Heat shock proteins (Hsps) constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD) at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes.
We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of 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.
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.
Imprints of stress response by rice seedlings in terms of expression levels of stress response gene HSP70 are characterised . The response to arsenic and/or heat shock are shown to be additive for the same stress or the combined stresses, indicating a commonality of signalling pathways.
Background and aims
Plants can withstand many abiotic stresses. Stress adaptation through retention of imprints of previous stress exposure has also been described in plants. We have characterized the imprint or memory of adaptive stress responses of rice seedlings to arsenic (As) and heat stress.
Two-week-old rice seedlings (both with and without As) were given a 45 °C heat shock for 3 h. While under heat shock, the leafy portion of the seedlings was harvested at regular intervals. Subsequently, the seedlings were kept at room temperature for recovery and sampling continued over 3 h. Total RNA and protein were extracted from the leafy portion of the seedlings and complementary DNA (cDNA) was prepared from total RNA. The cDNA was used as a template for the polymerase chain reaction to identify the transcription level of HSP70. Protein extracted from the seedlings was western-blotted. HSP70 and actin (loading control) antibodies were used to recognize the proteins on the same blot.
Our studies reveal that HSP70, a cellular chaperone gene, is over-expressed at the mRNA and protein levels when rice seedlings are exposed to As and heat. The effect is cumulative and increases with the duration of stress for 3 h. During 3 h recovery from heat stress at ambient temperatures for 3 h, the chaperone remains expressed at higher levels in plants pre-exposed to As.
Our findings demonstrate a retention of the imprint of previous stress exposure, perhaps through sustained activation of the signalling pathways upstream of over-expression of HSP70. Furthermore, stress-induced HSP70 expression was additive/cumulative for continued exposure to similar or different kinds of stress, indicating that a commonality of signal transduction networks is adopted when plants experience more than one stress.
Upland and lowland rice (Oryza sativa L.) represent two of the most important rice ecotypes adapted to ago-ecosystems with contrasting soil-water conditions. Upland rice, domesticated in the water-limited environment, contains valuable drought-resistant characters that can be used in water-saving breeding. Knowledge about the divergence between upland and lowland rice will provide valuable cues for the evolution of drought-resistance in rice. Genetic differentiation between upland and lowland rice was explored by 47 Simple Sequence Repeats (SSRs) located in drought responding expressed sequence tags (ESTs) among 377 rice landraces. The morphological traits of drought-resistance were evaluated in the field experiments. Different outlier loci were detected in the japonica and indica subspecies, respectively. Considerable genetic differentiation between upland and lowland rice on these outlier loci was estimated in japonica (Fst = 0.258) and indica (Fst = 0.127). Furthermore, populations of the upland and lowland ecotypes were clustered separately on these outlier loci. A significant correlation between genetic distance matrices and the dissimilarity matrices of drought-resistant traits was determined, indicating a certain relationship between the upland-lowland rice differentiation and the drought-resistance. Divergent selections occur between upland and lowland rice on the drought-resistance as the Qsts of some drought-resistant traits are significantly higher than the neutral Fst. In addition, the upland- and lowland-preferable alleles responded differently among ecotypes or allelic types under osmotic stress. This shows the evolutionary signature of drought resistance at the gene expression level. The findings of this study can strengthen our understanding of the evolution of drought-resistance in rice with significant implications in the improvement of rice drought-resistance.
Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors.
In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities.
Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-344) contains supplementary material, which is available to authorized users.
Rice (Oryza sativa L.); Heat shock proteins; Genome wide; Identification
Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods.
The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray.
The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.
MADS-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. In recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire MADS-box family in the model plant system, Arabidopsis. Though there have been some studies in rice as well, an analysis of the complete MADS-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. Furthermore, owing to the role of MADS-box family in flower development, an analysis involving structure, expression and functional aspects of MADS-box genes in rice and Arabidopsis was required to understand the role of this gene family in reproductive development.
