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
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.
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.
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.
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
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.
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.
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.
Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.
abiotic stress; development; gene expression; microarray analysis; phospholipase D
Reference genes are widely used to normalise transcript abundance data determined by quantitative RT-PCR and microarrays. However, the approaches taken to define reference genes can be variable. Although Oryza sativa (rice) is a widely used model plant and important crop specie, there has been no comprehensive analysis carried out to define superior reference genes.
Analysis of 136 Affymetrix transcriptome datasets comprising of 373 genome microarrays from studies in rice that encompass tissue, developmental, abiotic, biotic and hormonal transcriptome datasets identified 151 genes whose expression was considered relatively stable under all conditions. A sub-set of 12 of these genes were validated by quantitative RT-PCR and were seen to be stable under a number of conditions. All except one gene that has been previously proposed as a stably expressed gene for rice, were observed to change significantly under some treatment.
A new set of reference genes that are stable across tissue, development, stress and hormonal treatments have been identified in rice. This provides a superior set of reference genes for future studies in rice. It confirms the approach of mining large scale datasets as a robust method to define reference genes, but cautions against using gene orthology or counterparts of reference genes in other plant species as a means of defining reference genes.
Although the phytohormone auxin has been implicated primarily in developmental processes, some recent studies suggest its involvement in stress/defense responses as well. Recently, we identified auxin-responsive genes and reported their comprehensive transcript profiling during various stages of development and abiotic stress responses in crop plant rice. The analysis revealed tissue-specific and overlapping expression profiles of auxin-responsive genes during various stages of reproductive development. In addition, a large number of auxin-responsive genes were also found to be differentially expressed under various abiotic stress conditions. Here, we further analyze the expression profiles of auxin-responsive genes during various biotic stress conditions. Several auxin-responsive genes showed response to biotic stress as well. Our analysis provides evidence for role of auxin in plant defense responses and suggests cross-talk between auxin, abiotic stress and biotic stress signaling pathways.
auxin; auxin-responsive genes; biotic stress; rice (Oryza sativa); microarray
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.
Rice at reproductive stage is more sensitive to environmental changes, and little is known about the mechanism of heat response in rice panicle. Here, using rice microarray, we provided a time course gene expression profile of rice panicle at anther developmental stage 8 after 40°C treatment for 0 min, 20 min, 60 min, 2 h, 4 h, and 8 h. The identified differentially expressed genes were mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response. The predominant transcription factor gene families responsive to heat stress were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat stress. The motif co-occurrence analysis on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3 in response to heat stress. The regulation model central to ROS combined with transcriptome and ROS quantification data in rice panicle indicated the great importance to maintain ROS balance and the existence of wide cross-talk in heat response. The present study increased our understanding of the heat response in rice panicle and provided good candidate genes for crop improvement.
The phytohormone ethylene is a key signaling molecule that regulates a variety of developmental processes and stress responses in plants. Transcriptional modulation is a pivotal process controlling ethylene synthesis, which further triggers the expression of stress-related genes and plant adaptation to stresses; however, it is unclear how this process is transcriptionally modulated in rice. In the present research, we report the transcriptional regulation of a novel rice ethylene response factor (ERF) in ethylene synthesis and drought tolerance. Through analysis of transcriptional data, one of the drought-responsive ERF genes, OsDERF1, was identified for its activation in response to drought, ethylene and abscisic acid. Transgenic plants overexpressing OsDERF1 (OE) led to reduced tolerance to drought stress in rice at seedling stage, while knockdown of OsDERF1 (RI) expression conferred enhanced tolerance at seedling and tillering stages. This regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1 using the Affymetrix GeneChip, including the activation of cluster stress-related negative regulators such as ERF repressors. Biochemical and molecular approaches showed that OsDERF1 at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3 and OsAP2-39. Further investigations showed that OE seedlings had reduced expression (while RI lines showed enhanced expression) of ethylene synthesis genes, thereby resulting in changes in ethylene production. Moreover, overexpression of OsERF3/OsAP2-39 suppressed ethylene synthesis. In addition, application of ACC recovered the drought-sensitive phenotype in the lines overexpressing OsERF3, showing that ethylene production contributed to drought response in rice. Thus our data reveal that a novel ERF transcriptional cascade modulates drought response through controlling the ethylene synthesis, deepening our understanding of the regulation of ERF proteins in ethylene related drought 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.
Recently, we reported the genome-wide identification of 107 homeobox genes in rice and classified them into ten distinct subfamilies based upon their domain composition and phylogenetic analysis. Microarray analysis revealed the tissue-specific and overlapping expression profiles of these genes during various stages of floral transition, panicle development and seed set. Several homeobox genes were also found to be differentially expressed under abiotic stress conditions. Based on massively parallel signature sequencing (MPSS) data analysis, we report here that a large number of small RNA signatures are associated with rice homeobox genes, which may be involved in their tissue-specific/developmental regulation and stress responses. The association of a very large number of small RNA signatures suggested an unusually high degree of regulation of homeobox genes by small RNAs during inflorescence development.
homeobox genes; small RNA; microRNA; rice (Oryza sativa); developmental regulation; stress response
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.
