CA2336706A1 - Bending and/or leaning-induced plant promoter - Google Patents

Abstract

The present invention concerns the use of a promoter with SEQ.ID.NO.1 or with SEQ.ID.NO.2 or a functional fragment thereof, to obtain induction of gene expression by several kinds of mechanical stress, such as bending, elongation, compression, leaning, agitation or gravity and the like.

Description

Bending andlor leaning-induced plant promoter The present invention relates to the use of a promoter inducible by bending andlor leaning stress and is in addition very useful in the control of the synthesis and/or degradation of reaction wood. In poplar trees, said promoter is very useful in the control of the synthesis and/or degradation of tension wood.
Tension wood and compression wood are categories of the so called reaction wood.
Reaction wood has distinct anatomical, chemical, physical and mechanical characteristics; it is typically formed in bent twigs and branches as a reaction to the plant on the stress that is applied.
In gymnosperms, the reaction wood is generally formed at the ventral (lower, compressed) site of the bent twigs and branches. This tissue is exerting a longitudinal pressure that aims to raise up the spindle. This so called compression wood is typical for conifers.
In arborescent dicotyledons, the reaction wood is called tension wood.
Contrary to the compression wood, it is generally formed at the dorsal (upper, tensed) side of the twigs and branches. This tissue exerts a longitudinal traction that aims to reorient the spindle to its original direction. The fibres in this tissue are characterised by a shortage in lignification and, in most cases, by the presence of a gelatinous cellulose layer.
Although present in most trees, the formation of reaction wood can be far more pronounced in some species than in others. In poplar, which seems to be rather sensitive to bending stress, tension wood can even be found in straight trees and straight branches.
The presence of tension wood in trees causes several problems, both at harvesting (splitting) and during further processing (irreversible damage during drying, difficulties during sawing and veneer production, deformed wood surface, paper pulp CONFIRMATION COPY

of low quality). A high proportion of tension wood may make the wood unusable for other purposes than burning.
The current invention is intended to solve the above problem. Depending on the nature of the problem to be solved the production of tension wood can be accumulated or diminished according to the need to have more or less tension wood in a plant or tree. Thus the current invention concerns (plant) promoters inducible upon mechanical stress such as, but not limited to, bending and/or leaning at for instance the stem of a plant or tree. Said promoters, preferably plant promoters comprising the sequences as indicated in SEQ.ID.N0.1 and/or SEQ.ID.NO. 2 or functional parts thereof, can be used for instance in a recombinant gene construct comprising said promoter sequence in addition to coding sequences which are able (if present in a plant or tree cell) upon induction to provide an enhanced protection in the plant or tree against mechanical stress. As an example for said coding sequences can be used those genes coding for proteins involved in the synthesis and/or degradation of cell wall components in order to obtain more or less rigid cell wall structures. Alternatively coding sequences can be used in such a construct wherein the expression of those sequences as a consequence thereof is able to change, positively or negatively, the formation and/or content of tension wood in plants or trees in order to obtain more or less rigid stems of plants and trees, if required.
This can for instance be achieved by expression of the gene in another cell or tissue type by using cell or tissue specific promoters in conjunction with specific (such as consensus sequences) sequences of the bending and/or leaning inducible promoters as described in this invention.
The present invention thus concerns the use of a promoter comprising SEQ.ID.N0.1 or SEQ.ID.N0.2 or a promoter with at least 80% homology to said sequence or a functional fragment thereof, to obtain induction of gene expression by several kinds of mechanical stress, such as bending, leaning, elongation, compression, agitation or gravity and the like.