A genome-wide molecular characterization and microarray-based expression profiling of the genes encoding MADS-box transcription factor family in rice is presented. Using a thorough annotation exercise, 75 MADS-box genes have been identified in rice and categorized into MIKCc, MIKC*, Mα, Mβ and Mγ groups based on phylogeny. Chromosomal localization of these genes reveals that 16 MADS-box genes, mostly MIKCc-type, are located within the duplicated segments of the rice genome, whereas most of the M-type genes, 20 in all, seem to have resulted from tandem duplications. Nine members belonging to the Mβ group, which was considered absent in monocots, have also been identified. The expression profiles of all the MADS-box genes have been analyzed under 11 temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. Transcripts for 31 genes accumulate preferentially in the reproductive phase, of which, 12 genes are specifically expressed in seeds, and six genes show expression specific to panicle development. Differential expression of seven genes under stress conditions is also evident. An attempt has been made to gain insight into plausible functions of rice MADS-box genes by collating the expression data of functionally validated genes in rice and Arabidopsis.
Only a limited number of MADS genes have been functionally validated in rice. A comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of MADS-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies.
The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD), which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation.
Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only.
Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein orthologs from rice and sorghum. These similarities between ID1 and closely related genes in other grasses point to possible similarities in function.
Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12).
Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages.
The observed high conservation of interactions between proteins that serve as key regulators of the rice defense response suggests that the existing rice interactome can be used to predict interactions in wheat. Such predictions are less reliable for nodes that have undergone a different history of duplications and sub-functionalization in the two lineages.
Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use.
Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field.
This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.
Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests.
Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects.
In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.
Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control).
The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved cis regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development.
This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate family amino acids, and storage proteins. Some of the differentially expressed genes could be useful for the development of molecular markers if they are located in a known QTL region for milling yield or eating quality in the rice genome. Therefore, our comprehensive and deep survey of the developing seed transcriptome in five rice cultivars has provided a rich genomic resource for further elucidating the molecular basis of grain quality in rice.
Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or ‘expressology’, thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au).
Oryza sativa; rice; Arabidopsis thaliana; Arabidopsis; protein; subcellular location; transcript expression
In flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development.
Transcriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far.
Not only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation.
Pseudogenes are significant components of eukaryotic genomes, and some have acquired novel regulatory roles. To date, no study has characterized rice pseudogenes systematically or addressed their impact on the structure and function of the rice genome. In this genome-wide study, we have identified 11,956 non-transposon-related rice pseudogenes, most of which are from gene duplications. About 12% of the rice protein-coding genes, half of which are in singleton families, have a pseudogene paralog. Interestingly, we found that 145 of these pseudogenes potentially gave rise to antisense small RNAs after examining ∼1.5 million small RNAs from developing rice grains. The majority (>50%) of these antisense RNAs are 24-nucleotides long, a feature often seen in plant repeat-associated small interfering RNAs (siRNAs) produced by RNA-dependent RNA polymerase (RDR2) and Dicer-like protein 3 (DCL3), suggesting that some pseudogene-derived siRNAs may be implicated in repressing pseudogene transcription (i.e., cis-acting). Multiple lines of evidence, however, indicate that small RNAs from rice pseudogenes might also function as natural antisense siRNAs either by interacting with the complementary sense RNAs from functional parental genes (38 cases) or by forming double-strand RNAs with transcripts of adjacent paralogous pseudogenes (2 cases) (i.e., trans-acting). Further examinations of five additional small RNA libraries revealed that pseudogene-derived antisense siRNAs could be produced in specific rice developmental stages or physiological growth conditions, suggesting their potentially important roles in normal rice development. In summary, our results show that pseudogenes derived from protein-coding genes are prevalent in the rice genome, and a subset of them are strong candidates for producing small RNAs with novel regulatory roles. Our findings suggest that pseudogenes of exapted functions may be a phenomenon ubiquitous in eukaryotic organisms.
Pseudogenes are “defunct” copies of protein-coding genes that have been accumulated in a genome. They have conventionally been considered the junk byproducts of genome evolution, as they cannot code for proteins due to sequence degeneration. Recent important studies, however, have discovered that a subset of them have unforeseen roles as sources of non-coding RNA transcripts that can regulate the expression of functional coding genes. In this work, we have explored this perspective by studying to what extent rice pseudogenes can encode small RNAs, especially the antisense type. After cross-examining thousands of rice pseudogenes and several millions of small RNA sequences, we found that pseudogene-derived small RNAs are abundant in rice, and furthermore, many of them may function as natural antisense RNAs by interacting complementarily (based on sequence similarity) with sense RNAs from either parental genes or other pseudogenes. Some of these pseudogene RNAs may also be involved in repressing pseudogene transcription. Our findings shed new light on the multifaceted biological roles of pseudogene-derived siRNAs that may or may not affect their functional counterparts directly, indicating that more studies are required to explore in molecular detail the diverse functions of siRNAs originating from pseudogenes in both animals and plants.
