Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.

Generating high temperature tolerant transgenic plants: Achievements and challenges.

Production of plants tolerant to high temperature stress is of immense significance in the light of global warming and climate change. Plant cells respond to high temperature stress by re-programming their genetic machinery for survival and reproduction. High temperature tolerance in transgenic plants has largely been achieved either by over-expressing heat shock protein genes or by altering levels of heat shock factors that regulate expression of heat shock and non-heat shock genes. Apart from heat shock factors, over-expression of other trans-acting factors like DREB2A, bZIP28 and WRKY proteins has proven useful in imparting high temperature tolerance. Besides these, elevating the genetic levels of proteins involved in osmotic adjustment, reactive oxygen species removal, saturation of membrane-associated lipids, photosynthetic reactions, production of polyamines and protein biosynthesis process have yielded positive results in equipping transgenic plants with high temperature tolerance. Cyclic nucleotide gated calcium channel proteins that regulate calcium influxes across the cell membrane have recently been shown to be the key players in induction of high temperature tolerance. The involvement of calmodulins and kinases in activation of heat shock factors has been implicated as an important event in governing high temperature tolerance. Unfilled gaps limiting the production of high temperature tolerant transgenic plants for field level cultivation are discussed.

Gene expression analysis in response to low and high temperature and oxidative stresses in rice: combination of stresses evokes different transcriptional changes as against stresses applied individually.

Transcript expression profiles of rice seedlings were analyzed in response to (a) prior exposure with oxidative stress followed by heat or cold stress and (b) simultaneous exposure to oxidative stress along with heat stress or cold stress. The numbers of genes differentially regulated during stress combination of cold and oxidative stress as well as heat and oxidative stress treatments were higher when compared with the number of genes differentially regulated in response to individual stress conditions. A large number of transcript changes were noted unique to the stress combination mode as compared with when individual stresses were applied. Specific differences in the transcript expression profiles of OsHsf and OsClp gene family members were noted during combination of stresses as against individual stresses. For instance, OsHsf26 induction was specific to stress combinations, while OsHsfA2a, OsHsfA2f, and OsHsfA3 transcript levels were additively affected during combination of stresses. Unique promoter models and transcription factor binding sites (i.e. P$KNOX3_01, P$OSBZ8_Q6) were noted in the promoters of differentially regulated genes during combination of stresses. It is proposed that stress combinations represent a novel state of abiotic stresses for rice seedlings that might involve a different type of molecular response.

Genome-wide transcriptional profiles during temperature and oxidative stress reveal coordinated expression patterns and overlapping regulons in rice.

Genome wide transcriptional changes by cold stress, heat stress and oxidative stress in rice seedlings were analyzed. Heat stress resulted in predominant changes in transcripts of heat shock protein and heat shock transcription factor genes, as well as genes associated with synthesis of scavengers of reactive oxygen species and genes that control the level of sugars, metabolites and auxins. Cold stress treatment caused differential expression of transcripts of various transcription factors including desiccation response element binding proteins and different kinases. Transcripts of genes that are part of calcium signaling, reactive oxygen scavenging and diverse metabolic reactions were differentially expressed during cold stress. Oxidative stress induced by hydrogen peroxide treatment, resulted in significant up-regulation in transcript levels of genes related to redox homeostasis and down-regulation of transporter proteins. ROS homeostasis appeared to play central role in response to temperature extremes. The key transcription factors that may underlie the concerted transcriptional changes of specific components in various signal transduction networks involved are highlighted. Co-ordinated expression pattern and promoter architectures based analysis (promoter models and overrepresented transcription factor binding sites) suggested potential regulons involved in stress responses. A considerable overlap was noted at the level of transcription as well as in regulatory modules of differentially expressed genes.

OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.).

ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE(-273 to -280)) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.

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.

Cycloheximide-mediated superinduction of genes involves both native and foreign transcripts in rice (Oryza sativa L.).

Rice seedlings subjected to heat shock show rapid and transient induction of Oshsp17.4-CI, Oshsp17.9A-CI and OsClpB-cyt/hsp100 transcripts. When the seedlings were pre-treated with protein synthesis inhibitor cycloheximide, levels of the above transcripts during heat shock were more elevated than those seen with heat shock alone. Heat stress and cycloheximide co-treatment resulted in higher transcript accumulation in comparison to cycloheximide pre-treatment followed by heat stress. In transgenic plants raised with OsClpB-cyt/hsp100 promoter driving expression of the reporter gus gene, expression of the gus transcript was subjected to similar superinduction event as was seen with native OsClpB-cyt/hsp100 transcripts in untransformed plants. Yeast cells transformed with variably-sized rice ClpB-cyt/hsp100 promoter driving expression of the lacZ reporter transcript showed that specific sequences of the promoter region may be implicated in superinduction.

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

BACKGROUND: ClpB-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. RESULTS: Nine 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 Deltahsp104 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. CONCLUSIONS: Rice 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 ScDeltahsp104 mutant cells.

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.

Agroinfection of cloned Sri Lankan cassava mosaic virus DNA to Arabidopsis thaliana, Nicotiana tabacum and cassava.

Sri Lankan cassava mosaic virus (SLCMV) is a bipartite begomovirus infecting cassava in India and Sri Lanka. We have used Agrobacterium-mediated inoculation (agroinoculation) of cloned SLCMV DNA to inoculate additional hosts, Nicotiana tabacum and Arabidopsis. Although SLCMV infection in these hosts caused stunting, leaf deformation and developmental abnormalities, accumulation levels of viral DNA in the infected plants suggested that this virus was poorly adapted to them. In the natural host, cassava, agroinoculation produced infection at a low frequency. The monopartite nature of SLCMV, reported earlier in N. benthamiana, was maintained in the new hosts as well as in cassava.