Plant nutrition is one of the important areas for improving the yield and quality in crops as well as non-crop plants. Potassium is an essential plant nutrient and is required in abundance for their proper growth and development. Potassium deficiency directly affects the plant growth and hence crop yield and production. Recently, potassium-dependent transcriptomic analysis has been performed in the model plant Arabidopsis, however in cereals and crop plants; such a transcriptome analysis has not been undertaken till date. In rice, the molecular mechanism for the regulation of potassium starvation responses has not been investigated in detail. Here, we present a combined physiological and whole genome transcriptomic study of rice seedlings exposed to a brief period of potassium deficiency then replenished with potassium. Our results reveal that the expressions of a diverse set of genes annotated with many distinct functions were altered under potassium deprivation. Our findings highlight altered expression patterns of potassium-responsive genes majorly involved in metabolic processes, stress responses, signaling pathways, transcriptional regulation, and transport of multiple molecules including K+. Interestingly, several genes responsive to low-potassium conditions show a reversal in expression upon resupply of potassium. The results of this study indicate that potassium deprivation leads to activation of multiple genes and gene networks, which may be acting in concert to sense the external potassium and mediate uptake, distribution and ultimately adaptation to low potassium conditions. The interplay of both upregulated and downregulated genes globally in response to potassium deprivation determines how plants cope with the stress of nutrient deficiency at different physiological as well as developmental stages of plants.
Rice (Oryza sativa) varieties that are arsenate-tolerant (Bala) and -sensitive (Azucena) were used to conduct a transcriptome analysis of the response of rice seedlings to sodium arsenate (AsV) in hydroponic solution. RNA extracted from the roots of three replicate experiments of plants grown for 1 week in phosphate-free nutrient with or without 13.3 μM AsV was used to challenge the Affymetrix (52K) GeneChip Rice Genome array. A total of 576 probe sets were significantly up-regulated at least 2-fold in both varieties, whereas 622 were down-regulated. Ontological classification is presented. As expected, a large number of transcription factors, stress proteins, and transporters demonstrated differential expression. Striking is the lack of response of classic oxidative stress-responsive genes or phytochelatin synthases/synthatases. However, the large number of responses from genes involved in glutathione synthesis, metabolism, and transport suggests that glutathione conjugation and arsenate methylation may be important biochemical responses to arsenate challenge. In this report, no attempt is made to dissect differences in the response of the tolerant and sensitive variety, but analysis in a companion article will link gene expression to the known tolerance loci available in the Bala×Azucena mapping population.
Arsenate tolerance; candidate genes; glutathione-S-transferase; microarray; Oryza sativa
Plants face a variety of environmental stresses and have evolved molecular mechanisms to survive these challenges. One of these stresses is low oxygen conditions, which can occur under flooding conditions. Rice (Oryza sativa) is somewhat unique for its ability to tolerate and even germinate under low to no oxygen conditions. In this study, we examined global transcriptomic responses over the course of germination and in response to low oxygen and other abiotic stress in rice and Arabidopsis (Arabidopsis thaliana). Over 150 microarray datasets were analyzed in parallel to determine just how unique the low oxygen response is in rice. Comparison of aerobic germination in rice and Arabidopsis, with anaerobic germination in rice revealed conserved transcriptomic responses that are not only conserved across both species but also occur in the absence of oxygen in rice. Thus, these genes may represent functions necessary for the developmental progression of germination, whether or not oxygen is present in rice. Analysis of genes that responded differently in rice compared to Arabidopsis revealed responses specific to anaerobic germination in rice, including the down-regulation of genes encoding redox functions and up-regulation of receptor kinases. Comparison of a range of hypoxia/anoxia studies within and across Arabidopsis and rice revealed both conserved and species specific changes in gene expression (e.g., Arabidopsis specific up-regulation of WRKYs and rice specific down-regulation of heme), unveiling unique transcriptomic signatures of the low oxygen response. Lastly, a comparison of the low oxygen response with cold, salt, drought and heat stress revealed some similarity with the response to heat stress in Arabidopsis, which was not seen in rice. Comparison of these heat-responsive, abiotic stress marker genes in Arabidopsis with their rice orthologs revealed that while low oxygen may be perceived as an abiotic stress in Arabidopsis, this is not the case in rice.
low oxygen; transcriptomes; rice; Arabidopsis; microarrays
Widespread use of chromium (Cr) contaminated fields due to careless and inappropriate management practices of effluent discharge, mostly from industries related to metallurgy, electroplating, production of paints and pigments, tanning, and wood preservation elevates its concentration in surface soil and eventually into rice plants and grains. In spite of many previous studies having been conducted on the effects of chromium stress, the precise molecular mechanisms related to both the effects of chromium phytotoxicity, the defense reactions of plants against chromium exposure as well as translocation and accumulation in rice remain poorly understood.
