TY - JOUR
AU - Shinozaki, Kazuo
AB - Abstract Full‐length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. 155 144 RIKEN Arabidopsis full‐length (RAFL) cDNA clones were isolated. The 3′‐end expressed sequence tags (ESTs) of all 155 144 RAFL cDNAs were clustered into 14 668 non‐redundant cDNA groups, about 60% of predicted genes. The sequence database of the RAFL cDNAs is useful for promoter analysis and the correct annotation of predicted transcription units and gene products. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. RAFL cDNA microarrays were prepared, containing independent full‐length cDNA groups for analysing the expression profiles of genes under various stress‐ and hormone‐treatment conditions and in various mutants and transgenic plants. In this review, recent progress on transcriptome analysis using the RAFL cDNA microarray is highlighted. cDNA microarray, cold stress, drought stress, full‐length cDNA, gene expression, high‐salinity stress, RAFL cDNA. Received 21 March 2003; Accepted 22 July 2003 Introduction Arabidopsis thaliana has been adopted as a model organism in the study of plant biology because of its small size, short generation time, and high efficiency of transformation (Meinke et al., 1998). In order to sequence its small genome (125 Mb) (The Arabidopsis Genome Initiative, 2000), scientists in Japan, Europe and the USA collaborated in the Arabidopsis genome sequencing project (Bevan, 1997). Two of the five chromosomes (chromosomes 2 and 4, except for the nucleolar organizer regions and centromeres) were sequenced in 1999 (Lin et al., 1999; Mayer et al., 1999), and the remaining three chromosomes were sequenced in 2000 by the Arabidopsis Genome Initiative (AGI) (The Arabidopsis Genome Initiative, 2000). About 177 000 expressed sequence tags (ESTs) from Arabidopsis have been deposited in the EST database (dbEST) as of November 2002, including sequences from large‐scale EST projects in France (Höfte et al., 1993; Cooke et al., 1996), USA (Newman et al., 1994; White et al., 2000), and Japan (Asamizu et al., 2000). These projects have produced EST data from different tissues, organs, seeds, and developmental stages (Höfte et al., 1993; Newman et al., 1994; Cooke et al., 1996; Asamizu et al., 2000; White et al., 2000). However, most of these EST projects are based on cDNA libraries in which most of the inserts are not full‐length. ESTs are useful for making a catalogue of expressed genes, but not for further study of gene function. Consequently, genome‐scale collections of the full‐length cDNAs of expressed genes are important for the analysis of the structure and function of genes and their products in this functional genomics era. Arabidopsis full‐length cDNA libraries have been constructed from plants grown under different conditions as reported previously (Seki et al., 1998, 2002a) by the biotinylated CAP trapper method using trehalose‐thermoactivated reverse transcriptase (Carninci et al., 1996, 1997, 1998). 155 144 RIKEN Arabidopsis full‐length (RAFL) clones were isolated and clustered into 14 668 non‐redundant cDNA groups (Seki et al., 2002a). Microarrays are powerful tools for systematically analysing expression profiles of large numbers of genes, including stress‐inducible gene expression, tissue‐specific gene expression, and changes in the expression profiles of mutants and transgenic plants (Eisen and Brown, 1999; Richmond and Somerville, 2000). This DNA chip‐based technology arrays cDNA sequences on a glass slide at a density >1000 genes cm–2. These arrayed sequences are hybridized simultaneously to a two‐colour fluorescently labelled cDNA probe pair prepared from RNA samples of different cell or tissue types, allowing direct and large‐scale comparative analysis of gene expression. The full‐length cDNAs were used for the microarray analysis of expression profiles of Arabidopsis genes under various stress conditions and in various mutant and transgenic plants (Fig. 1) (Seki et al., 2001a, b). Full‐length cDNAs are also useful for determining the three‐dimensional structures of proteins by X‐ray crystallography and NMR spectroscopy and for studying the protein function by biochemical analyses and transgenic analyses (Fig. 1) (Seki et al., 2001b). In this review article, the present state and perspectives of the analysis of the RAFL cDNAs, such as their collection and microarray analysis, are summarized. Collection and functional annotation of RAFL cDNAs Until now, 19 full‐length cDNA libraries have been constructed from Arabidopsis plants grown under various stress, hormone and light conditions, from plants at various developmental stages, and from various plant tissues (Seki et al., 2002a). Single‐pass sequencing of the cDNA clones were performed from the 3′‐end. 155 144 3′‐end ESTs have been obtained as of February 2002 (Seki et al., 2002a). The 155 144 3′‐ESTs were clustered and then mapped onto the Arabidopsis genome. Finally, 14 668 non‐redundant RAFL cDNA clones were identified and mapped on the Arabidopsis genome (Seki et al., 2002a). The information of the 14 668 RAFL cDNA clones (the ‘RAFL cDNA’ genes) is available at http://www.sciencemag.org/content/vol0/issue2002/images/data/1071006/DC1/table_s1.zip (Seki et al., 2002a). Assuming that the total number of Arabidopsis genes is about 26 000, the RAFL clones isolated should account for about 60% of all Arabidopsis genes. From the 5′‐end sequences of mRNAs the promoter sequences can be obtained by comparison with the Arabidopsis genomic sequences. 