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Heat shock proteins or Hsps are critical in mounting plant resistance against heat stress. The complex Hsp spectrum of Arabidopsis thaliana plant contains over two hundred proteins belonging to six different families namely Hsp20, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100. Importantly, the cellular function(s) of most Hsps remains to be established. We aimed at phenotyping of stress and development response of the selected, homozygous hsp mutant lines produced by T-DNA insertional mutagenesis method. The heat stress phenotype was assessed for basal and acquired heat stress response at seed and seedling stages. Distinct phenotype was noted for the hot1-3 mutant (knockout mutant of Hsp101 gene) showing higher heat sensitivity and for the salk_087844 mutant (knockout mutant of Hsc70-2 gene) showing higher heat tolerance than the wild type seedlings. The homozygous cs808162 mutant (mutant of ClpB-p gene encoding for the chloroplast-localized form of Hsp101) did not survive even under unstressed, control condition. salk_064887C mutant (mutant of cpn60β4 gene) showed accelerated development cycling. The hot1-3 mutant apart from showing different heat response, exhibited development lesions like bigger size of seeds, buds, siliques, and pollen compared to the wild type plants. In response to controlled deterioration treatment of seeds, hot1-3 seeds showed higher accumulation of reactive oxygen species molecules, higher rates of protein and lipid oxidation and a faster decline in germination rate as compared to wild type seeds. Our findings show that Hsps perform diverse metabolic functions in plant response to stress, growth, and development.

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.

Caseinolytic protease (Clp)/Hsp100 proteins are members of the AAA+ (ATPase associated with a variety of cellular activities) family of proteins present in lower life forms and plants. These proteins represent a unique chaperone system that performs the crucial role of retrieving functional proteins from the aggregated state and are thus also referred to as disaggregases. Clp/Hsp100s were originally discovered in bacteria and yeast cells in late 1980s and early 1990s. Arabidopsis thaliana, in particular after the completion of its genome sequencing in the year 2000, has served as an excellent experimental system for investigating this important chaperone machine in plants. From the reverse and forward genetic analysis carried out with Arabidopsis Hsp101, it emerged that this protein is a powerful candidate gene in conferring heat tolerance. Importantly, this protein is implicated with heat tolerance both in vegetative and reproductive stages of the growth and development of Arabidopsis plants. In the data on genetic polymorphism in the amino acid sequence of Arabidopsis Hsp101 across diverse A. thaliana accessions sequenced in 1001 Arabidopsis genome sequencing project, we find that that there are sixty-three distinct AtHsp101 sequence types with variations in N-terminus, nucleotide binding and C-terminus domains in 855 analyzed Arabidopsis accessions. The in-depth analysis with these variant allelic forms may yield invaluable information on genetic variations in the regulatory regions which control the Hsp101 expression as well as those affecting Hsp101 disaggregase activity. Arabidopsis Hsp101 protein has turned out to be a prototype in search for homolog proteins and mechanisms of heat stress tolerance in rice as well as across diverse crops. Herein, we present the status of our understanding on the Arabidopsis Hsp101 genes, proteins, and their functional relevance.

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.

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.

The article is devoted to the scientific substantiation and development of the technology of minced meat products using a sodium alginate-based thermostable elastic emulsion. The functional, technological, rheological, heat-resistant, physico-chemical, structural-mechanical, organoleptic and consumer properties are investigated. It is predicted and experimentally confirmed that the use of structured thermostable emulsion in the composition of minced meat products allows a more rational use of meat raw materials, improve the organoleptic characteristics of minced meat products and nutritional value due to the enrichment of finished products with polyunsaturated fatty acids

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.

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.