Another aspect of this invention is the use of eukaryotic cells transformed with a recombinant gene placed under the control of said promoter to obtain gene expression upon mechanical stress.
To the invention also belongs as indicated above a method to control the synthesis and/or degradation of tension and/or compression wood. This is, for instance, achieved by placing a gene that can modify cell wall biosynthesis under the control of said promoter. Such a gene can be, as a non limitative example, a gene that is directly involved in the control of lignin, cellulose or hemicellulose biosynthesis, placed in sense or in anti-sense orieritation after said promoter; it can be a fragment of a gene involved in the control of lignin, cellulose or hemicellulose biosynthesis and/or degradation, placed in anti-sense orientation, to make anti-sense RNA, or alternatively it may be a gene that is able to modulate the levels of intermediates of the lignin, cellulose or hemicellulose biosynthesis.
Definitions The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
"Gene expression": any sequence of events that results in the synthesis of RNA
starting from DNA, known as "transcription" to the people skilled in the art, regardless if the resulting RNA is further translated into protein.
"Functional fragment" (of a promoter): any fragment or combination of at least two fragments derived from a promoter, that still can induce gene expression upon mechanical stress, regardless if this fragment or combination of fragments is used alone or in combination with another promoter and/or one or more fragments of one or more other promoters.
The term "fragment of a promoter (sequence) " means a truncated sequence of the originaE sequence referred to. The truncated sequence can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least WO 00/06752 PC'T/EP99/05490 a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or functions) of the original sequence.
"Mechanical stress": any stress induced by physical means such as compression, stretching, bending, leaning, agitation and/or gravity as non limiting examples.
With "bending" is meant the process of performing an external force at the stem at a certain location at said stem, whereupon as a result thereof the stem bends at the place of the external force. Subsequently the stem continues to grow at a normal original upright position.
With °leaning" is meant the. process of bringing the whole plant and the stem respectively under a certain angle (for instance 45°) as a whole.
Subsequently the stem continues to grow at a normal original upright position.
The terms "gene(s)", "polynucleotide", "nucleic acid sequence", "nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.
Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occuring nucleotides with an analog.
"Homology" means that the respective nucleic acid molecules or sequences are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described and that are derivatives of said nucleic acid molecules are, for example, variations of said nucleic acid molecules which represent modifications having the same biological function. They may be naturally occurring variations, such as sequences from other plant varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants.
The term "promoter" refers to regulatory DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated.
Alternatively the term "promoter" refers to a region located upstream or downstream from the start of transcription and which is involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells.
The term "plant" includes whole plants, plant organs (e.g. leaves, stems, flowers, roots etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the current invention includes higher plants, angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid and haploid.
The invention is further explained by some non-limiting examples.
Examples Expression of the caffeoyl-CoA-O-methyltransferase (CCoAOMT) genes in tension wood To study whether expression of the CCoAOMT genes is involved in the formation of tension wood in poplar, 3-month-old transgenic poplar stems transformed with a PCCoAOMT ~3-glucuronidase (GUS) reporter gene were bent and fixed mechanically.
Poplar plants used in stable transformation experiments corresponded to the INRA
clone 717-1 B4 (Populus tremula x Populus alba).
The chimeric promoter-GUS constructs (pBINPOPI and p8INPOP2, of which the promoter sequences itself, designated gPtCCoAOMT1 and gPtCCoAOMT2, respectively are provided in SEQ.ID.N0.1 and 2) were transferred to poplar, WO 00/06752 PC'T/EP99/05490 following the approach described by Lepl~ et al. (1992). In vitro plants were maintained on half MS medium at 24 °C with a photo-period of 16 h light and 8 h darkness. Two-month old plants were transferred to a greenhouse. The poplars were grown in the greenhouse at 21 °C with the same light cycle.
Reaction wood was induced by bending the young stems (at the internode 3 or 4 counted from the top) of 3-month-old transgenic plants, to a 90 ° angle for various periods. Alternatively reaction wood was induced by leaning the plants at a 45°
angle. The xylem and phloem both from the upper and lower part of the bent area as well as the pith were collected and frozen immediately into liquid nitrogen.
The control samples were extracted from the corresponding position of vegetatively propagated plant of the same transgenic lines that were not bent.
GUS staining and quantitative ffuorimetric assays were conducted on six independent tines for each transformant.
Histochemical staining for GUS activity was performed according to Jefferson et al.
(1987). Stems from transgenic plants were sectioned with a vibroslicer (Laborimpex, Brussels, Belgium), and fixed in 3% glutaraldehyde in 100 mM potassium phosphate buffer, pH 7.0, for 30 min at room temperature. GUS staining was carried out by incubating sections or samples with 2 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), 0.1 mM K3Fe(CN)6, and 0.1 mM K,Fe(CN)6. 3H20 in the same phosphate buffer. Staining was allowed to proceed at 37 °C until blue stain developed in the samples (1 to 4 h).
GUS activity was assayed by preparing crude plant extracts from stem tissue, and were assayed as described by Jefferson et al.(1987). The extracts were standardised by measuring the protein concentration using the method of Bradford (1976). GUS activity levels were assayed by enzymatic conversion of 4-methylumbelliferyl glucuronide to 4-methylumbelliferone which was quantified with a fluorimeter (365 nm excitation and 455 nm emission wavelengths). GUS activity is expressed as GUS U(unit)/h per Ng protein.
In the section of the stem which was bent for 5 days, histochemical assays showed that the GUS activity driven by the gPtCCoAOMT1 promoter was significantly induced in xylem fibres and phloem fibres for which no GUS activity was observed in the non-bent condition (Fig. 1 a). As shown in Figure 1 b and 1 c, when stems were bent for 9 days, GUS activity conferred by both promoters was dramatically induced in pith tissue where no GUS activity was detected in the non-bent plant.
Lignin was revealed by staining with phloroglucinol-HCI (P-HCI) according to Speer (1987). Sections and samples were incubated for 2 min in phloroglucinol solution (1 % in ethanol/water 92/8 v/v), then mounted in 25% HCI.
P-HCL staining revealed that these pith cells were signifying. Under higher magnification, GUS staining was visible in the differentiating xylem fibres, vessels and ray cells (Fig. 2). Figure 3 shows the levels of GUS activity measured in stems which were bent for 9 days. In the xylem tissue, GUS activity was increased from 0.141 U/h per Ng protein to 0.524 U/h per pg protein, whereas in the phloem tissues GUS activity was induced from 0.024 U/h per Ng protein to 0.132 U/h per Ng protein.
