A Cold-Shock Protein from the South Pole-Dwelling Soil Bacterium Arthrobacter sp. Confers Cold Tolerance to Rice.

Low temperature is a critical environmental factor restricting the physiology of organisms across kingdoms. In prokaryotes, cold shock induces the expression of various genes and proteins involved in cellular processes. Here, a cold-shock protein (ArCspA) from the South Pole-dwelling soil bacterium Arthrobacter sp. A2-5 was introduced into rice, a monocot model plant species. Four-week-old 35S:ArCspA transgenic rice plants grown in a cold chamber at 4 °C survived for 6 days. Cold stress significantly decreased the chlorophyll content in WT plants after 4 days compared with that in 35S:ArCspA transgenic plants. RNA-seq analysis was performed on WT and 35S:ArCspA transgenic rice with/without cold stress. GO terms such as "response to stress (GO:0006950)", "response to cold (GO:0009409)", and "response to heat (GO:0009408)" were significantly enriched among the upregulated genes in the 35S:ArCspA transgenic rice under normal conditions, even without cold-stress treatment. The expression of five cold stress-related genes, Rab16B (Os11g0454200), Rab21 (Os11g0454300), LEA22 (Os01g0702500), ABI5 (Os01 g0859300), and MAPK5 (Os03g0285800), was significantly upregulated in the transgenic rice compared with the WT rice. These results indicate that the ArCspA gene might be involved in the induction of cold-responsive genes and provide cold tolerance.

Rice protein-binding microarrays: a tool to detect cis-acting elements near promoter regions in rice.

MAIN CONCLUSION: The present study showed that a rice (Oryza sativa)-specific protein-binding microarray (RPBM) can be applied to analyze DNA-binding motifs with a TF where binding is evaluated in extended natural promoter regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements. Transcription factors (TFs) regulate gene expression at the transcriptional level by binding a specific DNA sequence. Thus, predicting the DNA-binding motifs of TFs is one of the most important areas in the functional analysis of TFs in the postgenomic era. Although many methods have been developed to address this challenge, many TFs still have unknown DNA-binding motifs. In this study, we designed RPBM with 40-bp probes and 20-bp of overlap, yielding 49 probes spanning the 1-kb upstream region before the translation start site of each gene in the entire genome. To confirm the efficiency of RPBM technology, we selected two previously studied TFs, OsWOX13 and OsSMF1, and an uncharacterized TF, OsWRKY34. We identified the ATTGATTG and CCACGTCA DNA-binding sequences of OsWOX13 and OsSMF1, respectively. In total, 635 and 932 putative feature genes were identified for OsWOX13 and OsSMF1, respectively. We discovered the CGTTGACTTT DNA-binding sequence and 195 putative feature genes of OsWRKY34. RPBM could be applicable in the analysis of DNA-binding motifs for TFs where binding is evaluated in the promoter and 5' upstream CDS regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements.

Recurrent Drought Conditions Enhance the Induction of Drought Stress Memory Genes in Glycine max L.

Plants remember what they have experienced and are thereby able to confront repeated stresses more promptly and strongly. A subset of the drought responsive genes, called stress memory genes, displayed greatly elevated levels under recurrent drought conditions. To screen for a set of drought stress memory genes in soybean (Glycine max L.), we designed a 180K DNA chip comprising 60-bp probes synthesized in situ to examine 55,589 loci. Through microarray analysis using the DNA chip, we identified 2,162 and 2,385 genes with more than fourfold increases or decreases in transcript levels, respectively, under initial (first) drought stress conditions, when compared with the non-treated control. The transcript levels of the drought-responsive genes returned to basal levels during recovery (watered) states, and 392 and 613 genes displayed more than fourfold elevated or reduced levels, respectively, under subsequent (second) drought conditions, when compared to those observed under the first drought stress conditions. Gene Ontology and MapMan analyses classified the drought-induced memory genes exhibiting elevated levels of transcripts into several functional categories, including those involved in tolerance responses to abiotic stresses, which encode transcription factors, protein phosphatase 2Cs, and late embryogenesis abundant proteins. The drought-repressed memory genes exhibiting reduced levels of transcripts were classified into categories including photosynthesis and primary metabolism. Co-expression network analysis revealed that the soybean drought-induced and -repressed memory genes were equivalent to 172 and 311 Arabidopsis genes, respectively. The soybean drought stress memory genes include genes involved in the dehydration memory responses of Arabidopsis.