Temperature is one of the key physical parameters that fine tunes plant growth and development. However, above the optimal range, it can negatively affect the physiology of plants. Supraoptimal temperature brings incongruity in cellular proteostasis resulting in the build-up of insoluble toxic protein aggregates. To prevent protein misfolding and aggregation, cells deploy different strategies including synthesis of heat shock proteins (Hsp) belonging to different families, like small Hsps (sHsps), Hsp40, Hsp60, Hsp70. Once these aggregates are formed, their dissolution and recovery of the functional proteins occurs by the action of Caseinolytic Protease B (ClpB)/Hsp100, which are evolutionarily conserved in bacteria, fungi and plants. ClpB function during heat stress (HS) is important and appears indispensable, as mutant bacteria, yeast as well as plants lacking ClpB protein fail to survive HS. Genetic expression of ClpB proteins is modulated both by high temperature as well as developmental cues. Plant contains three isoforms of ClpB/Hsp100, one each localized to cytoplasm (ClpB-C), chloroplast (ClpB-P) and mitochondria (ClpB-M), against one in bacteria and two in yeast. Among these, ClpB-C protein in particular governs the thermotolerance response in plants. This review introduces plant ClpB proteins, summarizes the knowledge gained hitherto in ClpB biology, critically analyzes the recent findings and brings forth the areas requiring thrust in the upcoming research on ClpBs.

Rice crop meets the calorific needs of the masses. High temperature stress has negative effects on the physiology and biochemistry of this crop. This is amply reflected from studies showing that heat stress has strong detrimental effects on photosynthetic efficiency, electrolyte leakage, lipid peroxidation, enzyme (superoxide dismutase, peroxidase, catalase, etc.) activities and transpiration rate at varied growth stages, ultimately affecting the total harvest of this crop. The macro cellular- and plant-level changes as of aboveare rooted inalterations at the molecular level, affecting the gene expression profiles, protein abundance and their interaction at the genomic level. With global warming at its pace, the cultivation of rice under field conditions is greatly threatened. Molecular analyses show that the response of rice to heat stress involves a host of genes including transcription factors, genes involved in calcium and hormonal signalling, reactive oxygen species metabolism genes and chaperones. Heat shock proteins (Hsps) are considered the key players with role in maintaining proteostasis under heat and related stresses. The expression of Hsps is centrally governed by heat shock factors (Hsfs). Hsps and Hsfs have emerged as the potential candidates for engineering stress tolerance in crop plants. Several attempts have been made in which rice genes have been incorporated in varied hosts and concurrently rice has been used as trans-host with various genes cloned from rice and other plant species. In this review, we provide an account of physiological, biochemical, transcriptomic and proteomic responses of rice crop to heat stress. The importance of ClpB-C/Hsp100 gene in heat stress tolerance and the potential heat shock factor(s) governing regulation of ClpB-C/Hsp100 for engineering heat stress tolerance in rice has been discussed.

Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses.

Engineering plant promoter sequence for optimal expression of a gene has been a long standing goal for plant scientists. In recent times, Sequence Specific Nucleases (SSNs) like CRISPR/Cas9 are enabling researchers to achieve this goal, in vivo in the genome. It is well known that SSNs have met with unprecedented success in rapid transgene free crop improvement largely by targeting the coding sequence. Here, we discuss the strategies being employed by plant scientists in targeting SSNs to non-coding promoter regions/Cis Regulatory Elements (CRE). We collectively refer all such endeavors as in vivo promoter engineering (IPE). We further classify the IPE efforts into CRE addition, CRE deletion/disruption, promoter swap/insertion and targeted promoter polymorphism. Till date, IPE has proven useful in altering plant architecture in tomato, developing resistance against Xanthomonas sp in rice and citrus, and engineering drought tolerance in maize. However it is quite challenging to achieve predictable changes in gene expression using IPE at this point. In future years, data generated from high throughput techniques to investigate non coding genome may immensely augment the efforts in this direction. As IPE does not involve addition of the transgene for modifying crop traits, it will be relatively more conducive to public acceptance in crop improvement programs.

Elevated temperatures affect the growth and reproduction of crop plants and thus have become concern worldwide. Hsp101/ClpB protein is a major molecular chaperone, performing dis-aggregation of protein aggregates formed during heat stress. In rice, OsHsfA6a binds to the promoter of OsHsp101/ClpB-C and regulates its expression. In this study, analysis of C-terminal domains of ClassA OsHsfs revealed the presence of aromatic, hydrophobic, acidic (AHA) and nuclear export signal (NES) motifs in all the members. Using deletion constructs, we show that the activation potential of OsHsfA6a is confined in the C-terminal activation domain comprising of AHA and NES sequences. The results obtained in yeast were complemented with transient expression of reporter in protoplast (TERP) based assay. Detailed analysis of OsHsfA6a splice variants shows the presence of one full version and a DBD truncated smaller version whose existence needs experimental evidences. Phylogeny analysis revealed that OsHsfA6a has diverged from A6a/A6b forms of Arabidopsis and tomato and has no expressologs. OsHsfA6a in-silico network was enriched in MAP kinases along with Hsp70 and Hsp90 proteins. Thus, it appears that regulation of OsClpB-C by HsfA6a is unique in rice and activation potential of OsHsfA6a resides in the single AHA motif located in the C-terminal domain.

