Journal of Experimental Botany, Vol. 54, No. 393, pp. 2709±2722, December 2003 DOI: 10.1093/jxb/erg304
RESEARCH PAPER
Transcriptional regulation of secondary growth in Arabidopsis thaliana Sookyung Oh, Sunchung Park and Kyung-Hwan Han* Department of Forestry, 126 Natural Resources, Michigan State University, East Lansing, MI 48824, USA Received 21 April 2003; Accepted 5 September 2003
Abstract Despite its economic and environmental signi®cance, understanding the molecular biology of secondary growth (i.e. wood formation) in tree species has been lagging behind that of primary growth, primarily due to the inherent dif®culties of tree biology. In recent years, Arabidopsis has been shown to express all of the major components of secondary growth. Arabidopsis was induced to undergo secondary growth and the transcriptome pro®le changes were surveyed during secondary growth using 8.3 K Arabidopsis Genome Arrays. Twenty per cent of the ~8300 genes surveyed in this study were differentially regulated in the stems treated for wood formation. Genes of unknown function made up the largest category of the differentially expressed genes, followed by transcription regulation-related genes. Examination of the expression patterns of the genes involved in the sequential events of secondary growth (i.e. cell division, cell expansion, cell wall biosynthesis, ligni®cation, and programmed cell death) identi®ed several key candidate genes for the genetic regulation of secondary growth. In order to gain further insight into the transcriptional regulation of secondary growth, the expression patterns of the genes encoding transcription factors were documented in relation to secondary growth. A computational biology approach was used to identify regulatory cis-elements from the promoter regions of the genes that were up-regulated in wood-forming stems. The expression patterns of many previously unknown genes were established and various existing insights con®rmed. The ®ndings described in this report should add new information that can lead to a greater understanding of the secondary xylem formation process.
Key words: Secondary growth, secondary xylem formation, wood formation.
Introduction Plant growth by means of apical meristems results in the development of sets of primary tissues such as epidermis, vascular bundles, and leaves. In addition to this primary growth, tree species undergo secondary growth and produce the secondary tissue `wood' (secondary xylem) from the vascular cambium (i.e. secondary meristem). The vascular cambium originates from the procambium and normally consists of 5±15 dividing cells. It occurs as a continuous ring of meristem cells that are located between the xylem and the phloem (the so-called `cambial zone') (Larson, 1994; Mauseth, 1998). The transition from procambium to cambium is not clearly understood. On the xylem side of the cambium, the cells ®rst go through stages of differentiation that involve cell division, expansion, maturation, ligni®cation, secondary cell wall thickening, and programmed cell death, in which all cellular processes are terminated (Chaffey, 1999). The growth of vascular cambium increases the diameter (by periclinal divisions) and the circumference (by anticlinal divisions) of an axis. Positional information appears to be required to co-ordinate this development of secondary xylem (Uggla et al., 1996, 1998). To achieve the patterned growth, each cell must express the appropriate sets of genes in a coordinated manner after receiving the necessary positional information. In other words, the control of cambial growth and differentiation is accomplished by changing the activity of key genes involved in developmental pathways. Recently, signi®cant progress has been made in the study of the genes and signalling mechanisms responsible for secondary wall formation, lignin and cellulose biosynth-
* To whom correspondence should be addressed. Fax: +1 517 432 1143. E-mail:
[email protected] Journal of Experimental Botany, Vol. 54, No. 393, ã Society for Experimental Biology 2003; all rights reserved
2710 Oh et al.
esis (Arioli et al., 1998), and xylem development (Fukuda, 1997; Ye, 2002). Secondary growth is one of the most important biological processes on Earth. Its product, wood, is of primary importance to humans as timber for construction, fuelwoods, and wood-pulp for paper manufacturing. It is also the most environmentally cost-effective renewable source of energy. However, despite its economic and environmental signi®cance, secondary growth has received little research interest, mainly because most agricultural products are derived from seeds or roots. Furthermore, the biology of wood formation is surprisingly understudied because of the inherent problems of tree species: long generation time, large size, and lack of genetically pure lines. Study of wood formation at the molecular level using real trees has begun in recent years. A genomics approach has been successfully used to examine global gene expression patterns in developing xylem tissues of black locust (Yang et al., 2003), pine (Allona et al., 1998; Lorenz and Dean, 2002), and poplar (Sterky et al., 1998; Hertzberg et al., 2001). However, current understanding of the molecular mechanisms of wood formation in trees is still limited. Recently, Arabidopsis, the most well-studied herbaceous model species, has been used as a model for the study of wood and ®bre production in trees (Lev-Yadun, 1994; Zhao et al., 2000). When kept from ¯owering by repeated removal of in¯orescences (i.e. decapitation) and grown at a low density, Arabidopsis produces a signi®cant quantity of secondary xylem (i.e. wood) that is suf®cient for various developmental studies (Lev-Yadun, 1994; Beers and Zhao, 2001). Zhao et al. (2000) characterized xylem-speci®c proteases in Arabidopsis. Chaffey et al. (2002) reported wood formation in the hypocotyls of shortday-grown Arabidopsis plants and demonstrated that the secondary xylem tissues produced in their study were structurally similar to those of an angiosperm tree (poplar). The primary objectives of this research were to identify xylogenesis-associated genes and to determine how they are regulated in Arabidopsis thaliana. Wood formation was induced as described by Lev-Yadun (1994). Then, using Arabidopsis Genome GeneChip (8.3K) Arrays, the global gene expression patterns were examined by comparing treatment versus control stems and xylem versus bark tissues, the genes were identi®ed that are differentially regulated for wood formation, and the differentially expressed genes were clustered into several groups based on their expression patterns. The expression pro®les of three types of transcription factors (AUX/IAA, R2R3MYB, and HD-containing) that have previously been shown to regulate the developmental processes involved in secondary growth were also documented. A computational biology approach was then used to identify several cisregulatory elements from the promoter regions of genes whose expression patterns could be associated with wood
