Amanjot Singh

Salt stress response in rice: genetics, molecular biology, and comparative genomics

Significant progress has been made in unraveling the molecular biology of rice in the past two decades. Today, rice stands as a forerunner amongst the cereals in terms of details known on its genetics. Evidence show that salt tolerance in plants is a quantitative trait. Several traditional cultivars, landraces, and wild types of rice like Pokkali, CSR types, and Porteresia coarctata appear as promising materials for donation of requisite salt tolerance genes. A large number of quantitative trait loci (QTL) have been identified for salt tolerance in rice through generation of recombinant inbred lines and are being mapped using different types of DNA markers. Salt-tolerant transgenic rice plants have been produced using a host of different genes and transcript profiling by micro- and macroarray-based methods has opened the gates for the discovery of novel salt stress mechanisms in rice, and comparative genomics is turning out to be a critical input in this respect. In this paper, we present a comprehensive review of the genetic, molecular biology, and comparative genomics effort towards the generation of salt-tolerant rice. From the data on comprehensive transcript expression profiling of clones representing salt-stress-associated genes of rice, it is shown that transcriptional and translational machineries are important determinants in controlling salt stress response, and gene expression response in tolerant and susceptible rice plants differs mainly in quantitative terms.

Heat shock factor gene family in rice: Genomic organization and transcript expression profiling in response to high temperature, low temperature and oxidative stresses

Binding of heat shock factors (HSFs) with heat shock element sequence is critical for the transcriptional induction of heat shock genes. Rice genome sequence shows 26 OsHsf genes out of which 25 possess various important domains noted in HSFs i.e. DNA binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES) and AHA type activation domain. OsHsf entry LOC_Os06g226100 has the oligomerization domain but lacks the above other domains. Also, there are no ESTs or full-length cDNA noted for this entry in database. Expression profiling showed that 22 OsHsf genes are induced by high temperature. Induction of 10 and 14 OsHsf genes was also noted against low temperature stress and oxidative stress, respectively. All OsHsf genes induced by oxidative stress were also induced by high temperature. On the other hand, induction of 6 and 1 OsHsf genes was noted to be exclusive to high and low temperature stresses, respectively. Seven OsHsf genes showed induced expression in response to all the three stresses examined. While in silico promoter analysis showed that OsHsf genes contain upstream regulatory elements corresponding to different abiotic stresses, there was lack of correlation noted between the in silico profiling of the elements and their corresponding transcript expression patterns. Apart from stress inducibility, EST database suggests that various OsHsf genes are developmentally regulated in diverse tissue types.

SUMO-conjugating enzyme (Sce) and FK506-binding protein (FKBP) encoding rice (Oryza sativa L.) genes: genome-wide analysis, expression studies and evidence for their involvement in abiotic stress response.

We report an in-depth characterization of two major stress proteins namely SUMO-conjugating enzyme (Sce) and peptidyl prolyl cis-trans isomerase (PPIase) in rice (Oryza sativa L.). Sce mediates addition of SUMO group to various cell proteins, through process referred to as SUMOylation. Rice nuclear genome has two putative genes encoding the Sce protein (OsSce1 and OsSce2). PCR-amplified full-length OsSce1 cDNA functionally complemented the growth defect in yeast cells lacking the equivalent Ubc9 protein (ScΔubc9). RT-PCR analysis showed that transcript levels of OsSce1 and OsSce2 in rice seedlings were regulated by temperature stress. OsSce1 protein was localized to the nucleus in onion epidermal cells as evidenced by the transient GFP expression analysis following micro-projectile gun-based shooting of an OsSce1-GFP fusion construct. PPIase proteins assist molecular chaperones in reactions associated with protein folding and protein transport across membrane. There are 23 putative genes encoding for FK506-binding proteins (FKBPs; specific class of PPIase) in rice genome. OsFKBP20 cDNA was isolated as a stress-inducible EST clone. Largest ORF of 561 bases in OsFKBP20 showed characteristic FK506-binding domain at N-terminus and a coiled-coil motif at C-terminus. RNA expression analysis indicated that OsFKBP20 transcript is heat-inducible. OsFKBP20 over-expression in yeast endowed capacity of high temperature tolerance to yeast cells. Yeast two-hybrid analysis showed that OsSce1 protein physically interacts with the OsFKBP20 protein. It is thus proposed that OsSce1 and OsFKBP20 proteins in concert mediate the stress response of rice plants.

Genome-wide analysis of rice ClpB/HSP100, ClpC and ClpD genes

BackgroundClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues.ResultsNine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300), 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770) and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210). Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Δhsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene imparted appreciably high level tolerance to the mutant yeast cells.ConclusionsRice class I Clp ATPase gene family is constituted of 9 members. Of these 9, only 3 belonging to ClpB group are heat stress regulated. Distribution of ClpB proteins to different cell organelles indicates that their functioning might be critical in different cell locations. From the complementation assays, OsClpD1 appears to be more effective than OsClpB-cyt protein in rescuing the thermosensitive defect of the yeast ScΔhsp104 mutant cells.

Genetic engineering for heat tolerance in plants.

High temperature tolerance has been genetically engineered in plants mainly by over-expressing the heat shock protein genes or indirectly by altering levels of heat shock transcription factor proteins. Apart from heat shock proteins, thermotolerance has also been altered by elevating levels of osmolytes, increasing levels of cell detoxification enzymes and through altering membrane fluidity. It is suggested that Hsps may be directly implicated in thermotolerance as agents that minimize damage to cell proteins. The other three above approaches leading to thermotolerance in transgenic experiments though operate in their own specific ways but indirectly might be aiding in creation of more reductive and energy-rich cellular environment, thereby minimizing the accumulation of damaged proteins. Intervention in protein metabolism such that accumulation of damaged proteins is minimized thus appears to be the main target for genetically-engineering crops against high temperature stress.

