AU2015202440B2 - Cambium/xylem-preferred promoters and uses thereof - Google Patents

Abstract

The present invention relates to nucleic acid molecules corresponding to regulatory portions of genes whose expression is predominant in cambium and/or xylem. The 5 invention also relates to compositions and methods of using the same to regulate the expression, in a cambium/xylem-preferred manner, of genes and/or any kind of nucleotide sequences in a plant. Nucleic acid molecules and its compositions include novel nucleotide 10 sequences for cambium/xylem-preferred promoters identified and isolated from poplar (Populus spp). Methods for expressing genes and/or any kind of nucleotide sequences in a plant using the promoter sequences disclosed herein are provided. The methods comprise stably incorporating 15 into the genome of a plant cell a nucleotide sequence operably linked to a cambium/xylem-preferred promoter of the present invention and regenerating a stably transformed plant that expresses the nucleotide sequence. 6469714_1 (GHMatters) P62336.AU.4 KAROLA

Description

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CAMBIUM/XYLEM-PREFERRED PROMOTERS AND USES THEREOF

[0001] ο ο (Ν ο (Ν ΙΟ Ο (Ν

5 FIELD OF THE INVENTION

[0002] The invention relates generally to the field of molecular biology, biochemistry and agriculture. More particularly, the invention relates to polynucleotides suitable for regulating gene expression in plants and 10 generation of transgenic plants with improved quality and productivity.

BACKGROUND AND PRIOR ART OF THE INVENTION

[0003] Modification of a plant trait through genetic 15 engineering depends upon the insertion into the plant genome of a polynucleotide construct containing the gene of interest, operably linked to a promoter that is functional in the transgenic plant. Within a plant genome, any single gene is, in general, operably linked to a 20 promoter that will determine when and where, within the plant tissues and organs, the gene should be expressed. Therefore if one wants to express a gene of interest in specific tissues or organs within a transgenic plant and in a temporally regulated manner, tissue-preferred 25 promoters must be used. On the other hand, expression in all plant tissues throughout the plant's life cycle could by achieved by using constitutive promoters.

[0004] In a number of situations the expression of particular genes in particular tissues or organs confers a 30 specific phenotype of interest to the plant. For example, if one wants to improve the nutritional quality of cereal seeds, a gene that confers such phenotype using seed-specific promoters is inserted, rather than using constitutive promoters that would allow the gene to be 35 expressed in all plant tissues causing, in some cases, undesirable phenotypes. In another example, if one wants to increase the amount of cellulose in the developing

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O (N O (N H O (N - 2 - vascular tissues of a forest tree, one would introduce into the plant genome a xylem- and/or cambium-preferred promoter operably linked to a heterologous gene encoding an enzyme involved in cellulose metabolism such that more 5 cellulose molecules could be produced in the developing plant xylem. In another example, the desired phenotype could be obtained by inhibiting the expression of an endogenous gene within a specific plant tissue. This could be done by introducing a construct comprising a 10 tissue-preferred promoter operably linked to a polynucleotide that would inhibit the expression of the endogenous gene, either by anti-sense hybridization or by RNA silencing (Matzke (ed.) et al. (2000) Plant Gene

Silencing. Kluwer Academic Publishers) . 15 [0005] Thus far, the production of genetically engineering plants expressing useful and/or desirable traits requires the availability of promoters that permit the gene or genes of interest to be expressed in a tissue-and timing -specific manner. Thus, isolation and 20 characterization of tissue-preferred, particularly cambium/xylem- preferred, promoters that can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in a tissue-preferred manner is essential for the genetic engineering of plants 25 exhibiting particular traits.

SUMMARY OF THE INVENTION

[0006] The present invention relates to isolated regulatory nucleic acid molecules from the genome of 30 Populus sp, and methods for regulating expression of heterologous nucleotide sequences in plant tissues, such as in a xylem and/or cambium- preferredmanner. The present invention also relates to isolated nucleic acid molecules which represent promoters able to direct tissue-35 specific expression of genes of interest. In a first aspect, the present invention provides an expression vector comprising: (i) an isolated nucleic acid molecule 9105329_1 (GHMatters) P62336.AU.5 r- o (N S'

(N

O (N O (N H O (N - 3 - that comprises a nucleotide sequence that is capable of initiating transcription of a gene in a cambium tissue-preferred manner, wherein said isolated nucleic acid molecule comprises: a nucleotide sequence as set forth in 5 SEQ ID N0.:5; (b) at least 20 contiguous nucleotides of a nucleotide sequence set forth in SEQ ID NO.:5 or a complement thereof; (c) a nucleotide sequence that has greater than 84% sequence identity to a nucleotide sequence set forth in SEQ ID NO.:5; or (d) a nucleotide 10 sequence that hybridizes under stringent conditions to a nucleotide sequence set forth in SEQ ID NO.:5 or a complement thereof, and (ii) a nucleic acid molecule which encodes a protein of interest, wherein (i) and (ii) are in operable linkage, wherein (i) does not normally regulate 15 (ii). Also disclosed is an isolated nucleic acid molecule comprising a nucleotide sequence that is capabale of initiating transcription of a agene in a plant cell, wherein said isolated nucleic acid molecule comprises: (i) a nucleotide sequence as set forth in SEQ ID No.: 5; (ii) 20 at least 20 contiguous nucleotides of a nucleotide sequence set forth in SEQ ID NO.: 5 or a complement thereof; (iii) a nucleotide sequence that has greater than 84% sequence identity to a nucleotide sequence set forth in SEQ ID NO.: 5; or (iv) a nucleotide sequence that 25 hybridizes under stringent conditions to a nucleotide sequence set forth in SEQ ID NO.: 5 or a complement thereof. The regulatory nucleic acid molecules of the present invention correspond to promoter sequences of genes which are preferably expressed in the cambium and/or 30 in the xylem of Populus sp. Genes encoding isoforms of sucrose synthase (SuSy), alphatubulin (TUB), arabinogalactan protein (ARAB), caffeic acid 3-0-methyltransferase (COMT), cinnamyl alcohol dehydrogenase (CAD), cinnamate 4-hydroxylase (C4H), cinnamoyl CoA 35 reductase (OCR), ferulate-5-hydroxylase (F5H), sinapyl alcohol dehydrogenase (SAD), UDP-D-glucuronate carboxy-lyase (UDP), lipid transfer protein (LTP) and ag-13 (AG13) 9105329_1 (GHMatters) P62336.AU.5

ο (N

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Ο (N Ο (N O (N 10 15 - 3a - were found to be expressed in the cambium/xylem tissue of Populus sp. And their promoters, which are the subject of the invention, have been isolated, cloned and validated. When these promoters are associated in a transgenic plant with genes other than those to which they were originally linked, the genes in question are prefereably expressed in the cambium and/or in the xylem of said transgenic plant. Methods of using the cambium/xylem-preferred promoters disclosed herein, for regulating expression of heterologous nucleotide sequences in cambium and/or xylem- preferred manner in a plant, are provided. [0007] The cambium/xylem-preferred promoters were identified through the analysis of a collection of Expressed Sequence Tags (ESTs) from Populus sp, representing apical shoot, bark, cambium, seed, xylem, leaf and root tissue. Based on the expression profile of 9105329_1 (GHMatlers) P62336.AU.5 Η Ο (Ν δ' Ο Ο (Ν Ο (Ν Ο (Ν - 4 - those ESTs among the different tissues, the twelve genes referred to supra were shown to be highly and preferably expressed in the cambium and/or in the xylem of Populus.

[0008] The cambium/xylem-preferred promoters of the 5 invention are set forth at SEQ ID NOS.: 1-12. Fragments of these nucleotide sequences, i.e., those set forth in SEQ ID NOS.: 1-12 comprising at least 20 consecutive nucleotides are also a feature of this invention. The smaller fragments, while not necessarily encoding 10 promoters or proteins with promoter activity, can function as antisense molecules and disable naturally occurring and expressed genes. The compositions of the invention further comprise nucleotide sequences having at least 65% Identity to the sequences set forth in SEQ ID NOS.: 1-12 15 or a fragment thereof, and nucleotide sequences that hybridize under high stringency conditions to any one of the aforementioned sequences.

[0009] "Stringent conditions" as used herein, refers to parameters with which the art is familiar, such as

20 hybridization in 3.5xSSC, IxDenhardt's solution, 25mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2mM EDTA for 18 hours at 65°C, followed by 4 washes of the filter at 65°C for 20 minutes, in 2xSSC, 0.1% SDS, and a final wash for up to 20 minutes in 0.5xSSC, 0.1% SDS, or O.SxSSC 25 and 0.1% SDS for greater stringency, and O.lxSSC, 0.1% SDS for even greater stringency. Other conditions may be substituted, as long as the degree of stringency is equal to that provided herein, using a 0.5xSSC final wash.

[0010] Other facets of the present Invention include 30 constructs, such as expression vectors comprising the promoters operably linked to a nucleotide sequence of interest, which encodes a desired protein. The promoters disclosed herein are capable of driving expression of polynucleotides of interest in a plant cell and said 35 promoters comprise any one of the nucleotide sequences of the present invention.

[0011] Also a part of the Invention are recombinant

646S749_1 (GHMatters) P62336.AU.5 KAROLA Η Ο (Ν - 5 - Ο Ο (Ν Ο (Ν Ο (Ν 10 15 20 plants or plant cells having stably incorporated into their genomes any one of the constructs described above or the promoter itself. [0012] Methods of the invention also include methods for stably incorporating the products of the invention into cells. [0012a] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. [0012b] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 schematically illustrates the plasmid vector pAPROM- ATG+promoter comprising the GUS reporter 25 gene operably linked to a promoter sequence. Promoters of the invention were cloned in this plasmid vector in substitution of the represented promoter sequence.

[0014] FIG. 2 shows the expression profile in a set of Populus tissues of SuSy, TUB, ARAB, UDP, LTP and AG13 30 genes, which are under the control of the promoters of the invention in Populus.

[0015] FIG. 3 shows the expression profile in a set of Populus tissues of COMT; CAD, C4H, CCR, F5H and SAD genes, which are under the control of the promoters of the 6469749_1 (GHMallers) P62336.AU.5 KAROLA

Ο (N Ο (Ν Ο (Ν Ο (Ν - 6 - invention in Populus.

[0016] FIG. 4 schematically illustrates the plasmid vector pALELLYXgi which is another embodiment of the invention. 5 [0017] FIGS. 5A and 5B show beta glucoronidase activity in the flowering stem of Arabidopsis plants, transformed in accordance with Example 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 10 [0018] The compositions of the present invention comprise novel nucleotide sequences for plant promoters, particularly cambium/xylem-preferred promoters for the Populus (woody aspen) genes encoding sucrose synthase (SuSy), alpha-tubulin (TUB); arabinogalactan protein 15 (ARAB), caffeic acid 3-0-methyltransferase (COMT), cinnamyl alcohol dehydrogenase (CAD), cinnamate 4-hydroxylase (C4H), cinnamoyl CoA reductase (CCR), ferulate-5-hydroxylase (F5H), sinapyl alcohol dehydrogenase (SAD), UDP-D-glucuronate carboxy-lyase 20 (UDP), lipid transfer protein (LTP) and ag-13 (AG13). The nucleotide sequences for these promoters are set forth in SEQ ID NOS.: 1-12, respectively. These promoters were isolated from the 5' untranslated region flanking the transcription initiation sites of their respective genes. 25 Methods for the isolation of the promoters are well known in the art and include bioinformatic tools for gene assembly such as Phred, Phrap, Consed (Gordon et al. (1998) Genome Research. 8:195-202), sequence alignment (Durbin et al. (1998) Biological sequence analysis - 30 probabilistic models of proteins and nucleic acids.

Cambridge University Press, Cambridge, UK), functional search (Altschul et al. (1997) Nucleic Acid Res: 25:3389- 3402) and PCR techniques (Sambrook and Russell (2001) Molecular Cloning - a laboratory manual. Cold Spring 35 Harbor Laboratory Press, Cold Spring Harbor, NY, USA) . Some of these methods are described in Example 1 supra, and all are incorporated by reference.

