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Plants grow in a dynamic environment. Their growth and development are influenced by abiotic (environmental) and biotic stresses. These stresses are the primary cause of crop loss worldwide. They can reduce the average yields of crop plants by more than 50% (Bartels and Sunkar 2005).
There are so many environmental stresses determining plant growth, distribution, and development. One of the most important environmental stresses is drought. This stress leads to cellular dehydration. It causes osmotic stress and water removal from cytoplasm into extracellular space, so the cytosolic and vacuolar volume are reduced (Bartels and Sunkar 2005).
Drought stress induces a range of physiological and biochemical responses in plants such as stomatal closure, repression of cell growth and photosynthesis, and activation of respiration (Lu, Chen et al. 2007). At cellular and molecular level, plants also respond and adapt to water deficit. For example, they accumulate specific osmolytes and proteins that are involved in stress tolerance.
Furthermore, Plants have developed various survival mechanisms to overcome continued exposure to drought stress. For example a stress-signal transduction which leads to various physiological and metabolic responses such as stress responsive gene expression (Lu, Chen et al. 2007). This mechanism will help plants survive if they subject to drought stress.
Numerous genes with diverse functions are induced or repressed by abiotic stress (Yamaguchi-Shinozaki and Shinozaki 2005). Most of their gene products may function in stress response and tolerance at the cellular level. Now, analyzing the functions of these genes is critical to further our understanding of the molecular mechanisms governing plant stress response and tolerance, ultimately leading to enhancement of stress tolerance in crops through genetic manipulation (Shinozaki and Yamaguchi-Shinozaki 2007).
Arabidopsis as model plant
Arabidopsis thaliana is a member of the Brassicaceae (mustard family). It is closely related to such economically important crop plants as turnip, cabbage, broccoli, and canola. It is not an economically important plant and considered a weed. However, it has several traits that make it very ideal to be organism for laboratory study (Anonymous 2004).
There are many advantages to use Arabidopsis as model plant. It has a fast life cycle. Its entire life cycle is completed in 6 weeks (Meinke, Cherry et al. 1998). It produces numerous self progeny, up to 10,000 seed per plant. It has very limited space requirements, and is easily grown in a greenhouse or indoor growth chamber. It possesses a relatively small, genetically tractable genome that can be manipulated through genetic engineering more easily and rapidly than any other plant genome . This fact, together with a newly developed means of creating gene knock out lines, has made many basic biologists realize that Arabidopsis may be the best model system for basic research in the biology of all multicellular eukaryotes. All together, these traits make Arabidopsis an ideal model organism for biological research (Anonymous 2002).
In the laboratory, Arabidopsis offers the ability to test hypotheses quickly and efficiently. With the knowledge we gain from the model plant thus established as a reference system, we can move forward with research and rapidly initiate improvements in plants of economic and cultural importance (Anonymous 2002).
One advantage offered to the plant researcher by Arabidopsis is its relatively small genome size. It has one of the smallest genomes in the plant kingdom, 115,409,949 base pairs of DNA distributed in 5 chromosomes (2n = 10) (Anonymous 2004). Many crop species have large genomes, often as a result of polyploidization events and accumulation of non-coding sequences during their evolution. Maize has a genome of approximately 2400 Megabase pairs (Mbp) - around 19 times the size of the Arabidopsis genome - with probably no more than double the number of genes, most of which occur in duplicate within the genome. The wheat genome is 16000 Mbp - 128 times larger than Arabidopsis - and it has three copies of many of its genes (Anonymous 2002). The large crop genomes make difficulties in sequencing, isolation and cloning of mutant loci. The results of the rice genome project prove that the Arabidopsis genome may be missing some homologs of genes present in the rice genome. However, most of the difference in gene number between Arabidopsis and crop species appears to result from polyploidy of crop species' genomes, rather than from large classes of genes present in crop species that are not present in Arabidopsis. Therefore, the genes present in Arabidopsis represent a reasonable model for the plant kingdom (Anonymous 2002).
Identification of drought-inducible genes in Arabidopsis by microarray
A number of stress-inducible genes have been identiï¬ed using microarray analysis in Arabidopsis. cDNAs or oligonucleotides microarray is a powerful tool for analyzing gene expression proï¬les of plants exposed to abiotic stresses such as drought, high salinity, or cold, or to ABA treatment (Seki, Narusaka et al. 2001). A 7000 full-length cDNA microarray was utilized to identify 299 drought-inducible genes, 54 cold-inducible genes, 213 high salinity-inducible genes, and 245 ABA-inducible genes in Arabidopsis (Seki, Narusaka et al. 2002). More than half of these drought-inducible genes were also induced by high salinity and/or ABA treatments. This indicated the existence of significant cross talk between the drought, high salinity, and ABA response pathways (Seki, Narusaka et al. 2002).