It was proposed that differentially-expressed genes, aside from genetic variations affecting protein processing and functioning, between hybrid and its parents provide essential candidates for studying heterosis or hybrid vigor. Based our serial analysis of gene expression (SAGE) data from an elite Chinese super-hybrid rice (LYP9) and its parental cultivars (93-11 and PA64s) in three major tissue types (leaves, roots and panicles) at different developmental stages, we analyzed the transcriptome and looked for candidate genes related to rice heterosis.
By using an improved strategy of tag-to-gene mapping and two recently annotated genome assemblies (93-11 and PA64s), we identified 10,268 additional high-quality tags, reaching a grand total of 20,595 together with our previous result. We further detected 8.5% and 5.9% physically-mapped genes that are differentially-expressed among the triad (in at least one of the three stages) with P-values less than 0.05 and 0.01, respectively. These genes distributed in 12 major gene expression patterns; among them, 406 up-regulated and 469 down-regulated genes (P < 0.05) were observed. Functional annotations on the identified genes highlighted the conclusion that up-regulated genes (some of them are known enzymes) in hybrid are mostly related to enhancing carbon assimilation in leaves and roots. In addition, we detected a group of up-regulated genes related to male sterility and 442 down-regulated genes related to signal transduction and protein processing, which may be responsible for rice heterosis.
We improved tag-to-gene mapping strategy by combining information from transcript sequences and rice genome annotation, and obtained a more comprehensive view on genes that related to rice heterosis. The candidates for heterosis-related genes among different genotypes provided new avenue for exploring the molecular mechanism underlying heterosis.
Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying embryogenesis. Here we report the transcriptome profiling analysis of rice developing embryos using RNA-Seq as an attempt to gain insight into the molecular and cellular events associated with rice embryogenesis. RNA-Seq analysis generated 17,755,890 sequence reads aligned with 27,190 genes, which provided abundant data for the analysis of rice embryogenesis. A total of 23,971, 23,732, and 23,592 genes were identified from embryos at three developmental stages (3–5, 7, and 14 DAP), while an analysis between stages allowed the identification of a subset of stage-specific genes. The number of genes expressed stage-specifically was 1,131, 1,443, and 1,223, respectively. In addition, we investigated transcriptomic changes during rice embryogenesis based on our RNA-Seq data. A total of 1,011 differentially expressed genes (DEGs) (log2Ratio ≥1, FDR ≤0.001) were identified; thus, the transcriptome of the developing rice embryos changed considerably. A total of 672 genes with significant changes in expression were detected between 3–5 and 7 DAP; 504 DEGs were identified between 7 and 14 DAP. A large number of genes related to metabolism, transcriptional regulation, nucleic acid replication/processing, and signal transduction were expressed predominantly in the early and middle stages of embryogenesis. Protein biosynthesis-related genes accumulated predominantly in embryos at the middle stage. Genes for starch/sucrose metabolism and protein modification were highly expressed in the middle and late stages of embryogenesis. In addition, we found that many transcription factor families may play important roles at different developmental stages, not only in embryo initiation but also in other developmental processes. These results will expand our understanding of the complex molecular and cellular events in rice embryogenesis and provide a foundation for future studies on embryo development in rice and other cereal crops.
Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3′UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.
Living cells must be able to withstand changes in the environment. For example, if there is a sudden increase in temperature—which could damage proteins or other molecules—most cells can respond with the ‘heat shock response’. In humans and other mammals, a single protein called heat shock factor 1 triggers the production of numerous heat shock proteins that protect the cell from the detrimental effects of high temperatures. Most heat shock proteins protect cells by binding to, and stabilizing, other molecules in the cell; this prevents these molecules from being damaged or from aggregating and allows them to continue to function as normal.