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying β-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3' end of the 5'untranslated region (5'UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5'UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5'UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5'UTR of OsClpB-C is dispensable for expression in seeds.

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.

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.

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.

Climate change is resulting in heightened incidences of plant heat stress episodes. Production of transgenic crops with enhanced heat stress tolerance is a highly desired agronomic trait for the sustainability of food production in 21st century. We review the current status of our understanding of the high temperature stress response of plants. We specifically deliberate on the progress made in altering levels of heat shock proteins (Hsp100, Hsp70/Hsp40 and sHsps), heat shock factors and specific metabolic proteins in improving plant tolerance to heat stress by transgenic approach.

Heat stress results in misfolding and aggregation of cellular proteins. Heat shock proteins (Hsp) enable the cells to maintain proper folding of proteins, both in unstressed as well as stressed conditions. Hsp70 genes encode for a group of highly conserved chaperone proteins across the living systems encompassing bacteria, plants, and animals. In the cellular chaperone network, Hsp70 family proteins interconnect other chaperones and play a dominant role in various cell processes. To assess the functionality of rice Hsp70 genes, rice genome database was analyzed. Rice genome contains 32 Hsp70 genes. Rice Hsp70 superfamily genes are represented by 24 Hsp70 family and 8 Hsp110 family members. Promoter and transcript expression analysis divulges that Hsp70 superfamily genes plays important role in heat stress. Ssc1 (mitochondrial Hsp70 protein in yeast) deleted yeast show compromised growth at 37 °C. Three mitochondrial rice Hsp70 sequences (i.e., mtHsp70-1, mtHsp70-2, and mtHsp70-3) complemented the Ssc1 mutation of yeast to differential extents. The information presented in this study provides detailed understanding of the Hsp70 protein family of rice, the crop species that is the major food for the world population.

Protein folding and disaggregation are crucial processes for survival of cells under unfavorable conditions. A network of molecular chaperones supports these processes. Collaborative action of Hsp70 and Hsp100 proteins is an important component of this network. J-proteins/DnaJ members as co-chaperones assist Hsp70. As against 22 DnaJ sequences noted in yeast, rice genome contains 104 J-genes. Rice J-genes were systematically classified into type A (12 sequences), type B (9 sequences), and type C (83 sequences) classes and a scheme of nomenclature of these proteins is proposed. Transcript expression profiles revealed that J-proteins are possibly involved in basal cellular activities, developmental programs, and in stress. Ydj1 is the most abundant J-protein in yeast. Ydj1 deleted yeast cells are nonviable at 37 °C. Two rice ortholog proteins of yeast Ydj1 protein namely OsDjA4 and OsDjA5 successfully rescued the growth defect in mutant yeast. As Hsp70 and J-proteins work in conjunction, it emerges that rice J-proteins can partner with yeast Hsp70 proteins in functioning. It is thus shown that J-protein machine is highly conserved.

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 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.

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.

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.

Glycosyltransferases (GTs) catalyze the reaction of glycosylation of various cellular macromolecules. Rice genome contains 609 potential GT genes which have been grouped into 40 gene families according to CAZy (Carbohydrate Active Enzyme) database. Phylogenetic tree showed that the members of GT61 family are related across genera. Close relationship among various rice GT61 proteins was also reflected from MEME analysis. Full-length cDNA corresponding to MSU rice locus Os02g22380 and encoding for xylosyltransferase was obtained (referred to as OsGT61-1). OsGT61-1 transcript was noted to be ABA- and NaCl-responsive and its expression was more prominent in root than in shoot tissues. Experiments involving fusion of OsGT61-1 promoter (1410 bp upstream to ATG) with GUS reporter gene showed that GUS expression was in consonance with the transcript expression. 1410 bp OsGT61-1 promoter contains two miniature inverted repeat transposable element sequences namely Gaijin (144 bp) and Wanderer (211 bp). Using yeast as trans-host, we noted that these sequences negatively influence the downstream transcript expression.