5′‐ESTs were also obtained of 14 034 RAFL cDNA clones and a promoter database was constructed (Seki et al., 2002a) using the Plant cis‐acting regulatory DNA elements (PLACE) database (Higo et al., 1999). The Arabidopsis promoter database constructed contains the information on the genomic sequences 1000 bp upstream from the 5′‐termini of each RAFL cDNA clone, and about 300 cis‐acting elements known from plants (Seki et al., 2002a). The Arabidopsis promoter database constructed is available at http://www.sciencemag.org/content/vol0/issue2002/images/data/1071006/DC1/table_s1.zip (Seki et al., 2002a, d). One of the interesting types of the microarray analysis is the identification of novel cis‐elements that regulate the expression of genes in response to various experimental treatments. By identifying subsets of the genes that have a common expression profile, it might be possible to identify conserved motifs in the promoter regions. This promoter database is expected to be useful for systematic analysis of cis‐acting elements in Arabidopsis (Seki et al., 2002a, d). Although many algorithms have been written to predict a transcription unit from genomic sequence data, the accuracy of their predictions is still limited. A more direct and efficient approach to identify coding sequences is to sequence full‐length cDNAs (Fig. 1). The results described demonstrate the power of full‐length cDNA sequences for improving the quality of multiple aspects of genome annotation. The full‐length sequences of the RAFL cDNA clones have been determined in collaboration with the Arabidopsis SSP group of the USA, which comprises the Salk Institute (principal investigator: Dr Joseph R Ecker), the Stanford Genome Technology Center (principal investigator: Dr Ronald W Davis), and the Plant Gene Expression Center (principal investigator: Dr Athanasios Theologis). Complete sequences of the Arabidopsis full‐length cDNAs will be used for gene annotation, such as UTRs, transcription start site and alternative splicing, and positional cloning. The RAFL cDNA clones are publicly available from the RIKEN Bioresource Center (http://www.brc.riken.go.jp/lab/epd/Eng/index.html). RAFL cDNA microarray analysis Recently, microarray technology has become a powerful tool for the systematic analysis of expression profiles of large numbers of genes (Eisen and Brown, 1999; Richmond and Somerville, 2000; Seki et al., 2001b). The full‐length cDNAs were used for the microarray analysis of expression profiles of Arabidopsis genes under various stress conditions, such as drought, cold and high‐salinity‐stresses (Seki et al., 2001a, 2002b), and high light stress (Kimura et al., 2003), various treatment conditions, such as abscisic acid (ABA) (Seki et al., 2002c), rehydration treatment after dehydration (Oono et al., 2003), ethylene (Narusaka et al., 2003), jasmonic acid (JA) (Narusaka et al., 2003), salicylic acid (SA) (Narusaka et al., 2003), reactive oxygen species (ROS)‐inducing compounds such as paraquat and rose bengal (Narusaka et al., 2003), UV‐C (Narusaka et al., 2003), proline (Pro) (Satoh et al., 2002), and inoculation with pathogens (Narusaka et al., 2003). The expression profiles in various mutants and transgenic plants have also been studied (Seki et al., 2001a; Osakabe et al., 2002; Abe et al., 2003; Dubouzet et al., 2003; Narusaka et al., 2003) (Table 1). A full‐length cDNA microarray is useful for analysing the expression pattern of Arabidopsis genes under various stress and hormone treatments. By combining the expression data with the genomic sequence data, target genes of stress‐related transcription factors and potential cis‐acting DNA elements can be identified. In this review article, recent progress on transcriptome analysis under abiotic stress conditions using RAFL cDNA microarray is summarized. Application of RAFL cDNA microarray analysis to identify drought‐, cold‐, high‐salinity‐stress‐ or ABA‐regulated genes Plant growth is greatly affected by environmental abiotic stresses, such as drought, high salinity and low temperature. Plants respond and adapt to these stresses in order to survive. These stresses induce various biochemical and physiological responses in plants. Several genes that respond to drought, high‐salinity or cold stress at the transcriptional level have been studied (Hasegawa et al., 2000; Shinozaki and Yamaguchi‐Shinozaki, 2000; Thomashow, 1999; Zhu, 2002). The products of the stress‐inducible genes have been classified into two groups: those that directly protect against environmental stresses and those that regulate gene expression and signal transduction in the stress response. The first group includes proteins that probably function by protecting cells from dehydration, such as the enzymes required for biosynthesis of various osmoprotectants, late‐embryogenesis‐abundant (LEA) proteins, antifreeze proteins, chaperones, and detoxification enzymes. The second group of gene products includes transcription factors, protein kinases, and enzymes involved in phosphoinositide metabolism. Stress‐inducible genes have been used to