cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit

Gene 314 (2003) 141 – 148 www.elsevier.com/locate/gene

cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit Ling Yang 1, Bingsong Zheng, Chuanzao Mao, Keke Yi, Feiyan Liu, Yunrong Wu, Qinnan Tao, Ping Wu * The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hua Jiachi Campus, Hangzhou 310029, PR China Received 11 March 2003; received in revised form 5 May 2003; accepted 16 May 2003 Received by G. Theissen

Abstract The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin (PIP2a), 9-cis-epoxycarotenoid dioxygenase (NCED1) and a negative regulator of gibberellin signal transduction (SPY); eight other genes participated in cell wall loosening or vesicle traffic. D 2003 Elsevier B.V. All rights reserved. Keywords: Oryza sativa L.; Root elongation; Water deficit; Inducible expression

1. Introduction Rice is the staple food for more than half of the world’s population. With limited water resources, future increases in rice production will largely rely on rainfed production. Upland rice, which relies strictly on rainfall as a source of water, is often exposed to drought stress and has developed drought-resistant traits (Yadav et al., 1997). It has been demonstrated that the growth of a deep root system in

Abbreviations: cDNA, DNA complementary to RNA; AFLP, amplified fragment length polymorphism; TDFs, transcript-derived fragments; LDPCR, long-distance polymerase chain reaction; EST, expressed sequence tag. * Corresponding author. Tel.: +86-571-86971130; fax: +86-57186971323. E-mail address: [email protected] (P. Wu). 1 Present address: College of Life and Environmental Science, Zhejiang Normal University, Jinhua 321004, P.R. China. 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00713-3

upland rice can be stimulated by water deficits, and upland rice often has a deeper root system than lowland rice under water deficit conditions (Yadav et al., 1997). The elongation of seminal roots is critical for the establishment of upland rice seedlings under water deficits (Sharp, 2002). An investigation of the regulation of genes in seminal root tips may provide insight into the molecular mechanism of this stimulation of root elongation by water deficits in upland rice. cDNA-amplified fragment length polymorphism (AFLP) is an efficient, sensitive and reproducible technology that offers several advantages over other methods of gene expression analysis (Bachem et al., 1996; Ditt et al., 2001). It does not require prior sequence information and enables the identification of novel genes. In the present study, we used cDNA-AFLP technology to identify water deficitinducible transcript-derived fragments (TDFs) in seminal root tips of an upland tropical rice variety at four time

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points within 72 h of water deficit treatment. One hundred and six upregulated genes showed four different expression patterns and were involved in several biochemical pathways. These TDFs constitute c2.4% of the total detectable TDFs from the 100 primer combinations used. The functions of these inducible genes in the water deficit-stimulated elongation of rice seminal roots are discussed.

2.2. Cell length measurements in the elongation zone in seminal roots

2. Material and methods

2.3. cDNA-AFLP analysis

2.1. Plant material and culture

Total RNA (400 Ag) was extracted from approximate 160 seminal root tips sampled at the four indicated time points by using Trizol reagent (GIBCO, Germany), and poly (A)+ RNA was isolated from 250 Ag RNA with an Oligotex mRNA Mini Kit (QIAGEN, Germany). Control RNA was prepared by pooling equal amounts of RNA from flooding seminal root tips at the four time points. Double-stranded cDNA was synthesized using a SMARTk cDNA Library Construction Kit (Clontech, USA) according to the manufacturer’s instructions. LD-PCR products were purified by a QIAquick PCR Purification Kit (QIAGEN, Germany) and digested by the TaqI/AseI enzyme combination. AFLP reactions were performed according to published methods (Bachem et al., 1996). Selective amplification products were separated on a 6% polyacrylamide gel run at 60 W until the xylene cyanole reached the bottom. DNA fragments were visualized by silver-staining according to the Silver Sequencek DNA Sequencing System Technical Manual (Promega, USA).