Mitral valve prolapse (MVP) is considered to be the most common valvular heart lesion in adult females of reproductive age. Our report reviews the obstetrical performance and outcome of 34 pregnancies in 15 women with MVP. In 12 (80%) patients cardiac lesion was suspected due to enigmatic dyspnoea and palpitation during antenatal period. Four mothers required propranolol for cardiac arrhythmias. However, all of them tolerated pregnancy and labour well. Four pregnancies (11.8%) ended in spontaneous abortion and 1 mother had intrauterine fetal death due to severe pregnancy-induced hypertension. There were 25 (73.5%) term and 3 (8.8%) preterm neonates without congenital abnormality. One preterm neonate had tracheo-oesophageal fistula and died 6 hours after corrective surgery. The mean birth weight of 2.8 kg was appropriate for mean gestation of 38.5 weeks when compared to the Institute's reference neonatal weight curve. Infective endocarditis prophylaxis was recommended in cases having mitral regurgitation and complicated delivery. Awareness of this common cardiac lesion, thorough cardiovascular examination during pregnancy, early detection and treatment of cardiac arrhythmias if any, are the essential steps to prevent rare but dreaded maternal complications.

Bilateral coronary artery to pulmonary artery fistulas is an uncommon congenital anomaly. These fistulas have a clinical and embryological significance. We report a rare case of combination of right coronary and left circumflex coronary fistula draining into main pulmonary artery, who presented in the emergency room with acute pain chest. The fistula had significant 1.7:1 left to right shunt and also myocardial ischemia as demonstrated by exercise Thallium(201) SPECT myocardial imaging. He was managed conservatively during the last 5 years without any percutaneous or surgical intervention.

Purpose Takayasu's arteritis (TA) is a non-specific, chronic, inflammatory panarteritis, affecting the aorta, its major branches and pulmonary arteries. The percutaneous or surgical revascularization in these patients is often difficult and challenging because of diffuse and multi-vessel involvement, difficulty in management of disease activity, and high rate of restenosis and re-intervention. Case report We report a 33-year-old man, a case of TA with fulminant active disease and multi-vessel involvement. His disease activity could not be suppressed with steroid treatment. He had multiple sequential bailout interventions of carotid, left main coronary and mesenteric arteries secondary to cerebro-vascular event, angina and mesenteric ischemia, respectively. Conclusion Despite comprehensive multi-specialty approach in management and also the global revascularization, he ultimately died of acute mesenteric ischemia. To our knowledge, there is no similar type of case being published in world literature till date.

Background:Doxorubicin-induced cardiotoxicity is widely known to occur at cumulative doses exceeding 450 mg/m2 . However, very few studies have reported incidence of cardiac dysfunction in patients on chemotherapy with lower cumulative doses. To the best of our knowledge, there is no study carried out so far that has reported the incidence of cardiac dysfunction in adult Indian patients receiving doxorubicin. This study was undertaken to determine the incidence of doxorubicin-induced cardiotoxicity by serial resting echocardiography in patients on chemotherapy and identify risk factors associated with cardiotoxicity.Materials and Methods:Patients that were started on doxorubicin-based chemotherapy in the period from January 2000 to June 2001 and had completed at least 300 mg/m2cumulative dose were taken in the study. Electrocardiography, chest X-ray and echocardiography were done at baseline, at 300 mg/m2and at 450 mg/m2cumulative doses of doxorubicin. All patients were evaluated for the presence of the following risk factors:Age>70 years, female sex, preexisting cardiac disease, hypertension, chest wall irradiation, body mass index (BMI)< 20 kg/m2 , Karnofsky performance status, combination chemotherapy with cyclophosphamide and presence of liver disease. Subclinical cardiac dysfunction was defined as ejection fraction fall greater than 10% on follow-up echocardiography.Results:Thirty patients satisfied the criterion for being considered for evaluation. One (3%) patient developed congestive cardiac failure, while 8 (27%) patients developed subclinical cardiac dysfunction. Concomitant use of cyclophosphamide significantly increased the risk of cardiac dysfunction ( P=0.048), while low BMI (< 20 kg/m2 ) and preexisting cardiac disease showed a trend towards increased risk of cardiac dysfunction ( P=0.07 for both).Conclusion:Twenty-seven percent of the patients developed subclinical cardiac dysfunction in the cumulative dose range of 300-450 mg/m2 . This entails regular monitoring for cardiac dysfunction by echocardiography during treatment.

