Journal of Integrative Plant Biology 2007, 49 (12): 1754–1762

Genome-wide Expression Profiling in Seedlings of theArabidopsis Mutant uro that is Defective in the

Secondary Cell Wall Formation

Zheng Yuan1, Xuan Yao2, Dabing Zhang2, Yue Sun3 and Hai Huang1∗

(1Graduate School of Chinese Academy of Sciences, National Laboratory of Molecular Genetics,

Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Shanghai 200032, China;2SJTU-SIBS-PSU Joint Center for Life Sciences, Key Laboratory of Microbial Metabolism, Ministry of Education,

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;3College of Life Sciences, East China Normal University, Shanghai 200062, China)

Abstract

Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economicusefulness. Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentiallyfor water and nutrient transport, respectively. On the other hand, products of plant secondary growth are important rawmaterials and renewable sources of energy. Although advances have been recently made towards describing molecularmechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood. Secondarycell wall formation in plants shares some common mechanisms with other plant secondary growth processes. Thus,studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms forplant secondary growth. We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant,upright rosette (uro), which is defective in secondary cell wall growth and has an unusually soft stem. Here, we show thatlignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem. We also show genome-wideexpression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes.Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling,cell division and plant secondary tissue growth. These results provide useful information for further characterizations ofthe regulatory network in plant secondary cell wall formation.

Key words: cell wall lignification; lignin; microarray; secondary growth; upright rosette.

Yuan Z, Yao X, Zhang D, Sun Y, Huang H (2007). Genome-wide expression profiling in seedlings of the Arabidopsis mutant uro that is defective inthe secondary cell wall formation. J. Integr. Plant Biol. 49(12), 1754–1762.

Available online at www.jipb.net

Plant tissues can be generally classified into two kinds;primary tissues and secondary tissues. Plant growth and differ-entiation of apical meristems result in the formation of primary

Received 4 Jan. 2007 Accepted 23 Mar. 2007

Supported by the Hi-Tech Research and Development (863) Program of

China (20060110Z1012) to H.H. and the National Natural Science Foundation

of China (30570159) to Y.S.∗Author for correspondence.

Tel: +86 (0)21 5492 4088;

Fax: +86 (0)21 5492 4015;

E-mail: <hhuang@sippe.ac.cn>.

C© 2007 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2007.00586.x

tissues such as epidermis, vascular bundles and piths. Someprimary tissues undergo secondary growth, leading to the pro-duction of secondary tissues, such as wood (Ko et al. 2004). Inthe primary tissues, a primary cell wall is comprised of cellulosemicrofibrils, hemicelluloses (or cross-linking glycans) and pecticpolymers. Some cell types, by contrast, develop secondary cellwalls that are comprised mostly of cellulose and cross-linkingglycans and can also be lignified (Cosgrove 2005).

Elucidation of regulatory mechanisms for plant secondarygrowth is of extreme importance, as most products of the sec-ondary growth can serve as widely used industry raw materials.However, our current understanding of genetic control of plantsecondary growth is limited. Secondary growth is predominantlyin trees, but the inherent problems of tree species, such as longgeneration time, large size and lack of genetically pure lines

uro Mutation and Secondary Cell Wall Growth 1755

have blocked the in-depth studies of the molecular genetic basisof plant secondary growth. In recent years, the model plantArabidopsis has been used in research of secondary growth(Chaffey et al. 2002), and progress has been made in the studyof gene expression and signaling mechanisms responsible forsecondary cell wall formation, lignin and cellulose biosynthe-sis, and xylem development (Chaffey et al. 2002; Cosgroveet al. 2002; Doblin et al. 2002; Boerjan et al. 2003; Oh et al.2003; Fukuda 2004; Cosgrove 2005; Ehlting et al. 2005; Zhaoet al. 2005; Groover and Robischon 2006). Plant secondarycell wall formation shares some common mechanisms withother secondary growth processes. For example, secondary cellwall formation and wood formation in trees both require ligninbiosynthesis and deposition. However, little is known abouta particular plant secondary growth question: the regulatorymechanism underlying the coordinated expression of genes forsecondary cell wall formation.