Our previous screening experiments revealed that the elongation of seminal roots of upland rice was stimulated by water deficit much faster than that of lowland rice. Based on these screening results, an upland tropical japonica (Oryza sativa L.) variety Azucena was used in this study. A sand growth medium culture experiment was performed using two water supply conditions, flooding and water deficit, for RNA extraction and root length measurements. In the flooding condition, the water level was kept 2 mm above the sand surface, while in the water deficit condition, pots were taken out of the water tanks, water was allowed to drain through the holes in the bottom of the pots and the sand was dried for 72 h. Germinated seeds were directly sown into the pots, which were then placed in tanks under flooding conditions for 5 days before the water deficit treatment was imposed. The experiment was conducted in a greenhouse under a diurnal photoperiod of 12-h light (158 Amol m 2 s 1). The daily maximum and minimum temperatures were 28 and 22 jC, respectively. The relative humidity ranged from 65% to 85%. Two separate experiments were carried out for RNA extraction. Seminal root tips (1 cm) were harvested at 4, 16, 48 and 72 h after drainage treatment. The water content of the sand at the four time points after treatment was also determined (Fig. 1).

The seminal root tips (1 cm) were fixed in formaldehyde fixative. Root cortical cells in the elongation zone were viewed under an inverted microscope (Axiovert 200, Zeiss, Germany), and cell lengths were measured using AxioVision 3.1 software. Twenty cells from each of six seminal root tips were measured.

2.4. Isolation and sequencing of TDFs The differentially accumulated TDFs were recovered by PCR under the same conditions as used for preamplification. Purified PCR products were ligated to the pUCm-T vector. The clones were sequenced using MegaBACE 1000 (Amersham Pharmacia, USA). 2.5. RNA gel blot analysis Total RNA (20 Ag) was separated by electrophoresis on a 1.2% formaldehyde agarose gel followed by blotting onto a nylon membrane (Hybond-N+, Amersham Pharmacia). Hybridization was performed as described previously (Cho and Kende, 1997). Hybridized membranes were scanned by a Typhoon 8600 scanner (Molecular Dynamics, USA). 2.6. Gene function analysis

Fig. 1. Water content of sand at different depths at four time points.

The MegAlign program in the DNASTAR package was used to identify redundant genes, where redundancy was defined as when they exhibited more than 95% identity over aligned regions or to the same database accession. Database searches were performed using the BLAST Net-

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3. Results 3.1. Water deficit stimulates rice seminal root elongation

Fig. 2. Stimulated seminal root elongation under 72-h water deficit treatment (.) compared with that under flooding condition (o). (A) Seminal root elongation ratio (mm/day). (B) Length of mature cortical cells in the seminal root elongation zone.

work Service (NCBI, National Center for Biotechnology Service) (http://www.ncbi.nlm.nih.gov/BLAST). The retrieved genomic sequences were further annotated at a web site (http://ricegaas.dna.affrc.go.jp/) or analyzed using the GENSCAN program (http://bioweb.pasteur.fr/seqanal/ interfaces/genscan.html). The functions of function-known genes by BLASTX and TBLASTX searches (E value cutoff=1e 4) (Ditt et al., 2001) were classified according to the putative function.

To assess the growth responses of seminal roots to a water deficit in an upland rice variety, Azucena, the 72h drain-off system was used in this study. As a result, the seminal root elongation rate was significantly stimulated ( P<0.01) during 72 h of water deficit, and peaked at 24 h (Fig. 2A). The lengths of mature cortical cells from the elongation zone in rice seminal root showed a rapid increase from 4 to 16 h of the water deficit, and decreased after 16 h (Fig. 2B). The results indicate that the stimulated elongation of cells in the elongation zone was at least partly responsible for the enhancement of seminal root growth under water deficit stress. 3.2. Differentially accumulated TDFs in rice seminal root tips within 72 h of the water deficit cDNA expression patterns were identified by selective amplification using 100 primer combinations, a total of 141 differentially accumulated TDFs from genes were identified. One hundred and twenty-two TDFs, ranging in length from 100 to 600 bp, were cloned and sequenced. These 122

Fig. 3. Comparison of expression patterns of 21 TDFs by both cDNA-AFLP (A) and Northern blotting (B). Templates derived from seminal root tips after water deficit treatment for 0, 4, 16, 48 and 72 h, respectively. Ribosomal RNAs were stained with ethidium bromide (EtdBr).