6469749_1 (GHMatters) P62336.AU.5 KAROLA

Ο (N Ό Ο Ο 'it (Ν Ο (Ν Ο (Ν - 7 - [0019] In various embodiments, the isolated nucleic acid molecules span 0.1 kb, 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb or 5 kb starting at the ATG start codon for the coding region of the genes in question. The isolated nucleic 5 acid molecules are referred to herein as promoters.

Promoters correspond to the nucleic acid molecules whose function is to regulate the expression of a gene. A promoter generally comprises specific signaling sequences called boxes, arranged along the promoter sequence, such 10 that its composition determines the temporal and spatial expression of a gene that is under its regulatory control. "Promoter" or "transcriptional initiation region" means a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA 15 synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence 20 the transcription initiation rate. It is recognized that, having identified the nucleotide sequences for the promoter regions disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5' untranslated region upstream from the 25 particular promoter regions identified herein.

[0020] Thus the promoter regions disclosed herein are generally further defined by additional upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers 30 and the like. In the same manner, the promoter elements, which enable expression in the desired tissue such a xylem and/or cambium, can be identified, isolated and used with other core promoters to confer cambium/xylem-preferred expression. 35 [0021] In the present invention, promoters that regulate the expression of genes specifically in the cambium and/or xylem were identified and isolated from

6469749_1 (GHMatters) P62336.AU.5 KAROLA

in ο (N Ο (Ν Ο (Ν ιη Ο (Ν - 8 -

Populus sp.

[0022] The SuSy gene encodes an isoform of sucrose synthase, an enzyme involved in the conversion of sucrose into UDP-glucose in the developing xylem. UDP-glucose is 5 the building block of cellulose that is synthesized and deposited in the plant cell wall. The SuSy gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 2). 10 [0023] The TUB gene encodes an isoform of alpha- tubulin, a structural globular protein involved in the formation of microtubules, which are part of the cytoskeleton. The TUB gene disclosed herein is preferentially expressed in the cambium and/or xylem of 15 Populus sp, although low levels of expression can be observed in other tissues (FIG. 2).

[0024] The ARAB gene encodes an isoform of arabinogalactan protein, member of a large family of plant cell wall-associated glycoproteins of unknown function. 20 The ARAB gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 2).

[0025] The COMT gene encodes an isoform of caffeic acid 3-0- methyltransferase implicated in the methylation 25 of both caffeic acid and 5- hydroxyferulic acid. These are intermediate compounds of lignin biosynthesis. The COMT gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3). 30 [0026] The CAD gene encodes an isoform of cinnamyl alcohol dehydrogenase, an enzyme that catalyzes the final step in the synthesis of monolignols, thereby converting the cinnamaldehydes to their corresponding alcohols. The CAD gene disclosed herein is preferentially expressed in 35 the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3).

[0027] The C4H gene encodes an isoform of cinnamate

$4$9749_1 (GHMatters) P6233$.AU.5 KAROLA

Ο (N Ο Ο (Ν Ο (Ν Ο (Ν - 9 - 4-hydroxylase, a member of the cytochrome P450 monooxygenase superfamily involved in the catalysis of the first oxidative reaction in the phenylpropanoid metabolism, namely the conversion of trans-cinnamic acid 5 to p-coumaric acid. The C4H gene disclosed herein is preferentially expressed In the camblum/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3).

[0028] The CCR gene encodes an isoform of cinnamoyl 10 CoA reductase, which catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e., the first specific step in the synthesis of lignin monomers. The CCR gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low 15 levels of expression can be observed in other tissues (FIG. 3) .

[0029] The F5H gene encodes a cytochrome P450-dependent monooxygenase that catalyzes the hydroxylation of ferulic acid in a biosynthesis directed towards sinapic 20 acid and syringyl lignin. The F5H gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3).

[0030] The SAD gene encodes a sinapyl alcohol 25 dehydrogenase that mediates the reduction of sinapaldehyde into syringyl monolignols in angiosperms. The SAD gene disclosed herein is preferentially expressed In the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3). 30 [0031] The UDP gene encodes the enzyme UDP-D- glucuronate carboxy-lyase involved in the breakdown of UDP-D-glucuronate into UDP-D-xylose and CO2. The UDP gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of 35 expression can be observed in other tissues (FIG. 3).

[0032] The DTP gene encodes an isoform of lipid transfer protein, a member of a family thought to 646974S_1 (GHMatters) P62336.AU.5 KAROLA ΙΟ Ο (Ν S' Ο Ο (Ν Ο (Ν ΙΟ Ο (Ν - 10 - participate in cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. The LTP gene disclosed herein is preferentially expressed 5 in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3).

[0033] The AG13 gene encodes an ag-13 protein of unknown function, whose expression has been associated with the ripening process in several plant species. The 10 AG13 gene disclosed herein is preferentially expressed in the cambium/xylem of Populus sp, although low levels of expression can be observed in other tissues (FIG. 3).

[0034] The cambium/xylem-preferred promoter sequences of the present invention drive the expression of operably 15 linked nucleotide sequences in a cambium/xylem-preferred manner. EXAMPLE 4 illustrates the expression of the GUS reporter gene in the cambium/xylem vessels/fiber complex of Arabldopsis thaliana transformed with a construct containing the GUS reporter gene operably linked to two 20 cambium/xylem-preferred promoters of the invention, i.e., the TUB (SEQ ID.: 2) and C4H (SEQ ID.: 6) promoters. EXAMPLE 4 also summarizes results showing expression of the GUS reporter gene in Arabldopsis plants transformed with constructs containing the GUS reporter gene operably 25 linked to each one of the promoter sequences disclosed herein. Thus, the cambium/xylem-preferred promoter sequences disclosed herein can be used to express an operably linked sequence of interest in the cambium and/or in the xylem. Hence, the cambium/xylem-preferred 30 promoters can be used to improve the wood quality of trees either by increasing the synthesis of cellulose or by decreasing the synthesis of lignin. "Decreasing lignin synthesis" means decreasing the total lignin content of woody trees by anywhere from 1-90%, preferably by about 35 80-90% relative to the lignin content in normal field

grown plants. "Increasing cellulose synthesis" means increasing the total cellulose content of woody trees by 6469749_1 (GHMatters) P62336.AU.5 KAROLA Η Ο (Ν δ' Ο Ο (Ν Ο (Ν Ο (Ν - 11 - 1-90%, preferably by about 80-90%, compared with normal field grown plants.

[0035] In addition, the cambium/xylem-preferred promoters can be used to inhibit the expression of genes 5 involved in the metabolism of developing xylem. The inhibition of such genes decreases the concentration of lignin and/or changes the relationship between guaiacyl and syringyl, the building blocks of lignins. The monomeric composition of lignins is an important 10 characteristic from the industrial point of view, because syringyl unit-rich lignins are more easily degraded during the pulping process, as they contain fewer strong 5-5' carbon bonds. Thus, the determination of the syringyl to guaiacyl (S/G) ratio is useful in evaluating wood quality 15 for cellulose production and papermaking (Boudet et al., 1998). "Changing the relationship between syringyl and guaiacyl" refers to increasing the syringyl/guaiacyl ratio by 1-90%, preferably from about 80-90% compared with normal field grown plants. 20 [0036] Other nucleic acid molecules within the invention are variants and/or fragments of the cambium/xylem-preferred promoter sequences such as those that encode fragments, analogs or derivatives of native cambium/xylem-preferred promoter sequences disclosed 25 herein. Such variants and/or fragments may be, e.g., naturally occurring variants of native cambium/xylem-preferred promoter sequences, or non-naturally occurring variants of cambium/xylem-preferred promoter sequences.

For example, the nucleotide sequence of such variants 30 and/or fragments can include deletions, additions, and/or substitutions of one or more nucleotides as compared to the native cambium/xylem-preferred promoter sequences.

Such variants and/or fragments may retain the biological activity and therefore drive, in a cambium/xylem-preferred 35 manner, the expression of operably linked nucleotide

sequences. Fragments of cambium/xylem-preferred promoter sequences comprise from about 10, to about 4000 6469749_1 (GHMallers) P62336.AU.5 KAROLA

ο (N Ο Ο (Ν Ο (Ν m ο (Ν - 12 - nucleotides or up to the number of nucleotides in the full-length cambium/xylem-preferred promoter sequences disclosed herein as, such as the 700-3500 nucleotides of SEQ ID NOS . :1-12. 5 [0037] "Variants" is intended to include substantially similar sequences. Naturally and non-naturally occurring "variants" of cambium/xylem-preferred promoter sequences within the invention are nucleic acid molecules having at least 65% sequence identity with the 10 native cambium/xylem-preferred promoter sequences disclosed herein, i.e., SEQ ID NOS: 1-12. "Variants" also include nucleic acid molecules that hybridize under stringent conditions, as defined herein, to the cambium/xylem-preferred promoter nucleic acid sequences of 15 SEQ ID NOS.: 1-12 or the complement of the sequences of SEQ ID NOS.: 1-12. For example, such "variants" may be nucleic acid molecules that hybridize to the sequence of SEQ ID NOS.: 1-12 or the complement of the sequences of SEQ ID NOS: 1-12 under low stringency conditions, moderate 20 stringency conditions, or high stringency conditions. Alternatively, such nucleic acids are those having a nucleotide sequence that is the complement of the full-length or portions of the sequences of SEQ ID NOS.: 1-12. Other variants of cambium/xylem-preferred promoter 25 sequences within the invention are polynucleotides that share at least 65% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, to the sequences of SEQ ID NOS: 1-12 or the complement of the sequences of SEQ ID NOS: 1-12. 30 [0038] "Stringent conditions", as used herein, refers to the parameters set forth supra.

[0039] For purposes of the present invention, sequence identity to any of the promoter sequences disclosed herein is preferably made using art known 35 methodologies such as the BLAST program, or any sequence alignment program that allows the alignment of identical nucleotides and verification of mismatches between non-

64$9749_1 (GHMatters) P62336.AU.5 KAROLA ΙΟ ο (Ν S' Ο (Ν Ο (Ν ΙΟ Ο (Ν - 13 - identical nucleotides so that the percentage of identity of compared sequences could be estimated.

[0040] The cambium/xylem-preferred promoters of the invention may be used to express a gene of interest. For 5 example, by using cambium/xylem-preferred promoters, the expression of native and/or non-native genes could be regulated in the cambium and/or xylem tissues of a plant, thus altering a plant's cellulose content, lignin content, pathogen or insect resistance, wood development, wood 10 quality, and the like. The native and/or non-native genes include those encoding enzymes, transporters, cofactors, transcription factors and a number of other genes that would affect cellulose and/or lignin deposition in the plant or pathogen or insect resistance. 15 [0041] For the present invention, "genes of interest" include those involved in cellulose metabolism and lignin metabolism. It is recognized that any gene of interest can be operably linked to the promoter of the invention and expressed in plant cambium and/or xylem tissues. 20 [0042] The cambium/xylem-preferred promoters of the present invention, when operably linked to a gene of interest and stably incorporated into a plant genome, drive cambium and/or xylem-preferred expression of the said gene of interest. Cambium and/or xylem-preferred 25 expression is intended to mean that expression of the gene of interest is most abundant in the cambium and/or in the xylem, although some level of expression of the gene of interest may occur in other plant tissues. Cambium encompasses any part of the cambial or procambial tissue 30 in any organ of the plant, including but not being limited to the root, shoot, stem, wood, leaf, petiole, and the like. Xylem means any part of the xylem tissue, including but not being limited to the tracheids, tracheary elements, vessels, fuse fibers and pith. Some of the 35 promoters disclosed herein may drive the expression of genes to the secondary xylem more prominently than to the primary xylem.

