[Show abstract][Hide abstract] ABSTRACT: Macronutrients are pivotal elements for proper plant growth and development. Although extensive gene expression profiling revealed a large number of genes differentially expressed under various nutrient deprivation, characterization of these genes has never been fully explored especially in rice. Coexpression network analysis is a useful tool to elucidate the functional relationships of genes based on common expression. Therefore, we performed microarray analysis of rice shoot under nitrogen (N), phosphorus (P), and potassium (K) deficiency conditions. Moreover, we conducted a large scale coexpression analysis by integrating the data with previously generated gene expression profiles of organs and tissues at different developmental stages to obtain a global view of gene networks associated with plant response to nutrient deficiency.
We statistically identified 5400 differentially expressed genes under the nutrient deficiency treatments. Subsequent coexpression analysis resulted in the extraction of 6 modules (groups of highly interconnected genes) with distinct gene expression signatures. Three of these modules comprise mostly of downregulated genes under N deficiency associated with distinct functions such as development of immature organs, protein biosynthesis and photosynthesis in chloroplast of green tissues, and fundamental cellular processes in all organs and tissues. Furthermore, we identified one module containing upregulated genes under N and K deficiency conditions, and a number of genes encoding protein kinase, kinase-like domain containing protein and nutrient transporters. This module might be particularly involved in adaptation to nutrient deficiency via phosphorylation-mediated signal transduction and/or post-transcriptional regulation.
Our study demonstrated that large scale coexpression analysis is an efficient approach in characterizing the nutrient response genes based on biological functions and could provide new insights in understanding plant response to nutrient deficiency.
[Show abstract][Hide abstract] ABSTRACT: Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.
[Show abstract][Hide abstract] ABSTRACT: Gene targeting (GT) is a technique used to modify endogenous genes in target genomes precisely via homologous recombination
(HR). Although GT plants are produced using genetic transformation techniques, if the difference between the endogenous and
the modified gene is limited to point mutations, GT crops can be considered equivalent to non-genetically modified mutant
crops generated by conventional mutagenesis techniques. However, it is difficult to guarantee the non-incorporation of DNA
fragments from Agrobacterium in GT plants created by Agrobacterium-mediated GT despite screening with conventional Southern blot and/or PCR techniques. Here, we report a comprehensive analysis
of herbicide-tolerant rice plants generated by inducing point mutations in the rice ALS gene via Agrobacterium-mediated GT. We performed genome comparative genomic hybridization (CGH) array analysis and whole-genome sequencing to evaluate
the molecular composition of GT rice plants. Thus far, no integration of Agrobacterium-derived DNA fragments has been detected in GT rice plants. However, >1,000 single nucleotide polymorphisms (SNPs) and insertion/deletion
(InDels) were found in GT plants. Among these mutations, 20–100 variants might have some effect on expression levels and/or
protein function. Information about additive mutations should be useful in clearing out unwanted mutations by backcrossing.
[Show abstract][Hide abstract] ABSTRACT: Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long-distance
oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic
soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin deposited in cell walls are thought
to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression
profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses
for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total
of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin
biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcription factor genes containing WRKY, AP2,
and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a
major constituent of the ROL barrier in roots of rice.
Journal of Experimental Botany 06/2014; DOI:10.1093/jxb/eru235 · 5.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Strigolactones (SLs) are a group of phytohormones that control plant growth and development including shoot branching. Previous
studies of the phenotypes of SL-related rice (Oryza sativa) dwarf (d) mutants demonstrated that SLs inhibit mesocotyl elongation by controlling cell division. Here, we found that the expression
of cytokinin (CK)-responsive type-A RESPONSE REGULATOR (RR) genes was higher in d10-1 and d14-1 mutants than in the wild type. However, CK levels in mesocotyls of the d mutants were not very different from those in the wild type. On the other hand, application of a synthetic CK (kinetin) enhanced
mesocotyl elongation in the d mutants and the wild type. d10-1 and d14-1 mesocotyls were more sensitive to CK than wild-type mesocotyls, suggesting that the up-regulation of the CK-responsive type-A
RR genes and the higher elongation of mesocotyls in the d mutants are mainly due to the increased sensitivity of the d mutants to CK. Co-treatment with kinetin and a synthetic SL (GR24) confirmed the antagonistic functions of SL and CK on mesocotyl
elongation. OsTCP5, which encodes a transcription factor belonging to the cell division-regulating TCP family, was also regulated by SL and
CK and its expression was negatively correlated with mesocotyl length. These findings suggest that OsTCP5 contributes to the
SL- and CK-controlled mesocotyl elongation in darkness.