formation. Elucidation of any commonality found in those regulatory elements frequently presented in wood formation-associated genes might provide some insight into the genetic regulation of secondary growth. Materials and methods Plant growth and treatment for wood formation in Arabidopsis Arabidopsis thaliana (L.) Heynh. Columbia plants were grown in a greenhouse using Baccto soil under 16/8 h light/dark conditions at 2363 °C. The plants in the wood formation treatment were grown at the density of one plant per 100 cm2 pot. Four weeks after germination, the in¯orescence was removed as previously described (Lev-Yadun, 1994). At that time, most of the plants had about ten rosette leaves and 4±5 cm long in¯orescences. The removal all newly emerging in¯orescences was continued for an additional 5 weeks. In addition, a 4-month-old poplar (Populus deltoides) stem was prepared as sample material to be used for an anatomical comparison of xylem structures found in Arabidopsis plant samples. For use in the control stem, 25 Arabidopsis plants were grown in a 100 cm2 pot. After 3 weeks without any treatment, 3±4 cm long young in¯orescence samples were harvested and used as control stems. Xylem and bark samples were also isolated from the treatment plant as described by Zhao et al. (2000). Brie¯y, about 1 cm of the root±hypocotyl junction region was excised from treatment plants and the lateral roots were trimmed from the primary root using a razor blade (VWR Co., West Chester, PA). Xylem and bark portions were separated by forceps and razor blade, quenched with liquid N2 and stored at ±80 °C until use. Cross-section samples were prepared by ®xation in 3% paraformaldehyde and 1.25% gluteraldehyde solution. After ®xation, the samples were dehydrated in a series of ethanol solutions (25, 50, 75, 95, and 100%), embedded in paraf®n (Sigma-Aldrich Co., St Louis, MO), cut using razor blade (VWR) and stained with 0.025% toluidene blue O. The sliced samples were observed under the microscope (American Optical Instruments, Buffalo, NY). RNA extraction and cDNA synthesis For treatment stem, control stem, xylem tissue, and bark tissue samplings, two biologically duplicate sets were prepared. At least 150 individual plants were harvested for each set. Total RNA was isolated using Qiagen RNeasy columns (Qiagen Co., Valencia, CA) and mRNA was isolated using Qiagen mRNA Midi kit (Qiagne Co.). The ®rst strand cDNA was synthesized from 800 ng of mRNA, in the reaction mixture using 100 pmol of an oligo dT (24) primer, containing a 5¢-T7 RNA polymerase promoter sequence, and 200 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) in 75 mM KCl, 3 mM MgCl2, 50 mM TRIS-HCl (pH 8.3), 10 mM dithiothreitol (DTT), and 0.5 mM dNTP. The second cDNA synthesis was performed in a reaction mixture with 25 mM TRIS-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)2SO4, 1 mM dNTP, 40 units E. coli DNA polymerase, 10 units E. coli ligase, and two units of RNase H. Double-stranded cDNA products were puri®ed by phenol/chloroform extraction and ethanol precipitation. cRNA synthesis Biotinylated cRNA were in vitro transcribed from synthesized cDNA by T7 RNA polymerase (BioArray high yield RNA Transcript Labelling Kit, Enzo Diagnostics Inc., Farmingdale, NY). The cRNAs were puri®ed using Qiagen RNeasy Spin Columns. 20 mg were then randomly fragmented via incubation at 94 °C for 35 min, in a buffer containing 30 mM magnesium acetate,
Secondary growth in Arabidopsis 2711 100 mM potassium acetate and 40 mM TRIS-acetate (pH 8.1), in order to produce molecules of approximately 35±200 base long cRNA GeneChip array hybridization The following hybridization was performed at the Genomics Technology Support Facility (GTSF) on the campus of Michigan State University. Brie¯y, fragmented cRNAs were denatured at 99 °C for 5 min in the mixture of 0.1 mg ml±1 sonicated herring sperm DNA and hybridization buffer containing 100 mM 2-N-morpholinoethanesulphonic acid (MES), 1 M NaCl, 20 mM EDTA, and 0.01% (w/v) Tween 20. Then, the hybridization mix was hybridized with GeneChipâ Arabidopsis Genome Arrays (Affymetrix, Santa Clara, CA) at 45 °C for 16 h on a rotisserie at 60 rpm. After hybridization, the array cartridge was rinsed and stained in a ¯uidics station (Affymetrix). The hybridized arrays were ®rst rinsed with wash buffer A (63 SSPE [0.9 M NaCl, 0.06 M NaH2PO4, 0.006 M EDTA], 0.01% [w/v] Tween 20, and 0.005% [w/v] Antifoam) at 25 °C for 10 min and then incubated with wash buffer B (100 mM MES, 0.1 M NaCl, and 0.01% [w/v] Tween 20) at 50 °C for 20 min. Next, the arrays were incubated with streptavidin phycoerythrin (SAPE) solution containing 100 mM MES, 1 M NaCl, 0.05% [w/v] Tween 20, 0.005% [w/v] Antiform, 10 mg ml±1 SAPE, and 2 mg ml±1 bovine serum albumin at 25 °C for 10 min, washed with wash buffer A at 25 °C for 20 min, and stained with biotinylated antistreptavidin antibody at 25 °C for 10 min in a ¯uidics station (Affymetrix). The probe array was scanned twice, and then the intensities were averaged with a Hewlett-Packard GeneArray Scanner. Data analysis Gene expression levels were measured by the calculated signal value which assigns a relative measure of abundance to the transcript, and the reliability of those data were evaluated using the P-value system as described in the Microarray Suite 5.0 (Affymetrix). A global scaling strategy that sets the average signal intensity of the array to a target signal of 500 was used for scaling and normalization, so all of the signal values are directly comparable. Expression data for all gene sequences on the GeneChip arrays were analysed using Microsoft Excel. The reproducibility of the array experiments was characterized by comparing each set of signal values from the duplicated experiments. Synthesis of cDNA and cRNA from each set of biological samples was performed independently. The labelled cRNA samples were then hybridized to two different GeneChip arrays. A coef®cient of determination was calculated between the duplicate experiments. The average signal value from the duplicated set and its standard deviations was calculated. The genes whose standard deviations exceed their average signal values were eliminated from the gene list. The average