; Ralph et al., 2001; Kim et al., 2003; Morreel et al., 2004; Ralph et al., 2008; Kim and Ralph, 2010). The inventors considered increasing C₆C₁end-group phenolics in lignin to reduce its polymerization degree and native branched structure. For this purpose, a hydroxycinnamoyl-CoA hydratase-lyase (HCHL, EC 4.2.2.101/EC 4.1.2.41) from Pseudomonas fluorescens was expressed in stems of Arabidopsis. HCHL is an enzyme that catalyzes the hydration of the double bond of the lignin precursor p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA thioesters, followed by a retro aldol cleavage reaction that produces the corresponding C₆C₁hydroxybenzaldehydes and acetyl-CoA (FIG. 1; Mitra et al., 1999). The promoter of a secondary cell wall cellulose synthase gene (Cesa4/IRX5) was used to restrict HCHL expression in lignified tissues of the stem (xylem and interfascicular fibers) and prevent depletion of hydroxycinnamoyl-CoAs in other tissues in which they are precursors of hydroxycinnamate conjugates and other derivates involved in plant defense and development (Gou et al., 2009; Luo et al., 2009; Buer et al., 2010; Milkowski and Strack, 2010). The data disclosed herein show that HCHL expression driven by the IRX5 promoter results for some lines in no significant changes in lignin content, plant development and biomass yields. It has also been demonstrated that C₆C₁phenolics accumulate as end-groups in the lignin of HCHL transgenics, which reduces lignin size and renders cell walls less recalcitrant to enzymatic hydrolysis.

III. Plants Having Reduced Lignification

A. Modification of Expression of an HCHL Enzyme
In one aspect, the present invention provides a method for engineering a plant having reduced lignification. This method includes these steps: first, introducing into the plant an expression cassette comprising a polynucleotide sequence encoding an HCHL enzyme and a promoter, with the coding sequence and the promoter being in an operably linked arrangement; and second, culturing the plant under conditions permissible for the expression of a functional HCHL to produce C₆C₁phenolics, which can be polymerized with other monolignols and thereby reducing lignification in the plant.
In particular, the present invention provides methods of engineering a plant having modified lignin polymers, which may have reduced size, molecular weight, and/or altered (especially reduced or less extensive) branching, that are substantially localized to the lignified tissue of the plant. This is achieved by first introducing into the plant an expression cassette as described above but in particular having a secondary cell wall specific promoter, and then culturing the plant under conditions in which the functional HCHL enzyme is expressed. This enzyme converts various hydroxycinnamoyl-coA into their respective hydroxybenzaldehydes that can be either directly incorporated or further modified (e.g., oxidation or reduction of the aldehyde group) by native enzymes prior to their incorporation into the lignin polymer by polymerization with native monolignols.
The expression cassette as described herein, when introduced into a plant, does not necessarily modify the lignin content. Vessel stays intact indicating that the lignin cell wall structure is still robust to prevent vessel collapse, but the lignin composition and properties are modified to a level that its recalcitrance is reduce.
One of skill in the art will understand that the HCHL that is introduced into the plant by an expression cassette described herein does not have to be identical to the Pseudomonas fluorescens HCHL, which was used in the experiments detailed in the example section of this disclosure. In some embodiments, the HCHL that is introduced into the plant by an expression cassette is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the Pseudomonas fluorescens HCHL. For example, a variant HCHL will have at least 80%, 85%, 90%, or 95% sequence identity in its amino acid residues as compared to SEQ ID NO:1, especially within one or more of the 8 highly conserved regions (shown in the 8 boxes in FIG. 8).
1. Hydroxycinnamoyl-CoA Hydratase-Lyase (HCHL)
In some embodiments, the expression cassette of this invention comprises a polynucleotide encoding an enzyme that produces modified monoligols that can cause reduced lignification. An example of such an enzyme is the Pseudomonas fluorescens HCHL, having the amino acid sequence set forth in SEQ ID NO:1. Additional examples of such HCHL suitable for use in the present invention include those shown in FIG. 8. Also appropriate for use in the present invention are variants HCHL, which may be naturally occurring or recombinantly engineered, provided the variants possess (1) substantially amino acid sequence identity to an exemplary HCHL (e.g., SEQ ID NO:1) and (2) the enzymatic activity to convert at least one lignin precursor p-coumaroy-CoA, caffeoyl-CoA, or feruloyl-CoA thioester into a corresponding C₆C₁hydroxylbenzaldehyde, as determined by an HCHL enzymatic assay known in the art by way of various scientific publications or described herein.
Examples of naturally occurring HCHL that can be used to practice the present invention includes, p-hydroxycinnamoyl CoA hydratase/lyase (HCHL), Enoyl-CoA hydratase/isomarase (ECH), Feruloyl-CoA hydratase/lyase (FCA, FerA), as well as those named in FIG. 8, the amino acid sequences for which are provided in SEQ ID NOs:4-34.
2. Secondary Cell Wall-Specific Promoters
In some embodiments, the polynucleotide encoding the HCHL is operably linked to a secondary cell wall-specific promoter. The secondary cell wall-specific promoter is heterologous to the polynucleotide encoding the HCHL, in other words, the promoter and the HCHL coding sequence are derived from two different species. A promoter is suitable for use as a secondary cell wall-specific promoter if the promoter is expressed strongly in the secondary cell wall, e.g., in vessel and fiber cells of the plant, but is expressed at a much lower level or not expressed in cells without the secondary cell wall.
In some embodiments, the promoter is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the native promoter of a gene encoding a secondary cell wall cellulose synthase Cesa4/IRX5, polynucleotide sequence set forth in Genebank Accession No. AF458083 _—1 and SEQ ID NO:35, and the promoter pIRX5 is contained in SEQ ID NO:3.
In some embodiments, the secondary cell wall-specific promoter comprises SEQ ID NO:3. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 or a variant thereof. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 comprising about 50 to about 1000 or more contiguous nucleotides of SEQ ID NO:3. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 comprising 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100; 75 to 1000, 75 to 900, 75 to 800, 75 to 700, 75 to 600, 75 to 500, 75 to 400, 75 to 300, 75 to 200; 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, or 100 to 200 contiguous nucleotides of SEQ ID NO:3.
Secondary cell wall-specific promoters are also described in the art. See, for example, Mitsuda et al 2005 Plant Cell; Mitsuda et al 2007 Plant Cell; Zhou et al 2009 plant cell; Ohtani et al 2011 Plant Journal. They are contained the polynucleotide sequences provided in this application as SEQ ID NO:36-61.
It will be appreciated by one of skill in the art that a promoter region can tolerate considerable variation without diminution of activity. Thus, in some embodiments, the secondary cell wall-specific promoter is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:3. The effectiveness of a secondary cell wall-specific promoter may be confirmed by an reporter gene (e.g., β-glucuronidase or GUS) assay known in the art or as described in the example section of this application.
B. Preparation of Recombinant Expression Vectors
Once the promoter sequence and the coding sequence for the gene of interest (e.g., a Pseudomonas fluorescens HCHL or any other HCHL as shown in FIG. 8) are obtained, the sequences can be used to prepare an expression cassette for expressing the gene of interest in a transgenic plant. Typically, plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a selectable marker. Such plant transformation vectors may also contain a promoter (e.g., a secondary cell wall-specific promoter as described herein), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
The plant expression vectors may include RNA processing signals that may be positioned within, upstream, or downstream of the coding sequence. In addition, the expression vectors may include regulatory sequences taken from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.
Plant expression vectors routinely also include selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin), herbicide resistance genes (e.g., phosphinothricin acetyltransferase), and genes encoding positive selection enzymes (e.g. mannose isomerase).
Once an expression cassette comprising a polynucleotide encoding the HCHL and operably linked to a promoter (especially a secondary cell wall specific promoter) has been constructed, standard techniques may be used to introduce the polynucleotide into a plant in order to express the HCHL and effectuate reduced lignification. See, e.g., protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.
Transformation and regeneration of plants is known in the art, and the selection of the most appropriate transformation technique will be determined by the practitioner. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence. Examples of these methods in various plants include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.
Following transformation, plants can be selected using a selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants or the ability to grow on a specific substrate, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic, herbicide, or substrate.
The polynucleotide sequence coding for an HCHL, as well as the polynucleotide sequence comprising a promoter (e.g., a secondary cell wall-specific promoter), can be obtained according to any method known in the art. Such methods can involve amplification reactions such as polymerase chain reaction (PCR) and other hybridization-based reactions or can be directly synthesized.
C. Plants in which Lignification can be Reduced
An expression cassette comprising a polynucleotide encoding an HCHL operably linked to a promoter, especially a secondary cell wall specific promoter, as described herein, can be expressed in various kinds of plants. The plant may be a monocotyledonous plant or a dicotyledonous plant. In some embodiments of the invention, the plant is a green field plant. In some embodiments, the plant is a gymnosperm or conifer.
In some embodiments, the plant is a plant that is suitable for generating biomass. Examples of suitable plants include, but are not limited to, Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, Jatropha, and Brachypodium.
In some embodiments, the plant into which the expression cassette of this invention is introduced is the same species of plant as the one from which the promoter is derived. In some embodiments, the plant into which the expression cassette is introduced is a different species of plant from the plant species the promoter is derived from.
D. Screening for Plants Having Reduced Lignification
After transformed plants are selected, the plants or parts of the plants may be evaluated to determine whether expression of the exogenous HCHL can be detected, e.g., by evaluating the level of RNA or protein, by measuring enzymatic activity of the HCHL, as well as by evaluating the size, molecular weight, content, or degree of branching in the lignin molecules found in the plants. These analyses can be performed using any number of methods known in the art.
In some embodiments, plants are screened by evaluating the level of RNA or protein. Methods of measuring RNA expression are known in the art and include, for example, PCR, northern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), and microarrays. Methods of measuring protein levels are also known in the art and include, for example, mass spectroscopy or antibody-based techniques such as ELISA, Western blotting, flow cytometry, immunofluorescence, and immunohistochemistry.
In some embodiments, plants are screened by assessing HCHL activity, and also by evaluating lignin size and composition. The enzymatic assays for HCHL are well known in the art and are described in this application. Lignin molecules can be assessed, for example, by nuclear magnetic resonance (NMR), spectrophotometry, microscopy, klason lignin assays, acetyl-bromide reagent or by histochemical staining (e.g., with phloroglucinol).

IV. Methods of Using Plants Having Reduced Lignification

Plants, parts of plants, or plant biomass material from plants having reduced lignification due to the expression of an exogenous HCHL in the secondary cell wall can be used for a variety of methods. In some embodiments, the plants, parts of plants, or plant biomass material generate less recalcitrant biomass for use in a conversion reaction as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used in a saccharification reaction, e.g., enzymatic saccharification, to generate soluble sugars at an increased level of efficiency as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase biomass yield or simplify downstream processing for wood industries (such as paper, pulping, and construction) as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase the quality of wood for construction purposes. In some embodiments the plants, parts of plants, or plant biomass material can be used in a combustion reaction, gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic or chemical). In further embodiments, the plants, parts of plants, or plant biomass is used as a forage crop and exhibit improved digestibility compared to wild-type plants.
Methods of conversion, for example biomass gasification, are known in the art. Briefly, in gasification plants or plant biomass material (e.g., leaves and stems) are ground into small particles and enter the gasifier along with a controlled amount of air or oxygen and steam. The heat and pressure of the reaction break apart the chemical bonds of the biomass, forming syngas, which is subsequently cleaned to remove impurities such as sulfur, mercury, particulates, and trace materials. Syngas can then be converted to products such as ethanol or other biofuels.
Methods of enzymatic saccharification are also known in the art. Briefly, plants or plant biomass material (e.g., leaves and stems) are optionally pre-treated with hot water, dilute alkaline, AFEX (Ammonia Fiber Explosion), ionic liquid or dilute acid, followed by enzymatic saccharification using a mixture of cell wall hydrolytic enzymes (such as hemicellulases, cellulases and beta-glucosidases) in buffer and incubation of the plants or plant biomass material with the enzymatic mixture. Following incubation, the yield of the saccharification reaction can be readily determined by measuring the amount of reducing sugar released, using a standard method for sugar detection, e.g. the dinitrosalicylic acid method well known to those skilled in the art. Plants engineered in accordance with the invention provide a higher saccharification efficiency as compared to wild-type plants, while the plants growth, development, or disease resistance is not negatively impacted.
Sugars generated from a saccharification reaction using plant biomass of the invention can be used for producing any product for which the sugars can serve as a carbon source. Examples of products include, but are not limited to, alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂and CO₂); antibiotics (e.g., penicillin and tetracycline); vitamins (e.g., riboflavin, B12, beta-carotene), fatty acids and fatty acid derivatives (as described, e.g., in PCT/US2008/068833); isoprenyl alkanoates (as described, e.g., PCT/US2008/068756, methyl butenol (as described, e.g., PCT/US2008/068831; fatty acid esters (as described, e.g., in PCT/US2010/033299), isoprenoid-based alternative diesel fuels (as described, e.g., in PCT/US2011/059784; a polyketide synthesized by a polyketide synthase, such as a diacid (see, e.g., PCT/US2011/061900), biofuels (see, e.g., PCT/US2009/042132) and alpha-olefins (see, e.g., PCT/US2011/053787).

EXAMPLES

The following examples are provided to illustrate but not to limit the claimed invention.

Example 1

Expression of Bacterial HCHL in Arabidopsis

I. Materials and Methods

Plant Material and Growth Conditions

Arabidopsis thaliana (ecotype Columbia, Col-0) seeds were germinated directly on soil. Growing conditions were 14 h of light per day at 100 mmol m⁻²s⁻¹, 22° C., 55% humidity. Selection of T1 and T2 homozygote transgenic plants was made on solid Murashige and Skoog vitamin medium (PhytoTechnology Laboratories) supplemented with 1% sucrose, 1.5% agar (Sigma-Aldrich) adjusted to pH 5.6-5.8, and containing 50 μg mL⁻¹kanamycin.

Chemicals

4-Hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, 5-hydroxyvanillin, 4-hydroxybenzyl alcohol, vanillyl alcohol, and 1-methyl-2-pyrrolidinone were purchased from Sigma-Aldrich. Vanillic acid, syringic acid, 3,4-dihydroxybenzaldehyde, syringaldehyde, and sinapyl alcohol were purchased from Alfa Aesar. 5-Hydroxyvanillic acid was obtained from Chromadex, and 3,4-dihydroxybenzyl alcohol from TCI America.
pIRX5:GUS Line and GUS Staining
Arabidopsis line CS70758 (ecotype Columbia, Col-2) was obtained from the Arabidopsis Biological Resource Center (ABRC). This line has a pMLBART plasmid containing an expression cassette consisting of the genomic fragment located upstream of the CESA4 start codon fused to the GUS gene. Histochemical GUS activity was performed as previously described (Eudes et al., 2006). Various organs of soil-grown line CS70758 were incubated for 1 h or 8 h at 37° C. in the GUS assay buffer using 5-bromo-4-chloro-3-indolyl-D-glucuronic acid (Indofine Chemical Company, Inc.) as a substrate. After staining, stem samples (1 cm) were cross-sectioned (80 μm) using a vibratome before observation under the microscope (Leica).

IRX5:HCHL Construct and Plant Transformation

For HCHL expression in Arabidopsis, the binary vector pTKan which is derived from pPZP212 was used (Hajdukiewicz et al., 2004). A Gateway cloning cassette (Invitrogen) was inserted between XhoI and PstI restriction sites to produce a pTKan-GW vector. The nucleotide sequence of the IRX5 promoter was amplified by PCR from Arabidopsis (ecotype Columbia, Col-0) genomic DNA using oligonucleotides 5′-CCCGGCGGCCGCATGAAGCCATCCTCTACCTCGGAAA-3′ and 5′-CCCGGCTAGCGGCGAGGTACACTGAGCTCTCGGAA-3′ (NotI and NheI restriction sites underlined), and inserted between the ApaI and SpeI restriction sites of pTKan-GW to produce a pTKan-pIRX5-GW expression vector. A codon-optimized nucleotide sequence encoding the HCHL enzyme from Pseudomonas fluorescens AN103 (accession number CAA73502) for expression in Arabidopsis was synthesized without stop codon (Genescript) and amplified by PCR using oligonucleotides 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTACTTACGAGGGAAGATG G-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCTCTTGTAAGCCTGGAGTCC-3′ (attb1 and attb2 sites underlined) for cloning into the Gateway pDONR221-f1 entry vector (Lalonde et al 2010). A sequence-verified HCHL entry clone was LR recombined with the pTKan-pIRX5-GW vector to generate the final IRX5:HCHL construct. The construct was introduced into wild type Arabidopsis plants (ecotype Col0) via Agrobacterium tumefaciens-mediated transformation (Bechtold and Pelletier, 1998).

RNA Extraction and RT-PCR

Total RNA (1 μg) extracted from inflorescence stems of IRX5:HCHL T1 transformants and wild type plants using the Plant RNeasy extraction kit (Qiagen) was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche applied Science). The obtained cDNA preparation was quality-controlled for PCR using tub8-specific oligonucleotides (5′-GGGCTAAAGGACACTACACTG-3′/5′-CCTCCTGCACTTCCACTTCGTCTTC-3′). Oligonucleotides 5′-ATGTCTACTTACGAGGGAAGATGG-3′ and 5′-TCTCTTGTAAGCCTGGAGTCC-3′ were used for the detection of HCHL expression by PCR.

Western Blot Analysis

For protein extraction, inflorescence stems of IRX5:HCHL T2 transformants and wild type plants were ground in liquid nitrogen, and 0.25 g of the resulting powder was homogenized with the extraction buffer [100 mM Tris-HCl pH 6.5, 2% (w/v) polyvinylpyrrolidone, 2% (v/v) β-mercaptoethanol, 1% (w/v) SDS] at 1400 rpm for 30 min. The mixture was centrifuged at 20,000 g for 5 min and the supernatant collected for protein quantification using the Bradford method (Bradford, 1976) and bovine serum albumin as a standard. For electrophoresis, soluble protein (5 μg) were mixed with 0.2 M Tris-HCl, pH 6.5, 8% (w/v) SDS, 8% (v/v) β-mercaptoethanol, 40% (v/v) glycerol, and 0.04% (w/v) bromophenol blue and incubated at 40° C. for 30 min. Proteins were separated by SDS-PAGE using 8-16% (w/v) polyacrylamide gradient gels (Invitrogen) and electrotransferred (100 volts, 45 min) onto PVDF membranes (Thermo Fisher Scientific). Blotted membranes were incubated 1 h in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6) containing 2% (w/v) non-fat milk powder, and incubated overnight with the universal antibody (1:20000) in TBS-T containing 2% (w/v) non-fat milk powder. Membranes were then washed in TBS-T for 30 min and incubated for 1 h with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:20000; Sigma-Aldrich) in TBS-T containing 2% (w/v) non-fat milk powder. Membranes were then washed in TBS-T for 30 min, and detection was performed by chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

HCHL Activity

For protein extraction, inflorescence stems of IRX5:HCHL T2 transformants and wild type plants were ground in liquid nitrogen, and 0.25 g of the resulting powder was homogenized with 25 mg of polyvinylpolypyrrolidone and 1.25 mL of extraction buffer (EB; 100 mM Tris-HCl, pH 8.5, 20 mM DTT, and 10 mM Na₂EDTA). Extracts were shaken at 1400 rpm for 15 min at 4° C., and centrifuged for 30 min at 20,000 g at 4° C. Supernatants were collected, adjusted to 2.5 mL with EB, and applied to PD10 columns (GE healthcare) pre-equilibrated with 25 mL of EB. Proteins were eluted with 3.5 mL of EB and quantified using the Bradford method (Bradford, 1976) and bovine serum albumin as a standard.
For HCHL activity, 5 μL of protein extract was incubated for 15 min at 30° C. with 150 μM feruloyl-CoA in 100 mM Tris-HCl pH 8.5 in a total volume of 50 μL. Total amounts of protein per reaction varied from 4 to 6.5 μg. Reactions were stopped with 50 μL of cold acidified methanol (12% glacial acetic acid/88% methanol, v/v) and stored at −70° C. until LC-MS analysis.

Biomass

For biomass measurements, IRX5:HCHL and wild type plants were grown until senescence and dried stems were collected without roots, leaves and siliques before weighing. Statistical analysis was performed using ANOVA followed by Scheffe post hoc test.

Microscopy

Five-week-old plants were use for microscopy. Stem segments cut between the first and second internodes were embedded in 4% agarose. Stem semi-thin sections (100-μm thickness) were obtained using a vibratome (Leica). For toluidine blue O (TBO) staining, sections were incubated in a 0.05% (w/v) solution of TBO (Sigma-Aldrich) in water for 30 sec and rinsed with water. For Wiesner lignin staining, sections were incubated for 3 min in phloroglucinol-HCl reagent (VWR International) and rinsed with water. For Mäulne lignin staining, sections were incubated in 4% KMnO₄for 5 min, rinsed with water, incubated in 37% HCl/H₂O (1:1, v/v) for 2 min, and observed after addition of a drop of aqueous ammonia. Sections were immediately observed using bright field light microscopy (Leica DM4000 B).

Soluble Phenolics Extraction

For extraction of methanol soluble phenolics, approximately 200 mg of frozen stem powder was mixed with 1 mL of 80% (v/v) methanol-water and shaken for 1 h at 1400 rpm. Extracts were cleared by centrifugation (5 min, 20,000 g), mixed with 400 μL of analytical grade water and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore) prior to LC-MS analysis. Alternatively, an aliquot of the filtered extracts was dried under vacuum, resuspended with 1 N HCl and incubated at 95° C. for 3 h for acid hydrolysis. The mixture was subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and resuspended in 50% (v/v) methanol-water prior to LC-MS analysis.

Cell-Wall Bound Phenolics Extraction

For extraction of cell-wall bound phenolics, mature senesced stems were collected without the leaves and siliques, and ball-milled to a fine powder using a Mixer Mill MM 400 (Retsch) and stainless steel balls for 2 min at 30 s⁻¹. Extract-free cell wall residues (CWR) were obtained by sequentially washing 60 mg of ball-milled stems with 1 ml of 96% ethanol at 95° C. twice for 30 min, and vortexing with 1 mL of 70% ethanol twice for 30 sec. The resulting CWR were dried in vacuo overnight at 30° C. Approximately 6 mg of CWR was mixed with 500 μL of 2 M NaOH and shaken at 1400 rpm for 24 h at 30° C. The mixture was acidified with 100 μL of concentrated HCl, and subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and suspended in 50% (v/v) methanol-water prior to LC-MS analysis.

LC-MS

Separation of C₆C₁phenolic acids and aldehydes was conducted on a Poroshell-120 column (150 mm length, 3 mm internal diameter, 2.7 μm particle size) using a 1200 Series HPLC system (Agilent Technologies Inc.). Analytes were separated using a gradient elution with mobile phase composition of 0.1% formic acid in water as solvent A, and 0.1% formic acid in acetonitrile-water (98:2, v/v) as solvent B. The elution gradient was 0-5 min 13% B, 5-7 min 50% B, 7-8 min 50% B, and 8-11 min 13% B, using a flow rate of 0.55 mL min⁻¹. The HPLC system was coupled to an Agilent 6210 time-of-flight (TOF) mass spectrometer (MS) via a 1:7 post-column split. Analyses were conducted using Electrospray ionization (ESI) in the positive ion mode. Detection of [M+H]⁺ ions was carried out in full scan mode at 0.85 spectra sec and a cycle time of 1.176 sec spectrum⁻¹using the following parameters: capillary voltage 3500 V, fragmentor 165 V, skimmer 50 V and OCT RF 170 V, drying gas flow rate 9 L min⁻¹, nebulizer pressure 15 psig, drying gas temperature 325° C. Separation of C₆C₁phenolic alcohols was conducted on the same HPLC and MS system using the same HPLC column. Analytes were separated using gradient elution with a mobile phase composition of 0.1% formic acid in water as solvent A, and 0.1% formic acid in methanol-water (98:2, v/v) as solvent B. Elution conditions were the same as described above. Analyses were conducted using atmospheric pressure chemical ionization (APCI) in the positive ion mode. Detection of [M−H₂O+H]⁺ ions was carried as described above except for the following parameters: capillary voltage 3200 V, corona current 4 μA, drying gas flow rate 12 L min⁻¹, nebulizer pressure 30 psig, vaporizer temperature 350° C. Quantification of compounds was made by comparison with standard curves of authentic compounds prepared in 50% (v/v) methanol-water.

Lignin Analysis

Extract-free samples (CWR) of ball-milled mature senesced stems were prepared using a Soxhlet apparatus by sequentially extracting the ground material with toluene:ethanol (2:1, v/v), ethanol, and water (Sluiter et al., 2008). The determination of lignin content using the standard Klason procedure (Dente, 1992) and the thioacidolysis procedure (Lapierre et al., 1995; 1999) were carried out on CWR. The lignin-derived monomers were identified by GC-MS as their trimethyl-silylated derivatives. All the lignin analyses were performed in duplicate.

Total and Hemicellulosic Sugar Analysis

For total sugar hydrolysis, CWR of ball-milled mature senesced stems (50 mg) were swelled in 500 μL H₂SO₄(72%, w/v) at 30° C. for 60 min, and autoclaved at 120° C. for 1 h in dilute H₂SO₄(4%, w/v) after addition of deionized water (14 mL). Samples were cooled down at room temperature and filtered using pre-weighted GF/C glass microfiber filters (Whatman). Filtrates were collected and diluted 100 times with deionized water prior to HPAEC-PAD analysis. For hemicellulose hydrolysis, CWR of ball-milled mature dried stems (5 mg) were hydrolyzed in 1 ml of 2 M trifluoroacetic acid (TFA) for 1 h at 120° C. TFA was removed by drying under vacuum and the residue suspended in deionized water (1 mL) prior to HPAEC-PAD analysis.

HPAEC-PAD Analysis

Monosaccharide composition was determined by HPAEC-PAD of hydrolyzed material. Chromatography was performed on a PA20 column (Dionex) at a flow rate of 0.5 mL min⁻¹. Before injection of each sample (20 μL) the column was washed with 200 mM NaOH for 10 min, then equilibrated with 10 mM NaOH for 10 min. The elution program consisted of a linear gradient from 10 mM NaOH to 5 mM NaOH from 0 to 1.5 min, followed by isocratic elution with 5 mM NaOH from 1.5 to 20 min, and a linear gradient up to 800 mM NaOH from 20 to 43 min. Monosaccharides were detected using a pulsed amperometric detector (gold electrode) set on waveform A according to manufacturer's instructions. A calibration curve of monosaccharide standards that includes L-Fuc, L-Rha, L-Ara, D-Gal, D-Glc, D-Xyl, D-GalA and D-GlcA (Sigma-Aldrich) was run for verification of response factors. Statistical analysis was performed using ANOVA followed by Tukey's test.

