Jitender Giri

Glycinebetaine and abiotic stress tolerance in plants

The accumulation of osmolytes like glycinebetaine (GB) in cell is known to protect organisms against abiotic stresses via osmoregulation or osmoprotection. Transgenic plants engineered to produce GB accumulate very low concentration of GB, which might not be sufficient for osmoregulation. Therefore, other roles of GB like cellular macromolecule protection and ROS detoxification have been suggested as mechanisms responsible for abiotic stress tolerance in transgenic plants. In addition, GB influences expression of several endogenous genes in transgenic plants. The new insights gained about the mechanism of stress tolerance in GB accumulating transgenic plants are discussed.

The Receptor-Like Cytoplasmic Kinase (OsRLCK) Gene Family in Rice: Organization, Phylogenetic Relationship, and Expression during Development and Stress

Receptor-like cytoplasmic kinases (RLCKs) in plants belong to the super family of receptor-like kinases (RLKs). These proteins show homology to RLKs in kinase domain but lack the transmembrane domain. Some of the functionally characterized RLCKs from plants have been shown to play roles in development and stress responses. Previously, 149 and 187 RLCK encoding genes were identified from Arabidopsis and rice, respectively. By using HMM-based domain structure and phylogenetic relationships, we have identified 379 OsRLCKs from rice. OsRLCKs are distributed on all 12 chromosomes of rice and some members are located on duplicated chromosomal segments. Several OsRLCKs probably also undergo alternative splicing, some having evidence only in the form of gene models. To understand their possible functions, expression patterns during landmark stages of vegetative and reproductive development as well as abiotic and biotic stress using microarray and MPSS-based data were analyzed. Real-time PCR-based expression profiling for a selected few genes confirmed the outcome of microarray analysis. Differential expression patterns observed for majority of OsRLCKs during development and stress suggest their involvement in diverse functions in rice. Majority of the stress-responsive OsRLCKs were also found to be localized within mapped regions of abiotic stress QTLs. Outcome of this study would help in selecting organ/development stage specific OsRLCK genes/targets for functional validation studies.

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.

Protein phosphatase complement in rice: genome-wide identification and transcriptional analysis under abiotic stress conditions and reproductive development

BackgroundProtein phosphatases are the key components of a number of signaling pathways where they modulate various cellular responses. In plants, protein phosphatases constitute a large gene family and are reportedly involved in the regulation of abiotic stress responses and plant development. Recently, the whole complement of protein phosphatases has been identified in Arabidopsis genome. While PP2C class of serine/threonine phosphatases has been explored in rice, the whole complement of this gene family is yet to be reported.ResultsIn silico investigation revealed the presence of 132-protein phosphatase-coding genes in rice genome. Domain analysis and phylogenetic studies of evolutionary relationship categorized these genes into PP2A, PP2C, PTP, DSP and LMWP classes. PP2C class represents a major proportion of this gene family with 90 members. Chromosomal localization revealed their distribution on all the 12 chromosomes, with 42 genes being present on segmentally duplicated regions and 10 genes on tandemly duplicated regions of chromosomes. The expression profiles of 128 genes under salinity, cold and drought stress conditions, 11 reproductive developmental (panicle and seed) stages along with three stages of vegetative development were analyzed using microarray expression data. 46 genes were found to be differentially expressing in 3 abiotic stresses out of which 31 were up-regulated and 15 exhibited down-regulation. A total of 82 genes were found to be differentially expressing in different developmental stages. An overlapping expression pattern was found for abiotic stresses and reproductive development, wherein 8 genes were up-regulated and 7 down-regulated. Expression pattern of the 13 selected genes was validated employing real time PCR, and it was found to be in accordance with the microarray expression data for most of the genes.ConclusionsExploration of protein phosphatase gene family in rice has resulted in the identification of 132 members, which can be further divided into different classes phylogenetically. Expression profiling and analysis indicate the involvement of this large gene family in a number of signaling pathways triggered by abiotic stresses and their possible role in plant development. Our study will provide the platform from where; the expression pattern information can be transformed into molecular, cellular and biochemical characterization of members belonging to this gene family.

Glycinebetaine‐induced water‐stress tolerance in codA‐expressing transgenic indica rice is associated with up‐regulation of several stress responsive genes

Rice (Oryza sativa L.), a non-accumulator of glycinebetaine (GB), is highly susceptible to abiotic stress. Transgenic rice with chloroplast-targeted choline oxidase encoded by the codA gene from Arthrobacter globiformis has been evaluated for inheritance of transgene up to R5 generation and water-stress tolerance. During seedling, vegetative and reproductive stages, transgenic plants could maintain higher activity of photosystem II and they show better physiological performance, for example, enhanced detoxification of reactive oxygen species compared to wild-type plants under water-stress. Survival rate and agronomic performance of transgenic plants is also better than wild-type following prolonged water-stress. Choline oxidase converts choline into GB and H2O2 in a single step. It is possible that H2O2/GB might activate stress response pathways and prepare transgenic plants to mitigate stress. To check this possibility, microarray-based transcriptome analysis of transgenic rice has been done. It unravelled altered expression of many genes involved in stress responses, signal transduction, gene regulation, hormone signalling and cellular metabolism. Overall, 165 genes show more than two-fold up-regulation at P-value < 0.01 in transgenic rice. Out of these, at least 50 genes are known to be involved in plant stress response. Exogenous application of H2O2 or GB to wild-type plants also induces such genes. Our data show that metabolic engineering for GB is a promising strategy for introducing stress tolerance in crop plants and which could be imparted, in part, by H2O2- and/or GB-induced stress response genes.