Detailed analysis of genome-wide transcriptome profiling in rice root is reported here, following Cr-plant interaction. Such studies are important for the identification of genes responsible for tolerance, accumulation and defense response in plants with respect to Cr stress. Rice root metabolome analysis was also carried out to relate differential transcriptome data to biological processes affected by Cr (VI) stress in rice. To check whether the Cr-specific motifs were indeed significantly over represented in the promoter regions of Cr-responsive genes, occurrence of these motifs in whole genome sequence was carried out. In the background of whole genome, the lift value for these 14 and 13 motifs was significantly high in the test dataset. Though no functional role has been assigned to any of the motifs, but all of these are present as promoter motifs in the Database of orthologus promoters.
These findings clearly suggest that a complex network of regulatory pathways modulates Cr-response of rice. The integrated matrix of both transcriptome and metabolome data after suitable normalization and initial calculations provided us a visual picture of the correlations between components. Predominance of different motifs in the subsets of genes suggests the involvement of motif-specific transcription modulating proteins in Cr stress response of rice.
Plant roots are important organs to uptake soil water and nutrients, perceiving and transducing of soil water deficit signals to shoot. The current knowledge of drought stress transcriptomes in rice are mostly relying on comparative studies of diverse genetic background under drought. A more reliable approach is to use near-isogenic lines (NILs) with a common genetic background but contrasting levels of resistance to drought stress under initial exposure to water deficit. Here, we examined two pairs of NILs in IR64 background with contrasting drought tolerance. We obtained gene expression profile in roots of rice NILs under different levels of drought stress help to identify genes and mechanisms involved in drought stress.
Global gene expression analysis showed that about 55% of genes differentially expressed in roots of rice in response to drought stress treatments. The number of differentially expressed genes (DEGs) increased in NILs as the level of water deficits, increased from mild to severe condition, suggesting that more genes were affected by increasing drought stress. Gene onthology (GO) test and biological pathway analysis indicated that activated genes in the drought tolerant NILs IR77298-14-1-2-B-10 and IR77298-5-6-B-18 were mostly involved in secondary metabolism, amino acid metabolism, response to stimulus, defence response, transcription and signal transduction, and down-regulated genes were involved in photosynthesis and cell wall growth. We also observed gibberellic acid (GA) and auxin crosstalk modulating lateral root formation in the tolerant NILs.
Transcriptome analysis on two pairs of NILs with a common genetic background (~97%) showed distinctive differences in gene expression profiles and could be effective to unravel genes involved in drought tolerance. In comparison with the moderately tolerant NIL IR77298-5-6-B-18 and other susceptible NILs, the tolerant NIL IR77298-14-1-2-B-10 showed a greater number of DEGs for cell growth, hormone biosynthesis, cellular transports, amino acid metabolism, signalling, transcription factors and carbohydrate metabolism in response to drought stress treatments. Thus, different mechanisms are achieving tolerance in the two tolerant lines.
Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the ATP-dependent phosphofructokinase (PFK) and the pyrophosphate-(PPi) dependent pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP). During oxygen deficiency ATP levels in rice seedlings are severely reduced, and it is hypothesized that PPi is used as an alternative energy source for the phosphorylation of fructose-6-phosphate during glycolysis. In this study, we analyzed the expression of 15 phosphofructokinase-encoding genes in roots and aerial tissues of anoxia-tolerant rice seedlings in response to anoxic stress and compared our data with transcript profiles obtained from microarray analyses. Furthermore, the intracellular localization of rice PFK proteins was determined, and the PFK and PFP isoforms were grouped in a phylogenetic tree. Two PFK and two PFP transcripts accumulated during anoxic stress, whereas mRNA levels of four PFK and three PFP genes were decreased. The total specific activity of both PFK and PFP changed only slightly during a 24-h anoxia treatment. It is assumed that expression of different isoforms and their catalytic properties differ during normoxic and anoxic conditions and contribute to balanced glycolytic activity during the low-oxygen stress. These characterizations of phosphofructokinase genes and the comparison to other plant species allowed us to suggest candidate rice genes for adaptation to anoxic stress.
