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

Beyond osmolytes and transporters: novel plant salt-stress tolerance-related genes from transcriptional profiling data

With recent advancements in DNA-chip technology, requisite software development and support and progress in related aspects of plant molecular biology, it is now possible to comprehensively analyze the expression of complete genomes. Global transcript profiling shows that in plants, salt-stress response involves simultaneous up and downregulation of a large number of genes. This analysis further suggests that apart from the transcripts that govern synthesis of osmolytes and ion transporters, two candidate systems that have attracted much of the attention thus far, transcripts encoding for proteins related to the regulation of transcriptional and translational machineries have a distinct role in salt-stress response. In particular, induction of transcripts of specific transcription factors, RNA-binding proteins, ribosomal genes, and translation initiation and elongation factors has recently been noted to be important during salt stress. There is an urgent need to examine cellular functionality of the above putative salt-tolerance-related genes emerging from the transcriptome analysis

Molecular characterization of a novel isoform of rice (Oryza sativa L.) glycine rich-RNA binding protein and evidence for its involvement in high temperature stress response

A novel full-length cDNA encoding for glycine rich (GR)-RNA binding protein (RBP) (Osgr-rbp4) is isolated from rice heat shock cDNA library. Amino acid sequence of the deduced protein reveals existence of RNA recognition motif (RRM) comprising of highly conserved RNA binding RNPI and RNPII domains at the N-terminus. C-terminus of this protein is rich in arginine and glycine residues. Blast search analysis on rice genome sequence database shows that GR-RBP protein family is constituted of multiple members with high level of amino acid conservation in RNA recognition motif and glycine domain regions. Similar analysis across wider biological systems from NCBI database indicated that rice GR-RBP4 has homologs in different living genera. Osgr-rbp4 transcript in rice seedlings is constitutively expressed as well as regulated by different abiotic stresses including high temperature stress. Ectopic over-expression of Osgr-rbp4 cDNA imparts high temperature stress tolerance to wild type yeast cells. It is shown that OsGR-RBP4 in rice leaf cells and its immunologically homologous protein in tobacco BY2 protoplasts are nuclear proteins. Upon heat shock, bulk of these proteins appears to be localized in the cytoplasm. We suggest that OsGR-RBP4 probably bind and stabilize the stress-inducible transcripts under HS conditions

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

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

Production of high temperature tolerant transgenic plants through manipulation of membrane lipids

This article does not have an abstract

Prospects of improving flooding tolerance in lowland rice varieties by conventional breeding and genetic engineering

Flooding is a recurrent phenomenon in several lowland rice-growing areas in India and elsewhere. Even though rice is a reasonably flooding-tolerant crop, the annual loss incurred by farmers due to floods is large. There are excellent traditional rice types with high level flooding tolerance. Combining high level flooding tolerance to high grain yield through conventional breeding has been successful to a limited extent so far but there are enormous opportunities for the same. There are also hopes that flooding tolerance can be genetically engineered in rice using a transgenic approach. We take a look on the prospects for improvement of rice to flooding stress through conventional breeding and through plant genetic engineering

Experimentation in biology of plant abiotic stress responses

During the course of growth under natural field conditions, crop plants are exposed to a number of different abiotic stresses (such as water stress, temperature stress, salt stress, flooding stress, chemical stress and oxidative stress). These stresses exert adverse effects on metabolism, growth and yield of the crops. The intensity of the abiotic stresses is on the rise, implying that various possible solutions for mitigating the damage caused by such stresses must be combined for future increase in crop production. At the level of plant genetics, there are indications that it may be possible to improve plants against such stress factors. However, the practical success in this regard depends on how well we understand the biochemistry. physiology and molecular biology of the plant abiotic stress responses. The cellular response of plants to abiotic stresses is of complex nature involving simultaneous interplay of several mechanisms. Although there is a great deal of progress in cataloguing the biochemical reactions that are associated with plant abiotic stress responses, precise understanding of the defense reactions leading to acquisition of stress tolerance remains a challenge. A number of different experimental systems including lower and higher plants as well as microbes have been analyzed for examining the plant abiotic stress responses. The molecular analysis of the stress response has been carried out at the level of stress proteins, stress genes, stress promoters, trans-acting factors that bind to stress promoters and signal transduction components involved in mediation of stress responses. The functional relevance of the stress - associated genes is being tested in different trans-systems including yeast as well as higher plant species. In this article, we discuss selective features of experimentation in biology of plant abiotic stress responses

Taming abiotic stresses in plants through genetic engineering: current strategies and perspective

During the last decade, major advances have been made in plant genetic engineering. The methods for stable genetic transformation as well as for regulation of introduced trans-genes have been optimised to a great deal. The major limiting factor in the widespread application of genetic engineering is the availability of the target genes. This is particularly true for engineering tolerance against abiotic stresses (such as those caused by high levels of salts in soils, reduced/excess availability of water and sub- and supra-optimal temperature regimes). In spite of this, the past 5 years (1993-1998) have witnessed significant achievements in terms of generating transgenics for enhanced tolerance to these stresses. For future work on producing plants with still higher level of tolerance, there is a need to expand the information on the stress-induced genes so that appropriate genes can be pyramided. The current upsurge in genomic research has the potential to catalyse efforts in elucidating new stress-responsive genes. There are also possibilities of engineering the whole cascade of multiple genetic changes through manipulation of the regulatory genes

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