We previously reported the identification and phenotypiccharacterizations of a semi-dominant Arabidopsis mutant, des-ignated upright rosette (uro) (Sun et al. 2000). The uro mutantplant displayed pleiotropic phenotypes, including a loss of apicaldominance with very soft stems. More detailed analyses showedthat processes of xylem and fiber differentiation were aberrantin the uro mutant, and thickening of the secondary cell wallwas delayed (Guo et al. 2004). These results imply that the uromutant could be used as a genetic source for exploring mech-anisms of secondary cell wall formation, and such studies mayprovide important information in understanding plant secondarygrowth.

In the present study, we extended our characterizations of theuro mutant by analyzing the lignification process in the uro cellwall. We found that the lignification level of the secondary cellwall in uro hypocotyl and stem was significantly reduced. Fora better understanding of mechanisms by which the plant sec-ondary cell wall is regulated, we carried out Arabidopsis whole-transcriptome profiling analyses. Functions of some genes thatmay be involved in secondary cell wall formation are discussed.

Results

uro mutation perturbs lignification in the secondary cellwall formation

To better understand the biology of the uro cell wall, we first car-ried out a series of sectioning with hypocotyls at seedling stages.On day 6 after germination, the overall phenotypes betweenwild-type Ler (Figure 1A) and uro (Figure 1B) hypocotyls of light-grown seedlings were nearly identical. Sections of hypocotylsat this stage consisted of a uniseriate epidermis, a cortexcontaining two layers of parenchyma and endodermis, and astele in the center (Figure 1A,B). The first indication of hypocotylsecondary cell growth was the appearance of anticlinal and

periclinal divisions in the procambial and pericycle cells, whichproliferate to generate the vascular cambium and the corkcambium (Chaffey et al. 2002). Although the cellular patternof uro hypocotyl (Figure 1D) at this stage was similar to thatof wild-type Ler (Figure 1C), the number of tracheary elementsof newly differentiated xylem in uro was obviously reduced. Inaddition, the secondary cell wall started to construct in wild-type hypocotyls at this stage, by lignin deposition in cell walls ofxylem tissues (Figure 1I), whereas this process was not notedin those of the same-stage uro mutant (Figure 1J).

During the subsequent hypocotyl growth, xylem and phloemtissues developed rapidly in wild-type seedlings and the wholehypocotyl became thick, as shown in the 25- and 38-day-oldplants, respectively (Figure 1E,G). Although xylem and phloemtissues also developed in uro hypocotyls, cell proliferationappeared restrained, resulting in less thickened hypocotyls(Figure 1F,H). Strikingly, lignification of xylem in hypocotylswas accelerated in the 25- and 38-day-old hypocotyls in thewild-type seedlings (Figure 1K,M). By contrast, this processwas dramatically delayed, with no or very minor lignificationappearing in the 25- and 38-day-old uro hypocotyls, respectively(Figure 1L,N). We also investigated the lignification processof cell walls in the inflorescence stem. Similarly, much lesslignification in the uro stem was noted (Figure 1P) as comparedwith that in the wild-type stem (Figure 1O). These resultsstrongly indicate that the gain-of-function uro mutation perturbssecondary cell wall formation.

Genome-wide analyses of altered gene expression in uroseedlings

To investigate the uro mutation that affects secondary cellwall formation, we analyzed global expression profiles of uroseedlings, using Affymetrix GeneChip that represents about24 000 genes of Arabidopsis. To avoid the indirect effectscaused by morphological changes from later uro development,we used 6-day-old seedlings from which morphological dif-ferences between wild-type and uro mutant were not visible(Figure 1A,B). Probe sets were normalized for each microarrayaccording to a previously reported method (Redman et al. 2004).From two biological replicates, 1 769 genes were identified withconsistent changes in transcript levels in uro, with the increasedand decreased transcription of 816 and 953 genes, respectively.These genes were grouped into four categories: (i) informationstorage and processing; (ii) cellular processes and signaling; (iii)metabolism; and (iv) genes with unknown functions, and couldbe further divided into 27 subgroups as shown in Figure 2.