signal values from the control stems, treatment stems, xylem samples, and bark samples were compared. Genes presenting more than a 2-fold difference in average signal values in each comparison were de®ned as differentially expressed genes. Selected genes were clustered according to their expression level by complete linkage hierarchical clustering using GeneSpringÔ (SiliconGenetics). Functional annotation of the genes were obtained from the Munich Information Center for Protein Sequences (MIPS) Arabidopsis database (MATDB, http://mips.gsf.de/proj/thal/db/) by using the AGI (Arabidopsis Gene Index, http://www.tigr.org/tdb/tgi/agi/) numbers provided by the GeneChip manufacturer (Affymetrix Co.). Northern blot analysis of selected R2R3-type MYB genes For probe DNA isolation in northern blot analysis, 50 ng of poly (A)+ RNA was isolated from the xylem tissue samples and used in the synthesis of cDNA as described above. Two microlitres of the reverse transcription reaction mixture was used as a template for
RT-PCR cloning with Taq polymerase (Promega, Madison, WI, USA) using gene-speci®c primers. The gene-speci®c primers were designed for MYB59 (accession no. AF062894), MYB48 (accession no. F272733), and MYB13 (accession no. Z97048). The sequences of primers used included the following: MYB59 (5¢-AGAGATGAAACTTGTGCAAG-3¢ and 5¢-ACAGAAGCTTCAAAAGTCTAT-3¢), MYB48 (5¢-ATGATGCAAGAGGAGGGAAA-3¢ and 5¢TTAACCTGACGACCACGGTGA-3¢), MYB13 (5¢AGATGGGGAGAAGACCATG-3¢ and 5¢-GGAAACGTAAACGACTTT-3¢). The reaction parameters were as follows: the ®rst cycle involved incubation at 94 °C for 5 min, which was followed by 30 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min; and a ®nal incubation at 72 °C for 7 min. The resulting PCR fragments were cloned into pGEM-T Easy vectors (Promega) and sequenced at the GTSF. Next, 4 mg of isolated total RNA from each sample was denatured with the mixture of 2.15 M formaldehyde and 50% formamide, fractionated by electrophoresis on 1.0% agar gels that contained 2.2 M formaldehyde according to the protocol as described in Oh et al. (2000) and then transferred to nylon membranes using 203 SSC. 32P-labelled probes were prepared using a random labelling kit according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). After hybridization, the membranes were washed with 23 SSC (13 SSC is 150 mM NaCl and 15 mM sodium citrate) and 0.1% (w/v) SDS at room temperature for 20 min and with 0.13 SSC and 0.1% SDS at 60 °C for 30 min. Analysis of cis-regulatory elements All of the Arabidopsis genomic sequences were obtained from TIGR (ftp://ftp.tigr.org). One kilo-base sequence was extracted from the 25 613 genes with known or predicted coding sequences found in the whole Arabidopsis genome. Then, that sequence was used to search for the known cis-elements listed at PLANTCARE (http:// oberon.rug.ac.be:8080/PlantCARE) or PLACE (http://www.dna. affrc.go.jp/htdocs/PLACE/wais.html) using a custom Perl script. Next, bootstrapping was performed by generating 1000 control promoter sets from whole Arabidopsis genes. Bootstrapped sets were generated using a custom Microsoft Excel macro and used to compare each of the selected gene groups (I and II) as well as the control sets. The DNA sequence fragments, frequently detected in the selected genes groups, were obtained from MotifSampler 2.0 (www.esat.kuleuven.ac.be/~thijs/Work/MotifSampler.html) and their frequencies were compared with a control group generated by the bootstrapping analysis.
Results and discussion Secondary xylem formation in Arabidopsis Rosette leaves of treatment plants grew much larger than those of the plants in the control group plants (Fig. 1A, B), as previously observed in similar studies (Lev-Yadun, 1994). The stems of treated plants formed a considerable amount of secondary xylem and had a rather thick cortex at the rosette level (Fig. 1D) with the development of interfascicular vascular cambium, when compared to the control plants that had no observable vascular cambium developed at the interfascicular region (Fig. 1C). The anatomy of the hypocotyls (Fig. 1F) from the treated Arabidopsis plants was similar to that of the 4-month-old poplar stem (Fig. 1E). This corroborates with the recent observation by Chaffey et al. (2002) that the anatomy of secondary xylem in Arabidopsis closely resembles the
2712 Oh et al.
the molecular mechanisms for secondary growth, mainly due to its wealth of genetic resources. The wood formation treatment procedure involves several weeks of repeated removal of in¯orescences, which leads to signi®cant plant wounding. Wounding might be a contributing factor for secondary xylem induction (Lev-Yadun, 2002). Mechanical wounding often induces transdifferentiation of parenchyma cells into tracheary elements (TE), which is demonstrated by vessel formation at the wounding site (Jacobs, 1952). The disturbed hormonal transport caused by wounding may result in new vascular tissue formation around the wound site. It was proposed that systemic wound signals might be involved in the initiation of transdifferentiation of parenchyma and epidermal cells into TEs (Fukuda, 1996). Cambial-like activity in the vascular bundle and interfascicular region was induced by wounding in Zinnia stems (Nishitani et al., 2002). Therefore, wounding caused by the repeated removal of in¯orescences in this experiment may lead to increased cambial activity, resulting in enhanced secondary xylem formation.
Fig. 1. Cross-sections of control and treatment stems of Arabidopsis thaliana. Cross-sections were stained with 0.025% toluidine blue O. (A) A 3-week-old Arabidopsis plant grown in high-density growth condition (control). (B) A 9-week-old Arabidopsis plant grown in a low-density growth condition with repeated removal of all newly emerging in¯orescences (treatment). The arrow bars in (A) and (B) indicate the stem samples used in this experiment. (C) Thin crosssections of paraf®n-embedded control stems was made with a razor blade. No secondary growth is observed. (D) Thin cross-section of treatment stem. Cambium zone was observed in vascular bundle and interfascicular region. Extensive secondary vascular tissue production was observed in treatment stems. Arrowheads and arrow bars in (C) and (D) indicate vascular bundle region and interfascicular region, respectively. (E) Thin cross-section of poplar stem (4-months-old) without any treatment. (F) Thin cross-section of hypocotyl region from treated plants. Vertical line in (E) and (F) indicates bark region. X, xylem; P, phloem region; PF, phloem ®bre; C, cortex; B, bark; CZ; cambial zone. Bars=0.7 cm (A, B), 80 mm (C, D), and 220 mm (E, F).