FT-Raman and FT-IR Spectral Analyses

FT-Raman spectroscopy was conducted on CWR of ball-milled mature senesced stems (2 mg) from three independent cultures. Raman spectra were collected using a Bruker MultiRAM FT-Raman system equipped with a 1064 nm diode laser (Bruker Optics Inc.). Five spectra were acquired for each sample with spectral resolution of 4 cm⁻¹using a laser power of 50 mW and 256 scans to achieve good signal-to-noise ratio. White light correction of the acquired spectra was performed to correct the influence of the optics on the spectrometer. Spectra in the range of 200-3500 cm⁻¹were smoothed and baseline corrected using OPUS software. Lignin and polysaccharides (cellulose and hemicellulose) content were determined using integrated intensities measured over the range of 1555-1690 cm⁻¹and 1010-1178 cm⁻¹, respectively. For FT-IR spectroscopy, analyses were carried out on xylem and interfascicular fibers tissues from 50-μm thick sections of the basal region of stems of five-week-old plants. For both wild type and IRX5:HCHL (line 2), five to six sections from three different plants were analyzed. FT-IR spectra were collected from a 50 μm×50 μm window targeting xylem vessels or interfascicular fibers, and normalization of the data and statistical analysis (Student's t-test) were performed as described (Mouille et al., 2003).

Isolation of Cellulolytic Lignin (CEL) and Size Exclusion Chromatography (SEC)

CEL lignin was purified from wild type and IRX5:HCHL (line 2) plants. One gram of ball-milled mature senesced stems was mixed with 50 mM NaCl (30 ml) and incubated overnight at 4° C. After centrifugation (2,800 g, 10 min), the biomass was extracted sequentially by sonication (20 min) with 80% ethanol (three times), acetone (one time), chloroform-methanol (1:1, v/v, one time) and acetone (one time). The obtained CWR were ball-milled for 3 h per 500 mg of sample (in 10 min on/10 min off cycles) using a PM100 ball mill (Retsch) vibrating at 600 rpm with zirconium dioxide vessels (50 mL) containing ZrO₂ball bearings (10×10 mm). Ball-milled walls (490 mg for wild type and 480 mg for IRX5:HCHL) were transferred to centrifuge tubes (50 mL) and digested four times over three days at 30° C. with crude cellulases (Cellulysin; Calbiochem; 60 mg g⁻¹of sample) in NaOAc pH 5.0 buffer (30 mL) under gentle rotation. The obtained CEL was washed 3 times with deionized water and lyophilized overnight. CEL recovered were 131 mg for wild type (27.3%) and 101 mg for IRX5:HCHL (20.6%). For SEC analysis, 1% (w/v) CEL lignin solutions were prepared in analytical-grade 1-methyl-2-pyrrolidinone-DMSO (1:1, v/v) and sonicated for 3 hours at 40° C.
Polydispersity of dissolved lignin was determined using analytical techniques SEC UV-F and SEC UV-A as described elsewhere (George et al., 2011, submitted). An Agilent 1200 series binary LC system (G1312B) equipped with FL (G1321A) and DA (G1315D) detectors was used. Separation was achieved with a Mixed-D column (5 mm particle size, 300 mm×7.5 mm i.d., linear molecular weight range of 200 to 400,000 u, Polymer Laboratories) at 80° C. using a mobile phase of NMP at a flow rate of 0.5 mL min⁻¹. Absorbance of material eluting from the column was detected at 300 nm (UV-A). Excitation 250 nm and emission 450 nm were used for UV-F detection. Intensities were area normalized and molecular mass estimates were determined after calibration of the system with polystyrene standards.

Cell Wall Pretreatments and Saccharification

Ball-milled mature senesced stems (10 mg) were mixed with 340 μL of water, 340 μL of H₂SO₄(1.2%, w/v), or 340 μL of NaOH (0.25%, w/v) for hot water, dilute acid, or dilute alkaline pretreatments, respectively, incubated at 30° C. for 30 min, and autoclaved at 120° C. for 1 h. After cooling down at room temperature, samples pretreated with dilute acid and dilute alkaline were neutralized with 5 N NaOH (25 μL) and 1.25 N HCl (25 μL), respectively. Saccharification was initiated by adding 635 μL of 100 mM sodium citrate buffer pH 6.2 containing 80 g ml⁻¹tetracycline, 5% w/w cellulase complex NS50013 and 0.5% w/w glucosidase NS50010 (Novozymes). After 72 h of incubation at 50° C. with shacking (800 rpm), samples were centrifuged (20,000 g, 3 min) and 10 μL of the supernatant was collected for reducing sugar measurement using the DNS assay and glucose solutions as standards (Miller, 1959).

Transcriptome Studies

Microarray analysis was performed on complete Arabidopsis thaliana transcriptome microarrays containing 24,576 gene-specific tags (GSTs) corresponding to 22,089 genes from Arabidopsis (Crowe et al., 2003; Hilson et al., 2004). RNA samples from three independent biological replicates were isolated and separately analyzed. For each biological replicate, RNA from the main inflorescence stem (first two internodes) of three plants were pooled. For each comparison, one technical replication with fluorochrome reversal was performed for each biological replicate (i.e. nine hybridizations per comparison). Reverse transcription of RNA was conducted in the presence of Cy3-dUTP or Cy5-dUTP (PerkinElmer-NEN Life Science Products), and hybridization and scanning of the slides were performed as described in Lurin et al. (2004).

Statistical Analysis of Microarray Data

Statistical analysis was performed with normalization based on dye swapping (i.e., four arrays, each containing 24,576 GSTs and 384 controls) as previously described (Gagnot et al., 2008). For the identification of differentially expressed genes, we performed a paired t test on log ratios, assuming that the variance of the log ratios was similar for all genes. Spots with extreme variances (too small or too large) were excluded. The raw P values were adjusted by the Bonferroni method, which controls the family-wise error rate (with a type I error equal to 5%) to minimize the number of false positives in a multiple-comparison context (Ge et al., 2003). Genes with a Bonferroni P value ≦0.05 were considered to be differentially expressed, as previously described (Gagnot et al., 2008).

Data Deposition

Microarray data from this article were deposited at GEO (http://www.ncbi.nlm.nih.gov/geo/) and at CATdb (http://urgv.evry.inra.fr/CATdb/) according to Minimum Information about a Microarray Experiment standards (MIME).

II. Results

Expression of a Bacterial HCHL Enzyme in Arabidopsis Stems

The tissue specific activity of the IRX5 promoter was verified using the beta-glucuronidase (GUS) as a reporter gene. Gus activity was essentially detected in the xylem vessels of the stem. After prolonged incubations, stem interfascicular fibers also showed strong GUS activity, and more moderate staining was observed in the vascular system of young seedlings, siliques, rosette and cauline leaves. No activity was detected in other organs or tissues except for the style and anthers (FIG. 9). A codon-optimized sequence encoding HCHL from Pseudomonas fluorescens AN103 was designed and cloned downstream of the IRX5 promoter for preferential expression in lignified tissues of Arabidopsis stems. Presence of HCHL transcripts in the main stem of five independent transformants was verified by RT-PCR in the T1 generation (FIG. 2A). Plants homozygous for the IRX5:HCHL construct were identified in the T2 generation, and used to analyze HCHL protein expression and activity in stems. Western blotting analysis using the ‘universal antibody’ allowed detection of HCHL in stem extracts of the five selected transgenic lines (FIG. 2B; Eudes et al. 2010). Furthermore, HCHL activity could be detected in the stem of these lines, ranging from 0.025 to 0.16 pkat vanillin μg⁻¹protein using feruloyl-CoA as substrate, whereas no detectable activity was observed in protein extracts of wild type plants (Table I). Two transgenic lines showing the highest and the lowest levels of HCHL activity, and two lines exhibiting intermediate activity level were selected for detailed analysis.

Growth Characteristics and Tissue Anatomy of IRX5:HCHL Lines

IRX5:HCHL plants had growth and development characteristics visually similar to the wild type from early rosette stage and until senescence (FIG. 10). However, mature senesced stems from lines IRX5:HCHL (4) and IRX5:HCHL (5) were little bit shorter (22% and 13% reduction) and had lower dry weight yield (30% and 16% reduction) compared to control plants, whereas those from lines IRX5:HCHL (1) and IRX5:HCHL (2) were not significantly different (Table II). Stem tissues of five-week-old IRX5:HCHL plants were inspected using light microscopy. Transverse stem cross-sections stained with Mäule and phloroglucinol-HCl reagents, which are indicative of S-units and hydroxycinnamaldehyde units in lignin, respectively, showed similar patterns between transgenic and wild type plants (FIGS. 3A and 3B). Similarly, lignin in stem sections stained with toluidine blue O did not revealed any quantitative differences between genotypes (FIG. 3C). A few collapsed xylem structures were, however, occasionally observed on some stem cross-sections of line IRX5:HCHL (4), but were absent in sections from other lines (FIG. 3C). Overall, these data suggest that lignin content is not drastically reduced in IRX5:HCHL plants.

IRX5:HCHL Lines Accumulate C₆C₁Soluble Phenolics

Methanol soluble fractions from stems of five-week-old wild type and IRX5:HCHL plants were extracted and analyzed by LC-MS. Analysis was performed to focus on hydroxybenzaldehydes, direct products of HCHL activity, and possible derivatives such as hydroxybenzoyl alcohols and hydroxybenzoic acids and their glucose conjugates. Trace amounts of 4-hydroxybenzaldehyde (HBAld), 3,4-dihydroxybenzaldehyde (3,4-DHBAld), and 4-hydroxybenzoic acid (HBA) were detected in IRX5:HCHL stem soluble extracts but not in wild type (Table III). Notably, much larger quantities of 4-hydroxybenzoic acid glucoside (HBAGlc) and 4-hydroxybenzoic acid glucose ester (HBAGE) were detected in IRX5:HCHL plant soluble extracts (ranging from 0.48 to 0.57 mg g⁻¹FW for HBAGlc, and from 0.96 to 1.65 mg g⁻¹FW for HBAGE), whereas trace amounts of these HBA-glucose conjugates were present in wild type extracts (Table III).
Considering that other soluble C₆C₁phenolics could be glycosylated, acid hydrolysis of the soluble fractions was performed to release aglycones from conjugated forms. This procedure brought down HBAGE and HBAG pools to undetectable levels, and concomitantly increased free HBA content in samples (Table IV). HBA content in the IRX5:HCHL lines ranged between 1.59 and 2.49 mg g⁻¹FW, which represents a 113 to 179 fold increase compared to values observed in wild type samples, and indicates that 88-94% of HBA accumulated in transgenic lines is glycosylated. In addition to HBA, other C₆C₁phenolics quantified in acid-treated extracts include vanillin (Van), 5-hydroxyvanillin (5OH-Van), syringaldehyde (Syrald), 5-hydroxyvanillic acid (5OH-VA), and syringic acid (SyrA), which are only detected in IRX5:HCHL extracts, as well as HBAld, 3,4-DHBAld, 3,4-dihydroxybenzoic acid (3,4-DHBA), and vanillic acid (VA), which are on average 14, 119, 1.6, and 40 times more abundant in IRX5: HCHL extracts compared to wild type, respectively (Table IV).

IRX5:HCHL Lines Show Enrichment in Cell Wall-Bound C₆C₁Phenolics

Extract-free cell wall residues (CWR) obtained from mature senesced stems of wild type and IRX5:HCHL plants were subjected to mild alkaline hydrolysis for the release of loosely-bound phenolics. This procedure released from the cell wall samples some HBAld, 3,4-HBAld, Van, 5OH-Van, SyrAld, HBA, VA, and SyrA, which were quantified using LC-MS analysis. 5OH-Van, undetectable in wild type cell wall, was present in that of IRX5:HCHL samples and HBAld, SyrAld, HBA, VA, and SyrA were increased on average by approx 2, 6, 68, 2 and 5 fold in cell walls of IRX5:HCHL plants compared to the wild type, respectively (Table V). These results indicate that larger amounts of C₆C₁phenolics are loosely-bound to cell walls in IRX5:HCHL plants. On the other hand, amount of ferulate and coumarate released from cell walls using this procedure did not differ between transgenic and wild type samples.

Spectral Analysis of IRX5:HCHL Plant Stems

Line IRX5:HCHL (2), which showed no defective xylem structures and biomass yield similar to wild type plants, was selected for further analyses. Fourier transformed Raman (FT-Raman) spectroscopy was used to determine the chemical composition of CWR obtained from senesced stems of IRX5:HCHL plants. Compared to the wild type, data showed that lignin content and amount of polysaccharides (cellulose and hemicellulose) in IRX5:HCHL plants were not significantly different (FIG. 4A). Moreover, Fourier transformed infrared (FT-IR) spectral analysis conducted on lignified tissues (xylem and interfascicular fibers) of transverse stem sections of five-week-old IRX5:HCHL and wild type plants revealed differences between the two genotypes (FIG. 4B). In particular, significant changes in spectra were observed for bands assigned to different bending or stretching of lignin (Agarwal and Atalla, 2010, Fackler et al., 2010). For example, absorptions at wavelengths 1589 cm⁻¹and 1506 cm⁻¹(aryl ring stretching), 1464 cm⁻¹(C—H group deformation), 1425 cm⁻¹(methoxyl C—H group deformation), 1379 cm⁻¹(aromatic skeletal vibrations combined with C—H group in plane deformation), and 1268 cm⁻¹(aryl ring breathing with C=O group stretch) were modified in fibers, whereas the most significant difference for xylem cell walls was observed at band 1367 cm⁻¹(methoxyl C—H group deformation). Overall, spectral analyses suggested compositional modifications of lignin in plants expressing HCHL.

Monosaccharide Content and Composition in IRX5:HCHL Plant Stems

Monosaccharide composition was determined after sulfuric acid hydrolysis of total cell wall polysaccharides from mature senesced stems of line IRX5:HCHL (2) and wild type plants. Although both genotypes had similar amount of total monosaccharides, IRX5:HCHL plants showed reduction in glucose (−12%) and increase in xylose (+22%) and arabinose (+16%) compared to wild type plants (Table VI). Moreover, hemicellulosic monosaccharides released from CWR using trifluoroacetic acid showed that total amount of sugar quantified in this hydrolysate was 23% higher in IRX5:HCHL stems which corresponds to higher xylose (+23%) and arabinose (+22%) contents compared to wild type (Table VI).
Incorporation of Unusual C₆C₁Monomers into the Lignin of IRX5:HCHL Plants
Lignin content and monomeric composition in mature senesced stems from wild type and IRX5:HCHL (2) plants was determined on CWR. In two independent cultures, klason lignin (KL) was identical and accounted for about 20% of the CWR for both wild type and IRX5:HCHL plants (Table VII). Lignin monomer composition was evaluated by thioacidolysis, a chemical degradative method that generates thioethylated monomers from lignin units involved in labile β-O-4 bonds. Data showed that total amount of conventional H, O, and S monomers released from CWR after thioacidolysis (or total yield) was reduced by 25% and 16% in the two independent cultures of IRX5:HCHL plants compared to the wild type, indicating that fewer of these three monolignols are crosslinked as β-O-4 bond in transgenics (Table VII). Considering identical KL values for both wild type and IRX5:HCHL CWR, these data indicate higher frequency of thioacidolysis-resistant bonds between lignin monomers in transgenic plants. The relative amount of G and S units recovered from this lignin fraction was unchanged, both wild type and transgenic samples showing an S/G ratio ranging between 0.34-0.36, however, molar frequency of H units was significantly higher in IRX5:HCHL plants (Table VII). Furthermore, the content of non-conventional units such as Van, Syrald, and SyrA released by thioacidolysis showed on average a 1.44-, 20.8-, and 1.65-fold increase in IRX5:HCHL plants compared to wild type plants, respectively. Interestingly, two new lignin units were released from the lignin of transgenics plants, which were identified as C₆C₁vanillyl alcohol (Vanalc) and syringyl alcohol (Syralc) (Table VIII). On the other hand, the content of coniferaldehyde end-groups (Cald) and VA was unchanged between the two genotypes (Table VIII). Overall, these data showed higher amount of C₆C₁phenolic end-groups among monomers released by thioacidolysis from IRX5:HCHL stem cell walls compared to wild type.

Lignin of IRX5:HCHL Plants has Reduced Molecular Mass

The polydispersity of cellulolytic lignin purified from wild type and IRX5:HCHL (2) stems was determined using size exclusion chromatography (SEC). Elution profiles acquired by monitoring UV-A absorbance (SEC UV-A₃₀₀) and UV-F fluorescence (SEC UV-F _ex250/_em450) of the dissolved lignin revealed differences between wild type and IRX5:HCHL plants (FIG. 6). First, total area corresponding to the largest mass peak detected between 7 min and 13.5 min was severely reduced in transgenics due to significant diminution of the largest lignin fragments which elute between 7 min and 9 min. Similarly, smaller molecular mass material which elutes later in a second peak between 13.5 min and 19.5 min was more abundant (increased by 27% and 16% using UV-A and UV-F detections) in IRX5:HCHL samples. Finally, the amount of the smallest lignin fragments detected between 19.5 min and 26.5 min using UV-F is increased by 55% in transgenics (FIG. 6). These results demonstrate smaller chains and reduced polymerization degree in lignin purified from IRX5:HCHL plants.

IRX5:HCHL Lines Show Increased Saccharification Efficiency

To examine impact lignin size reduction on cell wall digestibility caused by the expression of the HCHL enzyme in lignifying tissues, saccharification assays were conducted biomass derived from mature senesced stems pretreated with hot water, dilute alkaline, and dilute acid. After a 72-h incubation with cellulase and glucosidase, pretreated biomass of IRX5 HCHL plants released more reducing sugars compared to wild type (FIG. 7). In particular, improvement of saccharification efficiency observed for the different IRX5:HCHL lines ranged from 34% to 77% after hot water, from 43% to 71% after dilute alkaline, and from 15% to 31% after dilute acid pretreatments (FIG. 7).

III. Discussion

Expression of HCHL in plants has originally been considered for in planta production of valuable and soluble compounds such as Van and HBA. Due to strong ectopic HCHL expression, however, adverse phenotypes such as chlorotic and senescing leaves, stunting, low pollen production, male sterility, collapsed xylem vessels, and reduction of biomass were observed in transgenic tobacco, and sugarcane (Mayer et al., 2001; Merali et al., 2007; McQualter et al., 2005). In this study, the inventors selected the promoter of a secondary cell wall cellulose synthase to preferentially express HCHL in the lignifying tissues of Arabidopsis stems (FIG. 9). Successfully, plants transformed with the IRX5:HCHL construct were not dwarf or sterile, and young rosette leaves did not show reduced epidermal fluorescence which is symptomatic of alteration in phenylpropanoid-derived soluble phenolic pools. Although two IRX5:HCHL lines showed reduced biomass, and in one case some occasional collapsed xylem vessels caused by stronger HCHL activity and possibly modification of call wall integrity, some other IRX5:HCHL lines were comparable to wild-type plants.
As expected, the transgenic lines show increased amount of soluble C₆C₁aldehydes (HBAld, 3,4-DHBAld, and Van), which are produced upon HCHL activity after cleavage of hydroxybenzoyl-CoA, 3,4-dihydroxybenzoyl-CoA, and feruloyl-CoA (FIG. 11). HCHL has no activity against sinapoyl-CoA, suggesting that Syrald is a conversion product of Van, which is supported by the identification of the new intermediate 5OH-Van (Mitra et al., 1999; FIG. 11). Similarly, the data presented herein cannot exclude that some of the 3,4-DHBald and Van accumulated in transgenics derive from HBAld after successive hydroxylation and methoxylation on the C-3 position of the phenyl ring. Interestingly, several genes encoding monooxygenases are upregulated in plants expressing HCHL, but no known or predicted O-methyltransferase showed altered expression level (Table IX). Analysis of soluble aromatics in transgenics also shows that C₆-C₁aldehydes are oxidized into their respective acid forms. This conversion could be a response to reduce the amount of these chemically reactive compounds since several genes from the short-chain dehydrogenase/reductase (SDR), aldo-keto reductase (AKR), and aldehyde dehydrogenase (ALDH) families are upregulated in plants expressing HCHL, (FIG. 11; Kirch et al., 2004; Kavanagh et al., 2008). In particular, AKR4C9 (At3g37770) encodes an enzyme known to metabolize a range of hydroxybenzaldehydes (Simpson et al., 2009). In addition, soluble C₆C₁phenolics predominantly accumulate as conjugates in transgenics since we showed that glucose conjugates (phenolic glucoside and glucose ester) represented around 90% of the HBA soluble pool, presumably for vacuolar storage as previously described for other C₆C₁phenolics (Eudes et al., 2008). This C₆C₁acid glucoside accumulation is in agreement with what was observed in tobacco, sugar beet, datura and sugar cane plants expressing HCHL (Mayer et al., 2001; Mitra et al., 2002; McQualter et al., 2005; Rahman et al., 2009). Interestingly, expression analysis of HCHL plants revealed seven up-regulated genes of the UDP-glucosyltranferase (UGT) family and among them UGT75B1 and UGT73B4 were previously shown to catalyze glucose esterification and phenolic glucosylation of benzoates (Table IX; Lim et al, 2002; Eudes et al., 2008).
Furthermore, this study showed that some C₆C₁phenolics are released from extract-free cell wall fractions of senesced stems upon mild alkaline hydrolysis. Higher amounts of HBAld, 5OH-Van, SyrAld, HBA, VA, and SyrA were measured in the ‘loosely wall-bound’ fraction of IRX5:HCHL lines compared to wild type. Although the type of linkages involved is unclear, loosely attached C₆C₁phenolics were previously extracted from cell walls of Arabidopsis leaves and roots (Tan et al., 2004; Forcat et al., 2010).
The lignin from plants expressing HCHL shows increased content of C₆C₁phenolics. Notably, analysis of lignin monomers released after thioacidolysis identified two novel units (Vanalc and Syralc) and showed large amounts of Syrald, Van, and SyrA. This suggests part of C₆C₁aldehydes are converted into alcohols and acids and demonstrates that they are incorporated into the lignin as β-O-4-linked C₆C₁monomer end-groups in lignin (FIG. 11). Due to the absence of phenyl propanoid tail, these new monolignols when incorporated in lignin end chains, should block further polymerization of the polymer and act as condensation terminator or stopper molecules. Interestingly, transgenic plants also show higher content of conventional H-units (+30%), which preferentially distribute as terminal end-groups in lignin and contribute to modifications of lignin size and structure (Lapierre, 2010; Ziebell et al., 2010). In addition, plants overproducing C₆C₁monolignols and with similar lignin content as wild type plants show a lower thioacidolysis release of monolignols, indicating a reduction in the availability of free propanoid tail in lignin end-chain for polymer elongation. It also indicates higher carbon-carbon linkages and increased lignin condensation degree.
It was postulated that higher incorporation of end-group units in lignin would hinder more frequently chain elongation and ultimately reduce lignin branching and polymerization degree. This hypothesis is further supported by the analysis the polydispersity of lignin in plants overproducing theses “stopper” molecules, which shows significant reduction of high molecular masses and significant increase of low molecular masses, hence supporting smaller lignin chain length. These observations are relevant for understanding the higher susceptibility of the biomass from HCHL lines to polysaccharide enzymatic hydrolysis. Although saccharification efficiency of biomass is determined by several characteristics of cell walls, the observed saccharification efficiency improvement after different pretreatments suggests that less ramified lignin would reduce cross-linkages and embedding of cell wall polysaccharides (cellulose and hemicellulose) and would favor their accessibility to hydrolytic enzymes. This hypothesis is supported by the fact that total sugar content is unchanged in cell walls of plants overproducing theses C₆C₁monomers.
it is concluded that in planta the over-production of lignification “stopper” molecules can be used to modify the lignin structure in order to reduce lignocellulosic biomass recalcitrance. Since this approach does not require any particular genetic background, it should be easily transferable to various energycrops. Restricting the biosynthesis of these lignification “stopper” molecules in supporting lignified tissues (i.e. schlerenchyma fibers) as well as avoiding strong production in conductive tissues (i.e. vessels) should limit the risk of adverse effects on plant development and biomass yield.

