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.
Jasmonates (JA) are well-known phytohormones which play important roles in plant development and defense against pathogens. Jasmonate ZIM domain (JAZ) proteins are plant-specific proteins and act as transcriptional repressors of JA-responsive genes. JA regulates both biotic and abiotic stress responses in plants; however, its role in nutrient deficiency responses is very elusive. Although, JA is well-known for root growth inhibition, little is known about behavior of JAZ genes in response to nutrient deficiencies, under which root architectural alteration is an important adaptation. Using protein sequence homology and a conserved-domains approach, here we identify 10 novel JAZ genes from the recently sequenced Chickpea genome, which is one of the most nutrient efficient crops. Both rice and chickpea JAZ genes express in tissue- and stimuli-specific manners. Many of which are preferentially expressed in root. Our analysis further showed differential expression of JAZ genes under macro (NPK) and micronutrients (Zn, Fe) deficiency in rice and chickpea roots. While both rice and chickpea JAZ genes showed a certain level of specificity toward type of nutrient deficiency, generally majority of them showed induction under K deficiency. Generally, JAZ genes showed an induction at early stages of stress and expression declined at later stages of macro-nutrient deficiency. Our results suggest that JAZ genes might play a role in early nutrient deficiency response both in monocot and dicot roots, and information generated here can be further used for understanding the possible roles of JA in root architectural alterations for nutrient deficiency adaptations.
jasmonates; nutrient deficiency; root; gene expression; jas degron; TIFY
Duplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. New genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization. Rice is a model for functional genomics research, an important crop for human nutrition and a target for biofortification. Increased zinc and iron content in the rice grain could be achieved by manipulation of metal transporters. Here, we describe the ZINC-INDUCED FACILITATOR-LIKE (ZIFL) gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication.
Sequences of sixty-eight ZIFL genes, from nine plant species, were comparatively analyzed. Although related to MSF_1 proteins, ZIFL protein sequences consistently grouped separately. Specific ZIFL sequence signatures were identified. Monocots harbor a larger number of ZIFL genes in their genomes than dicots, probably a result of a lineage-specific expansion. The rice ZIFL paralogs were named OsZIFL1 to OsZIFL13 and characterized. The genomic organization of the rice ZIFL genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar ZIFL gene pairs. Most rice ZIFL promoters are enriched for the core sequence of the Fe-deficiency-related box IDE1. Gene expression analyses of different plant organs, growth stages and treatments, both from our qPCR data and from microarray databases, revealed that the duplicated ZIFL gene pairs are mostly co-expressed. Transcripts of OsZIFL4, OsZIFL5, OsZIFL7, and OsZIFL12 accumulate in response to Zn-excess and Fe-deficiency in roots, two stresses with partially overlapping responses.
We suggest that ZIFL genes have different evolutionary histories in monocot and dicot lineages. In rice, concerted evolution affected ZIFL duplicated genes, possibly maintaining similar expression patterns between pairs. The enrichment for IDE1 boxes in rice ZIFL gene promoters suggests a role in Zn-excess and Fe-deficiency up-regulation of ZIFL transcripts. Moreover, this is the first description of the ZIFL gene family in plants and the basis for functional studies on this family, which may play important roles in Zn and Fe homeostasis in plants.
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
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.
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.
Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families.
Rice, a model monocot, is the first crop plant to have its entire genome sequenced. Although genome-wide transcriptome analysis tools and genome-wide, gene-indexed mutant collections have been generated for rice, the functions of only a handful of rice genes have been revealed thus far. Functional genomics approaches to studying crop plants like rice are much more labor-intensive and difficult in terms of maintaining the plants than when studying Arabidopsis, a model dicot. Here, we describe an efficient method for dissecting gene function in rice and other crop plants. We identified light response-related phenotypes for ten genes, the functions for which were previously unknown in rice. We also carried out co-expression analysis of 72 genes involved in specific biochemical pathways connected in lines carrying mutations in these ten genes. This analysis led to the identification of a novel set of genes likely involved in these pathways. The rapid progress of functional genomics in crops will significantly contribute to overcoming a food crisis in the near future.
