Aba

, ABA-dependent

ABA-independent

ABA-independent

|zfhdrs|-[nacrs i |mycrs[{mybrs

ERDl RD22 RD29B

Phosphorylation

|zfhdrs|-[nacrs i |mycrs[{mybrs

ERDl RD22 RD29B

ABA-independent

ABA-independent

Post-translational regulation

DRE/CRT RD29A

ABA-independent

Post-translational regulation

DRE/CRT RD29A

Figure 6.1 Specificity and cross talk of regulatory networks of gene expression in response to osmotic and cold stresses. cis elements and transcription factors (TFs) are shown in boxes and ovals, respectively. Black circles indicate the modification of TFs. Dotted arrow indicates possible regulation. Dotted double arrows indicate possible cross talk.

Identification of stress-inducible genes Differential screening of cDNA Homology-based PCR cloning Microarray

Characterization of stress-inducible genes

Structural analyses and classification of genes

Studies of gene expression under various stress conditions using Northern blot analysis or real-time PCR analysis

Studies of developmental expression Northern blot analysis or real-time PCR analysis

Identification of cis-acting elements in promoter region of gene of interest

Isolation of promoter region (ca. 2-2.5 kb from ATG)

• PCR-based cloning if the genomic sequence data are available

• Cloning of genomic DNA from genomic library if the genomic sequence data is not available in silico analysis of promoter region

• To search for known cis-elements

• To search for conserved sequence(s) if a number of genes are under investigation Deletion and base-substitution analyses using GUS- or LUC- containing binary pBI-based or pGreen-based vector

Identification of tran-acting elements (transcription factors)

Homology-based PCR cloning Yeast one-hybrid system DNA-ligand binding

Verification of the binding specificity both in vivo and in vitro using yeast one-hybrid system and EMSA

Chracterization of transcription factors

Structural analyses and classification

Localization of DNA-binding domain using yeast one-hybrid system and/or EMSA

Localization of activation domain using protoplast transactivation assay

Possible interaction of transcription factors using yeast two-hybrid system and pull-down assay

Functional analysis of transcription factors in planta

Possible modification of transcription factors such as phophorylation Gain-of-function study of transcription factor in plant

• Morphological phenotype of transgenic plants overexpressing the transcription factor

• Stress-tolerant ability of transgenic plants overexpressing the transcription factor

• Response to ABA of transgenic plants overexpressing the transcription factor

• Microarray

Loss-of-function study of transcription factor

• Morphological phenotype of the mutant plant

• Stress-tolerant ability of the mutant plant

• Response to ABA of the mutant plant

• Microarray

Figure 6.2 Scheme for study of regulatory networks of gene involved in stress signaling.

is hoped that it can be used to achieve improvements in crop production. This chapter highlights the methods used to study signaling pathways operating in stress-affected cells and mutual interactions between these pathways. The steps are summarized in a flow diagram as illustrated in Fig. 6.2.

2. Identification of Stress-Responsive Genes

As the first step for studying signaling pathways, we initially used differential hybridization to screen stress-responsive genes. More than 50 independent cDNAs have been identified from Arabidopsis, which were confirmed to respond to osmotic and cold stresses by RNA gel blot hybridization (Shinozaki and Yamaguchi-Shinozaki, 1997, 2000). With the help of powerful microarray technology, a large array of genes—299 drought-inducible genes, 213 high salinity stress-inducible genes, and 54 cold-inducible genes—induced by the stress conditions were discovered by transcriptome analysis using a cDNA microarray containing approximately 7000 independent full-length Arabidopsis cDNA clones (Seki et al., 2004; Shinozaki et al., 2003). Upon release of the Affimetrix 22K Gene Chip, more stress-inducible genes were identified, and data are now available on numerous public Web sites, for example, TAIR (http://www.arabidopsis. org/) and Genevestigator (https://www.genevestigator.ethz.ch/).

The products ofstress-inducible genes have been classified into two groups by their functions: (i) the group of functional proteins that directly protect cells from stresses by the production of important metabolic proteins and (ii) the group of regulatory proteins that regulate gene expression and signal transduction in the stress response. The first group includes enzymes required for biosynthesis of various osmoprotectants and enzymes for fatty acid metabolisms, late embryogenesis-abundant proteins, antifreeze proteins, chaperones, water channel proteins, sugar and proline transporters, detoxification enzymes, proteinase inhibitors, ferritin, and lipid-transfer proteins. The second group consists of transcription factors (TFs), protein kinases, enzymes involved in phosphoinositide metabolism, and enzymes required for the synthesis of the plant hormone abscisic acid (Nakashima and Yamaguchi-Shinozaki, 2006; Yamaguchi-Shinozaki and Shinozaki, 2006). As for the timing of the induction of stress-inducible genes, those encoding the regulatory proteins are logically induced rapidly and transiently in response to drought, high salinity, and cold stresses. Meanwhile, those encoding functional proteins accumulate slowly and gradually within 10 h subsequent to stress treatments (Fowler and Thomashow, 2002; Kreps et al., 2002; Seki et al., 2002; Vogel et al., 2005).

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