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.

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.

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.

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.

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.

Mapping of domains of heat stress transcription factor OsHsfA6a responsible for its transactivation activity.

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.

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.

Functional analysis of Hsp70 superfamily proteins of rice (Oryza sativa).

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.

Functional relevance of J-protein family of rice (Oryza sativa).

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.

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.

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data.

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.