Oryza sativa; anoxia; submergence; phosphofructokinase; pyrophosphate
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.
Gene expression in response to Cu stress in rice leaves was quantified using DNA microarray (Agilent 22K Rice Oligo Microarray) and real-time PCR technology. Rice plants were grown in hydroponic solutions containing 0.3 (control), 10, 45, or 130 μM of CuCl2, and Cu accumulation and photosynthesis inhibition were observed in leaves within 1 d of the start of treatment. Microarray analysis flagged 305 Cu-responsive genes, and their expression profile showed that a large proportion of general and defence stress response genes are up-regulated under excess Cu conditions, whereas photosynthesis and transport-related genes are down-regulated. The Cu sensitivity of each Cu-responsive gene was estimated by the median effective concentration value (EC50) and the range of fold-changes (F) under the highest (130 μM) Cu conditions (|log2F|130). Our results indicate that defence-related genes involved in phytoalexin and lignin biosynthesis were the most sensitive to Cu, and that plant management of abiotic and pathogen stresses has overlapping components, possibly including signal transduction.
Copper-sensitivity; DNA microarray; excess copper stress; gene expression; Oryza sativa L
Plants are simultaneously exposed to multiple stresses resulting in enormous changes in the molecular landscape within the cell. Identification and characterization of the synergistic and antagonistic components of stress response mechanisms contributing to the cross talk between stresses is of high priority to explore and enhance multiple stress responses. To this end, we performed meta-analysis of drought (abiotic), bacterial (biotic) stress response in rice and Arabidopsis by analyzing a total of 386 microarray samples belonging to 20 microarray studies and identified approximately 3100 and 900 DEGs in rice and Arabidopsis, respectively. About 38.5% (1214) and 28.7% (272) DEGs were common to drought and bacterial stresses in rice and Arabidopsis, respectively. A majority of these common DEGs showed conserved expression status in both stresses. Gene ontology enrichment analysis clearly demarcated the response and regulation of various plant hormones and related biological processes. Fatty acid metabolism and biosynthesis of alkaloids were upregulated and, nitrogen metabolism and photosynthesis was downregulated in both stress conditions. WRKY transcription family genes were highly enriched in all upregulated gene sets while ‘CO-like’ TF family showed inverse relationship of expression between drought and bacterial stresses. Weighted gene co-expression network analysis divided DEG sets into multiple modules that show high co-expression and identified stress specific hub genes with high connectivity. Detection of consensus modules based on DEGs common to drought and bacterial stress revealed 9 and 4 modules in rice and Arabidopsis, respectively, with conserved and reversed co-expression patterns.
Arabidopsis thaliana is clearly established as the model plant species. Given the ever-growing demand for food, there is a need to translate the knowledge learned in Arabidopsis to agronomically important species, such as rice (Oryza sativa). To gain a comparative insight into the similarities and differences into how organs are built and how plants respond to stress, the transcriptomes of Arabidopsis and rice were compared at the level of gene orthology and functional categorisation.
Organ specific transcripts in rice and Arabidopsis display less overlap in terms of gene orthology compared to the orthology observed between both genomes. Although greater overlap in terms of functional classification was observed between root specific transcripts in rice and Arabidopsis, this did not extend to flower, leaf or seed specific transcripts. In contrast, the overall abiotic stress response transcriptome displayed a significantly greater overlap in terms of gene orthology compared to the orthology observed between both genomes. However, ~50% or less of these orthologues responded in a similar manner in both species. In fact, under cold and heat treatments as many or more orthologous genes responded in an opposite manner or were unchanged in one species compared to the other. Examples of transcripts that responded oppositely include several genes encoding proteins involved in stress and redox responses and non-symbiotic hemoglobins that play central roles in stress signalling pathways. The differences observed in the abiotic transcriptomes were mirrored in the presence of cis-acting regulatory elements in the promoter regions of stress responsive genes and the transcription factors that potentially bind these regulatory elements. Thus, both the abiotic transcriptome and its regulation differ between rice and Arabidopsis.
