Introduction

Drought, salt loading, and freezing are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plants respond to these stresses at molecular and cellular levels as well as physiological levels. Expression of a variety of genes has been demonstrated to be induced by these stresses.1,2 The pro ducts of these genes are thought to function not only in stress tolerance but also in the regulation of gene expression and signal transduction in stress response.3 Thus, these gene products can be classified into two groups. The first group includes proteins that probably function in protecting cells from dehydration. The second group of gene products contains protein factors that are involved in further regulation of gene expression and signal transduction, and that function in stress response.3

Genetic engineering is thought to be useful for improving the stress tolerance of plants. Recently, several different approaches were attempted to improve the stress tolerance of plants by gene transfer. 4 The genes selected for transformation were those involved in encoding enzymes required for the biosynthesis of various osmoprotectants.5-7 Other genes that have been selected for transformation include those that encoded enzymes for modifying membrane lipids, LEA protein, and detoxification enzyme.8-11 In all these experiments, a single gene for a protective protein or an enzyme was overexpressed under the control of the 35S cauliflower mosaic virus

(CaMV) constitutive promoter in transgenic plants, although a number of genes have been shown to function in environmental stress tolerance and response. The genes encoding protein factors that are involved in regulation of gene expression and signal transduction and function in stress response seem to be useful in improving the tolerance of plants to stresses by gene transfer, as they can regulate many stress inducible genes involved in stress tolerance.

Drought is one of the most severe environmental stresses, and affects almost all the plant functions. Abscisic acid (ABA) is produced under water deficit conditions and plays important roles in tolerance against drought. Most of the drought inducible genes that have been studied to date are also induced by ABA. 3 It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Several reports have described genes that are induced by dehydration but are not responsive to exogenous ABA treatments. 3 These findings suggest the existence of ABA independent, as well as ABA dependent, signal transduction cascades between the initial signal of drought stress and the expression of specific genes. 3 To understand the molecular mechanisms of gene expression in response to drought stress, cis- and trans-acting elements that function in ABA-independent and ABA-responsive gene expression by drought stress have been precisely analyzed. 3 In this article, we summarize recent progress of our research on cis- and trans-acting factors involved in ABA-independent gene expression in drought stress response. We also report stress tolerance of transgenic plants that overexpress a single gene for a stress inducible transcription factor, using Arabidopsis as a model.

Function of Water Stress-Inducible Genes

A variety of genes are induced by drought stress, and functions of their gene products have been predicted from sequence homology with known proteins. Genes induced during drought stress conditions are thought to function not only in protecting cells from dehydration by the production of important metabolic proteins but also in the regulation of genes for signal transduction in the drought stress response.1, 3 Thus, these gene products are classified into two groups (Fig. 20.1).

The first group includes proteins that probably function in stress tolerance:3 water channel proteins involved in the movement of water through membranes, the enzymes required for the biosynthesis of various osmoprotectants (sugars, proline, and betaine), proteins that may protect macromolecules and membranes (LEA protein, osmotin, antifreeze protein, chaperon, and mRNA binding proteins), proteases for protein turnover (thiol proteases, Clp protease, and ubiquitin) and the detoxification enzymes (glutathione S-trans-ferase, soluble epoxide hydrolase, catalase, superoxide dismutase, and ascorbate peroxi-dase). Some of the stress inducible genes that encode proteins, such as a key enzyme for proline biosynthesis, were overexpressed in transgenic plants to produce a stress tolerant phenotype of the plants.6

The second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response: protein kinases, transcription factors and enzymes in phospholipid metabolism.3 Now it becomes more important to elucidate the role of these regulatory proteins for further understanding of plant responses to water stress and for improving the tolerance of plants by gene transfer. The existence of a variety of drought inducible genes suggests complex responses of plants to drought stress. Their gene products are involved in drought stress tolerance and stress responses.

Expression of Dehvdration-Induced Genes in Response to Envitonmental Stresses and ABA

The expression patterns of genes induced by drought were analyzed by RNA gel blot analysis. Results indicated broad variations in the timing of induction of these genes under drought conditions. Most of the drought inducible genes respond to treatment with exogenous ABA, whereas some others do not.1,3 Therefore, there are not only ABA-dependent but also ABA-independent regulatory systems of gene expression under drought stress. Analysis of the expression of ABA inducible genes revealed that several genes require protein biosynthesis for their induction by ABA, suggesting that at least two independent pathways exist between the production of endogenous ABA and gene expression during stress.

As shown in Figure 20.2, we identified at least four independent signal pathways which function under drought conditions: Two are ABA-dependent (pathways I and II) and two are ABA-independent (pathways III and IV). One of the ABA-independent pathways overlaps with that of the cold response (pathway IV). One of the ABA-dependent pathways requires protein biosynthesis (pathway I).1,3 The existence of complex signal transduction pathways in drought response gives a molecular basis for the complex physiological responses of plants to drought stress.

Identification of Cis-Acting Element, DRE, Involved in Drought Responsive Expression

A number of genes are induced by drought, salt and cold in aba (ABA deficient) or abi (ABA insensitive) Arabidopsis mutants. This suggests that these genes do not require ABA for their expression under cold or drought conditions. Among these genes, the expression of a drought inducible gene for rd29A/lti78/cor78 was extensively analyzed. 12 At least two separate regulatory systems function in gene expression during drought and

Fig. 20.1. Drought stress inducible genes and their possible functions in stress tolerance and response. Gene products are classified into two groups. The first group includes proteins that probably function in stress tolerance (Functional Proteins), and the second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response (Regulatory Proteins).

Drought Response Gene

Fig. 20.1. Drought stress inducible genes and their possible functions in stress tolerance and response. Gene products are classified into two groups. The first group includes proteins that probably function in stress tolerance (Functional Proteins), and the second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response (Regulatory Proteins).

cold stress; one is ABA independent (Fig. 20.2, pathway IV) and the other is ABA dependent (pathway II).

To analyze the cis-acting elements involved in the ABA-independent gene expression of rd29A, we constructed chimeric genes with the rd29A promoter fused to the p-glucuronidase (GUS) reporter gene and transformed Arab-idopsis and tobacco plants with these constructs. The GUS reporter gene driven by the rd29A promoter was induced at significant levels in transgenic plants by conditions of dehydration, low temperature, or high salt or by treatment with ABA.13 The deletion, the gain of function and the base substitution analysis of the promoter region of rd29A gene revealed that a 9 bp conserved sequence, TACCGACAT (DRE, Dehydration Responsive

Element), is essential for the regulation of the expression of rd29A under drought conditions. Moreover, DRE has been demonstrated to function as a cis-acting element involved in the induction of rd29A by either low temperature or high salt stress. 12 Therefore, DRE seems to be a cis-acting element involved in gene induction by dehydration, high salt or low temperature, but does not function as an ABA responsive element in the induction of rd29A.

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