Co-expression networks systematically constructed from large-scale transcriptome data reflect the interactions and functions of genes with similar expression patterns and are a powerful tool for the comprehensive understanding of biological events and mining of novel genes. In Arabidopsis (a model dicot plant), high-resolution co-expression networks have been constructed from very large microarray datasets and these are publicly available as online information resources. However, the available transcriptome data of rice (a model monocot plant) have been limited so far, making it difficult for rice researchers to achieve reliable co-expression analysis. In this study, we performed co-expression network analysis by using combined 44 K agilent microarray datasets of rice, which consisted of 33 laser microdissection (LM)-microarray datasets of anthers, and 143 spatiotemporal transcriptome datasets deposited in RicexPro. The entire data of the rice co-expression network, which was generated from the 176 microarray datasets by the Pearson correlation coefficient (PCC) method with the mutual rank (MR)-based cut-off, contained 24,258 genes and 60,441 genes pairs. Using these datasets, we constructed high-resolution co-expression subnetworks of two specific biological events in the anther, “meiosis” and “pollen wall synthesis”. The meiosis network contained many known or putative meiotic genes, including genes related to meiosis initiation and recombination. In the pollen wall synthesis network, several candidate genes involved in the sporopollenin biosynthesis pathway were efficiently identified. Hence, these two subnetworks are important demonstrations of the efficiency of co-expression network analysis in rice. Our co-expression analysis included the separated transcriptomes of pollen and tapetum cells in the anther, which are able to provide precise information on transcriptional regulation during male gametophyte development in rice. The co-expression network data presented here is a useful resource for rice researchers to elucidate important and complex biological events.
Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice.
We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors.
These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots.
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Rice; Arabidopsis; Plant hormone; Cytokinin; Transcriptomics
Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants.
Using a combination of oligonucleotide microarrays and data mining pipelines, we examined daily rhythms in gene expression in one monocotyledonous and one dicotyledonous plant, rice and poplar, respectively. Cycling transcriptomes were interrogated under different diurnal (driven) and circadian (free running) light and temperature conditions. Collectively, photocycles and thermocycles regulated about 60% of the expressed nuclear genes in rice and poplar. Depending on the condition tested, up to one third of oscillating Arabidopsis-poplar-rice orthologs were phased within three hours of each other suggesting a high degree of conservation in terms of rhythmic gene expression. We identified clusters of rhythmically co-expressed genes and searched their promoter sequences to identify phase-specific cis-elements, including elements that were conserved in the promoters of Arabidopsis, poplar, and rice.
Our results show that the cycling patterns of many circadian clock genes are highly conserved across poplar, rice, and Arabidopsis. The expression of many orthologous genes in key metabolic and regulatory pathways is diurnal and/or circadian regulated and phased to similar times of day. Our results confirm previous findings in Arabidopsis of three major classes of cis-regulatory modules within the plant circadian network: the morning (ME, GBOX), evening (EE, GATA), and midnight (PBX/TBX/SBX) modules. Identification of identical overrepresented motifs in the promoters of cycling genes from different species suggests that the core diurnal/circadian cis-regulatory network is deeply conserved between mono- and dicotyledonous species.
The response regulators represent the elements of bacterial two-component system and have been characterized from dicot plants like Arabidopsis but little information is available on the monocots, including the cereal crops. The aim of this study was to characterize type-A response regulator genes from rice, and to investigate their expression in various organs as well as in response to different hormones, including cytokinin, and environmental stimuli.
By analysis of the whole genome sequence of rice, we have identified ten genes encoding type-A response regulators based upon their high sequence identity within the receiver domain. The exon-intron organization, intron-phasing as well as chromosomal location of all the RT-PCR amplified rice (Oryza sativa) response regulator (OsRR) genes have been analyzed. The transcripts of OsRR genes could be detected by real-time PCR in all organs of the light- and dark-grown rice seedlings/plants, although there were quantitative differences. The steady-state transcript levels of most of the OsRR genes increased rapidly (within 15 min) on exogenous cytokinin application even in the presence of cycloheximide. Moreover, the expression of the OsRR6 gene was enhanced in rice seedlings exposed to salinity, dehydration and low temperature stress.