wood of a poplar tree. While the xylem tissue similarity suggests a similar xylem formation process occurring in the two species, Arabidopsis stems do not have the ray parenchyma cells that are present in poplar and other angiosperm tree species. Another limitation in the Arabidopsis system is the lack of a perennial nature in stem growth. Therefore, very important aspects of secondary growth, such as seasonal cycle of cambial activity cannot be studied in Arabidopsis. Nonetheless, Arabidopsis offers an outstanding model system to study
Differential gene expression in treatment stem, bark and xylem The reproducibility of the experiments was tested by calculating the coef®cient of determination between the two biological replicates. All of the experiments were highly reproducible with R2=0.92 for the control, 0.89 for the treatment group, and 0.86 for the bark and xylem experiments. A total of 1658 genes (20% of the ~8300 genes on the array) had differential expressions with 2-fold or more change (for a list of these genes, see Table S1±S4 at Journal of Experimental Botany online). Of those, 543 genes were up-regulated in the treated stem group, while 530 genes were up-regulated in the control stem group, 304 genes in the xylem tissue group, and 281 genes in the bark tissue group (Fig. 2). Of the 304 xylem up-regulated genes, 66 genes were also up-regulated in the treatment stem group (Fig. 2). Furthermore, 108 of the genes up-regulated in the bark tissue group were also up-regulated in the treatment stem group (Fig. 2). In order to gain functional inference of the up-regulated genes in the treatment, control, xylem, and bark groups, the differentially expressed genes were assigned to functional categories according to the annotation by AGI (Arabidopsis Gene Index, http://www.tigr.org/tdb/tgi/agi/) numbers in the Munich Information Center for Protein Sequences (MIPS) Arabidopsis database (MATDB, http:// mips.gsf.de/proj/thal/db/). Across all four samples, unclassi®ed genes made up the largest category (about 18±24%) (Fig. 3). A higher number of transcription regulation-related genes were up-regulated in the treatment stem group and xylem tissue group, when compared to those in the control stem and bark tissue groups (Fig. 3). These results support the hypothesis that secondary growth
Secondary growth in Arabidopsis 2713
Fig. 2. Venn diagram showing up-regulated (>2-fold) genes in control and treatment stems, xylem, and bark from the Arabidopsis Genome array analyses. A list of these differentially regulated genes is provided in supplementary tables.
in a plant species is a matter of regulation, and does not result from the presence of structural genes necessary for secondary growth. In fact, many non-woody plant species, with the exception of monocots, can be induced to undergo secondary growth as it has been demonstrated in Arabidopsis. As expected, defence-related genes were highly represented among the genes up-regulated in bark tissue (16%), but not in xylem tissue (4%), further demonstrating that plant defence responses occur within the sieve element±companion cell complex of phloem (Ruiz-Medrano et al., 2001). When comparing treatment and control stems, higher numbers of photosynthetic genes were up-regulated in control stems than in treatment stems (Fig. 3). In Zinnia, the transdifferentiation of in vitrocultured mesophyll cells to xylem cells reduced the expression levels of photosynthetic genes and increased the expression levels of protein synthesis-related genes (Demura et al., 2002). Cell division
During secondary xylem formation, the cells on the xylem side of the cambium ®rst go through stages of differentiation that involve a division zone where the xylem mother cells continue to divide, then an expansion zone where the derivative cells expand to their ®nal size, next a maturation zone where ligni®cation and secondary cell wall thickening occurs, and ®nally through a zone of programmed cell death where all cellular processes are terminated (Chaffey, 1999). The cell division for second-
Fig. 3. Functional classi®cation of the up-regulated genes in control and treatment stems, bark and xylem. The differentially expressed genes in each sample were assigned to functional categories following those of the Munich Information Center for Protein Sequences (MIPS) Arabidopsis database (MATDB, http://mips.gsf.de/proj/thal/db/). (A) Comparison between control and treatment stems; (B) comparison between bark and xylem.
ary xylem is initiated in the 1~2 layers of the cambium region (Mellerowicz et al., 2001). In the xylem and bark tissue comparison, four cell cycle-related genes (CYCD3;1, SKP1, CDC20, and CDC20-like protein) were signi®cantly up-regulated in bark (Table 1). CYCD3;1 is a G1 type cyclin and is involved with the induction of cell division during multicellular trichome development (Schnittger et al., 2002). SKP1 is a homologue of yeast kinetochore protein that is required for cell cycle progression at both the S1 and M phases. SKP1 has been suggested as a marker for actively dividing cells and meristemic activity in Arabidopsis (Porat et al., 1998). Auxin is thought to be involved in the regulation of cell division (Hagen and Guilfoyle, 2002). Three auxin biosynthesis-related genes (nitrilase 4, indole-3-acetaldehyde oxidase, and CYP78B2 mono-oxygenase) were upregulated in the bark. The Kip-related protein 4 (KRP4) gene was up-regulated in the xylem. This cyclin-dependent kinase inhibitor is a negative regulator of cell cycle progression in leaf primordial cells of Arabidopsis (Veylder et al., 2001). These results show that the genes promoting cell division were up-regulated in bark, while the cell division inhibitor was up-regulated in xylem. It is likely that the vascular cambium cells might have come off with the bark tissue when it was separated from the xylem tissue. Therefore, the gene expression pro®le of bark tissue might also include cambium cells. Beers and Zhao (2001) have separated bark tissue from the xylem of Arabidopsis
2714 Oh et al. Table 1. Expression patterns of selected xylogenesis-related genes Functional category
AGI No.
Fold changea
Putative ID
X/B Cell division
Cell elongation
Cell wall biosynthesis
Ligni®cation
Cell death
At4g34160 At1g10230 At5g40880 At4g33260 At2g32710 At5g22300 At3g43600 At4g39950 At3g47380 At1g05310 At2g47550 At2g45220 At3g14310 At1g14720 At2g06850 At5g23860 At5g12250 At1g64740 At5g17420 At4g18780 At4g24010 At5g49720 At1g13930 At1g62500 At2g05520 At2g30210 At4g34050 At4g36220 At2g21890 At1g51680 At2g40370 At2g37040 At5g54160 At2g30490 At3g53260 At1g02500 At4g01850 At4g37930 At1g20850 At4g35350 At2g45040 At4g00230 At4g11320 At4g16190
CYCD3;1 SKP1 Putative CDC20 CDC20 protein±like KRP4 Nitrilase 4 Indole±3±acetaldehyde oxides CYP79B2 monooxygenase Putative PME (pectin methyl esterase) Putative PME Putative PME Putative PME Putative PME XTH28 (xyloglucan endotransglycosylases) XTH4 Beta±8 tubulin Beta±6 tubulin Alpha±1 Tubulin IRX3 (CesA, cellulose synthase) Putative CesA Putative CesA Cellulase homolog HRGP (hydroxyprolin±rich glyco proteins) Proline±rich protein Glycine±rich protein (GRP) Putative laccase PutativeCCoAMT FAH1 (ferulate±5±hydroxylase) CAD (cinnamyl alcohol dehydrogenase) At4CL1 (4 coumarate±CoA ligase) Putative laccase PAL1 O±methyltransferase C4H PAL SAMS (S±adenosyl methionine synthetase) SAMA2 Glycine HRMT like XCP2 (cystein peptidases) XCP1 Metalloproteinase XSP1 (serine peptidase) Cysteine proteinase Cysteine proteinase
±2.4 ±2 ±2.6 ±2.8 2.4 ±2 ±3 ±2.6 7.3 4 2.5 2 ±2.5 3 ±3.7 5.4 2.8 9.9 4.9 2.6 2 3.5 ±20 ±7.1 ±2.7 2.5 10.3 8.9 4.8 2.7 2.6 2.4 2.4 2.1 2.5 2.3 28.8 25.4 9.1 2.3 3.5 ±26
T/C
±3.5
±2.8
±6.3 ±4.7 ±3.3 3 ±2.8
10.5 ±2.7 2.4
2 ±3.2 ±2 ±7.9 ±3.9 ±2.3
5.9
a
Fold change: X/B, fold change between xylem versus bark; T/C, treatment versus control. Negative values in fold change mean down-regulation. For example, `-2' in T/C means `2-fold down-regulation' in treatment stems (or 2-fold up-regulation in control stems).