6469749_1 (GHMallers) P62336.AU.5 KAROLA

ο (N Ο (Ν Ο (Ν Ο (Ν - 14 - [0043] The constructs containing the cambium/xylem-preferred promoters disclosed in the present invention and an operably linked gene of interest may be provided in expression cassettes as depicted in the figures. Such 5 expression cassettes comprise the cambium/xylem-preferred promoters of the present invention, or variants or fragments thereof, operably linked to a gene of interest whose expression is directed to the cambium and/or xylem. Such an expression cassette may contain restriction sites 10 for insertion of the gene of interest under the transcriptional control of the cambium/xylem-preferred promoters. The expression cassette may additionally contain a number of other nucleic acid sequences, including selectable marker genes, transcriptional and 15 translational initiation sequences, and a plant transcriptional and translational termination sequence.

The termination region may be native with the DNA sequence of interest or may be from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline 20 synthase termination regions (Gielen et al., EMBO J. 3:835-846 (1984), Depicker et al., Mol. and Appl. Genet., 1:561-573 (1982)).

[0044] Reporter genes or selectable marker genes may be included in the expression cassettes. Examples of 25 suitable reporter genes known in the art can be found in, for example, Jefferson et al. (1991) in Plant Molecular

Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1-33. Selectable marker genes for selection of transformed cells or tissues can include 30 genes that confer herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15:127-136), bromoxynil (Stalker et al. (1988) Science 242:419-423), 35 glyphosate (Shaw et al. (1986) Science 233:478-481) and phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513-2518) .

6469749_1 (GHMatters) P62336.AU.5 KAROLA Ο (Ν δ' Ο Ο (Ν Ο (Ν ΙΟ Ο (Ν - 15 - [0045] The expression cassettes of the present invention operably linked to a gene of interest are useful for the transformation of a variety of plants. Such plants, include, but are not limited to. Eucalyptus 5 species (E. alba^ E. alhens^ E. amygdalina^ E. aromaphloia, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E. brevistylis, E. brockwayi, E. camaldulensis, E. ceracea, E. cloeziana, E. coccifera, E. cordata, 10 E. cornuta^ E. corticosa, E. crebra^ E. croajingolensis, E. curtisii, E. dalrympleana^ E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. divers!folia ^ E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E. erythrocorys, E. erythrophloia, 15 E. eudesmoides, E. falcata, E. gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis x urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, 20 E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua, E. obtusiflora, E. occidentalis, 25 E. optima, E. ovata, E. pachyphylla, E. pauciflora, E. pellita, E. perriniana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata, E. regnans, 30 E. risdonii, E. robertsonii, E. rodwayi, E. rubida, E. rubiginosa, E. saligna, E. salmonophloia, E. scoparia, E. sieberi, E. spathulata, E. staeri, E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragons, E. tetrodonta, E. tindaliae, E. torquata, E. umbra, 35 E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetarensis, E. willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, E. woodward!!.

6469749_1 (GHMatters) PS233$.AU.5 KAROLA

ο (N Ο Ο (Ν Ο (Ν Ο (Ν - 16 -

Populus species (Ρ. alba^ Ρ. alba χ Ρ. grandidentata^ Ρ. alba χ Ρ. tremula ^ Ρ. alba χ Ρ. tremula var. glandulosa, Ρ. alba χ Ρ. tremuloldes^ Ρ. balsam!fera ^ Ρ. balsam!fera subsp. tr!chocarpa, P. balsam!fera subsp. tr!chocarpa x 5 P. delto!des, P. dHata^ P. delto!des ^ P. euphrat!ca ^ P. euramer!cana, P. k!takam!ens!s, P. las!ocarpa ^ P. laur!fol!a^ P. max!mow!cz!! ^ P. max!mow!cz!! x P. balsam!fera subsp. tr!chocarpa, P. n!gra, P. s!ebold!! x P. grand!dentata, P. suaveolens^ P. szechuan!ca, 10 P. tomentosa r P. tremula^ P. tremula x P. tremuloldes ^ P. tremuloldes, P. wllsonl!^ P. canadensis^ P. yunnanensls) and Conifers as, for example, loblolly pine (Plnus taeda) ^ slash pine (Plnus elliotii) ^ ponderosa pine (Plnus ponderosa)^ lodgepole pine (Plnus contorta)^ 15 and Monterey pine (Plnus radlata); Douglas-fir (Pseudotsuga menzlesl!); Western hemlock (Tsuga canadensis); Sitka spruce (Plcea glauca)/ redwood (Sequoia sempervlrens); true firs such as silver fir (Abies amabllls) and balsam fir (Abies balsamea); and cedars such 20 as Western red cedar (Thuja pllcata) and Alaska yellow-cedar (Chamaecyparls nootkatensls).

[0046] The expression cassettes may be stably incorporated into plant genomes by Agrobacterium-mediated transformation (Fraley et al. (1983) Proc. Natl. Acad. 25 Scl. USA. 80:4803-4807) or by the biobalistics method (Klein et al. (1987) Nature. 327:70-73).

[0047] All technical terms used herein are terms commonly used in biochemistry, molecular biology and agriculture, and can be understood by one of ordinary

30 skill in the art to which this invention belongs. Those technical terms can be found in: Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Current Protocols in Molecular 35 Biology, ed. Ausubel et al., Greene Publishing Associates and Wiley-Interscience, New York, 1988 (with periodic updates); Short Protocols in Molecular Biology: A

64$9749_1 (GHMatters) P6233$.AU.5 KAROLA

Ο (N Ο Ο (Ν Ο (Ν Ο (Ν 30 35 - 17 -

Compendium of Methods from Current Protocols in Molecular Biology, 5th ed., vol. 1-2, ed. Ausubel et al., John Wiley & Sons, Inc., 2002/ Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al., Cold Spring Harbor Laboratory 5 Press, Cold Spring Harbor, N.Y., 1997. Methods involving plant biology techniques are described herein and are described in detail in methodology treatises such as Methods in Plant Molecular Biology: A Laboratory Course Manual, ed. Maliga et al., Cold Spring Harbor Laboratory 10 Press, Cold Spring Harbor, N.Y., 1995. Various techniques using PCR are described, e.g., in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, 2nd ed.. Cold Spring 15 Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs can be derived from known sequences by using computer programs intended for that purpose (e.g.. Primer, Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). Methods for chemical 20 synthesis of nucleic acids are discussed, for example, in Beaucage and Caruthers (1981) Tetra. Lett. 22:1859-1862 and Matteucci and Caruthers (1981) J. Am. Chem. Soc. 103:3185.

[0048] The present invention is further illustrated 25 by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way. EXAMPLE 1

Expression Profile of Genes Preferably Expressed in

Cambium/Xy1em [0049] Expressed Sequence Tags (ESTs) from Populus sp. were clustered using the CAPS program (Huang and Madan (1999) Genome Res. 9:868-877) . Such ESTs were obtained from libraries representing the following tissues: apical

6469749_1 (GHMatters) P62336.AU.5 KAROLA

ο (N Ο Ο (Ν Ο (Ν Ο (Ν - 18 - shoot, bark, cambium, seed, xylem, leaf and root. The set of clusters thus generated was searched for those clusters composed of at least 90% of ESTs from libraries representing Populus cambium and xylem tissues. Twelve 5 clusters were chosen based on their high and preferred level of expression in the cambium and/or in the xylem of Populus. A BLASTX search against the non-redundant GenBank database was then performed with each one of the twelve clusters, and it was concluded that they represent 10 expressed sequences from the following genes, sucrose synthase (SuSy), alpha-tubulln (TUB), ablnogalactan protein (ARAB), caffeic acid 3-0-methyltransferase (COMT), cinnamyl alcohol dehydrogenase (CAD), clnnamate 4-hydroxylase (C4H), cinnamoyl CoA reductase (CCR), 15 ferulate-5-hydroxylase (F5H), slnapyl alcohol dehydrogenase (SAD), UDP-D-glucuronate carboxy-lyase (UDP), lipid transfer protein (DTP) and ag-13 (AG13). FIGs 2 and 3 show the expression profile in several tissues of Populus for each of the clusters representing 20 the genes whose promoters are disclosed herein. The series of histograms in FIGs 2 and 3 ultimately depict the relative abundance of each gene in cDNA libraries representing the aforementioned tissues (apical shoot, bark, cambium, seed, xylem, leaf and root). Thus, the 25 histograms compose a set of digital expression data which is an approximation of the relative level of expression for the twelve genes whose promoters are disclosed herein. EXAMPLE 2 30

Isolation of Promoter Sequences [0050] BLASTN was performed for each one of the twelve clusters against the genomic sequences from Populus 35 trichocarpa made available by the Joint Genome Institute, US Department of Energy as part of the "Populus Genome Sequencing Project" (http://genome.jgi-

6469749_1 (GHMallers) P$233$.AU.5 KAROLA

Ο (N Ο Ο (Ν Ο (Ν Ο (Ν -19- psf.org/poplarO/poplarO.info.html). Selected nucleotide regions from each cluster corresponding to putative exons were used as driver sequences in the retrieval of genomic sequence reads comprising the transcription initiation 5 region and adjacent upstream promoter sequences. These genomic reads were assembled using the PHRAP (Gordon et al. (1998) Genome Res. 8:195-202) program to obtain a contig encompassing approximately 700 to 3500 nucleotides of putative promoter region upstream from the 10 transcription initiation point (+1 nucleotide, which corresponds to the beginning of the respective mRNA).

These contigs contain the promoter regions for each of the genes encoding the mRNAs represented by the twelve clusters concluded to be preferably expressed in the 15 cambium and/or in the xylem tissues of Populus. These twelve promoter regions correspond to sequences disclosed herein under SEQ ID NOS.: 1-12.

[0051] For isolation of specific promoter regions, pairs of gene-specific primers (usually 30 nt in length) 20 were designed from the sequences of the promoter contigs described above to amplify by PCR a fragment of 700 to 3500 nucleotides from the promoter region of each one of the twelve genes whose promoter sequences are disclosed herein. The first round of PCR was performed on genomic 25 DNA sample from Populus deltoldes or P. trichocharpa, which was prepared from leaves using the cetyltrimethyl-ammonium bromide (CTAB) extraction method (Aldrich and Cullis (1993) Plant Mol. Biol. Report. 11:128-141). The primers were designed to amplify the region upstream of 30 the coding sequence, i.e., the 5' untranslated region and promoter region of the chosen gene. The sequences of the primers used are given below for each promoter:

6469749_1 (GHMallers) P62336.AU.5 KAROLA Η Ο (Νδ' sucrose synthase (SuSy) - 20 - Ο Ο (Ν Ο (Ν Ο (Ν 10 15 20 25 30 35 5' - GCCATAGCTCCTTAAGAGAAACAGAAAGCAA - 3' ( SEQ ID NO : 13) 5' - CAATATAGAATCAATGAACAGCACTAGTTTGC - 3' ( SEQ ID NO : 14) 5' - TCATGTCCTATCCAACGGCG - 3' ( SEQ ID NO : 15) alpha-tubulin (TUB) 5 ’ - CTCATTTTCTCTCAAAGCTCAAAG - 3' (SEQ ID NO: 16) 5 ’ - GACAACTAGTCTAAAGTTAAAACTTAGACC - 3' (SEQ ID NO: 17) 5 ’ - CCCTGGAGGTTGGGGTGAGT - 3' (SEQ ID NO: 18) arabinogalactan protein (ARAB) 5 ’ - GCGTTCATCTACAAAACCCTCCTCC - 3' (SEQ ID NO: 19) 5 ’ - TTCATCCTTATTTTTTTGGGATA - 3' (SEQ ID NO: 20) 5 ’ - CAAAGGATCATGGAGTTGGA - 3' (SEQ ID NO: 21) caffeic acid 3-0-methyltransferase (COMT) 5 ’ - TATACTAATATGACCTAATAACTTAGAAGTGTGG - 3’ (SEQ ID NO: 22) 5 ’ - CATCTTGATCAAGATTGAATTC - 3' (SEQ ID NO: 23) 5 ’ - CATAATATCAAAACTTAAGC - 3' (SEQ ID NO: 24) cinnamyl alcohol dehydrogenase (CAD) 5’ - TGAATTGATGACGTAGGAAACATGATAAACATG - 3' (SEQ ID NO: 25) 5’ - CATTTTCTTGAAACAATGAGGCTAAGAG - 3' (SEQ ID NO: 26) cinnamate 4-hydroxylase (C4H) 5' - GACATGAGAAACTAACGTTGCTTGAATTC - 3' (SEQ ID NO: 27) 5' - CATAATATTGGAACTGGTTTCTTTGTCAGAAAG - 3' (SEQ ID NO: 28) cinnamoyl CoA reductase (CCR) 5' - GCGCTCGGGTTGTCACCATAGTTTC - 3' (SEQ ID NO: 29) 5' - CATGTTGTTATATTTAGATAAATGTA - 3' (SEQ ID NO: 30) ferulate-5-hydroxylase (F5H) 5’ - TTCATCAAGCAATAATAATAAGGTGAGGC - 3' (SEQ ID NO: 31) 5’ - CATGGATGCAGATTTTTGTGTTTGTG - 3' (SEQ ID NO: 32) 5’ - TTCAGTGAACATGCTGCCACAATGAC - 3' (SEQ ID NO: 33)