[Show abstract][Hide abstract] ABSTRACT: The plant secondary cell wall is the major source of lignocellulosic biomass, a renewable energy resource which can be used for bioethanol production. To comprehensively identify transcription factors (TFs), glycosyltransferase (GT), and glycosyl hydrolase (GH) involved in secondary cell wall formation in rice (Oryza sativa), co-expression network analysis was performed using 68 microarray data points for different rice tissues and stages. In addition to rice genes encoding orthologs of Arabidopsis thaliana TFs known to regulate secondary cell wall formation, the network analysis suggested many novel TF genes likely to be involved in cell wall formation. In the accompanying paper (Hirano et al.), several of these TFs are shown to be involved in rice secondary cell wall formation.Based on a comparison of the rice and Arabidopsis networks, TFs were classified as common to both species or specific to each plant species, suggesting that in addition to a common transcriptional regulatory mechanism of cell wall formation, the two plants may also use species-specific groups of TFs during secondary wall formation. Similarly, genes encoding GT and GH were also classified as genes showing species-common or species-specific expression patterns. In addition, genes for primary or secondary cell wall formation were also suggested.The list of rice TF, GT, and GH genes provides an opportunity to unveil the regulation of secondary cell wall formation in grasses, leading to optimization of the cell wall for biofuel production.
[Show abstract][Hide abstract] ABSTRACT: Through co-expression network analysis, we identified 123 rice transcription factors (TFs) as candidate rice secondary cell wall regulators (Hirano et al., the accompanying paper). To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to either the MYB, NAC or homeodomain containing TF families were over-expressed or down-regulated in rice. With the exception of OsMYB58/63-RNAi plants, all transgenic plants showed phenotypes possibly related to secondary cell wall alteration, such as dwarfism, narrow and dark green leaves, and also altered rice cinnamyl alcohol dehydrogenase 2 (OsCAD2) gene expression and lignin content. These results suggest that many of the 123 candidate secondary cell wall regulating TFs are likely to function in rice secondary cell wall formation.Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, one representing TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and one for which no previous involvement in cell wall formation has been reported even in Arabidopsis (BLH6). OsMYB55/61 and OsBLH6-GFP fusion proteins localized to the nucleus of onion epidermal cells. Moreover, expression of a reporter gene driven by the OsCAD2 promoter was enhanced in rice calli when OsMYB55/61 or OsBLH6 were transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential rice secondary cell wall TFs through the rice co-expression network analysis.
[Show abstract][Hide abstract] ABSTRACT: The genetic improvement of drought resistance is essential for stable and adequate crop production in drought-prone areas. Here we demonstrate that alteration of root system architecture improves drought avoidance through the cloning and characterization of DEEPER ROOTING 1 (DRO1), a rice quantitative trait locus controlling root growth angle. DRO1 is negatively regulated by auxin and is involved in cell elongation in the root tip that causes asymmetric root growth and downward bending of the root in response to gravity. Higher expression of DRO1 increases the root growth angle, whereby roots grow in a more downward direction. Introducing DRO1 into a shallow-rooting rice cultivar by backcrossing enabled the resulting line to avoid drought by increasing deep rooting, which maintained high yield performance under drought conditions relative to the recipient cultivar. Our experiments suggest that control of root system architecture will contribute to drought avoidance in crops.
[Show abstract][Hide abstract] ABSTRACT: Deficiency of the three essential macronutrients, nitrogen, phosphorus and potassium, leads to large reduction in crop growth and yield. To characterize the molecular genetic basis of adaptation to macronutrient deprivation, we performed microarray analysis of rice root at 6 and 24 h after nitrogen, phosphorus and potassium deficiency treatments. The transcriptome response to nitrogen depletion occurred more rapidly than corresponding responses to phosphorus and potassium deprivation. We identified several genes important for response and adaptation to each nutrient deficiency. Furthermore, we found that signaling via reactive oxygen species is a common feature in response to macronutrient deficiency and signaling via jasmonic acid is associated with potassium depletion response. These results will facilitate deeper understanding of nutrient utilization of plants.
[Show abstract][Hide abstract] ABSTRACT: Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs) where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, β-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis Rab5 isoforms to the GTP-bound form. Therefore, loss of function mutations in GEF or Rab5 disrupt normal transport of proglutelin from the Golgi to PSV, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP and GDP bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSV and in the maintenance of the general structural organization of the endomembrane system in rice seeds.
[Show abstract][Hide abstract] ABSTRACT: Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.
Proceedings of the National Academy of Sciences 12/2012; 110(2). DOI:10.1073/pnas.1216151110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Determining the drivers of gene expression patterns is more straightforward in laboratory conditions than in the complex fluctuating environments where organisms typically live. We gathered transcriptome data from the leaves of rice plants in a paddy field along with the corresponding meteorological data and used them to develop statistical models for the endogenous and external influences on gene expression. Our results indicate that the transcriptome dynamics are predominantly governed by endogenous diurnal rhythms, ambient temperature, plant age, and solar radiation. The data revealed diurnal gates for environmental stimuli to influence transcription and pointed to relative influences exerted by circadian and environmental factors on different metabolic genes. The model also generated predictions for the influence of changing temperatures on transcriptome dynamics. We anticipate that our models will help translate the knowledge amassed in laboratories to problems in agriculture and that our approach to deciphering the transcriptome fluctuations in complex environments will be applicable to other organisms.