hypocotyls, but did not attempt to locate the cambial cells. In poplar, cell-cycle control genes were highly expressed in the phloem and meristematic region of a stem crosssection (Hertzberg et al., 2001). The present study does not provide any gene expression patterns at cell-type resolution. A transgenic approach using marker proteins (e.g. GUS or GFP) or in situ hybridization with selected genes is needed to obtain such information. Cell elongation
Pectin is related to cell expansion. The degree of its methylation in¯uences cell wall extensibility for increasing the cell size (Goldberg et al., 1996). Pectin methyl
esterase (PME) catalyses dimethylesteri®cation of cell wall polygalacturonans. Depending on the cell wall properties, PME promotes the action of pH-dependent cell wall hydrolysis and contributes to cell wall loosening. It causes the cell wall to become rigid by blocking free carboxyl groups that interact with bivalent ions like Ca2+ (Micheli, 2001). In this study, four PMEs were upregulated in xylem, but only one putative PME was upregulated in bark (Table 1), suggesting the differential involvement of different PMEs in cell expansion in both xylem and bark tissue. Xyloglucan endotransglycosylases (XETs) are thought to be involved in the restructuring of cell wall cross-links by cutting a xyloglucan at an internal
Secondary growth in Arabidopsis 2715
site and then ligating its end to a different xyloglucan chain. In poplar, XETs have a multifunctional role in cell wall construction (Bourquin et al., 2002). In pine, an XET gene was speci®cally found in xylem and juvenile wood tissues, indicating its role in the structural modi®cation of xylem cell walls (Allona et al., 1998; Whetten et al., 2001). In the current study, an Arabidopsis XET (XTH28) showed xylem abundant expression patterns compared with bark and no differential expression between treatment and control stem groups (Table 1). In xylem tissue, XETs might be involved in a shuf¯ing of xyloglucan chains for xylem cell expansion. It is notable that the XTH4 gene was up-regulated in bark, but down-regulated in the stems treated for wood formation. The meristematic cambium region is suggested to be a major site of XET activity in poplar stem (Bourquin et al., 2002). XTH4 had a higher (>2-fold) level of expression in the ¯ower than in the stem of a 4-week-old Arabidopsis plant (Yokoyama and Nishitani, 2001). Thus, the members of the XET gene family as a whole seem to be expressed in versatile cell types and have various functions including secondary xylem formation-related activities. Cell wall synthesis Major constituents of the cell wall are cellulose, hemicelluloses and pectins. Microtubules, which are strands formed by alpha-beta tubulin heterodimers, control the orientation of cellulose micro®brils in xylem cells (Chaffey and Barlow, 2002). Successive changes in microtubule density and orientation have been observed in developing ®bres of hybrid aspen (Mellerowicz et al., 2001). Several tubulin genes were up-regulated in xylem or treatment stems (Table 1). The primary-walled developing xylem and phloem tissues of Populus tremuloides were comprised of 47% pectin, 22% cellulose and others (Simson and Timell, 1978). By contrast, the mature wood from P. nigra contains large amounts of cellulose (48%) and lignin (19%) (McDougall et al., 1993). Cellulose has been the subject of wood formation studies using poplar (Allona et al., 1998) and pine (Hertzberg et al., 2001). In the present study, there was no cellulose synthase gene (CesA) up-regulated in bark, while three CesA genes had higher expression levels in the xylem tissue when compared with the bark tissue (Table 1). One of the three genes, IRX3, was about 10-fold up-regulated in xylem. IRX3 is thought to be important in xylogenesis because Arabidopsis plants with a mutation in the gene (irx3) show a severe de®ciency of cellulose deposition in secondary cell walls, resulting in collapsed xylem cells (Turner and Somerville, 1997). Poplar CesA gene, having high sequence homology (78% DNA identity) with IRX3, has been isolated from developing xylem and was shown to be induced by stem banding and mechanical stress (Wu et al., 2000). However, it is notable that the expression level of CesA genes did not vary between the treatment and
control stem groups, with exception of one CesA gene (At4g18780) that was up-regulated in the control stems (3fold). Several classes of structural proteins may serve a structural role, eventually becoming solidly cross-linked in response to growth termination or pathogen attack. A list of such cell wall proteins includes hydroxyproline-rich glycoproteins (HRGPs), proline-rich proteins (PRPs), and glycine-rich proteins (GRPs) (Cassab and Varner, 1988). The expression of HRGP genes is regulated both developmentally and environmentally by signals such as wounding and infection (Fukuda, 1996). Bao et al. (1992) isolated an extensin-like HRGP from the xylem of loblolly pine and showed that the protein was present in secondary cell walls of xylem cells during ligni®cation. In this study, a putative HRGP gene was up-regulated in xylem, but remained constant between treatment and control stems (Table 1). GRPs are a class of proteins that have a 60% glycine residue arranged predominantly in (Gly-X)n repeats (Keller et al., 1988). GRP1.8 had been localized in the cell walls of primary phloem in many plant species (Keller et al., 1988; Ye et al., 1991). A GRP gene (At2g05520) was found that was up-regulated in both the bark and treatment stem groups, suggesting its involvement in bark secondary wall formation. Ligni®cation Lignin is a heterogeneous phenolic polymer that is deposited in secondary cell walls along with cellulose and hemicelluloses. It was found that 4 coumarate-CoA ligase (4CL, At1g51680), cinnamyl alcohol dehydrogenase (CAD, At2g21890), ferulate-5-hydroxylase genes (FAH1, At4g36220) and putative laccase (At2g40370) were highly expressed in xylem, but not in bark (Table 1), suggesting their roles in the polymerization of lignin in secondary xylem formation. However, there was one putative lignin biosynthesis-related gene (At2g30210) that was up-regulated in bark. Interestingly, the expression levels of most lignin biosynthesis-related genes did not differ between treatment and control stems, except the FAH1 that had a higher expression in treatment stems. One possible explanation for this is that lignin biosynthesis occurs in both treatment and control stems, but mainly in xylem tissues. This explanation is supported by the observation that young Arabidopsis plants that do not undergo secondary growth still undergo extensive ligni®cation (Dharmawardhana et al., 1992). The methylation of the lignin precursors is carried out by S-adenosyl methionine synthetase (SAMS) and CCoAOMT enzymes (Zhong et al., 1998). SAMS catalyses the transfer of an adenosyl group from ATP to the sulphur atom of methionine, resulting in the synthesis of SAM, a common methyl group donor. Although SAMS is a housekeeping enzyme, its activity has been found to occur more frequently in xylem than in bark or other poplar tissues (Vander et al., 1996). In the poplar