Binding affinities and interactions among different heat shock element types and heat shock factors in rice (Oryza sativa L.)

Binding of heat shock factors (Hsfs) to heat shock elements (HSEs) leads to transcriptional regulation of heat shock genes. Genome‐wide, 953 rice genes contain perfect‐type, 695 genes gap‐type and 1584 genes step‐type HSE sequences in their 1‐kb promoter region. The rice genome contains 13 class A, eight class B and four class C Hsfs (OsHsfs) and has OsHsf26 (which is of variant type) genes. Chemical cross‐linking analysis of in vitro synthesized OsHsf polypeptides showed formation of homotrimers of OsHsfA2c, OsHsfA9 and OsHsfB4b proteins. Binding analysis of polypeptides with oligonucleotide probes containing perfect‐, gap‐, and step‐type HSE sequences showed that OsHsfA2c, OsHsfA9 and OsHsfB4b differentially recognize various model HSEs as a function of varying reaction temperatures. The homomeric form of OsHsfA2c and OsHsfB4b proteins was further noted by the bimolecular fluorescence complementation approach in onion epidermal cells. In yeast two‐hybrid assays, OsHsfB4b showed homomeric interaction as well as distinct heteromeric interactions with OsHsfA2a, OsHsfA7, OsHsfB4c and OsHsf26. Transactivation activity was noted in OsHsfA2c, OsHsfA2d, OsHsfA9, OsHsfC1a and OsHsfC1b in yeast cells. These differential patterns pertaining to binding with HSEs and protein–protein interactions may have a bearing on the cellular functioning of OsHsfs under a range of different physiological and environmental conditions.

Chymotrypsin protease inhibitor gene family in rice: Genomic organization and evidence for the presence of a bidirectional promoter shared between two chymotrypsin protease inhibitor genes.

Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Full-length cDNA encoding for a putative subtilisin-chymotrypsin protease inhibitor (OCPI2) was obtained from Pusa basmati 1 (indica) rice seedlings. 620 bp-long OCPI2 cDNA contained 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDa subtilisin-chymotrypsin protease inhibitor (CPI) cytoplasmic protein. Expression analysis by semi-quantitative RT-PCR analysis showed that OCPI2 transcript is induced by varied stresses including salt, ABA, low temperature and mechanical injury in both root and shoot tissues of the seedlings. Transgenic rice plants produced with OCPI2 promoter-gus reporter gene showed that this promoter directs high salt- and ABA-regulated expression of the GUS gene. Another CPI gene (OCPI1) upstream to OCPI2 (with 1126 bp distance between the transcription initiation sites of the two genes; transcription in the reverse orientation) was noted in genome sequence of rice genome. A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment. Expression of both GFP and GUS from the same epidermal cell showed that this sequence represents a bidirectional promoter. Examples illustrating gene pairs showing co-expression of two divergent neighboring genes sharing a bidirectional promoter have recently been extensively worked out in yeast and human systems. We provide an example of a gene pair constituted of two homologous genes showing co-expression governed by a bidirectional promoter in rice.

Plant Hsp100/ClpB-like proteins: poorly-analyzed cousins of yeast ClpB machine

ClpB/Hsp100 proteins act as chaperones, mediating disaggregation of denatured proteins. Recent work shows that apart from cytoplasm, these proteins are localized to nuclei, chloroplasts, mitochondria and plasma membrane. While ClpB/Hsp100 genes are essentially stress-induced (mainly heat stress) in vegetative organs of the plant body, expression of ClpB/Hsp100 proteins is noted to be constitutive in plant reproductive structures like pollen grains, developing embryos, seeds etc. With global warming looming large on the horizon, ways to genetically engineer plants against high temperature stress are urgently needed. Yeast mutants unable to synthesize active ClpB/Hsp100 protein show a clear thermosensitive phenotype. ClpB/Hsp100 proteins are implicated in high temperature stress tolerance in plants. We herein highlight the selected important facets of this protein family in plants.

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.

Glycine-rich RNA binding protein of Oryza sativa inhibits growth of M15 E. coli cells.

BackgroundPlant glycine-rich RNA binding proteins have been implicated to have roles in diverse abiotic stresses.FindingsE. coli M15 cells transformed with full-length rice glycine-rich RNA binding protein4 (OsGR-RBP4), truncated rice glycine-rich RNA binding protein4 (OsGR-RBP4ΔC) and rice FK506 binding protein (OsFKBP20) were analyzed for growth profiles using both broth and solid media. Expression of OsGR-RBP4 and OsGR-RBP4ΔC proteins caused specific, inhibitory effect on growth of recombinant M15 E. coli cells. The bacterial inhibition was shown to be time and incubation temperature dependent. Removal of the inducer, IPTG, resulted in re-growth of the cells, indicating that effect of the foreign proteins was of reversible nature. Although noted at different levels of dilution factors, addition of purified Os-GR-RBP4 and OsGR-RBP4ΔC showed a similar inhibitory effect as seen with expression inside the bacterial cells.ConclusionsExpression of eukaryotic, stress-associated OsGR-RBP4 protein in prokaryotic E. coli M15 cells proves injurious to the growth of the bacterial cells. E. coli genome does not appear to encode for any protein that has significant homology to OsGR-RBP4 protein. Therefore, the mechanism of inhibition appears to be due to some illegitimate interactions of the OsGR-RBP4 with possibly the RNA species of the trans-host bacterial cells. The detailed mechanism underlying this inhibition remains to be worked out.