improve the stress tolerance of plants by gene transfer (Hasegawa et al. 2000; Shinozaki and Yamaguchi‐Shinozaki, 2000; Thomashow, 1999). Although hundreds of genes have been found to be involved in abiotic stress responses (Shinozaki and Yamaguchi‐Shinozaki, 1999, 2000; Xiong and Zhu, 2001, 2002; Xiong et al., 2002; Zhu, 2002), many genes have not been identified. Therefore, the full‐length cDNA microarray containing c. 1300 RAFL cDNAs was applied to identify new drought‐ or cold‐inducible genes (Seki et al., 2001a). Forty‐four and 19 cDNAs for drought‐ and cold‐inducible genes, respectively, were isolated, 30 and 10 of which were novel stress‐inducible genes that have not been reported as drought‐ or cold‐inducible genes previously. As described above, overexpression of the DREB1A/CBF3 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter or the stress‐inducible rd29A promoter in transgenic plants gave rise to strong constitutive expression of the stress‐inducible DREB1A target genes and increased tolerance to freezing, drought, and salt stresses (Kasuga et al., 1999; Liu et al., 1998). Kasuga et al. (1999) identified six DREB1A target genes. However, it is not clearly understood how overexpression of the DREB1A cDNA in transgenic plants increases stress tolerance to freezing, drought and high salinity stresses. The full‐length cDNA microarray containing c. 1300 RAFL cDNAs was applied in order to identify new target genes of DREB1A (Seki et al., 2001a). Twelve stress‐inducible genes were identified as target stress‐inducible genes of DREB1A, and six of them were novel. All DREB1A target genes identified contained DRE (Yamaguchi‐Shinozaki and Shinozaki, 1994) or DRE‐related CCGAC core motif sequences in their promoter regions (Seki et al., 2001a). Recently, a new full‐length cDNA microarray was prepared containing c. 7000 independent Arabidopsis full‐length cDNA groups, including drought‐inducible genes, responsive to dehydration (rd) and early responsive to dehydration (erd) (Taji et al., 1999), as positive controls, the PCR‐amplified fragment from the lambda control template DNA fragment (Takara) as an external control, and the mouse nicotinic acetylcholine receptor epsilon‐subunit (nAChRE) gene and the mouse glucocorticoid receptor homologue gene, which have no substantial homology to any sequences in the Arabidopsis database, to assess for non‐specific hybridization as negative controls (Seki et al., 2002b). The RAFL cDNA microarray containing c. 7000 Arabidopsis full‐length cDNA groups was applied to identify new drought‐, cold‐, high‐salinity‐ or ABA‐inducible genes. In this study, the PCR‐amplified fragment from the lambda control template DNA fragment (Takara) was used as an external control gene to equalize hybridization signals generated from different samples, and the genes with expression ratios (dehydration/unstressed, cold/unstressed, high‐salinity/unstressed, or ABA/unstressed) greater than five times that of the lambda control template DNA fragment in at least 1 time‐course point as dehydration‐, cold‐, high‐salinity‐stress, or ABA‐inducible genes were selected. 299 drought‐inducible genes, 54 cold‐inducible genes, 213 high‐salinity‐stress‐inducible genes, and 245 ABA‐inducible genes were identified (Seki et al., 2002b, c). Information on each stress‐inducible gene is available at http://pfgweb.gsc.riken.go.jp/pub_data/seki002/supplemental1.xls and http://pfgweb.gsc.riken.go.jp/pub_data/seki003/supplemental1.xls. Venn diagram analysis indicated the existence of significant cross‐talk between drought and high‐salinity stress signalling processes (Seki et al., 2002b). Many ABA‐inducible genes are induced after drought‐ and high‐salinity‐stress treatments, which indicates the existence of significant cross‐talk between drought and ABA responses (Seki et al., 2002c). These results indicate the presence of strong overlap of gene expression in response to drought, high‐salinity and ABA (Shinozaki and Yamaguchi‐Shinozaki, 2000) and partial overlap of gene expression in response to cold and osmotic stress. Stress‐inducible genes and functions of their gene products identified by RAFL cDNA microarray The products of the drought‐, high‐salinity‐ or cold‐stress‐inducible gene products can be classified into two groups (Seki et al., 2002b; Shinozaki and Yamaguchi‐Shinozaki, 1999, 2000). The first group includes functional proteins or proteins that probably function in stress tolerance. They are LEA proteins, heat shock proteins, KIN (cold‐inducible) proteins, osmoprotectant‐biosynthesis‐related proteins, carbohydrate‐metabolism‐related proteins, water channel proteins, sugar transporters, potassium transporters, detoxification enzymes, proteases, senescence‐related genes, protease inhibitors, ferritin, and lipid transfer proteins (Seki et al., 2002b). LEA proteins and heat shock proteins have been shown to be involved in protecting macromolecules such as enzymes and lipids (Shinozaki and Yamaguchi‐Shinozaki, 1999). Proline, sugars and raffinose family oligosaccharides (RFO) probably function as osmolytes in protecting cells from dehydration (Cushman and Bohnert, 2000; Taji et al., 2002). KIN proteins may have a unique ability to neutralize ice nucleators and inhibit ice recrystallization (Holmberg and Bülow, 1998). Water channel