Recently, Arabidopsis whole-genome tiling array analysis under drought-, cold-, high-salinity-stress or ABA treatments also showed the consistent result. In drought analysis, 1,188 genes were up-regulated and 217 genes were down-regulated after 2 hours treatment. After 10 hours treatment, 2,059 genes were up-regulated and 2,075 genes were down-regulated. These genes included many reported drought genes, such as RD29A (responsive to dehydration), RD20, DREB1A (dehydration-responsive element/DRE-binding protein), BREB2A and AtMYC2 (Matsui, Ishida et al. 2008).
The products of the drought-inducible genes identiï¬ed through the recent microarray analyses in Arabidopsis can be classiï¬ed into two groups (Shinozaki and Yamaguchi-Shinozaki 2007); (Matsui, Ishida et al. 2008). The ï¬rst group is functional transcript/protein. These transcript/proteins probably function in stress tolerance. These include molecules such as chaperones, late embryogenesis abundant (LEA) proteins, osmotin, antifreeze proteins, mRNA-binding proteins, key enzymes for osmolyte biosynthesis, water channel proteins, sugar and praline transporters, detoxiï¬cation enzymes, and various proteases (Shinozaki and Yamaguchi-Shinozaki 2007); (Matsui, Ishida et al. 2008). The second group is regulatory transcripts/proteins. These transcripts/proteins function in further regulation of signal transduction and gene expression that probably function in the stress responses. These include various transcription factors, protein kinases, protein phosphatases, enzymes involved in phospholipid metabolism, and other signalling molecules such as calmodulin-binding protein (Shinozaki and Yamaguchi-Shinozaki 2007); (Matsui, Ishida et al. 2008).
Source: (Shinozaki and Yamaguchi-Shinozaki 2007)
Figure 1. Functions of drought stress-inducible genes in stress tolerance and response. There are two groups, the first group is functional proteins (probably function in stress tolerance) and the second group is regulatory protein (probably function in stress response)
Many transcription factor genes were stress inducible. This indicated that various transcriptional regulatory mechanisms may function in regulating drought stress signal transduction pathways. Some of these transcription factors could regulate expression of stress-inducible genes independently. The others transcription factors could regulate expression of stress-inducible genes cooperatively and may constitute gene networks in Arabidopsis.
Regulation of gene expression in response to drought
In Arabidopsis, at least there are four independent regulatory systems (independent pathways) for gene expression in response to water stress. Two are abscisic acid (ABA)-dependent and two are ABA-independent regulatory systems (Shinozaki and Yamaguchi-Shinozaki 2000).
Modified from: Shinozaki and Yamaguchi-Shinozaki (2007)
Figure 2. Regulatory system for gene expression in response to drought stress in Arabidopsis. At least four regulatory system are exist; two are ABA-dependent and two are ABA-independent
One of the ABA-independent regulatory systems involves dehydration-responsive element/C-repeat (DRE/CRT) (Fig.2). It has been identiï¬ed as a cis-acting element with a 9-bp conserved sequence, TACCGACAT. DRE is an essential cis-acting element for regulation of RD29A (responsive to dehydration) gene (Yamaguchi-Shinozaki and Shinozaki 1994). Stockinger et al. (1997) and Liu et al.(1998) have isolated and identified three cDNAs encoding DRE binding protein, CBF1, DREB1A, and DREB2A. However, only DREB2 involve in signal transduction pathways under dehydration condition (Liu, Kasuga et al. 1998).
Although DREB2 are induced by dehydration stress and may activate other genes involved in drought stress tolerance, over-expression of DREB2 in transgenic Arabidopsis plants did not improve stress tolerance. It suggests the involvement of post-translational activation of DREB2 proteins (Liu, Kasuga et al. 1998).
Recently, an active form of DREB2 was reported to transactivate target stress-inducible genes and improve drought tolerance in transgenic Arabidopsis (Sakuma, Maruyama et al. 2006). The DREB2 protein is expressed under normal growth conditions and activated by osmotic stress through post-translational modiï¬cation in the early stages of the osmotic stress response.