2716 Oh et al.
developing xylem EST library, SAMS was clearly presented as a xylem tissue abundant gene (Sterky et al., 1998). In the current study, it was found that two SAMS were up-regulated in xylem not in bark (Table 1). In addition, putative glycine hydroxymethyltransferase, which mediates the conversion of 5,10-methylene-tetrahydrofolate to and from tetrahydrofolate (Mijnsbrugge et al., 2000), was highly up-regulated (>28-fold) in xylem (Table 1), indicating that the methyl cycle for ligni®cations is more active in xylem than in bark. Cell death
Cell death is initiated by the disruption of vacuole membranes that results in the release of hydrolytic enzymes into the cytosol (Groover and Jones, 1999). Such hydrolytic enzymes as cystein proteinases, serine proteinases, and nucleases are highly induced during xylogenesis (Fukuda, 1996). Papain-type cysteine peptidases (XCP1 and XCP2) and putative subtilisin-type serine peptidase (XSP1) have been identi®ed from Arabidopsis xylem (Zhao et al., 2000). In their report, XCP1 and XCP2 genes were expressed abundantly in xylem when compared with bark tissue, but less abundantly in the young stem when it was compared with the ¯ower. The authors suggested that these proteinases had specialized functions (e.g. autolysis of xylem TE) associated with plant growth and differentiation. In the present study, it was found that the two genes (XCP1 and XCP2) were more highly expressed in xylem than in bark, and in control over treatment stems (Table 1). These ®ndings are similar to those previously reported by Zhao et al. (2000). It is possible for these enzymes to have multiple functions in the programmed cell death (PCD) process during xylogenesis as well as in the developmental process. Recently, ectopic expression of XCP1 resulted in early senescence in Arabidopsis (Funk et al., 2002). However, the exact mechanism by which XCP1 or XCP2 activate the cell death process as a ®nal step of xylem formation is unknown. Another PCD-related protein, metalloproteinase, gene was up-regulated in xylem compared with bark. A cucumber metalloproteinase, having a 38.6% identity with Arabidopsis metalloproteinase, expressed at the boundary of senescence and PCD (Delorme et al., 2000). Zhao et al. (2000) were unable to ®nd any bark abundant endopeptidase in their cDNA library screening. On the contrary, one cysteine proteinase gene (At4g11320) that was 26-fold up-regulated in bark when compared with xylem was discovered. However, its signal intensity was low (1140) when compared with the average signal intensity of the other genes (2620). Transcriptional regulation of secondary xylem formation
Transcription factors were more highly expressed in xylem than in bark and in the treatment than in the control stem
(Fig. 3). This could partially explain what kind of transcriptional activities are promoted in the xylem and the treatment groups, where secondary xylem formation events have occurred. The possibility is presented that transcription factors can be involved in secondary xylem formation. AUX/IAA expression: Auxin plays diverse roles in cellular and developmental regulation such as cell division, expansion, differentiation, and patterning of vascular tissue (Reed, 2001). Auxin is a key signal for secondary xylem formation (Sundberg et al., 2000). AUX/IAA genes, which are induced rapidly by auxin, have been indicated in auxin signal transduction (Worley et al., 2000). Moyle et al. (2002) recently isolated eight Aux/IAA genes (PttIAAs) from a hybrid aspen (Populus tremula 3 P. tremuloides) and described tissue-speci®c expression patterns of the genes, having ®ve Aux/IAA genes (PttIAA1, 2, 3, 4, and 8) up-regulated in xylem. Arabidopsis is estimated to have 29 genes encoding Aux/ IAA proteins with highly conserved domains (Liscum and Reed, 2002). The 8.3 k Arabidopsis GeneChip contains 19 Aux/IAA genes. It was found that eight of the Aux/IAA genes (IAA19, IAA28, IAA22, IAA2, IAA12, IAA8, IAA13, and IAA26) were up-regulated (>2-fold) in xylem compared with bark. IAA19 was highly up-regulated (7.5-fold) in xylem compared with bark and in the control stems (5.5-fold) compared with the treatment stems. It should be noted that it was not possible to detect any Aux/ IAA genes up-regulated in bark. R2R3-MYB transcription factors: It is estimated that there are about 1600 transcription factor genes and 131 of them are classi®ed as R2R3-type MYB transcription factors (Riechmann et al., 2000). It has been proposed that the MYB family genes are involved in plant-speci®c processes because their presence is limited to plants (Martin and PazAres, 1997; Stracke et al., 2001). Several MYB family genes have been implicated in the regulation of ligni®cation and ¯avonoid biosynthesis in Antirrhinum species (Tamagnone et al., 1998). It has also been suggested that xylem-abundant MYB proteins might be involved in the transcriptional regulation of secondary xylem formation (Newman and Campbell, 2000). In the present study, it was found that eleven R2R3-type MYB genes were upregulated in xylem compared with bark (Fig. 4A). The poplar orthologue of MYB52 (AL 164087) was expressed abundantly in the late cell expansion and late cell maturation region of stem cross-sections (Hertzberg et al., 2001). In Arabidopsis, MYB52 was up-regulated in xylem and down-regulated in bark. Also, two phylogenetically close MYB genes (MYB59 and MYB48) (Romero et al., 1998), were up-regulated in xylem and treatment stems. Northern blot analysis of MYB59 and MYB48 con®rmed their GeneChip expression patterns (Fig. 5). Two MYB
Secondary growth in Arabidopsis 2717
Fig. 4. R2R3-type MYB transcription factor genes up-regulated in xylem (A) or bark (B). The promoter region sequences (1 kb upstream) were obtained from the TIGR web site (ftp://ftp.tigr.org). The cis-elements survey was performed using the tools available at PLANTCARE (http://oberon.rug.ac.be:8080/PlantCARE). X/B, signal intensity of xylem over that of bark; B/X, bark/xylem; T/C, treatment/ control. Negative values mean down-regulation. For example, `-2' in T/C means `2-fold down-regulation' in treatment stems (or 2-fold upregulation in control stems). Asterisk: ratio based on non-passing values (below detection level). Black triangles: ABA response motif (ABRE, CCGAC) (Baker et al., 1994). Black squares: droughtresponse element (TACCGACAT) (Yamaguchi-Shinozaki and Shinozaki, 1994). Grey squares: c-repeat drought-response element (TGGCCGAC) (Baker et al., 1994). (Black circles) MYB binding motif (MBS, CAACTG) (Yamaguchi-Shinozaki and Shinozaki, 1994). (Black inverted triangles), bZIP protein recognition site (TGACGTCA) (Cheong et al., 1998).