6469749.1 (GHMallers) P62336.AU.5 KAROLA ΙΟ Ο (ΝS' Ο (Ν Ο (Ν ΙΟ Ο (Ν 10 15 - 21 - sinapyl alcohol dehydrogenase (SAD) 5' - AATCGAAACCGATCGATTTGAACTGG - 3' 5' - CATGGTGCTTGCTTCAGATAG - 3' UDP-D-glucuronate carboxy-lyase (UDP) 5’ - GGAAATGTCAACACTTGTGTGACCACAC - 3’ 5’ - GACATTCTTGTCCAATTTCTGAA - 3’ lipid transfer protein (LTP) 5’ - GGAGCCTCCATATTTCTGTATCTC - 3’ 5’ - CAAGACGATGAAATGAAGAACTGATAGC - 3’ ag-13 (AG13) 5' - GACATTCCTTGACTTAATATGATGCT - 3’ 5' - GAATTCGCATCCATGCGGTGAGTTCG - 3’ :SEQ ID NO: 34; ;SEQ ID NO: 35) ;SEQ ID NO: 36) ;SEQ ID NO: 37) ;SEQ ID NO: 38) ;SEQ ID NO: 39) ;SEQ ID NO: 40) ;SEQ ID NO: 41) 20 25 [0052] PCR was performed using commercially available reagents and cycle parameters of 5 min at 94° C followed by 35 cycles of 94° C for 1 min, then a varying annealing temperature, as described infra for 1 min, then 72° C for 3 min. The annealing temperature (T) was adjusted for each primer pair and ranged from 50° C to 59° C. Finally, the samples were held at 72° C for 7 min, then at 4° C until further analysis. Ten μ1 of each of the resulting amplified DNA fragments were run on a 0.8% agarose gel, purified using the GFX Gel Purification Kit (Amersham), subcloned into pGEM-T-Easy vector (Promega) and then into EcoRI and Bglll sites of the pAPROM-ATG vector. Final sequences were determined on the resulting plasmids. 30

Figure 1 schematically illustrates the expression cassette pAPROM-ATG comprising the GUS gene operably linked to a promoter disclosed herein. Figure 4 schematically illustrates the plasmid vector comprising a gene of interest operably linked to a promoter of the invention. 35

$4$9749_1 (GHMallers) P62336.AU.5 KAROLA ΙΟ ο (Ν S' Ο Ο (Ν Ο (Ν ΙΟ Ο (Ν - 22 - EXAMPLE 3

Transformation of Arabidopsis Plants 5 [0053] Arabidopsis thaliana Columbia plants were transformed using an Agrobacterium tumefaciens mediated transformation protocol (Bechtold et ai., (1993) C. R.

Acad. Sci. Paris 316:1194-1199; Bent et ai., (1986) Mol. Gen. Genet. 204:383-396) with individual constructs 10 containing any one of the promoters of the invention operably linked to a gene of interest. The constructs also contained the selectable marker gene Bar that confers resistance to herbicidal phosphinothricin analogs like ammonium gluphosinate (Thompson et al. (1987) EMBO J. 15 9:2519-2523). In this example, the gene of interest operably linked to the cambium/xylem-preferred promoters of the invention is the reporter gene Gus encoding the enzyme beta-glucuronidase (GUS) (Jefferson (1987) Plant Mol. Biol. Rep. 5: 387-405) that facilitates visual 20 inspection of the desirable phenotype, i.e., expression of GUS in a cambium/xylem-preferred manner.

[0054] Seeds of Arabidopsis thaliana ecotype Columbia were sown in pots containing vermiculite. Plants were grown under a 16/8 hours dark/light regime at 22°C. After 25 4-5 weeks, plants were transformed with the Agrobacterium tumefaciens strain GV3101 in accordance with Bent et al., (1986) Mol. Gen. Genet. 204:383-396, which harbors the plasmid vector comprising the gene of interest operably linked to each one of the promoters of the invention. 30 [0055] For plant transformation, 1 liter of LB medium containing rifampicin, gentamycin and kanamicin was inoculated with an aliquot of overnight starter Agrobacterium culture. The culture was then grown overnight at 28°C in a rotary shaker, until OD600 is >8.0. 35 The Agrobacterium was precipitated by centrifugation and the bacterial pellet was resuspended in ~300 ml of 5% sucrose and 0.03% Silwet L-77. This Agrobacterium

6469749_1 (GHMatters) P62336.AU.5 KAROLA Ο (Nδ' Ο Ο (Ν Ο (Ν Ο (Ν 10 15 - 23 - suspension was sprayed onto the plants. The pots were placed in a tray which was covered with plastic wrap to maintain humidity and the plants were grown at the above regime, in order to mature and to set seeds. [0056] Seeds were harvested and surface sterilized in a solution containing 50% bleach and 0.02% Triton X-100 for 7 minutes. Seeds were then rinsed 3 times in sterile distilled water and plated out in MS medium containing 6 mg/1 of Finale as a selection agent. After 5 to 7 days, transformants were visible as green plants. Transformed plants were transferred onto new selection plates and after 6-10 days were transferred to pots containing vermiculite and grown under conditions of 16 hours light/ 8 hours dark at 22°C. EXAMPLE 4 GUS expression assay in Arabidopsis plants 20 [0057] Inflorescence stems of the transformed plants described in EX7\MPLE 3 were cut and histologically stained for GUS activity. Subsequent cuttings induced the formation of secondary xylem at the basis of plants that could also be histologically stained for GUS activity. 25 [0058] In figures 5A and 5B, activity of beta-

glucuronidase in flowering stems of transgenic Arabidopsis plants is shown. These transgenic Arabidopsis plants were transformed with a construct containing the gene Gus operably linked to cambium/xylem-preferred promoters of 30 the invention, namely TUB (SEQ ID.: 2) (A) and C4H (SEQ ID.: 6) (B). Darker bands along the longitudinal axis of the stem (arrowheads) represent primary vascular bundles stained blue after the chromogenic assay, indicating the functionality and tissue-specificity of the respective 35 promoter in each transgenic line.

[0059] The table below summarizes GUS assay data obtained through the analysis of inflorescence stem 6469749_1 (GHMallers) P62336.AU.5 KAROLA 20 25 IT) - 24 - Ο (N σ3 Ο Ο (Ν Ο (Ν Ο (Ν 10 cuttings of Tirabidopsis thaliana plants transformed with expression constructs according to EXAMPLE 2 comprising the Gus gene under the control of promoter sequences disclosed herein. For all promoters tested, vascular GUS expression pattern was observed. In some cases, GUS activity was markedly high in specific vascular cell types such as vessel elements, as for example in plants transformed with constructs comprising the LTP (SEQ ID.: II), C4H (SEQ ID.: 6) or TUB (SEQ ID.: 2) promoters. In other cases, a vascular pattern was observed but no specific cell type therein could be pinpointed as the main GUS expression site. 15

Expression pattern Promoter No. of events analyzed Total of GUS-positive events vessel elements only Vessel elements+ other vascnlar cell types Non-vascnlar cell types SUSY 92 21 1 17 3 LTP 75 38 19 14 5 C4H 89 43 14 28 1 TUB 78 20 9 10 1 COMT 72 24 2 15 7 CAD 79 37 8 16 13 SAD 75 30 4 13 13 UDP 72 20 4 14 2 CCR 74 22 4 14 4 [0060] Other aspects of the invention will be clear to the skilled artisan, and need not be reiterated here.

[0061] The terms and expression which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expression of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible with the scope of the invention.

[0062] The entire disclosure in the complete specification of our Australian Patent Application Nos 2005230212, 2010201471 and 2012203911 are by this cross-reference incorporated into the present application. 30

6469749_1 (GHMatters) P62336.AU.5 KAROLA ΙΟο (Ν Ο (Ν Ο (Ν Ό Ο (Ν

- 25 - SEQUENCE LISTING <110> PAPES, Fabio GERHARDT, Isabel Rodrigues ARRUDA, Paulo <120> CAMBIUM/XYLEM-PREFERRED PROMOTERS AND USES THEREOF <130> ALEL 202.1 PCT 10 <140> <141> <150> US 60/560,227 <151> 2004-04-06 15 <160> 41 <170> Patentin version 3.2 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH; 3035

<212> TYPE; DNA <213> ORGANISM: Populus sp. 25 <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(3035) <223> OTHER INFORMATION: Sucrose synthase (SUSY) promoter <400> SEQUENCE: 1 30 35 tcatgtccta ctatatatct ttatattatt gcaatacata gttcttaaaa ataataataa tacaaagagt tccaacggcg ttttttttta ttgatatatt aaaaataata ccaacaaaag taataataat gtaactcaac atgcaaactt atttaaattg acatcaagaa gaacccacca gaagcaaagt aataataata tagtcatgtt cgctgtcccg ttaattcttt taaattttaa acctaagaat agatctctga aaattaattt ctaaatttat cactttttca atatttttat aaaaataatt acttgtcaat gtcaaaaacc aacttggtgt tctctagaga taggacgagg attcttttaa tttaaaattt gcatagaagt agaggaaacc aataataaaa ttactagttt tgaagtttag 70 ttttatattt 140 acttaaccac 210 acacctgcta 280 atagaaacac 350 ttaatttaat 420 gagttttaca 490