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Table 1 Accumulated expression of TDFs in rice seminal root tips subjected to water deficit TDFa

Size (bp)

Accumulationb 4h

16 h

48 h

Sequence similarity

Accession no.

GenBank Hit

CB238397 CB238399 CB238400 CB238401 CB238402 CB238403

AL513005 CAA08997 BI808607 BAA94239 AP005710 BAA92952

CB238404 CB238405

NP_198445 AB012046

CB238406 CB238407

D10957 AAC36055

CB238408 CB238409

AC084320 BAC10549

CB238410

AF262215

CB238411 CB238412 CB238413 CB238414

U09450 U30477 JE0156 NP_567009

CB238415 CB238416 CB238417 CB238418

AU057092 NP_180858 AF062393 D29693

72 h

Induced within 4 h of water deficit treatment: 31 (2)c T41# 242 + T16# 175 + + T23# 143 + + T36 267 + + T52 316 + + T63 179 + +

+ + + + +

L13 A3#

275 231

+ +

+ +

+ +

+ +

T1# T49#

441 414

+ +

+ +

+ +

+ +

T62# L22#

241 236

+ +

+ +

+ +

+ +

T18#

302

+

+

+

+

T31# T37# T40# T47#

135 140 196 423

+ + + +

+ + + +

+ + + +

+ + + +

T51# T71# T79# T83# Unknown proteins (8)

136 174 479 290

+ + + +

+ + + +

+ + + +

+ + + +

Polyprotein from transposon TNT MAP3K beta 1 protein kinase Fruit-ripening protein similar to ASR Lipase Reverse transcriptase Similar to Homo sapiens mRNA for KIAA0039 Serine/threonine protein kinase Adenine phosphoribosyltransferase (APRT) O-acetylserine (thiol)-lyase (OASTL) Gibberellin action negative regulator (SPY) Actin-depolymerizing factor (ADF) 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) Guanine nucleotide exchange protein (GEP2) Enolase Expansin (OsEXP2) Xyloglucan endotransferase (XET) Vacuolar protein sorting protein 33a (VPS33a) GTP-binding protein rab11b (Rab11b) Endo-glucanase family 9 (EGase) Aquaporin (PIP2a) Calmodulin (CaM)

CB238429 CB238430 CB238431

BAB09011 NP_197180 NP_189916

+ + + + + + +

Autophagocytosis protein-like (APG) Similar to SART1 protein Guanine nucleotide exchange-like protein (GEP) Disease resistance protein I2 Peroxidase Pyruvate dehydrogenase kinase Integral membrane protein (OsNramp3) S-adenosyl-methionine methyltransferase Xaa-Pro aminopeptidase 1 Pathogenesis-related protein 1 Deoxyguanosine kinase Drought-induced protein Di19

CB238432 CB238433 CB238434 CB238435 CB238436 CB238437 CB238438 CB238439 CB238440

BAB89710 NP_567919 AF323611 U60767 NP_196057 CAC59823 AU075829 AAG51141 AU101147

+ + + + + + + + + +

Glycine-rich protein bHLH protein Regulator of chromosome condensation Cytochrome P450 monooxygenase Cytochrome c oxidase subunit 5c Retroelement Co-repressor protein GTPase SR1 induced by sucrose starvation xABC transporter-like protein Receptor-like protein kinase precursor Stomatin-like protein DNA-binding protein