Altered expression levels of the plant hormone-relatedgenes

Our previous phenotypic characterizations of the mutant led usto propose that the dominant uro phenotypes may be related to

1756 Journal of Integrative Plant Biology Vol. 49 No. 12 2007

Figure 1. Secondary cell wall formation is defective in the uro mutant.

(A–H) Sections are from the central part of hypocotyls and were stained with 0.05% toluidine blue O.

(A) A section from 6-day-old wild-type Ler .

(B) A section from 6-day-old uro.

(C) A section from 12-day-old Ler .

(D) A section from 12-day-old uro.

(E) A section from 25-day-old Ler .

(F) A section from 25-day-old uro.

(G) A section from 38-day-old Ler .

(H) A section from 38-day-old uro hypocotyl. Note that the cell division and differentiation of xylems in uro were retarded (arrow).

(I–P) Sections are from the central part of hypocotyls and were stained with 1% phloroglucinol-HCl for lignin content analyses.

(I) A section from 12-day-old Ler .

(J) A section from 12-day-old uro.

(K) A section from 25-day-old Ler .

(L) A section from 25-day-old uro.

(M) A section from 38-day-old Ler .

(N) A section from 38-day-old uro showing that a few tracheary elements started to be lignified.

(O–P) Sections from central inflorescence stems of 5-week-old plants.

(O) A section from Ler .

(P) A section from uro. Note that the lignin deposition was blocked in both uro hypocotyl and stem (arrows). co, cortex; cop, cortex parenchyma; cz,

cambial zone; e, epidermis; en, endodermis; if, interfascicular fiber; ip, interfascicular fiber precursor; pc, procambium; pe, pericycle; ph, phloem; pi,

pith; st, stele; te, tracheary elements; v, vessel element; x, xylem.

(A–P) Bars, 50 µm.

uro Mutation and Secondary Cell Wall Growth 1757

Figure 2. Functional categorizations of genes that showed altered expression levels in the uro mutant.

The software Clusters of Orthologous Groups (COG) from website http://www.ncbi.nlm.nih.gov/COG/ was used for gene functional categorization.

1, Translation, ribosomal structure and biogenesis; 2, RNA processing and modification; 3, Transcription; 4, Replication, recombination and repair;

5, Chromatin structure and dynamics; 6, Cell cycle control, cell division, chromosome partitioning; 7, Nuclear structure; 8, Defense mechanisms; 9,

Signal transduction mechanisms; 10, Cell wall/membrane/envelope biogenesis; 11, Cell motility; 12, Cytoskeleton; 13, Extracellular structures; 14,

Intracellular trafficking, secretion, and vesicular transport; 15, Posttranslational modification, protein turnover, chaperones; 16, Energy production and

conversion; 17, Carbohydrate transport and metabolism; 18, Amino acid transport and metabolism; 19, Nucleotide transport and metabolism; 20,

Coenzyme transport and metabolism; 21, Lipid transport and metabolism; 22, Inorganic ion transport and metabolism; 23, Secondary metabolites

biosynthesis, transport and catabolism; 24, General function prediction only; 25, Unnamed genes; 26, Function unknown; 27, genes that are not found

in this category system.

the defective auxin action in Arabidopsis (Guo et al. 2004). OurGeneChip results further supported this idea that auxin contentand signaling in uro may be abnormal. It is known that theYUCCA genes encoding a favin monooxygenase-like enzymecatalyze hydroxylation of the amino group of tryptamine, a rate-limiting step in the tryptophan dependent auxin biosynthesis(Zhao et al. 2001; Cheng et al. 2006). We found that thelevel of one YUCCA gene, YUCCA1, was markedly elevatedin uro (Table 1). On the other hand, levels of several genesinvolving auxin signaling, including IAA19 and a number of auxinresponsive genes, were decreased (Table 1). These resultsstrongly suggest that auxin signaling in uro mutant is abnormal,consistent with the uro auxin defective phenotypes such as lossof apical dominance and delayed vascular differentiation (Guoet al. 2004).