6469749.1 (GHMallers) P62336.AU.5 KAROLA - 26 - o (N aattttaagg ccactgaaga tttatatagt cattaatttc agaatatata agattagttg agttacgtat 560 >> aaattgatta aaaaatcata ttaataaaaa taaaaaaatt aatttaaagg tttaagaaat caaattaaga 630 ci 2 gaaaagagtg gtgttttatt tttcatcgtg ccctctctca acagacaagt agaatgatga gagagagagg 700 gtaaagaaat ggatttatga gaacattgac cacagggaaa gagagaagcg gttttgtgaa aggaacaatg 770 O 5 aaaccacagg aaggtaaagc ggtaatgata tatttcacga atactaaaac tagaacaaca agttttttaa 840 tcaaattaaa ccacgagtgc aaggccgtct tctctgtgta taaaagggtc cttcttcttt ctcatttccc 910 O attctcatct gcaaacttct cctttgcaat ctttctttct tgcgttctgt gtgttcgttg tgatttgtgt 980 >1 tcattcttct tgtctattag cttgtccccc cgtccgactg ctttctgtat ttattctggc attaagctta 1050 (N O aggtaaagat ccctcaacta tcccaagcaa tttattctgt ttttatgtga tcttgaggga tcttcctctt 1120 CN 10 ggatgcgctt tttatttttt cttcctcctt cttcctgctc cttcttacct tgtatctgat cccccagacg 1190 O aaaatgtttt ttgttttttt aattagctca acaaatcaaa aacattcaca taataacaca gctcgaaaga 1260 (N aatctgatac agttttaatc tgttgtattt taaaaatcat tacagttcat gcatgctgat actttaccat 1330 gtcatgaaat taaatcccag catccttttc catagccaaa gaaggatcag cagcatgctg atagtttacc 1400 atgtcatgaa attaaatccc agcatccttt tccatagcca aagaaagatc agcagcatgc ttgcttatac 1470 15 aaggtcttcg cttgcttatc aaggccactg aaacatcatc atcgtcataa ctatgataga acccgcctac 1540 tgccggcatt gaaaacatca tcactagtgt ctctacatta aaaaacaccc actgtctaat ttcctatttt 1610 tttactctta aaatgtcttt cggcttgagc tcctcgggct ccacggatgg caactgctgt attatatata 1680 tatatatata tatatatata tatatatata tatatatata tatatatata tatatttccc tgttggctac 1750 atagacctgt taataccgta taaatagata atattaatat atagaattca tgtatctttc cgagattaag 1820 20 cgatgccgta taaataatat taatatcttt gaatcagtat gtatattaat taaaattaat ttttttcaaa 1890 gtaattttaa gagcgcattt tcaacatcca tttagttttt ttttaataat aaatctctct ttgcattaat 1960 cctaacgttt gaacttagta aattaaaaaa aggaaaatac ctttttcacc aatatagaat caatgaacag 2030 cactagtttg cttgaaataa aaataaaaat aaaatctaat aagacatttc gaaatcatcc ttatccgcaa 2100 atcactacat tagtatagta tcttgaaaga taagcaagga tcatgcaagt ttataataat taaacttaaa 2170 25 acgtactatg acgtgtgcat cattcattca ttctgcatga aactctccac aagtctagcc tttgcatcat 2240 tcattctact tcattttatt ttttcctcta atggtttcga ttgatttttc tttcttagag tctggtcttt 2310 tagttcaact ttacatgttt taggctcgta ttttgagaga aaaaaaagaa aaaagtatgc agatcatgat 2380 tctgcaaaat actgaactag tgttctgatg aattaacatg tagcatgtat aatgctggaa gaactaaaga 2450 gcagttgggc tgccatgacc aaaagaaact tcgactgatt ataaatgtca aaacttgggc ccattctttg 2520 30 gtttctgtct gttgttttat gccatggcaa aactctgctt atttttcaac gtccaacgtc aaatgggaga 2590 ggtttaaatt ctattgttat gtctaaacca cgtggttgtt atctatatct gaccgaacat tcaagctttt 2660 ggtattccac aagaagggtt ttctctcttc tttcttttca taattgtaat gtgtttaatt tgtttcttgc 2730 ccaataatct tctctgcttc aaactaactt taattgttcg atctcttgcg ttattttaga catgtgcaat 2800 cacctttcac tgttgaaaaa atggttggtg aggtgaggtg gtaggttttg aagtcttcta gaataatgtg 2870 35 gtttctctgt tgctcttgac ttcttcttgt agatcatttc tggctggcta agctatccat acccccccgc 2940 ccctacaaat aatattgagt tgttgctggt cttaattcct attatctgtt attactccca ctgattgctt 3010 tctgtttctc ttaaggagct atggc 3035

6469749_1 (GHMatters) P62336.AU.5 KAROLA iT) - 27 - ccctggaggt tggggtgagt gaaataagag ggttaaatat tttttttgga ttaaaccatt caaagtgaat 70 tttttaataa aatctcatag gctgattaaa tgaaattcct ttagagtcat catacggtaa atttgatgtt 140 agtttggtgt tatagtgcaa attacttttt aattaaaaga tagcaatgct tccagcatgg tggactcgtt 210 tttcaaatcg aaagctgctt cttcttcttt gttttttttt tttaatcttg tttttctaat ttcataaaaa 280 ccaatcatta tttcgcaggt caggtagtta aatttgttag gctaattgat ccagaaacct ccggaaagtc 350 aaactcaaat aaactgctga cctttttatt tatttttatt ttttgaattc taattcgtcg gactatctgg 420 tcaagataat ccacctctca tgcgaatact tcttagagtg ccatccatta taccctgtta agttgccggt 490 gattgcacat gtttgaccac cctccctccc ctaattttca cggcggaaag gggcttgttt gggcttgttt 560 taaattataa taatagtgat gatttaaagt attttttatt taaaaatata ttaaaataat tttttttatt 630 ttttaaaaat tatttttaac atcaaaacaa catgaaaaca taaaaaaatt gttttcattc tttttaaaaa 700 tatttttttt ctatttttat tcaatattat tatatagttt tcttattttt atttttctat taagtattat 770 taggtttttc tgtttttttt tttaatttaa aggaaataat tttttttcta ttcaatatta ttagaaattt 840 ctaatttttt ctatataaag gattttaaaa ttgtaataac attttgacaa gaaatttaat gaataaaaat 910 taaatattct agatatctct tcacagttat gacattcttg gttttaattt ataataaatc gcattatcat 980 taaccctcgg ctaaattatc tatttattta tgaccatgga aacacaagtg cgtgtgtatt tggggaggtg 1050 tgggtttaaa gcctgcaata taattgaaga aaaaatttaa gaatttttcc gcgttgatga aaccctgatt 1120 gaaggttgga gcatgcctca ataggcagac gggcgaaact tagaaaccag gaataaacgt gaaacacggg 1190 attcacacga atttggaaat ccacgcttgt aaagaaaacc aaaccgcata attttatttc ctatttgttt 1260 tcgcgtcttg tttttaaaaa atttaaattt tattttattt ttttttcttt aaattaatat ttttttgata 1330 attttagatc attttaatat gctgatatca aaaataaatt ttaaaaaata aaaaaaatat attattttaa 1400 tatatttcta aataaaaaac acttcaaaaa acaattataa ccatattttc aaacaagtac tattaaaaaa 1470 gtgatggaca agagaaatca aggggtcgcg gatgcgcttc agcaatagtg aatgacaact agtctaaagt 1540 taaaacttag acctcctcgc gtaaatttta tatttatatt tttaatatta atacattaaa ataattaaaa 1610 aataatttaa aaatcattaa ttcatacaaa atttttaaag catattaaaa agagaataaa cggcaaaaac 1680 Ο

(N σ3 S <200> <210> o 5 <211> <212> o <213> <220> (N O <221> CN 10 <222> O <223> (N <400>

15 20 25 30 35 6469749_1 (GHMatters) P62336.AU.5 KAROLA - 28 - r-H O (N aaacctacgc taattgtgaa ataaaagatt aatctatgca cacggtatcg ttttacttca ctggtcggtg 1750 >> taataatttc tctaacctta tgacccaaca attcactatt ttgaaaccct tgttattatt ttttttatca 1820 2 accattttct taatctccat ttcactcatt ccagttgcct ggacagtgga catggtggcg gtgcctcttg 1890 atcttttcta gttgggccac atgaatacac ttcaagggat ttgaaactag gcctaatcga ttgaaacgta I960 O 5 gaatccactc tctaattgag aggacggccc accctcctgg gcgacgtgcc ctctcatcca ccaggaccac 2030 cgccatcatg ccttctctgc tccttcctca cgcctcccaa cagaatgaca ttattagcct ccatcccaac 2100 O 'st" tatagaccgg cagtggcaca actgcaattt cctacaaccc aagacgatcc ccaaaactaa attcaaaaat 2170 caaaatggag cgggcaacta accatggtta aaataacgat tcggccaacc tggcaaaatc aagaattagg 2240 (N O tggcttggga aacggcatca ttggcatgca cctaatttga cccgtggtta aactaaccct ggttagctaa 2310 (N 10 accacacact ccctccgtcc cctaatttct ctccctctga aagtatataa accccatact cacagaccta 2380 2450 O aaagctcacc cctgaaattt cataggcgtc ttgataaacg ccaccctccc tcagcatcaa ttccaattgt (N ctttgctttc gattttctct tcttttaata tctgttgatc tttgtgcttt gagagaaaat gag 2513 <200> SEQUENCE CHARACTERISTICS: 15 20

<210> SEQ ID NO 3 <211> LENGTH: 2041 <212> TYPE: DNA <2I3> ORGANISM: Populus sp. <220> FEATURE: <22I> NAME/KEY: promoter <222> LOCATION: (1)...(2041) <223> OTHER INFORMATION: Arabinogalactan protein (ARAB) promoter <400> SEQUENCE: 3 25 caaaggatca tggagttgga atccccacca tccctatttt atttgataaa aattaagcac cagggtggta 70 gggatctatg caagttccaa gttcaaagga cttttcactg gaagtgatat gtcagagaat aatatataaa 140 ttatttcttg gaatctcacc aatccctatt tatttgataa aaattaagta caaggtagtg cgaaacctgt 210 acaagtttta agcctaaagg gctttcactt gaagaggtgt gttagagaat aatataaatc atatcttaga 280 accttaccta acatcttaag ctattgagat gagatgattc tttgacatgg tatcagaact ttaatgacca 350 aacagtcatg agtttgaatc tcaccatccc tatttatttg ataaaaatta agcacaagat agtgtgggca 420 tgtgcaagtt tcaagcttaa ttgactttta cttgaggggt gtgtgttaga gaatgatata aatcatatct 490 tggaatctta cctaataact taagttattg gattgagatg attatttgac gatcagagaa gacaaagcat 560 gcattaagga gggtagagag aaaggaaaag gaggttgcag gacaatggtg aaagcaaata tttcattaca 630 agtttttgaa gtggttggaa tcaaaatgtt gttctcttta atctgtaaga ttatatatgg ttctgctgac 700 aacatttgaa tgcgaggctg aacataatgc aaaagagtag aaaatgctaa ttatcaagaa atcaggcttc 770 tgaaacagaa ctacctttac taggttatct cttgaacttc tactaaactt aatgtgaaca aatctgctgt 840 30 35

6469749_1 (GHMatlers) P62336.AU.5 KAROLA ΙΟο (ΝS' - 29 - Ο Ο (Ν Ο (Ν ιη Η Ο (Ν 10 15 attgctctca cacaggaacc ttttaagttt cctcagaatg aatttttctc tagtttaagc aatcccacat 910 caggttaagt tcttttctcc tgtttcaaaa ctgctggtgt tgataattag agaaaagaga gtgttagaga 980 gcataggatt gttactttaa gcttgaggaa gtggattcca atcagtaaaa ttgtcgaggt tatatcacaa 1050 ttttcataaa ctgaatgtga cagacgactg ccagaaaaac ccttctatga tttgctgcat tatggaggaa 1120 aatcatggtt ttggtggaag catgatccat tcatcctagt acgtttaaca tgaataaaag gcttgagctc 1190 tagtacagaa tcccttgcct caactccctt catccttcct cctccgcgtt catctacaaa accctcctcc 1260 accgcctttt ctttcatcct ctccatgaat aaaagactat tatgccattc aacatcatgt aaaagaacac 1330 aattcctttt acttcgaaat ggctatctta aagtttcaag acttgcgttt gcatactgca aaatcacttt 1400 tatcaatagc atgacctcta cgggctcatg tacataaggt aagtgtttct tcatgaagtt gtgttaagtg 1470 atggtctggt gtgagatttg atttctgagc gtgcgaatct agaaaattag tgatctatca atgtctgtca 1540 aggattaagg atgtaaatat tcgttctttt aagctaaaag agcaaagact tggctattta cgatacaaag 1610 gtcagtttag atcgcttgtc taaatcttct gtcattatag atgatttgtt ttgatgttaa gaagcatgct 1680 cagctgttct gctagtgatg attcacaatc atggacatct ttatttgttg tcacagccac ttgaaatcta 1750 ccttttagaa cctttttttt ttgcctgctt ttccaaggaa agtagttgct gcagcattgt taaatttccc 1820 tctccattga tgaccctaca gcttttggag tgagataagg tactagcaat ctagttgtat aactaaaatt 1890 gtatattgca cctaacttga tcctctgtcc actactataa aaacctcact ctatctcatc tttacacatc 1960 aaacacttta tgattgaaat caatttgcat tagtatattt gaattgtttc gagcatttta tcccaaaaaa 2030 ataaggatga a 2041 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 2422