[Show abstract][Hide abstract] ABSTRACT: Similarity of gene expression across a wide range of biological conditions can be efficiently used in characterization of gene function. We have constructed a rice gene coexpression database, RiceFREND (http://ricefrend.dna.affrc.go.jp/), to identify gene modules with similar expression profiles and provide a platform for more accurate prediction of gene functions. Coexpression analysis of 27 201 genes was performed against 815 microarray data derived from expression profiling of various organs and tissues at different developmental stages, mature organs throughout the growth from transplanting until harvesting in the field and plant hormone treatment conditions, using a single microarray platform. The database is provided with two search options, namely, 'single guide gene search' and 'multiple guide gene search' to efficiently retrieve information on coexpressed genes. A user-friendly web interface facilitates visualization and interpretation of gene coexpression networks in HyperTree, Cytoscape Web and Graphviz formats. In addition, analysis tools for identification of enriched Gene Ontology terms and cis-elements provide clue for better prediction of biological functions associated with the coexpressed genes. These features allow users to clarify gene functions and gene regulatory networks that could lead to a more thorough understanding of many complex agronomic traits.
[Show abstract][Hide abstract] ABSTRACT: A wide range of resources on gene expression profiling enhance various strategies in plant molecular biology particularly in characterization of gene function. We have updated our gene expression profile database, RiceXPro (http://ricexpro.dna.affrc.go.jp/), to provide more comprehensive information on the transcriptome of rice encompassing the entire growth cycle and various experimental conditions. The gene expression profiles are currently grouped into three categories, namely, 'field/development' with 572 data corresponding to 12 data sets, 'plant hormone' with 143 data corresponding to 13 data sets and 'cell- and tissue-type' comprising of 38 microarray data. In addition to the interface for retrieving expression information of a gene/genes in each data set, we have incorporated an interface for a global approach in searching an overall view of the gene expression profiles from multiple data sets within each category. Furthermore, we have also added a BLAST search function that enables users to explore expression profile of a gene/genes with similarity to a query sequence. Therefore, the updated version of RiceXPro can be used more efficiently to survey the gene expression signature of rice in sufficient depth and may also provide clues on gene function of other cereal crops.
[Show abstract][Hide abstract] ABSTRACT: Rice (Oryza sativa) is the most aluminum (Al)-tolerant crop among small-grain cereals, but the mechanism underlying its high Al resistance is still not well understood. To understand the mechanisms underlying high Al-tolerance, we performed a comparative genome-wide transcriptional analysis by comparing expression profiling between the Al-tolerance cultivar (Koshihikari) and an Al-sensitive mutant star1 (SENSITIVE TO AL RHIZOTOXICITY 1) in both the root tips and the basal roots. Exposure to 20 µM AlCl(3) for 6 h resulted in up-regulation (higher than 3-fold) of 213 and 2015 genes including 185 common genes in the root tips of wild-type and the mutant, respectively. On the other hand, in the basal root, genes up-regulated by Al were 126 and 2419 including 76 common genes in the wild-type and the mutant, respectively. These results indicate that Al-response genes are not only restricted to the root tips, but also in the basal root region. Analysis with genes up- or down-regulated only in the wild-type reveals that there are other mechanisms for Al-tolerance except for a known transcription factor ART1-regulated one in rice. These mechanisms are related to nitrogen assimilation, secondary metabolite synthesis, cell-wall synthesis and ethylene synthesis. Although the exact roles of these putative tolerance genes remain to be examined, our data provide a platform for further work on Al-tolerance in rice.
PLoS ONE 10/2012; 7(10):e48197. DOI:10.1371/journal.pone.0048197 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Magnesium (Mg)-mediated alleviation of aluminum (Al) toxicity has been observed in a number of plant species, but the mechanisms underlying the alleviation are still poorly understood. When a putative rice (Oryza sativa) Mg transporter gene, Oryza sativa MAGNESIUM TRANSPORTER1 (OsMGT1), was knocked out, the tolerance to Al, but not to cadmium and lanthanum, was decreased. However, this inhibition could be rescued by addition of 10 μm Mg, but not by the same concentration of barium or strontium. OsMGT1 was expressed in both the roots and shoots in the absence of Al, but the expression only in the roots was rapidly up-regulated by Al. Furthermore, the expression did not respond to low pH and other metals including cadmium and lanthanum, and was regulated by an Al-responsive transcription factor, AL RESISTANCE TRANSCRIPTION FACTOR1. An investigation of subcellular localization showed that OsMGT1 was localized to the plasma membrane. A short-term (30 min) uptake experiment with stable isotope (25)Mg showed that knockout of OsMGT1 resulted in decreased Mg uptake, but that the uptake in the wild type was enhanced by Al. Mg concentration in the cell sap of the root tips was also increased in the wild-type rice, but not in the knockout lines in the presence of Al. A microarray analysis showed that transcripts of genes related to stress were more up- and down-regulated in the knockout lines. Taken together, our results indicate that OsMGT1 is a transporter for Mg uptake in the roots and that up-regulation of this gene is required for conferring Al tolerance in rice by increasing Mg concentration in the cell.