proteins and sugar transporters are thought to function in the transport of water and sugars through plasma membranes and tonoplast to adjust the osmotic pressure under stress conditions. Potassium transporters may function in the transport of K+ which is an essential cofactor for many enzymes (Hasegawa et al., 2000) or may control K+ uptake and regulate Na+ uptake, which can be an important determinant of salinity tolerance (Bray, 1997). Detoxification enzymes, such as glutathione S‐transferase are thought to be involved in the protection of cells from active oxygens. Proteases including cysteine proteases, Clp protease, and ubiquitin‐conjugating enzyme are thought to be required for protein turnover and recycling of amino acids. Drought stress has been shown to accelerate leaf senescence which is characterized by many subcellular changes, including an increase in protease activities (Thomas and Stoddart, 1980). The protease inhibitors may perform a defensive role against the proteases. Ferritin may have a function in protecting the cells from oxidative damage caused by various stresses by sequestering intracellular iron involved in the generation of various reactive hydroxyl radicals through a Fenton reaction (Bajaj et al., 1999). Lipid transfer proteins and fatty acid‐metabolism‐related genes may have a function in the repair of stress‐induced damage in membranes or changes in the lipid composition of membranes, perhaps to regulate the permeability to toxic ions and the fluidity of the membrane (Holmberg and Bülow, 1998; Torres‐Schumann et al., 1992). The second group contains regulatory proteins, that is, protein factors involved in the further regulation of signal transduction and gene expression that probably function in the response to stress (Seki et al., 2002b, c; Shinozaki and Yamaguchi‐Shinozaki, 1999, 2000). They include various transcription factors, protein kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and other signalling molecules, such as calmodulin‐binding protein (Seki et al., 2002b, c). Among the drought‐, cold‐ or high‐salinity‐stress‐inducible genes identified, c. 40 (corresponding to c. 11% of all stress‐inducible genes identified) transcription factor genes were found, suggesting that various transcriptional regulatory mechanisms function in the drought‐, cold‐ or high‐salinity‐stress signal transduction pathways (Seki et al., 2002b, c). Among these stress‐inducible transcription factors, there are six DREB family cDNAs, two ethylene‐responsive element binding factor (ERF) family cDNAs, 10 zinc finger family cDNAs, four WRKY family cDNAs, three MYB family cDNAs, two basic helix‐loop‐helix (bHLH) family cDNAs, four bZIP family cDNAs, five NAC family cDNAs, and three homeodomain‐leucine zipper (HD‐ZIP) transcription factor family cDNAs. These transcription factors probably regulate various stress‐inducible genes co‐operatively or separately. Among six protein kinase genes, two receptor‐like protein kinase genes were found. These regulatory proteins are thought to function in further regulating various functional genes under stress conditions. Functional analysis of these stress‐inducible transcription factors or protein kinase genes should provide more information on the signal transduction in the responses to drought, cold and high‐salinity stresses. Various genes involved in the metabolism of ABA, ethylene, JA, and auxin, and JA‐ or auxin‐regulated genes have been identified as drought‐inducible genes (Seki et al., 2002b), suggesting the link between ethylene, JA, and auxin, and drought‐stress‐signalling pathways. Also, aldehyde dehydrogenase genes, genes related to secondary metabolism, genes involved in various cellular metabolic processes, genes encoding membrane proteins and cytochrome P450 were identified as drought‐ or high‐salinity‐stress‐inducible genes (Seki et al., 2002b). At present, the functions of most of these genes are not fully understood. Furthermore, the functions of many drought‐, cold‐ or high‐salinity‐stress‐inducible genes that were found remain unknown. Cold‐inducible genes, stress‐down‐regulated genes and rehydration‐inducible genes identified using the RAFL cDNA microarray Among the 54 cold‐inducible genes identified, the promoter sequence was obtained for 41 genes (Seki et al., 2002b). Among the 41 genes, 32 contained either the DRE or DRE‐related CCGAC core motif in the promoters, suggesting that DRE is a major cis‐acting element involved in cold‐inducible gene expression (Seki et al., 2002b). Among 41 cold‐inducible genes, nine did not contain the DRE or DRE‐related CCGAC core motif in their promoters. These results suggest the existence of novel cis‐acting elements involved in cold‐inducible gene expression (Seki et al., 2002b). Analysis of the expression profiles of cold‐inducible genes during cold treatment showed the existence of at least two groups that show different expression profiles (Seki et al., 2002b). In one group containing the DREB1A gene, gene expression was rapid and transient in response to cold treatment, reached a maximum at 2 h, and then decreased (Seki et al., 2002b). In the other group containing DREB1A target genes, such as rd29A, erd10, cor15A, rd17, kin2, and RAFL06‐16‐B22 genes, their expression increased slowly and gradually after cold treatment