Ten type-A response regulator genes identified in rice, the model monocot plant, show overlapping/differential expression patterns in various organs and in response to light. The induction of OsRR genes by cytokinin even in the absence of de novo protein synthesis qualifies them to be primary cytokinin response genes. The induction of OsRR6 in response to different environmental stimuli indicates its role in cross-talk between abiotic stress and cytokinin signaling. These results provide a foundation for further investigations on specific as well as overlapping cellular functions of type-A response regulators in rice.
Virus infection induces and suppresses host gene expression on a global level. Rice stripe virus (RSV) is the type species of the genus Tenuivirus and infects rice and Arabidopsis plants. Microarray-based and next generation sequencing-based transcriptomic approaches have been used to study rice-RSV interactions. However, our knowledge of the response of Arabidopsis plants to RSV infection is limited, and it requires further investigation to determine the similarities (or differences) in virus-host interactions between monocot and dicot hosts infected with RSV.
We characterized transcriptome changes in Arabidopsis thaliana infected with rice stripe virus (RSV) with RNA-seq based digital gene expression (DGE) analysis. The transcriptomes of RSV-infected samples were compared to those of mock-treated samples at 14 and 21 days post-infection (dpi) during different stages of symptom development.
We identified 624 differentially expressed genes (DEGs) in Arabidopsis influenced by RSV at 14 dpi and 21 dpi, among which at 14 dpi, 255 transcripts were induced, and 38 were repressed; at 21 dpi, 146 were induced, and 237 were repressed. Functional annotation indicated that these DEGs were related to multiple biological functions, including defense response, secondary metabolism, protein amino acid phosphorylation and response to abiotic stress.
Importantly, the transcription of genes related to host defense systems was activated by RSV infection at an early stage of symptom development (14 dpi), whereas over the infection period (21 dpi), the host defense response systems were suppressed. A total of 52 genes were continuously differentially expressed between the two time points, indicating that the majority of DEGs were transient and unique to a particular time point during symptom development. The DEGs, particularly the defense response genes, identified in this study are candidates suitable for further functional analysis during the RSV-Arabidopsis interaction.
Electronic supplementary material
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Rice stripe virus; RNA-seq; Digital gene expression (DGE); Defense response
MADS-box transcription factors, besides being involved in floral organ specification, have also been implicated in several aspects of plant growth and development. In recent years, there have been reports on genomic localization, protein motif structure, phylogenetic relationships, gene structure and expression of the entire MADS-box family in the model plant system, Arabidopsis. Though there have been some studies in rice as well, an analysis of the complete MADS-box family along with a comprehensive expression profiling was still awaited after the completion of rice genome sequencing. Furthermore, owing to the role of MADS-box family in flower development, an analysis involving structure, expression and functional aspects of MADS-box genes in rice and Arabidopsis was required to understand the role of this gene family in reproductive development.
A genome-wide molecular characterization and microarray-based expression profiling of the genes encoding MADS-box transcription factor family in rice is presented. Using a thorough annotation exercise, 75 MADS-box genes have been identified in rice and categorized into MIKCc, MIKC*, Mα, Mβ and Mγ groups based on phylogeny. Chromosomal localization of these genes reveals that 16 MADS-box genes, mostly MIKCc-type, are located within the duplicated segments of the rice genome, whereas most of the M-type genes, 20 in all, seem to have resulted from tandem duplications. Nine members belonging to the Mβ group, which was considered absent in monocots, have also been identified. The expression profiles of all the MADS-box genes have been analyzed under 11 temporal stages of panicle and seed development, three abiotic stress conditions, along with three stages of vegetative development. Transcripts for 31 genes accumulate preferentially in the reproductive phase, of which, 12 genes are specifically expressed in seeds, and six genes show expression specific to panicle development. Differential expression of seven genes under stress conditions is also evident. An attempt has been made to gain insight into plausible functions of rice MADS-box genes by collating the expression data of functionally validated genes in rice and Arabidopsis.