CB238450 CB238451 CB238452 CB238453 CB238454 CB238455 CB238456 CB238457 CB238458 CB238459 CB238460 CB238461 CB238462

AC113334 NP_563749 AC111016 NM_115968 AB027123 AC108761 BAB78631 NP_463213 U16256 NP_200882 AL662944 AAF68388 NM_101067

Induced after 16 h of water deficit treatment: 21 (7)c L9# 274 + L16# 234 + T65# 381 + L1 T26 A2 T21 T27 T57 T70 T75 T90 Unknown proteins (2)

283 276 366 400 303 364 99 327 199

+ + + + + + + + +

Induced after 48 h of water deficit treatment: 26 (4)c T77 276 T82 318 T84 208 D2 247 D4 227 L5 211 L10 314 L14 207 L15 469 T4 234 T6 202 T7 298 T9 532 Unknown proteins (9)

+ + + + + + + + +

+ + + + + + + + + + + + +

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Table 1 (continued ) TDFa

Size (bp)

Accumulationb 4h

16 h

48 h

Sequence similarity

Accession no.

GenBank Hit

Late embryogenesis abundant protein Low-temperature-induced protein Myosin-like protein Aspartyl tRNA synthetase Inositol 1,3,4-trisphosphate kinase Similar to gene for Pib NBS-LRR type resistance protein Alpha-mannosidase Potential cadmium/zinc-transporting ATPase ABC transporter-like protein FtsH-metalloproteinase Glutamyl-tRNA amidotransferase subunit A H1 histone NADH dependent glutamate synthase

CB238476 CB238477 CB238478 CB238479 CB238480 CB238481 CB238482 CB238483 CB238484

NP_174694 AB011367 NP_173599 D23307 AA753305 BAA89577 AP005811 AAL73068 BM419260

CB238485 CB238486 CB238487

BAB09847 NP_566889 BAB11605

CB238488 CB238489

CAA44318 AB008845

72 h

Induced after 72 h of water deficit treatment: 28 (5)c A1 451 D6 114 T11 278 T24 222 T28 127 T34 319 T54 300 T56 274 T72 222

+ + + + + + + + +

T73 T74 T78

224 267 577

+ + +

T81 T91 Unknown proteins (9)

215 341

+ +

a TDF with # showed a maximal accumulation at 4 or 16 h of water deficit. An unknown protein (accession no. CB238421) and an unknown gene (accession no. CB238444) are also included. b (+) means accumulation at indicated time point after water deficit treatment. c The total number of TDFs is indicated, with the number of unknown genes shown in parentheses.

clones represent a nonredundant set of 106 genes. To validate the cDNA-AFLP expression patterns, 21 TDFs were selected among the different expression pattern groups for RNA gel blot analysis. The comparisons showed that 16 of the 21 cDNAs examined had the same expression profiles as on the cDNA-AFLP analysis. The remaining five cDNAs, CaM, NCED1, PIP2a, EGase and GEP2, showed differential expression patterns (Fig. 3). These results indicate that the original cDNA-AFLP pattern was validated in 76% of the cases, so the general approach is a reliable method for identifying upregulated genes in rice seminal root tips during the water deficit. The 106 unique genes could be classified into four groups with different upregulation patterns during the time course of the water deficit. Thirty-one genes were upregulated within 4 h of the stress, 21 genes after 16 h, 26 after 48 h and 28 after 72 h (Table 1). In group 1, 14 genes showed upregulated during the time course of the treatment, with peak expression at the 16 h time points. 3.3. Functional classification of induced genes by the water deficit in rice seminal root tips Among the 106 upregulated genes, 60 were genes of known function which could be grouped into seven functional categories, including transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element (Table 2). Some of 60 genes with known function are first reported in rice, such as MAP3K beta 1 protein kinase (MAP3K), 9-cis-epoxycarotenoid dioxygenase 1 (NCED1),

a negative regulator of gibbrellin signal transduction (SPY), an SR-related protein essential for spliceosome assembly (SART1) (Bolland and Hewitt, 2001). Seven genes for signal transduction components and six for expression regulators were upregulated in the water-deficient root tips (Table 1). Among the seven signal trans-