<212> TYPE: DNA 25 <213> ORGANISM: Populus sp. <220> EEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(2422) 30 <223> OTHER INEORMATION: Caffeic acid 3-0-methyltransferase (COMT) promoter <400> SEQUENCE: 4 cataatatca aaacttaagc agatcaaatt gaaatatatt tgtaattttt atataaatta gcactgatat 70 gtcaaaataa agacttcaaa ttcaaaactt aagtagacca aactgaaata tatttgtaat tcctatagaa 140 atcaacattg gtataccaaa ataaagagtt tagatttctg atctagcctg cagcagcaga gtaaaacaaa 210 35 aataaagtct gaataggaat cacgaaataa aatgaaatga agaattgcaa aatcataatt aaatgaagtc 280 tgaagtttca aaatcctgac caggtataaa attaagatgc aaaaaacaaa atcttatcag aactaaagtt 350

agataatcga aagtaaagta gaatctagat ttaattaatg tattggaggg gaacaattgt tcatattcga 420 6469749_1 (GHMatters) P62336.AU.5 KAROLA -SO o (N tcaaggaaat taacacctaa ttaaataaaa aggctcgaag atgagaagga cggtgcatgg atggtcaaaa 490 aacgaagcag cagaagagaa tggtcggtgg tgcacagtca tgttaaatgt ccaaattaaa aacaaaaaaa 560 aggtttaatt atgaaaatat ttcattctta acgaatatat caaactgcca aaccccccac cggttccatt 630 Ό tatatgggag gagtgattga tatttttatt aaactcaatt tatttataat ttaatttaaa atctgattga 700 O 5 tgtcttataa taaattttaa aaaaatatat agataaaggt tgatctagtc aattcaagag tcaataatga 770 ttttatcaaa atttaattta atttttttaa aaacaaaaca taattccaaa acaatgttgt ttggattttt 840 O tttttaaaaa aaaacataat ccacccatgt cattaattta ccaaactcct aacacaatca tgtttaataa 910 \J cccttcaatt ttcaaaaata atttcagttc ttatatttat ttttatttgc aaattagtcc ttgtttgaat 980 (N O tttcttttta gttcttatac tttacaaaaa ttatagttta tttttttatt gtgattcttt ttattataat 1050 CN 10 taaggtccct acatgctttt ttttttatgt aatgcttttt aatgtaataa atcattctga ttgtaatcat 1120 O caattatata attattttga caattacata attaaatata gaaatataat aaattattac gttacatgat 1190 (N ctattactaa gtacccaagt ctctacgtca atgttcaatt ttcagcaggt ggttctgtta gaatgtccca 1260 tccaaaatat ggattcattg atacgatttt taagtccaaa caaccctcat attaagcaaa accctcatat 1330 taagcaaaag attattatta ttattattat tattattatt tattattatt attattgttt ttgttgttgt 1400 15 gcttcttctt tttctcaatc aacaaaattt ttaccaactt caagattttt ttttttatgg ttaaaggtat 1470 actaatatga cctaataact tagaagtgtg gattatagat aaaattagca attcgtgcta tatagtgggt 1540 tggatattta tttatataaa aaaattatat atataagttt ttttttatgc atacttgtac aaaaaaaaaa 1610 tataaataca aatcaaatat ttattcaatc aaatgataat agaaccagat atatatgaaa ttgattaaaa 1680 aaaatatatc atgttaggtc aacatattag aaatactata caaaaataaa tatttatatg tatataacac 1750 20 atacaaagat tttctatagc gtgtgtttat tcagtgagtt tcatttatat taactttaaa atcattagtt 1820 ttataggatg taaatttatc ttttattaat tttaaatgtg ttcaataaat acaatcgggt gaatgtatca 1890 ttatgtgatt gaatatctta atctgcattt atctcttaat tttttcagtt ttttttttgt tattgttaat 1960 gaattttttt ttatttatat aaatgattat tgatttattt aattagatgc tttatacttt aattttttat 2030 atataaaaaa acatattaaa acaatctata tacctgatat ttttattttt aaaaattata acccatgata 2100 25 aagaagtttt ataaacctac ctgcttgaca tattacatca tgttccaata gtctcccctg aaacaggtta 2170 aaaaaaaaaa agtttggcaa ataagacgag gaaaaatata tagaaaaaaa ggtagggagt cagttctagg 2240 aagaagacat ttgtgcatca agtagagagg agggaccaac cacaaggtgg ttgagcactt caccatatat 2310 agcaccactt tgcaacctct ttttcagtat tctcatatcc tcttcacttc ttttcttttc accttcttca 2380 accttttgtt tccttaaaga attcaatctt gatcaagatg gg 2422

SO 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 793 <212> TYPE: DNA <213> ORGANISM: Populus sp. <220> FEATURE:

6469749_1 (GHMatters) P62336.AU.5 KAROLA - 31 -

ο (N Ο <221> ΝΑΜΕ/ΚΕΥ: promoter <222> LOCATION: (1)...(793) <223> OTHER INFORMATION: cinnamyl alcohol dehydrogenase (CAD) promoter <400> SEQUENCE: 5 Ο (Ν Ο (Ν Ο (Ν 10 15 ttgaattgat gacgtaggaa acatgataaa catgtaatct aaatatatct catgtctagg tcatgggttt 70 cacgtattag tccagcttta tccaaaataa tttttttatt tgttattatt gttaccttat tttttcatca 140 tattattaaa ttaattaaaa tttaatcaaa acattaattt tttcttactt ttttttaaaa tataatcttc 210 tcttaaattt cttttttcat gtttaaaaaa atttcagtcg acggcacaac aatccagtaa ataccaaggg 280 tatattgtcg ccactcacca ccaactacgt caattaagca aataatataa ttaggcaact gtgtaaccac 350 catggaaatt aagatattcc tttcatgaaa tacttaatta gtgacgtata catgatgctc caaacctcat 420 cacagattca gtgttcttaa ctattatgtt cccttttgtt tcccaagaac catgagttaa tcaggaccat 490 cgatactact gaggccccac caatgttttg atcatgtgga caatgttcac ttgattttca actttgaaga 560 aatgacccat ggttgtggaa gcagaggatg gcgccactcc atcacatttc acctaccacc acccgtaaaa 630 tatgcggagc tgtccttgtc ttttttgttg ccaagtaacc tttgccattc tttattgtgc ttttgtatat 700 atactcatcc atagtggctt ataattcttc aactctccac agaaactcca taggtctctc ttagcctcat 770 tgtttcaaga aaatggtaga tct 793 20 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 984 <212> TYPE: DNA <213> ORGANISM: Populus sp. <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(984) <223> OTHER INFORMATION: cinnamate 4-hydroxylase (C4H) promoter <400> SEQUENCE: 6 30 35 tgatatgaga aactaacgtt gcttgaattc aagatagaaa ttgaccttgc aagaagacaa acgtattctt 70 ggaaacacgt attaataaat acaaagtagt ttgtcacact acgggagaaa atatctaata aaagtaagac 140 cttatagttt caggaggtta ggttgatatt taaagagaga tttcttttat taacttttta tatatgttga 210 aatcttgaaa ttaatattaa aaagatttgt taatcctttt ctcttgaata ctttggattg atgtgaggga 280 ttcacattta aactattctt aaatgaatct tgaagctgta tgtttgatat tgtgttttta aaatgtattt 350 atctttaaaa aatatcaaat taatgatttt ttaatgtttt ttaaagattt gaaagtatta atttaaaaaa 420 taaaataaaa ttattttaat atatttttaa ataaaaaata tttttgaaga gcagactgca ccctatactt 490

6469749_1 (GHMatters) P62336.AU.5 KAROLA - 32 - (N gatctcaatt ttaaagagat ttggagaaca caagaattaa aaaagaaaag gataggaaaa aaaaactttc 560 ttgtttgata gccttattac ttgaagctga aatcatcata gattagtggc gcccacatta catcttgtat 630 S agaaatatag aaaggcctgg caaattaatt aatatgatga ccatatgaca ttttcggcca ccaacccgcc 700 ttacctacta ctatccatga tcatcaatgt cactctccta ccacctcaaa tgtaacgccg ttaactcccc 770 o 5 cccccccaca cacacacaca accctagcta gtagccacac gctccaccac ctaacgtgtg aaattcaact 840 tcatttcctc tctaattttt gtagcttata aaacccaagc tctcctcgtc ctgttgctcc catccaacaa 910 o ccatcactct tcttacctca aaaatcccca cctctttctg acaaagaaac cagttccaat attatggtag 980 atct 984

(N o (N in O (N 10 <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 7 <2II> LENGTH: 1007

<212> TYPE: DNA 15 20 <213> ORGANISM: Populus sp. <220> EEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(1007) <223> OTHER INEORMATION: cinnamoyl CoA reductase (CCR) promoter <400> SEQUENCE: 7 tgcgctcggg ttgtcaccat agtttcattt cttaatttat taagttaaat taagatacaa taagttggtc 70 acgttttaaa gcaaagagaa acaggaaatg ggtaaaaagc aacataaatt ctctttcaca tttttttgtc 140 accaggttct ttgttggtct aggagtatta attaattaat gctttgacat tgatttattc gttaattctt 210 ttaaaacact gaattaaatc caatccacac acaaaatgaa atgggggtag gtgatgtggg tgattatttt 280 ttattcggtt tgatttttat taaaaaaaat aaccaaactg aattattata tttttaaaaa aactaaaacc 350 ggttcaaacc ggtcggtttc aattcggttt tttaggacaa caaccggttc aaaccacttt ggctcggttt 420 aggtttgatt cggttcgatt tttttgattt taggtttata aaacggaaat tgaactgaac cggttaattt 490 tttaaaaatt ttaaatttaa ttttttaatt attttctttt taattttttg attttatcag tttttcaaat 560 ttttttttca cttaagagag gccatggtca tcatgtacct tcaaagaaga gagagaaata gcaaagcaca 630 tggtgacgtt gtgttgacga ttcacattac aaagacccat actcctactt cacaaacctt aataataata 700 ataataataa taataataat aatagtaata agagaaaaaa ctagaaaaac aaaaacaaag agagaagaat 770 ctctttcctc tctctcagag gcgaatattt accagtagta ggtgaggatg gtaacttcta accttataaa 840 tacatccact ccaccatgtc tttccttgta acatccactt ttcaagccaa gataagaaga aaagacatct 910 cctctcctct ttctctctgt ctgttctcca ctttcccagt caccaaactc gtatacatat aattacattt 980 atctaaatat aacaacatgg tagatct 1007 25 30 35