Silencing of class I small heat shock proteins affects seed-related attributes and thermotolerance in rice seedlings.

MAIN CONCLUSION: Silencing of CI-sHsps by RNAi negatively affected the seed germination process and heat stress response of rice seedlings. Seed size of RNAiCI-sHsp was reduced as compared to wild-type plants. Small heat shock proteins (sHsps) are the ATP-independent chaperones ubiquitously expressed in response to diverse environmental and developmental cues. Cytosolic sHsps constitute the major repertoire of sHsp family. Rice cytosolic class I (CI)-sHsps consists of seven members (Hsp16.9A, Hsp16.9B, Hsp16.9C, Hsp17.4, Hsp17.7, Hsp17.9A and Hsp18). Purified OsHsp17.4 and OsHsp17.9A proteins exhibited chaperone activity by preventing formation of large aggregates with model substrate citrate synthase. OsHsp16.9A and OsHsp17.4 showed nucleo-cytoplasmic localization, while the localization of OsHsp17.9A was preferentially in the nucleus. Transgenic tobacco plants expressing OsHsp17.4 and OsHsp17.9A proteins and Arabidopsis plants ectopically expressing OsHsp17.4 protein showed improved thermotolerance to the respective trans-hosts during the post-stress recovery process. Single hairpin construct was designed to generate all CI-sHsp silenced (RNAiCI-sHsp) rice lines. The major vegetative and reproductive attributes of the RNAiCI-sHsp plants were comparable to the wild-type rice plants. Basal and acquired thermotolerance response of RNAiCI-sHsp seedlings of rice was mildly affected. The seed length of RNAiCI-sHsp rice plants was significantly reduced. The seed germination process was delayed and seed thermotolerance of RNAiCI-sHsp was negatively affected than the non-transgenic seeds. We, thus, implicate that sHsp genes are critical in seedling thermotolerance and seed physiology.

Strigolactone Signaling Genes Showing Differential Expression Patterns in Arabidopsis max Mutants.

Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching. The molecular mechanism underlying SL biosynthesis, perception, and signal transduction is vital to the plant branching phenotype. Some aspects of their biosynthesis, perception, and signaling include the role of four MORE AXILLARY GROWTH genes, MAX3, MAX4, MAX1, and MAX2. It is important to identify downstream genes that are involved in SL signaling. To achieve this, we studied the genomic aspects of the strigolactone biosynthesis pathway using microarray analysis of four max mutants. We identified SL signaling candidate genes that showed differential expression patterns in max mutants. More specifically, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 4 (ACC4) and PROTEIN KINASE 3 (PKS3) displayed contrasting expression patterns, indicating a regulatory mechanism in SL signaling pathway to control different phenotypes apart from branching phenotype.

A WUSCHEL Homeobox Transcription Factor, OsWOX13, Enhances Drought Tolerance and Triggers Early Flowering in Rice.

Plants have evolved strategies to cope with drought stress by maximizing physiological capacity and adjusting developmental processes such as flowering time. The WOX13 orthologous group is the most conserved among the clade of WOX homeodomain-containing proteins and is found to function in both drought stress and flower development. In this study, we isolated and characterized OsWOX13 from rice. OsWOX13 was regulated spatially in vegetative organs but temporally in flowers and seeds. Overexpression of OsWOX13 (OsWOX13-ov) in rice under the rab21 promoter resulted in drought resistance and early flowering by 7-10 days. Screening of gene expression profiles in mature leaf and panicles of OsWOX13-ov showed a broad spectrum of effects on biological processes, such as abiotic and biotic stresses, exerting a cross-talk between responses. Protein binding microarray and electrophoretic mobility shift assay analyses supported ATTGATTG as the putative cis-element binding of OsWOX13. OsDREB1A and OsDREB1F, drought stress response transcription factors, contain ATTGATTG motif(s) in their promoters and are preferentially expressed in OsWOX13-ov. In addition, Heading date 3a and OsMADS14, regulators in the flowering pathway and development, were enhanced in OsWOX13-ov. These results suggest that OsWOX13 mediates the stress response and early flowering and, thus, may be a regulator of genes involved in drought escape.