Crosstalk of different hormone signaling pathways has be-come clear in recent years in higher plants (Gazzarrini andMcCourt 2003). Our data showed that in addition to the auxinpathway, expression of genes in other hormone pathways wasalso altered. The IPT genes, encoding adenylate isopentenyl-transferase, may play important roles in the cytokinin biosyn-thesis (Sakakibara 2005). However, the transcription level ofthe IPT3 gene in uro was increased (Table 1). Similarly, theArabidopsis AMP1 gene, which plays roles in shoot apicalmeristems maintenances, cell proliferation, photomorphogen-esis, and flower timing, is also required for the regulation ofthe endogenous cytokinin level (Helliwell et al. 2001), whereaswas upregulated in uro (Table 1). These results suggest thatthe altered gene expressions might affect hormone levels and

responses in the uro mutant, and thus cause the severe uromorphological changes.

Expression of genes relating to the secondary tissuegrowth

As shown in the phenotypic analyses of uro hypocotyls andstems, secondary tissue growth was markedly delayed andlignification level was reduced in the uro mutant (Figure 1). Con-sistent with the uro phenotypes, our microarray data revealedthat expression of a number of genes involved in cell division andcell wall deposition was changed in uro (Table 1). Interestingly,177 genes (more than 10% of all expression altered genes)correlating to the secondary tissue growth (Ehlting et al. 2005;Zhao et al. 2005) changed their expression levels in the uro mu-tant (see supplementary Table 1). These expression changes inthe uro mutant were consistent with our cellular observations ofhypocotyls and stems, suggesting that the uro soft stem may bemainly caused by defects in the secondary cell wall formation(Figure 1).

Validation of gene expression in profiling experimentsby RT-PCR

To test the reliability of our microarray results, we carriedout reverse transcription-polymerase chain reaction (RT-PCR)analyses using cDNAs from the same-stage wild type and uroseedlings. Genes that represent different altered levels were

1758 Journal of Integrative Plant Biology Vol. 49 No. 12 2007

Table 1. Selected genes from the initial 1 769 genes that showed altered expression levels in the uro seedling

