Functional Studies on Two Positive Osmotic Stress Regulators, AtBG2 and AtANAC096

Abstract

AbstractThe phytohormone abscisic acid (ABA) plays critical roles in plant growth and development as well as biotic and abiotic stress responses in plants. As abiotic stress is one of most serious challenges for plants, it is of great importance to understand genetic traits that are specifically responsible to abiotic conditions as well as for further conduct of genetic engineering. In this study, by using forward genetic screening, that are, firefly luciferase reporter (LUC) activity suppression screening, in which LUC reporter is driven by the osmotic stress-responsive RD29A promoter, in the presence of NaCl stress conditions and cotyledon stay green screening in the presence of ABA, we isolated two mutants, AtBG2 as well as AtANAC096, respectively, and further conduct the functional studies.In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via AtBG1-mediated hydrolysis of glucose-conjugated ABA (ABA-GE). However, it is not known how many different -glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered -glucosidase homolog, AtBG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. AtBG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. AtBG2 hydrolyzed ABA-GE to ABA in vitro. In addition, AtBG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of AtBG2 rescued the atbg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis atbg2 alleles with a T-DNA insertion in AtBG2 were more sensitive to dehydration and NaCl stress, whereas AtBG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific -glucosidases in response to abiotic stresses.Diverse signaling pathways are used to respond to ever-changing environmental conditions. In these signaling circuits, various types of transcription factors are a core component by activating expression of a large number of genes involved in the response reactions. Accordingly, the action mechanism of transcription factors has been one of the main focuses in research on abiotic stress responses. However, it is still far from full understanding on the physiological roles and action mechanism of theses transcription factors involved in abiotic stress responses. In this study, we provide evidence that AtANAC096, a member of the NAC transcription factor family, plays a positive role in the abiotic stress responses in Arabidopsis. An Arabidopsis mutant that has a T-DNA insertion at AtANAC096 exhibited hyposensitivity to exogenous ABA in germination and growth, delayed stomatal closure and increased water loss, whereas transgenic plants overexpressing AtANAC096 exhibited hypersensitivity to exogenous ABA and decrease in water loss. At the molecular level, AtANAC096 activated transcription of a subset of ABA and dehydration stress-inducible genes including RD29A and NCED3. Moreover, AtANAC096 interacted with ABF2 and ABF4 and exhibited a synergistic relationship with ABFs in transcription of common target genes. Expression of AtANAC096 itself is induced by ABA and dehydration stress. Based on these results, we propose that AtANAC096 is involved in transcription of genes regulated by ABA-dependent signaling pathways by acting alone or together with ABFs in a synergistic manner in response to dehydration stress.