Alterations in lipid metabolism and genetic predisposition are major risk factors for coronary artery disease (CAD). Variations in genes involved in lipid metabolism may act synergistically to confer risk or protection against CAD. The objective of the present study was to determine such interactions in variants of apolipoprotein E and apolipoprotein A1 genes. One hundred and forty subjects with clinically confirmed CAD and 100 unrelated normal subjects participated in the study. Multiple regression analysis was used to relate lipid and apolipoprotein profiles with genotypes. Odd ratios were calculated for various combinations of ApoE and ApoA1 genotypes. Prevalence of ApoE ‘E4’ and ApoA1 ‘A’ and ‘T’ alleles was significantly higher in patients than controls. Serum apolipoprotein E and apolipoprotein A1 levels were significantly lower in CAD patients than controls. When lipid parameters were related to genotypes, the polymorphisms associated to various markers were in agreement with previous reports. ApoE 2/4 genotype in combination with either ApoA1 heterozygous GA or CT genotype conferred higher risk of CAD. E3 allele in homozygous or heterozygous state in combination with ApoA1+83 CC genotype conferred highest protection (P

Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) enzymes are responsible for the operation of ethanolic fermentation pathway that appears to correlate to an extent with anoxia tolerance in plants. This study was undertaken with the objective of (a) analysing the rice pdc gene family and (b) altering the efficacy of the ethanolic fermentation process, through production of transgenic rice plants over- and under-expressing pyruvate decarboxylase (employing Ospdc1 gene from rice) as well as over-expressing alcohol dehydrogenase (employing Ghadh2 gene from cotton) proteins. Correlations noted in this study between the pattern of expression of the Pdc alpha-subunit and Ospdc2 transcript as well as between the Pdc beta-subunit and Ospdc1 transcript suggest the possibility that alpha-subunit is encoded by Ospdc2 and that beta-subunit is encoded by Ospdc1. The fact that levels of Pdc beta-subunit were particularly high in pUH-sPdc1 (plasmid construct designed for over-expression of Ospdc1) seedlings while levels of beta-subunit levels were negligible or lower in pUH-asPdc1 (plasmid construct designed for under-expression of Ospdc1) seedlings also support these observations. Transgenics raised for over-expression of Pdc and Adh and under-expression of Pdc were confirmed for the transgene presence and effects by PCR, Southern blotting, Northern blotting, Western blotting and isozyme assays. Pdc and Adh over-expressing rice transgenics at early seedling stage under unstressed control growth conditions showed slight, consistent advantage in root vigour as compared to that of wild-type seedlings.

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.

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.

We cloned and characterized the full-length coding sequence of a small heat shock protein 17.9 gene from faba bean encoding 160 amino acids and containing the conserved α-crystallin domain at the C-terminus. Homology and phylogenetic analysis suggested its proximity with the class II sHsp members of fabaceae family. Therefore, we name this gene as VfHsp17.9-CII. The VfHsp17.9-CII transcript showed a clear heat stress induction pattern in leaves of young seedlings and flowering plants. Transient expression of VfHsp17.9-CII fused with green fluorescent protein reporter indicated its nuclear localization. Overexpression of recombinant VfHsp17.9-CII protein in Escherichia coli cells increased tolerance of the bacterial cells to heat and arsenic stresses. The reduction of faba bean pollen viability in response to heat stress correlated with the accumulation pattern of VfHsp17.9-CII transcript in heat stressed pollen. It is suggested that VfHsp17.9-CII protein plays a key role in heat and heavy metal stress tolerance.

