Insights into genomic variations in rice Hsp100 genes across diverse rice accessions.

MAIN CONCLUSION: The Hsp101 gene is present across all sequenced rice genomes. However, as against Japonica rice, Hsp101 protein of most indica and aus rice contain insertion of glutamic acid at 907th position. The understanding of the heat stress response of rice plants is important for worldwide food security. We examined the presence/absence variations (PAVs) of heat shock proteins (Hsps)/heat shock transcription factor (Hsf) genes in cultivated rice accessions. While 53 Hsps/Hsfs genes showed variable extent of PAVs, 194 genes were the core genes present in all the rice accessions. ClpB1/Hsp101 gene, which is critically important for thermotolerance in plants, showed 100% distribution across the rice types. Within the ClpB1 gene sequence, 40 variation sites consisting of nucleotide polymorphisms (SNPs) and short insertion/deletions (InDels) were discerned. An InDel in ClpB1 leading to an in-frame insertion of 3 nucleotides (TCC) thereby an additional amino acid (glutamic acid) at 907th amino acid position was noted in most of the indica and aus as against japonica rice types. Three rice types namely Moroberekan (japonica), IR64 (indica) and N22 (aus) were further analyzed to address the question of ClpB1 genomic variations and its protein levels with the heat tolerance phenotype. The growth profiling analysis in the post heat stress (HS) period showed that N22 seedlings were most tolerant, IR64 moderately tolerant and Moroberekan highly sensitive. Importantly, the ClpB1 protein sequences of these three rice types showed distinct differences in terms of SNPs. As the ClpB1 protein levels accumulated post HS were generally higher in Moroberekan than N22 seedlings in our study, it is proposed that some additional gene loci in conjunction with ClpB1 regulate the overall rice heat stress response.

AtHsc70-1 negatively regulates the basal heat tolerance in Arabidopsis thaliana through affecting the activity of HsfAs and Hsp101.

Heat shock protein 70 (Hsp70) chaperones are highly conserved and essential proteins with diverse cellular functions, including plant abiotic stress tolerance. Hsp70 proteins have been linked with basal heat tolerance in plants. Hsp101 likewise is an important chaperone protein that plays a critical role in heat tolerance in plants. We observed that Arabidopsis hsc70-1 mutant seedlings show elevated basal heat tolerance compared with wild-type. Over-expression of Hsc70-1 resulted in increased heat sensitivity. Hsp101 transcript and protein levels were increased during non-heat stress (HS) and post-HS conditions in hsc70-1 mutant seedlings. In contrast, Hsp101 was repressed in Hsc70-1 over-expressing plants after post-HS conditions. Hsc70-1 showed physical interaction with HsfA1d and HsfA1e protein in the cytosol under non-HS conditions. In transient reporter gene analysis, HsfA1d, HsfA1e and HsfA2 showed transcriptional response on the Hsp101 promoter. HsfA1d and HsfA2 transcripts were at higher levels in hsc70-1 mutant compared with wild-type. We provide genetic evidence that Hsc70-1 is a negative regulator affecting HsfA1d/A1e/A2 activators, which in turn regulate Hsp101 expression and basal thermotolerance.

Hsp70, sHsps and ubiquitin proteins modulate HsfA6a-mediated Hsp101 transcript expression in rice (Oryza sativa L.).