within 10 h (Seki et al., 2002b). The expression of the DREB1A gene during cold stress preceded that of the DREB1A target genes. These results support the view that DREB1A regulates the expression of the DREB1A target genes, such as rd29A,erd10, cor15A, rd17, kin2, and RAFL06‐16‐B22 genes (Kasuga et al., 1999; Seki et al., 2001a). Analysis of stress‐down‐regulated as well as stress‐up‐regulated genes is important in understanding the molecular responses to abiotic stresses. Many drought‐, high‐salinity‐, cold‐stress‐, or ABA‐down‐regulated genes were identified by microarray analysis (Seki et al., 2002b, c). The list and the expression data on these drought‐, cold‐, high‐salinity‐stress‐ or ABA‐down‐regulated genes is available at http://pfgweb.gsc.riken.go.jp/pub_data/seki003/supplemental5.xls and http://pfgweb.gsc.riken.go.jp/pub_data/seki002/supplemental4.xls. Among the drought‐, cold‐, high‐salinity‐stress‐, or ABA‐down‐regulated genes, many photosynthesis‐related genes were found, such as ribulose 1,5‐bisphosphate carboxylase small subunit (rbcS), chlorophyll a/b‐binding protein (cab), and the components of photosystem I and II. These results are consistent with the previous report that water stress inhibits photosynthesis (Tezara et al., 1999). Analysis of genes involved in the recovery process from drought stress as well as drought‐stress‐inducible genes is also important, not only to understand the molecular responses to abiotic stresses but also to improve the stress tolerance of crops by gene manipulation. The cDNA microarray analysis was applied to identify genes that are induced during the rehydration process after dehydration stress treatment and 152 rehydration‐inducible genes were identified (Oono et al., 2003). These genes can be classified into the following three major groups: (1) regulatory proteins involved in further regulation of signal transduction and gene expression, (2) functional proteins involved in the recovery process from dehydration‐induced damage, (3) functional proteins involved in plant growth (Oono et al., 2003). Venn diagram analysis also showed that among the rehydration‐inducible genes, at least two gene groups, that is, genes functioning in adjustment of cellular osmotic conditions and those functioning in the repair of drought‐stress‐induced damage existed and that most of the rehydration‐down‐regulated genes are dehydration‐inducible (Oono et al., 2003). Application of RAFL cDNA microarray to study the expression profiles under abiotic stress conditions Simpson et al. (2003) showed that a 14 bp region (CACTAAATTGTCAC; site‐1‐like sequence) from –599 to –586, and a myc recognition element (CATGTG) from –466 to –461 in the promoter region of the erd1 gene encoding a regulatory subunit of Clp protease (Kiyosue et al., 1993, 1994; Nakashima et al., 1998) are responsible for gene expression during dehydration. To assess the frequency with which the sequence with homology to the core sequence of the site‐1 motif, and the myc recognition element (CATGTG), occur together in the promoter regions of dehydration‐inducible genes, an homology search for these two sequences within the promoter regions of dehydration‐inducible genes was performed. Of the 100 drought‐, cold‐, high‐salinity‐stress‐ or ABA‐inducible genes (Seki et al., 2002b, c) that show the greatest degree of induction by dehydration, 22 contained both the putative core motif of the site‐1‐like sequence (in either forward or complementary orientation) and the putative myc recognition sequence in their promoter regions (Simpson et al., 2003). Examination of the data revealed that just under 50% of the 22 genes show similar pattern of expression in response to dehydration, high salinity, ABA, and cold treatment as that of the erd1 gene; such that induction by dehydration > high salinity > ABA > cold, and that 21 have low levels of induction in response to cold stress. These results suggest that these sequences may also function as novel cis‐acting elements in stress‐responsive gene expression (Simpson et al., 2003). The transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA (Abe et al., 2003). Abe et al. (2003) studied the expression profiles in the transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs using the RAFL cDNA microarray. mRNAs prepared from 35S:AtMYC2/AtMYB2 and wild‐type plants were used for the generation of Cy3‐labelled and Cy5‐labelled cDNA probes, respectively. Microarray analysis of the transgenic plants revealed that several ABA‐inducible genes were up‐regulated in the 35S:AtMYC2/AtMYB2 transgenic plants. Abe et al. (2003) searched for the MYC recognition sequence (CANNTG) and the MYB recognition sequences (A/TAACCA and C/TAACG/TG) located within the 10–600 bp upstream region from each putative TATA box in the promoter regions of the 32 up‐regulated genes identified. Abe et al. (2003) found that 29 genes have the MYC recognition sequence, 29 genes have the MYB recognition sequence, and 26 genes have both MYC and MYB recognition sequences in their promoter regions. Ds insertion mutant of the AtMYC2 gene was less sensitive to ABA and showed significantly decreased ABA‐induced gene expression of rd22 and AtADH1. These results indicated that both AtMYC2 and AtMYB2 function as transcriptional activators in