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.
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.
The interaction between phytohormones is an important mechanism which controls growth and developmental processes in plants. Deciphering these interactions is a crucial step in helping to develop crops with enhanced yield and resistance to environmental stresses. Controlling the expression level of OsAP2-39 which includes an APETALA 2 (AP2) domain leads to phenotypic changes in rice. Overexpression of OsAP2-39 leads to a reduction in yield by decreasing the biomass and the number of seeds in the transgenic rice lines. Global transcriptome analysis of the OsAP2-39 overexpression transgenic rice revealed the upregulation of a key Abscisic Acid (ABA) biosynthetic gene OsNCED-I which codes for 9-cis-epoxycarotenoid dioxygenase and leads to an increase in the endogenous ABA level. In addition to OsNCED-1, the gene expression analysis revealed the upregulation of a gene that codes for the Elongation of Upper most Internode (EUI) protein, an enzyme that catalyzes 16α, 17-epoxidation of non-13-hydroxylated GAs, which has been shown to deactivate gibberellins (GAs) in rice. The exogenous application of GA restores the wild-type phenotype in the transgenic line and ABA application induces the expression of EUI and suppresses the expression of OsAP2-39 in the wild-type line. These observations clarify the antagonistic relationship between ABA and GA and illustrate a mechanism that leads to homeostasis of these hormones. In vivo and in vitro analysis showed that the expression of both OsNCED-1 and EUI are directly controlled by OsAP2-39. Together, these results reveal a novel mechanism for the control of the ABA/GA balance in rice which is regulated by OsAP2-39 that in turn regulates plant growth and seed production.
Hormones play an important role in controlling plant growth and development through a dynamic and complicated set of interactions. ABA and GA are well-known as antagonistic partners although the mechanism through which this occurs still needs further elucidation. In this project, we found that a transcription factor isolated from rice and coding for the AP2 domain (OsAP2-39) directly controls a key ABA biosynthetic gene (OsNCED-1) and also a gene that codes for a GA deactivation protein (EUI). In addition, we show that ABA induces the expression of EUI which in turn would lead to GA deactivation. ABA also suppresses OsAP2-39 expression which would lead to a reduction in ABA synthesis. Therefore, OsAP2-39 links the ABA production and GA deactivation processes which results in ABA/GA balance and homeostasis.
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.
Pollen development from the microspore involves a series of coordinated cellular events, and the resulting mature pollen has a specialized function to quickly germinate, produce a polar-growth pollen tube derived from the vegetative cell, and deliver two sperm cells into the embryo sac for double fertilization. The gene expression profiles of developing and germinated pollen have been characterised by use of the eudicot model plant Arabidopsis. Rice, one of the most important cereal crops, has been used as an excellent monocot model. A comprehensive analysis of transcriptome profiles of developing and germinated pollen in rice is important to understand the conserved and diverse mechanism underlying pollen development and germination in eudicots and monocots.
We used Affymetrix GeneChip® Rice Genome Array to comprehensively analyzed the dynamic changes in the transcriptomes of rice pollen at five sequential developmental stages from microspores to germinated pollen. Among the 51,279 transcripts on the array, we found 25,062 pollen-preferential transcripts, among which 2,203 were development stage-enriched. The diversity of transcripts decreased greatly from microspores to mature and germinated pollen, whereas the number of stage-enriched transcripts displayed a "U-type" change, with the lowest at the bicellular pollen stage; and a transition of overrepresented stage-enriched transcript groups associated with different functional categories, which indicates a shift in gene expression program at the bicellular pollen stage. About 54% of the now-annotated rice F-box protein genes were expressed preferentially in pollen. The transcriptome profile of germinated pollen was significantly and positively correlated with that of mature pollen. Analysis of expression profiles and coexpressed features of the pollen-preferential transcripts related to cell cycle, transcription, the ubiquitin/26S proteasome system, phytohormone signalling, the kinase system and defense/stress response revealed five expression patterns, which are compatible with changes in major cellular events during pollen development and germination. A comparison of pollen transcriptomes between rice and Arabidopsis revealed that 56.6% of the rice pollen preferential genes had homologs in Arabidopsis genome, but 63.4% of these homologs were expressed, with a small proportion being expressed preferentially, in Arabidopsis pollen. Rice and Arabidopsis pollen had non-conservative transcription factors each.
Our results demonstrated that rice pollen expressed a set of reduced but specific transcripts in comparison with vegetative tissues, and the number of stage-enriched transcripts displayed a "U-type" change during pollen development, with the lowest at the bicellular pollen stage. These features are conserved in rice and Arabidopsis. The shift in gene expression program at the bicellular pollen stage may be important to the transition from earlier cell division to later pollen maturity. Pollen at maturity pre-synthesized transcripts needed for germination and early pollen tube growth. The transcription regulation associated with pollen development would have divergence between the two species. Our results also provide novel insights into the molecular program and key components of the regulatory network regulating pollen development and germination.
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