AGI ID Gene description Log2 ratio

Plant hormone-related

At5g18060 Auxin-responsive protein, putative −1.69

At1g29460 Auxin-responsive protein, putative −1.40

At4g14560 Auxin-responsive protein (IAA1) −1.28

At5g01990 Auxin efflux carrier family protein −1.20

At4g34760 Auxin-responsive family protein −1.17

At3g15540 Indoleacetic acid-induced protein 19 (IAA19) −1.04

At4g32540 Flavin-containing monooxygenase (YUCCA1) 2.36

At2g34550 Gibberellin 2-oxidase (GA2OX3) 1.26

At3g63110 Cytokinin synthase (IPT3) 1.73

At3g54720 Glutamate carboxypeptidase (AMP1) 1.36

Cell cycle/division-related

At1g75780 Tubulin beta-1 chain (TUB1) −2.23

At2g16700 Actin depolymerizing factor 5 −1.85

At1g01750 Actin-depolymerizing factor, putative −1.52

At5g65420 D-type cyclin −2.22

At1g15570 Cyclin, putative −1.40

At2g26760 Cyclin, putative −1.22

At2g44740 Cyclin family protein −1.11

Cell wall deposition-related

At2g37090 Glycosyl transferase family 43 protein (IRX9) −4.50

At2g43050 Putative pectinesterase −4.18

At2g06850 Endo-xyloglucan transferase (EXT) (EXGT-A1) −1.60

At4g30290 Similar to xyloglucan endotransglycosylase (XTH19) −1.59

At5g48070 Similar to xyloglucan endotransglycosylase (XTH20) −1.4

At2g36870 Putative xyloglucan endo-transglycosylase −1.43

At1g65310 Similar to xyloglucan endotransglycosylase (XTH17) −1.25

At4g14130 Endo-xyloglucan transferase, putative (XTR7) −1.15

At3g61000 Glycosyl hydrolase family protein 85 4.01

At4g25810 Endo-xyloglucan transferase, putative (XTR6) 1.91

At1g80170 Polygalacturonase 1.15

selected in the RT-PCR experiments. These genes includedGA2 (At1g79460), IPT6 (At1g25410), RGL3 (At5g17490),IAA1 (At4g14560), IAA19 (At3g15540), FMO (YUCCA1)(At4g32540), AMP1 (At3g54720), CYC2b (At4g35620), CYC1(At4g37490), CYC2a (At2g17620), EXPL3 (At3g45960), XTR9(At4g25820), XTR6 (At4g25810), and genes encoding glycosylhydrolase family protein 85 (At3g61000) and glycosyl trans-ferase family protein 48 (At1g06490), respectively. All genesexamined using RT-PCR showed the expression consistencywith those from genome-wide transcript profiles (Figure 3),indicating the reliability of our microarray data.

Discussion

Despite the prominent roles of plant secondary growth inthe economy as well as in plant development, the molecularmechanisms are well understudied when trees are used as ex-

perimental materials. In recent years, several research groupshave examined the whole-genome gene expression pattern indeveloping xylem tissues and have revealed some componentsthat might be important for secondary tissue growth (Oh et al.2003; Ko and Han 2004; Ko et al. 2004; Ehlting et al. 2005;Zhao et al. 2005). These studies provided important informationin elucidating the regulatory mechanisms by which the plantsecondary growth is controlled. However, we are still far fromfully understanding the complete process of plant secondarygrowth. In this study, we demonstrate an alternative approachby characterizing the Arabidopsis uro mutant, in which thesecondary cell wall formation is defective.

Hypocotyl is a tissue that was often used as a model forstudying the mechanism of cell development and a varietyof biological events that participate in its developmental con-trol, because of the simplicity of its cell types and structures(Vandenbussche et al. 2005). Different from wild-type plant,cell proliferation and cell wall lignification in the uro hypocotyl

uro Mutation and Secondary Cell Wall Growth 1759

Figure 3. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of selected genes with altered expression levels in the uro seedlings.

ACTIN transcripts were used as the internal control. RT-PCR was carried out using cDNA from the same stage seedlings as those used in GeneChip

arrays. Genes were selected from three clusters, including the hormone-correlated, cell cycle/division-related, and cell wall deposition-related clusters.

G, genomic DNA.

is severely blocked, resulting in reduced numbers of vascularcells and much less accumulated lignin in the secondary cellwalls of both hypocotyl and stem. Cellulose is known to beanother important composition in forming secondary cell walls.In addition to lignin, cellulose contents in the secondary cellwall of uro may also be reduced, as transcription level of theIRX9 gene which encodes an important glycosyl transferasefor cellulose synthesis (Pena at al. 2007) was dramaticallydownregulated (Table 1). Furthermore, a group of genes, which

encode putative xyloglucan endotransglycosylase (XTH) andplay a critical role in secondary cell wall formation (Vissenberget al. 2005a, 2005b) were also negatively regulated in the uromutant (Table 1).

We previously reported that the uro mutant has a very softinflorescence stem, due to the poorly developed interfascicularfiber cells (Guo et al. 2004). In the present study, we proposethat the cellular base of the soft uro stem is due to a lack oflignin and cellulose accumulation in the secondary cell walls.

1760 Journal of Integrative Plant Biology Vol. 49 No. 12 2007

Plant cell lignification is a complex process, undergoing fromlignin biosynthesis in the cell to deposition to the place wherethe plant secondary cell wall forms (Sederoff et al. 1999). Basedon the uro phenotypic analyses, we propose that cell states inuro may protect cells from lignification. It is known that dividingcells or cells in the young stages are not lignified. Growth patternanalyses showed that hypocotyls of uro were in a retardedcell division and differentiation manner, which may result in anelongated primary cell growth phase without secondary cell wallformation. This hypothesis was supported by the observationthat cell division was slowed and xylem and phloem tissueswere delayed to form in the uro hypocotyl.