A protein called eEF1A1 is involved in the final stages of protein production and also enhances the function of heat shock factor 1 during the heat shock response. To make a protein, an enzyme called RNA polymerase transcribes DNA in the nucleus of the cell into a messenger RNA molecule that then exits the nucleus and binds to a ribosome. This molecular machine then translates the messenger RNA sequence into a protein by joining together individual building blocks called amino acids in the correct order. Like other elongation factors, eEF1A1 helps to select the amino acids that match the sequence of the messenger RNA template. However, it was unclear how eEF1A1 helped to protect cells during the heat shock response.
Nudler et al. have now engineered cells—from humans and mice—that make less of the eEF1A1 protein than normal. These cells had enough of this protein to support their growth and development under normal conditions, but not enough to help during the heat shock response. When these cells are subjected to a sudden increase in temperature, they fail to produce a sufficient amount of major heat shock proteins. Heat shock factor 1 is needed to transcribe the genes that encode these heat shock proteins, and Nudler et al. found that eEF1A1 must bind to heat shock factor 1 and then to a moving RNA polymerase for these genes to be transcribed efficiently. Moreover, the eEF1A1 protein was shown to bind to and stabilize the heat shock proteins' messenger RNAs, and aid their export from the nucleus and their binding to the ribosome.
These newly discovered roles for eEF1A1 during the heat shock response highlight this elongation factor as a promising drug target for treating diseases where protein folding goes awry, for example in Alzheimer's or Parkinson's disease. In adults, neurons do not make enough eEF1A1, and Nudler et al. suggest that enabling these cells to make more of this protein could help to treat a range of neurodegenerative conditions.
transcription factors; RNA stability; nuclear export; human; mouse
The effects of viral infection involve concomitant plant gene variations and cellular changes. A simple system is required to assess the complexity of host responses to viral infection. The genome of the Rice yellow mottle virus (RYMV) is a single-stranded RNA with a simple organisation. It is the most well-known monocotyledon virus model. Several studies on its biology, structure and phylogeography have provided a suitable background for further genetic studies. 12 rice chromosome sequences are now available and provide strong support for genomic studies, particularly physical mapping and gene identification.
The present data, obtained through the cDNA-AFLP technique, demonstrate differential responses to RYMV of two different rice cultivars, i.e. susceptible IR64 (Oryza sativa indica), and partially resistant Azucena (O. s. japonica). This RNA profiling provides a new original dataset that will enable us to gain greater insight into the RYMV/rice interaction and the specificity of the host response. Using the SIM4 subroutine, we took the intron/exon structure of the gene into account and mapped 281 RYMV stress responsive (RSR) transcripts on 12 rice chromosomes corresponding to 234 RSR genes. We also mapped previously identified deregulated proteins and genes involved in partial resistance and thus constructed the first global physical map of the RYMV/rice interaction. RSR transcripts on rice chromosomes 4 and 10 were found to be not randomly distributed. Seven genes were identified in the susceptible and partially resistant cultivars, and transcripts were colocalized for these seven genes in both cultivars. During virus infection, many concomitant plant gene expression changes may be associated with host changes caused by the infection process, general stress or defence responses. We noted that some genes (e.g. ABC transporters) were regulated throughout the kinetics of infection and differentiated susceptible and partially resistant hosts.
We enhanced the first RYMV/rice interaction map by combining information from the present study and previous studies on proteins and ESTs regulated during RYMV infection, thus providing a more comprehensive view on genes related to plant responses. This combined map provides a new tool for exploring molecular mechanisms underlying the RYMV/rice interaction.
Genome dynamics of pathogenic organisms are driven by pathogen and host co-evolution, in which pathogen genomes are shaped to overcome stresses imposed by hosts with various genetic backgrounds through generation of a variety of isolates. This same principle applies to the rice blast pathogen Magnaporthe oryzae and the rice host; however, genetic variations among different isolates of M. oryzae remain largely unknown, particularly at genome and transcriptome levels. Here, we applied genomic and transcriptomic analytical tools to investigate M. oryzae isolate 98-06 that is the most aggressive in infection of susceptible rice cultivars. A unique 1.4 Mb of genomic sequences was found in isolate 98-06 in comparison to reference strain 70-15. Genome-wide expression profiling revealed the presence of two critical expression patterns of M. oryzae based on 64 known pathogenicity-related (PaR) genes. In addition, 134 candidate effectors with various segregation patterns were identified. Five tested proteins could suppress BAX-mediated programmed cell death in Nicotiana benthamiana leaves. Characterization of isolate-specific effector candidates Iug6 and Iug9 and PaR candidate Iug18 revealed that they have a role in fungal propagation and pathogenicity. Moreover, Iug6 and Iug9 are located exclusively in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, suggesting that they might participate in biotrophy by inhibiting the SA and ET pathways within the host. Thus, our studies identify novel effector and PaR proteins involved in pathogenicity of the highly aggressive M. oryzae field isolate 98-06, and reveal molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions.