Table 2 Functional categories of genes induced by the water deficit in rice seminal root tips Major functional categoriesa Transport facilitation Metabolism and energy Stress- and defense-related protein Cellular organization and cell-wall biogenesis Signal transduction Expression regulator Transposable element Unclassified protein Other rice EST No hit Total

Numberb

Percentage

11 (6) 14 (3) 10 (1)

12.3 12.3 9.4

9 (4)

8.5

7 6 3 28 2 16 106

(3) (2) (1) (1) (0) (1) (22)

6.6 5.7 2.8 25.5 1.9 15.1 100

a Unclassified protein denotes sequence that is homologous to unknown, hypothetical or putative protein with unknown function in other organisms. Other rice EST indicates matches to unannotated rice ESTs. No hit indicates identity only to unannotated genomic sequences or low similarity to existing nucleotide sequences. b The total number of TDFs with a maximal accumulation before 16 h of water deficit treatment are indicated in parentheses. The accession number of an unknown protein in unclassified protein category and an unknown gene in no hit category are CB238421 and CB238444, respectively.

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duction components, four genes for calmodulin (CaM), MAP3K, NCED1 and serine/threonine protein kinase were rapidly induced (Table 1) and may be involved in the early signal transduction under water deficit stress. The expression of three other genes for GTPase, receptor-like protein kinase precursor and inositol 1,3,4-trisphosphate kinase were increased in the later stage of the response (Table 1). Six expression regulators were upregulated with different inducible pattern, including SPY, SART1, a bHLH protein, a regulator of chromosome condensation, a corepressor protein and a DNA-binding protein (Table 1). The fact that these 13 genes were induced at different stages of the response suggests that they may be involved in the control of different processes in the water deficit response. Ten stress- and defense-related genes were upregulated, including SR1 (induced by sucrose starvation), low-temperature-induced protein and fruit-ripening protein similar to ASR (ABA- and stress-induced protein) as well as four defense-related genes (Table 1), suggesting an overlap in function between water deficit and other biotic and abiotic stresses. A total of 27 of the 106 genes encode proteins with unknown function, and the remaining 16 sequences showed similarity only to unannotated genomic sequences or no match in the nucleotide database (Table 2), which suggests that there are several new genes responsive to the water deficit in rice seminal root tips.

4. Discussion The peak of seminal root elongation rate occurs within 24 h of the water deficit treatment (Fig. 2). Therefore, it

could be suggested that the genes with a maximal expression at 4 or 16 h of water deficit may be related to the root elongation stimulated by the water deficit at the transcriptional level. Twenty-two of the 106 genes were identified in this case as potential candidates, including 14 genes, whose expression was consistent with the changes in root length (Tables 1 and 2). Among the 22 candidates, 10 genes were previously recognized as cell elongation-related genes, including aquaporin (PIP2a) (Malz and Sauter, 1999), endo-glucanase (EGase) (Shani et al., 1997), xyloglucan endotransferase (XET) (Uozu et al., 2000), expansin (OsEXP2) (Cho and Kende, 1997), actin-depolymerizing factor (ADF) (Chen et al., 2002), SPY (Swain et al., 2002), GTP-binding protein rab11b (Rab11b) (Ueda and Nakano, 2002), guanine nucleotide exchange protein (GEP and GEP2) (Geldner et al., 2003) and adenine phosphoribosyltransferase (APRT) (Zhang et al., 2002). However, relatively little is known about their roles in root elongation under water deficits. Three genes encoding enolase in glycolysis, O-acetylserine (thiol)-lyase (OASTL) in S-assimilation (Barroso et al., 1999) and APRT in the main adenine salvage pathway (Zhang et al., 2002), may supply material and energy for growing cells (Fig. 4). ADF plays a crucial role in the regulation of actin remodeling in cell growth process (Chen et al., 2002). The expression of ADF was rapidly induced under the water deficit (Fig. 3) indicating that the rate of actin turnover may be enhanced (Fig. 4) (Chen et al., 2002). As for ASR, SART1, polyprotein from transposon TNT, an unknown protein and an unknown gene (Table 1), no available information indicated that they were related to root elongation. Other genes whose expression reached a maximum at 48 or 72 h of the water deficit treatment, such as SR1 and pyruvate dehydrogenase kinase (Fig. 3), are likely involved in tolerance to severe stress.