Hsp100 chaperones disaggregate the aggregated proteins and are vital for maintenance of protein homeostasis. The level of Hsp100 synthesised in the cells has a bearing on the survival of plants under heat stress. The Hsp100 transcription machinery is activated within minutes of the onset of heat stress. The heat shock factor HsfA6a plays a major role in the transcriptional regulation of the Hsp101 gene in rice plants. Through yeast-2-hybrid library screening, we identified small heat shock proteins (sHSPs), Hsp70 and ubiquitin as HsfA6a interacting proteins (HIPs). The bimolecular fluorescence complementation assays showed the physical interaction of HsfA6a with Hsp16.9A-CI and Hsp18.0-CII in the cytosolic region and with cHsp70-1 in the nucleus. With the Hsp101 promoter: reporter gene assays, using yeast cells and rice protoplasts, we show that CI-sHsps and CII-sHsps are negative regulators and Hsp70 positive regulator of the HsfA6a activity in modulation of Hsp101 transcription. We also noted that the HsfA6a interactors, Hsp70 and CI-sHsps and CII-sHsps, physically interact with each other. We noted that HsfA6a binds with the CI-sHsp and Hsp70 promoters, implying that HsfA6a has a role in transcriptional regulation of its interacting proteins. Furthermore, we noted that the mutation of the ubiquitin/sumoylation acceptor site lysine 10 to alanine (K10A) of HsfA6a enhanced its DNA binding potential on the Hsp101 promoter, implying that these modifiers are possibly involved in modulation of HsfA6a activity. Our work shows that Hsp70, CI-sHsps and CII-sHsp, and ubiquitin proteins coordinate with HsfA6a in mediating the Hsp101 transcription process in rice.

Silencing of class I small heat shock proteins affects seed-related attributes and thermotolerance in rice seedlings.

MAIN CONCLUSION: Silencing of CI-sHsps by RNAi negatively affected the seed germination process and heat stress response of rice seedlings. Seed size of RNAiCI-sHsp was reduced as compared to wild-type plants. Small heat shock proteins (sHsps) are the ATP-independent chaperones ubiquitously expressed in response to diverse environmental and developmental cues. Cytosolic sHsps constitute the major repertoire of sHsp family. Rice cytosolic class I (CI)-sHsps consists of seven members (Hsp16.9A, Hsp16.9B, Hsp16.9C, Hsp17.4, Hsp17.7, Hsp17.9A and Hsp18). Purified OsHsp17.4 and OsHsp17.9A proteins exhibited chaperone activity by preventing formation of large aggregates with model substrate citrate synthase. OsHsp16.9A and OsHsp17.4 showed nucleo-cytoplasmic localization, while the localization of OsHsp17.9A was preferentially in the nucleus. Transgenic tobacco plants expressing OsHsp17.4 and OsHsp17.9A proteins and Arabidopsis plants ectopically expressing OsHsp17.4 protein showed improved thermotolerance to the respective trans-hosts during the post-stress recovery process. Single hairpin construct was designed to generate all CI-sHsp silenced (RNAiCI-sHsp) rice lines. The major vegetative and reproductive attributes of the RNAiCI-sHsp plants were comparable to the wild-type rice plants. Basal and acquired thermotolerance response of RNAiCI-sHsp seedlings of rice was mildly affected. The seed length of RNAiCI-sHsp rice plants was significantly reduced. The seed germination process was delayed and seed thermotolerance of RNAiCI-sHsp was negatively affected than the non-transgenic seeds. We, thus, implicate that sHsp genes are critical in seedling thermotolerance and seed physiology.

Analysis of transactivation potential of rice (Oryza sativa L.) heat shock factors.