Example 2

Expression of Bacterial HCHL in Rice

This example illustrates expression of bacterial HCHL in a monocot, rice. Rice plants were transformed with the DNA constructs described in Example 1. Rice lines were engineered (FIG. 12) that expressed the HCHL gene, as demonstrated by RT-PCR (FIG. 13). Furthermore, evaluation of rice lines demonstrated that they accumulated pHBA (para-hydroxybenzoate) (FIG. 14), which is generated from the conversion of p-coumaroyl-CoA by HCHL.
This experiment additionally demonstrated that a secondary wall promoter, pIRX5, from a dicot (Arabidopsis in this example), can be used in a monocot (rice in this example).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, accession numbers, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

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ILLUSTRATIVE SEQUENCES

SEQ ID NO: 1

Amino acid sequence for Pseudomonas fluorscens HCHL

(GenBank Accession No. CAA73502)

MSTYEGRWKTVKVEIEDGIAFVILNRPEKRNAMSPTLNREMIDVLETLEQDPAA

GVLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKP

TIAMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTV

GHRQSLYYIMTGKTFGGQKAAEMGLVNESVPLAQLREVTIELARNLLEKNPVVL

RAAKHGFKRCRELTWEQNEDYLYAKLDQSRLLDTEGGREQGMKQFLDDKSIKPG

LQAYKR

SEQ ID NO: 2

Polynucleotide sequence encoding SEQ ID NO: 1

(codon-optimized by GenScript)

ATGTCTACTTACGAGGGAAGATGGAAGACTGTTAAGGTTGAGATCGAGGATGGA

ATCGCTTTCGTTATCCTCAACAGACCTGAGAAGAGAAACGCTATGTCTCCTACT

CTCAACAGAGAGATGATCGATGTTCTCGAGACTCTCGAGCAGGATCCTGCTGCT

GGAGTTCTCGTTCTCACTGGAGCTGGAGAGGCTTGGACTGCTGGTATGGATCTC

AAGGAGTACTTCAGAGAGGTTGATGCTGGACCTGAGATCCTCCAGGAGAAGATC

AGAAGAGAGGCTTCTCAGTGGCAGTGGAAGCTCCTCAGAATGTACGCTAAGCCT

ACTATCGCTATGGTTAACGGATGGTGCTTCGGAGGAGGATTCTCTCCTCTCGTT

GCTTGCGATCTCGCTATCTGCGCTGATGAGGCTACTTTCGGACTCTCTGAGATC

AACTGGGGAATCCCTCCTGGAAACCTCGTTTCTAAGGCTATGGCTGATACTGTT

GGACATAGACAGTCTCTCTACTACATCATGACTGGAAAGACTTTCGGAGGACAG

AAGGCTGCTGAGATGGGACTCGTTAACGAGTCTGTTCCTCTCGCTCAGCTCAGA

GAGGTTACTATCGAGCTCGCTAGAAACCTCCTCGAGAAGAACCCTGTTGTTCTC

AGAGCTGCTAAGCATGGATTCAAGAGATGCAGAGAGCTCACTTGGGAGCAGAAC

GAGGATTACCTCTACGCTAAGCTCGATCAGTCTAGACTCCTCGATACTGAGGGA

GGAAGAGAGCAGGGTATGAAGCAGTTCCTCGATGATAAGTCTATCAAGCCTGGA

CTCCAGGCTTACAAGAGA

SEQ ID NO: 3

Polynucleotide sequence containing IRX5 promoter (pIRX5)

ATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATG

GCATGGATGCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTT

ACCACAAGAGCAACGGTTGTTTCGGGTAAATAAACTAAACTTAACCATATACAT

TAGCCTTGATTCGGTTTTTGGTTTGATTTATGGATATTAAAGATCCGAATTATA

TTTGAACAAAAAAAAATGATTATGTCACATAAAAAAAAATTGGCTTGAATTTTG

GTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTCATTTGTTTTCTGGTTAG

CTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGATTTGAAAC

AGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAG

GCACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGT

TTCAAGATCTTTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTC

GAAAATCTTATTGAAGCCGGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTC

GAATCATATTTAATAACCGCATTAAACCGAAATTTAAATTCTAATTTCACCAAA

TCAAAAAGTAAAACTAGAACACTTCAGATAAATTTTGTCGTTCTGTTGACTTCA

TTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAATAAAAACCCTAAA

AACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTTTAGGTA

AGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTC

ATTAATTTGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTT

GTGAAATCATATTTAAAACATTAATAGGTATTTATGTCTAATTTGGGGACAAAA

TAGTGGAATTCTTTATCATATCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTT

ATGATTATGTTACATGCATTGGTCCATATAAATCTATGAGCAATCAATATAATT

CCGAGCATTTTGGTATAACATAATGAGCAAGTATAACAAAAGTATCAAACCTAT

GCAGGGGAGAAGATGATGAAAAGAAGAGTGTGAGCCAATACAAAGCAGATTTGA

GGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCACAAT

GGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACT

CCATCTACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCA

GAGTTTCATTCTGATTCTTGAGTCTTTTTTTTCTCTCTCCCTCTTTTCATTTCT

GGTTTATATAACCAATTCAAATGCTTATGATCCATGCATGAACCATGATCATCT

TTGTGTTTTTTTTTCCTTCTGTATTACCATTTTGGGCCTTTGTGAAATTGATTT

TGGGCTTTTGTTATATAATCTCCTCTTTCTCTTTCTCTACCTGATTGGATTCAA

GAACATAGCCAGATTTGGTAAAGTTTATAAGATACAAAATATTAAGTAAGACTA

AAGTAGAAATACATAATAACTTGAAAGCTACTCTAAGTTATACAAATTCTAAAG

AACTCAAAAGAATAACAAACAGTAGAAGTTGGAAGCTCAAGCAATTAAATTATA

TAAAAACACTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCTG

TCTCTTGAAGCTTTCTTATGACACAAACCTTAGACCCAATTTCACTCACAGTTT

GGTACAACCTCAGTTTTCTTCACAACAAATTCAAACATCTTACCCTTATATTAC

CTCTTTATCTCTTCAATCATCAAAACACATAGTCACATACATTTCTCTACCCCA

CCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCC

SEQ ID NO: 4

Sagittula_stellata_E-37__ZP_01746375 (amino acid sequence)

MTATEATLPANDPDLSGDNVAVAFEDGIAWVKLNRPEKRNAMSVSLAEDMNVVLD

KLEIDDRCGVLVLTGEGSAFSAGMDLKDFFRATDGVSDVERMRAYRSTRAWQWRT

LMHYSKPTIAMVNGWCFGGAFTPLICCDLAISSDDAVYGLSEINWGIIPGGVVSK

AISTLMSDRQALYYVMTGEQFGGQEAVKLGLVNESVPADKLRERTVELCKVLLEK

NPTTMRQARMAYKYIREMTWEESAEYLTAKGDQTVFVDKEKGREQGLKQFLDDKT

YRPGLGAYKR

SEQ ID NO: 5

Saccharopolyspora_erythraea_NRRL_2338_YP_001105000

(amino acid sequence)

MSTPTTDPGTTTTPWGDTVLVDFDDGIAWVTLNRPEKRNAMNPAMNDEMVRTLDA

LEADPRCRVMVLTGAGESFSAGMDLKEYFREVDQTADPSVQIRVRRASAEWQWKR

LAHWSKPTIAMVNGWCFGGAFTPLVACDLAISDEEARYGLSEINWGIPPGGVVSR

ALAAAVSQRDALYFIMTGETFDGRRAEGMRLVNEAVPAERLRERTRELALKLAST

NPVVLRAAKVGYKIAREMPWEQAEDYLYAKLEQSQFLDAERGREKGMAQFLDDKS

YRPGLSAYSTD

SEQ ID NO: 6

Solibacter_usitatus_Ellin6076_YP_821552

(amino acid sequence)

MDQYEEKWQTVKVEVDAEGIAWVIFNRPAKRNAMSPTLNREMAQVLETLELDAAA

KVLVLTGAGESWSAGMDLKEYFREVDGQPESHQEKIRREASLWQWKLLRMYAKPT

IAMVNGWCFGGAFSPLVACDLAIADEKAVFGLSEINWGIPPGNLVSKAVADTMGH

RKALHYIMTGETFTGAQAAEMGLVNAAVPTSELREATRTLALKLASKNPVILRAA

KHGFKRCRELTWEQNEDYLYAKLDQALHRDPEDARAEGMKQFLDEKSIKPGLQSY

KRS

SEQ ID NO: 7

Ralstonia_solanacearum_GMI1000_NP_521786

(amino acid sequence)

MATYEGRWNTVKVDVEDGIAWVTLNRPDKRNAMSPTLNREMIDVLETLELDGDAQ

VLVLTGAGESWSAGMDLKEYFRETDGQPEIMQERIRRDCSQWQWKLLRFYSKPTI

AMVNGWCFGGAFSPLVACDLAIAADDAVFGLSEINWGIPPGNLVSKAVADTMGHR

AALHYIMTGETFTGREAAEMGLVNRSVPRERLREAVTELAGKLLAKNPVVLRYAK

HGFKRCRELSWEQNEDYLYAKVDQSNHRDPEKGRQHGLKQFLDDKTIKPGLQTYK

RA

SEQ ID NO: 8

Xanthomonas_albilineans_YP_003377516 (amino acid sequence)

MSNYQDRWQTVQVQIDAGVAWVTLNRPEKRNAMSPTLNREMIDVLETLELDSAAE

VLVLTGAGESWSAGMDLKEYFREIDGKEEIVQERMRRDCSQWQWRLLRFYSKPTI

AAVNGWCFGGAFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAVADTMGHR

NAMLYIMTGRTFTGTEAAQMGLVNASVPRAQLRAEVTKLAQELQQKNPVVLRFAK

HGFKRCRELTWEQNEDYLYAKVDQSNHRDPEKGRQQGLKULDDKTIKPGLQTYKR

SEQ ID NO: 9

Acinetobacter_baumannii_ATCC_17978_YP_001084143

(amino acid sequence)

MKMSYENRWETVDVKVEDGIAWVTLNRPEKKNAMSPTLNREMIDVLETLELDQNA

KVLVLTGAGDSWTAGMDLKEYFREVDTQPEIFQERIRRDSCRWQWQLLRMYSKPT

IAMVNGWCFGGGESPLVACDLAIAADEATEGLSEINWGIPPGNLVSKAMADTVGH

RASLYYIMTGKTFSGKEAETMGLVNKSVPLAQLKAEVTELANCLLEKNPVVLRTA

KNGFKRCRELTWDQNEDYLYAKLDQCIHRDTENGRQEGLKQFLDEKSIKPGLQSY

KRTG

SEQ ID NO: 10

Acinetobacter_sp._ADP1_YP_046390 (amino acid sequence)

MTYDKRWETVDVQVEHGIAWVTLNRPHKKNAMSPTLNREMIDVLETLELDSEAKV

LVLTGAGDSWTAGMDLKEYFREVDAQPEIFQERIRRDSCRWQWQLLRMYSKPTIA

MVNGWCFGGGFSPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRA

SLYYIMTGKTFTGKEAEAMGLINKSVPLAQLKAEVTELAQCLVEKNPVVLRTAKN

GEKRCRELTWDQNEDYLYAKLDQCNHRDTEGGRQEGLKQFLDEKSIKPGLQSYKR

TG

SEQ ID NO: 11

Chromohalobacter_salexigens_DSM_3043_YP_572340

(amino acid sequence)

MSDYTNRWQTVKVDVEDGIAWVTLNRPEKRNAMSPTLNREMIDVLETIELDQDAH

VLVLTGEGESFSAGMDLKEYFREIDASPEIVQVKVRRDASTWQWKLLRHYAKPTI

AMVNGWCFGGAFSPLVACDLAIAADESVFGLSEINWGIPPGNLVSKAMADTVGHR

QALYYIMTGETFTGPQAADMGLVNQSVPRAELRETTHKLAATLRDKNPVVLRAAK

TGFKMCRELTWEQNEEYLYAKLDQAQQLDPEHGREQGLKQFLDDKSIKPGLESYR

R

SEQ ID NO: 12

Burkholderia_cenocepacia_AU_1054_ZP_04942909

(amino acid sequence)

MSKYDNRWQTVEVKVEAGIAWVTLNRPEKRNAMSPTLNREMLEVLDAVEFDDEA

KVLVLTGAGAAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP

TIAMVNGWCFGGGFSPLVACDLAIAADDAVFGLSEINWGIPPGNLVSKAMADTV

GHRRALHYIMTGDTFTGAEAAEMGLVNSSVPLAELRDATIALAARLMDKNPVVL

RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKTIKPG

LQAYKR

SEQ ID NO: 13

Burkholderia_ambifaria_MC40-6_YP_776799

(amino acid sequence)

MSKYDNRWQTVEVNVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA

KVLVLTGAGSAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP

TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV

GHRRALHYIMTGDTFTGVEAAEMGLVNSSVPLAGLRDATIALAARLMDKNPVVL

RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG

LQAYKR

SEQ ID NO: 14

Burkholderia_cepacia_AMMD_YP_776799 (amino acid sequence)

MSKYDNRWQTVEVNVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA

KVLVLTGAGSAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP

TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV

GHRRALHYIMTGDTFTGVEAAEMGLVNSSVPLAGLRDATIALAARLMDKNPVVL

RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG

LQAYKR

SEQ ID NO: 15

Burkholderia_thailandensis_MSMB43_ZP_02468311

(amino acid sequence)

MSKYDNRWQTVEVKVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA

KVLVLTGAGTAWTAGMDLKEYFREIDGGPDALQEKVRRDASEWQWRRLRMYGKP

TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV

GHRCALHYIMTGDTFTGVEAADMGLVNRSVPLAELRDATIALAARLIDKNPVVL

RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG

LQAYKR

SEQ ID NO: 16

Burkholderia_ubonensis_Bu_ZP_02382374

(amino acid sequence)

MSKYENRWQTVEVKVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA

KVLVLTGAGAAWTAGMDLKEYFREIDGGPDALQEKVRRDASEWQWRRLRMYGKP

TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV

GHRRALHYIMTGDTFTGVEAADMGLVNRSVPLAELRDATIALAARLIDKNPVVL

RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG

LQAYKR

SEQ ID NO: 17

Azotobacter_vinelandii_AvOP_YP_002798614

(amino acid sequence)

MNKYEGRWKTVIVEIEGGIAWVTLNRPDKRNAMSPTLNREMRDVLETLEQDPAAR

VLVLTGAGSAWTAGMDLKEYFREVDAGPEILQEKIRREACEWQWKLLRMYAKPTV

AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR

QALYYIMTGKTFDGRQAAEMGLVNQSVPLAQLRETVATLCQDLLDKNPVVLRAAK

NGFKRCRELTWEQNEDYLYAKLDQSRLLDEEGGREEGMRQFLDEKSIKPGLQAYK

R

SEQ ID NO: 18

Pseudomonas_putida_KT2440_NP_745498 (amino acid sequence)

MSKYEGRWTTVKVELEAGIAWVTLNRPEKRNAMSPTLNREMVDVLETLEQDADAG

VLVLTGAGESWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRLYAKPTI

AMVNGWCFGGGFSPLVACDLAICANEATFGLSEINWGIPPGNLVSKAMADTVGHR

QSLYYIMTGKTFDGRKAAEMGLVNDSVPLAELRETTRELALNLLEKNPVVLRAAK

NGFKRCRELTWEQNEDYLYAKLDQSRLLDTTGGREQGMKQFLDDKSIKPGLQAYK

R

SEQ ID NO: 19

Pseudomonas_fluorescens_SBW25_YP_002872871

(amino acid sequence)

MSNYEGRWTTVKVEIEEGIAWVILNRPEKRNAMSPTLNREMIDVLETLEQDPAAG

VLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKPTI

AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR

QSLYYIMTGKTFGGQKAAEMGLVNESVPLAQLREVTIELARNLLEKNPVVLRAAK

HGFKRCRELTWEQNEDYLYAKLDQSRLLDTEGGREQGMKQFLDDKSIKPGLQAYK

R

SEQ ID NO: 20

Pseudomonas_syringae_NP_792742 (amino acid sequence)

MSKYEGRWTTVKVEIEQGIAWVILNRPEKRNAMSPTLNREMIDVLETLEQDPEAG

VLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKPTI

AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR

QSLYYIMTGKTFDGKKAAEMGLVNESVPLAQLRQVTIDLALNLLEKNPVVLRAAK

HGFKRCRELTWEQNEDYLYAKLDQSRLLDKEGGREQGMKQFLDDKSIKPGLEAYK

R

SEQ ID NO: 21

Ralstonia_eutropha_JMP134_YP_299062 (amino acid sequence)

MANYEGRWKTVKVSVEEGIAWVMFNRPEKRNAMSPTLNSEMIQVLEALELDADAR

VVVLTGAGDAWTAGMDLKEYFREVDAGPEILQEKIRRDACQWQWKLLRMYAKPTI

AMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHR

QALHYIMTGDTFTGQQAAAMGLVNKSVPRSQLREHVLELAGKLLEKNPVVLRAAK

HGFKRSRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKSIKPGLQAYK

RA

SEQ ID NO: 22

Burkholderia_glumae_BGR1_YP_002908688

(amino acid sequence)

MSYEGRWTTVKVTVEAGIGWVVLNRPEKRNAMSPTLNKEMIDVLETLELDDEAQV

LVLTGEGDAWTAGMDLKEYFREVDAASDVVQERIRRDASRWQWQLLRMYSKPTIA

MVNGWCFGGGESPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRQ

ALYYIMTGDTFTGKQAAQMGLVNQSVPRAALREATVALAAKLLDKNPVVLRNAKH

GFKRSRELTWEQNEDYLYAKLDQANYRDKEGGREKGLKQELDDKSIKPGLQAYKR

SEQ ID NO: 23

Burkholderia_phytofirmans_PsJN_YP_001887778

(amino acid sequence)

MSYEGRWKTVKVDVAEGIAWVSFNRPEKRNAMSPTLNKEMIEVLEAVELDAEAQV

LVLTGEGDAWTAGMDLKEYFREVDAGPEILQEKIRRDACRWQWQLLRMYSKPTIA

MVNGWCFGGGFSPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRQ

ALYYIMTGETFTGQEAAQMGLVNKSVPRAELREATRALAGKLLEKNPVVLRAAKH

GFKRCRELTWDQNEDYLYAKLDQAQLRDPEGGREQGLKQFLDDKAIKPGLQTYKR

SEQ ID NO: 24

Burkholderia_mallei_ATC_23344_YP_105383

(amino acid sequence)

MSYEGRWKTVEVIVDGAIAWVTLNRPDKRNAMSPTLNAEMIDVLEAIELDPEARVL

VLTGEGEAWTAGMDLKEYFREIDAGPEILQEKIRRDASRWQWQLLRMYAKPTIAMV

NGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQALY

YIMTGETFTGAQAAQMGLVNRSVPRAQLRDAVRALAAKLLDKNPVVLRNAKHGFKR

CRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKTIKPGLQAYRR

SEQ ID NO: 25

Burkholderia_pseudomallei_Pasteur_ZP_01765668

(amino acid sequence)

MSYEGRWKTVEVIVDGAIAWVTLNRPDKRNAMSPTLNAEMIDVLEAVELDPEARV

LVLTGEGEAWTAGMDLKEYFREVDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA

MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQ

ALYYIMTGETFTGAQAAQMGLVNRSVPRAQLRDAVRALAAKLLDKNPVVLRNAKH

GEKRCRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKTIKPGLQAYRR

SEQ ID NO: 26

Burkholderia_multivorans_ATCC_17616_YP_001583186

(amino acid sequence)

MSYEGRWKTVKVAVEGGIAWVTLNRPEKRNAMSPTLNAEMIDVLEAIELDPEAQV

LVLTGEGDAWTAGMDLKEYFREVDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA

MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQ

ALYYIMTGDTFTGQQAAQMGLVNKSVPRAQLRDEVRALAAKLLDKNPVVIRNAKH

GFKRCRELTWEQNEDYLYAKLDQANYRDPEGGREQGLKQFLDEKSIKPGLQAYKR

SEQ ID NO: 27

Burkholderia_vietnamiensis_G4_YP_001116289

(amino acid sequence)

MGYEGRWKTVKVEVAGGIAWVTLNRPEKRNAMSPTLNTEMIDVLEAIELDADAQV

LVLTGEGDAWTAGMDLKEYFREIDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA

MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRE

ALYYIMTGDTFTGQQAARMGLVNKSVPRAQLRDEVRALAAKLLDKNPVVIRNAKH

GFKRCRELTWEQNEDYLYAKLDQANYRDPEGGREQGLKQFLDDKSIKPGLQAYKR

SEQ ID NO: 28

Sphingobium_japonicum_UT26S_YP_003543683

(amino acid sequence)

MSEYLTEGPDLSRTCVDVMFDEGIAWVTLNRPEKRNAMSPTLNSEMLAILEQLELD

PRCGVVVLTGAGDSFSAGMDLKEYFRETDGLPPAQVRRIRQTAQAWQWRTLQHFGK

PTIAMVNGWCFGGAFTPLVACDLAIAANEAVEGLSEINWGIIPGGNVTKAIQERLR

PQDAALYIMTGRNFTGEKAAQMGLVAEAVPLTDLRDHTRALALELLSKNPVVLNAA

KIALKKVADMTWDVAEDYLVAKGAQTRVADKTDGRNKGITQFLDEKSYKPGLEGYR

RDK

SEQ ID NO: 29

Xanthomonas_axonopodis_NP_641235 (amino acid sequence)

MNEHDVVSVRIENRIAWVKFARPDKRNAMSPALNRRMMDVLDELEFDDNVGVLV

LGGEGTAWSAGMDLKEYFRETEAQGLRGVRRSQRESYGWERRLRWYQKPTIAMV

NGWCFGGGFGPLFACDLAIAADEAQFGLSEINWGILPGGGVTKVAVELLSMRDA

MWMTLTGEMVDGKKAAEWRLVNESVPLERLEARTREVAELLLRKNPVALKYAKD

AVRRVGTMTYDEAEDYLVRMQEAANSFDNNARKEGIRQFIDEKSYKPGLGEYDL

SKHSA

SEQ ID NO: 30

Xanthomonas_campestris_ATCC_33913_NP_636201

(amino acid sequence)

MNEHDVVSVHVENRIAWVKFARPDKRNAMSPALNRRMLDVLDELEFDDNVGVLVL

GGEGTAWSAGMDLKEYFRETEAQGLRGVRRSQRESYGWFRRLRWYQKPTIAMVNG

WCFGGGEGPLFACDLAIAADEAQFGLSEINWGILPGGGVTKVAVELLSMRDAMWM

TLTGELVDGRKAAEWRLVNESVPLERLETRTREVAELLLKKNPVALKYAKDAVRR

VGTMTYDEAEDYLVRMQEAANSFDNNARKEGIRQFIDEKRYKPGLGAYEPDAGTN

SEQ ID NO: 31

Azospirillum_sp._B510_YP_003451575 (amino acid sequence)

MTQQQAAARTGTAEDVVTVELDNGVAWVTLNRPDKRNAMNPALNARMHGVLDD

LEVDDRCQVLVLTGAGESFSAGMDLKEYFRETEAKGHMATRRAQRDSYGWWRR

LRWFEKPSIAMVNGWCFGGAFSPLFACDLAVAADEAQFGLSEINWGIIPGGNV

TKVVADLMSQREAMYYILTGETFDGRKAAEMKLVNFSVPHAELRAKVRAIADN

LLEKNPQTLKAAKDAFKRVVEMPFDAAEDYLVVRQESLNYLDKSEGRKQGIKQ

FIDDKTYRPGLGAYKR

SEQ ID NO: 32

Agrobacterium_vitis_S4_YP_002549228 (amino acid sequence)

MTVAEKSDADTVLVDIEDRIAFVTFNRPEKRNAMNPALNIRMAEVLEELEADDRC

GVLVLRGAGTSWSAGMDLQQYFRDNDDKPRHATLKSRRQSGGWWQRLTYFEKPTI

AMVNGWCFGGAFNPLVACDLAIAANEATFGLSEINWGILPGGNVTRAVAEVMNHR

DSLYYIMTGEPFGGEKARDMGLVNESVPLEELETRVRKLCASLLEKNPVTMKAAK

DTFKRVRNMPWELADDYIYAKLEQMLLLDKTRGRDEGLKQFLDDKTYRPGLGAYK

RK

SEQ ID NO: 33

Rhizobium_etli_Brasil_5_YP_001985541 (amino acid sequence)

MTENTSPVLVEFDGGIAFVTLNRPEKRNAMNPALNARMLEVLDELEGDERCGVLV

LRGAGQSWSAGMDLKEYFRDNDDKPRDATLKARRQSGGWWGRLMYFEKPTIAMVN

GWCFGGAFTPLVSCDLAIAAEEANFGLSEINWGILPGGNVTRAVAEVMRHRDALY

YIMTGELFGGRKAAEMGLVNEAVPLVDLETRVRKICASLLEKNPVTLKAAKDTYK

RVRNLPWDLADDYIYAKLEQMLFLDKTKGRDEGLKQFLDDKTYQPGLGAYKRGR

SEQ ID NO: 34

Rhizobium_leguminosarum_bv_trifolii_WSM1325_YP_002973001

(amino acid sequence)

MTEDKSPVLVEFDSGIAFVTLNRPEKRNAMNPALNIRMLEVLDELEGDERCGVL

VLRGAGESWSAGMDLKEYFRDNDDKPRDVTLKARRQSGNWWGRLMYFEKPTIAM

VNGWCFGGAFTPLVSCDLAIAAEEANFGLSEINWGILPGGNVTRAVAEVMRHRD

ALYYIMTGELFGGRKAAEMGLVNEAVPLAELEPRVRKICASLLEKNPVTLKAAK

DTYKRVRNLPWDLADDYIYAKLEQMLFLDKTKGRDEGLKQFLDDKTYQPGLGAY

KRGR

SEQ ID NO: 35

Amino acid sequence for IRX5

(GenBank Accession No. AF458083_1)