ABA‐inducible gene expression under drought‐stress conditions in plants. In rice, Dubouzet et al. (2003) isolated five cDNAs for DREB homologues: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high‐salinity stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Overexpression of OsDREB1A in transgenic Arabidopsis plants resulted in improved tolerance to drought, high‐salinity and freezing stresses, indicating that OsDREB1A has functional similarity to DREB1A (Dubouzet et al., 2003). Several OsDREB1A target genes were identified by the cDNA microarray and RNA gel blot analyses. Computer analysis showed that the seven OsDREB1A target genes have at least one core GCCGAC sequence as the DRE core motif in their promoter regions. Some of the DREB1A target genes such as kin1, kin2 and erd10, containing ACCGAC, but not GCCGAC, as the DRE core motifs in their promoter regions, were not overexpressed in the 35S:OsDREB1A plants. These results indicated that the OsDREB1A protein binds more preferentially to GCCGAC than to ACCGAC in the promoter regions, whereas the DREB1A protein binds to both GCCGAC and ACCGAC efficiently (Dubouzet et al., 2003). Proline (Pro) is one of the most widely distributed osmolytes in water‐stressed plants. l‐Pro is metabolized to l‐Glu via Δ1‐pyrroline‐5‐carboxylate (P5C) by two enzymes, Pro dehydrogenase (ProDH) and P5C dehydrogenase (Strizhov et al., 1997; Yoshiba et al., 1997). The ProDH gene in Arabidopsis is up‐regulated not only by rehydration after dehydration, but also by l‐Pro and hypo‐osmolarity (Kiyosue et al., 1996; Nakashima et al., 1998). Satoh et al. (2002) analysed the promoter regions of ProDH to identify cis‐acting elements involved in l‐Pro‐induced and hypo‐osmolarity‐induced expression in transgenic tobacco and Arabidopsis plants. Satoh et al. (2002) found that a 9 bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to l‐Pro and hypo‐osmolarity and that the ACTCAT sequence is a core cis‐acting element. To elucidate whether the promoter region of the other l‐Pro‐inducible genes have the ACTCAT sequence, Satoh et al. (2002) used the Arabidopsis full‐length cDNA microarray, and identified 50 l‐Pro‐inducible genes by cDNA microarray analysis. Satoh et al. (2002) also found that 27 l‐Pro‐inducible genes identified have the ACTCAT sequence in their promoter regions. 21 genes among the 27 genes showed l‐Pro‐inducible expression based on RNA gel blot analysis. These results suggest that the ACTCAT sequence is conserved in many l‐Pro‐inducible promoters and plays a key role in l‐Pro‐inducible gene expression. The microarray analysis also showed that some l‐Pro‐inducible genes do not have the ACTCAT sequences in their promoter regions, suggesting the existence of other cis‐acting elements for l‐Pro‐inducible gene expression. Recently, promoter analysis of the rehydration‐inducible genes also suggested that the ACTCAT sequence is a major cis‐acting element involved in rehydration‐inducible gene expression and that some novel cis‐acting elements involved in rehydration‐inducible gene expression existed (Oono et al., 2003). Advantages and disadvantages of the RAFL cDNA microarray There are several advantages and disadvantages to using the RAFL cDNA microarrays for the systematic analysis of expression profiles of large numbers of genes. Using a full‐length cDNA microarray, it is easy to isolate full‐length cDNAs for further functional analysis. Biochemical characteristics of the gene products are easily analysed from the overexpression of the full‐length cDNAs in a bacteria, yeast or wheat germ cell‐free protein synthesis system. Functions of the gene products in planta can be analysed by the overexpression of full‐length cDNAs in transgenic plants. Moreover, promoter sequences and putative cis‐acting elements of each gene can be predicted by comparison of full‐length cDNA sequences with the Arabidopsis genomic sequence. However, cross‐hybridization between highly related sequences may occur in the full‐length cDNA microarray (Richmond and Somerville, 2000). To avoid cross‐hybridization problems, oligonucleotide microarrays such as the Agilent Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA, USA) and Affymetrix GeneChip Array (Affymetrix, Inc., Santa Clara, CA, USA) is thought to be a powerful tool in the future. Microarray data analysis There are many sources of systematic variation in microarray experiments which affect the measured gene expression levels (Finkelstein et al., 2002a). To remove the systematic variation, it is necessary to normalize the microarray data. Normalization of extracted data is a requirement for all gene expression profiling. A number of normalization approaches can be employed, including the use of housekeeping genes that are hypothesized to show a constant expression between samples, the use of spiking (external) control genes and global approaches, where the total level of gene expression for a large portion of the total genes under study is considered to stay constant (Donson et al., 2002). To compare measurements from gene expression array experiments, quantitative data are commonly normalized using reference genes such as housekeeping genes or spiking control genes, or global normalization methods based on mean or median values. These methods are based on the assumption