Particularly, in pith tissue where GUS activity is barely detectable in the non-bent condition, the GUS activity was elevated more than 10-fold by bending. When stems were bent for one month, P-HCI staining revealed the formation of special tension wood tissues (Fig. 4). The GUS staining was more intense in the differentiating xylem of the upper side compared to the lower side of the leaning stem (Fig. 5).
Expression of CCoAOMT protein is altered upon bending and leaning To confirm the induced expression of CCoAOMT during the formation of tension wood under stress conditions, two kinds of stress were applied to poplar stems. In a first experiment, the stem was mechanically bent at an angle of 90 degrees for 9 days. In a second experiment, the plant was leaned at 45 degrees for 9 days. Subsequently, immunolocalisations were carried out on sections from both stem pieces harvested from the site of bending or leaning stems. These immunolocalisations were carried out with polyclonal antibodies against alfalfa CCoAOMT (Kersey ef al., 1999). The results show that upon bending and leaning, the cell-specific expression was altered as compared to the non-stressed condition.
As shown in Figure 6, upon bending and leaning, CCoAOMT was significantly induced in all cell types of differentiating xylem with strong expression in the ray cells. Together with the promoter-GUS assay, these data demonstrate that the expression of CCoAOMT is regulated at the transcriptional level.
A 456 by fragment of the gPtCCoAOMTI promoter and a 497 by fragment of the gPtCCoAOMT2 promoter are sufficient to induce gene expression upon bending and leaning In order to localize promoter regions potentially involved in the transcriptional control of the CCoAOMT genes in response to abiotic stresses, a series of promoter deletions were generated by removing part of the 5' flanking sequences of both CCoAOMT genes (Fig. 7). Subsequently, their effect on gene expression in transgenic poplar was analyzed. Multiple independent poplar transformants were generated for each construct. The primary transformants were assayed histochemically for GUS expression. GUS staining in each tissue was scored as positive (+) or negative (-). Although some variation in the levels of GUS
activity in each tissue type was observed, there were clear differences in the cell- or tissue-specific GUS expression patterns between the different constructs, as shown in Table 1.
For the deletions of the gPtCCoAOMT1 gene, differences were found in the tissue specific expression between plants transformed with a construct containing the full-length promoter (p8INPOP1 ) and the various promoter deletions (Table 1 ).
As indicated in Table 2, GUS activity was undetectable in the transgenic lines transformed with the pBINIDD1 (-114) promoter-GUS construct, which corresponds to a promoter fragment without TATA box. In the transgenic lines with the deletion p81N1DC2 (-184), no GUS activity could be detected in the xylem tissue.
However, faint GUS staining was detected in the bark tissue including phloem fibres, cortex and periderm. Transgenic lines transformed with the pBIN1D83 (-199} construct, which is only 15 by longer than p8lN1DC2, failed to direct expression in any tissue, indicating that this additional 15 nucleotides, which correspond to an AC-II
element, constitute a cis-negative regulatory element for controlling the expression in bark WO 00/06752 PC'T/EP99/05490 tissue. Transformants containing the p8lN?DA5 (-456) construct, conferred expression in cambial ray initials and in differentiating xylem rays.
For the gPtCCoAOMT2 promoter, the deletion of pBIN2DD? (-110) and pBIN2DB3 (-195) resulted in a complete loss of gene expression. PBIN2DA4 (-497) however directed GUS activity in the phloem fibres and in contact rays associated with vessel, similar to the full-length gPtCCoAOMT2 promoter. This -497 by fragment of the gPtCCoAOMT2 promoter is thus sufficient to control the cell-specific expression whereas in addition it shows that cis-acting positive regulatory elements, which control the expression in xylem vessels and in adjacent ray cells and in phloem fibres, are located between nucleotides -497 and -195.
Subsequently, the transgenic poplars containing the promoter deletions were analyzed upon mechanical bending and leaning. GUS staining revealed that pBIN?DA5(-456) and pBIN2DA4(-497) were significantly induced in all xylem cells types and strongest in ray cells in response to mechanical bending. The other deletions did not shown any GUS activity upon bending stress. Thus, the region -456 to -199 of the promoter of gPtCCoAOMT1 and the region -X97 to -195 in the promoter of gPtCCoAOMT2 are necessary and sufficient to drive gene expression upon mechanical bending and contain therefore positive cis-acting regulatory elements to drive gene expression upon bending.
Finally, several consensus regions are identified by aligning the sequences of these two fragments.
Expression of CCoAOMT genes in response to bending and leaning is involved in determining lignin heterogeneity Mechanical stress caused by leaning stems results in compression wood in gymnosperms and tension wood in angiosperms (Timell, 1986; Castera et al.
1994).
Both compression and tension wood have been shown to have an altered lignin content and composition as compared to normal non-stressed wood (Timely 1986;
Rolando et al., 1992). In contrast to the intensive study of lignin biosynthesis enzymes such as 4CL (4-cinnamate-CoA ligase), PAL (phenylalanine ammonia lyase), COMT
(caffeic acid-O-methyltransferase), CAD (cinnamylalcohol dehydrogenase), and cinnamoyl-coenzyme A reductase in compression wood (Kutsuki and Higuchi, 1981;
Popko, 1993; Zhang and Chiang, 1997), little information is available on gene expression in tension wood. In this invention a gene is reported that is induced upon bending in a hardwood species. More specifically the promoter is responsive to signals caused by relevant stress. The expression of both chimeric CCoAOMT genes was upregulated in all lignifying tissues of the stem, 9 days after mechanical bending; there was a pronounced increase in GUS activity in xylem and phloem and in the pith cells concomitantly with the deposition of lignin in the pith. It is noteworthy that in response to mechanical bending and leaning, the cell-specific expression pattern directed by both CCoAOMT promoters in the stem was lost. The immunodetection of CCoAOMT
upon bending and leaning confirms the promoter-GUS data, indicating that induced expression of CCoAOMT is regulated at the transcriptional level. It may be assumed that induced expression of CCoAOMT in xylem tissue probably has an impact on the production of lignin units which are incorporated into the lignin polymer and thus on the lignin heterogeneity in tension wood. It has been shown that lignin for tension wood and normal wood is different (Rolando et al. 1992). Thus the regulation of expression CCoAOMT upon mechanical stresses is involved in at least part of the control of wood quality, during the formation of tension wood.
A Materials and Methods section is disclosed hereafter wherein an explanation is provided for the methods and materials used in the current invention.
Plant material and transformation Poplar plants used in stable transformation experiments corresponded to the INRA clone 717-1B4 (Populus tremula x Populus alba).
The chimeric promoter-GUS constructs were transferred to poplar, following the approach described by Lepl~ et al. (1992). In vitro plants were maintained on half MS medium at 24 °C with a photo-period of 16 h light and 8 h darkness.
Two-month old plants were transferred to a greenhouse. The poplars were grown in the greenhouse at 21 °C with the same light cycle.