The plant lignification process is known to be closely related tothe plant hormone actions (Biemelt et al. 2004; Zhao et al. 2005;Groover and Robischon 2006). In our microarray analyses, anumber of genes that are involved in the hormone biosynthesisand signaling in uro were changed in their expression levels.We previously proposed that the uro mutation may affect auxinregulatory pathways, because the uro single mutant showedsome auxin action defective phenotypes such as the loss ofapical dominance. In addition, uro mutation can geneticallyinteract with pin1, a mutant with a defective auxin polar trans-porter, and uro pin1 double mutant plants had very severepleiotropic phenotypes (Guo et al. 2004). Several functionalcategories of auxin, including signal perception and/or trans-duction, auxin transport, and auxin-mediated transcriptionalregulation and cellulose biosynthesis, were identified to beinvolved in secondary growth program (Zhao et al. 2005). Thelignification defect in the secondary cell wall formation, togetherwith other auxin action defective phenotypes in the uro mutant,is consistent with expression changes of the auxin-action relatedgenes. Additionally, our GeneChip array data also showed thatexpressions of a number of genes that associate with otherhormone pathways were changed. It was previously reportedthat several other plant hormones can influence secondary cellwall formation. For example, cytokinins, ethylene, gibberellins(GAs), brassinosteroids and xylogan all demonstrated theirroles in affecting vascular differentiation, though the underly-ing mechanisms are not clear (Sachs 2000; Fukuda 2004).Since crosstalk among hormone pathways occurs in manyplant growth and developmental processes and the uro mutantdemonstrated several auxin defective phenotypes (Guo et al.2004), it is possible that the altered expression levels of otherhormone-related genes may be caused by an indirect effect.

Our data also revealed the altered expression levels of cellcycle and cell division related genes as well as the cell wall de-position related genes in the uro mutant. These results might ex-plain some of the uro mutant phenotypes, including the aberrantxylem and phloem cell division and the abnormal lignin deposi-tion in the secondary cell walls. However, we cannot exclude apossibility that the expression changes of these genes may oc-cur downstream of the hormone regulation. Since the uro pheno-types were caused by a single nuclear gene mutation (Sun et al.

2000), isolation of the URO gene would help further elucidationof molecular mechanisms of the secondary cell wall formation.

Materials and Methods

Plant materials and growth conditions

The Arabidopsis mutant uro, which is in the Landsberg erecta(Ler) genetic background, was generated previously from a T-DNA mutagenesis experiment (Sun et al. 2000). For histolog-ical and microarray analyses, wild-type and uro seeds weresterilized with 70% ethanol for 15 min, followed by five washesusing distilled water. The sterilized seeds were sowed on platescontaining 1% agar and PNS (plant nutrition medium with 0.5%[w/v] sucrose) salts (Estelle and Somerville 1987), kept in 4 ◦Cfor 3 days and then moved to a growth chamber in 22 ◦C underwhite fluorescent light (200 µmol/m2 per s) with a photo periodof 16 h light and 8 h dark.

Histology and microscopy

Six- to 38-day-old plants were used for hypocotyl and stemsection observations. Hypocotyls of Ler and uro mutant werefixed in FAA (formalin : acetic acid; 50% ethanol = 5:5:90, v/v),and then dehydrated through a graded series of ethanol. Thehypocotyl specimens were embedded in resin overnight accord-ing to a previously described method (Chaffey et al. 2002),and sliced into 10–15-µm sections, which were subsequentlystained with 0.05% toluidine blue O (TBO) for 1.5 min andrinsed with water. The resultant sections were then sealed forobservation. For lignin examination, resin sections of hypocotyls(Ruellanda et al. 2003) or manual sections of stems (Guoet al. 2004) were stained with 1% phloroglucinol containing 70%ethanol for 2 min, followed by treatment with the concentratedHCl. Photos were taken according to our previous method (Guoet al. 2004).

