Breeding objectives and requirements for producing transgenics for major field crops of India

To identify crop improvement objectives in twelve important field crops (rice, wheat, maize, sorghum, pearlmillet, pigeonpea, chickpea, mungbean, cotton, potato, mustard and soybean) that are grown extensively in India, we conducted a survey amongst plant breeders, pathologists, entomologists and agronomists specializing in each of these identified crops. A questionnaire was sent to around fifteen scientists actively involved with each crop with the following queries: (1) Identification of problems with the crop at the regional level in terms of priority, (2) Identification of problems with the crop at the national level in terms of priority, (3) Which are the most extensively grown cultivars of the crop at the regional and at the national levels?, (4) What steps could be taken to raise the yield of the crop (heterosis breeding, pure-line breeding)?, Do you know of combiners that would give high heterosis in the crop?, (5) Do you know of germplasm sources that could be used for meeting some of the breeding objectives?, (6) What is your assessment of need for transgenics (a) for nutritional enhancement, (b) for resistance to biotic stresses, (c) for resistance to abiotic stresses, (d) for herbicide resistance and (e) for value addition

Intergenic sequence between Arabidopsis caseinolytic protease B-cytoplasmic/heat shock protein100 and choline kinase genes functions as a heat-inducible bidirectional promoter

In Arabidopsis (Arabidopsis thaliana), the At1g74310 locus encodes for caseinolytic protease B-cytoplasmic (ClpB-C)/heat shock protein100 protein (AtClpB-C), which is critical for the acquisition of thermotolerance, and At1g74320 encodes for choline kinase (AtCK2) that catalyzes the first reaction in the Kennedy pathway for phosphatidylcholine biosynthesis. Previous work has established that the knockout mutants of these genes display heat-sensitive phenotypes. While analyzing the AtClpB-C promoter and upstream genomic regions in this study, we noted that AtClpB-C and AtCK2 genes are head-to-head oriented on chromosome 1 of the Arabidopsis genome. Expression analysis showed that transcripts of these genes are rapidly induced in response to heat stress treatment. In stably transformed Arabidopsis plants harboring this intergenic sequence between head-to-head oriented green fluorescent protein and beta-glucuronidase reporter genes, both transcripts and proteins of the two reporters were up-regulated upon heat stress. Four heat shock elements were noted in the intergenic region by in silico analysis. In the homozygous transfer DNA insertion mutant Salk_014505, 4,393-bp transfer DNA is inserted at position 2517 upstream of ATG of the AtClpB-C gene. As a result, AtCk2 loses proximity to three of the four heat shock elements in the mutant line. Heat-inducible expression of the AtCK2 transcript was completely lost, whereas the expression of AtClpB-C was not affected in the mutant plants. Our results suggest that the 1,329-bp intergenic fragment functions as a heat-inducible bidirectional promoter and the region governing the heat inducibility is possibly shared between the two genes. We propose a model in which AtClpB-C shares its regulatory region with heat-induced choline kinase, which has a possible role in heat signaling

Genetic engineering for high-level tolerance to abiotic stresses through over-expression of transcription factor genes: the next frontier

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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

Rice sHsp genes: genomic organization and expression profiling under stress and development

<p>Abstract</p> <p>Background</p> <p>Heat shock proteins (Hsps) constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD) at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes.</p> <p>Results</p> <p>We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of <it>Arabidopsis</it>. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in <it>Arabidopsis</it>. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed.</p> <p>Conclusion</p> <p>We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed that these genes are differentially expressed under stress and at different stages in the life cycle of rice plant.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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. <it>OsClpB-cyt </it>transcript is shown to be enriched at milk and dough stages of seed development. While expression of <it>OsClpB-m </it>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 <it>Δhsp104 </it>mutant yeast cells showed that <it>OsClpB-cyt</it>, <it>OsClpB-m</it>, <it>OsClpC1 </it>and <it>OsClpD1 </it>have significantly positive effects. Remarkably, <it>OsClpD1 </it>gene imparted appreciably high level tolerance to the mutant yeast cells.</p> <p>Conclusions</p> <p>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 <it>ScΔhsp104 </it>mutant cells.</p

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

Current initiatives in proteomics research: the plant perspective

The recent upsurge in structural genomics is leading to the accumulation of a huge wealth of literature on nucleotide sequences. After the nucleotide sequence of a given stretch of DNA is obtained (by manual or robotic methods), the next step is to use some software program to distinguish the possible open reading frames in the thick of sequences. However, the acid test, whether the new sequence corresponds to any functionality in terms of transcription and translation is to identify the protein which it encodes. Functional genomics and proteomics are the buzzwords in modern-day genomics. The science of proteomics is a possible approach to relate the skeletal nucleotide sequence information to functional attributes of the cell. The identification and isolation of novel genes with potential biotechnological applications warrant that genomics and proteomics must go hand in hand. Three major steps in proteome analysis are the separation of complex protein mixtures by two-dimensional protein gel electrophoresis (2D), characterization of the separated proteins by mass spectrometer (MS) and database searching. The power of 2D is such that it allows even minor changes in gene expression caused by internal or external cues to be effectively scored. Most proteins resolved by 2D have high purity, which can facilitate their identification by MS. In recent years, methods for automated proteomics based on incorporation of new ideas in both hardware and software development have been optimized to a great deal. We discuss the progress and applications of the proteomics science, with special reference to plants