Plant bending and leaning Young stems (at the internode 3 or 4 counted from the top) of three-month-old transgenic plants were bent and fixed to a 90 degree angle for various periods.
The xylem and phloem from the bent area as well as the pith were collected and frozen immediately into liquid nitrogen. The control samples were taken from the corresponding position of non-bent plants from the same transgenic lines.
Tension wood was induced by leaning three-month-old poplars to 45 degree for 9-days. The upper part of stem (about 20 cm) grows vertically after leaning the plant.
The samples for immunolocalization and GUS assays were taken from the curved part of the plant, i.e. between the oblique and the vertical part of the stem.
Immunolocalization Stem internodes from control poplar and from poplars that were bent or leaned were cut into 1-mm pieces and fixed in 3% glutaraldehyde for 4 h at room temperature and followed by 14 h at 4 degrees C. The embedding in LR white resin (Polysciences, Waarington, Pa., USA), sectioning, and subsequent immunogold-labeling were carried out as described previously by De Clercq et al. (1990) using a polycfonal antibody against alfalfa CCoAOMT. Semi-thin sections (3~m) for light microscopy were made using an Ultracut microtome (Reichert-Jung) using glass knives. Immunogold silver staining was performed using the manufacturer's protocol (Amersham, Aylesbury, UK). The CCoAOMT antibody was diluted 1:5000.
Plasmid constructions The characterization of gPtCCoAOMTI and gPtCCoAOMT2 has been described previously (Chen et al., 1998). To fuse the 5' UTR sequences of both CCoAOMT genes to the coding sequence of the gus gene, an Ncol site was generated by PCR, three codons downstream of the CCoAOMT start codon by using an oligo complementary to the 5' flanking vector DNA in combination with the 21-mer oligonucleotide 5'-CTCTCCCATGGTGGCCATTAT-3' and 5'-CTCTCCCATGGCGGCCATTAT-3' for gPtCCoAOMT9 and gPtCCoAOMT2, respectively (the single line indicates the Ncol site and the double lines mark the reversed original start codon). By using these oligonucleotides, the 1,994 by and 1,363 by promoter fragments of gPtCCoAOMTI and gPtCCoAOMT2 were generated by PCR, respectively. Subsequently, both PCR products were digested with Ncol and Sacl and cloned into the Ncol/Sacl site of pGUS1 (Peleman et al., 1989) yielding the plasmids pGUSPOP1 and pGUSPOP2. Subsequently, both chimeric PCCoAOMT1-GUS and PCCoAOMT2-GUS genes were isolated from pGUSPOP1 and pGUSPOP2, respectively, by an Xbal digest, and cloned into the Xba! site of the binary vector p81N19 (Bevan, 1984), resulting in the plasmids p8lNPOPI and p8lNPOP2, respectively.