MEPNTMASFDDEHRHSSFSAKICKVCGDEVKDDDNGQTFVACHVCVYPVCKPCYE

YERSNGNKCCPQCNTLYKRHKGSPKIAGDEENNGPDDSDDELNIKYRQDGSSIHQ

NFAYGSENGDYNSKQQCRPNGRAFSSTGSVLGKDFEAERDGYTDAEWKERVDKWK

ARQEKRGLVTKGEQTNEDKEDDEEEELLDAEARQPLWRKVPISSSKISPYRIVIV

LRLVILVFFFRFRILTPAKDAYPLWLISVICEIWFALSWILDQFPKWFPINRETY

LDRLSMRFERDGEKNKLAPVDVFVSTVDPLKEPPIITANTILSILAVDYPVNKVS

CYVSDDGASMLLFDTLSETSEFARRWVPFCKKYNVEPRAPEFYFSEKIDYLKDKV

QTTFVKDRRAMKREYEEFKVRINALVAKAQKKPEEGWVMQDGTPWPGNNTRDHPG

MIQVYLGKEGAFDIDGNELPRLVYVSREKRPGYAHHKKAGAMNAMVRVSAVLTNA

PFMLNLDCDHYINNSKAIRESMCFLMDPQLGKKLCYVQFPQRFDGIDHNDRYANR

NIVFFDINMRGLDGIQGPVYVGTGCVFNRPALYGYEPPVSEKRKKMTCDCWPSWI

CCCCGGGNRNHHKSKSSDSSSKKKSGIKSLLSKLKKKNKKKSDDKTMSSYSRKRS

ATEAIFDLEDIEEGLEGYDELEKSSLMSQKNFEKRFGMSPVFIASTLMENGGLPE

ATNTSSLIKEAIHVISCGYEEKTEWGKEIGWIYGSVTEDILTGFRMHCRGWKSVY

CMPKRPAFKGSAPINLSDRLHQVLRWALGSVEIFFSRHCPLWYAWGGKLKILERL

AYINTIVYPFTSIPLLAYCTIPAVCLLTGKFIIPTINNFASIWFLALFLSIIATA

ILELRWSGVSINDLWRNEQFWVIGGVSAHLFAVFQGLLKVLFGVDTNFTVTSKGA

SDEADEFGDLYLFKWTTLLIPPTTLIILNMVGVVAGVSDAINNGYGSWGPLFGKL

FFAFWVIVHLYPFLKGLMGRQNRTPTIVVLWSILLASIF

SLVWVRIDPFLPKQTGPLLKQCGVDC

SEQ ID NO: 36

Polynucleotide sequence PATCESA7_PATIRX3

TGGGAACTTTCGGTACATTTTCCAATAAAATCTATATACTATAAGATATTAAAT

ATACACAAATATATCTAAGTGAATCATACAAATTATGTAGGCACACAGGAAGAG

GCTGCTGAGGCTTATGACATTGCAGCCATTAAATTCAGAGGATTAAGCGCAGTG

ACTAACTTCGACATGAACAGATACAATGTTAAAGCAATCCTCGAGAGCCCGAGT

CTACCTATTGGTAGTTCTGCGAAACGTCTCAAGGACGTTAATAATCCGGTTCCA

GCTATGATGATTAGTAATAACGTTTCAGAGAGTGCAAATAATGTTAGCGGTTGG

CAAAACACTGCGTTTCAGCATCATCAGGGAATGGATTTGAGCTTATTGCAGCAA

CAGCAGGAGAGGTACGTTGGTTATTACAATGGAGGAAACTTGTCTACCGAGAGT

ACTAGGGTTTGTTTCAAACAAGAGGAGGAACAACAACACTTCTTGAGAAACTCG

CCGAGTCACATGACTAATGTTGATCATCATAGCTCGACCTCTGATGATTCTGTT

ACCGTTTGTGGAAATGTTGTTAGTTATGGTGGTTATCAAGGATTCGCAATCCCT

GTTGGAACATCGGTTAATTACGATCCCTTTACTGCTGCTGAGATTGCTTACAAC

GCAAGAAATCATTATTACTATGCTCAGCATCAGCAACAACAGCAGATTCAGCAG

TCGCCGGGAGGAGATTTTCCGGTGGCGATTTCGAATAACCATAGCTCTAACATG

TACTTTCACGGGGAAGGTGGTGGAGAAGGGGCTCCAACGTTTTCAGTTTGGAAC

GACACTTAGAAAAATAAGTAAAAGATCTTTTAGTTGTTTGCTTTGTATGTTGCG

AACAGTTTGATTCTGTTTTTCTTTTTCCTTTTTTTGGGTAATTTTCTTATAACT

TTTTTCATAGTTTCGATTATTTGGATAAAATTTTCAGATTGAGGATCATTTTAT

TTATTTATTAGTGTAGTCDTAATTTAGTTGTATAACTATAAAATTGTTGTTTGT

TTCCGAATCATAAGTTTTTTTTTTTTTTGGTTTTGTATTGATAGGTGCAAGAGA

CTCAAAATTCTGGTTTCGATGTTAACAGAATTCAAGTAGCTGCCCACTTGATTC

GATTTGTTTTGTATTTGGAAACAACCATGGCTGGTCAAGGCCCAGCCCGTTGTG

CTTCTGAACCTGCCTAGTCCCATGGACTAGATCTTTATCCGCAGACTCCAAAAG

AAAAAGGATTGGCGCAGAGGAATTGTCATGGAAACAGAATGAACAAGAAAGGGT

GAAGAAGATCAAAGGCATATATGATCTTTACATTCTCTTTAGCTTATGTATGCA

GAAAATTCACCTAATTAAGGACAGGGAACGTAACTTGGCTTGCACTCCTCTCAC

CAAACCTTACCCCCTAACTAATTTTAATTCAAAATTACTAGTATTTTGGCCGAT

CACTTTATATAATAAGATACCAGATTTATTATATTTACGAATTATCAGCATGCA

TATACTGTATATAGTTTTTTTTTTGTTAAAGGGTAAAATAATAGGATCCTTTTG

AATAAAATGAACATATATAATTAGTATAATGAAAACAGAAGGAAATGAGATTAG

GACAGTAAGTAAAATGAGAGAGACCTGCAAAGGATAAAAAAGAGAAGCTTAAGG

AAACCGCGACGATGAAAGAAAGACATGTCATCAGCTGATGGATGTGAGTGATGA

GTTTGTTGCAGTTGTGTAGAAATTTTTACTAAAACAGTTGTTTTTACAAAAAAG

AAATAATATAAAACGAAAGCTTAGCTTGAAGGCAATGGAGACTCTACAACAAAC

TATGTACCATACAGAGAGAGAAACTAAAAGCTTTTCACACATAAAAACCAAACT

TATTCGTCTCTCATTGATCACCGTTTTGTTCTCTCAAGATCGCTGCTAATCTCC

GGCCGTCCCT

SEQ ID NO: 37

Polynucleotide sequence PATCESA8_PATIRX1

TTTAGTGCAGTCTAGGAAGACGGATCCAAAGGAGATAAACAGAGTTCAAGAAGCT

CTTAACTACTATACAATCGAATCGTCAGCCGCGCTTTTTGTTTCGTTCATGATCA

ATTTGTTTGTAACTGCGGTTTTCGCGAAAGGGTTTTATGGAACCAAACAAGCTGA

TAGTATAGGACTGGTTAACGCGGGATATTACCTACAAGAGAAATATGGCGGTGGT

GTTTTCCCGATACTATACATTTGGGGGATTGGTTTATTAGCTGCTGGACAAAGCA

GTACTATAACCGGGACTTATGCTGGACAGTTTATAATGGAAGGGTTCTTAGATCT

TCAAATGGAACAATGGCTATCAGCTTTTATAACGAGAAGCTTTGCTATTGTACCT

ACTATGTTTGTTGCTATTATGTTTAACACATCCGAGGGCTCGCTCGATGTTTTAA

ACGAATGGCTTAACATTCTTCAGTCGATGCAGATTCCTTTCGCGGTTATTCCTCT

TTTGACTATGGTTTCTAATGAACATATCATGGGTGTCTTCAAGATCGGACCTTCG

CTTGAGGTAAAGCAATTTTTTGTCATCTCTCTTTATTGTTATGTGCTTTTGATTG

TAACGAGTTAGTTGGGATCTTTGCAGAAGCTAGCTTGGACTGTGGCGGTGTTTGT

GATGATGATAAATGGGTATCTTCTTCTAGATTTCTTCATGGCTGAAGTGGAAGGG

TTTCTTGTTGGGTTTCTGGTTTTTGGTGGAGTAGTTGGATACATCAGTTTCATCA

TCTATCTTGTTTCTTATAGAAGCTCACAATCTTCTTCCTGGTCGAGTTTAGAAAT

GTCAGAGAGAGTTGTTTCCACAGAGACGTAGAAACCCATAACTTTAGTATTCTTC

AACCCTTACAACTTATCTGAGCAAAATCAGAAGGTCGAATTTGATGGATGGTTTT

GCTGTATTTGGTCAACGGTTTTATTTGAGACAGTAGACCAGAGGAAACTCAGATG

TGATGATGCAAAGACTGAATTGGTTAAGAGTGTAGATTGATTTGTTCTAACATTG

CAAATGTAGAGTAGAATTATGCAAAAAACGTTAATGAACAGAGAAGTGATTAAGC

AGAAACAAAATTAGAGAAGTGATATTATATCTCAAAATTTATTTTTGGTACAGCT

AAAGCTCAAATTGTTATAGAGATTAGAGATATTAAACCAAATGACGAGTGTTTTC

TTTAGTAGTAAACGGTGAAAATTCTCTTCTGACAAAGACAATTAAAATTTTAGGT

TTAAGACTTTAATATTTGTCACAAATTGTCATTTACCTAAATAAAAAAAAAACTA

AATATTTTTTTTAGATACATATGTGTCTTATAATTTTAACTATAAATTTTAATTT

TATGTCTTAAATAATTGTTTACACTATAAATTTAAATATTTTAATGCTAAAATTA

ATTTGATTCAAAAAAGTGATTTTAATTCTTATTTTTCTTATAGAAAGTTGGTGAT

TGAAAAGATTTACTTAAAAATTATAACAACTTCAATGGTGAATAACCCGACCCGA

ATAAACCGGATATAACAACTTCAATGTTAGCTTGATATAGAAAGTACGGTGACGC

TTAGGAGGCAAGCAAGCTAGTATCTGCCGCTGGTTAGAGACAAAGAACATGTGTC

ACTCCTCTCAACTAAAACTTTCCTTCACTTTCCCGCAAAATCATTTCAAAAAAGC

TCCAAATTTAGCTTACCCATCAGCTTTCTCAGAAAACCAGTGAAAGAAACTTCTC

AACTTCCGATTTTTCACAATCCACCAAACTTTTTTTAATAACTTTTTTTCCTCTT

ATTACAAAACCTCCACTCTCATGGCTTCTCAAACTTGTTATCCATCCAAATCTCA

ATCCCTAATTAGGGTTCATTTCTCTGTTTCTCCAAACAGGGGAATTCGAAG

SEQ ID NO: 38

Polynucleotide sequence PATNST1

GTTTGTAGAGTTGGATCAGCATCCAGATTTAAACCCTTATTTTTGTTTTTGCCAA

GCATCCAGACTTAATCCTATATTAGATACTGTATATGCATCTTGATGGAATATAG

ACTATATAGAAAGACCAAAAATGGAAGAGTACGAATAAAAATGCATAATATACCT

TGGAAATTATTCTTGGTTATTGTGAAACTTAAAACATTTCAACGAAGTCATATAC

TATTATTTAATCATTGATTTAAAATTGCTAATCAAATCACGTGTTGTTGTTATAT

ATGGATAAAGAGTTAAACTATAACACAACTGAGAAAAAAATAAAGTTATCAATTT

TGTTAAGAATCAATGAAGGTTTCACAAGACTGGGAAGAAAAAAAAATAGATATAT

GGAGTACATAAAACATTAAAATTTTGCTAAATTTTACTTTTGAACTCTATTGATT

CGGGTTGACATGATGATAATGTTACATTCGTACAATTTCACAATGAAAAAAACGA

GTACTAAATATTGTCAATCAAACATATGAATGTACAAAAATCCATAAACTCTACC

AAAATAGAATGAAGATTCTGAAATCAAACCTACTTTTTCTTTTTAATTATAAATT

CAACTATATTATAAATTTATTTATCACAAATAATAGAGGAGTGAGAATATTTTAG

ACAACGCAAATTTCTTTTATTTAGTTCTTATACTTTATTTTTTACCAAACGTTAA

TTAAAAAAATCACACATACATAATTTCTAAAAAAAATGTATTCTTCAAGTAATAT

ATCTTTCTGAGTACTAGTTTATCTATTTATCTCCGTATTTAATAATCAAAAGTTA

CGTTTAAAATAGAAACAACTTTTATCAAACAAAATATATTAGAAAACGCATGGTA

CTGGCTACTGGAAAGAATCATGACCTGTAAATTTCTACAGTTTTCCCGTTTTATA

TAGTACTTAGAAACTTTGGATTTTCATAGCGCAACCAATAAACACATGGACTTAA

GACACAAAAAAAGTTGGGTGCAATGTCATTAATCAAACTAAAAAAATAATGATTA

AAAGCATGGAATTCCGAAAACGCAACAAAATGATTCTGTGTTTAGACAAATGCAG

AAAGGCCTCTTAACTAATCTTAAATAAAGTCTTAGTTCCAACCACATAAACACTC

CTTAGCTCCATTAATTTTGGTTTTCTTAATTACGTTTCTACACAAGTACACGTAC

TTACACATACAATTCCACAGTCTAAATGATAAAACTATGTGGTTTTTGACGTCAT

CGTTACCTTTCTGTCGTCTCACCTTTATATAGTGTCTCTAACAGAACGTAACAAC

CAAATGTTTAAAAAAATAAAAACAGCACCCCTTAATTAGGCTCATTCGTTTTGCA

CTAACCATACTACAAATCATCTCGAACGATCGAGCAAAGATTTGAAAAATAAATA

AACGTATAACTCTAGAGATTTTCATTAGCTAAGAAAAGTGAAATCGATTGTTAAT

CCTATTTCAGACGGGACAGGAACACTCATTACCCAACTCTATCATCTCTCGAACA

CCAAACTATATCTACCGTTTGGGGCATTATTTCCCACTTTCTTTCGAAGACAATT

TCCCATATATAACATATACACATTATTACTAATATATTTTTATAAATTTTCGTCA

CATCCCAAAAAAAAACACTCTTTGTCACATCAACTAGTTTTTTTGTAACGATCAA

ACCTTTTCGTTTAAAAAAAAAAAACTTTTGTAGTGTAAACGTTTATTTATCGATG

AAAAAAGCCACATCTTCCGGAGGGAAACTTTTTAAGACACCCTATTTCGACTTTA

TTTTGTAAATACAGTGTGCATGTGCATATAAAGAGAGATATCATTTGTATAAATA

TCAAGAATTAGAAGAGAAAAAGAGAGAAGAAGACAATCTATTACTATTACGATGT

GTGGGTTGTTAATTTGTTTAAAGGGAGCTTTTCTATAGAGATTTTTAAGGTCAAG

GGTCATCGTTCGATGTGGGCTTGCTTCCTACAATCTAGTTGCCTTACGGGGCCTA

CTCTTTTTCTTTTGATAACTACATCACCTTTTTTTTCTCCGACAACTATATATCA

CTTTTTTTATGTTTTCCTTTTTTTCTTCACAATAATTCTTTACTCGTTGCAAATG

TAAAGATACACAAAGTTACTTATTTTGTTTACGATGGTTCTTAGTAGTTTAAAGA

ATTAATGAATAAGATAAACCTAAACTTTGAAAAGACTAAAAAAAATGTATAACAA

CATACATTATACGTATTTGAAATAGTCCAAGTGATATTATGTCATTGATATTAGC

ACAAATAATTACGATGCCTGATATTGTCACATTTGATGATTTTAAGTTCTTGTAA

AAGATAAGTGTAACTAAATCACTATAGTGAGGCCCACGTTTTAATTTCTAAACTA

ATTACAATGACAATAAAATAGCAAAACTATTTAAAACTAGACGCCAAAAAAAATT

GAAACTAATAATTGTGAAAAAAGAACAAGAGAATAATAATCATTAATAATTGACA

AGTGAAATTAATATATTGCTCTTGGAGGGTTATATTTTAATTTTCAAACTAAATA

ATGAATACAAATGGAAAAGCTAATGATAAGAGTTGAATTTTAATAATTAAGAAAA

ACAAAAAAAGGTGTACAAGGAGACACATGCGTTTTCCTCATGCATCTTGTTTTTA

TACAACAATATATATATATATATTGAGTCATTCTCTGCTAGCTCTCTCATCTCCA

ACTTTCAGTATGATATATAGTTACAATTAAATAAACCTCACATGCTCTATTCTTG

CTTGATTTTTGAGTTAATCTTGAATCTCTTTG

SEQ ID NO: 39

Polynucleotide sequence PATCESA4_PATIRX5

ATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATG

GCATGGATGCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTT

ACCACAAGAGCAACGGTTGTTTCGGGTAAATAAACTAAACTTAACCATATACAT

TAGCCTTGATTCGGTTTTTGGTTTGATTTATGGATATTAAAGATCCGAATTATA

TTTGAACAAAAAAAAATGATTATGTCACATAAAAAAAAATTGGCTTGAATTTTG

GTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTCATTTGTTTTCTGGTTAG

CTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGATTTGAAAC

AGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAG

GCACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGT

TTCAAGATCTTTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTC

GAAAATCTTATTGAAGCCGGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTC

GAATCATATTTAATAACCGCATTAAACCGAAATTTAAATTCTAATTTCACCAAA

TCAAAAAGTAAAACTAGAACACTTCAGATAAATTTTGTCGTTCTGTTGACTTCA

TTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAATAAAAACCCTAAA

AACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTTTAGGTA

AGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTC

ATTAATTTGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTT

GTGAAATCATATTTAAAACATTAATAGGTATTTATGTCTAATTTGGGGACAAAA

TAGTGGAATTCTTTATCATATCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTT

ATGATTATGTTACATGCATTGGTCCATATAAATCTATGAGCAATCAATATAATT

CGAGCATTTTGGTATAACATAATGAGCCAAGTATAACAAAAGTATCAAACCTAT

GCAGGGGAGAAGATGATGAAAAGAAGAGTGTGAGCCAATACAAAGCAGATTTGA

GGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCACAAT

GGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACT

CCATCTACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCA

GAGTTTCATTCTGATTCTTGAGTCTTTTTTTTTCTCTCTCCCTCTTTTCATTTC

TGGTTTATATAACCAATTCAAATGCTTATGATCCATGCATGAACCATGATCATC

TTTGTGTTTTTTTTTCCTTCTGTATTACCATTTTGGGCCTTTGTGAAATTGATT

TTGGGCTTTTGTTATATAATCTCCTCTTTCTCTTTCTCTACCTGATTGGATTCA

ATATTAAGTAAGACTAAAGTAGAAATACATAATAACTTGAAAGCTACTCTAAGT

TAGAACATAGCCAGATTTGGTAAAGTTTATAAGATACAAAATACAAATTCTAAA

GAACTCAAAAGAATAACAAACAGTAGAAGTTGGAAGCTCAAGCAATTAAATTAT

ATAAAAACACTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCT

GTCTCTTGAAGCTTTCTTATGACACAAACCTTAGACCCAATTTCACTCACAGTT

TGGTACAACCTCAGTTTTCTTCACAACAAATTCAAACATCTTACCCTTATATTA

CCTCTTTATCTCTTCAATCATCAAAACACATAGTCACATACATTTCTCTACCCC

ACCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCCT

SEQ ID NO: 40

Polynucleotide sequence PATGAUT8_PATIRX8

ACGAGCTGACTTGTACCGATGAGCTGGCTCTTCTGGGCGAGCTGGCTGATCTTGA

CGAGCAGACTTCTCCCGACGAGCTGACTTGTGTCGATGAGCTGGCTCTTCTGGGC

GAGTTGGCTGATCTTGACGAGCAGACTTCTCCCGACGAGCTGACTTGTGTCGATG

AGCTGGCTCTTCTGGGCGAACTGGCTGATCTTGACGAGCAGACTTCTCCCGACGA

GCTGACTTGTGCTATCCTTTCTCCAGGTCTCGAAAAAGTCCCCTTTCCCGAGACT

TTCTATTCCTTATTTATACCCGTCCGTATAGTAGGGTACGCAAGGTGAATTCTCG

AGAGTGCCCCTTTTCTACGCAGCCGAACTCACATCCTGACCAGGCCGGGCTTCGG

CCTGGTGGGCCGGCTCGAGTTCTAAAGTGATGGTCGGGGCTGGGTCGTTATTCCT

TGAAATGGGCCGGTTGATCACTGAGGCCCAATTGATGTATCAACATGTGGTTTTT

ATAAAAAGAGTCGTGAGAAGAGTTTTCTCTAAAAATCCCTTGTGTTTGGTAATCA

AACTTCATTCAACCAACGAATTCCAAAAAAACAACTAAATTGTTCGGGTATATAA

AATGATTGGTAATGATATATCCCATAGAGGCCGTAGACATAGGCCCAAAAAGTTT

CCATAACTAGCAGAAATTGAAACTTGCAAGTTGCAAATATTATTACACTGGAAAG

GCAACAAGTCTTGAAGTACAAACTACAAAGACTTCTTGTTTGGATGGGGACGACT

GACGAGTTTGAATAACTTAAGAGAAAAGGGTCGCAATCGAAATTAGACAAGAAAT

TAGTCCTCAAAAAGTAAATTCTGAAGTTGAAGCTCCAATGTCTTTGTTCAAAGAC

TTTATTTAGATGTAAAGTTATGTCTTGTAACCACCAAACAGCTCCTTTTCATCTA

CACTCCCAATTTTTTTAACATCTATGTTTTGCATTGCCTTTGACTTGTCTTTCTC

TCTCCAACTTCTCTCCTTCAACATAAAGCCAAATCCTAAATCCAAATCCCTTAAA

CCGAACCGAATTAAACCGAAGCTGTTGAACTATCGCAAAATTTCAGATCTTACTA

ATCATAAACATGTGACGTTTAATTCATTTTAAGAGTTTCATGATTTGCACTGAAT

GGTATTCCGAGTCCACCGGAAAAAAACTTTTCCTACAAGTAGAAAAAGGATAACC

CCATAAATCCAAATAACCTAACCGATCAAACATATACCAATATAAACCAAAACAA

GATTCAGATTCATCGGTTTAGTAATCGAAGTAATGTACTAATGTGTAATATTGAT

TCCACCACCAGCTTAGAGATTCGAACCAAAAACCGAATAGCGCATAACCGAGAAA

ACCCAAAGCTTCCTAACAAATACATAAAACCGTGGTGTTTCTAATTCTAACCAAC

ACACGTTTCCTTTTTATTCACAAGAAACATCAGAGTTATGATCTGCCATTAATAA

CCTAAACACAAAGCAAGGTTAGGTAAATGATATGGACCCCTAATGAATAATCATA

CAATACATAACAACGTAAGATCCAGTTTCCCTCTTCG

SEQ ID NO: 41

Polynucleotide sequence PATNST2

AACGGTGGCGTGATGGAGCTTCATCCTCCCATCTTCGCCGAATTCATCACCAACG

AATTTCCCGGCCATGTCATCCACGACTCTTTAAGCCTCCGCCACTCATCTCCACC

GCTTCTCCACGGCGAAGAACTCTTTCCCGGTAACATCTACTACCTCCTTCCTCTT

TCTTCTTCCGCAGCCGCGACCGCTCAACTGGATTCCTCCGACCAACTATCAACGC

CGTACAGAATGTCTTTCGGGAAGACGCCGATAATGGCGGCTTTGAGTGGCGGTGG

TTGTGGAGTGTGGAAGGTGAGGCTTGTGATAAGTCCGGAGCAGTTGGCGGAAATT

CTTGCGGAGGATGTGGAAACGGAAGCGTTGGTGGAAAGTGTGAGGACGGTGGCGA

AGTGTGGCGGTTACGGCTGCGGCGGAGGAGTTCATTCGAGAGCGAATTCAGACCA

GCTAAGCGTTACGAGTAGCTTTAAAGGGAAATTGTGGTAAAATTTCGAATTATGA

ATAAACTACGTTTATGTTTTAATCTGTTTCACGATTTAAGCATTTAAATTAGTAT

GTTGATTTCCGTATTCATTGAAGACTTGGAACGATTATATAAGTTTATCAACGTA

GATATATTTGAAATATCATTGTTATCTCTCATGAAACAATTAATTTATGAAGTCG

TAGACTCGTAGTTAGAGATTATTTAATCTTCCCTATTCAATGCCAAAAGTCTAGA

AGAGCAAAACAAAAGGGAGAAACTCTTTTATTTCAGGCCCAATGACACAAAGCTG

GCCAGAAACAGTTTAAGATTAGGCTAAAGTTATAAGTCCGACAAGCACGAGTGCT

AATATATATAGTTATATGACGTCTCACCATTAAGGGTTTAATAAATTTTGAAACA

CCTCAAATTAAGATTGCTTCCCATGCAAACTTCCTTCATCTTCTAGAAAAATTAC

GATTTGTAATACTTCAATTATATCATTTTAGTTTTTTGTCACTAATTATCATCAA

TTTATCATAGCTCCGTGCCGCAACAACGTTCGTTTTAATCAGATTATATATTACT

CTGCTATAAACTCAGAACCATGTTAGAAAAATGAAAAAGACATTTCAGAATATTC

ATTAACTCAAAATTTTAATCTCATGATTTAATTTTTTATTAACAATGTTATCCTA

TAGCACATGGCAAATTTGAACGGCCCTTGCGTATTAATCTATTATAATCTCAAAA

CCATGTGTAAGAAAAAGGAAATTCAGAAAATAACCTTTTGTAAATAGGCCCCCAC

AAAATCTACAACATACGTAGATACCTCCTCGCTTACAGTTGTAAACAACTGTTCA

TCTAGATTCATGCCGTCATTCAAGTTTAAATTAATACAATAATTTAAAATTTTAA

TTTGGATGAATCGAATCCACCGTCGTTTCCTGAATACCAGATAGGTTAACTTTAT

GATTAGTTCGAGTGAACCACATGCACAATATTCGAATCTTAGACATTCGTTGCAA

TGTTAACTTCACATATATTTGATAAACGCTTCTTGAATCAGATCTTAATCTCTTT

CTTTCTCTCCATCTTCTAAGGAGGTTGTGGATTATCATGTAGTATATCATTATCT

TCGCATCACCTTCAACAAGAACAAGCTACGAGCTTTAAAGTCGTATTTAACACAA

TAATGTATAAAGTCTTTCTTCATCACATCACATACATTTTTTGTTGCCATCACCC

TTCATTCACTTTTTTTGTTAACACTATTCGTTTCTATATAAAATAAAAATAAAAT

GAGGAATGTCTTGTCCATAGAGATTTTTAAGGTCGAGGGTCATCGGAGCGATGTG

GGCTTGCTTCCTACATTATAGTTGATATGTGGATCCCGCGTGGACCATATTTTTA

CCCAATAGCTACGTGCATGGTCCCACCGCTCTCTCTCACGCACTATTCCGAAATT

GCCATAAACAATTTCACCGGACAAAAAGAGCAAATAATTTCGATGTTTAATAAAG

AGACCATTAGTATATTTGACCCAAAAAAAAATAAAAAAAAAAGAGAGACATTACT

ATAACTTTTATTAGATGAAATATTGCAACATTGTATTTATAACGGATCTAATTTA

CTGAATCATATTTTTTTTCTTTGTTAAAGAGATACTGAATCATGCAGAAAAATAG

ATAGATTTTTAAATACTAGGTGAACTCATGACGAATCAACCATTACGAGAGATTT