6469749_1 (GHMatters) P62336.AU.5 KAROLA IT) - 33 - ttcagtgaac atgctgccac aatgacatat atatcatcac aaattaatta atgtctactt taatgctgat 70 atatcttttg tttattattt tttttcctat catgggaaat gagatcaact ttttcagatg aaaattacta 140 attaaactat catatttcca gtttaatcaa agatatggaa tctttatttc actaaagata ttattattca 210 taagaatttg atgagttctt gcattatttg ttagattatc ttcaccctct tgcaattagt gcttcatgga 280 ctcctttttt tcttgtgaaa gtagtttgcc atttaaatat agaaatatct catgctttac aaaatataat 350 aatctcccct aagatataat aaattgaact gagatgcaat taagtcggtt aaaaggcctg gatactgcca 420 gtgaataaga tttacacaaa atattggatt ttttcccgtc ctgaaagcta attattgtca gaaaaatacg 490 ttttgaaata gttgattttt attgatatgg tggaataaaa acatcaatgg ttccaatgtc taaccacgaa 560 aatgacttgt aaaatttata ataaggtcta tttttttcat caagcaataa taataaggtg aggcatcaaa 630 atctctcact ttttgcttct gatcaaagat cactaagcag aacttgcatg gaacctcatc tctctctctc 700 tccccctctc tctctctccc cctctccctc tctatatata tatatatata tatatatata tatgcaagta 770 ttagtcacat tgcatgagta cgtggcagtt ttggatatgc tttgataacg gataacaccg agagtacaaa 840 acaaaatctg ggtaggtagc tggctcaatt gcaaccaaat aataataaga aattttagct gcaagcaatt 910 aagaaaatga aagattgcac ctatgtcaac cactgggtta atatttatga tcttaatctt ttttttttgt 980 ataatttctt ttatatgccg tgaaatgaag tcagccctta agttttacat aaatgtttag gttaattaga 1050 aaggagttaa ttctatatat aataagttgt tgattgaaac aaaatatggt ctgtcactct atttttgggt 1120 tgctttttat tgcatagtac ttctgcccta ttgattcagt gaacGCtttc gtatttataa tataataaag 1190 tagaccttga ataaatattg acatgtaact taaaacatta attgtcctcg ttttgacaac ataaaatctg 1260 tatcaacgta cgtgctcttg tttagggttt tctttagaca actttatatc tagaaaacgt aattcaatca 1330 aaaaagatat atatatatat atatatatat atatatatat atatatatat atatatagac agacgacata 1400 acaaaaatgt tcgggtcaga actctggact actgatcgaa gttgtttcaa atatattgaa tggtatatct 1470 taccatagta attaactgag ttatttcaag atattacaca gacataacat attttgttct tgatcaaaat 1540 atattttatt taaaaatata ttaaaataat atatttttta tttttaaaaa tatattttta atatcaatac 1610 attaaaataa tttaaaatat aaaaatacaa aaatattttt taaccacaaa aaaaaaaaac tatgaaaatt 1680 aatgttctta aatattgttc tccatccaga ttttggtacg tatgcgttcc cagtgtgtac ttgtttatga 1750 Ο

(N δ- <200> <210> <211> o 5 <212> <213> o <220> <221> (N o <222> CnI 10 in <223> <400>

(N

15 20 25 30 35 6469749_1 (GHMatters) P62336.AU.5 KAROLA O (N S'

O

o 'it 'it (N O (N o (N taatcgaaac cgatcgattt gaactggttt cttttttttt ttaattttgg tttggttgct tttttttgtc 70 acccctaata attatatata ataatataaa taaaattatt taccattatt tgtctgagat tttttttaat 140 agaatgatta aaatgatatt gtaaaaaaaa cctaataata ccatactttt caaataatat tttttactat 210 tattagtgat tggtttgctg tcaaagttgt tttttttttt tttactattc ttaggagttt gtttctttta 280 ccctagtcta caggagtttg ttagttacta tcatttcttt aaaaaggaaa ctcatatgga aaaggaaaaa 350 ttgattaaat acaaaaaatt ataaaattac atagagtttt tatttatttg aacgattgag tttaatttta 420 acttaataaa atataattaa ttacaggtaa aacaagtact tatcaatcat tataagtata ttataaaaca 490 tattaattat gagttcagca aagatttgtg ctgatttctt gtctcttcta aactacatgt gacaagatag 560 aaaaaacatc taaatgctaa tgattcttta atatatgact atgcaagtca tttatcttat ttaaatacat 630 taatttaaat caaacttaat tttaaattat tggattctaa tataattgtg ttttaaaaca cttaggtagc 700 ttccttgttg gacccgaaac tggttcatga actgaaataa tctatgcgaa taacgttttc ccacaaaaag 770 aagaacgact tgctttttta gcgacaatca tgcctccttc gacctcaccg atgacaccac ctgtgagtgc 840 tgtttgccag taacatcacc tccttgtccc tatgtgtata tagaaagaca aacttgccaa gcataaaaaa 910 gaagaagaag aagtcatact atatatttcc tgccttcctt ctcgacgata tttctctatc tgaagcaagc 980 accatggtag atcta 995 - 34 - aagtctactc ttatttttca acttttctca agacattgaa ttagtaaacc aatgttttac gaattggata 1820 cgaaaccttc caaaataata tatatatata tatatatata tatatatata tatatatata tatatatata 1890 tatatatata tatatatata aagagggagg gagggggtgg gggaggtcac aaaaaacctg tatataaagc 1960 cccgtaatat ctttctcagc ttagcaacat ctgaaagttg caattaatca gtggtgtgta ctgtgatgca 2030 cacaatacaa tacataccat agacacaaac acaaaaatct gcatccatgg a 2081 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 10 <211> LENGTH: 995 <212> TYPE: DNA <213> ORGANISM: Populus sp. <220> FEATURE: <221> NAME/KEY: promoter 15 <222> LOCATION: (1)...(995) <223> OTHER INFORMATION: sinapyl alcohol dehydrogenase (SAD) promoter <400> SEQUENCE: 9 20 25 30 35 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10

6469749_1 (GHMallers) P62336.AU.5 KAROLA

ο (N Ο Ο (Ν Ο (Ν Ο (Ν 10 15 20 25 - 35 - <211> LENGTH: 1269 <212> ΤΥΡΕ: DNA <213> ORGANISM: Populus sp. <220> FEATURE: <221> ΝΑΜΕ/ΚΕΥ: promoter <222> LOCATION: (1)...(1269) <223> OTHER INFORMATION: UDP-D-glucuronate carboxy-lyase (UDP) promoter <400> SEQUENCE: 10 ggaaatgtca acacttgtgt gaccacacgc acactgtaga cgctacctta cctggccaga ccccgtcgcc 70 cagggattac aatttaattt gaatttgata atatcatctc aactaacttg aatgaatatt ctttttttaa 140 cagttgtatt gcttcatgga aaataaatat tgtatatatt aggatattta atttgaaata aatattatca 210 aatatgactc aaaacccagt ctaatatatt tatattttga atatgataca atataaacct ttttagtatt 280 aacataatgc atgtgttgaa taaatatttt tttttattaa ataataaata tggattgaat gtcgaaaaga 350 gaaataaata gtgtactcat agttacccca tgtacaagtt gagtacaaca acagatgtag tcaaaataaa 420 agaaaactcg gtctgacgtg tcgttaccat tactgtcatt ggacagtaaa gtctttcgat tgtaacagaa 490 catgttctcc ttctctctgg ccagtaacga ccgcgaatta cgcttcctcg aaatttcaat ctaaccttga 560 acactatata agtatatgcc ctgtctctca tcatccgctg tccttaaatc ccttcaaaat actacaacaa 630 aatatttttt tccctcaatt tatttcagca gcaaaagtct acgtggtaat taaatctcaa tttccattcg 700 tttttatagg gatttttggt tgtctggaga aaaaaataat ggtcatggga ttgagagatt ttgagattca 770 gatctgaagt ttgtttttaa ttttttcaat aactggtggg gtatggtttt tcgttgattt gaagcattgt 840 acatttcgtg tttttgaagt ctcatttaat ttatgcgtcc ctccttttct ctctcactag ctggtgttgt 910 ttgttggtgt gtttattatc atgattagtt gttaaccatc tattttttaa tctaatttgg ttacaatcga 980 gttctttata taaagctgta gtctttgagt ttcatgactc gcagcgaaaa aagtttgaga ttttgactct 1050 attttttcac accactcagg tgaactggat ttattatcat gtttttaatt gaaacttgtt ggctggtttg 1120 atttaaggtt tttgatttgt gggttattta tgaatgtgag gattatgcaa tgttttgttt ctgggttgtt 1190 tttacaattt atggtggatt gatttttttt tttaattttc atgattttca gaaattggac aagaatgtca 1260 gatctgata 1269 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 1025 <212> TYPE: DNA <213> ORGANISM: Populus sp. 35 <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(1025)

6469749_1 (GHMallets) P62336.AU.5 KAROLA

Ο (N - 36 - <223> OTHER INFORMATION: lipid transfer protein (LTP) promoter <400> SEQUENCE: 11

O ’d" (No (N O (N 10 15 gaattcgatt acgatgaaat gaagaactga tagcataatc aatcagaaga ttgataatta ttcaaaataa 70 tttttcgaac aatattcaat gcatgatgat tatatgtcgg atcaataaat aatcaattta atgtaaaaaa 140 ggggtactta agtaaataat aataataata ataatgaatg ccttagcatc taaaattcgc tatttttaga 210 agaatcacat tccaagcttc atgaacaatc taatgttcaa tgacatttga tatttttaat aattcaagaa 280 tctcaacaat acaagaatca ttggcatcgc aagatatttt ccctaagcaa gctctaaaat ccccgtacaa 350 aacatccttt aaggtatata tattagttcg aaaataatta tgtgttaatc ttcatgtgca gtggtgagta 420 tttcggccat tcaggcgggt gacccgggat cgttccccag caacggcgtc agttttaatt tttatgtttt 490 cttgaaagtt ttcttaattc ttggcgctgg ctttttgggt ggaaggaacg cggtgttgcg aaaggtaatg 560 gccactaatt gggcaagata atggcatgtc tgtgttgcgg tagttggctc aaaggggagc tttgtggtgg 630 tggtaatatt ggagttctag tcttctagag acccactgag atggctggat aatgagcttc aagggttaat 700 tttgcgctgt cattaaaatg gtaacatctg gatatatgca atggaatggg atgatatggc acccaaatca 770 ccaacctttg attggactgg aaagaactat aatttacaac actaattttc taaagccaag tgctgcaata 840 atatcaactt gtctcttgtt gtagtgctag ccccattttg attagtggac tgggcatcga gttgaggttc 910 atcttgcagt ataaaagctg tccataggag taggagcatt gcattcccat acagcaagaa aatcaatttg 980 ttcatatata tagttgagat acagaaatat ggaggctcca gatct 1025 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <21I> LENGTH: 2341

<212> TYPE: DNA <213> ORGANISM: Populus sp. 25 <220> FEATURE: <221> NAME/KEY: promoter <222> LOCATION: (1)...(2341) <223> OTHER INFORMATION: ag-13 (AG13) promoter <400> SEQUENCE: 12 30 35 gaattcgcat ccatgcggtg agttcgcatt ggtttgatcc aagtggaaca tttccatacc cacaccccca 70 ttagcataac aatcctttat taaaccacta gctagacatg caagattcaa cctacacaca agaacccact 140 agatagactt ccactggaac catgcagcat tctcccgtga tgacctcatt actcagtctt ttctactggg 210 gtttctgttt caaccttctc ctctgtttca acaggcttct gttcttcctt ttcttcttct tcctttgggg 280 cttcgactgc aacctccgct tcttctgccg gtgcctcacc aggccctgta gtctctttag cctcctcgac 350 aacaggctct acgggtatat ccggctcctc ttttgtctcc tcaacaaccg gctctggtgt ttccttaggt 420 gtctcctcct cagttttctc tagtaccgtt ggctcttctg cagcgatctt ggtctcttcg agcacttctt 490