Description of the figures Ficqure 1: (a) Transverse section of a bent stem (B) and a non-bent stem (N) stained for GUS activity for the pBINPOP1 construct (b) Section of bent stems double stained for GUS activity and for lignin using P-HCI for the pBINPOP1 construct (c) same double staining of bent stems for the pBINPOP2 construct.
Figure 2: Staining for GUS activity in the stem section of transgenic poplar transformed with the pBINPOP1 construct (a) Staining for GUS activity of tension stem: the staining was found in all kinds of xylem cells, such as fibres, vessels and ray cells.
(b) Staining for GUS activity in normal, non-bent stem: the staining is localized in xylem vessels and in the adjacent ray cells.
Figure 3: GUS activity in stems of transgenic poplar transformed with the pBINPOP1 construct which were bent for 9 days (a) Comparison of the GUS activity in xylem, phloem and pith for bent and non-bent stems.
(b) Ratio of increase in GUS activity (expressed as x-fold) upon bending.
Fi_ '4ure 4: Formation of tension wood tissue in poplar stem which had been bent for one month, as revealed by P-HCI staining.
Fi ure 5: Staining for GUS activity in non-bent and bent stems of transgenic poplar, transformed with the pBINPOP1 construct.
(a) Longitudinal section of a non-bent stem, stained for GUS activity.
(b) Longitudinal section of a bent stem, after bending for one month double stained for GUS activity and for lignin, using P-HCI.
Fi ure 6: Immunodetection of CCoAOMT in poplar stem.
lJigiure 7: CCoAOMT promoter-GUS constructs (detailed description in WO
99/09188 as published 25 February 1999).