Microarray and data analyses

Seedlings of 6-day-old wild-type and homozygous uro plantswere collected for total RNA extraction, using the RNAisoReagent (TaKaRa, Dalian, China). RNAs from two duplicateplant materials were used for labeling and hybridization, carriedout by Gene Tech Biotechnology Company (Shanghai, China),according to the standard Affymetrix protocol (Affymetrix, SantaClara, CA, USA, http://www.affymetrix.com/support/technical/manual). The Affymetrix GeneChip ATH1 array was used inthe array experiment. A coefficient of log 2 was used asthe selection criterion for differential gene expression betweenwild-type Ler and uro mutant plants. The value for geneexpression is the average of the two corresponding duplicates,and changes of transcript levels greater than twice were usedfor functional category analyses, using the COG software

uro Mutation and Secondary Cell Wall Growth 1761

(http://www.ncbi.nlm.nih.gov/COG/), with manual adjustmentwhen necessary (Tatusov et al. 2001).

RT-PCR

Reverse transcription-polymerase chain reaction was carriedout according to our previous method (Xu et al. 2003),with ACTIN as an internal control. Genes and their specificprimers that were used in the RT-PCR are listed below: forent-kaurene synthase gene, 5-CGGTTGCTTCTGGTTTCTTT-ATGTCTATCA-3 and 5-TGGTTTCTGTATGGTTTCATCAGT-GAC-3; for adenylate isopentenyltransferase 6 gene,5-CCTTGTTATCACCACCACTCTCTCATTCTT-3 and 5-CTC-CTCTACTCCTATCGTCTTCCGAA-3; for gibberellin responsemodulator gene, 5-GACGATGAAACGAAGCCATCAAGA-3and 5-TTCAGGTAGGGACACGAGTCGTAG-3; for auxin-responsive protein gene, 5-GTGAGAGAATATGGAA-GTCACCAATG-3 and 5-AGACAATGGATCATAAGGCAG-TAGG-3; for indole- acetic acid-induced protein 19 gene,5-CAAGAAATGGAGAA GGAAGGACTC-3 and 5-TGTTC-AAGTCATCATCACTCGTCTAC-3; for flavin-containingmonooxygenase gene, 5-CTGGAGAGTAAAGACTCATGA-TAACACAGA-3 and 5-TGACCATCCATAAACTTTGCTCC-3; for putative glutamate carboxypeptidase gene, 5-GGT-GCTAGAGCTCCGTTGGAGT-3 and 5-CTACATTAAGATAAG-CAACAGCACTTGCA-3; for cyclin 2b gene, 5-CAATGG-TTAATCCAGAGGAGAACA-3 and 5-ATCCTCCATCTCAA-CTTCTTCC-3; for G2/mitotic-specific cyclin gene, 5-AGAG-AACACTAAGATGATGACTTCTCGTTC-3 and 5-TGCGAGGT-CATTCTCAACATCAGC-3; for putative cyclin gene, 5-CTTCG-TTAAACCCACTTCAACGA-3 and 5-CCACTGTTACATCCTC-CATCTCAACT-3; for expansin family protein gene, 5-CTATG-GCTACGAGCTTCTTTGC-3 and 5-TGACCTCCTTGGTACAA-TAGCTTTATC-3; for endo-xyloglucan transferase gene, 5-C-TTCTCACTTGTACTCTTGACAAGGTCT-3 and 5-TCTCGTT-GTTCTTGAACACCCG-3; for putative endo-xyloglucan trans-ferase gene, 5-CAATCACTTAGCAATGGCGATGAT-3 and5-GAATTCTCTTATGGGAGTTCCATCCA-3; for glycosyl hyd-rolase family protein 85 gene, 5-ATGCTGCTCTATTTGCTGAA-GAG-3 and 5-CCATGTTTAGAGGTTTCATCTGATGT-3; forglycosyl transferase family 48 protein gene, 5-GGAACTTCG-TACCGTAGCTCA-3 and 5-TAGTTTGGAACTCAGACACAAA-TG-3; and for cyclin family protein, 5-ATGGCGGAACTTGAGA-ATCCAAG-3 and 5-CAACAACAACAACTCGCTGTTTGA-3.

Acknowledgements

The authors thank B. Xu for critical reading of this manuscript.

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(Handling editor: Yu-Xin Hu)