Functional Identification of Arabidopsis Stress Regulatory Genes Using the Controlled cDNA Overexpression System    

Abstract

Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress-and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained highlevel ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.

Waterproofing Crops: Effective Flooding Survival Strategies

“…Evaluation of single and combinatorial mutants or RNA interference lines for several of these genes indicated that they are important for low-oxygen survival but overlap in function. Although RAP2.12 overexpression enhances lowoxygen survival, neither RAP2.12, RAP2.2, HRE1, nor HRE2 overexpression enhances the accumulation of hypoxia-responsive mRNAs under nonstress conditions (Papdi et al, 2008;Hinz et al, 2010;Licausi et al, 2010). This paradox is explained by the instability of the Arabidopsis group VII ERF proteins under oxygen-replete conditions.…”

The Arabidopsis ZINC FINGER PROTEIN3 Interferes with Abscisic Acid and Light Signaling in Seed Germination and Plant Development

“…Screening for ABA-insensitive seed germination of COS-transformed Arabidopsis plants led to the identification of numerous transgenic lines showing different degrees of ABA insensitivity (Papdi et al, 2008). Germination of line A44 was insensitive to 5 mM ABA in the presence of an estradiol inducer of the chemically regulated promoter in the pER8-GW vector, while it was similar to the wild type in the absence of ABA Figure 1.…”

“…A single PCR fragment was amplified from genomic DNA of A44 plants using ER8A and ER8B primers flanking the cDNA insert (Papdi et al, 2008). The nucleotide sequence of the recovered fragment showed 100% identity with the full-length cDNA of the AT5G25160 gene, encoding ZFP3, the function of which was unknown.…”

“…AtPP2CA was identified in a library of complementary DNA (cDNA)-overexpressing lines, which were screened for the ability to germinate in the presence of inhibitory concentrations of ABA (Kuhn et al, 2006). A similar gain-of-function strategy was employed in the conditional cDNA-overexpressing system (COS) to identify novel components in ABA signal transduction (Papdi et al, 2008). While the ectopic overexpression of regulatory genes may result in dominant dwarfism or reduced fertility (Kasuga et al, 1999;Dinkins et al, 2002), the chemically induced expression in the COS circumvents such disadvantages and permits the generation of fertile transgenic plants (Rigó et al, 2012).…”

“…While the ectopic overexpression of regulatory genes may result in dominant dwarfism or reduced fertility (Kasuga et al, 1999;Dinkins et al, 2002), the chemically induced expression in the COS circumvents such disadvantages and permits the generation of fertile transgenic plants (Rigó et al, 2012). Regulated overexpression of cDNAs was employed in the identification of the small heat shock protein gene HSP17.6A, which could confer ABA insensitivity to Arabidopsis (Arabidopsis thaliana), pointing to a novel function of this gene (Papdi et al, 2008).…”