Only a limited number of MADS genes have been functionally validated in rice. A comprehensive annotation and transcriptome profiling undertaken in this investigation adds to our understanding of the involvement of MADS-box family genes during reproductive development and stress in rice and also provides the basis for selection of candidate genes for functional validation studies.
The bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [32P]ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice.
Northern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em1a, the strongest ABA Responsive Element till reported from the upstream of EmBP1, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5'upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar.
The bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars.
The protein phosphatase 2Cs (PP2Cs) from various organisms have been implicated to act as negative modulators of protein kinase pathways involved in diverse environmental stress responses and developmental processes. A genome-wide overview of the PP2C gene family in plants is not yet available.
A comprehensive computational analysis identified 80 and 78 PP2C genes in Arabidopsis thaliana (AtPP2Cs) and Oryza sativa (OsPP2Cs), respectively, which denotes the PP2C gene family as one of the largest families identified in plants. Phylogenic analysis divided PP2Cs in Arabidopsis and rice into 13 and 11 subfamilies, respectively, which are supported by the analyses of gene structures and protein motifs. Comparative analysis between the PP2C genes in Arabidopsis and rice identified common and lineage-specific subfamilies and potential 'gene birth-and-death' events. Gene duplication analysis reveals that whole genome and chromosomal segment duplications mainly contributed to the expansion of both OsPP2Cs and AtPP2Cs, but tandem or local duplication occurred less frequently in Arabidopsis than rice. Some protein motifs are widespread among the PP2C proteins, whereas some other motifs are specific to only one or two subfamilies. Expression pattern analysis suggests that 1) most PP2C genes play functional roles in multiple tissues in both species, 2) the induced expression of most genes in subfamily A by diverse stimuli indicates their primary role in stress tolerance, especially ABA response, and 3) the expression pattern of subfamily D members suggests that they may constitute positive regulators in ABA-mediated signaling pathways. The analyses of putative upstream regulatory elements by two approaches further support the functions of subfamily A in ABA signaling, and provide insights into the shared and different transcriptional regulation machineries in dicots and monocots.
This comparative genome-wide overview of the PP2C family in Arabidopsis and rice provides insights into the functions and regulatory mechanisms, as well as the evolution and divergence of the PP2C genes in dicots and monocots. Bioinformatics analyses suggest that plant PP2C proteins from different subfamilies participate in distinct signaling pathways. Our results have established a solid foundation for future studies on the functional divergence in different PP2C subfamilies.
WRKY III genes have significant functions in regulating plant development and resistance. In plant, WRKY gene family has been studied in many species, however, there still lack a comprehensive analysis of WRKY III genes in the woody plant species poplar, three representative lineages of flowering plant species are incorporated in most analyses: Arabidopsis (a model plant for annual herbaceous dicots), grape (one model plant for perennial dicots) and Oryza sativa (a model plant for monocots).
In this study, we identified 10, 6, 13 and 28 WRKY III genes in the genomes of Populus trichocarpa, grape (Vitis vinifera), Arabidopsis thaliana and rice (Oryza sativa), respectively. Phylogenetic analysis revealed that the WRKY III proteins could be divided into four clades. By microsynteny analysis, we found that the duplicated regions were more conserved between poplar and grape than Arabidopsis or rice. We dated their duplications by Ks analysis of Populus WRKY III genes and demonstrated that all the blocks were formed after the divergence of monocots and dicots. Strong purifying selection has played a key role in the maintenance of WRKY III genes in Populus. Tissue expression analysis of the WRKY III genes in Populus revealed that five were most highly expressed in the xylem. We also performed quantitative real-time reverse transcription PCR analysis of WRKY III genes in Populus treated with salicylic acid, abscisic acid and polyethylene glycol to explore their stress-related expression patterns.