Fig. 4. A simple model of root elongation stimulated by the water deficit. Genes in italics were identified in this study. Those labeled with * mean that ABRE or CE3 sequences were observed in 2000 bp immediately upstream from the ATG. PK, protein kinase; PP, protein phosphase; TF, transcription factor; 2nd SM, second messenger molecules.

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4.1. Four genes and their possible roles in signal transduction and expression regulation during root elongation stimulated by the water deficit Under water stress, a general signal transduction pathway starts with signal perception, followed by the synthesis and release of short-lived second messengers (Neill and Burnett, 1999). Second messengers can modulate intracellular Ca2+ level, often initiating a protein phosphorylation cascade (Xiong et al., 2002). CaM is the best known Ca2+ sensor. Recent studies suggest that CaM perceives local Ca2+ and activates a MAPK pathway (Xiong et al., 2002). In this case, the upregulation pattern of CaM and MAP3K (Table 1) suggest that both of components mediate water deficitinduced signal transduction pathways. These signal molecules may activate the expression of certain genes encoding transcription factors (TFs) (Fig. 4) (Neill and Burnett, 1999). The TFs subsequently activate the expression of stress- or ABA-inducible genes during root elongation stimulated by the water deficit (Fig. 4) (Neill and Burnett, 1999; Xiong et al., 2002). When root cells experience changes in their external water status, enhanced ABA synthesis in root tips is probably the key response (Neill and Burnett, 1999; Sharp, 2002). The formation of xanthoxal by NCED is the key limiting step in ABA biosynthesis (Neill and Burnett, 1999; Sharp, 2002). In this study, rice NCED1 was identified. The fact that its upregulated expression pattern matched the time course of changes in root length (Fig. 3) supports the idea that an increased concentration of endogenous ABA may function to promote root growth under water deficits (Sharp, 2002). In addition, ABA induces the expression of hundreds of genes (Neill and Burnett, 1999). cis-acting elements involved in ABA-inducible gene expression, such as the ABA responsive element (ABRE, PyACGT(T/G)C) (Neill and Burnett, 1999) and coupling elements (CE3, CGCGTG(T/G)C) (Hobo et al., 1999), have been reported. The 5Vregion of the genes were retrieved by performing BLASTN queries of the respective TDFs against the complete genome sequence. The sequences of the 5Vregions (2000 bp immediately upstream from the ATG ) were used to obtained shared motifs by finding ABRE and CE3. These motif search algorithms were performed using the Motif Sampler algorithms, which can be accessed through the PlantCARE database web site (http://www.esat.kuleuven. ac.be/~thijs/Work/MotifSampler.html). Among the 22 genes identified in this case, the 5Vsequences were found in 20 genes. Five genes (MAP3K, NCED1, OsEXP2, XET and ADF) contained ABRE or CE3 in their promoters (Table 3), suggesting that they may be ABA-inducible. Interestingly, the promoter of NCED1 also contains an ABRE, suggesting that the possible positive feedback regulation of ABA biosynthesis by ABA exists. SPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter

147

Table 3 Presence and position of motifs in 5Vregion of five genes Gene MAP3K beta 1 protein kinase (MAP3K) 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) Actin-depolymerizing factor (ADF) Expansin (OsEXP2) Xyloglucan endotransferase (XET)

ABRE

CE3 CGCGTGGC ( 528)

CACGTGGC ( 552)

CGCGTGTC ( 1684) TACGTGGC ( 1574) TACGTGTC ( 1726)