binding cis-elements (MBS) were found in the 1 kb upstream region of the MYB48 gene, suggesting that the expression of MYB48 could be regulated by other MYB genes (Fig. 4A). However, how MYB48 regulates the transcriptional events during secondary xylem formation is currently not known. There were four MYB genes upregulated in bark (Fig. 4B). MYB34 carries three ABA (abscisic acid) response cis-elements (ABREs±CCGAC) in 1 kb upstream region of the gene and was previously described as drought-inducible (Kranz et al., 1998). ABREs are usually responsive to environmental stress such as cold, drought and salt stress, and act to transfer the ABA signal or its corresponding molecule (Baker et al., 1994; Straub et al., 1994). This gene was up-regulated in bark and treatment stems. Another bark up-regulated MYB gene (MYB28) also has three ABRE motifs in its promoter region. MYB13 is regulated by dehydration, exogenous ABA and wounding, and can be detected at the shoot apex and base of developing ¯owers (Kirik et al., 1998; Jin and Martin, 1999), suggesting its potential role in shoot
Fig. 5. Northern blot analysis of selected R2R3-type MYB genes that were highly up-regulated in xylem (MYB59 and MYB48) or bark (MYB13). Total RNA (4 mg) was isolated from control stems (C), treatment stems (T), xylem (X), and bark (B) tissues. Immobilized RNA was hybridized with 32P-labelled probes of selected MYB genes. The probes were ampli®ed from xylem tissue using gene-speci®c primers. The bottom panel shows the EtBr-stained rRNA under UV illumination before blotting, indicating equal amount of total RNA loading.
morphogenesis. In this study, MYB13 was up-regulated in bark and treatment stems. Its expression pattern was con®rmed by northern blot analysis (Fig. 5). Homeodomain (HD) genes: Homeodomain (HD) transcription factors play key roles in developmental processes, cell fate and pattern formation (Affolter et al., 1990). One Arabidopsis HD-leucine zipper protein, ATHB-8, can be an early marker of vascular development because it is active in the provascular cells of embryos (Baima et al., 1995). Overexpression of the gene in transgenic Arabidopsis and tobacco plants resulted in high amounts of primary and secondary xylem production (Baima et al., 2000), suggesting its role in the regulation of vascular development. A poplar orthologue of ATHB-8 was expressed in the cambial meristem and the expansion region of poplar stems, but not in phloem and maturation region (Hertzberg et al., 2001). In Arabidopsis, ATHB-8 was highly up-regulated in xylem compared with bark (Fig. 6A). However, there was no signi®cant difference in its expression level between control and treatment stems. ATHB-15 was phylogenetically very close to ATHB-8 at the amino acid level (Fig. 7). ATHB-15 was also upregulated in xylem. However, unlike ATHB-8 it was up-regulated in treatment stems compared with control stems. This suggests that these structurally similar genes might be involved in different stem developmental stages. Two other HD genes (ATHB-9 and ATHB-14) that are close to ATHB-8 in the phylogenetic tree (Fig. 7) were also up-regulated in xylem (Fig. 6A). It is implicated that the Arabidopsis PHABULOSA gene (ATHB-14) and PHAVOLUTA gene (ATHB-9) have roles in the perception of radial positional information when determining
2718 Oh et al.
Fig. 6. Homeodomain (HD) genes up-regulated in xylem (A) or bark (B). The promoter region sequences (1 kb upstream) were obtained from the TIGR web site (ftp://ftp.tigr.org) (www.arabidopsis.org). The cis-elements survey was performed using the tools available at PLANTCARE (http://oberon.rug.ac.be:8080/PlantCARE). X/B, signal intensity of xylem over that of bark; B/X, bark/xylem; T/C, treatment/ control. Negative values mean down-regulation. For example, `-2' in T/C means `2-fold down-regulation' in treatment stems (or 2-fold upregulation in control stems). Asterisk: ratio based on non-passing values (below detection level). Black triangles: ABA response motif (ABRE, CCGAC). Black squares: drought-response element (TACCGACAT). Grey squares: c-repeat drought-response element (TGGCCGAC). Black circles: MYB binding motif (MBS, CAACTG).