These results reveal significant divergence between Arabidopsis and rice, in terms of the abiotic stress response and its regulation. Both plants are shown to employ unique combinations of genes to achieve growth and stress responses. Comparison of these networks provides a more rational approach to translational studies that is based on the response observed in these two diverse plant models.
The stimulation effect that some beneficial agronomic qualities have exhibited in present-generation plants have also been observed due to ion implantation on plants. However, there is relatively little knowledge regarding the molecular mechanism of the stimulation effects of ion-beam implantation. In order to extend our current knowledge about the functional genes related to this stimulation effect, we have reported a comprehensive microarray analysis of the transcriptome features of the promoted-growth rice seedlings germinating from seeds implanted by a low-energy N+ beam. The results showed that 351 up-regulated transcripts and 470 down-regulated transcripts, including signaling proteins, kinases, plant hormones, transposable elements, transcription factors, non-coding protein RNA (including miRNA), secondary metabolites, resistance proteins, peroxidase and chromatin modification, are all involved in the stimulating effects of ion-beam implantation. The divergences of the functional catalog between the vacuum and ion implantation suggest that ion implantation is the principle cause of the ion-beam implantation biological effects, and revealed the complex molecular networks required to adapt to ion-beam implantation stress in plants, including enhanced transposition of transposable elements, promoted ABA biosynthesis and changes in chromatin modification. Our data will extend the current understanding of the molecular mechanisms and gene regulation of stimulation effects. Further research on the candidates reported in this study should provide new insights into the molecular mechanisms of biological effects induced by ion-beam implantation.
implantation with low-energy ion beam; stimulation effect; analysis of microarray
Rice blast disease is a major threat to rice production worldwide, but the mechanisms underlying rice resistance to the causal agent Magnaporthe oryzae remain elusive. Therefore, we carried out a transcriptome study on rice early defense response to M. oryzae. We found that the transcriptional profiles of rice compatible and incompatible interactions with M. oryzae were mostly similar, with genes regulated more prominently in the incompatible interactions. The functional analysis showed that the genes involved in signaling and secondary metabolism were extensively up-regulated. In particular, WRKY transcription factor genes were significantly enriched among the up-regulated genes. Overexpressing one of these WRKY genes, OsWRKY47, in transgenic rice plants conferred enhanced resistance against rice blast fungus. Our results revealed the sophisticated transcriptional reprogramming of signaling and metabolic pathways during rice early response to M. oryzae and demonstrated the critical roles of WRKY transcription factors in rice blast resistance.
Plant growth depends on synergistic interactions between internal and external signals, and yield potential of crops is a manifestation of how these complex factors interact, particularly at critical stages of development. As an initial step towards developing a systems-level understanding of the biological processes underlying the expression of overall agronomic potential in cereal crops, a high-resolution transcriptome analysis of rice was conducted throughout life cycle of rice grown under natural field conditions.
A wide range of gene expression profiles based on 48 organs and tissues at various developmental stages identified 731 organ/tissue specific genes as well as 215 growth stage-specific expressed genes universally in leaf blade, leaf sheath, and root. Continuous transcriptome profiling of leaf from transplanting until harvesting further elucidated the growth-stage specificity of gene expression and uncovered two major drastic changes in the leaf transcriptional program. The first major change occurred before the panicle differentiation, accompanied by the expression of RFT1, a putative florigen gene in long day conditions, and the downregulation of the precursors of two microRNAs. This transcriptome change was also associated with physiological alterations including phosphate-homeostasis state as evident from the behavior of several key regulators such as miR399. The second major transcriptome change occurred just after flowering, and based on analysis of sterile mutant lines, we further revealed that the formation of strong sink, i.e., a developing grain, is not the major cause but is rather a promoter of this change.
Our study provides not only the genetic basis for functional genomics in rice but also new insight into understanding the critical physiological processes involved in flowering and seed development, that could lead to novel strategies for optimizing crop productivity.