This study highlighted the duplication and diversification of the WRKY III gene family in Populus and provided a comprehensive analysis of this gene family in the Populus genome. Our results indicated that the majority of WRKY III genes of Populus was expanded by large-scale gene duplication. The expression pattern of PtrWRKYIII gene identified that these genes play important roles in the xylem during poplar growth and development, and may play crucial role in defense to drought stress. Our results presented here may aid in the selection of appropriate candidate genes for further characterization of their biological functions in poplar.
This article was reviewed by Prof Dandekar and Dr Andrade-Navarro.
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WRKY III; Microsynteny; Gene duplication; Expression; Populus
Alternative splicing (AS) contributes significantly to protein diversity, by selectively using different combinations of exons of the same gene under certain circumstances. One particular type of AS is the use of alternative first exons (AFEs), which can have consequences far beyond the fine-tuning of protein functions. For example, AFEs may change the N-termini of proteins and thereby direct them to different cellular compartments. When alternative first exons are distant, they are usually associated with alternative promoters, thereby conferring an extra level of gene expression regulation. However, only few studies have examined the patterns of AFEs, and these analyses were mainly focused on mammalian genomes. Recent studies have shown that AFEs exist in the rice genome, and are regulated in a tissue-specific manner. Our current understanding of AFEs in plants is still limited, including important issues such as their regulation, contribution to protein diversity, and evolutionary conservation.
We systematically identified 1,378 and 645 AFE-containing clusters in rice and Arabidopsis, respectively. From our data sets, we identified two types of AFEs according to their genomic organisation. In genes with type I AFEs, the first exons are mutually exclusive, while most of the downstream exons are shared among alternative transcripts. Conversely, in genes with type II AFEs, the first exon of one gene structure is an internal exon of an alternative gene structure. The functionality analysis indicated about half and ~19% of the AFEs in Arabidopsis and rice could alter N-terminal protein sequences, and ~5% of the functional alteration in type II AFEs involved protein domain addition/deletion in both genomes. Expression analysis indicated that 20~66% of rice AFE clusters were tissue- and/or development- specifically transcribed, which is consistent with previous observations; however, a much smaller percentage of Arabidopsis AFEs was regulated in this manner, which suggests different regulation mechanisms of AFEs between rice and Arabidopsis. Statistical analysis of some features of AFE clusters, such as splice-site strength and secondary structure formation further revealed differences between these two species. Orthologous search of AFE-containing gene pairs detected only 19 gene pairs conserved between rice and Arabidopsis, accounting only for a few percent of AFE-containing clusters.
Our analysis of AFE-containing genes in rice and Arabidopsis indicates that AFEs have multiple functions, from regulating gene expression to generating protein diversity. Comparisons of AFE clusters revealed different features in the two plant species, which indicates that AFEs may have evolved independently after the separation of rice (a model monocot) and Arabidopsis (a model dicot).
Heat shock proteins (Hsps) constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD) at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes.
We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed.
We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed that these genes are differentially expressed under stress and at different stages in the life cycle of rice plant.
Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium.
We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice.
We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0749-9) contains supplementary material, which is available to authorized users.
Brachypodium distachyon; Phytohormone; Salicylic acid; Jasmonic acid; Ethylene; Plant disease resistance; Defense mechanism; Immunity system; Marker gene
Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress.
Phosphate is an important nutrient for all living organisms. This chemical group forms part of the backbone of DNA molecules, and has a crucial role in many chemical reactions that occur inside cells. Plants in particular need a source of phosphate to grow. This is why agricultural fertilizers are rich in phosphate, but unfortunately, the use of fertilizers is not sustainable. Many researchers are now looking for new ways to maintain high crop yields without chemical fertilizers, and understanding how crops are affected in times of shortage will be pivotal to achieving this goal.
DNA contains coded information in the form of genes, which can either be switched on or off. Chemical marks added to the DNA can earmark genes for activation or inactivation, a bit like handwritten annotations in an instruction manual. One example is the addition of a chemical tag called a methyl group to one of the letters of the DNA code—so-called ‘cytosine methylation’. However, little is known about how the pattern of these chemical marks on DNA changes in response to changes in the environment.