radial patterning in shoots (McConnell et al., 2001). It is notable that three HD genes (ATHB-5, ATHB-6, and ATHB-16) were also structurally similar to each other (Fig. 7) and were up-regulated in bark (Fig. 6B). A Zinnia HD gene, ZeHB3, has high sequence homology with the three genes. Because of its immature phloem cell-speci®c expression pattern, it has been suggested as a marker for early stages of phloem differentiation (Nishitani et al., 2001). The fact that these genes were up-regulated in bark suggests that they might act as transcriptional regulators in the development of primary and/or secondary phloem. Identi®cation of regulatory cis-elements for secondary growth
Cluster analysis was performed for the 585 differentially expressed genes (304 xylem up-regulated and 281 bark upregulated genes) using average signal values (Fig. 8). From the cluster analysis, two groups were chosen for further study. Group I comprised 25 genes that were up-regulated in both xylem and treatment stems, but down-regulated in both the bark and control stem (i.e. secondary xylem formation-associated genes) (Fig. 8). It includes two MYB genes (MYB59 and MYB48) and two lignin-biosynthesis related genes (FAH1 and putative CAD). Group II contains 25 genes that are up-regulated in both the bark and treatment stems together, but down-regulated in both the xylem and control stems (Fig. 8). Two HD genes (ATHB-6
Fig. 7. Phylogenetic tree of homeodomain (HD) genes. Amino acid sequences from the 19 selected HD genes were used to generate the phylogenetic tree using the GeneBee program (http:// www.genebee.msu.su/services). The corresponding AGI numbers with each HD gene are follows: ATHB-1, At3g01470; ATHB-2, At4g16780; ATHB-3, At5g15150; ATHB-4, At2g44910; ATHB-5, At5g65310; ATHB-6, At2g22430; ATHB-7, At2g46680; ATHB-8, At4g32880; ATHB-9, At1g30490; ATHB-10, At1g79840; ATHB-12, At3g61890; ATHB-13, At1g69780; ATHB-14, At2g34710; ATHB-15, At1g52150; ATHB-16, At4g40060; ATHB-17, At2g01430; BLH2, At4g36870; BLH4, At2g23760; HD-like, At4g34610.
and ATHB-16) and one MYB gene (MYB14) are included in Group II. In an attempt to identify putative cis-elements for secondary xylem formation signalling, the promoter region of the genes from the two groups was surveyed for known cis-elements listed at PLANTCARE or PLACE as described in the Materials and methods section. Frequency of cis-element motifs in the promoter regions of 1000 randomly selected Arabidopsis gene sets were used as a control. The frequency of a putative ABRE3 ciselement sequence (CAACGTG) was signi®cantly high in Group I at a 95% con®dence interval given from 1000 control promoter sets (Table 2). ABRE3 motif (GCAACGTGTC) is found in the promoter region of this gene, which encodes a Class 3 late embryogenesisabundant protein (HVA1) in barley (Straub et al., 1994). The extA cis-element sequence (AACGTGT) was frequently presented in Group I (Table 2). ExtA ciselement is located in the promoter region of an extensin gene that responds to wounding and tensile stress in Brassica napus (Elliott and Shirsat, 1998). Wounding and tensile stress stimulate secondary xylem formation in
Secondary growth in Arabidopsis 2719
Fig. 8. Hierarchical clustering of differentially regulated genes and selection of xylem (Group I) and bark (Group II) up-regulated genes. Red colour indicates the gene carrying a putative ABRE3 motif (CAACGTG) in its promoter region, green for extA motif (AACGTGT), purple colour for SAUR motif (CATATG), and blue for the genes carrying both ABRE3 and extA motifs.
poplar (Wu et al., 2000; Demura et al., 2002). The AACGTGT motif of extA cis-element is similar to the G box motif (CACGTG), a binding site for transcriptional
activators in the promoter regions of many plant genes (Holdsworth and Laties, 1989). While experimental veri®cation is needed, the presence of the two functional cis-
2720 Oh et al. Table 2. Regulatory cis-element motifs identi®ed from the promoter regions of the genes up-regulated in wood-forming stems Function
ABA Activator Unknownf Unknown Unknown Unknown IAA Unknown Unknown Unknown Unknown
Motif IDb
ABRE3 extA I_1 I_2 I_3 I_4 SAUR II_1 II_2 II_3 II_4
Motif sequence
CAACGTG AACGTGT ATA[GC]AA[AT]C TATTTGTT ATTGTTAT GAAT[AC]GT CATATG CACACAC[AG] ATATACA[AG] [AT]AGCCAT ACGTAAA
Per cent occurrencea Controlc
Group Id
5 8 23 19 10 20 29 9 18 17 14
24 24 56 40 28 44
95% CIe
Reference
0~12 0~20 8~36 8~32 0~20 8~36 16~44 0~20 4~32 4~32 4~28
Straub et al., 1994 Elliott and Shirsat, 1998
Group IId
60 23 50 42 31
Xu et al., 1997
a Percent occurrence, frequency of the motifs among the gene in each group. For example, SAUR motif it occurred in 16 genes of Group II while it occurred in 290 genes of 1 000 randomly selected genes (control). b Motif IDs were from PLACE or PLANTCARE. c Control, the promoter regions of 1 000 randomly selected Arabidopsis genes were used for calculation of average occurrence of the motifs. d Group I and II, promoter regions of 25 genes in each group were used to identify the frequent motifs. e CI, con®dence interval at 95% were calcurated from bootstrapping analysis. f Unknown motifs were identi®ed using MotifSampler 2.0.
elements (putative ABRE3 and extA) in the promoter regions of Group I genes suggests that the expression of those genes involved in secondary xylem formation might be regulated by such signals as ABA, tensile stress, and wounding. This Arabidopsis system might provide a model for the study of stress-induced secondary xylem formation in trees (e.g. reaction and tension wood). Soybean Small Auxin-Up RNA (SAUR) genes are transcriptionally induced by exogenous auxin within a few minutes (Hagen and Guilfoyle, 2002). The promoter of a SAUR gene (SAUR 15A) has been shown to contain multiple auxin response motifs (Xu et al., 1997), and is necessary and suf®cient for auxin induction (Li et al., 1994). The frequency of CATATG (SAUR) motif was present in signi®cantly higher numbers (60 versus 29 in control) in the promoter regions of Group II genes (Table 2), suggesting that auxin may play a signi®cant role in secondary growth, especially secondary phloem and bark formation. The promoter sequence analysis using Motifsampler 2.0 identi®ed several previously not described putative cis-elements that were present in signi®cantly high numbers in the promoter regions of the selected genes (Table 2). For example, a motif (`ATA[GC]AA[AT]C') was present about twice as often in the promoter regions of Group I genes than in the control. Careful evaluation of the functional roles of these unknown motifs might provide some new insight into the transcriptional regulation of secondary growth in plants. Supplemetary data The lists of differentially expressed genes in xylem (Table S1), bark (Table S2), wood formation treatment
(Table S3), and control (Tble S4) can be found at Journal of Experimental Botany online. Acknowledgements We thank Merilyn Ruthig for her technical assistance, Drs Susanne Kleff, Jeff Landgraf, and Annette Thelen for their help with Affymetrix GeneChip analysis. This work was supported by the USDA grant (no. 2001-34158-11222).
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