CTGGATAAAAGCAAAAACAAAACAAAACTAACATGCTAATCTAGGCAATTAGTAG

AGCGAAAAGTCGGCAAAACCAAAGGCCGAAGAAGCTTGATCGATATACTTTTTTT

TTTTTGTTTTGGCTGGATATACTTGGTATGAACTAAGAATTAAGTAAAAACTCAT

AGGGAGTAATTTTTCGAGAAGTGCATTCACTATGAGTATAAAACAGACATTTTCA

AATTATTAAAACAAGCTCTTAGAGGCTCATATGTTTAATTGTAAGTGGCGGCTCA

TGCGAACTTATAATGAAAACATCAAATATTCGGAAAAATAATACTCCACTGTTAA

AAAGAAAACTTAACAAAGGAATTAAAAATATGAGAGCAAAAGAACACATGCATTT

TCTCATGCATGTACTATTATTTATTTTTTTGCAGAGTTGATGTAAAAAATATACA

CATATATATAGACATACTTTGGTTAGTTATAAACTCGTTCTATTTTCTTCTCCTT

TTTCTATCTTTAGCA

SEQ ID NO: 42

Polynucleotide sequence PATNST3

ATTCTACACATTCACAAAGTTTACTACACTATATATAATTTACCCAACAAACACT

TATTTTACTGCATTATTCAGTATATTATCTTACCTATAAATGTGTATCATCATCA

TCAATAACGCGATTATTTGTGCTGAAGGATTATATATTCAAAATGATCTAGTTAT

ATATGTCACATGATTGCCGTTAACAAGACACATTTGAAGAAGCTAAGCAAGAAAA

ACGGACACTITTGCGACTTGTTACATAATTTAACTTATAGGTCAAAAGAATTTGA

TTAGTCATTGCAACTACGTGTGGATGTCACTTTCTATTCAACCAAAACTCACAAT

ATTATATGATCTAGTTTTGTCGTATTACTGATTTGTATTATAAAATGTTATTTAA

TTTGAATTCTACGTAGATATTGCTCATGCATGATAGTATGTATCTAAACTATTCA

AATAACTAACTACGTGGATATTTTATAATCCAAGTAAAAAGCAGAAAGTGGGTAA

CTACGTCAGTATGACTATACTTTTATCGGAATTGCTTGACATCCAAACTTTTGCT

ATGCTTCACCAACCAATGCAGTTTCACTTAATTATTAACTATTGACTATGTCTTA

TTAAGTTAGCACTAATTCGTTAATCATTCAAAACGTTATTTGATTGAATTACATA

TTACACTCTCTTTCTGCATCACCACTCACACCATATGCAACTATAACCAACTCAT

ATTCAAATGTATTAATTGGATTTTGGTGCGAGATTAAAAATTGAAAGGAAACAAC

ACAATATGATAATGGGATAAAATCTTGAACGGAAACTCAAACTAATCCTCATAAG

GTATAACAAAATAACAATTTAAGCTAAGCACAACAACATACAAGTTCGACCTTTT

CCTTTGATGATCCAGCCCAACAGTTCTCTTATATCTCAAACCATTCGACCATTTG

AGCCAAACTAGCTAAACCTGCAGGAATCAAAACCAACAAAGATTCAGATTAGCTA

AACCGGTTTCATCCCTTTGTCACATGACTCACATCCGTCTTCTACATAACGATTT

CTAATGATGTGAGCTCTTAACTTGCTCCAGCAAGATCATCAACTTTGGAGCACCT

TCAATGATTTAGTTAACATGTTAGATAAATTAAATATTCTTGTTTCAATATATAT

CAACTTTAGTGTAAAAGCCTTAACATTCTCTTGAATATTTAATTTATTTCTCCTT

ATTTCGATTTAATGACAAATGTGAATTAATTTTTGTGATATTTTTGTTCGAAATT

AGTTTTCAGTTAATAACATACATGTGAGCATGGGACACACATGATTTAACAAAAG

GGAATGACGAAATGATATATCAAAATATTAGTATGGGAACAAATTACGAGGTGAA

ACTTCACACTCAACTCAATTAAAACTAGAATAAAGAAATGGAAAAAGTGAAAGAA

TGAGAGGTCAAATGTGGTTAATCATTATGTGGTATTAGTTAATCCATCAATTGTG

TACCCAAAAGCATGATTAAGCATAGAATTTAGAGAAACAAAACATCATTATTAAT

GTTGAAACACAAAGATCCCATCAACAGACAAATGATAAGTACAGTGCATGTAGGG

TAACAACTTTTATGTACATGTTATATACTTATATTATATAATAAGAAAACGATTA

AAGTGTCATTGCTCCAGCCTCTATTTGTAAATCATATTATATCAGTATGCTTAAT

TCCAATAATTAAGTCCATAACTAAAATATATACACATATATGTATGTTAAATGGT

TGAATATATACATATATTTTCATAAACAAATATTGCTAATTAATTCAGTTATTTG

TGTACATAATCCAACTATCACCTTTTTAGCTGGAAGTGGATATTCCAACATGTCA

GTCTGTCACTCCCACATTCATACTCTCTATTCTTTTTAGCTATTTCAATATCTAC

GGTTAAATATTAATGGCTATATAGCCTTACCCTTCATTTTAGTTTTTTTTTGGTA

TTCGCATAACCATCGAATACTCAAACTTACTATGTAAGATGGTCTGAATAACTAT

TTCCGATTTAAGATGAATAGCTAGATTGAAATATACATGCACTAATTGGACATGC

ACTAAAGGCAGAGGTGAATTAAATGATGAAATGAAGATGAAGTGTCACACTTGTG

CAAAAAGCATGTCCCCTGCTCTTCTCCGCTTGTTTCAATTTCTTTGACTTTCATC

ACGTTTTTGTCACTTAAATACACCAAAAAATATAGTACAATTAAACATCGAAAAT

CGTCCAAAAAGAAGAAAAAAAATCATGGAAAGTTCTTTCGTTAATGTTACACACA

TTATCTTGATTAGGTGACACCAGATATTAGAATAAAAATGATAGATTATGAAAAG

AAAAAAAAAATTGATGTATTTTTAGGATACATCGAAAGGAATGAACATACCAAAA

ACATGGGAAAAAATAGATAACTAATTAACATGGTAGAATGTAGATGACGTAGATC

ATGAAACGAGTGTGTGATATATTAATGAAAATTATTTTAATATACGTAGCTATAT

TAGAAAATAATTTACATTTATTTTCTTCTAAACAAATCTATACTTTATATTTACA

TACATTAGTAAAGACCAAAACACATGGAATTCAAATTCTGCAATAAGTAATTGCA

AGAAAACACAAAGATTAATCCCCCACTAAACCCGTTTATTTACGTTAGTATTTTT

CCGTTTTATACATTACACATGACATGACATTACACGTCAAAAGAAATATGTCTTA

CGTCAGAACTTACGTATGATCAAACTCGATTTAAACATAGAAACATCTGTTTACT

AAATTATACTAATTTCATAAAGACACTTTAATGCATGAACTTCTTTGTTTAAATA

ACAATTTCCCCCTTTTGGGGGCTATGTCTCGTCGAGTCCTACCACCATTATAAAT

TATCTCATCGTTTGCTTTCTTTTTTTTAAGTTGTAACCATTTCCACTCGTAATCA

TACAACTTCTCTACTCTTCTAGAGCAAAAACCCAAAAATATATTGCTATCTTCGT

TA

SEQ ID NO: 43

Polynucleotide sequence PATFRA8_PATIRX7

CTTCAAATCTCTTGTATCATTAAATAGTAACGTTTTAAATATTTTCTGGATAAGCA

TAAGTTTCTTTGAAAACTATTTTGTATATATTCCTACTTCTCCATTTTTCTAAATT

ATTTTATATTATACATAGTTTTCCAAATTATCAAACATTTTTACATGTTTTGACTA

ATAAATAAACATATTACTGCGAATTAATTAAAAATAAATATTCCACACAATAATTA

CCTTACAAGCGAATAAACTTTTACTATGTTTTCGATGTAAATTTTTCTTACATATT

TGTAACTGAAATTTCTAACTTGTTGTTTCATAAGTTTTAAAATTTATTATCTAATT

ATCTACTTTTATGTGTTCTAGAGCAAAGTGCTAAATGTATATATACTTAGATGTTG

TGTTGTAATCCAATGTCAATATAATCAATGATTTAGCTATTTGTAAACATACTAAA

TAGTATTCCACCAAAAAAAAAACATACTAAACAGTAAACAAACAGCAAAAACAAAA

TCCACATGTCCTAAAAGATAGTCTGATTTTCGTTCATAATGCTCTGGTTTTTGAAA

GATAATAATTGTGTTGTATGAGTGTATGACAAATATTCATTGGTTTGAGAAGTTAA

CAAAATTTGGTGGCTACAAATGGTTTCCTATTCGAGTTGGGTCCATTATCCCTTGG

CGTGTACGGAAATAATACCTACCCATCATAATCTGATCAAAGATGAGGTAGTCTTT

AAATAAATTTTGCGGCTTATATCAATCTTTATGTACTATAAACTGTGAACTTTTTG

TTCTTCAGGACTTCCACATCATTGCCCAATCCGGTTATACCTTCGCTAGTTAATAT

GTTAATTAACATTAAATTAAAGAGCTAACATTTCTTAGGTAGTAAAATAGAAGTTT

TGAACTACTATACTACTAACATGTGAAAATACTTTAGTCACAAATATGACAATATA

CAAATTTATTGGAATGCAAATTCTTGAATTTCAATTGTTTGAAAATTATATATTTC

TACATAACAATTCTTTATAAACTAAAAATATTAATTTTCCATGGCTATGCGTTATA

CGTATATGTCAAATATTTTTATTATTTATATAATTTTACGATAAATTAGTACTCCT

ACTTTACTATATTACTCAACACTAAAAGACCTCTTTAACTCCGCCTAACAAGATAT

GTTTTCTTTTGAATGTTTCGGTTAAACATGACAGAGATTTGTITTCTTGCTTTCGC

TCAATACATATTTGTGCTCCTTTAGAAAAGTAGTATTTCCTAACAATCCAACATTT

TCATATTTATTATATCTTTTAAATATTATCATGGTTCTTTTTCTTTCGTCATGTTT

GGCCTCTTTAAAATAATTCTTGAATTGTATGAGCATTAATCCAATAACGTCCTGAT

CCCAAAAACCTCATATTAGGTTTGAGAGTCCGAAAATATACTTTTCACATAAAGCA

CCTAAGGTGTCATACTTTAACAACTTCACAAAATATGCAAAATTTGTCATTGTCAC

TTTGAGATGTAAGTTTTTTTTTACATGCAAATAGATTGAGTCTCTTTACGTGTAAA

TTCATTTAATAAAATTGTATGGAATATCTATTTATATCATATATTTCTAACATATA

TATAAATATCTATACAAAAATACGACTTTTTGGCACATGTAATTAGAAAAATCCAC

AAGAAACAGAAAAAAGAAACACCAAATACAACGAAATGAAGAAATTATTATAAATT

TGAATGGCTTAACATCTCTTAAGAGTCAACAAGGTAAAGGATTAATTAGTAGTCTT

CATCAATCTTTCTCCACCTTCTTCTATTCCTTAATCTCCACTTTATCTCCCAAACC

CGAAAACTCCTCTICACCAACTTAAACCCTATTAACTAATCCCAACAATCAGATGT

TTCGAATTCAACAACCAGCTCAGGCCATAAGATTCATCCCGGAGAAACAAGAACG

SEQ ID NO: 44

Polynucleotide sequence PATIRX9

CGGGTTTTCGGTTCGACCCGGACTCGAAACGGGTCTAGATGAAGAAAACCTCATCT

CTTTTTGTGTCTAAGGATTTTTTGGTACTGAAACTCTCACTCTTTTTTTTGGTTCC

TCTGGTCCCTCTCTATATGATTCAGATCGAACACTGTGGTTTTATATTTTTTAATG

TTTTGTTATGTTCACACGTTGGGTTCAGAAAAATTGACGGCCGAGATCTTTTCTAT

AAGAGGAAATCGGTGGTTCTACTTAGCTAATCCTTTTTACTAGAAAAGTTTAACAT

TTTGTACTTTTTGTCTGTATGCTCTCTAGTTGTTTGTTGAGATCTCTTGCTGCTAG

ATTCACTTTTTGGGACACATTGCTTTGTATTTGAAGCTAGAAAGTTTATATCAACA

TGATCTAAAAAAGTATTTTAAGAGAACTACATTGAGGTAGTTATTTCTTTTCCTAA

ATTAGTCATTGGTAAATTACATCGTGACATTTATAGAACATTGCAGAGCATAAAAG

ATTGAAAAAAAAATGAGCTGAGATTTGTATGTATATAAAGAAAACGTATTAGCATA

GCTTTCTTTCAGATTTAACGGTGGAAATCATACAAAACTTTCTTGCAGAACAATGA

GTATATATATGAAGGACTCGTTAACGAAAATATTAGTTTAAATCTAGATATCTTCC

AGTAAAATATGAGTTTCGCCTTCGTATATGATACGGCAATAACTTTGGGACCAACT

AATTTGCATATCACATGTTGATATCTCTTTCAGTTCTACTCATTCTTTTTTTTTGA

AAACAACAAATTATTGGCTGCAAATGTTTTTTGGTTTAACTAGTGCTTCTCTAATT

GTCAAGTATCTTAGTCTAGAGTTAATTACTTAAATACTAAAAGGCTGTCGACAAAA

TCAAGCTTGAATCTCCTTGTGGTATCTTCAACTCTTCGTTGTCTGCTTACGAGTGG

TTTACTCAGTAATTATCTATAATATGTTATTTTTTTTCCCTCATCTTTTAGTTGTT

GTTTCATTACATTGAAAAGCTTGTAATGTCTTTATATGGTATATATGGATCTTATG

AGTGAGGCAAGATCCATGATGTTTTTGATCTTAGAATGTATATGATGATCTTAGAA

TGTATTTGACCGCCCACAAATTATTGTTCATTGGGATTATATCTCTAGTCCAACTC

CAAGCAATCGAAATGGGTCCTGCTTTTAAGAACAACAGTATATGTTTAAGAATAAT

AACTTTATATATTCTCGATTTTAAGATCTTTTGACAAAACCTCCTTTTCGTTAGGA

GCGTACTAATTTCCAAGTGTTTGATTAGTGGGGTCTCCGTAAATTTATTTAGAGTT

TCTATCTATTTATTAATAGCTCAATTAATTAATCTATACTGTATCTAAACATCAAT

TTATATATTTACTCTTGAGACCAAAACTGTCAATTTATAACATTGGATAGTTTCTT

AATTCTTATTATATATTTTTCAAACACTTTTCAAGACTAATCTCCACATTAGGTAC

TCTCTCTAGAGATAAAAATATTTATCAAAAACATTTTTATTTATTTATTAAGTAGT

AGATAAACTACTGTGGCAAAATCGTAAATGTCTAAATGCTGATGAATTTTTTTTGC

TGCTCCAATCTGGTTTAGTGCTCCATATACATCCACGGCCAAAATGAATCTATGGC

GGCATTAAGATTCATTAGTAAGCAACGATTATATTAATATAATTGTTTTTAGCAAT

GATTTTCCGTAATTTCCCAAATATGTTTCAGTTAATGTGTTCCAATCCCAACAACT

GGTTGTTGCAAAAGACCACCAACGCAAGCAATCATCAAACATCAAAATAATCTTAC

CTTAGCGAACAAACAATAACTACACAATTCTCATAAAGCTCTTATATATCACTAAC

TTCACACATTTTGTTTTCCACAAAAATAAAAACGGAACTCACTCAAGAAACCTTCT

TCCTTGAAGAGAGGGTT

SEQ ID NO: 45

Polynucleotide sequence PATGUT1_PATIRX10

AATAACAACCACTTAAGTTACTGCAAGTTACCACAAAGAAAAATGATCTAGCAA

ATGAGTAGCATCATATTGATCAAAGACACTGCAAGATAAAAGTCACCTTGCTAA

TGTTCGAGATAATGATAAAGTGTAGACTTGGAGCAAGAAGCCATTTAAACTAAC

AACTTCCTAATTGAGACCTTTCATGTAACTTAATGTCAAAATCACAAGCAACTA

GAGGAAGAAATAAAAATGTACCAGGTAGCTTCTTGGGCTTCCTCATGGGAACAA

ATTTGGCACCAATAGCCAACGCAATAGGAGGGCCAAAAATGAAACCTCTAGCTT

CAACACCTGCATTTACCACAACATCAATTTAGGCAGAACCAAAAATCATCCACC

AATTCATTTCAACTTTTCAGTTTAAGCTAAAGCACTCAGTATCTAAAAAGGCCA

AAAGAAACTAAATCCACAAGCTGTTAATCGATTGGAGTACCAAACAGAACCATA

CGAGTTGTTACCTGCAACAACAGATATGCCTTTATCTTTGTATCTATCAACAAA

CAAAGCAATAGTATCCTTAAAGGCCTCAGTGTCGAGAAGAAGCGTCGTTATGTC

CTGAAACATGATTCCTGCCAAGTATCCAAATTAAAACCTTAAGATCCCAACGCA

GATCAAGACTAGAGACGATATTAATCGGTATAAATGGAAAAAATGGAGACCTGG

TTTAGGGAAGTCGGGGATGACTCTAATGGAAGAGGCAATCTTAGCGATTCTGGG

ATCTTGCACATCTTCAGTCGCCATTTCACTGTCCCGACTGGCTGCTGCTTTAGC

AAAATACTCGGCGTCAGATTTGCAAACACAGAGAGACCCTAAAGACTCAATAGA

GAGACACAGTGATGAAAAAATGACCAATTTATCCCGAATGGTAACGCTTTGACG

GAATTGCCCCACGCAAGCAAAATATCTTTTTCAAAAGGAAACAAAAAGTTTAAA

AGGGAAATAGAAGGTGGTGGGGTCTACCGGCGGAGGAGAAGAGGCGGAGTGAGG

TGGTTGAACGGTGGTTTGAGAGGCGGATCGAAGGAGGAGCACGGTGGTGGTTGT

TGAGAAGACGGTTGCAAGGAACAGCACGAGCAAGACAGAGACGATGAGAAACAA

GTGGAGAAATTATTATTGTTTGCATTGTCTTTGGACTGAGAGATCTTAAAAGAG

AATGTAAATTACTTTAAACACGGAATAATGGACAAAAGCCGTGATCAATGACTT

TTCAAGTCTTAACCAAACCTATAACTCATCCATTGTTTGTTTTTTCTACATATT

TCTTCACATAAAATTGGATGATTTAGAATCTTTCAGAGTGTTCACACTCCAACA

GATTATTATCCACAATGTTATGGTTACATTTAGAGATATATAACAATGTTCATT

TCATCGTTGCTAATGACATAAAACGATCAAAAACTGAATCATAGTACTTCTTTT

ACAGTGATCTCAAATATATTAATCGCTAATCAATGAATTATGTCACCTATAATT

GTCGTATTACCAACAACTATAAAACATATATATAAAAAATTGTTGTCGTTAACT

AGTTGTTGATAGTGGCCACTCTAAAACGATCATGACCTACTACGGAAGTTATAA

CTAGTCAACGTTGGACGTTAGCAAGGCCCAATGGACATTAACTCAGCCCATAAT

AGCACGCGCCTTGTGATGTGCACCAGTTTCCGTCTTTGGTCGTTGAATTCAAGG

AAAAAAAAAGTACATCACAAGCAATTTCTTACTTATCTGTGACTTGAAGCTATT

TCTCCAATTTCGTTTTCCATCGACACTCTATTTCATTTTCACCATTCACGTCTT

CCTTCTGAATAAAATAAACCCTAAAACCTAATACCGAAGTAAACTCGTCAACCA

CTGCGCCCATGACCTCCCAACGATACTCTTCCCTTATATTCTTCCTCTTCCTTC

TTCCTTTCTGCGATCCAAACCTTCAAACACATCTCCGGTAGAT

SEQ ID NO: 46

Polynucleotide sequence PATIRX14

ACCTGCATCGAATTTATATAAATTTAAAACACATTATCATCATCTCTAACTTGAA

CTTTTAAACAAGTTTATCTTTTTGTTTCACAAAAAAAAACAAGTTTATCTTTATG

TCCCTCCTGAGACATATAAAACAAGATTATCTTTCTTCTTAGTAGGGATATAGCA

AGTCCGGACGAGATCAAAAGTAGATTGACTCTTAAGATCTTACTAAGTTTGAGCT

TGCTTTGGTTCCCACCTCTAAAAACCAGTTTTGCATAGTCTGAGACTCGTGTTAA

ATTCGATCAAATCTCTCTTTCAACGACGGTTAACTATGGACGTATTCGCAAAACA

TCACATAAAAACATCTCTAAAGTATTTGGCTATTTGCATAAATATTTCACTCTTA

CAGTCGTCAAAAGTATGAATGAACTCTACATATCGGCCCAATATGAACCAATTTG

TAAGACCATAATGGAAAGCCCATGTTTCTCTTGTGCTTGTTTTAGTTGCAGAATC

ATTAGTTCACATATTGACCGGATTATATTAGTTTTTAAAAACGCATGTATGATGT

AGTCACTGTATCATACCCAAGTTACTGTATTCATTACCCAAGTTCAAACTCGATA

AAATGCATAAACTAAACATACGTTCTTTAGCCTTTTGTTTTCACTTCAATTAACT

CATTTTGTGCGTTGTATATTTTTTTTCTTTCCAACAGCTACTTTTCTCACGTCTA

TATTTTTTACCGTTTGTGATTTTTGAGTCTCAAATATATGGAATTGTTTTTTTTA

AATGGCTACTTTCCAAAGTCTTATATTTTTTACCGTTGTAAATGTTCAGTTTCAG

ATATATATGGATTTCTTTTTCTAATGGCTACTTCTCTAACGTCTATATCTTTTAC

CGTTGTAAATTTTAAATTCTGAAATATATTACCGTTTGTGATTGAGTTCACTTGA

CACACCTTCGTTAAAAATTACACAACAAAAAGCGTTCACAATAAGCCCAATGGGC

CTAAAAGACCCTAACAATCGAACATACCCTTCTGACCAACACATTTTCTTAAGGA

GACACTGTTGGTCCATTTACTCATTTAAGTAGGATTCATAACACTTGTCATGGTC

GTCATTTCTTGTTCAAATGCCTTTTTAAGTAATAACGCAATGGAAGCATATATAT

ACTTTAAACCCACAAATTAATAATGCATATGTATCTATTTTTCTTGCATATACTA

AACATGTCTAAGTATGATATAAACTTTGACACTTTGGTGGTGCTGAGTAATCATC

ATATTTATGCTTTGTGTGCAAGTGAAAACGAACCGATAACAATCTTTAAGACTTC

CCTACCAAACCGGTTTAACCTTCACAACAAACAAACCTAGATCAATTATCTCTAA

ACCAAAACCCTTCAAACCATGTCTTTTGTCGGACCAAACTGTACTCTTATATATG

ACATGCAGATACGTCGTTTTCATGGGCCTTACTAATGGCCCATTAAAAACATTCG

TAATCAATTATTTTGGTTAGTCTTTCCCAAATTCGTCTACATTCCTCCTCGATAA

TCACTTTTAATTAAAACCATATGAATTTACGAAAAAAACAAAAACACAATTATCA

TTATGCAAAACATTTAATTCAATAAATTGAGGGATGTTTAATGTTAACACCAAAA

ATTATTACCAAAAATTGACTTCAATTAGAGACATATTAAAACGACCCTGATTTTA

CTCAAAACTTAATTGAAAGATTTAATTATCCAATAATAAAACGACACGTGTACCT

CCTTGTCGCTTTCCTCTGCTTTCTTCGATGGCGTTGCATCGAAGCATCAGAGAGA

TTGGTATGGTGGTGGTGGTGAGAGAGCAGCAACAACAGCAAGAAGAGAAAGCGAT

AATCGAACTGATTAAGATCGTGAAATCCAAGTAATCTCTGTTGCTTAATCTCAGA

TCTTTTTGATAAGGAGAAGGAAGCAGAAGAAAGAGGTCAACGAAGAAG

SEQ ID NO: 47

Polynucleotide sequence PATMYB46

GTTACACTAACGGTTTCTTGTTAGATTTAGCTGACGTGTCTTTATGAATATATAT

AGAGTTAAATTTTAATATTTTAAGAGTAGTATTACTTCATTAAAAGCTTAGTTGT

AAAATTACTAAAGATTTTCATATATTATAAACTATTTTTTCCTGGCAAACTTATA

TTATAAAATTTGTTGAGCGATTGTGTGATTCTTTCATCCACAATTAGATTAAAAA

AAATCGCAAAAAGTAATACAAGAAAAAATAATAATTTTACAAATTAATAATGATT

GTTTCTTTGGCTAAGAGTTCAGATTTGCAGAGTGTTTTTTGGTCCTTGGGCGATA

TTACGAAAAGTGAATTGTAAAGATATGTATAGATTGTGAGGAAAATGCGAGAATA

ACTGAGAGCTAGGGCTATGCATGAGATGATTGAAATATCATGAACCAAATGGTTA

GATGAGAGCTTGGAGTGAGAGGTGACACTTGTTTGAGATGGGGAATAGCGGATTA

ATGTGCTTGCATGACCTTGGTTCTGAATTTTCGATTGATGAAATCTTGCATTTCG

TTATTTTCAAACTTTGTCCACGAGTTTTACATAACTAGGTTCATTCAAGTTACAA

CTTAAATTGGTTAGCTGACGTCTTTTTTCATGCATATACAAGAGGTTGCATTTGC

AAGCTTCAAAAGAGATTACACCAAAAACAATTTCCCCTAAAGGTTAAGATATATC

TTTGGCCITCAATTCGACATTAGGAATTATGITCAAGATTCAAGATTCAGTACTA

TTCTAACTTCTTTTGTACTTTATCTATGGATGTCTTGTTTATGATTGTATAAAAA

GTTTTGTTTTTTCGGATGGGTGGGCTATTAATATTATAAATCATATAATATGAGT

GTTCTGTAAAAAAATAAAAATGATATGAGTGTAAATCGAGAACTTAAAAAATCAT

GACACACGTTTATATATTAAAGAAAAAACGAATATAAAATATATGGATAAAAGGA

GTATAACATTTTCTTCATTACAATAATTAGATTTCTTCAAGTATACGTGTTGGTG

CGCGAGAGGTGGTTGTGTGAAGCCGAAGCAAAACTTCTTGCTCGCTAAGCCTCAT

ATAACACAAAAAAAGGGTTCTGTGACACACGTCGATTTATTTTATACAATTGAAA

TATGCTTACATACGTATACAATTAATTAAATAACACAACATTTGCTTACCTTGAA

AATGAAGACATCTTTGAATAGAAATAGACATGCTCATGAATATATATATTAATGT

TATATACTATCATATATCAATGTTATATATCATATATATACACACGTAAGGTTAA

CGAATTAGATATGTCTGTAATGTATACCTTGTGAATGAAGAAACTAATAGAAATG

AGTTATATATTCAAAAAGAACAAGAAAAGAAGAAAATAAAATTAAGAACAAGTGA

AGAGCACTTCTCCTTTTTTTCTTTGATGTTTTGCATATCGGGTCTTTTTCAAAAC

CGTTTTCGTCCATGACCGATCAACTAACGTTTCTTCATTTCGTCAAATTAGTTAT

ATACAAAACATACATTTGTTGTTGGTGTATTTTATTTTATTTACCTTACACAATA

TATGCCGACAAAAAAAATGTGTTTAATTTGAAAAAGAGCCAGGGTTCGGATGTTT

TTCTTTTATGTTTTAAAACAAAGCAACACTATATTATAAATATAATATATACAAT

AAAAATATAATTAAGGAATAGAGATTAAAAAGGAAGAAGTGCAAATGGTTTTTCT

TCCCAGAATTGTAAGCAAACCATACAACCATCCCTTTCTCATCATCATCATTCTC

CCTTCATCAAGTCTTCTCTCTTTTCTCTCTCTATTATAAAACAAACTTCACTCGT

TCACATCAATGGATCCTTGAGAAAGACAAACAAATTGAAGAGAAATAATAACAAT

TAACTCAACCAAAAAT

SEQ ID NO: 48

Polynucleotide sequence PATMYB58

CAAAGACTAGAGACAGAGGCGTGCCAATAGCAACACGTTTGCTTTCGTCATGCA

AATTGGGATATTTCAACTTTCTTCCATTTTTTCAACCTAGTTTACTAAACTTTT

CTTTTTCCAGTGCGAACCTAATTGGTTCTAGTTAAAATAACATTTTCGTAAGTT

GTTCACCAAACAAAGGAACATATGATTATAACTTTACTAGAGATGCATGCACAA

TAATGCTATTGTCGAATAAATACTTATATCTTCTCCAAAAAAGTTTCTTTATTA

TGTTAGAAGATCCATCAATATACTAATTGATTTTTGGTTATATGTTTTGATTTA