Advances in Transgenic Rice Biotechnology

Rice is the most amenable crop plant for genetic manipulation amongst monocots due to its small genome size, enriched genetic map, availability of entire genome sequence, and relative ease of transformation. Improvement in agronomic traits of rice is bound to affect a sizeable population since it is a primary source of sustenance. Recent advances like use of ‘clean gene’ technology or matrix attachment regions would help improve rice transformation. Function of several novel genes and their promoters has been analyzed in transgenic rice. Significant progress has been made in introducing traits like herbicide, biotic stress and abiotic stress tolerance. Attempts also have been made to enhance nutritional characteristics of the grain and yield. Identification of genes controlling growth and development can be used to modify plant architecture and heading period. Transgenic rice can serve as a biofactory for the production of molecules of pharmaceutical and industrial utility. The drive to apply transgenic rice for public good as well as commercial gains has fueled research to an all time high. Successful field trials and biosafety of transgenic rice have been reported. This would act as a catalyst for greater acceptance of genetically modified food crops. The lessons learnt from rice can be extended to other cereals thereby opening new opportunities and possibilities.

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 SAPs are responsive to multiple biotic stresses and overexpression of OsSAP1, an A20/AN1 zinc-finger protein, enhances the basal resistance against pathogen infection in tobacco.

Eukaryotic A20/AN1 zinc-finger proteins (ZFPs) play an important role in the regulation of immune and stress response. After elucidation of the role of first such protein, OsSAP1, in abiotic stress tolerance, 18 rice stress associated protein (SAP) genes have been shown to be regulated by multiple abiotic stresses. In the present study, expression pattern of all the 18 OsSAP genes have been analysed in response to different biotic stress simulators, in order to get insights into their possible involvement in biotic stress tolerance. Our results showed the upregulation of OsSAP1 and OsSAP11 by all biotic stress simulator treatments. Furthermore, the functional role of OsSAP1 in plant defence responses has been explored through overexpression in transgenic plants. Constitutive expression of OsSAP1 in transgenic tobacco resulted into enhanced disease resistance against virulent bacterial pathogen, together with the upregulation of known defence-related genes. Present investigation suggests that rice SAPs are responsive to multiple biotic stresses and OsSAP1 plays a key role in basal resistance against pathogen infection. This strongly supports the involvement of rice SAPs in cross-talk between biotic and abiotic stress signalling pathways, which makes them ideal candidate to design strategies for protecting crop plants against multiple stresses.

Improvement in phosphate acquisition and utilization by a secretory purple acid phosphatase (OsPAP21b) in rice

Summary Phosphate (Pi) deficiency in soil system is a limiting factor for rice growth and yield. Majority of the soil phosphorus (P) is organic in nature, not readily available for root uptake. Low Pi‐inducible purple acid phosphatases (PAPs) are hypothesized to enhance the availability of Pi in soil and cellular system. However, information on molecular and physiological roles of rice PAPs is very limited. Here, we demonstrate the role of a novel rice PAP, OsPAP21b in improving plant utilization of organic‐P. OsPAP21b was found to be under the transcriptional control of OsPHR2 and strictly regulated by plant Pi status at both transcript and protein levels. Biochemically, OsPAP21b showed hydrolysis of several organophosphates at acidic pH and possessed sufficient thermostability befitting for high‐temperature rice ecosystems with acidic soils. Interestingly, OsPAP21b was revealed to be a secretory PAP and encodes a distinguishable major APase (acid phosphatase) isoform under low Pi in roots. Further, OsPAP21b‐overexpressing transgenics showed increased biomass, APase activity and P content in both hydroponics supplemented with organic‐P sources and soil containing organic manure as sole P source. Additionally, overexpression lines depicted increased root length, biomass and lateral roots under low Pi while RNAi lines showed reduced root length and biomass as compared to WT. In the light of these evidences, present study strongly proposes OsPAP21b as a useful candidate for improving Pi acquisition and utilization in rice.

JAZ Repressors: Potential Involvement in Nutrients Deficiency Response in Rice and Chickpea

Jasmonates (JA) are well-known phytohormones which play important roles in plant development and defense against pathogens. Jasmonate ZIM domain (JAZ) proteins are plant-specific proteins and act as transcriptional repressors of JA-responsive genes. JA regulates both biotic and abiotic stress responses in plants; however, its role in nutrient deficiency responses is very elusive. Although, JA is well-known for root growth inhibition, little is known about behavior of JAZ genes in response to nutrient deficiencies, under which root architectural alteration is an important adaptation. Using protein sequence homology and a conserved-domains approach, here we identify 10 novel JAZ genes from the recently sequenced Chickpea genome, which is one of the most nutrient efficient crops. Both rice and chickpea JAZ genes express in tissue- and stimuli-specific manners. Many of which are preferentially expressed in root. Our analysis further showed differential expression of JAZ genes under macro (NPK) and micronutrients (Zn, Fe) deficiency in rice and chickpea roots. While both rice and chickpea JAZ genes showed a certain level of specificity toward type of nutrient deficiency, generally majority of them showed induction under K deficiency. Generally, JAZ genes showed an induction at early stages of stress and expression declined at later stages of macro-nutrient deficiency. Our results suggest that JAZ genes might play a role in early nutrient deficiency response both in monocot and dicot roots, and information generated here can be further used for understanding the possible roles of JA in root architectural alterations for nutrient deficiency adaptations.