MAIN CONCLUSION: Based on yeast one-hybrid assays, we show that the presence of C-terminal AHA motifs is not a prerequisite for transactivation potential in rice heat shock factors. Transcriptional activation or transactivation (TA) of heat stress responsive genes takes place by binding of heat shock factors (Hsfs) to heat shock elements. Analysis of TA potential of thirteen rice (Oryza sativa L.) Hsfs (OsHsfs) carried out in this study by yeast one-hybrid assay showed that OsHsfsA3 possesses strong TA potential while OsHsfs A1a, A2a, A2b, A4a, A4d, A5, A7b, B1, B2a, B2b, B2c and B4d lack TA potential. From a near complete picture of TA potential of the OsHsf family (comprising of 25 members) emerging from this study and an earlier report from our group (Mittal et al. in FEBS J 278(17):3076-3085, 2011), it is concluded that (1) overall, six OsHsfs, namely A3, A6a, A6b, A8, C1a and C1b possess TA potential; (2) four class A OsHsfs, namely A3, A6a, A6b and A8 have TA potential out of which A6a and A6b contain AHA motifs while A3 and A8 lack AHA motifs; (3) nine class A OsHsfs, namely A1a, A2a, A2b, A2e, A4a, A4d, A5, A7a and A7b containing AHA motif(s) lack TA function in the yeast assay system; (4) all class B OsHsfs lack AHA motifs and TA potential (B4a not analyzed) and (5) though all class C OsHsf members lack AHA motifs, two members C1a and C1b possess TA function, while one member C2a lacks TA potential (C2b not analyzed). Thus, the presence or absence of AHA motif is possibly not the only factor determining TA potential of OsHsfs. Our findings will help to identify the transcriptional activators of rice heat shock response.

ClpB/Hsp100 proteins and heat stress tolerance in plants.

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.

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 ß-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 -517 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.

Response of different genotypes of faba bean plant to drought stress.

Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant-1, fresh weight (FW) and dry weight (DW) plant-1, area leaf-1, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H2O2) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes "C5" and "Zafar 1" were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype "C5" and "Zafar 1" were found to be relatively tolerant to drought stress and genotypes "G853" and "C4" were sensitive to drought stress.

Coexpression network analysis associated with call of rice seedlings for encountering heat stress.

Coexpression network analysis is useful tool for identification of functional association of coexpressed genes. We developed a coexpression network of rice from heat stress transcriptome data. Global transcriptome of rice leaf tissues was performed by microarray at three time points--post 10 and 60 min heat stress at 42 °C and 30 min recovery at 26 °C following 60 min 42 °C heat stress to investigate specifically the early events in the heat stress and recovery response. The transcriptome profile was significantly modulated within 10 min of heat stress. Strikingly, the number of up-regulated genes was higher than the number of down-regulated genes in 10 min of heat stress. The enrichment of GO terms protein kinase activity/protein serine threonine kinase activity, response to heat and reactive oxygen species in up-regulated genes after 10 min signifies the role of signal transduction events and reactive oxygen species during early heat stress. The enrichment of transcription factor (TF) binding sites for heat shock factors, bZIPs and DREBs coupled with up-regulation of TFs of different families suggests that the heat stress response in rice involves integration of various regulatory networks. The interpretation of microarray data in the context of coexpression network analysis identified several functionally correlated genes consisting of previously documented heat upregulated genes as well as new genes that can be implicated in heat stress. Based on the findings on parallel analysis of growth of seedlings, associated changes in transcripts of selected Hsps, genome-wide microarray profiling and the coexpression network analysis, this study is a step forward in understanding heat response of rice, the world's most important food crop.

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Heat-induced proteomic changes in anthers of contrasting rice genotypes under variable stress regimes.

Heat stress drastically affects anther tissues resulting in poor plant fertility, necessitating an urgent need to determine the key proteome regulation associated with mature anther in response to heat stress. We identified several genotype - specific protein alterations in rice anthers of Moroberekan (Japonica, heat sensitive), IR64 (Indica, moderately heat tolerant), and Nagina22 (Aus, heat tolerant) in the short-term (ST_HS; one cycle of 42°C, 4 hours before anthesis) and long-term (LT_HS; 6 cycles of 38°C, 6 hours before anthesis) heat stress. The proteins upregulated in long-term heat stress in Nagina22 were enriched in biological processes related to unfolded protein binding and carboxylic acid metabolism, including amino acid metabolism. In short-term heat stress, Nagina22 anthers were enriched in proteins associated with vitamin E biosynthesis and GTPase activator activity. In contrast, downregulated proteins were related to ribosomal proteins. The expression of different Hsp20 and DnaJ was genotype specific. Overall, the heat response in Nagina22 was associated with its capacity for adequate metabolic control and cellular homeostasis, which may be critical for its higher reproductive thermotolerance. This study improves our understanding of thermotolerance mechanisms in rice anthers during anthesis and lays a foundation for breeding thermotolerant varieties via molecular breeding.