AAGACAAAACTATACAGGACATGCATGTGAGAACAAAAATTGTTGTTGTTGTAG

TTGCTAGTTGAGTTTTATTTATGTTGCCAAAATAACACCATGTCAACTTTAATT

TTCGTCATATAATTTAACGTAAGCATGATGTGTTTCGTCATATCTTGTTTGGCA

TATGGAATATAAATCATACTATTGATTTGGAATCTTTAACTTAACTTCCTATTA

AGTAAGCGATTGATGCTGATATGTATGTTTCTTTAGATTGATGAACGTAATATT

AATCAGTAGTGGATATACATTGTATCTTTAGAATTTAGGTTAGTATATTATGGC

CAAAATGACTAAATTGAGTACCATAAACTAAAGTTAAAGTAGTGGTAAAAGCTT

ACGATATTGTTTTATAACAATTTTCAAAAAGTAAAAGATATATAAATGTTAGAG

GTTTTGGATAACCATATTGTTCTATAACATTTTAAACATATGTCATATATGTTT

CGTTTATAATATTTATGACTTGACCAAATAATTTGTGTATGTTATTTAAATCCA

AATATATATGAGAAATATATAGACGACATGATTAAAATTATTTAAAAGAGTCAT

GATGAGAGGGATGGAGACTAAAAAAAAGAGGAGAAAAAGATAGAACGTCGAGAA

ATGTTGTGTGTGTATAAAGTAAAGGAAAGCTAATTTGATCATTGTATTCGAAGA

AAACAAAAAAGTATACACATGTTACAGGGTTATAGGACCCATTTTCTTTAAAAT

AAATCCACTATGGACTGATGTACATATTTTTTCTTACTGTTCTTAAGCATGATT

TTATATGTATAATATGGTTATAGATTAGAATTTTATTCAGCCTTCCACGATTCT

TAACCCTAACCAGTCAATTTTTTCTTCCTTATAAATATGAGTGCCAATCGGAAG

GTGATAGCATCCTTACGTCTTGTTTGGTAGATTACTAAGTCAAGTTTTATTCAT

GAAATTTCCACTTATCAAACTTTCTCATTTTGTTAAAATTTAAAACCGTTTTTC

AAAAGTTGGTATAGCCATAGACAGAAAAAAATTATTACAATCCTATCTGATTTG

ACTCAGACACCCTAATTAGTCAAATCTCAAAATTAGCTAATATTAACTAACGAG

TTGCGCATTTTGCAGCAGTACAACAAAATTAGTCAAAATAATTTAGGATAACAG

CACTAATCACAGGAACAGGTATTTTTTTTTTTCCTTTCTTTTGACATCATAAAG

ATGGATTCAACTTATAGATTGGTCAGAGGCAATCTTTATAGGTTTCATGATTGA

ATAAAAAATATGAGACTCAGTATCTAAGTTTCAAACATGTTTCATCTGTGTTTA

GTTGATTACATTTTCATAATAGTTTATTAATGACATATAGAAATGCGAACTATA

CAATTATAAAAAAGATGTGAATTTTGCCAGATACTCATCCACAATATAGACAAG

TTTTTAACCTCAACAAATCTGATGTGACATTTGTCAATGTCTGTGGTTTATAAC

ATGTTTCTCAATGTCAGGATCACACACACCACTTCTCATGTATAAATACACATA

AAAGCAATTGGATTTGGTAAGAGGGAATCTCAAAAGTGTGTGTCTGTGAGAGAG

GAGAGAGAGAAT

SEQ ID NO: 49

Polynucleotide sequence PATMYB63

GTTGATATATATTAATATGTGTCCCTATTATGATCACACAAAACATACACATGCA

GAGCTTTATTCCAATAGCTAAAATCTGAACTTTAAAGTCAGTACACTCGAAATTG

ATATTGACGTATGTATTACTAATAGCAACATGTGTTCTTTCATCATAAGTTTACA

TAATTTTTTAATTTTATTCTACTTAATTAATGTCACAGTTTCCATCGTTTTGATA

AGGTCCATACTCCATAGGGACGTTGAAAATTTAATTTAATTTTTTCCACTCATAG

TTGTCCTTTTTTTCTTAGTAAAGTTTGGGAAAGTTTTCCCACTCATACTTGTTTG

TTCACCAACCTTCTGATTACCAAGAGTCGTATAAAAATGCAAAACTAATAGATCG

TCATTTATATATGTTGCTCCTATAGACTTTTATCGACAAAATTTTCCGAATTAAT

CATTTTGTAACTTCAATACATATACGTCCAGATATTTACCCTAGTGAAAAATATT

TCTTCTTTTTCAAACCTCTTTTCCTCTCTATTCCTCCTAAGAGCTTGTTAACGTA

ACAAAATTGTTGGGTTTATTAACTTCAATTATTGTCGATACTTAGTACTTTAAAA

TATTTGGAGTAATAGATGTAGTGATGGCTGTGTCGTAATTGCTTGAATAATTTTG

GATGGGTACAGAGGAATTAATTAAGTAATGAAGGTTTGGTGGAATTAAGTAATTA

ACGTAGCCAAGAGCCAACAACAACACCAAACCCACCAAACATTAAAAAAGTCAAA

AAGACGTAAGTCTTTGACCTCTTCCACTCTCTTTGGTCTTTAGTTTGGTGAGTTC

GTGCACTATGCTCACACACACTCCTTACGCCTTTTGGTGTTTTCGGATGTGATTA

GAAATGACTTTTTAACAGTTTTTTTTTTTTTCTGTCTCTCATTTTAATGTTATAT

TTAAGGATTATATATATTTCTGCTTTTTTGTATACAAAATATGAAAATATTCCAT

GGAGTGACGTATGGAGTGACTGCGTACTTAGTAAAACAGCATTATTAGTGAGAGT

TCATTTTTCTCGTGTTACACTGTATCTACATGATGATCACGGGACTATCTATTAT

TCAAAAGTTGGTAATTATACACTGAGCCTGATTACAGAAGACTCGCAGACAAAAA

CTAATATAATCAATTCCTCCTATGTATACCTTAAGCTAATTCTTAATTAACAAGT

TGCAGATTTACAATCTTATTTTAGTCAAAACACTACCTAATATTTTGCCACTTTA

TAACTATATATTCTTACTCCTCCAAAGTATTTTTATTAAGAAATACATAAAACTC

TTATCATTACCGCTGTAAATTCCTAAGACCATTTCAATTAACACTCGTCGACATG

TAGTAGTTTCTTACATTAGCGAAATTTATTTCAGACAATTTTATAAGATATGTCA

AATCTGATAATATTTTTAACACGAGATGCTAGTTTCCATTATTACTTGATGTCAA

AAAAGAAGAAAATATTATTTAAGGATTTTGGTTTCTAAAAACGAATGTGAAATAT

TCATGCATCGGTGTTAGAAGGAAAGATAAGTTGCATGCATCATAAGGATGCCAAA

TGAAGTAAAAATGAGAAAATGGAATCATACCAAATAATCAACCATACCACAGACA

GACAACCTTTTCCCACTCAACAAATCTGATTTGACATTTATCAATTCCTCTGTTT

ACATATTCATCTTTTCTCATGTCAAGATCACACACTCTTACCTCTCATATATATA

AAACAGAACCAAATTATCTTTGGTAAAAGTGAATCTCATCAGGAACTGAGTGATA

TAAAGTTATATATATAGAGGAGAGAGGGAGTGAGAGGGAGTGAGAGAGAGAGA

SEQ ID NO: 50

Polynucleotide sequence PATMYB83

TTTGATACAGCAACAGAAAAAAAATAAAAATACAGAAGAACATTAAGAATGATC

TTCTACCATCTGAGAATGGCAAATCCAGAAAGGATGAGAGAAGAGATGATCATG

ATAATAGACATTCCTGGGAAACAAGAGGCTCCTGTCTGTGACTCTGAATCGTAC

TGTGCGGAGGCGGTAAAGACAGAGGAGAGCGTCATTGTGGCGAAAACAGCCATC

GCTGTGTATTGATTGTAGCCAGAAGCCATGTTTACTAAATTTGACCCTCTCAAA

ACCAATTATGTCACCTTTGGCTTTGGCTTTACCAATGTTGTTGTTTTATAGGGA

AAGAAGAAGTTCGTGGGGACGTGAAGAGCATAAGGTTAATGCTCATTTCATAAA

ACCCCACTTTCTGTTTGTTGGTCAACGATTGTTATTGTAATGACTAATGACCTA

TAGAACAAAACCCATCTAACATGAATCTTCTTTTAAATGGATTTGGTGAAAAGA

CCAAGTTTTAAAATCATCATACGTGCGATGAAAGAATACCCAATTTGAAGCATG

AGCCCAATGATAGTTTATAGGCCCAAATAATTTTGATTTATAGTCACAGACAGG

ACAGGAGCCTCTTGTTTTATGAGTTGAATTGGGCCGAAGATGATACAATATAAA

GCATGAGACCAATAGAGGACTGACCAGTTTCTTACCTTCGTTCGTCGAAGAATC

GAACAGTCCCTTAATTTTTCCAGATTCAGATTAATAGCCTATGTATCATCTGTT

TGGATGTGTTAGGCTCTTTTGAATTTCTTAAAATTAGTCTAGATTTTGATTTGT

GATATCCTTGTTATACAAAATTTGAATTTTTCAGAAAATTCATACTTAATTTCA

TGGTAGACTTGTCGAACACTGTGATTTGTTTGGGAAAAAAAAGGTTTAGTTTAT

ATTCATTACGTACGTGATGCATGATGCTTAGTATGCATTAAGATAGAGTATATG

ATCCGTGCTCCATCATTACTTGCTATTATCGATCGATACTTACTATTATTGATC

CTTAAAAGCTGATTTTTGCATGCGCATTATTTTCAATATGCTATTTTGAAAATA

TTTTTTGATGATGATGATTGTTTTATTTCGGTTATAAGTTATAAACGGACTCGT

TTTTGTGATTGAATTATGGGCTTTTGATATCACATCAAATGTTATTTATGTGGA

AATGAATTGAGAAAAAATGATGATTTTATCTTGCACCTATTCTTAAGTTTGGCT

TTGATGTGTTTGGCTTTTGATGCTATATTTCTGTCAAAGAATCCTGAATTTATT

TATTTATTTAGATTCGGTTGATTGTGTCGTAAAATGGAAGTTACTTCAAAATAA

GCCTCCTTGCAAGAGTATATATACTATATTACTTTTAGATAGTGAAAATTGGTT

ATTAGTTGTCGTTTAGAAAGAAGGAAATTTTAAGAAAAAATACTGATCGTAAAC

TATAACCAATGTATGTATTAGTATACTTTGATACTTCAAACACACGTGTGTGGT

GCGGGGATGAGACAGAGAAAGAGGTTGGTCTTGTTCTTGTCTTTGACTCTAAAC

CAGTCTTTTTCGATACATTTTTCTTCACTCACAAGTCTATCATCATGTTTCTAA

CGAAGACATTTATTTTATTTATATTTTGTAACAAAAAAATGAAGACCCACCTCT

TGCTTCTTCTTCACATCCCCATTTCATCTTCTCTCTGTCTCTCTCTATTAGAGA

CTCTCTCTACTCTACCCATCAATCTCAACAAACACTACTTTCTATCTCTTTCTC

TCTTTGTCATATCCATTACGCATATTCGTATCATTCCAAAGCAATCCCCACAAA

TCATATCATCCTCTCCATCTTTCCTTGCTTCTTACAATCTCTCCAATTTCAAAT

CTGTATACTCTTCTTCAAAAAGGCTCCACCAGTCCAAA

SEQ ID NO: 51

Polynucleotide sequence PATMYB85

CTTAGCATACAATCTTTAATTTTTCATGGAAGATTTTTAAAACATTTCCGATCC

GATTAAACAAAGAAGCGAGCGAGCCACATTCTGACAATAATTAAGTAGACACTA

TGATACGACGAAGAATATTAATTTAATATTGAAAGATAGATCAATTTGTAGCAA

AACCATGAAGCCAAAATTGCAAGTCACCCACAAGTCGCAAAGATTAGAAACATA

TATTGATACAGTGATCTATACGTGTACACCATGTGTCAAATGGATATTCGTCTA

TTATTTTTCGTATCGGCGACAAAGTATTTTGTGCGGCAATTCATTATTGAAGCT

TTTACTAAGTTTCTTCTATGTTATGTAAAAAACAAATCTTACCAAAATTAGAGA

CTCGTATATAATATACTTAATAGGTTTGTTAGGGTTGCCAAAAAAAATGGTTTA

TCGCAATGGACTAAAGATCTCAATTCTCAAAACTTATCGGATTTTGCCATAGTT

GAACCGGACCAAGCCAATTATTTGAGATTCTGAAAAGAGTATTATTATGGGCAA

ATTCTGAATATTTTATGTAAAATCGGTTTTGTAAAGACTGGATCATATTTTTAT

TCGTGTTTATTTCACAGCTGATAGCGACAACAATGAAAAATTCATTTTTTTTGT

GTGTGTCATCAACTATTAGAGTCGGTGATTTATATACAGTTTTGGTGACAGAAT

AAGTGCCTACAACTTAAAACTACACTAGTTTTAGTTATCAAGATCCTTAGTACT

TAATGTTGAAATTAATACATTTTTTAATAAATAAATACAAAGTATATATTTATT

TGAAACTTGAGCAAGTATTTGAGTAAAAAAGGTATGAATCGCACGTGTGATTGC

GTACATTCGCACGCATCCTATCCTTTCACATTAGTTCCAAAGTCATTTTCACCA

ACCAAATGCGACATCTCCAATACTCCTTTCTATGATCCTACTAGCAACAGATTT

GACAAAGTAAGACAAATTATATTTCTTAACCTTAATCATTTCTGACCAAAAAAA

ACCTGAATCATTATTTATTAGAATAATCTTATTTTATCAGAATTCGTAATTCTT

TAGCTGACTAACTCCTAATTAAAATGAACCATTCAATATAAAAATATAAACGAA

CGTATTATGTATAAAGTCAGATACAGAAGATCTTCTTTGAAACTGTTGTAATTT

CCCCATCATGACACCTGTATATACATACGTACCTTAAAAAAATTCTGATCTATA

TGTACTTTTGTATGAACGAGTAATGCATAATTCTTATTTAGATTAGACATTCTT

TAATGATAAAATAGTGAAGACGGGTATTATACATATATTAAGTCACTATTAGGG

TGATTAATTGTATTTATATACCAAGAAATCTCTAAGTGACAACATTATGAGGGT

GATTAGTAGTCCGTACTGTTTTTCATTCTAACCAATCACATAAAAGAATACTAA

AAGCGACAAAAAAAACTATTATCAGCTTTTTATACCATTTTATATGTTCGTTAT

TTATACCGTTTTTAATTATTTATATGTTATCAATTACTTTTTTCATATCGACAA

AAGATTTTATAATTTTTTGTGTTACCAATCGAACCATGTATATATATATAACCG

TTACTAGTTAAAATGCTTTGCCATAATGCCACTAGAATTTTTAATAAAAGTTAC

TAAAACAATTTCGAAAATATTAAGATGTAAAGTTATTTTTTCCTGAAACCAATT

GTGGGGGAAAGGTGTGAGAAGGTTATATATAGGTGGGTGAGCTTTGGTAAGCTT

TTGACATAACTTGCAAGCTGTTGAGATTTTCCATCCTCGATAACTTTATTCTTC

CATATCTCTTCCATTTCGCTCTCTATTTCACATCCCCATATAACATAATATACA

ATCACACATATCATTTCTATATAGTATTTA

SEQ ID NO: 52

Polynucleotide sequence PATMYB103

TGGTGCCCTGGTCTACAGTTCCCTAGTTAAGATTCTATTTTGACAACAAAATTGA

GTATTCCAATCATCCATATTTGTTATAGGGAGAAATTGAGCATGCTATATACGGT

GATATATATGATATTTATTTAATATATTGAATAACAAACACAGAACTAGTGTTAT

AGGTGCAGGTATGTAAATATAATGTGATAAACATTTTTTATATAGATTGGAAAGA

ATACGAGATTGTTGTTGCTCTGTTAGAACGAACAAACAGAACTAGTGTTATAGGT

GCAGGTAAGGTAAATATAATGTGATAAACATTTTTTATATAGATTGGAACAAATA

AACATTTTTCTGTTAGAACGAACAAAGGCCTGTCAAAAAGAACAAACCTGATGTG

ACATATTACATATATGATTATAATTGATTATTGTATATATAGTATTGCATGACTA

TCTTTACAAGATTTCAATACGAAAATAAATTAAAAGGAGAAAATTTATAAACGAA

GCGATTTCATTCTCGGTAAGGTTTTCCGATATGTCTCCTAACTAAATCAAAGCCT

TGTAATTGAAACTTGTAATGATTGTTATTCTATATATCTTTGAAGAAAGCTTCGG

TATTGGACGTACTTAATATAATTGGATTTTATTTAAATTACAAAAATCACTGTAT

AATTCGGCTACATGACTTAATCAATTATTTCACGTTGAAAACAATACTATATCAA

CTTCAAATACACTCCTTGTGTATGGATTCCACAAGTTCTTTTCTATCTATAGAAA

TATAGAATCCACAAGTTCTATCTACTTTTATTAGAATTTTTTATTGTTCGTTGTT

GTTAACATAATTATAAGCAATAAATTCAAAAAAAAAAAAATCTAAAAGACACAAA

ATTTCCATCTTTGATAGGGCTTCGGAATCATTAATTACTTTTTACAAACAAAAAA

GAAGAAGATAGGGCTTCGGAAATTATTAGAAAGATGGAAGGATATTGTATTAAAT

TTGGCTTCATATTTTCCTTTGGTTTGCGGCCATCAAGTACTAGTACTACTCAGTA

CCCACAGGCCACAGGAAAAAAAGTAGTACTGCTTTATTAAGTGTGTTACGATAAA

TGGAAAGCGTTTTAGTATGTGATTACAAATTGTTGTATGTGATCATTAATTAGTT

ATTGGTCCGACTTCTAAGTTTAAATATTTTCAGAATTCAGTTAGTTTAATTATAC

ATGTTAGACGAAATGACTCTTTTTGAGCCAATATATAATGTATCTGAATTTTTCA

TTTTGAAAAATCTTTTTATAAAATAATAGGTCAACCTCGAATTATTATAATAAAA

TAAATAATTTGCGTTACTATGAAAATTAATTTACTGAATACAGTATATAGAGAGA

GATAGAAATAGAGGAAAACAGTGGATAATACATGATTAGTTGATACTCATGTGCA

GCGAGTCTATATATATTATATACTCATGATTAGTTGATACATATGTGACGATTAG

TTCTACGAATCAATCTCTAGTTTTCGTCTTAAAATCATTTGGTTTTATAGAATAT

AAGAAACAAAGAAAACAGGAACTTTCGCGGGAGACAGAAGGGTACGTGAAGAGAA

AAACACATAAAAGTGATAAGGGCTTAACGTAATAATACTACAACAAAACCTCTCT

ACGTACAACGAGTAATAACACATGAAAATAGAAAGTCGATGAGACATCGTTTTAA

GGTTAGATCGATGAAGAAATATCTCAGGCCCCACCCCTGGGACCCGACCCGACCC

GACCCGACCCGACCTTTGTCTCCTCTCCTTTTAAAAACTCTCCATTGCTTCTTTG

TCTTCTCTCTTCTATAACATAACTCAAGAAATTAAAGAAGATAGATAGAGAGAGA

GAGAGAGAGTAAAAACCTAAAGGGTGATATACTTATATAAAAATTAATTTAAGAT

TGTGATTAAGTGGTTCACTATATTTAAGTTACTTTGAGGAGCTACTAATC

SEQ ID NO: 53

Polynucleotide sequence PATCADC

AAAGCAAATCGATCTGCCAAACATATCACAGCTCTTGGAGAAAATGCAGGTCTC

TTCAGACTCTGATATTTCGGATCTCGATAGCCTTAAATTCGATGCTCCATTGCC

TAGTCATATGCAACTAAGCTTTAATTTGTTGAAATCTAGAGTCGAAACTTGTGA

CAAAAATTAGATTTTTTTTCTTACCGAGCTTTCTTCTTTGTGTTCATTGAGGCC

CAAGTATTTGTGTATTTGGACCTGAATATTCTCATACAAAGATAAATAATTATA

ATTAAATGATTTTTCGCATATAATCATTATTGTGGTATGATTAACACAGTTGGT

GTGATGACTGATTGACACAATAATCACCGTTTGGATTCGATTCCTTTAATACTT

GTCACTAGAGTTGTTTGACTAAACAGCTAACTTGTCACTAGAGTTATTGTGTTT

GTATTTTGATCTGTTATTAATCTGATTGGGTATAATTACAGATAGAGAGACATC

TATATTGTAATTAAGACAATCTTAAAGTGTAAACTAAAAAGATCTCTCTGACCT

CTGGAAAACGAAAGGTGGGTGACACATCACTCTAGCTATGAATATGATGAATAT

TCAGTACCTAACCGAACAAAGACTGGTTTGGTATTTTTATTGGAAAAAAGAGAT

AAATAATTGTGAATGTGAATTATCCTGTCTGAAAGGTAAGCTGATGACATGGCG

TTATATGATTGGACGAGCTTCAGAACAAAAGAGTAGCGTCGAATCGAATCTTTA

CCTACTACACTTTGAACTTTGAAGTACATTACCTACTTCCTCCTTGATCGAACG

TCTTTTCTCAAAACTATTTTATTTCCCCAATTAAAGTAGTGGTGATAAATTCAC

AAAAATACAAACACTTTTATTTTTGACGTCAAAAACAAATACTTCTTTGAACAG

GCTATTACAATATTTTTAAGAAAAAAGTAAGCAAAATAGTCCACAAACCAAAAT

CTGTAACATATTAAACGATTTATGTTTTTTTTTTTTTTTCTTAACTAGAGAACA

ATTCGGGCTTTTACTAAGGATGATGAGTGTAGTTACCGAATAGTGTATTCATAT

AATCTTTTAATGAGCTTAAGATATGATATTATTTCGACTAATCAGATAAGAGTA

GTTAGATAATTTCGTAATAGAGCAACTCTTTCGCAAATAAAACCATTGTAAACA

TTACCAATTAGTTTTTCTTTTTTTTTGGTCACAACCAATTAGTTTGTTTGTTCT

ATTTTATGAAGTGCGTATTAAAGCTAACGTGTTTACAGTAACGCCACACAAATA

AAAATAAAAATAATTATGTACTTTATGGATTTATAGAAAAAACAAGAATAGTCA

CCAAAAATTGATTGTGTCATATATCTTTTGTCAACTATTTTATCTTATTTTTCT

ATGGATATGTATGTCCAAAATGTTAGACAAAAAACCAAAAAATCATGTCCAAAA

TTTCGTTAGGCTGCCGATATCTCTGTTTCCCTTTCAACGACTATCTATTTAATT

ACCGTCGTCCACATTGTTTTTAATATCTTTATTCGAGGTTGGTTTAGTTTTTTT

TACCAAACTCACTTTGCTACGTTTTTGCCTTTTTGGTATGGTTGTATTTGTACC

ACCGGGAAAAAAAAGATAAGAGGTTTGGTTGGTCGAGCTTACTGATTAAAAAAT

ATACACGTCCACCAAATATTAAAACAATATATCCCATTTTTCCTCCTCTCTTTT

GGTATTACATTAATATTTTATTATTTCCCCATTTGCTCTGTATATATAAACATA

TGTCAATAGAGTGCCTCTACAGTCATGTTTCCATAGACATAATCTCTCACCATT

GTTTTTCTCTGCAAAACTAAAGAAACAAAAAAAGAAAAATCGGAGAAACCAAGA

AAAAAGAA

SEQ ID NO: 54

Polynucleotide sequence PATCADD

GCTTCGGTGATGCATTTCTCCTTCTCATCAATCATCCTAGCAATGTTTTGAAGC

TGAGAAATTCTCCACTCGTAGCTCTTCGTTCTGCCAGAGTTGAAGTTGCTTCTG

AGCTCATCTACAAGCAAAGCTGCTTCTTTTCCACTAAAGTCTGATGCTTGCTCC

TTTACCACAGCAGATAGTGTTGCATAACAAGTACTGATTCAAGACACCAAAACC

GCAATGTGAGAGACTTTAAGACTAAAAATCATGGATAAGACTAAAAAAACATGG

ATAAGTATCAACTGTTCTCACGATTATTTATTCATACCACTGTACTTAAACTTA

AAACCCACTATACTAAATAGAAAGGTAATCATCAAAAAATCAGTATGTAAAAAC

CACTTTTGTGAATAAAATATGTAAAATGGGTGAATAAAGAAATGTGCTTACAAT

TTCAACCGATAAGGGATACAAGCATTGCTGCAATATCCACCACCACCACGACGA

GATATCCGAAAAGGTGAAGTTGCAACATTTAATCTGCAACAAAAGAGGCCATTC

ATTAAAATGGTACTAATTAGATCTAATCATATCATATTGAATGACCAAATCATT

CACAGAAGCATCCATTGCTCCAATTAACATTCTAGACCAAATTCAACTTAAAGG

TAACTCTTTTATACAGGAAACCGAGAAACCGAAAACGCAATTCACATAAAAAGG

AAGGCTTGTTTGGAGAAGCAGAATCGAACAAGTCAATCTCAAACCCTGATGAGC

AGGTTTTTCAAGTTACCTGGCAGGAGAAAAACCCTTGGCAAAACAAAGGGTTTG

AATATGATTAATCTCTAGAAGCTTCGTCATGACTTGGGTTCAGTTAAAAATCTC

AAATTGGAGACATTATTGGTGTTTATATATTTGAGAGAGAGAGCCAGAGAGGAG

ACGTTGAATTGAATGAAGGGTGTGGTCGGAAGAGAAGACGTGTAGAAGAGACGA

GACAAGTAAATTTAAGCATTGGCCCCATTTACAGCCACAAGTCCGCTACAACAA

ATTATTTCCAAGAAACTCTGAGATAACGTCGTGATGAAACGGCTCATGCTGCTG

TTGTGATTCGTGAATTAGAGGTTTATCTTTTGGGTTTTTGAATGTTACTTAATT

GGACGGTCGATTTTTCAAACTGGGTGTGAAATGTGAATGGGTCATTCATAATGG

GCTTTTGTTTTAATGTGAAGCCATTCACACACTCTTTGTCCTTCTTTTCTATTA

TTCATAACTGTCACTCTTTGTTCTTCGAAATAGTAAAGAGCAAATCGATTCTTT

GTTGATCTGGGCCGTAAAATTTCCATGGTTGTGGGAAGTATTCTCGCAGCTGAT

CTGGGCCGTCAATGCTACAGTTTCATGTCAGAGAGAGGTCAAGAATCAACACGT

GGCCAACCATGATTTTAAACCAAAGCAAACACACGATTAGACCCCACATTGTTT

GTTCACCAACCCCCGTGGACCCTCCTTTAGCCGACGTGTCCACGTCAATAGTGG

TTTTTCTTCCTTTCAAAGTACACAAATTCCATTCTTTCTCATTTTACTTTTTGG

ATTACGTTGTTGTTATAAACTGGTAAAATGAATTATGAATGCAAATAAATTTCA

TTTAAGTTTTGTIGGCTICTAATATTTTTTTCACCTAAAATTCTAATAAACTAC

ACAGCCATGAGCCATCGTATGAAAAGAAGAAGAAAAAAAATGTCTTTTTCTAGA

AGGATCTTTCAACGACTAAAAAAGATTTTAAGCTTTTGACTAATTTTGTCAATA

ATATACACAAATTTACACTCAATTATAGCCATCAAATGTGTGCTATGCAGAAAC

ACCAATTATTTCATCACACATACGCATACGTTACGTTTCCAACTTTCTCTATAT

ATATATATAGTAATACACACACATAAACAGCAAAAGCGTGAAAGCAGCAGATCA

AGATAAGAAAGAAGAAAGAATCATCAAAAA

SEQ ID NO: 55

Polynucleotide sequence PATPAL1

TTTTCCCAATGATACAACTATAAATCAAAAAGAAAAAATGTACTGATAAACGAA

ACTAAACGTATAAATTAATATATTTCTTGACATAAATAGGAGGCTTTTGCCTGC

TAGTCTGCTACGATGGAAGGAAAAATGCATGCACACATGACACATGCAAAATGT

TTCAATGAAGACGCATTGCCCAATTAACCAACACACCACTTCTTCCATTCCACC

CATATTATTTATTTCTACCATTTTCTTTAATTTATTGTTTTTTCTTTGATTCAT

ACACTGTTTATGACTATTACATTTTCCCTTTCGACTAATATTAACGCGTTTAAA

CCAAAGAATGGATTTGATAATGAAATTTTATTTTATTAGCATATAGATAATGGA

TGGCTTCATGCTTGGTTTCCATGACAAGGAATGACACAAGATAATTATTTTGAA

TAAAATCATAAATATGATAATACTAGTTGTAAAAAAACTTGAGTGTTTCGTGTG

TTATTTTTCGGTTTCTTGACTTTTTATATTTCTCGTTTTTGTAATTTTAGGATG