Genetic variations in pathogens, such as the causal agent of rice blast Magnaporthe oryzae, often lead to circumvention of disease-resistance cultivars. Previous genome-wide analyses of model organisms suggest that pathogen effectors are also rapidly evolving, especially in regions with high genome plasticity. However, genetic variations among different isolates remain largely unknown in M. oryzae, particularly at the genome and transcriptome levels. In this study, we provided a systematic genomic and interaction transcriptome profile for a dominant rice blast field isolate, resulting in identification of 134 candidate effectors. Two effectors, Iug6 and Iug9, and one pathogenicity-related (PaR) gene product, Iug18, were subjected to functional characterization. We found that Iug6 and Iug9 are located in the biotrophic interfacial complex (BIC) and their overexpression leads to suppression of defense-related gene expression in rice, while Iug18 appears to be a novel PaR protein. Our studies support the hypothesis that isolate-unique genes may serve as a source of genetic variability in the M. oryzae population encountering different environments. Our studies also facilitate further understanding of effectors and genomic variations in pathogenicity of M. oryzae.
Brewers’ rice, is locally known as temukut, is a mixture of broken rice, rice bran, and rice germ. The current study is an extension of our previous work, which demonstrated that water extract of brewers’ rice (WBR) induced apoptosis in human colorectal cancer (HT-29) cells. We also identified that brewers’ rice was effective in reducing the tumor incidence and multiplicity in azoxymethane (AOM)-injected colon cancer rats. Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells. To further determine whether the in vitro mechanisms of action observed in the HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers’ rice in regulating the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats.
The mRNA levels of several upstream-related genes in the Wnt signaling pathway in HT-29 cells treated with WBR were determined by quantitative real-time PCR analyses. Caspase-3 and −8 were evaluated using a colorimetric assay. Male Sprague–Dawley rats were administered two intraperitoneal injections of AOM in saline (15 mg/kg body weight) over a two-week period and received with 10, 20, and 40 % (w/w) brewers’ rice. The expressions and protein levels of cyclin D1 and c-myc were evaluated by immunohistochemical staining and western blotting, respectively.
The overall analyses revealed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the casein kinase 1 (CK1) and adenomatous polyposis coli (APC) mRNA levels. We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and −8 activities compared with the control (P < 0.05). In vivo analyses indicated that brewers’ rice diminished the β-catenin, cyclin D1, and c-myc protein levels.
We provide evidence that brewers’ rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo. We suggest that brewers’ rice may be a useful dietary agent for colorectal cancer.
Brewers’ rice; Colorectal cancer; Wnt signaling; Cyclin D1; c-myc
GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes.
In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the possible biological functions of the rice OsGELP genes.
Our current genomic analysis, for the first time, presents fundamental information on the organization of the rice OsGELP gene family. With combination of the genomic, phylogenetic, microarray expression, protein motif distribution, and protein structure analyses, we were able to create supported basis for the functional prediction of many members in the rice GDSL esterase/lipase family. The present study provides a platform for the selection of candidate genes for further detailed functional study.
The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.
Glutathione S-transferases (GSTs) are the ubiquitous enzymes that play a key role in cellular detoxification. Although several GSTs have been identified and characterized in various plant species, the knowledge about their role in developmental processes and response to various stimuli is still very limited. In this study, we report genome-wide identification, characterization and comprehensive expression analysis of members of GST gene family in crop plant rice, to reveal their function(s).