References Jefferson R.A., Kavanagh T.A., and Bevan M.W. {1987). GUS fusions: ~i-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO J. 6, 3901-3907.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of micrograrn quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 72, 248-254.
Speer, E.O. (1987). A method of retaining phloroglucinol proof of lignin.
Stain TechnoL 62, 279280.
Monties, B. (1989} Lignins. In Methods in Plant Biochemistry, Volume 1 (Dey, P.P:
and Harbome, J.B., eds). London: Academic Press, pp. 113-157.
Xing-Hai Zhang and Vincent L. Chiang (1997) Molecular cloning of 4-coumarate:
coenzyme a ligase in loblolly pine and the role of this enzyme in the biosynthesis of lignin in compression wood. Plant Physiol, 113:65-74.
Popko JL (1993) Regulatory enzymes of lignin biosynthesis in norinal and compression wood of loblolly pine. M.S. thesis, Michigan Technological University, Houghton, pp 51-60.
Kutsuki.H and Higuchi.T (1981 ) Activities of some enzymes of lignin formation in reaction wood of Thuja orienfalis, Metasequoia glypfostroboides and Robinia pseudoacacia. Planta 152:365-368.
Timell TE (1986) Compression wood in gymnosperms. Springer-Verlag, Heidelberg, Germany, pp 360-379.
Cote, W.A. (1977) Wood ultrastructure in relation to chemical composition. In:
The structure, Biosynthesis, and Degradation of Wood, (recent advances in Phytochemistry, Vol. 11 ). pp. 1-44. Loewus, F.A. and Runeckels, V.C., Eds., Plenum Press, New York.
Rolando, B. Monties, and C. Lapierre {1992) Methods in Lignin Chemistry (Edited by S.Y. Lin and C.W. Dence) Springer Series in Wood Science, Springer-Verlag Berlin Heidelberg.
Lepl~, J.C., Brasileiro, A.C.M., Michel, M.F., Delmotte, F., and Jouanin, L.
(1992).
Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell Rep. 11, 137-141.

IS
De Clercq A, Vandewiele M, De Rycke R, Van Damme J, Van Montagu M, Krebbers E, Vandekerckhove J. (1990) Expression and processing of an Arabidopsis 2S
albumin in transgenic tobacco. Plant Physiol. 92, 899-907.
R. Kersey, K. Inoue, K. R. Schubert, and R. A. Dixon (1999) Immunolocalization of two lignin O-methyltrasferases in stems of alfalfa (Medicago sativa L.).
PROTOPLASMA.