GATTATTTAGCTTGCTTTTCTCTTTTATTACTTTCTAAAATTTTATTTATAAAC

TCATTTTTAATATATTGACAATCAATAAATGAGTTATCTTTTAATTAATAAAAA

ATTTGTAAACTCTTGTAAACAGATCATAGTCACTAAAAGCTATTATAAGTTATT

TGTAGCTATATTTTTTTATTTCATGAACTTAGGATAAGATACGAAAATGGAGGT

TATATTTACATAAATGTCACCACATTGCCTTTGTCATGCAAACGGCGTGTTGCG

TCACTCGCCTCCTATTGGGAATCTTATAATCGCGTGAATATTATTAGAGTTTGC

GATATTTCCACGTAATAGTTATCTTTCACAAATTTTATACTCAATTACAAAATC

AACGAAAATGTACATTTGTATCTTTAACTATTTACGTTTTTTTTACGTATCAAC

TTTCAGTTATATGTTTTGGATAATATATTTTTTTACTTTTGACTTTTCAGTTTT

CACCTAATGATTGGGATATACATATGCATGCATAGTTCCCATTATTTAAATGTA

AGCTAAGTGCATATGAACTGTTAGTCAAAATTACGAAGTTTATTTGTACATATA

TATAGTTATAACAAAATGGTACAGTAAATTAAACAGAACATCAAGAAAGTACAA

AAGACTGAACACAATAATTTACATGAAAACAAAACACTTAAAAAATCATCCGAT

AAAATCGAAATGATATCCCAAATGACAAAAATAACAATATAGAAAATACAAAAA

CAAAAACAAAATATGAAAGAGTGTTATGGTGGGGACGTTAATTGACTCAATTAC

GTTCATACATTATACACACCTACTCCCATCACAATGAAACGCTTTACTCCAAAA

AAAAAAAAAAAACCACTCTTCAAAAAATCTCGTAGTCTCACCAACCGCGAAATG

CAACTATCGTCAGCCACCAGCCACGACCACTTTTACCACCGTGACGTTGACGAA

AACCAAAGAAATTCACCACCGTGTTAAAATCAAATTAAAAATAACTCTCTTTTT

GCGACTTAAACCAAATCCACGAATTATAATCTCCACCACTAAAATCCATCACTC

ACTCTCCATCTAACGGTCATCATTAATTCTCAACCAACTCCTTCTTTCTCACTA

ATTTTCATTTTTTCTATAATCTTTATATGGAAGAAAAAAAGAAACTAGCTATCT

CTATACGCTTACCTACCAACAAACACTACCACCTTATTTAAACCACCCTTCATT

CATCTAATTTTCCTCAGGAACAAATACAATTCCTTAACCAACAATATTACAAAT

AAGCTCCTATCTTCTTTCTTTCTTTTAGAGATCTTGTAATCTCCTCTTAGTTAA

TCTTCTATTGTAAAACTAAGATCAAAAGTCTAA

SEQ ID NO: 56

Polynucleotide sequence PATPAL2

TTTCCCTGTTTTTTTTCCCCTCTTTCTGTTTCCCATTTGAAAGTAAAAGATCATTT

AAGCACCTAACTCAATTTTATTTTATTTTAAACACCTAATGTCATGCTCCTTGGCT

CCTTGTAATTAGTTGATCGTTTCAATTTAGACCAGCAAAACATTTTAGTATGTTCG

TAAATATTGCGTACATGCCATTTCGTTTGTCATGCAAACGGTGTGTGTTTCTTTAC

TTAGCTTCTAGTTGGTGTATATTGCGTCGCATTAATATCGGTTTACCTTCCTCCTG

TCTACGTAATGATATATTCTCCACCACAAATTTAAATTCTTATTGAAATTTCCTAA

TTTTTTAGGTAGCTCAAGGTCTCAAGTATACTACGTACCCTATTTTTTTGAATATC

TATCTATATTATAACAAGAGTTTTTCTGAGCTAGTTAATGAGATGACAATATTCTA

CATAAATAAATGACCCTCGAAAGTTTCAAGTACTTTAGGATCTGACCAAATCGGGG

TAAAACATTTTGAAACTAATTACGTTCACATCTACCATCGATGATTGACAAGCTTA

TTGTCACCTTTTATGTTAAAGTGACATGGTCTTGACGTTAATTTGCATGTTATTCT

ACATCTATAGTCCAAAGATAGCAAACCAAAGAAAAAAATTGTCACAGAGGGTTCAA

TGTTACTTAGATAGAAATGGTTCTTTACAATAATAAATTTATGTTCCATTCTTCAT

GGACCGATGGTATATATATGACTATATATATGTTACAAGAAAAACAAAAACTTATA

TTTTCTAAATATGTCTTCATCCATGTCACTAGCTCATTGTGTATACATTTACTTGC

TTCTTTTTGTTCTATTTCATTTCCTCTAACAAATTATTCCTTATATTTTGTGATGT

ACTGAATTATTATGAAAAAAAACCTTTACACTTGATAGAGAAGCATATTTGGAAAC

GTATATAATTTGTTTAATTGGAGTCACCAAAATTATACAAATCTTGTAATATCATT

AACATAATAGCAAACTAATTAAATATATGTTTTGAGGTCAAATGTTCGGTTTAGTG

TTGAAACTGAAAAAAATTATTGGTTAATAAAATTTCAAATAAAAGGACAGGTCTTT

CTCACCAAAACAAATTTCAAGTATAGATAAGAAAAATATAATAAGATAAACAATTC

ATGCTGGTTTGGTTCGACTTCAACTAGTTAGTTGTATAAGAATATATTTTTTTAAT

ACATTTTTTTAGCAACTTTTGTTTTTGATACATATAAACAAATATTCACAATAAAA

CCAAACTACAAATAGCAACTAAAATAATTTTTTGAAAACGAAATTAGTGGGGACGA

CCTTGAATTGACTGAACTACATTCCTACGTTCCACAACTACTCCCATTTCATTCCC

AAACCATAATCAATCACTCGTATAAACATTTTTGTCTCCAAAAAGTCTCACCAACC

GCAAAACGCTTATTAGTTATTACCTTCTCAATTCCTCAGCCACCAGCCACGACTAC

CTTTTCGATGCTTGAGGTTGATATTTGACGGAACACACAAATTTAACCAAACCAAA

CCAAAACCAAACGCGTTTTAAATCTAAAAACTAATTGACAAACTCTTTTTGCGACT

CAAACCAAATTCACGTTTTCCATTATCCACCATTAGATCACCAATCTTCATCCAAC

TGGTCATCATTAAACTCTCACCCACCCCTCATACTTCACTTTTTTCTCCAAAAAAT

CAAAACTTGTGTTCTCTCTTCTCTCTTCTCTTGTCCTTACCTAACAACAACACTAA

CATTGTCCTTCTTATTTAAACGTCTCTTCTCTCTTCTTCCTCCTCAGAAAACCAAA

AACCACCAACAATTCAAACTCTCTCTTTCTCCTTTCACCAAACAATACAAGAGATC

TGATCTCATTCACCTAAACACAACTTCTTGAAAACCA

SEQ ID NO: 57

Polynucleotide sequence PATC3H

ATCGTAAGTTTTTTTGTGTGTGTGTTAACAATGTACTCACTACTCACTGTTCCAT

ATTTTTGATGTACGTATATCGAAAACATTCTGCCAACAAATGCAAACATAACAAA

AGTCAAAAACAATAACATAACCGGGAATTAAACCAAAATGTAATTGCTTTTTATT

AGTGTCAGGCCTTCTGCTTAAAAATATTCTCGGCCCAGAGCCCATTAACACCTAT

CTCAATTCATATTGAAGAAAATGACTATATTACTTGACAAAAACTTTAGTCAGAA

AAATATGGAATCTCTTTCGGTACTGCTAAGTGCTAACCTTAAATAGTATAGAATT

CTTAGTTCATTCTCAAAAACATAGCTATATGTAGATTATAAAAGTTCGATATTAT

TTCCTGCAAAAGATGTTATAATGTTACAACTTACAAGAAAATGATGTATATGTAG

ATTTTATAAACTGGTACCGTAATTCATAAAAGATGGTGGTGGGTATGTATCAGTA

ACGGAACTTACATATGCGTGTGTATTACTATGTCTATATGGTGTATTCCTTTGTG

TGGAACAATGCACGTCAGAGTTGTTTATTTTCTTATAGAATTTAAGGAATCAATT

ATTGGATTTCTCAAGGTGAAAGTGGACTTCTTTGCACGCAAGGTCTAGTTGCCGA

CTTGCCGTTGCATGTAACATGATTGTTGAAATAAAGTGAATTGAGAGAAGTTTGG

CCAGACATTTTAAATTTAACCCAAAAAAAGTAGGGCCTAACACAAAATATAACCT

CTCTTTGTTCAAAGGAAATAACACCTACGTCTTATAATTGAACCAAACATTGAAT

CATTGAACTCACCTATAATAATTATAATAACACGAATTCACAAGACACCTAAAAG

AAAAAGTTCACAAAAACAAATAAAAATTTACCTCTCACCAAACACACTCACCTAC

CCGTCTGGTCCCACTGACCCCAACATACAACACCGACTCTCTCCCACACCAATTT

TTTTTTTTGGCGTTTTAAAACAAATAAACTATCTATTTTTTTTTCTTACCAACTG

ATTAATTCGTGAATAATCTATTATCTTCTTCTTTTTTTTGTGACGGATGATTAGT

GCGTGGGGAAATCAAAATTTACAAAATTTGGGATGATTCCGATTTTTGCCATTCG

ATTAATTTTGGTTAAAAGATATACTATTCATTCACCAAGTTTTCAGATGAGTCTA

AAAGATAATATCATTTCACTAGTCACTTAAAAAAAGGGTTAAAAGAACATCAATA

ATATCACTGGTTTCCTTAGGTGACCCAAAAAAAGAAGAAAAAGTCACTAGTTTCT

TTTTGGAAATTTTACTGGGCATATAGACGAAGTTGTAATGAGTGAGTTTAAATTT

ATCTATGGCACGCAGCTACGTCTGGTCGGACTATACCAAGTTACCAACTCTCTCT

ACTTCATGTGATTGCCAATAAAAGGTGACGTCTCTCTCTCTCTCACCAACCCCAA

ACCACTTTCCCCACTCGCTCTCAAAACGCTTGCCACCCAAATCTATGGCTTACGG

GGACATGTATTAACATATATCACTGAGTGAAAAGAAGGGTTTATTACCGTTGGAC

CAGTGATCAAACGTGTTTTATAAAAATTTGGAATTGAAAACATGATTTGACATTT

TTAATGATGGCAGCAGACGAAACCAACAACACTAAGTTTAACGTTCGTGGAGTAT

ACTTTTCTATTTTCGAAGAAGACATATAACTAAGCTGATTGTTATTCTTCATAGA

TTTCTTTTCACTGCGAATAAAAGTTTGTGAACATGTCACCGTTTGAACACTCAAC

AATCATAAGCGTTTTACCTTTGTGGGGTGGAGAAGATGACAATGAGAAAGTCGTC

GTACATATAATTTAAGAAAATACTATTCTGACTCTGGAACGTGTAAATAATTATC

TAAACAGATTGCGAATGTTCTCTACTTTTTTTTTGTTTACATTAAAAATGCAAAT

TTTATAACATTTTACATCGCGTAAATATTCCTGTTTTATCTATAATTAATGAAAG

CTACTGAAAAAAAACATCCAGGTCAGGTACATGTATTTCACCTCAACTTAGTAAA

TAACCAGTAAAATCCAAAGTAATTACCTTTTCTCTGGAAATTTTCCTCAGTAGTT

TATACCAGTCAAATTAAAACCTCAAATCTGAATGTTGAAAATTTGATATCCAAGA

AATTTTCTCATTGGAATAAAAGTTCAATCTGAAAATAGATATTTCTCTACCTCTG

TTTTTTTTTTTCTCCACCAACTTTCCCCTACTTATCACTATCAATAATCGACATT

ATCCATCTTTTTTATTGTCTTGAACTTTGCAATTTAATTGCATACTAGTTTCTTG

TTTTACATAAAAGAAGTTTGGTGGTAGCAAATATATATGTCTGAAATTGATTATT

TAAAAACAAAAAAAGATAAATCGGTTCACCAACCCCCTCCCTAATATAAATCAAA

GTCTCCACCACATATATCTAGAAGAATTCTACAAGTGAATTCGATTTACACTTTT

TTTTGTCCTTTTTTATTAATAAATCACTGACCCGAAAATAAAAATAGAAGCAAAA

CTTC

SEQ ID NO: 58

Polynucleotide sequence PATCCR1_PATIRX4

AAAATTGTGTCTAAGAATGTGGAACCGAGTAGTTCTCCAGAAGTCAGGTATGAA

AGTATATAAGAATTCTAGTTTTAGTTGTTTGAAAGTTTGATCCGTGAGTGAATT

AGTTCACAATTATGGATGTAGATCCTCTATGCAAACAATGAAGAAGAAAGACTC

TGTAACAGACTCCATTAAGCAAACAAAAAGAACCAAAGGTGCACTGAAGGCTGT

AAGCAATGAACCAGAAAGCACTACAGGGAAAAATCTTAAATCCTTGAAAAAGCT

GAATGGTGAACCTGATAAAACAAGAGGCAGAACTGGCAAAAAGCAGAAGGTGAC

TCAAGCTATGCACCGGAAAATCGAAAAAGATTGTGATGAGCAGGAAGACCTCGA

AACCAAAGATGAAGAAGACAGTCTGAAATTGGGGAAAGAATCAGATGCAGAGCC

TGATCGTATGGAAGATCACCAAGAATTGCCTGAAAATCACAATGTAGAAACCAA

AACTGATGGAGAAGAGCAGGAGGCAGCGAAAGAGCCAACGGCAGAGTCTAAAAC

TAATGGAGAGGAGCCAAATGCAGAACCCGAAACTGATGGAAAAGAGCATAAATC

ATTGAAGGAGCCAAATGCAGAGCCCAAATCTGATGGAGAAGAGCAGGAGGCAGC

AAAAGAGCCAAATGCTGAGCTCAAAACTGATGGAGAAAATCAGGAGGCAGCAAA

AGAGCTAACTGCAGAACGCAAAACTGATGAGGAAGAGCACAAGGTAGCTGATGA

GGTAGAGCAAAAGTCACAGAAAGAGACAAATGTAGAACCGGAAGCTGAGGGAGA

AGAGCAAAAGTCAGTGGAAGAGCCAAATGCAGAACCCAAGACCAAGGTAGAAGA

GAAAGAGTCAGCAAAAGAGCAAACTGCAGACACAAAATTGATTGAGAAGGAGGA

TATGTCTAAGACAAAGGGAGAAGAGATTGATAAAGAAACATATTCAAGCATCCC

TGAGACTGGTAAAGTAGGAAACGAAGCTGAAGAAGATGATCAGAGAGTGATTAA

GGAACTGGAAGAAGAGTCTGACAAGGCAGAAGTCAGTACTACGGTGCTTGAGGT

TGATCCATGAATGAAGGATTGTTAGGTAAATGTTAATCCAGGAAAAAAAGATTG

GTTCTTGTGGTTTAGGTAACTTATGTATTAAGTGAAGCTGCTTGTTTAGAGACT

AATGGTGTGTTTTATGAGTAGATTCTTCTGACCTATGTCTCGTTATGGAACTAG

TTTGATCTTATGTCACCTTGCTAGCAGCAGATATTGATATTTATATATTTAAGA

GACATGCGCATGAGAATGAGGGTATGGAAAAGTCCATATCAGATGACACAAACA

ATGATCGTATGTGTAGTCACTTGTGCATTTCCAGTTTTGGACATAAAATTCTGA

TATTGCATAGAAATGTTTTTAAATAACACTAATCCAAACCTAAATAAAATATCT

CTATACATCATCTAGAAATGTATGGCTTGATCAAGAATTGTAGATAATAATACC

CTGAGTTAAATGATTGTAGGTATTATTTCAGTTTTCAAAATTGTCCAAATTTAT

GAGCTATATTAAAGATAATATTTTCAATAAGGTGTGTAGTTCTAAATGTTTCTT

CTTCTTCCACCAACCCCTCTTTCTATATGTATGTTCTTTTTTCTAAAATAATTG

TTTGTTCTTTTTTAGATATATCAAATTAAATATAAAAAATATTGACAAAACTTA

TTTACCATTGTTAGGTGAACTTGGCAAGTGTGTAAATATAAAGATAACATTCCT

TTTCGTTCTTTATATATACGAAACGTACCACAAATTTCTAACTAAAGCATTCAT

AGTCTCTCGAAAGCCTCTTTTCAGAACCGAAGCTCTTTACTTTCGTCCACCGGG

AAAT

SEQ ID NO: 59

Polynucleotide sequence PATF5H

AAATTTTTGTATGAAATATTTCTTTAACGAAAATAAATTAAATAAAATTTAAAA

TTTATATTTGGAGTTCTATTTTTAATTTAGAGTTTTTATTGTTACCACATTTTT

TGAATTATTCTAATATTAATTTGTGATATTATTACAAAAAGTAAAAATATGATA

TTTTAGAATACTATTATCGATATTTGATATTATTGACCTTAGCTTTGTTTGGGT

GGAGACATGTGATTATCTTATTACCTTTTTATTCCATGAAACTACAGAGTTCGC

CAGGTACCATACATGCACACACCCTCGTGAAACGAGCGTGACTTAATATGATCT

AGAACTTAAATAGTACTACTAATTGTGTCATTTGAACTTTCTCCTATGTCGGTT

TCACTTCATGTATCGCAGAACAGGTGGAATACAGTGTCCTTGAGTTTCACCCAA

ATCGGTCCAATTTTGTGATATATATTGCGATACAGACATACAGCCTACAGAGTT

TTGTCTTAGCCCACTGGTTGGCAAACGAAATTGTCTTTATTTTTTTATGTTTTG

TTGTCAATGTGTCTTTGTTTTTAACTAGATTGAGGTTTAATTTTAATACATTTG

TTAGTTTACAGATTATGCAGTGTAATCTGATAATGTAAGTTGAACTGCGTTGGT

CAAAGTCTTGTGTAACGCACTGTATCTAAATTGTGAGTAACGACAAAATAATTA

AAATTAAAGGGACCTTCAAGTATTATTAGTATCTCTGTCTAAGATGCACAGGTA

TTCAGTAATAGTAATAAATAATTACTTGTATAATTAATATCTAATTAGTAAACC

TTGTGTCTAAACCTAAATGAGCATAAATCCAAAAGCAAAAATCTAAACCTAACT

GAAAAAGTCATTACGAAAAAAAGAAAAAAAAAAGAGAAAAAACTACCTGAAAAG

TCATGCACAACGTTCATCTTGGCTAAATTTATTTAGTTTATTAAATACAAAAAT

GGCGAGTTTCTGGAGTTTGTTGAAAATATATTTGTTTAGCCACTTTAGAATTTC

TTGTTTTAATTTGTTATTAAGATATATCGAGATAATGCGTTTATATCACCAATA

TTTTTGCCAAACTAGTCCTATACAGTCATTTTTCAACAGCTATGTTCACTAATT

TAAAACCCACTGAAAGTCAATCATGATTCGTCATATTTATATGCTCGAATTCAG

TAAAATCCGTTTGGTATACTATTTATTTCGTATAAGTATGTAATTCCACTAGAT

TTCCTTAAACTAAATTATATATTTACATAATTGTTTTCTTTAAAAGTCTACAAC

AGTTATTAAGTTATAGGAAATTATTTCTTTTATTTTTTTTTTTTTTTAGGAAAT

TATTTCTTTTGCAACACATTTGTCGTTTGCAAACTTTTAAAAGAAAATAAATGA

TTGTTATAATTGATTACATTTCAGTTTATGACAGATTTTTTTTATCTAACCTTT

AATGTTTGTTTCCTGTTTTTAGGAAAATCATACCAAAATATATTTGTGATCACA

GTAAATCACGGAATAGTTATGACCAAGATITTCAAAGTAATACTTAGAATCCTA

TTAAATAAACGAAATTTTAGGAAGAAATAATCAAGATTTTAGGAAACGATTTGA

GCAAGGATTTAGAAGATTTGAATCTTTAATTAAATATTTTCATTCCTAAATAAT

TAATGCTAGTGGCATAATATTGTAAATAAGTTCAAGTACATGATTAATTTGTTA

AAATGGTTGAAAAATATATATATGTAGATTTTTTCAAAAGGTATACTAATTATT

TTCATATTTTCAAGAAAATATAAGAAATGGTGTGTACATATATGGATGAAGAAA

TTTAAGTAGATAATACAAAAATGTCAAAAAAAGGGACCACACAATTTGATTATA

AAACCTACCTCTCTAATCACATCCCAAAATGGAGAACTTTGCCTCCTGACAACA

TTTCAGAAAATAATCGAATCCAAAAAAAACACTCAAT

SEQ ID NO: 60

Polynucleotide sequence PATLAC4

CAATTATATTTGGTTTCGATTGAAATTCAATCTAATGTGGTTAGATGAGTCCTA

TATTACCATGTCATTGTTAATACCCATTGCCAAAAATAAAAGTGAAGCAGAAGG

AGAAATTGTTTTTGTATACCCGAAGGAATTAAGATGTACGATCTTAAAATAGAC

ATTTCGGCCATCTATCAAAATAAATGTCTAAAAGTTTTGTGGTCGTCTTAAATA

CTACTTCGAGTTCAGACGTATACGTCTCACCAAAGTAATGCACATACTTGATGT

TAAGTTTATCTCTTTTTACTATTTCAAATTTCGCGTTTGACAACACTTTAAGTC

TACATTATCCATAGAGAATATAACATAAAGATCATGAACTTCTCATGAATGTAT

AAGACAAATCAAGCTTATATATGAGATCTATTTAGTAATTTGATATGTATGTAA

TATATGATAAATCTTTGATGCAATATTTTATTATGATTATTAGATATACACTAG

TCAACTTTAACTTTAGAAGATTAATCATTCCGTCGCAAACCATACCATAAATTA

GCAAGGGATCGACTTAATATCTCCGATCCGCTATATATTTAAGAAGCATTTAGA

TTGTTTATAATACATGTCATGATTTTATAATTATGTATATATAAATACTAATTG

ATGTATGAAGTACGTAGATAATGTTACGATCTATTAATCTATTTACATTAACTT

TTAATTAGTGTTGAGTAGGGAAAATTAACATATAAACCTTTAGCAGTTGGTTGT

ATTATTAAAAATAATTTGAACTTAAAATCCACCTTCGAAAAGATAAATCAAACA

AGTATAAAAAATGCTATAAATCCAGAATATTTACCTAAGGTTTTTATTCTTCTA

CTTAATAATGTAAGATAAAACCGGCACAATACTTGTTACGTATGCATGGTAGGT

ACCGCAATTGTGTAAGCAAATCGGCACAATACTAAGGTTACATATACTAACTAA

ATAAAACAATCTGATTTCAGTGACACCGTATATCTAACCTTTATTCAAATCCAA

GGGAACATGACTTGACTTCTTCTGTTGGAACTAACTCGATCCCTCAACCATCTC

CAGGGATAGAAGAGTTAGTAAAATCAAACTTGAAGTGAGGAAGTAAGCAGTTTA

ACGACTCCATATGACTACAGTTATATACAAAGTTGGGCACAAAGTACAAGTACT

AAATACTCAAAGTCAGATAATAATTTTAATAAGTACAAACTATATATATGCAGT

ACAATTATTGAGTATATATAAACGAGACTGGTGATTTGGGGCATTGTCCACCAG

GGTGTTATATCCCAATTGAAATTTGAAAATTTAAGTGTGTGAGTGTTACGACAA

AAAAAAGTGTGTGAATTGTAGGCGCGGTGAAAAGGTAAATTAAGATTGGAACTA

GAAAAATAGTTGAATATCCTTTACTAAAAGTTGTCAATTCCGGTTTTAGTAAAA

AAAAATTTTAAAATAGAAATTTTATCCAAAAGACTTCAAACACACATATTCGCA

TATATAACATAAGATATCATTTTTTGTAAACAGTTAAAAAGAAAAACACATGTT

TTTTTITTTAATTTAGAAAAAAACATGTTATTATACAAAACAGAGTTTTGCCCA

CTTTTAATATGTTATGAAAAGAAAAATGATTTTCTTGGGTTTGGTCAGAGAGAT

TGGTTGTGGTAAGAATGGGAATCTTAATTACAAAGAATTGGATTTTGGGTCGAC

CTACCACCTAAAACGACGTCGCCTCCATCTCTGGTTTCCAAATCTCTTTCTCCT

CTCCCTTTATAAGCTTGCGTTGGCCAGTCGCTCATCTCGAAAACAGAGAGAAAA

AGACTAAAAACACAGTTTAAGAAGAAGGAGAGATAGAGAGAGAAGAGAAAGATA

GAGAGGGAG

SEQ ID NO: 61

Polynucleotide sequence PATLAC17

TAAGTTTAAGTCCAATAATTTCATTTTACTAGTAAAGATCACAATGTCATTTACC

GCATTCACTTAATAATTGCTGAATTCACATAGTGCCTGTAAATTAAGACTAATTT

TAGGTTTCAAATAATTTTTCTTTTTTACATAACTTACGATCGATATTTTAAATGG

TATTGGTAAGTTTAAGGTATATAGATAGTGTGTCTAAACTAGAGTTCGTTGAAAT

TGGTCTGAGGTATAAATACCTAAAAGGTTATATATGTTTTTAGTTTAATGTAATT

CGATAAATTTTAGTCGAAACCGTTAAGAGATATCAGAATTTCGTTTTCAAATAAT

ATGGGATATAATTACCCGGGATTAACCGTACCTGATAAAATATAGCTCTCGTACG

TGTCACATGCCTAATGCCTAGTTAAACTTAAAACGAATATCTATATTTACTGTTA

TTGATTGTGAGTTACCAACTAAAATATTGTTAAAAGACATTGTAAAACTACAAAT

GGTTCGAACTGTATACTAATGATGTAAACTCGTGTTTCATCGTTATGTCCGATAT

TTTTTTCATTCAACCATTATTCAATTTCAAGATTTCTTTATTGTCTTTTTTTCTT

TCTAGAAAGCCTATATATTTAATTACCCACTTTGCATATTCAGAGGATAAGTTGA

TACGTACTTGTTAGCAACCTGTCTAGATCATCTTTTGATTGTAGATTTGACTTTA

AATTTCTCACAATTATAAATATGAAAAATAACAAGCAAAGAATTTACAAATGTAT

ATAATTATATACACGCATTGATGAATAAACATATTTAGAAAATAATGTGTTCTAA

GGAAATTTTGTGGCATTTTTTAAAAAATAATTAAACAAATAAGAATAGTGTAAAG

TTGTTTAAATATGTATGTATAAGTGGCATGCCTTTGAGGATACGAACTTAAAAGG

GAGTTAGGTAACTTGCTTGGGAAATAAAATAGCCAACCTTAATTTGAGGTTTCCT

CAATGTTCTTATCAAAAAGAATAAAAATTTCGGAAATTCCCTTCATGGATTTTGA

TATCTAACCCTAATCGTGACCTTCTTTGATAGCTACAATCTCCCTCTCTTTGCTT

ATTCCCCAAGCAATTTTAGCTTACGAATGTTTTGACTAACTCCACATCGGTTTAT

CTCTTAAGTTCCCCACCTACAAATATACAAAAAAAGAAGTAAAATAAAAATAATT

ATTAACAAACCGATGAAGTACTTATCATTTATAAACATGCTTATGAAATGTATTT

TCTAAAACATAACCGCTAACCAGAGAAGTTTCCTAGAGTTCTGCTTCAGACTCTT

TTGGTCGATCAAGAAGTCTCCAAGAGTTGTTTTTGTTGGGTCTAAACAAAACTTG

GCCAGGGAACAAATCAAACTATATTATTAATCTTCTACATCTGGTCCTAAGTTCC

TTACTATCTCATGTTAAAATTTGAAGTCTAATATACTCAAAGCTGTCAAAGAAGC

AGAACATGGAAGAGGAACTGTCATATCTGAGAAACCAAAATTGGCAATCTTGCAT

TTCATATTTAGAATCTACGCCATAGTATTGAGATGGAAACAAAGAGTTTTCGAAG

AGGGTCAAAGAGTTTGACTTATCTTTGACACCACTCATACATTAGCTGTTCATAT

AATCTAACAACTAGTCAATATCAAGTGTCTCCAAATTACGGAGAGTACTTCTCTA

CCAATTATCTTTTTGTTTTTCATAAACATTTTACTAATTGTTTTTTCTATATCTC

CTGCTCAAGCAAACACCTAACTCTCCTTTCCTATATATACACTAAAGGTTGAAAA

CAATGAATCCACAATCTACAGCAAAACATAAGCGAGGCAGAGTCTTCAGAAAACT

TACCTGCTCTAAACAACGCCTCCGTGTCCAAGCTCACTTCA

TABLE I

In-vitro HCHL enzyme activities in
stems of five-week-old wild type (WT) and
IRX5:HCHL plants. Values are means of three
biological replicates.