that (i) selected reference genes are expressed at a standard level in all experiments or (ii) that a mean or median signal of expression will give a quantitative reference for each individual experiment. Note that normalization by reference genes is dependent on an accurate measure of RNA quantity. Recently, Yang et al. (2002) proposed new normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. Many useful normalization strategies have been reported so far (Bowtell and Sambrook, 2002; Donson et al., 2002; Finkelstein et al., 2002a; Kroll and Wolfl, 2002). Global normalization may work well for similar samples and normalization by reference genes may have an adavantage for more divergent samples (Donson et al., 2002). However, there is no single standard normalization method. In the initial study on the expression profiling using c. 1300 independent RAFL cDNAs (Seki et al., 2001a), the Arabidopsis α‐tubulin gene was used (Ludwig et al., 1987) with the same expression level in these experimental conditions as an internal control gene. However, a universal set of housekeeping genes that is stable under any potential stress, tissue comparison, developmental stage, mutational background, and subcellular fractionation is required for a general‐use genome‐wide array analysis. Therefore, the spiking control genes were used to study the expression profiles in various environmental conditions in the next step. Using c. 7000 RAFL cDNA microarray, the expression profiles of Arabidopsis genes were studied under various stress and hormone treatment conditions, such as drought, cold and high‐salinity‐stresses (Seki et al., 2001a, b), and ABA (Seki et al., 2002c) as described above. In these experiments, the PCR‐amplified lambda control template DNA fragment (Takara) was used as a spiking control gene to equalize hybridization signals generated from different samples. For the identification of drought, cold, high‐salinity‐ stress or ABA‐inducible genes, the results were compared by the external control method with those by the intensity‐dependent normalization (Yang et al., 2002). In this comparison, genes with expression ratios (dehydration/unstressed, cold/unstressed, high‐salinity/unstressed or ABA/unstressed) greater than five times in at least one time‐course point as dehydration, cold, high‐salinity stress‐ or ABA‐inducible genes were selected. Genes showing a signal value <1000 (typically twice the mean background value) in both Cy3 and Cy5 channels were not considered for the analyses. The list of the drought, cold, high‐salinity‐ stress or ABA‐inducible genes obtained by the external control method was almost identical to that obtained by intensity‐dependent normalization (Yang et al., 2002) in each time‐course treatment (data not shown). The best method of microarray data analysis has not yet been determined. Until this is resolved, the researchers should run each microarray data set through a series of quality tests and verify the expression results of key genes by northern blot or RT‐PCR analyses. Conclusions and perspectives The cDNA microarray analysis is useful for analysing the expression pattern of Arabidopsis genes under drought‐, cold‐, or high‐salinity stresses and for identifying the target genes of stress‐related transcription factors. Potential cis‐acting DNA elements can be identified by combining the expression data with the genomic sequence data. By the expression profiling approach, more than 300 drought‐, cold‐, or high‐salinity‐stress‐inducible genes and 40 drought‐, cold‐, or high‐salinity‐stress‐inducible transcription factor genes have been identified, suggesting that various transcriptional regulatory mechanisms function in these stress signal transduction pathways. Functional analysis of these drought‐, cold‐ or high‐salinity‐stress‐inducible genes should provide more information on the signal transduction in these stress responses. By genetic approaches and biochemical analyses of signal transduction and stress tolerance of drought, cold and high‐salinity stress, several mutants on the signal transduction and stress tolerance of these stresses have been identified (Browse and Xin, 2001; Finkelstein et al., 2002b; Knight and Knight, 2001; Xiong et al., 2002; Zhu, 2002). In a genetic screen using a firefly luciferase reporter gene (LUC) under the control of the RD29A promoter, Zhu’s group isolated several Arabidopsis mutants with altered induction of stress‐responsive genes under drought, high‐salinity, cold, and ABA treatments (Ishitani et al., 1997). Compared with wild‐type RD29A‐LUC plants, mutants exhibited either a constitutive (cos), high (hos), or low (los) level of RD29A‐LUC expression in response to various stress or ABA treatments (Ishitani et al., 1997; Xiong and Zhu, 2001, 2002; Xiong et al., 2002; Zhu, 2002). These mutants might be involved in the activation of the DRE/CRT class genes. The Arabidopsis salt overly sensitive (sos) mutants (sos1, sos2, sos3, and sos4) were also identified by genetic screening for seedlings that were hypersensitive to growth inhibition by NaCl stress (Liu and Zhu, 1998; Liu et al., 2000; Shi et al., 2000, 2002). The sos1, sos2 and sos3 mutants are hypersensitive to salt stress, but activation of the DRE/CRT class of genes seems to be unchanged in them. Reverse genetic