Tango between Ethylene and HSFA2 Settles Heat Tolerance.

The phytohormone ethylene has roles in senescence, fruit ripening, and biotic and abiotic stress responses. However, the detailed mechanism(s) by which ethylene affects the plant heat stress response (HSR) is not well understood. Two recent studies by Huang et al. and Shekhawat et al. now reveal that ethylene signaling converges on HSFA2 to bring about heat stress (HS) tolerance in plants.

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.

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.

Heat-Induced Oxidation of the Nuclei and Cytosol.

The concept that heat stress (HS) causes a large accumulation of reactive oxygen species (ROS) is widely accepted. However, the intracellular compartmentation of ROS accumulation has been poorly characterized. We therefore used redox-sensitive green fluorescent protein (roGFP2) to provide compartment-specific information on heat-induced redox changes of the nuclei and cytosol of Arabidopsis leaf epidermal and stomatal guard cells. We show that HS causes a large increase in the degree of oxidation of both compartments, causing large shifts in the glutathione redox potentials of the cells. Heat-induced increases in the levels of the marker transcripts, heat shock protein (HSP)101, and ascorbate peroxidase (APX)2 were maximal after 15 min of the onset of the heat treatment. RNAseq analysis of the transcript profiles of the control and heat-treated seedlings revealed large changes in transcripts encoding HSPs, mitochondrial proteins, transcription factors, and other nuclear localized components. We conclude that HS causes extensive oxidation of the nucleus as well as the cytosol. We propose that the heat-induced changes in the nuclear redox state are central to both genetic and epigenetic control of plant responses to HS.

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

BACKGROUND: 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 alpha-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. RESULTS: We identified 40 alpha-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 Arabidopsis. 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 Arabidopsis. 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. CONCLUSION: 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.

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.

Cpn60ß4 protein regulates growth and developmental cycling and has bearing on flowering time in Arabidopsis thaliana plants.

Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60ß subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60ß subunit. We noted that the cpn60ß4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60ß4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60ß4 mutant plants than WT. Importantly, cpn60ß4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60ß4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60ß4 gene was complemented in the mutant background. The overexpression of Cpn60ß4 gene using CaMV35 promoter in wild type background (OE-Cpn60ß4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60ß4 mutant plants compared to Col-0. The results of this study suggest that Cpn60ß4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis.

Safety of intrauterine contraceptive device (copper T 200 B) in women with cardiac disease.

BACKGROUND: Women with cardiac disease have need for effective long-lasting reversible contraception. Women with cardiac disease are at increased risk for bacterial endocarditis. There is limited research regarding the use of intrauterine contraceptive devices (IUD) in women with cardiac disease. STUDY DESIGN: In a prospective study, the IUD copper (Cu T200B) was inserted in 40 women with cardiac disease. Infective endocarditis prophylaxis was given 1 h before IUD insertion. The IUD was inserted under aseptic conditions. Ten milliliters of venous blood was obtained for culture of aerobic and anerobic bacteria within 1 h of insertion of the copper T IUD. Women were contacted for follow-up at frequent intervals. RESULTS: There was no incidence of uterine perforation, hemorrhage or spontaneous expulsion of the IUD. All blood cultures were sterile. There were no cases of infective endocarditis. Four women (10%) had menorrhagia at the 6-month follow-up which responded to medical management. One woman had PID for which antibiotics were given. Five women had mild cramps and five had spotting after insertion of the IUD. Patient adherence was excellent as none returned for removal for reasons other than desire for another pregnancy. CONCLUSION: The Cu T200B IUD is a reasonably safe and effective method of temporary contraception in women with cardiac disease who are not receiving anticoagulant therapy.