To identify novel elements of plant salt stress adaptation, salt-induced transcript accumulation was compared in the model crop plant rice and in the halotolerant grass Festuca rubra ssp. litoralis by cDNA-array hybridizations. Results of the study show major differences in transcript expression profiles between the salt sensitive rice and the naturally halotolerant grass species. The data indicate that the salt tolerance strategy of F. rubra ssp. litoralis involves activated expression of genes with functions in osmotic and ion homeostasis, in metabolism, and general stress defense that is missing in rice. In addition, transcripts with a function in regulation of transcription, translation, signal transduction and protein turnover showed different transcriptional responses. Among other signaling elements that were regulated by salt in the halotolerant F. rubra ssp. litoralis but not in rice, the putative serine/threonine protein kinase SnRK1b (sucrose non-fermenting-1-related kinase 1) was identified. It is hypothesized that modification of signal transduction pathways and transcriptional control in salt-sensitive species according to regulatory mechanisms identified in related halophytes can activate the complex network of molecular processes that lead to an improved tolerance of salinity.
halophyte; protein kinase; rice; salt tolerance; signaling; transcriptome
Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H2O2) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H2O2-responsive proteins in the apoplast of rice seedling roots.
14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H2O2 for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H2O2 were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H2O2 conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H2O2-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H2O2 stress.
The present study is the first apoplast proteome investigation of plant seedlings in response to H2O2 and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H2O2 response.
• Background and Aims Mineral nutrient deficiencies and salinity constitute major limitations for crop plant growth on agricultural soils. 14-3-3 proteins are phosphoserine-binding proteins that regulate the activities of a wide array of targets via direct protein–protein interactions and may play an important role in responses to mineral nutrients deficiencies and salt stress. In the present study, the expression profiling of the 14-3-3 gene family in response to salt stress and potassium and iron deficiencies in young tomato (Solanum lycopersicum) roots was investigated in order to analyse the 14-3-3 roles of the proteins in these abiotic stresses.
• Methods Sequence identities and phylogenetic tree creation were performed using DNAMAN version 4.0 (Lynnon Biosoft Company). Real-time RT–PCR was used to examine the expression of each 14-3-3 gene in response to salt stress and potassium and iron deficiencies in young tomato roots.
• Key Results The phylogenetic tree shows that the 14-3-3 gene family falls into two major groups in tomato plants. By using real-time RT–PCR, it was found that (a) under normal growth conditions, there were significant differences in the mRNA levels of 14-3-3 gene family members in young tomato roots and (b) 14-3-3 proteins exhibited diverse patterns of gene expression in response to salt stress and potassium and iron deficiencies in tomato roots.
• Conclusions The results suggest that (a) 14-3-3 proteins may be involved in the salt stress and potassium and iron deficiency signalling pathways in young tomato roots, (b) the expression pattern of 14-3-3 gene family members in tomato roots is not strictly related to the position of the corresponding proteins within a phylogenetic tree, (c) gene-specific expression patterns indicate that isoform-specificity may exist in the 14-3-3 gene family of tomato roots, and (d) 14-3-3 proteins (TFT7) might mediate cross-talk between the salt stress and potassium and iron-deficiency signalling pathways in tomato roots.
Solanum lycopersicum; real-time RT–PCR; expression; gene family; 14-3-3; salt stress and potassium deficiency; iron deficiency
'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip® microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts.
Following cross-hybridisation of Musa gDNA to the Rice GeneChip® Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments.
Our results demonstrate that despite the general paucity of nucleotide sequence data in Musa and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip® is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species.
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.
Phospholipase A (PLA) is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice.
A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A1 (PLA1), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A2 (sPLA2) based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling.
The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants.
Nitrogen [N] is a critical limiting nutrient for plants and has to be exogenously supplied to many crops, to achieve high yield with significant economic and environmental costs, specifically for rice. Development of low-input nitrogen sustainable crop is necessary for sustainable agriculture. Identification of regulatory elements associated with low-N tolerance is imperative for formulating innovative approaches for developing low-N tolerant crop plants, using gene manipulation. MicroRNAs (miRNAs) are known to play crucial roles in the modulation of gene expression in plants under various environmental conditions.