approaches, such as transgenic analyses, have also become useful for studying the function of the signalling components (Apse and Blumwald, 2002; Finkelstein et al., 2002b; Gong et al., 2002; Guo et al., 2002; Hasegawa et al., 2000; Iuchi et al., 2001; Xiong et al., 2002). The availability of the Arabidopsis genome sequence will not only greatly facilitate the isolation of mutations identified by the above genetic screen, but also offer many other useful opportunities to study stress signal transduction. Genome‐wide expression profiling of the stress‐resistant or stress‐sensitive mutants, and mutants on the stress signal transduction should help identify more genes that are regulated at the transcriptional level by the signalling components. Moreover, full‐length cDNAs (Seki et al., 2002a) are useful resources for transgenic analyses, such as overexpression, antisense suppression, and double‐stranded RNA interference (dsRNAi) and biochemical analyses to study the function of the encoded proteins. T‐DNA‐ or transposon‐knockout mutants also offer the opportunity to study the function of the genes. Genome‐wide protein interaction studies will help to identify the interactions among signalling components and to construct the signal networks ‘dissected’ with the above genetic analysis. The information generated by focused studies of gene function in Arabidopsis will be the springboard for a new wave of strategies to improve the tolerance to dehydration, salt and cold in agriculturally important crops. Acknowledgements This work was supported in part by a grant for Genome Research from RIKEN, the Program for Promotion of Basic Research Activities for Innovative Biosciences, the Special Coordination Fund of the Science and Technology Agency, and a Grant‐in‐Aid from the Ministry of Education, Culture, Sports and Technology of Japan (MEXT) to KS. It was also supported in part by a Grant‐in‐Aid for Scientific Research on Priority Areas (C) ‘Genome Science’ from MEXT to MS. View largeDownload slide Fig. 1. Application of full‐length cDNAs to functional genomics. View largeDownload slide Fig. 1. Application of full‐length cDNAs to functional genomics. Table 1. Expression profiling using RIKEN Arabidopsis full‐length (RAFL) cDNA microarray Reference  Microarray type and scale  Aim of microarray analysis  Seki et al. (2001a)  Ver. 1; c. 1300 full‐length cDNAs  Expression profiles under drought or cold stress      Identification of genes up‐regulated in the DREB1A/CBF3 overexpressor  Seki et al. (2002b)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under drought, high‐salinity or cold stress  Seki et al. (2002c)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under ABA treatments  Satoh et al. (2002)  Ver. 2; c. 7000 full‐length cDNAs  Identification of Pro‐inducible genes  Osakabe et al. (2002)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the ARR4/ATRR1/IBC7 overexpressor  Abe et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the overexpressor of AtMYC2/rd22BP1and AtMYB2  Dubouzet et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the OsDREB1A overexpressor  Simpson et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Promoter analysis of genes up‐regulated by drought, high‐salinity,cold stress or ABA treatment  Kimura et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under high light stress  Narusaka et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under JA, SA, ethylene, paraquat, rose bengal orUV treatments       Expression profiles in wild type and pad3‐1 plants after the inoculation withAlternaria brassicicola  Oono et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under recovery process from dehydration to rehydration  Reference  Microarray type and scale  Aim of microarray analysis  Seki et al. (2001a)  Ver. 1; c. 1300 full‐length cDNAs  Expression profiles under drought or cold stress      Identification of genes up‐regulated in the DREB1A/CBF3 overexpressor  Seki et al. (2002b)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under drought, high‐salinity or cold stress  Seki et al. (2002c)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under ABA treatments  Satoh et al. (2002)  Ver. 2; c. 7000 full‐length cDNAs  Identification of Pro‐inducible genes  Osakabe et al. (2002)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the ARR4/ATRR1/IBC7 overexpressor  Abe et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the overexpressor of AtMYC2/rd22BP1and AtMYB2  Dubouzet et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Identification of genes up‐regulated in the OsDREB1A overexpressor  Simpson et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Promoter analysis of genes up‐regulated by drought, high‐salinity,cold stress or ABA treatment  Kimura et al. (2003)  Ver. 2; c. 7000 full‐length cDNAs  Expression profiles under high light stress  Narusaka et al. 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TI - RIKEN Arabidopsis full‐length (RAFL) cDNA and its applications for expression profiling under abiotic stress conditions
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erh007
DA - 2004-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/riken-arabidopsis-full-length-rafl-cdna-and-its-applications-for-TUDg9k9aLi
SP - 213
EP - 223
VL - 55
IS - 395
DP - DeepDyve
ER -