A systematic analysis revealed the presence of at least 79 GST genes in the rice genome. Phylogenetic analysis grouped GST proteins into seven classes. Sequence analysis together with the organization of putative motifs indicated the potential diverse functions of GST gene family members in rice. The tandem gene duplications have contributed a major role in expansion of this gene family. Microarray data analysis revealed tissue-/organ- and developmental stage-specific expression patterns of several rice GST genes. At least 31 GST genes showed response to plant hormones auxin and cytokinin. Furthermore, expression analysis showed the differential expression of quite a large number of GST genes during various abiotic stress (20), arsenate stress (32) and biotic stress (48) conditions. Many of the GST genes were commonly regulated by developmental processes, hormones, abiotic and biotic stresses.
The transcript profiling suggests overlapping and specific role(s) of GSTs during various stages of development in rice. Further, the study provides evidence for the role of GSTs in mediating crosstalk between various stress and hormone response pathways and represents a very useful resource for functional analysis of selected members of this family in rice.
Alternative splicing (AS) contributes significantly to protein diversity, by selectively using different combinations of exons of the same gene under certain circumstances. One particular type of AS is the use of alternative first exons (AFEs), which can have consequences far beyond the fine-tuning of protein functions. For example, AFEs may change the N-termini of proteins and thereby direct them to different cellular compartments. When alternative first exons are distant, they are usually associated with alternative promoters, thereby conferring an extra level of gene expression regulation. However, only few studies have examined the patterns of AFEs, and these analyses were mainly focused on mammalian genomes. Recent studies have shown that AFEs exist in the rice genome, and are regulated in a tissue-specific manner. Our current understanding of AFEs in plants is still limited, including important issues such as their regulation, contribution to protein diversity, and evolutionary conservation.
We systematically identified 1,378 and 645 AFE-containing clusters in rice and Arabidopsis, respectively. From our data sets, we identified two types of AFEs according to their genomic organisation. In genes with type I AFEs, the first exons are mutually exclusive, while most of the downstream exons are shared among alternative transcripts. Conversely, in genes with type II AFEs, the first exon of one gene structure is an internal exon of an alternative gene structure. The functionality analysis indicated about half and ~19% of the AFEs in Arabidopsis and rice could alter N-terminal protein sequences, and ~5% of the functional alteration in type II AFEs involved protein domain addition/deletion in both genomes. Expression analysis indicated that 20~66% of rice AFE clusters were tissue- and/or development- specifically transcribed, which is consistent with previous observations; however, a much smaller percentage of Arabidopsis AFEs was regulated in this manner, which suggests different regulation mechanisms of AFEs between rice and Arabidopsis. Statistical analysis of some features of AFE clusters, such as splice-site strength and secondary structure formation further revealed differences between these two species. Orthologous search of AFE-containing gene pairs detected only 19 gene pairs conserved between rice and Arabidopsis, accounting only for a few percent of AFE-containing clusters.
Our analysis of AFE-containing genes in rice and Arabidopsis indicates that AFEs have multiple functions, from regulating gene expression to generating protein diversity. Comparisons of AFE clusters revealed different features in the two plant species, which indicates that AFEs may have evolved independently after the separation of rice (a model monocot) and Arabidopsis (a model dicot).
Peptide transporter (PTR) family whose member can transport di-/tripeptides and nitrate is important for plant growth and development. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, a genomic survey, characterization and expression profile analysis of the PTR family in this species has not been reported.
In this study, we report a comprehensive identification, characterization, phylogenetic and evolutionary analysis of 84 PTR family members in rice (OsPTR) as well as their whole-life expression patterns. Chromosomal distribution and sequence analysis indicate that nearly 70% of OsPTR members are involved in the tandem and segmental duplication events. It suggests that genome duplication might be a major mechanism for expansion of this family. Highly conserved motifs were identified in most of the OsPTR members. Meanwhile, expression profile of OsPTR genes has been analyzed by using Affymetrix rice microarray and real-time PCR in two elite hybrid rice parents, Minghui 63 and Zhenshan 97. Seven genes are found to exhibit either preferential or tissue-specific expression during different development stages of rice. Under phytohormone (NAA, GA3 and KT) and light/dark treatments, 14 and 17 OsPTR genes are differentially expressed respectively. Ka/Ks analysis of the paralogous OsPTR genes indicates that purifying selection plays an important role in function maintenance of this family.
These investigations add to our understanding of the importance of OsPTR family members and provide useful reference for selecting candidate genes for functional validation studies of this family in rice.