SEQUENCE LISTING
<110> VLAAMS INTERUNIVERSITAIR INSTITUUT VOOR BIOTECHNOL
<120> BENDING AND/OR LEANING-INDUCED PLANT PROMOTER
<130> V2/002-V027 <190>
<191>
<150> 98202518.1 <151> 1998-07-27 <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 3800 <212> DNA
<213> Populus trichocarpa cv trichobel <220>
<221> promoter <222> (1)..(1999) <220>
<221> CART signal <222> (1843)..(1897) <220>
<221> TATA-signal <222> (1871)..(1876) <220>
<221> misc_signal <222> (1799)..(1806) <223> AC-element II
<400> 1 tctagagttc gtggtttatt tttatatttt taaagtattt aaaaaaaaat aattttttta 60 attttttatt tggtttaaat taattttttt ttgtattttt atattatttt gatgtgatga 120 tataaaaaat aaaattttaa aagtaaccct tgtctaaata caaaataggc tctatatata 180 agccagagga ggtgatattt tgattatttt ctttaagact acagatgacc cgataacatg 240 aaacaaaaat ataaaaacaa ggtcaaccgt gtgacttgtt ccgcccccgt cccgggttcg 300 accctctatg tgcacgcctg tcacccccgc ggtgccttac ctgctcctgg gcttgcagga 360 tgtccagtgg gccgtgggga atagtcgtgg tgcgcgtaag ctggcccgga caccccatgt 920 aaatcaaaaa aaaaaaaaaa aaaaaaacaa ggtcacgcgg gcattcctag cttctctata 480 ggcattttgc tttgagcctc cgaccattcc cccctgctct tttcttattc acgatagaag 590 tctgttcagt taaagaaaac atggcctgtt ttcctagaaa tttatctata aaaaaggaaa 600 ttcttgttaa aataaagaaa aaaattcaat tgaaatacac ttcccagctt tcgaatgttt 660 ctattatatt ttggaaaaca tgtgaaaaat attttattaa tgttcacttg aaaaaatggt 720 tatggttgct tttcaaagtg ttttttactt ggaaatgctt taaaataata tttttttatt 780 ttttaaaaat catttgtgat atcagcgcat caaaatgatt tgaaaacatt aaaaaatatt 840 aatttaaaac aaaaaaataa aataaaataa tttaaatttt tttaaatact tttaaaacat 900 aaaaaaaaaa cagaatagaa tttagacata aaaagtgtta ttctcgtcgt ttaatagcgg 960 gaggtggtaa gaaacatgct aagcattcac agttttggat attgattatc catgtgttac 1020 cctaaaaaat gcctttttcc atttcatgaa atcctcattt aatagcaatt ctttaaagga 1080 ggagactaga gacagaggtg gctgctgtca acctagtcgg tgaatttaaa cttcaaccgg 1140 cacatatatg cataccaagt atacccttta catctgccct aattaagact gtaaaacgga 1200 tttggatttt tgccgacaaa ggctagtttg tggagaaaaa acaacgaaga taaattttta 1260 gatgacaaag tcaacaatag ttcgagagat tctttaaagg actcatccgt tgacggaggt 1320 ggccatatgc taccaactct tggacgtgga gtccctttgg taatttcacc tatccctcac 1380 ccaatttcta ttagcagtta gcacatgtaa tttatgattg gtgagcccag cacaaatctt 1940 ttccagttaa acacatatat taatttatga ttaattattt aattctctcc actcttaaca 1500 aattaatcat acatggcata acattttagc ttttgatctc gagaatctct acctaaccat 1560 tgacttcttt actgttcagg aatcttagaa aaaaggagga caaaaaaaaa tatgcccaat 1620 aaattattta agaatttgaa ccgatatttg gtgtcataga tcccaaaaat gacgccagcg 1680 atgcctaagg gaaggagtac cactagccca cagcacgata cgatcaccaa caaggtgggt 1740 cccatatttg gtgggccaaa aacccacatt atccttcgtc ctaactacag gaacctcacc 1800 aaccccctcc cggttggtag ccggtccagc ctccccgcta ctccaattca aaccgggctc 1860 tcatttccaa taaataccac ccgcccttta ccattttcga tcaggttagg catcactacc 1920 atcatcaaca aaaaaaaaaa aaaaatccaa ggccaagaaa gagatcgtag tttaattaga 1980 agatatacac aataatggcc accaacggag aggaacagca aagccaggca ggaaggcacc 2040 aggaagttgg ccacaagagc cttttgcaaa gtgatgctct ttaccaggta atttaaacgg 2100 taaatccttg ttcttgtgca agttttcccc cttttcttgt tgttgtttgc tttttaacat 2160 tttcttttta tatttggatt attttttagt atattctcga gactagtgtg tatccaagag 2220 agcctgaatg catgaaggag ctcagggagg tgactgccaa gcatccttgg tatgtttctt 2280 gattttcaca tacatttaaa tacataaaca taagagattt tgtctgatat taatgcttat 2340 ggtgtgatgt taataaattt gacgtgaatg atgggaaaca ggaacatcat gaccacatct 2900 gctgatgaag ggcaattctt gaatatgctt ttgaagcttg tcaatgccaa gaacaccatg 2960 gagatcggtg tttacactgg ctattctctc ttggccactg ccctggctat ccctgaggat 2520 ggcaaggtaa actaaaaact taaaatgtct cggtcccaaa tcaaatttta ttaagtaaaa 2580 taaataaata aataaataaa ttcatgtgat gataaaaaat aaatgggatt ttgtttcttg 2690 cagatcttgg ctatggacat caacagagaa aactatgaat tgggtctccc agtaattcag 2700 aaagctggtg ttgcgcacaa gattgatttc aaggaaggcc ctgctctacc agttcttgat 2760 caaatgattg aagatgtaag aaatactcta tgttcgacaa aaaatgaaaa tggaagaggg 2820 aaaaacatcc ttttttgtct acttgtatga gaaaaagaaa cgatgctcgt tttttaactt 2880 gatatataaa ttatatgact attactaatc ttactattct gtgtgggaaa acgcagggga 2940 agtgccatgg aagttttgat ttcatctttg tggatgctga caaggacaat tatataaatt 3000 atcacaagag gttgattgag cttgtaaaag ttggtgggct gattgggtac gacaacactc 3060 tgtggaatgg atctgtggtg gcaccacctg atgcaccaat gaggaagtat gtgaggtact 3120 acagggactt tgttttggag ctcaacaagg cacttgctgc tgaccccagg attgaaattt 3180 gcatgcttcc tgttggtgat ggcatcactc tctgccgtcg gatccaatga gggacctgcc 3240 agtattgtta tctgatgttg accattgaaa tggtcactta caagaacaag ggagatgcaa 3300 tagttgtttt tacccacttt gtatttcaat ggcttataat ttgtgtactt gaacagaatg 3360 gtgtatgatt gagaaattcc tctcttaaat ttctgtaagt ggatttttta tgcacttaat 3420 caatattgtt cggtggctaa atacttgtta gttgttatgc attgctaaga tggagatttc 3480 tcatctatct caggccatca tagtttaacc agtttacaac taaatctcga gaaaggtttg 3590 ttccaattaa gtgttctaga cattatgaat gattgtatct aaaatggttc caaaacttct 3600 aatccgttgg acttcttttt gtgcaaaatg ttttttatgt ttcaagattg ttttgtttag 3660 acggtgagaa aacaagaagc gtgtacgatg tacctactag ttgctaacta gtcactttag 3720 agtttagaga cagacattga ggttgtggcg aggaaaatcc gaaaattatc ttgtaaacaa 3780 gtctaacttt tcacacgaat 3800 <210> 2 <211> 2983 <212> DNA
<213> Populus trichocarpa cv trichobel <220>
<221> TATA signal <222> (1244)..(1249) <220>
<221> promoter <222> (1)..(1363) <220>
<221> misc-signal <222> (1067)..(1179) <223> AC-element II