		Enzyme activity ± SE
	Plant line	(pkat vanillin μg⁻¹protein)

	WT	nd^a
	IRX5:HCHL (1)	0.112 ± 0.026
	IRX5:HCHL (2)	0.075 ± 0.022
	IRX5:HCHL (3)	0.042 ± 0.006
	IRX5:HCHL (4)	0.160 ± 0.038
	IRX5:HCHL (5)	0.025 ± 0.002

	^and, not detected.

TABLE II

Height of the main inflorescence stem and total stem
dry weight of senesced wild type (WT) and IRX5:HCHL
plants. n, number of plants analyzed. Asterisks indicate
significant differences from the wild-type (, P < 0.05, *,
P < 0.01, ***, P < 0.001).

	Height (cm)	Dry weight (mg)
Plant line	Mean ± SE	Mean ± SE	n

WT	62.4 ± 4.6	477.7 ± 51.3	16
IRX5:HCHL (1)	60.3 ± 5.0	501.6 ± 62.8	14
IRX5:HCHL (2)	56.0 ± 4.6	435.3 ± 62.5	12
IRX5:HCHL (4)	48.3 ± 4.4***	335.7 ± 63.4***	15
IRX5:HCHL (5)	54.1 ± 7.6**	399.1 ± 61.1*	16

TABLE III

Quantitative analysis of soluble phenolics in stems from five-week-old wild type (WT) and
IRX5:HCHL plants. Values are means of four biological replicates.

Mean ± SE (μg g⁻¹fresh weight)

Plant line	HBAld		3,4-DHBAld	HBA	HBAGlc	HBAGE

WT	nd^a	nd^a	nd^a	2.32 ± 0.20	1.34 ± 0.41
IRX5:HCHL (1)	1.02 ± 0.07	0.33 ± 0.02	5.53 ± 0.36	544.87 ± 157.79	1653.74 ± 504.38
IRX5:HCHL (2)	0.62 ± 0.08	0.23 ± 0.02	4.77 ± 0.41	569.23 ± 138.73	1046.97 ± 439.35
IRX5:HCHL (4)	0.83 ± 0.18	0.29 ± 0.03	4.64 ± 0.57	484.06 ± 74.23	959.79 ± 189.25
IRX5:HCHL (5)	1.04 ± 0.09	0.34 ± 0.02	5.59 ± 0.27	531.29 ± 51.13	1360.03 ± 178.03

^and, not detected

TABLE IV

Quantitative analysis of acid-hydrolyzed soluble phenolics in stems from five-week-old
wild type (WT) and IRX5:HCHL plants. Values are means of four biological replicates.

Mean ± SE (μg g⁻¹fresh weight)

		3,4-					3,4-
Plant line	HBAld	DHBAld	Van	5OH-Van	SyrAld	HBA	DHBA	VA	5OH-VA	SyrA

WT	0.6 ± 0.1	0.1 ± 0.0	nd^a	nd^a	nd^a	14.0 ± 2.8	10.2 ± 2.7	5.0 ± 0.9	nd^a	nd^a
IRX5:HCHL	11.8 ± 2.1	14.3 ± 2.0	11.9 ± 3.8	24.3 ± 2.0	1.7 ± 0.0	2492.4 ± 534.9	17.3 ± 2.4	226.9 ± 32.6	8.1 ± 0.7	44.7 ± 7.6
(1)
IRX5:HCHL	5.7 ± 1.5	10.4 ± 2.6	3.9 ± 1.28	12.4 ± 6.1	1.6 ± 0.1	1726.1 ± 706.7	13.7 ± 3.4	175.9 ± 37.1	6.2 ± 1.5	45.9 ± 10.9
(2)
IRX5:HCHL	7.2 ± 0.8	9.9 ± 0.6	6.4 ± 1.26	10.7 ± 1.7	1.7 ± 0.1	1588.3 ± 181.1	15.4 ± 1.7	183.6 ± 19.0	5.8 ± 0.3	31.3 ± 2.4
(4)
IRX5:HCHL	9.9 ± 1.2	12.8 ± 0.7	8.0 ± 1.73	16.9 ± 2.5	1.9 ± 0.1	2061.3 ± 336.2	16.4 ± 1.2	202.3 ± 9.2	7.0 ± 0.5	39.5 ± 3.2
(5)

^and, not detected.

TABLE V

Quantitative analysis of cell wall-bound phenolics in stems from extract-free senesced mature
dried wild type (WT) and IRX5:HCHL plants. Values are means of four biological replicates.

Mean ± SE (μg g⁻¹dry weight)

		3,4-					3,4-
Plant line	HBAld	DHBAld	Van	5OH-Van	SyrAld	HBA	DHBA	VA	5OH-VA	SyrA

WT	5.8 ± 0.6	1.1 ± 0.0	59.4 ± 6.5	nd^a	17.8 ± 1.0	6.2 ± 0.9	nd^a	24.2 ± 2.0	nd^a	10.6 ± 0.3
IRX5:HCHL	11.1 ± 0.4	0.6 ± 0.0	36.9 ± 2.7	0.8 ± 0.1	107.8 ± 6.4	486.4 ± 28.2	nd^a	42.2 ± 3.2	nd^a	47.5 ± 2.3
(1)
IRX5:HCHL	8.9 ± 0.4	0.6 ± 0.0	25.7 ± 5.9	0.6 ± 0.1	99.9 ± 4.6	427.9 ± 49.3	nd^a	39.6 ± 1.9	nd^a	43.3 ± 0.9
(2)
IRX5:HCHL	9.1 ± 0.9	0.7 ± 0.0	29.9 ± 2.7	0.8 ± 0.1	122.2 ± 14.8	421.8 ± 28.2	nd^a	36.8 ± 1.4	nd^a	54.1 ± 6.1
(4)
IRX5:HCHL	9.1 ± 0.7	0.7 ± 0.0	45.6 ± 6.2	0.7 ± 0.0	122.4 ± 5.9	349.6 ± 27.6	nd^a	47.7 ± 3.0	nd^a	59.3 ± 3.1
(5)

^and, not detected.

TABLE VI

Chemical composition of total and hemicellulosic cell wall sugars in senesced
mature dried stems from wild type (WT) and line IRX5:HCHL (2). Values are
means ± SE of three biological replicates. Asterisks indicate significant
differences from the wild type (P < 0.001).

Mean ± SE (mg g⁻¹CWR)

Total Sugars

Hemicellulosic Sugars

Sugar	WT	IRX5:HCHL	WT	IRX5:HCHL

Fucose	2.23 ± 0.08	2.21 ± 0.05	1.44 ± 0.03	1.49 ± 0.05
Rhamnose	10.83 ± 0.35	11.71 ± 0.19	9.12 ± 0.23	9.76 ± 0.26
Arabinose	16.01 ± 0.56	18.58 ± 0.54*	10.15 ± 0.30	12.40 ± 0.38*
Galactose	23.06 ± 0.66	22.69 ± 0.82	15.34 ± 0.33	16.49 ± 0.50
Glucose	442.76 ± 7.09	388.66 ± 7.58*	10.09 ± 0.34	11.25 ± 0.33
Xylose	201.63 ± 1.71	245.20 ± 3.31*	114.39 ± 0.97	141.16 ± 4.20*
Galacturonic acid	93.74 ± 2.56	99.96 ± 1.52	37.13 ± 1.86	40.58 ± 1.12
Glucuronic acid	4.10 ± 0.16	4.60 ± 0.39	2.66 ± 0.17	3.12 ± 0.09
Total	794.36 ± 13.17	793.61 ± 14.85	191.32 ± 4.23	236.25 ± 6.93*

TABLE VII

Lignin content and main H, G and S lignin-derived monomers obtained by thioacidolysis
of extract-free senesced mature dried stems from wild-type (WT) and line
IRX5:HCHL (2). Values are means ± SE from duplicate analyses.

		Total yield
	Klason lignin	(H + G + S)
Plant line	KL % of CWR	μmol g⁻¹KL	% H	% G	% S	S/G

Culture

1	WT	20.42 ± 0.14	1356 ± 40	0.98 ± 0.00	73.2 ± 0.3	25.9 ± 0.3	0.35 ± 0.01
	IRX5:HCHL	20.12 ± 0.15	1014 ± 5	1.48 ± 0.04	73.7 ± 0.5	25.2 ± 0.3	0.34 ± 0.01
Culture 2	WT	20.32 ± 0.25	1238 ± 13	1.09 ± 0.00	73.8 ± 0.3	25.2 ± 0.3	0.34 ± 0.01
	IRX5:HCHL	21.29 ± 0.14	1041 ± 7	1.47 ± 0.00	72.7 ± 0.1	25.9 ± 0.1	0.36 ± 0.00

TABLE VIII

Minor monomers obtained by thioacidolysis of extract-free mature senesced dried stems from wild-type
(WT) and line IRX5:HCHL (2). Values are means ± SE of duplicate analyses. Values are expressed in μmol
g⁻¹KL and as a relative percentage of the total main H, G and S monomers released by thioacidolysis.

	Vanalc	Syralc	Van	Syrald	Cald	VA	SyrA
	μmol g⁻¹KL	μmol g⁻¹KL	μmol g⁻¹KL	μmol g⁻¹KL	μmol g⁻¹KL	μmol g⁻¹KL	μmol g⁻¹KL
Plant line	(% H + G + S)	(% H + G + S)	(% H + G + S)	(% H + G + S)	(% H + G + S)	(% H + G + S)	(% H + G + S)

Culture 1	WT	nd*	nd*	4.3 ± 1	0.9 ± 0.3	7.2 ± 0.6	6.7 ± 0.2	1.4 ± 0.0
	IRX5:HCHL	5.0 ± 0.1	2.6 ± 0.2	(0.31)	(0.06)	(0.53)	(0.49)	(0.10)
		(0.49)	(0.25)	6.5 ± 1.4	18.7 ± 3.5	7.9 ± 0.3	6.8 ± 0.2	2.2 ± 0.0
				(0.64)	(1.84)	(0.77)	(0.67)	(0.21)
Culture 2	WT	nd*	nd*	4.6 ± 0.7	0.8 ± 0.3	6.9 ± 0.1	6.2 ± 0.2	1.2 ± 0.0
	IRX5:HCHL	5.3 ± 0.1	2.9 ± 0.1	(0.37)	(0.06)	(0.55)	(0.50)	(0.09)
		(0.50)	(0.28)	6.3 ± 0.7	16.7 ± 1.9	6.8 ± 0.1	7.0 ± 0.0	2.1 ± 0.0
				(0.60)	(1.60)	(0.66)	(0.65)	(0.20)

*nd, not detected.

TABLE IX

Comparative transcriptomics of IRX5:HCHL stems and WT. Positive and negative ratios are indicative of
upregulation and downregulation of the gene in plants expressing HCHL.

AGI Gene ID	Annotated Function	log2 ratio	P value

	MONOOXYGENASES
AT1G62570	flavin-containing monooxygenase family protein		0.00E+0
AT3G28740	cytochrome P450 family protein		0.00E+0
AT4G15760	monooxygenase, putative (MO1)	0.86
AT4G37370	CYP81D8	0.72	1.20E−7
AT3G28740	cytochrome P450 family protein	0.70	5.58E−7
AT2G12190	cytochrome P450, putative	0.65	8.60E−6
AT1G69500	CYP704B1	0.58	7.38E−4
AT3G14610	CYP72A7	0.51	2.96E−2
	DEHYDROGENASES/REDUCTASES
AT4G13180	short-chain dehydrogenase/reductase (SDR) family protein		0.00E+0
AT2G37770	aldo/keto reductase family protein, Transcript variant 1		0.00E+0
AT2G37770	aldo/keto reductase family protein, Transcript variant 2	0.96	0.00E+0
AT2G29350	SAG13 (Senescence-associated gene 13); short-chain dehydrogenase/reductase (SDR) family protein	0.83
AT1G14130	2-oxoglutarate and Fe(II)-dependent oxygenese superfamily protein	0.72	9.59E−8
AT1G72680	cinnamyl-alcohol dehydrogenase, putative	0.90	0.00E+0
AT1G60730	aldo/keto reductase family protein	0.65	8.30E−6
AT1G18020	FMN-linked oxidoreductases superfamily protein, Transcript variant 1	0.62	7.14E−5
AT1G18020	FMN-linked oxidoreductases superfamily protein; Transcript variant 2	0.59	4.38E−4
AT2G47130	short-chain dehydrogenase/reductase (SDR) family protein, Transcript variant 1	0.58	7.60E−4
AT2G47130	short-chain dehydrogenase/reductase (SDR) family protein, Transcript variant 2	0.58	8.36E−4
AT1G18020	FMN-linked oxidoreductases superfamily protein, Transcript variant 3	0.54	6.80E−3
AT5G14780	FDH (FORMATE DEHYDROGENASE); NAD binding/oxidoreductase, acting the CH—OH group of donors	0.53	1.24E−2
AT1G54100	ALDH7B4 (ALDEHYDE DEHYDROGENASE 7B4); 3-chloroallyl aldehyde dehydrogenase	0.51	3.00E−2
	UDP-GLUCOSYLTRANSFERASES
AT1G05560	UGT75B1		0.00E+0
AT2G15490	UGT73B4		0.00E+0
AT4G34138	UGT73B1		0.00E+0
AT2G30140	UGTB7A2	0.79
AT4G34131	UGT73B3	0.58	8.09E−4
AT3G11340	UGT76B1	0.58	8.97E−4
AT4G01070	UGT72B1	0.52	2.00E−2
	TRANSPORTERS
AT3G23560	ALF5 (ABERRANT LATERAL ROOT FORMATION 5), antiporter/transporter		0.00E+0
AT2G36380	PDR6 (PLEIOTROPIC DRUG RESISTANCE 6) ATPase, coupled to transmembrane movement of substances		0.00E+0
AT3G51860	CAX3 (cation exchanger 3); cation; cation antiporter		0.00E+0
AT5G65380	Multidrug and toxic compound extrusion (MATE) efflux family protein	0.92	0.00E+0
AT1G79410	ATOCT5 (organic cation/carnitine transporter 5)	0.89	0.00E+0
AT5G13750	ZIFL1 (ZINC INDUCED FACILITOR-LIKE 1); tetracycline:hydrogen antiporter/transporter	0.78
AT1G76520	auxin efflux carrier family protein	0.70	4.27E−7
AT1G76530	auxin efflux carrier family protein	0.69	8.91E−7
AT4G18197	AT4G18200/PUP7 (purine permease 7); purine transporter	0.64	1.62E−5
AT4G28390	AAC3 (ADP/ATP CARRIER 3); ATP:ADP antiporter/binding	0.62	6.57E−5
AT5G45380	DUR3 (DEGRADATION OF UREA 3); sodium:solute:symporter family protein	0.61	1.05E−4
AT3G18830	PLT5 (POLYOL TRANSPORTER 5)	0.57	1.56E−3
AT2G17500	auxin efflux carrier family protein	0.55	3.26E−3
	DETOXIFICATION
AT1G17170	ATGSTU24 (Glutathione S-transferase (class tau) 24)		0.00E+0
AT2G29420	ATGSTU7 (GLUTATHIONE S-TRANSFERASE 25)		0.00E+0
AT2G47730	ATGSTF8 (GLUTATHIONE S-TRANSFERASE 8)		0.00E+0
AT4G02520	ATGSTF2 (Glutathione S-transferase (class phi) 2)	0.78
AT3G09270	ATGSTU8 (Glutathione S-transferase (class tau) 8)	0.65	1.43E−5
AT2G29490	ATGSTU1 (GLUTATHIONE S-TRANSFERASE 19)	0.54	7.21E−3
AT4G19880	unknown protein, Glutathione S-transferase family protein	0.76
AT5G39050	ATPMaT1 (phenolic glucoside malonyltransferase 1); transferase family protein	0.77
AT5G39090	ATPMaT1-like; transferase family protein	0.52	2.13E−2
	JASMONIC ACID METABOLISM
AT1G76680	OPR1 (12-oxophytodienoate reductase 1)		0.00E+0
AT5G54206	12-oxophytodienoate reductase-related	0.99	0.00E+0
	STRESS INDUCIBLE/DEFENSE/SENESCENCE
AT5G49480	ATCP1 (CA2+-BINDING PROTEIN 1); calcium ion binding, NaCl stress inducible		0.00E+0
AT1G35260	Bet v I allergen family protein, defense response	0.88	0.00E+0
AT3G62550	universal stress protein (USP) family protein, Adenine nucleotide alpha-like protein	0.87	0.00E+0
AT1G73500	ATMKK9 (Arabidopsis thaliana MAP kinase kinase 9)	0.80
AT4G02380	SAG21 (SENESCENCE-ASSOCIATED GENE 21)	0.77
AT3G04720	PR4 (PATHOGENESIS-RELATED 4), similar to the antifungal chitin-binding protein hevein	0.64	2.04E−5
AT1G75270	DHAR2; glutathione dehydrogenase (ascorbate)	0.61	1.17E−4
AT1G70530	CRK3 (CYSTEINE-RICH RLK (RECEPTOR-LIKE PROTEIN KINASE) 3), protein kinase family protein	0.60	2.36E−4
AT3G50970	LTI3O/XERO2 (LOW TEMPERATURE-INDUCED 30); dehydrin stress-related	0.58	8.28E−4
AT5G27760	hypoxia-responsive family protein	0.54	6.87E−3
AT3G56710	SIB1 (SIGMA FACTOR BINDING PROTEIN 1); binding	0.51	2.30E−2
	MISCELLANEOUS
	Transcription factor
AT5G63790	ANAC102 (Arabidopsis NAC domain containing protein 102); transcription factor. Transcript variant 1		0.00E+0
AT1G77450	ANAC032 (Arabidopsis NAC domain containing protein 32); transcription factor		0.00E−0
AT5G63790	ANAC102 (Arabidopsis NAC domain containing protein 102); transcription factor, Transcript variant 2	0.65	1.26E−5
AT1G01720	ATAF1 (Arabidopsis NAC domain containing protein 2); transcription factor	0.54	7.23E−3
	Glycine-rich protein
AT2G05380	GRP3S (GLYCINE-RICH PROTEIN 3 SHORT ISOFORM) Transcript variant 1		0.00E+0
AT2G05380	GRP3S (GLYCINE-RICH PROTEIN 3 SHORT ISOFORM) Transcript variant 2		0.00E+0
AT2G05530	glycine-rich protein	0.96	0.00E+0
AT2G05540	glycine-rich protein	0.90	0.00E+0
	Auxin metabolism
AT3G44300	NIT2 (NITRILASE 2)		0.00E+0
AT3G44310	NIT1 (NITRILASE 1)	0.51	3.32E−2
	Other
AT5G30870	transposable element gene; pseudogene, hypothetical protein		0.00E+0
AT3G14990	4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, putative		0.00E+0
AT1G65280	heat shock protein binding/unfolded protein binding		0.00E+0
AT4G16190	cysteine proteinase, putative	0.89	0.00E+0
AT1G02850	glycosyl hydrolase family 1 protein BGLU11	0.86
AT1G17860	trypsin and protease inhibitor family protein/Kunitz family protein	0.86
AT3G49780	ATPSK4 (PHYTOSULFOKINE 4 PRECURSOR); growth factor	0.82
AT2G41380	embryo-abundant protein-related, methyltransferase activity	0.82
AT3G24420	hydrolase, alpha/beta fold family protein	0.79
AT5G52810	ornithine cyclodeaminase/mu-crystallin family protein	0.67	2.65E−6
AT5G17380	pyruvate decarboxylase family protein	0.64	1.84E−5
AT1G23890	NHL repeat-containing protein	0.59	3.35E−4
AT4G28380	leucine-rich repeat family protein, zinc ion binding	0.59	3.52E−4
AT4G01870	tolB protein-related	0.59	4.41E−4
AT1G37130	NIA2 (NITRATE REDUCTASE 2)	0.62	6.75E−5
AT1G24610	SET domain-containing protein, unknown protein	0.58	9.14E−4
AT4G11600	ATGPX6 (GLUTATHIONE PEROXIDASE 6); glutathione peroxidase	0.52	1.83E−2
	UNKNOWN
AT5G61820	unknown protein		0.00E+0
AT1G76600	unknown protein		0.00E+0
AT1G76960	unknown protein	0.71	2.01E−7
AT4G17840	unknown protein	0.67	2.77E−6
AT1G21680	unknown protein	0.61	1.38E−4
AT5G40960	unknown protein, DUF3339	0.59	5.08E−4
AT4G08555	unknown protein	0.58	8.16E−4
AT2G30690	unknown protein, DUF593	0.53	8.86E−3
AT5G66052	unknown protein	0.50	4.05E−2
	CARBOHYDRATE METABOLISM
AT2G06850	EXGT-A1 (ENDO-XYLOGLUCAN TRANSFERASE); hydrolase, acting on glycosyl bonds	−0.51	2.51E−2
AT3G52840	BGAL2 (beta-galactosidase 2), Glycoside hydrolase family 35, putative lactase	−0.52	1.83E−2
AT3G01345	Glycoside hydrolase family 35, beta-galactosidase putative	−0.53	1.13E−2
AT3G53190	pectate lyase family protein	−0.56	1.69E−3
AT5G03350	legume lectin family protein, carbohydrate binding	−0.57	1.28E−3
AT1G26810	GALT1 (galactosyltransferase 1), Glycoside transferase family 31	−0.61	1.02E−4
AT1G19600	pfkB-type carbohydrate kinase family protein	−0.63	3.41E−5
AT4G28250	ATEXPB3 (ARABIDOPSIS THALIANA EXPANSIN B3)	−0.79
AT3G30720	unknown protein, QUA-QUINE STARCH (QQS)	−1.08	0.00E+0
	MISCELLANEOUS
AT4G27440	PORB (PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B); protochlorophyllide reductase	−0.50	4.45E−2
AT5G02890	HXXXD-type acyl-transferase family protein	−0.50	3.86E−2
AT1G18950	aminoacyl-tRNA synthetase family	−0.51	3.29E−2
AT5G47330	palmitoyl protein thioesterase family protein	−0.53	1.07E−2
AT1G03870	FLA9 (FLA9)	−0.54	4.82E−3
AT1G20530	unknown protein, DUF630 and DUF632	−0.55	4.67E−3
ATCG00470	ATP SYNTHASE EPSILON CHAIN, rotational mechanism	−0.55	3.12E−3
AT5G51720	unknown protein, 2 iron, 2 sulfur cluster binding	−0.56	2.02E−3
ATCG00330	RPS14, CHLOROPLAST RIBOSOMAL PROTEIN S14	−0.58	8.70E−4
ATCG00340	D1 subunit of photosystem I and II reaction centers, Transcript variant 1	−0.62	5.42E−5
AT2G38870	serine-type endopeptidase inhibitor activity, pathogenesis-related peptide of the PR-6 proteinase inhibitor family	−0.64	1.54E−5
ATCG00340	D1 subunit of photosystem I and II reaction centers, Transcript variant 2	−0.71	2.75E−7

Claims

1. An engineered plant comprising a heterologous hydroxycinnamoyl-CoA hydratase-lyase (HCHL) operably linked to a promoter.

2. The engineered plant of claim 1, wherein the promoter is a secondary cell wall-specific promoter.

3. The engineered plant of claim 2, wherein the secondary cell wall-specific promoter is an IRX5 promoter.

4. The engineered plant of claim 1 wherein the HCHL is Pseudomonas fluorescens HCHL.

5. (canceled)

6. The engineered plant of claim 1, wherein the plant is a dicot.

7. The engineered plant of claim 1 wherein the plant is selected from the group consisting of Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.

8. (canceled)

9. A part of the plant of claim 1.

10. (canceled)

11. Biomass comprising the plant, or part of the plant, of claim 1.

12. An isolated expression cassette comprising a polynucleotide sequence encoding a hydroxycinnamoyl-CoA hydratase-lyase (HCHL) and a heterologous promoter, the promoter operably linked to the polynucleotide sequence, wherein the promoter is a secondary cell wall-specific promoter.

13. The expression cassette of claim 12, wherein the HCHL is Pseudomonas fluorescens HCHL.

14. The expression cassette of claim 12, wherein the promoter is an IRX5 promoter.

15. A plant cell comprising the expression cassette of claim 12.

16. A method for engineering a plant having reduced lignification, the method comprising:

(1) introducing a polynucleotide encoding a hydroxycinnamoyl-CoA hydratase-lyase (HCHL) into a plant, wherein the polynucleotide in the plant is operably linked to a promoter; and

(2) culturing the plant under conditions in which the HCHL is expressed, thereby reducing lignification in the plant.

17. The method of claim 16, wherein the promoter is heterologous to the plant.

18. (canceled)

19. The method of claim 16, wherein the promoter is a secondary cell wall-specific promoter.

20. The method of claim 19, wherein the secondary cell wall-specific promoter is an IRX5 promoter.

21. The method of claim 16, wherein the HCHL is Pseudomonas fluorescens HCHL.

22. (canceled)

23. The method of claim 16, wherein the plant is a dicot.

24. The method of claim 16, wherein the plant is selected from the group consisting of Arabidopsis, poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.

25. A method of obtaining soluble sugars, the method comprising incubating the biomass of claim 11 in a saccharification reaction.