(Robertson et al., 1998). An investigation of the spy mutant phenotype suggested that SPY may play a role in root growth (Swain et al., 2002), but the detailed mechanisms of its role in water deficit-stimulated root elongation are not well understood. The upregulation of SPY during water deficit may result in the activation of ABA-responsive genes (Fig. 4). 4.2. Upregulation of PIP2a and three genes encoding cellwall-loosening proteins during root elongation stimulated by the water deficit Cell expansion is initiated by stress relaxation of the cell wall (Lee et al., 2001). We identified three genes for cell wall-loosening proteins, OsEXP2, XET and EGase, which were rapidly induced in rice seminal root tips by the water deficit and showed expression patterns matching the time course changes of root length (Fig. 3). Expression of OsEXP2 in elongating rice internodes (Cho and Kende, 1997), EGase in tobacco root elongating zones (Shani et al., 1997) and XET in elongating rice internodes (Uozu et al., 2000) were investigated, the results indicating that they are correlated with root elongation. Under water deficit, upregulation of OsEXP2, XET and EGase, probably make the cell walls in the elongation zone more extensible, thereby stimulating root elongation (Wu and Cosgrove, 2000; Lee et al., 2001) (Fig. 4). Cell expansion is driven by the uptake of water into the vacuole (Lee et al., 2001). A significant component in cellular water transport is aquaporin, which is enriched in zones of rapid cell division and expansion (Malz and Sauter, 1999; Javot and Maurel, 2002). Roots that show a remarkable capacity to alter their water permeability over the short term in response to stress are also mediated by aquaporins (Javot and Maurel, 2002). We identified an aquaporin in the plasma membrane, PIP2a, which showed an inducible expression pattern that paralleled that of root length (Fig. 3), indicating a possible role in the root elongation stimulated by the water deficit (Fig. 4). Altogether, cell growth requires coordinated water uptake and irreversible cell wall enlargement (Wu and Cosgrove, 2000).

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4.3. Vesicle traffic may be implicated in the root elongation stimulated by the water deficit Five genes, VPS33a (vacuolar protein sorting protein), Rab11b, GEP, GEP2 and APG (autophagocytosis protein) (Table 1) are involved in vesicle traffic. VPS33a is a component of the Class C VPS complex that plays essential roles in the processes of membrane docking and fusion at Golgi-to-vacuole protein transport (Sato et al., 2000). Rab11 has been shown to play a role in the late recycling of transferrin receptors and in trans-Golgi network-to-plasma membrane transport. Transgenic studies have shown that Rab11 homologues are involved in cell elongation (Ueda and Nakano, 2002). Both GEP and GEP2 encode a guanine nucleotide exchange factor for small G proteins of the ADP ribosylation factor class, which are localized in endosomes and act as a regulator of intracellular vesicle trafficking. ARF-GEP is also required for the recycling of auxin transport components, thereby affecting root elongation (Geldner et al., 2003). APG was proposed to be involved in a transport system from the cytoplasm to vacuole, leading to dynamic rearrangements of cellular membranes (Klionsky and Emr, 2000). As shown in Table 1 and Fig. 3, the expression changes of VPS33a, Rab11b and GEP2 was consistent with the changes in root length, whereas the expression of GEP and APG was upregulated at the 16 h time point. This indicates that vesicle traffic is essential for plant cell growth, and delivers precursor material and membrane necessary for the cell growth stimulated by the water deficit (Fig. 4) (Ueda and Nakano, 2002). In summary, we identified 106 upregulated genes in an upland rice seminal root tips within 72 h of the water deficit. Among them, 22 genes may be related to root elongation stimulated by the water deficit at the transcriptional level. A simple model outlining the sequences of cellular events linking perception of a water-deficit signal to root elongation is shown in Fig. 4. The data obtained here will provide the first clues for guiding further functional studies of root cell growth stimulated by water deficit in rice. Acknowledgements The research was supported by the National Key Basic Research Special Foundation of China (G 1999011700). References Bachem, C.W.B., van der Hoeven, R.S., de Bruijn, S.M., Vreugdenhil, D., Zabeau, M., Visser, R.G.F., 1996. Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9, 745 – 753. Barroso, C., Romero, L.C., Cejudo, F.J., Vega, J.M., Gotor, C., 1999. Salt-