MiRNAs associated with low-N tolerance have not been identified so far. In this study, we investigated microarray-based miRNA expression in low-N tolerant and low-N sensitive rice genotypes under low N condition. Expressions of 32 miRNAs differed significantly in the two genotypes. Of these 32 miRNAs, expressions of nine miRNAs were further validated experimentally in leaves as well as in roots. Of these differentially expressed miRNAs, six miRNAs (miR156, miR164, miR528, miR820, miR821 and miR1318) were reported in leaves and four (miR164, miR167, miR168 and miR528) in roots. Target genes of all the 32 miRNAs were predicted, which encode transcription factors, and proteins associated with metabolic processes or stress responses. Expression levels of some of the corresponding miRNA targets were analysed and found to be significantly higher in low N-tolerant genotype than low-N sensitive genotype. These findings suggested that miRNAs played an important role in low-N tolerance in rice.
Genome-wide differences in expression of miRNA in low N-tolerant and low N-sensitive rice genotypes were reported. This provides a platform for selection as well as manipulation of genotypes for better N utilization efficiency.
Rice is highly sensitive to drought, and the effect of drought may vary with the different genotypes and development stages. Genome-wide gene expression profiling was used as the initial point to dissect molecular genetic mechanism of this complex trait and provide valuable information for the improvement of drought tolerance in rice. Affymetrix rice genome array containing 48,564 japonica and 1,260 indica sequences was used to analyze the gene expression pattern of rice exposed to drought stress. The transcriptome from leaf, root, and young panicle at three developmental stages was comparatively analyzed combined with bioinformatics exploring drought stress related cis-elements.
There were 5,284 genes detected to be differentially expressed under drought stress. Most of these genes were tissue- or stage-specific regulated by drought. The tissue-specific down-regulated genes showed distinct function categories as photosynthesis-related genes prevalent in leaf, and the genes involved in cell membrane biogenesis and cell wall modification over-presented in root and young panicle. In a drought environment, several genes, such as GA2ox, SAP15, and Chitinase III, were regulated in a reciprocal way in two tissues at the same development stage. A total of 261 transcription factor genes were detected to be differentially regulated by drought stress. Most of them were also regulated in a tissue- or stage-specific manner. A cis-element containing special CGCG box was identified to over-present in the upstream of 55 common induced genes, and it may be very important for rice plants responding to drought environment.
Genome-wide gene expression profiling revealed that most of the drought differentially expressed genes (DEGs) were under temporal and spatial regulation, suggesting a crosstalk between various development cues and environmental stimuli. The identification of the differentially regulated DEGs, including TF genes and unique candidate cis-element for drought responsiveness, is a very useful resource for the functional dissection of the molecular mechanism in rice responding to environment stress.
Plant diurnal oscillation is a 24-hour period based variation. The correlation between diurnal genes and biological pathways was widely revealed by microarray analysis in different species. Rice (Oryza sativa) is the major food staple for about half of the world's population. The rice flag leaf is essential in providing photosynthates to the grain filling. However, there is still no comprehensive view about the diurnal transcriptome for rice leaves. In this study, we applied rice microarray to monitor the rhythmically expressed genes in rice seedling and flag leaves. We developed a new computational analysis approach and identified 6,266 (10.96%) diurnal probe sets in seedling leaves, 13,773 (24.08%) diurnal probe sets in flag leaves. About 65% of overall transcription factors were identified as flag leaf preferred. In seedling leaves, the peak of phase distribution was from 2:00am to 4:00am, whereas in flag leaves, the peak was from 8:00pm to 2:00am. The diurnal phase distribution analysis of gene ontology (GO) and cis-element enrichment indicated that, some important processes were waken by the light, such as photosynthesis and abiotic stimulus, while some genes related to the nuclear and ribosome involved processes were active mostly during the switch time of light to dark. The starch and sucrose metabolism pathway genes also showed diurnal phase. We conducted comparison analysis between Arabidopsis and rice leaf transcriptome throughout the diurnal cycle. In summary, our analysis approach is feasible for relatively unbiased identification of diurnal transcripts, efficiently detecting some special periodic patterns with non-sinusoidal periodic patterns. Compared to the rice flag leaves, the gene transcription levels of seedling leaves were relatively limited to the diurnal rhythm. Our comprehensive microarray analysis of seedling and flag leaves of rice provided an overview of the rice diurnal transcriptome and indicated some diurnal regulated biological processes and key functional pathways in rice.