<900> 2 tctagagaac acggtttcaa ccgcgtttcc aaacatgatt taaatataca cggtatattt 60 ttgcatttca atagggtttt tgaaaaagaa atgaatttta tttatttatt ttgttttaaa 120 ttatttttta gtatttttag atcgttttaa catgtcaatg tcaaaaaaaa ttaagaaaaa 180 tattatttca atttatttgt aagaaaaaaa accttaaaaa acaattatta ccaaaacatc 240 aaactggctc tgaaagtatc tcatagcata atgcactaac caattattta aattttccat 300 cctgtcatgg agaaagattc catggttgaa gactgtatga taaggaaaaa tgtcatgaac 360 tcatggtata agtaatttcc tatccaataa cagcaagctt gatgttaggt tagggttgat 920 ggttgtcttc tttcatggaa atgttttgcc atgcccacac gaaacaggca agaaaaccag 980 acaatattag gaattgtttc aatgtattga tattaaaaat aaattttaaa attaaaaaaa 590 tattatttta atatatttat aaataaaaaa tacttaaaaa aaacatctat tacacttaaa 600 aaaaacatta attaattact gcctagcttt actagaaaat ccacacacta actgggcgat 660 tgaaactcca gccattttta tatatttgtc ctgtgattat catagacggt aaaacgaaat 720 tggatttttt tattttgttg gagaaaaaaa aaagaaaata aatattgtca gcagtaagac 780 ggagagattc ttaaaaggag tcatccattg tcaatgcggt ggctacgagc caccaactcc 840 cgtggagtca aattcttgag gacacctcac caacccctta cccactttct attagcagca 900 catgtagcca tccccaacaa caaagtggtg agcccaccac aattttctac tctctacgat 960 ttaaatcaat tacacgtggc ataaaatgtc gagcctttta tttcaagaaa ccaaacctaa 1020 caccgtgaac ttaatttctt tcgcaaatat ctaaaataag ggtacatgaa ttaaatgtat 1080 agaaattgaa ttagtgtccg aaacctaaaa tgaccactgg acaaacaccg ataagtgggt 1190 cccaaaatac ccacggtgtc ctaagaactc accaaccccc acccggttgg aagccggtcc 1200 aaccacccca ctactccggc tcaaaccgga ctctcatctc caataaatac cacctgccct 1260 tgccattttc aatcaggtca gacatcctta ccatcgtcgc ccccagaaaa accttccaac 1320 gccaggaaag agagtatagt tttgttataa gatatacaaa ataatggccg ccaacggaga 1380 ggaacagcag actcaggccg gaaggcatca agaagttggc cacaagagcc ttttgcaaag 1490 tgatgctctt taccaggtaa tttaaccgag aaacccctga tcttggtgcg agttttgttt 1500 ttttttcctt ggtttgtttt ttttttaaca ttttttgtat atatttggat tggtttttag 1560 tatattcttg agaccagtgt gtacccaaga gagcctgaat gcatgaagga gcttagagag 1620 ttgactgcca agcatccttg gtatgtttgt tgaatcctca catgcatttt aaatacatca 1680 acatgagaga ttttattttc ataaaaaaaa aaaaagggtt ttgttttcta tatattgatg 1790 tttatggcgt ggtgttaata aatctgatgt gaatgatggg aaacaggaac atcatgacca 1800 catctgctga tgaagggcaa ttcttgaaca tgcttttaaa gcttatcaat gccaagaaca 1860 ccatggagat tggtgttttc actggctatt ctctcttggc cactgctctt gctatccctg 1920 aggatggaaa ggtaaaaact aaagacctga gatttctcgg tcccaaatca gtcaaaagaa 1980 attaggtgac ggtaagaaaa taattgggat cttgtgactt gcagatcttg gctatggaca 2090 tcaacagaga aaactatgaa ctgggtctcc cggtgattca gaaagctggt ctggaacaca 2100 agattgagtt caaggaaggc cctgctctgc cagttctcga tcaaatgatt gaagatgtaa 2160 gaaatattct gtctttgaca aaaaaaaaaa cattttttgt ttgctcgtat gagaaaagag 2220 atgatattca tttaagaaaa ttgatgtgct attactaagc ttactattct gtgtgtggcg 2280 aactacaggg aaagtaccat ggaacttatg acttcatctt tgtggatgct gacaaggaca 2340 attatattaa ctaccacaag aggttgattg agcttgtcaa agtcggaggg ttgattgggt 2400 atgacaacac cctgtggaat ggatctgtgg tggcaccagc cgatgcgcca atgaggaagt 2960 atgtgaggta ctatagggac tttgttctgg agctcaataa ggcacttgca gctgacccca 2520 ggattgaaat ctgcatgctt tcctgttggt gatggtatca ctctctgccg tcggatcaag 2580 tgaggggctg cattccctgc cgatattatg tacctgaaag aatggtgaat ggccgagaaa 2640 ctccattctt gaatttctgt ctaagtggat tttttacgca cttaatccat attgttcttt 2700 aaccaagtac ttatcacgcg gtgctgagaa tgttcatggt ttaaggagtt tgcttcaaca 2760 ttcaaaatta agaagcacgg ggtcccttgc tcccttacga agaccactag atgcggttcc 2820 cattcagtta atggccttaa ggacgagttt atttcctttt cgttctgact ttctgtacat 2880 gcagcccgca ctctcctcgt aaataaaaac tgctaaccgc aagtaaaaga atgtgtatcg 2940 agaaagggct tttgcaagct tggccttgaa ctagcgtgga aaa 2983 <210> 3 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: 21-mer oligonucleotide (for gPtCCoAOMTl) <220>
<221> misc signal <222> (6)..(11) <223> NcoI site WO 00/06752 PC'T/EP99/05490 <220>
<221> misc signal <222> (16)..(18) <223> reversed original start codon <400> 3 ctctcccatg gtggccatta t 21 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: 21-mer oligonucleotide (for gPtCCoAOMT2) <220>
<221> misc signal <222> (6)..(11) <223> NcoI site <220>
<221> misc_signal <222> (16) .(18) <223> reversed original start codon <400> 9 ctctcccatg gcggccatta t 21

Claims (7)

Claims

1. The use of a promoter according to SEQ.ID.NO. 1 or a promoter with at least 80%
homology to said sequence or a functional fragment thereof in the construction of eukaryotic cells transformed with a recombinant gene placed under the control of said promoter to obtain gene expression upon mechanical stress.

2. The use of a promoter according to SEQ.ID.NO. 2 or a promoter with at feast 80%
homology to said sequence or a functional fragment thereof in the construction of eukaryotic cells transformed with a recombinant gene placed under the control of said promoter to obtain gene expression upon mechanical stress.

3. The use of a promoter according to claim 1 or 2 wherein said stress is caused by bending and/or leaning.

4. The use of a promoter according to claim 1 or 2 wherein said stress is caused by compression.

5. The use of a promoter according to claim 1 or 2 wherein said stress is caused by stretching.

6. The use of a promoter according to claim 1-5 to modulate lignin synthesis in plants.

7. The use of a promoter according to claim 6 wherein said plant is a poplar.