6469749_1 (GHMallets) P62336.AU.5 KAROLA - 37 - i-H O (N tagtttcagc ttcagctggg gcctcgggct ctggtgccac gggctcctca gatgctgcaa ctttctctgc 560 δ' ttcttttggc tcttcatgag ttactgcctc tggtgctgca gtgaccgctt cttctgtggt ggtctcaacc 630 ttgattggtt gttcattttt ttcctctaca agtgcattct gcgctgacac aacctgcagg atacgttatt 700 aaaagaaaag aatgttcacc aaaatgctga tgaggtctta ccatttgtta tatatataga gatgaatata 770 o 5 cgaattttca aatatgaaca tccacgaatt aaagatcata attaagatgg aggtgttgat cttgatgtac 840 attccatcag cataaaactt atcagagtta tatatataaa tatatttaat gacttggaag aagtaataga 910 o 'sl- tgaaatctgt taaataaact tctcaagagg gagattaaat cattcttagt gaatgagtta cctcaacagt 980 (N ggccattgga actagaagga aaataaagca cagctgggat gcaaaagaaa actgtaagaa gcaaaaaggt 1050 o (N acgttggagt aattatcaca gaagaggatg aagaaattgc tttgagtatt tgatgcagag tactgatgaa 1120 10 cgagggtgga tttatataga gatgtagggg gctcactcga gcgagggagg gagtgagtga gagaagagag 1190 O ctaccgtccg aggaatcttg ggatctgaca ccatagctga tgtcattaaa gaattgttgg aagtgaattc 1260 (N ctttttagaa tattttttat ttataaatat attataataa tattttttat tttttaaaat ttattttgat 1330 atatgtatat taaaaagaat aaaaataaaa attaaatttt aacaaatctc catttgggca cacgatttaa 1400 tttgaaaagg ctaaaataat ggaggccatt ttcatcttag ccatcatctt cttttggtcg cgtgtgctga 1470 15 tgtgctttgt gcagtcggtc atgtaggtga ttatcatcca ttcatgttct caacttgcca ttcgtcatta 1540 acaactcctc cctttttttt cttttttttt taaggataaa tgaattaatt ttttaagaaa ataatgaaaa 1610 taatttgtca aaaattttag aaataaaaaa ttccaacaat gctgggtcac taaaattatt aataatattt 1680 aagaaataaa agcaattgac caaaagaact ttcaaaaaaa gctatcttta tttttttttt taatatttct 1750 caatatttgc ttgcactata aactagtact gtgattttct catgttaaat aataataata ataataataa 1820 20 tcacccttaa ccaataggca taatttactt caaacaagcg aataaaactc tgacgtggaa atttaagttg 1890 gtcccacgct ctctctcggc cattgcttta tcaattatgg tatttcataa aaaatttaat tttttttaaa 1960 tagttttaat atattaatat taaaaataat ttttaaaata aaaaatatta ttttaatata tctttaaatt 2030 aaaactactt taataaacaa gctatcacat tatcaaacgc tatttaaagt cggcggatcc cacgagatgc 2100 agggatagca acattagtgt aggactggat cagctgagct ggagctggtg gacggccatg tccacggatt 2170 25 tcgtcgctgt cgattacgtg tcaacagttt ttttttatat tattttcttc tacttttcca gatggatcca 2240 agcctccaag aacgaaacat tggctacagt ttgaaaactc ttaaaaatgt taagattaat aagattagca 2310 gcatcatatt aagtcaagga atgtcagatc t 2341 <200> SEQUENCE CHARACTERISTICS: 30 <210> SEQ ID NO 13

<21I> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oligonucleotide 35 <400> SEQUENCE: 13 5'- GCCATAGCTC CTTAAGAGAA ACAGAAAGCA A -3'

6469749_1 (GHMatlers) P62336.AU.5 KAROLA

Ο (N - 38 - Ο (Ν Ο (Ν Ο (Ν 10 15 <200> SEQUENCE CHARACTERISTICS: <2Ι0> SEQ ID NO 14 <2ΙΙ> LENGTH: 32 <2Ι2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oligonucleotide <400> SEQUENCE: 14 5'- CAATATAGAA TCAATGAACA GCACTAGTTT GC -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 15 <2II> LENGTH: 20 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 15 5'- TCATGTCCTA TCCAACGGCG - 3' <200> SEQUENCE CHARACTERISTICS: 20 <2I0> SEQ ID NO 16

<2II> LENGTH: 24 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide 25 <400> SEQUENCE: 16 5'- CTCATTTTCT CTCAAAGCTC AAAG -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 17 30 <2II> LENGTH: 30

<2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 17 35 5'- GACAACTAGT CTAAAGTTAA AACTTAGACC -3' <200> SEQUENCE CHARACTERISTICS:

6469749_1 (GHMatters) P62336.AU.5 KAROLA 30 35

Ο (N - 39 - Ο (Ν Ο (Ν Ο (Ν 10 15 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> ΤΥΡΕ: DNA <213> ORGANISM/SOURCE: synthetic <221> ΝΑΜΕ/ΚΕΥ: primer/oligonucleotide <400> SEQUENCE: 18 5'- CCCTGGAGGT TGGGGTGAGT - 3' <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 19 5'- GCGTTCATCT ACAAAACCCT CCTCC -3' <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 20 <211> LENGTH: 23

<212> TYPE: DNA <213> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 20 25 5'- TTCATCCTTA TTTTTTTGGG ATA -3' <200> SEQUENCE CHARACTERISTICS:

<210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 21 5'- CAAAGGATCA TGGAGTTGGA - 3' <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 22

6469749_1 (GHMatters) P62336.AU.5 KAROLA

Ο (N - 40 - Ο (Ν Ο (Ν Ο (Ν 10 15 <211> LENGTH: 34 <212> ΤΥΡΕ: DNA <213> ORGANISM/SOURCE: synthetic <221> ΝΑΜΕ/ΚΕΥ: primer/oligonucleotide <400> SEQUENCE: 22 5'- TATACTAATA TGACCTAATA ACTTAGAAGT GTGG -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 23 <2II> LENGTH: 22 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 23 5'- CATCTTGATC AAGATTGAAT TC -3' <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 24 <2II> LENGTH: 20 2 0 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 24 5 ' - CATAATATCA AAACTTAAGC - 3 ' 25 <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 25 <2II> LENGTH: 33 <2I2> TYPE: DNA 30 <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 25 5'- TGAATTGATG ACGTAGGAAA CATGATAAAC ATG -3' 35 <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 26 <2II> LENGTH: 28 6469749_1 (GHMatters) P62336.AU.5 KAROLA

Ο (N - 41 -

o (N O (N O (N

<212> ΤΥΡΕ: DNA <213> ORGANISM/SOURCE: synthetic <221> ΝΑΜΕ/ΚΕΥ: primer/oligonucleotide <400> SEQUENCE: 26 5'- CATTTTCTTG AAACAATGAG GCTAAGAG -3' <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 27 <2II> LENGTH: 29 10 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 27 5'- GACATGAGAA ACTAACGTTG CTTGAATTC -3' 15 <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 28 <2II> LENGTH: 33 <2I2> TYPE: DNA 20 <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 28 5'- CATAATATTG GAACTGGTTT CTTTGTCAGA AAG -3' 25 <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 29 <2II> LENGTH: 25 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic 30 <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 29 5'- GCGCTCGGGT TGTCACCATA GTTTC -3' <200> SEQUENCE CHARACTERISTICS: 35 <2I0> SEQ ID NO 30

<2II> LENGTH: 26 <2I2> TYPE: DNA 6469749_1 (GHMatters) P62336.AU.5 KAROLA

Ο (N - 42 - Ο (Ν Ο (Ν Ο (Ν 10 <213> ORGANISM/SOURCE: synthetic <221> ΝΑΜΕ/ΚΕΥ: primer/oligonucleotide <400> SEQUENCE: 30 5'- CATGTTGTTA TATTTAGATA AATGTA -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 31 <2II> LENGTH: 29 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 31 5'- TTCATCAAGC AATAATAATA AGGTGAGGC -3' 15 <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 32 <2II> LENGTH: 26 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic 20 <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 32 5'- CATGGATGCA GATTTTTGTG TTTGTG -3' <200> SEQUENCE CHARACTERISTICS: 25 <2I0> SEQ ID NO 33

<2II> LENGTH: 26 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide 30 <400> SEQUENCE: 33 5'- TTCAGTGAAC ATGCTGCCAC AATGAC - 3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 34 35 <2II> LENGTH: 26

<2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic

6469749_1 (GHMatters) P62336.AU.5 KAROLA 35

Ο (N - 43 - Ο (Ν Ο (Ν Ο (Ν 10 <221> ΝΑΜΕ/ΚΕΥ: primer/oligonucleotide <400> SEQUENCE: 34 5'- AATCGAAACC GATCGATTTG AACTGG -3' <200> SEQUENCE CHARACTERISTICS: <2Ι0> SEQ ID NO 35 <2ΙΙ> LENGTH: 21 <2Ι2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oligonucleotide <400> SEQUENCE: 35 5'- CATGGTGCTT GCTTCAGATA G -3' <200> SEQUENCE CHARACTERISTICS: 15 <2I0> SEQ ID NO 36

<2II> LENGTH: 28 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oligonucleotide 20 <400> SEQUENCE: 36 5'- GGAAATGTCA ACACTTGTGT GACCACAC -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 37 25 <2II> LENGTH: 23

<2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oligonucleotide <400> SEQUENCE: 37 30 5'- GACATTCTTG TCCAATTTCT GAA -3' <200> SEQUENCE CHARACTERISTICS:

<2I0> SEQ ID NO 38 <2II> LENGTH: 24 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <221> NAME/KEY: primer/oligonucleotide

6469749_1 (GHMatters) P62336.AU.5 KAROLA

Ο (N - 44 - Ο (Ν Ο (Ν Ο (Ν <400> SEQUENCE: 38 5'- GGAGCCTCCA TATTTCTGTA TCTC -3' <200> SEQUENCE CHARACTERISTICS: 5 <2Ι0> SEQ ID NO 39 <2ΙΙ> LENGTH: 28 <2Ι2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oligonucleotide 10 <400> SEQUENCE: 39 5'- CAAGACGATG AAATGAAGAA CTGATAGC -3' <200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 40 15 <2II> LENGTH: 26 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 40 2 0 5'- GACATTCCTT GACTTAATAT GATGCT -3'

<200> SEQUENCE CHARACTERISTICS: <2I0> SEQ ID NO 41 <2II> LENGTH: 26 25 <2I2> TYPE: DNA <2I3> ORGANISM/SOURCE: synthetic <22I> NAME/KEY: primer/oiigonucieotide <400> SEQUENCE: 41 5'- GAATTCGCAT CCATGCGGTG AGTTCG -3' 30 25697179.I I

6469749_1 (GHMatters) P62336.AU.5 KAROLA

Claims (15)

1. The claims defining the invention are as follows:

   1. An expression vector comprising: (i) an isolated nucleic acid molecule that comprises a nucleotide sequence that is capable of initiating transcription of a gene in a cambium tissue-preferred manner, wherein said isolated nucleic acid molecule comprises: a nucleotide sequence as set forth in SEQ ID NO.:5; (b) at least 20 contiguous nucleotides of a nucleotide sequence set forth in SEQ ID NO.:5 or a complement thereof; (c) a nucleotide sequence that has greater than 84% sequence identity to a nucleotide sequence set forth in SEQ ID NO.:5; or (d) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence set forth in SEQ ID NO.:5 or a complement thereof, and (ii) a nucleic acid molecule which encodes a protein of interest, wherein (i) and (ii) are in operable linkage, wherein (i) does not normally regulate (ii).
2. 2. An expression vector according to claim 1, wherein said expression vector is a plasmid.
3. 3. A method of making a protein encoded by an expression vector according to claim 1 or claim 2, comprising transforming or transfecting a cell with said expression vector, and culturing said cell under conditions favorable for the expression of said protein .
4. 4. A recombinant host cell, wherein said recombinant host cell is transformed or transfected with an expression vector according to claim 1 or claim 2.
5. 5. A recombinant host cell according to claim 4, wherein said expression vector is stably incorporated in said recombinant host cell's genome.
6. 6. A recombinant host cell according to claim 4 or claim 5, wherein said recombinant host cell is a plant cell.
7. 7. A method of making a recombinant host cell, said method comprising transforming or transfecting a cell with an expression vector according to claim 1 or claim 2.
8. 8. A method according to claim 7, wherein said recombinant host cell is a plant cell.
9. 9. A method for making a protein, said method comprising culturing a plant or plant part which comprises a recombinant host cell according to claim 6 or claim 8, under conditions favoring production of said protein by said plant or plant part.
10. 10. A plant or plant part comprising a recombinant plant cell according to claim 6 or claim 8.
11. 11. A method according to claim 9 or a plant part according to claim 10, wherein said plant part is selected from the group consisting of a root, a stem, a leaf, a flower, a fruit, a seed, a pistil, a stigma, a style, an ovary, an ovule, an stamen, an anther, and a filament.
12. 12. A method according to claim 9 or a plant part according to claim 10, wherein said plant is a dicot.
13. 13. A method or plant according to claim 12, wherein said dicot is Eucalyptus.
14. 14. A method according to claim 9 or a plant according to claim 10, wherein said plant is a monocot.
15. 15. A method according to claim 9 or a plant according to claim 10, wherein said plant is a gymnosperm.