Secco et al. investigated changes in cytosine methylation in both rice and Arabidopsis plants that had been deprived of phosphate. Arabidopsis, or thale cress, is a model plant that is often studied by plant biologists because it is small and grows quickly. The experiments showed that when rice plants were not given enough phosphate, the pattern of DNA methylation changed. This was particularly true around certain genes that help the plants survive in these difficult conditions. Notably, in the absence of phosphate, methylation also occurs more often in DNA sequences called transposable elements that sit close to these useful genes, and less often around other genes.
Transposable elements, also known as ‘jumping genes’ can move within the genome and thus potentially have damaging effects through altering the DNA sequence. However, DNA methylation normally prevents this from happening. Therefore, the extra methylation observed by Secco et al. may be a cautionary measure to inactivate these transposable elements and limit their potential deleterious effects. Further experiments went on to show that these useful genes seem to be switched on before the DNA of these transposable elements is methylated, implying that the extra methylation observed in these transposable elements is a consequence of the activation of these nearby useful genes.
By contrast, similar experiments performed in Arabidopsis reveal a very limited change in DNA methylation when the plants are grown under stressful conditions. This might be because Arabidopsis has considerably fewer transposable elements than rice. The next challenge will be to explore how significant the environmentally induced silencing of transposable elements is to the stress responses and genome integrity of crop plants.
epigenome; DNA methylation; nutrient stress; transcriptome; phosphate starvation; rice; Arabidopsis; other
Phosphorus (P), an essential macronutrient, is often limiting in soils and affects plant growth and development. In Arabidopsis thaliana, Low Phosphate Root1 (LPR1) and its close paralog LPR2 encode multicopper oxidases (MCOs). They regulate meristem responses of root system to phosphate (Pi) deficiency. However, the roles of LPR gene family in rice (Oryza sativa) in maintaining Pi homeostasis have not been elucidated as yet.
Here, the identification and expression analysis for the homologs of LPR1/2 in rice were carried out. Five homologs, hereafter referred to as OsLPR1-5, were identified in rice, which are distributed on chromosome1 over a range of 65 kb. Phylogenetic analysis grouped OsLPR1/3/4/5 and OsLPR2 into two distinct sub-clades with OsLPR3 and 5 showing close proximity. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed higher expression levels of OsLPR3-5 and OsLPR2 in root and shoot, respectively. Deficiencies of different nutrients ie, P, nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) exerted differential and partially overlapping effects on the relative expression levels of the members of OsLPR family. Pi deficiency (−P) triggered significant increases in the relative expression levels of OsLPR3 and 5. Strong induction in the relative expression levels of OsLPR3 and 5 in osphr2 suggested their negative transcriptional regulation by OsPHR2. Further, the expression levels of OsLPR3 and 5 were either attenuated in ossiz1 and ospho2 or augmented in rice overexpressing OsSPX1.
The results from this study provided insights into the evolutionary expansion and a likely functional divergence of OsLPR family with potential roles of OsLPR3 and 5 in the maintenance of Pi homeostasis in rice.
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The online version of this article (doi:10.1186/s12870-016-0853-x) contains supplementary material, which is available to authorized users.
Rice; Phosphate deficiency; OsLPR family; OsLPR3; OsLPR5; Phosphate homeostasis
Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.
The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. In Arabidopsis thaliana, the transcriptional regulator DNA polymerase II subunit B3‐1 (DPB3‐1)/nuclear factor Y subunit C10 (NF‐YC10) has been reported as a positive regulator of Dehydration‐responsive element binding protein 2A (DREB2A), and the overexpression of DPB3‐1 enhances heat stress tolerance without growth retardation. Here, we show that DPB3‐1 interacts with DREB2A homologues in rice and soya bean. Transactivation analyses with Arabidopsis and rice mesophyll protoplasts indicate that DPB3‐1 and its rice homologue OsDPB3‐2 function as positive regulators of DREB2A homologues. Overexpression of DPB3‐1 did not affect plant growth or yield in rice under nonstress conditions. Moreover, DPB3‐1‐overexpressing rice showed enhanced heat stress tolerance. Microarray analysis revealed that many heat stress‐inducible genes were up‐regulated in DPB3‐1‐overexpressing rice under heat stress conditions. However, the overexpression of DPB3‐1 using a constitutive promoter had almost no effect on the expression of these genes under nonstress conditions. This may be because DPB3‐1 is a coactivator and thus lacks inherent transcriptional activity. We conclude that DPB3‐1, a coactivator that functions specifically under abiotic stress conditions, could be utilized to increase heat stress tolerance in crops without negative effects on vegetative and reproductive growth.