Nakashima et al. (1997) have identified several drought-inducible genes that do not respond to ABA treatment. It indicates the existence of another ABA-independent pathway regulating the dehydration stress response (Fig. 2). These genes include ERD1 (early responsive to dehydration), which encodes a Clp protease regulatory subunit, ClpD. However, the ERD1 gene is not only induced by dehydration. It is also up-regulated during natural senescence and dark-induced senescence.
Abscisic acid (ABA) regulates the expression of many genes. These gene might function in dehydration tolerance. Most of these ABA-inducible genes contain a conserved sequence, PyACGTGGC, in their promoter regions (Seki, Narusaka et al. 2002). This sequence functions as a cis-acting element and named ABA responsive element (ABRE).
ABRE are important cis-acting elements controlling ABA-responsive expression of the Arabidopsis RD29B gene (Fig. 2). Promoter analysis of this gene indicated that two ABREs as cis-acting element are require for the dehydration-responsive expression of RD29B gene (Uno, Furihata et al. 2000).
The RD22 gene is a dehydration-responsive gene induced by the application of exogenous ABA to Arabidopsis plants. Unlike most of ABA-inducible genes, RD22 gene does not contain any typical ABRE conserved sequence in its promoter (Yamaguchi-Shinozaki and Shinozaki 1993). This result indicates the existence of another regulatory system for gene expression in response to ABA under drought stress.
The cis-regulatory region of the RD22 promoter was investigated by monitoring the expression of beta-glucuronidase (GUS). A 67-bp nucleotide fragment contains the sequences of the recognition sites for some transcription factors such as MYC and MYB has been identified (Iwasaki, Yamaguchi-Shinozaki et al. 1995).
Transcriptional factors, such as the MYC and MYB proteins, are transcriptional activators in one of the ABA-dependent pathways (Fig. 2) (Abe, Urao et al. 2003). A MYC transcription factor, AtMYC2 (RD22BP1), and a MYB transcription factor, AtMYB2, were shown to bind cis-elements in the RD22 promoter and subsequently activate RD22 (Abe, Urao et al. 2003). The synthesis of MYC and MYB proteins follow the accumulation of endogenous ABA. This fact deï¬ned their role in later stage stress responses. Microarray analysis has revealed the target genes of MYC/MYB in over-expressing transgenic plants such as alcohol dehydrogenase and ABA- or jasmonic acid (JA)-inducible (Abe, Urao et al. 2003). Over-expression of both AtMYC2 and AtMYB2 not only resulted in an ABA-hypersensitive phenotype but also improved osmotic stress tolerance of the transgenic plants.
Improving drought stress tolerance in Arabidopsis via gene transfer
Many scientists have attempted to improve drought stress tolerance by gene transfer (Table 1.). Several stress-inducible genes have reported to increase drought stress tolerance in transgenic plant significantly. These particular genes encode key enzymes which regulate biosynthesis of specific substances related to drought tolerance such as proline, polyamines, variety of sugar and sugar alcohol (Shinozaki and Yamaguchi-Shinozaki 2007).
Genes encoding LEA protein, heatshock protein and galactinol synthase (GolS were introduced to improve drought-stress tolerance in transgenic Arabidopsis (Taji, Ohsumi et al. 2002; Umezawa, Fujita et al. 2006).
Table 1. Improved drought stress tolerance in plants via gene transfer
Kang et al. (2002)
Liu et al. (2008)
Cui et al. (2008)
Lee and Hwang (2009)
Wang et al. (2008)
Abe et al. (2003)
Liu et al. (1998)
Haake et al. (2002)
Arabidopsis Rice and tobacco
Zhang et al. (2007) Zhang et al (2008)
Dai et al. (2007)
Transcription factors have also proven quite useful in improving stress tolerance in transgenic plants, through inï¬‚uencing expression of a number of stress-related target genes (Shinozaki et al., 2003; Yamaguchi-Shinozaki & Shinozaki, 2005).
Many genes from others species have also been use to improve drought tolerance in Arabidopsis. Transgenic Arabidopsis plants containing VfPIP1 gene (a putative aquaporin gene from Vicia faba) exhibited faster growth rate, a lower transpiration rate, and greater drought tolerance. In addition, the stomata of this plants closed signiï¬cantly faster than those of the control plants under ABA or dark treatment. These results suggest that VfPIP1 expression may improve drought resistance of the transgenic plants by promoting stomatal closure under drought stress (Cui, Hao et al. 2008).
Transcriptome analyses have provided powerful tools to discover stress-responsive genes in Arabidopsis. Arabidopsis has also been an excellent model plan for drought stress response and tolerance study. It has helped us to understand how plant responses to drought stress.