Iron (Fe) is the most limiting micronutrient element for crop production in alkaline soils. A number of transcription factors involved in regulating Fe uptake from soil and transport in plants have been identified. Analysis of transcriptome data from Oryza sativa grown under limiting Fe conditions reveals that transcript abundances of several genes encoding transcription factors are altered by Fe availability. These transcription factors are putative regulators of Fe deficiency responses.
Transcript abundance of one nuclear located basic helix-loop-helix family transcription factor, OsIRO3, is up-regulated from 25- to 90-fold under Fe deficiency in both root and shoot respectively. The expression of OsIRO3 is specifically induced by Fe deficiency, and not by other micronutrient deficiencies. Transgenic rice plants over-expressing OsIRO3 were hypersensitive to Fe deficiency, indicating that the Fe deficiency response was compromised. Furthermore, the Fe concentration in shoots of transgenic rice plants over-expressing OsIRO3 was less than that in wild-type plants. Analysis of the transcript abundances of genes normally induced by Fe deficiency in OsIRO3 over-expressing plants indicated their induction was markedly suppressed.
A novel Fe regulated bHLH transcription factor (OsIRO3) that plays an important role for Fe homeostasis in rice was identified. The inhibitory effect of OsIRO3 over-expression on Fe deficiency response gene expression combined with hypersensitivity of OsIRO3 over-expression lines to low Fe suggest that OsIRO3 is a negative regulator of the Fe deficiency response in rice.
Abiotic stresses such as drought, salinity, and adverse temperatures are major limiting factors for plant growth and reproduction. Plant responses to these stresses are coordinated by arrays of regulatory networks including the induction of endogenous abscisic acid (ABA), a well documented phytohormone for stress responses. However, whether or how these abiotic stresses affect the endogenous biosynthesis or metabolism of other phytohormones remains largely unknown. Here, we report the changes of endogenous indole-3-acetic acid (IAA) and jasmonic acid (JA) levels and expression of genes related to the biosynthesis or signaling of these hormones in rice under various abiotic stress conditions. The IAA content was decreased after drought stress, but it was significantly increased under cold and heat stresses. And the auxin-regulated gravitropism of root tip was inhibited by cold stress. Many genes involved in the IAA biosynthesis and signaling were changed in transcript level under these stresses, and the changes were essentially in agreement with the change of endogenous IAA level. Interestingly, the endogenous JA content was increased markedly under drought and cold stresses, but it was reduced by heat stress. Accordingly, many genes involved in JA biosynthesis and signaling were induced by drought and cold treatment but these genes were significantly suppressed by heat stress. We concluded that endogenous levels of IAA and JA were differentially regulated by abiotic stresses in rice, implying diverse roles of these hormones in stress responses.
Oryza sativa; abiotic stress; auxin; jasmonic acid
Plants employ two distinct strategies to obtain iron (Fe) from the soil. In Strategy I but not Strategy II plants, Fe limitation invokes ethylene production which regulates Fe deficiency responses. Oryza sativa (rice) is the only graminaceous plant described that possesses a Strategy I-like system for iron uptake as well as the classic Strategy II system. Ethylene production of rice roots was significantly increased when grown under Fe-depleted conditions. Moreover, 1-aminocyclopropane-1-carboxylic acid (ACC) treatment, a precursor of ethylene, conferred tolerance to Fe deficiency in rice by increasing internal Fe availability. Gene expression analysis of rice iron-regulated bHLH transcription factor OsIRO2, nicotianamine synthases 1 and 2 (NAS1 and NAS2), yellow-stripe like transporter 15 (YSL15) and iron-regulated transporter (IRT1) indicated that ethylene caused an increase in transcript abundance of both Fe (II) and Fe (III)-phytosiderophore uptake systems. RNA interference of OsIRO2 in transgenic rice showed that ethylene acted via this transcription factor to induce the expression of OsNAS1, OsNAS2, OsYSL15, and OsIRT1. By contrast, in Hordeum vulgare L. (barley), no ethylene production or ethylene-mediated effects of Fe response could be detected. In conclusion, Fe-limiting conditions increased ethylene production and signalling in rice, which is novel in Strategy II plant species.
Ethylene; gene expression; iron homeostasis; rice; signalling pathway