Rice Stress Associated Protein 1 (OsSAP1) Interacts with Aminotransferase (OsAMTR1) and Pathogenesis-Related 1a Protein (OsSCP) and Regulates Abiotic Stress Responses.

Stress associated proteins (SAPs) are the A20/AN1 zinc-finger containing proteins which can regulate the stress signaling in plants. The rice SAP protein, OsSAP1 has been shown to confer abiotic stress tolerance to plants, when overexpressed, by modulating the expression of endogenous stress-related genes. To further understand the mechanism of OsSAP1-mediated stress signaling, OsSAP1 interacting proteins were identified using yeast two-hybrid analysis. Two novel proteins, aminotransferase (OsAMTR1) and a SCP/TAPS or pathogenesis-related 1 class of protein (OsSCP) were found to interact with OsSAP1. The genes encoding OsAMTR1 and OsSCP were stress-responsive and showed higher expression upon abiotic stress treatments. The role of OsAMTR1 and OsSCP under stress was analyzed by overexpressing them constitutively in Arabidopsis and responses of transgenic plants were assessed under salt and water-deficit stress. The OsAMTR1 and OsSCP overexpressing plants showed higher seed germination, root growth and fresh weight than wild-type plants under stress conditions. Overexpression of OsAMTR1 and OsSCP affected the expression of many known stress-responsive genes which were not affected by the overexpression of OsSAP1. Moreover, the transcript levels of OsSCP and OsAMTR1 were also unaffected by the overexpression of OsSAP1. Hence, it was concluded that OsSAP1 regulates the stress responsive signaling by interacting with these proteins which further regulate the downstream stress responsive gene expression.

OsiSAP1 overexpression improves water-deficit stress tolerance in transgenic rice by affecting expression of endogenous stress-related genes.

KEY MESSAGE: OsiSAP1, an A20/AN1 zinc-finger protein, confers water-deficit stress tolerance at different stages of growth by affecting expression of several endogenous genes in transgenic rice. Transgenic lines have been generated from rice constitutively expressing OsiSAP1, an A20/AN1 zinc-finger containing stress-associated protein gene from rice, driven by maize UBIQUITIN gene promoter and evaluated for water-deficit stress tolerance at different stages of growth. Their seeds show early germination and seedlings grow better under water-deficit stress compared to non-transgenic (NT) rice. Leaves from transgenic seedlings showed lesser membrane damage and lipid peroxidation under water-deficit stress. Relatively lower rate of leaf water loss has been observed in detached intact leaves from transgenic plants during late vegetative stage. Delayed leaf rolling and higher relative water content were also observed in transgenic plants under progressive water-deficit stress during reproductive developmental stage. Although reduction in grain yield is observed under unstressed condition, the relative water-deficit stress-induced yield losses are lower in transgenic rice vis-à-vis NT plants thereby resulting in yield loss protection. Transcriptome analysis suggests that overexpression of OsiSAP1 in transgenic rice results in altered expression of several endogenous genes including those coding for transcription factors, membrane transporters, signaling components and genes involved in metabolism, growth and development. A total of 150 genes were found to be more than twofold up-regulated in transgenic rice of which 43 genes are known to be involved in stress response. Our results suggest that OsiSAP1 is a positive regulator of water-deficit stress tolerance in rice.

SAPs as novel regulators of abiotic stress response in plants.

Stress associated proteins (SAPs), novel A20/AN1 zinc-finger domain-containing proteins, are fast emerging as potential candidates for biotechnological approaches in order to improve abiotic stress tolerance in plants - the ultimate aim of which is crop-yield protection. Until relatively recently, such proteins had only been identified in humans, where they had been shown to be key regulators of innate immunity. Their phylogenetic relationship and recruitment of diverse protein domains reflect an architectural and mechanistic diversity. Emerging evidence suggests that SAPs may act as ubiquitin ligase, redox sensor, and regulator of gene expression during stress. Here, we evaluate the new knowledge on SAPs with a view to understand their mechanism of action. Furthermore, we set an agenda for investigating hitherto unexplored roles of these proteins.

Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants.

• The inbuilt mechanisms of plant survival have been exploited for improving tolerance to abiotic stresses. Stress-associated proteins (SAPs), containing A20/AN1 zinc-finger domains, confer abiotic stress tolerance in different plants, however, their interacting partners and downstream targets remain to be identified. • In this study, we have investigated the subcellular interactions of rice SAPs and their interacting partner using yeast two-hybrid and fluorescence resonance energy transfer (FRET) approaches. Their efficacy in improving abiotic stress tolerance was analysed in transgenic Arabidopsis plants. Regulation of gene expression by genome-wide microarray in transgenics was used to identify downstream targets. • It was found that the A20 domain mediates the interaction of OsSAP1 with self, its close homolog OsSAP11 and a rice receptor-like cytoplasmic kinase, OsRLCK253. Such interactions between OsSAP1/11 and with OsRLCK253 occur at nuclear membrane, plasma membrane and in nucleus. Functionally, both OsSAP11 and OsRLCK253 could improve the water-deficit and salt stress tolerance in transgenic Arabidopsis plants via a signaling pathway affecting the expression of several common endogenous genes. • Components of a novel stress-responsive pathway have been identified. Their stress-inducible expression provided the protection against yield loss in transgenic plants, indicating the agronomic relevance of OsSAP11 and OsRLCK253 in conferring abiotic stress tolerance.

Modulation of transcription factor and metabolic pathway genes in response to water-deficit stress in rice.

Water-deficit stress is detrimental for rice growth, development, and yield. Transcriptome analysis of 1-week-old rice (Oryza sativa L. var. IR64) seedling under water-deficit stress condition using Affymetrix 57 K GeneChip® has revealed 1,563 and 1,746 genes to be up- and downregulated, respectively. In an effort to amalgamate data across laboratories, we identified 5,611 differentially expressing genes under varying extrinsic water-deficit stress conditions in six vegetative and one reproductive stage of development in rice. Transcription factors (TFs) involved in ABA-dependent and ABA-independent pathways have been found to be upregulated during water-deficit stress. Members of zinc-finger TFs namely, C2H2, C2C2, C3H, LIM, PHD, WRKY, ZF-HD, and ZIM, along with TF families like GeBP, jumonji, MBF1 and ULT express differentially under water-deficit conditions. NAC (NAM, ATAF and CUC) TF family emerges to be a potential key regulator of multiple abiotic stresses. Among the 12 TF genes that are co-upregulated under water-deficit, salt and cold stress conditions, five belong to the NAC TF family. We identified water-deficit stress-responsive genes encoding key enzymes involved in biosynthesis of osmoprotectants like polyols and sugars; amino acid and quaternary ammonium compounds; cell wall loosening and structural components; cholesterol and very long chain fatty acid; cytokinin and secondary metabolites. Comparison of genes responsive to water-deficit stress conditions with genes preferentially expressed during panicle and seed development revealed a significant overlap of transcriptome alteration and pathways.