specific regulation of the cytosolic O-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid. Plant Mol. Biol. 40, 729 – 736. Bolland, D.J., Hewitt, J.E., 2001. Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis. Gene 271, 43 – 49. Chen, C.Y., Wong, E.I., Vidali, L., Estavillo, A., Hepler, P.K., Wu, H.M., Cheung, A.Y., 2002. The regulation of actin organization by actindepolymerizing factor in elongating pollen tubes. Plant Cell 14, 2175 – 2190. Cho, H.T., Kende, H., 1997. Expression of expansin genes is correlated with growth in deepwater rice. Plant Cell 9, 1661 – 1671. Ditt, R.F., Nester, E.W., Comai, L., 2001. Plant gene expression response to Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. U. S. A. 98, 10954 – 10959. Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W., Muller, P., Delbarre, A., Ueda, T., Nakano, A., Jurgens, G., 2003. The Arabidopsis GNOM ARF-GEP mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112, 219 – 230. Hobo, T., Asada, M., Kowyama, Y., Hattori, T., 1999. ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J. 19, 679 – 689. Javot, H., Maurel, C., 2002. The role of aquaporins in root water uptake. Ann. Bot. 90, 301 – 313. Klionsky, D.J., Emr, S.D., 2000. Autophagy as a regulated pathway of cellular degradation. Science 290, 1717 – 1721. Lee, Y., Choi, D., Kende, H., 2001. Expansins: ever-expanding numbers and functions. Curr. Opin. Plant Biol. 4, 527 – 532. Malz, S., Sauter, M., 1999. Expression of two PIP genes in rapidly growing internodes of rice is not primarily controlled by meristem activity or cell expansion. Plant Mol. Biol. 40, 985 – 995. Neill, S.J., Burnett, E.C., 1999. Regulation of gene expression during water-deficit stress. Plant Growth Regul. 29, 23 – 33. Robertson, M., Swain, S.M., Chandler, P.M., Olszewski, N.E., 1998. Identification of a negative regulator of gibberellin action, HvSPY, in barley. Plant Cell 10, 995 – 1007. Sato, T.K., Rehling, P., Peterson, M.R., Emr, S.D., 2000. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell 6, 661 – 671. Shani, Z., Dekel, M., Tsabary, G., Shoseyov, O., 1997. Cloning and characterization of elongation specific endo-1,4-beta-glucanase (cel1) from Arabidopsis thaliana. Plant Mol. Biol. 34, 837 – 842. Sharp, R.E., 2002. Interaction with ethylene: changing views on the role of abscisic acid in root and shoot growth responses to water stress. Plant Cell Environ. 25, 211 – 222. Swain, S.M., Tseng, T.S., Thornton, T.M., Gopalraj, M., Olszewski, N.E., 2002. SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant. Plant Physiol. 129, 605 – 615. Ueda, T., Nakano, A., 2002. Vesicular traffic: an integral part of plant life. Curr. Opin. Plant Biol. 5, 513 – 517. Uozu, S., Tanaka-Ueguchi, M., Kitano, H., Hattori, K., Matsuoka, M., 2000. Characterization of XET-related genes of rice. Plant Physiol. 122, 853 – 860. Wu, Y., Cosgrove, D.J., 2000. Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. J. Exp. Bot. 51, 1543 – 1553. Xiong, L., Schumaker, K.S., Zhu, J.K., 2002. Cell signaling during cold, drought, and salt stress. Plant Cell Suppl., S165 – S183. Yadav, R., Courtois, B., Huang, N., McLaren, G., 1997. Mapping genes controlling root morphology and root distribution in a doubled-haploid population of rice. Theor. Appl. Genet. 94, 619 – 632. Zhang, C., Guinel, F.C., Moffatt, B.A., 2002. A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3. Protoplasma 219, 59 – 71.