Towards Production of Abiotic Stress Tolerant Transgenic Rice Plants: Issues, Progress and Future Research Needs

Towards Developing Transgenic Rice Plants Tolerant to Flooding Stress

Crop plants are adversely affected by abiotic stresses. Drought is the most widespread and damaging of all environmental stresses. At the global level, significant proportion of cultivable land masses is affected by high salt levels. Heat and cold stresses profoundly affect agricultural yields of major crops. Also, the level of abiotic stresses is on the rise due to both natural and man-made interventions. The ambient temperature is gradually increasing due to the increased levels of CO2 and other greenhouse gases. The episodes of drought and flooding stress have become more erratic over the years. The production of transgenic crops that can withstand increased level of abiotic stresses is a silver lining to sustain and increase food production in future. Techniques of producing transgenic crops need to be improvised to achieve high frequency transformation. Current experiments deploying nanotechnology tools for gene delivery are extremely relevant in production of new generation of transgenic plants. With such tools, it would be possible to experimentally produce higher number of transgenic lines and screen out the transgenic lines showing desired phenotype in higher numbers. In the ensuing paragraphs, we delve on the current status of abiotic stress tolerant transgenic crops and also project how nanotechnology tools can help in future endeavors.

Coexistence of both congenital and atherosclerotic coronary heart disease is rare. We report a 52-year-old man who had severe valvular pulmonic stenosis and significant atherosclerotic disease involving coronary, subclavian and renal arteries. This combination of congenital heart disease and atherosclerotic arterial disease is probably the first report in published English literature. He underwent successful staged percutaneous interventions, which included balloon pulmonic valvuloplasty and angioplasty and stenting of right renal, left subclavian and right coronary arteries. On follow-up at 8 months, a check angiogram revealed patent stents and insignificant gradient across pulmonic valve.

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.

Transhiatal esophagectomy (THE) is a common operative procedure for carcinoma esophagus. Complications of this procedure include arrhythmias and hypotension during blunt dissection of the esophagus from posterior mediastinum. In the literature, exact incidence and type of arrhythmias have not been reported. We employed Holter monitoring during mediastinal manipulation in patients undergoing THE, for this purpose.This prospective study was carried out in 20 consecutive American Society of Anesthesiologists grade I-II patients undergoing THE. Anesthetic technique included induction with thiopentone and maintenance with morphine, vecuronium, and isoflurane. In addition to routine parameters, Holter monitoring was undertaken to record the exact incidence and types of arrhythmias. "Premanipulation" or control period included duration of 30 minutes preceding mediastinal manipulation, while "during manipulation" or study period included the duration of mediastinal manipulation. The incidence of arrhythmias was studied for 48 hours in the postoperative period. The Fisher exact test was applied to analyze incidence of arrhythmias and hypotension.Out of 20 patients, only 2 had arrhythmias in the premanipulation period, while 13 had arrhythmias during the manipulation period (p0.01). During the manipulation period, arrhythmias included supraventricular ectopics and ventricular ectopics in 2 patients each and a combination of both in 9 patients. Arrhythmias were transient and had no correlation with either duration or degree of hypotension in all the patients. However, there was a linear relationship between hypotension and duration of mediastinal manipulation. Two patients (10%) had atrial arrhythmias in the postoperative period.In transhiatal esophagectomy, there is a significant incidence of both arrhythmias and hypotension during mediastinal manipulation. The incidence of arrhythmias can be minimized by limiting the duration of the manipulation. The incidence of postoperative arrhythmias was not significant.

The present study was addressed to understand as to how the expression of genes, that play crucial role in both inflammation and autoimmune process, within blood mononuclear cells are effected by the molecular mimicry between streptococcal antigen and heart tissue recognized as main contributor towards the genesis of rheumatic heart disease (RHD). Such a study for the first time revealed that as compared to genomic profile within normal blood mononuclear cells, the cells derived from rheumatic heart disease patients exhibited significantly higher expression of genes coding for IL-8, IFN-gamma and CX3CR1 coupled with significant downregulation of CD36 mRNA expression. Based upon these results, we propose that maintenance of such a pathognomonic transcriptome within blood mononuclear cells may be responsible for the initiation and progression of rheumatic heart disease.

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. High soil salinity, contributed largely by NaCl, is one of the important environmental factors that limits distribution and productivity of major crops. Salinity affects approximately 20% of world’s arable land and approximately 40% of irrigated land to various degrees. Transgenic research provides much-needed flexibility in manipulation of crops by altering the expression levels of native genes or by incorporating alien genes for a desired trait, in a relatively shorter time-frame. In the past one decade of research, production of salt-stress tolerant transgenic plants by genetic engineering has been claimed in over 100 research publications (Grover et al. 2003).