DNA polymerase II subunit B3‐1/nuclear factor Y subunit C10; heat stress tolerance; Oryza sativa; transcriptional regulation; dehydration‐responsive element binding protein 2; microarray analysis
In plants, complex regulatory mechanisms are at the core of physiological and developmental processes. The phytohormone abscisic acid (ABA) is involved in the regulation of various such processes, including stomatal closure, seed and bud dormancy, and physiological responses to cold, drought and salinity stress. The underlying tissue or plant-wide control circuits often include combinatorial gene regulatory mechanisms and networks that we are only beginning to unravel with the help of new molecular tools. The increasing availability of genomic sequences and gene expression data enables us to dissect ABA regulatory mechanisms at the individual gene expression level. In this paper we used an in-silico-based approach directed towards genome-wide prediction and identification of specific features of ABA-responsive elements. In particular we analysed the genome-wide occurrence and positional arrangements of two well-described ABA-responsive cis-regulatory elements (CREs), ABRE and CE3, in thale cress (Arabidopsis thaliana) and rice (Oryza sativa).
Our results show that Arabidopsis and rice use the ABA-responsive elements ABRE and CE3 distinctively. Earlier reports for various monocots have identified CE3 as a coupling element (CE) associated with ABRE. Surprisingly, we found that while ABRE is equally abundant in both species, CE3 is practically absent in Arabidopsis. ABRE-ABRE pairs are common in both genomes, suggesting that these can form functional ABA-responsive complexes (ABRCs) in Arabidopsis and rice. Furthermore, we detected distinct combinations, orientation patterns and DNA strand preferences of ABRE and CE3 motifs in rice gene promoters.
Our computational analyses revealed distinct recruitment patterns of ABA-responsive CREs in upstream sequences of Arabidopsis and rice. The apparent absence of CE3s in Arabidopsis suggests that another CE pairs with ABRE to establish a functional ABRC capable of interacting with transcription factors. Further studies will be needed to test whether the observed differences are extrapolatable to monocots and dicots in general, and to understand how they contribute to the fine-tuning of the hormonal response. The outcome of our investigation can now be used to direct future experimentation designed to further dissect the ABA-dependent regulatory networks.
The development of rice (Oryza sativa) seed is closely associated with assimilates storage and plant yield, and is fine controlled by complex regulatory networks. Exhaustive transcriptome analysis of developing rice embryo and endosperm will help to characterize the genes possibly involved in the regulation of seed development and provide clues of yield and quality improvement.
Our analysis showed that genes involved in metabolism regulation, hormone response and cellular organization processes are predominantly expressed during rice development. Interestingly, 191 transcription factor (TF)-encoding genes are predominantly expressed in seed and 59 TFs are regulated during seed development, some of which are homologs of seed-specific TFs or regulators of Arabidopsis seed development. Gene co-expression network analysis showed these TFs associated with multiple cellular and metabolism pathways, indicating a complex regulation of rice seed development. Further, by employing a cold-resistant cultivar Hanfeng (HF), genome-wide analyses of seed transcriptome at normal and low temperature reveal that rice seed is sensitive to low temperature at early stage and many genes associated with seed development are down-regulated by low temperature, indicating that the delayed development of rice seed by low temperature is mainly caused by the inhibition of the development-related genes. The transcriptional response of seed and seedling to low temperature is different, and the differential expressions of genes in signaling and metabolism pathways may contribute to the chilling tolerance of HF during seed development.
These results provide informative clues and will significantly improve the understanding of rice seed development regulation and the mechanism of cold response in rice seed.