Analysis of Genes with Alternatively Spliced Transcripts in the Leaf, Root, Panicle and Seed of Rice Using a Long Oligomer Microarray and RNA-Seq.

Pre-mRNA splicing further increases protein diversity acquired through evolution. The underlying driving forces for this phenomenon are unknown, especially in terms of gene expression. A rice alternatively spliced transcript detection microarray (ASDM) and RNA sequencing (RNA-Seq) were applied to differentiate the transcriptome of 4 representative organs of Oryza sativa L. cv. Ilmi: leaves, roots, 1-cm-stage panicles and young seeds at 21 days after pollination. Comparison of data obtained by microarray and RNA-Seq showed a bell-shaped distribution and a co-lineation for highly expressed genes. Transcripts were classified according to the degree of organ enrichment using a coefficient value (CV, the ratio of the standard deviation to the mean values): highly variable (CVI), variable (CVII), and constitutive (CVIII) groups. A higher index of the portion of loci with alternatively spliced transcripts in a group (IAST) value was observed for the constitutive group. Genes of the highly variable group showed the characteristics of the examined organs, and alternatively spliced transcripts tended to exhibit the same organ specificity or less organ preferences, with avoidance of 'organ distinctness'. In addition, within a locus, a tendency of higher expression was found for transcripts with a longer coding sequence (CDS), and a spliced intron was the most commonly found type of alternative splicing for an extended CDS. Thus, pre-mRNA splicing might have evolved to retain maximum functionality in terms of organ preference and multiplicity.

RapaNet: A Web Tool for the Co-Expression Analysis of Brassica rapa Genes.

Accumulated microarray data are used for assessing gene function by providing statistical values for co-expressed genes; however, only a limited number of Web tools are available for analyzing the co-expression of genes of Brassica rapa. We have developed a Web tool called RapaNet (http://bioinfo.mju.ac.kr/arraynet/brassica300k/query/), which is based on a data set of 143 B rapa microarrays compiled from various organs and at different developmental stages during exposure to biotic or abiotic stress. RapaNet visualizes correlated gene expression information via correlational networks and phylogenetic trees using Pearson correlation coefficient (r). In addition, RapaNet provides hierarchical clustering diagrams, scatterplots of log ratio intensities, related pathway maps, and cis-element lists of promoter regions. To ascertain the functionality of RapaNet, the correlated genes encoding ribosomal protein (L7Ae), photosystem II protein D1 (psbA), and cytochrome P450 monooxygenase in glucosinolate biosynthesis (CYP79F1) were retrieved from RapaNet and compared with their Arabidopsis homologues. An analysis of the co-expressed genes revealed their shared and unique features.

Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice.

BACKGROUND: Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by gene promoters. Therefore, it is important to identify the binding motifs of transcription factors to better understand the networks associated with embryogenesis. RESULTS: Here, we used a protein-binding microarray (PBM) to identify the binding motifs of OsSMF1, which is a basic leucine zipper transcription factor involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1 or OsbZIP58) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage protein synthesis, and it functions as a key regulator of starch synthesis. Quadruple 9-mer-based PBM analysis and electrophoretic mobility shift assay revealed that OsSMF1 bound to the GCN4 (TGA(G/C)TCA), ACGT (CCACGT(C/G)), and ATGA (GGATGAC) motifs with three different affinities. We predicted 44 putative OsSMF1 target genes using data obtained from both the PBM and RiceArrayNet. Among these putative target genes, 18, 21, and 13 genes contained GCN4, ACGT, and ATGA motifs within their 1-kb promoter regions, respectively. Among them, six genes encoding major grain filling proteins and transcription factors were chosen to confirm the activation of their expression in vivo. OsSMF1 was shown to bind directly to the promoters of Os03g0168500 (GCN4 motif), patatin-like gene (GCN4 motif), α-globulin (ACGT motif), rice prolamin box-binding factor (RPBF) (ATGA motif), and ONAC024 (GCN4 and ACGT motifs) and to regulate their expression. CONCLUSIONS: The results of this study suggest that OsSMF1 is one of the key transcription factors that functions in a wide range of seed developmental processes with different specific binding affinities for the three DNA-binding motifs.

A Large-Scale Functional Analysis of Putative Target Genes of Mating-Type Loci Provides Insight into the Regulation of Sexual Development of the Cereal Pathogen Fusarium graminearum.

Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces sexual progeny (ascospore) as an important overwintering and dissemination strategy for completing the disease cycle. This homothallic ascomycetous species does not require a partner for sexual mating; instead, it carries two opposite mating-type (MAT) loci in a single nucleus to control sexual development. To gain a comprehensive understanding of the regulation of sexual development in F. graminearum, we used in-depth and high-throughput analyses to examine the target genes controlled transcriptionally by two-linked MAT loci (MAT1-1, MAT1-2). We hybridized a genome-wide microarray with total RNAs from F. graminearum mutants that lacked each MAT locus individually or together, and overexpressed MAT1-2-1, as well as their wild-type progenitor, at an early stage of sexual development. A comparison of the gene expression levels revealed a total of 1,245 differentially expressed genes (DEGs) among all of the mutants examined. Among these, genes involved in metabolism, cell wall organization, cellular response to stimuli, cell adhesion, fertilization, development, chromatin silencing, and signal transduction, were significantly enriched. Protein binding microarray analysis revealed the presence of putative core DNA binding sequences (ATTAAT or ATTGTT) for the HMG (high mobility group)-box motif in the MAT1-2-1 protein. Targeted deletion of 106 DEGs revealed 25 genes that were specifically required for sexual development, most of which were regulated transcriptionally by both the MAT1-1 and MAT1-2 loci. Taken together with the expression patterns of key target genes, we propose a regulatory pathway for MAT-mediated sexual development, in which both MAT loci may be activated by several environmental cues via chromatin remodeling and/or signaling pathways, and then control the expression of at least 1,245 target genes during sexual development via regulatory cascades and/or networks involving several downstream transcription factors and a putative RNA interference pathway.

A single module type I polyketide synthase directs de novo macrolactone biogenesis during galbonolide biosynthesis in Streptomyces galbus.

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed ß-keto group modifications.

Genome-wide transcriptome analysis of two contrasting Brassica rapa doubled haploid lines under cold-stresses using Br135K oligomeric chip.

Genome wide transcription analysis in response to stresses is important to provide a basis of effective engineering strategies to improve stress tolerance in crop plants. We assembled a Brassica rapa oligomeric microarray (Br135K microarray) using sequence information from 41,173 unigenes and analyzed the transcription profiles of two contrasting doubled haploid (DH) lines, Chiifu and Kenshin, under cold-treatments. The two DH lines showed great differences in electrolyte leakage below -4°C, but similar patterns from 4°C to -2°C. Cold-treatments induced 885 and 858 genes in Chiifu and Kenshin, respectively. Overall, 134, and 56 genes showed an intrinsic difference in expression in Chiifu and Kenshin, respectively. Among 5,349 genes that showed no hit found (NHF) in public databases, 61 and 24 were specifically expressed in Chiifu and Kenshin, respectively. Many transcription factor genes (TFs) also showed various characteristics of expression. BrMYB12, BrMYBL2, BrbHLHs, BrbHLH038, a C2H2, a WRKY, BrDREB19 and a integrase-type TF were induced in a Chiifu-specific fashion, while a bHLH (Bra001826/AT3G21330), bHLH, cycling Dof factor and two Dof type TFs were Kenshin specific. Similar to previous studies, a large number of genes were differently induced or regulated among the two genotypes, but many genes, including NHFs, were specifically or intrinsically expressed with genotype specificity. Expression patterns of known-cold responsive genes in plants resulted in discrepancy to membrane leakage in the two DH lines, indicating that timing of gene expression is more important to conferring freezing tolerance rather than expression levels. Otherwise, the tolerance will be related to the levels of transcripts before cold-treatment or regulated by other mechanisms. Overall, these results indicate common signaling pathways and various transcriptional regulatory mechanisms are working together during cold-treatment of B. rapa. Our newly developed Br135K oligomeric microarray will be useful for transcriptome profiling, and will deliver valuable insight into cold stresses in B. rapa.

A pepper MSRB2 gene confers drought tolerance in rice through the protection of chloroplast-targeted genes.

BACKGROUND: The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. RESULTS: A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. CONCLUSIONS: Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice.

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Coexpression network analysis associated with call of rice seedlings for encountering heat stress.

Coexpression network analysis is useful tool for identification of functional association of coexpressed genes. We developed a coexpression network of rice from heat stress transcriptome data. Global transcriptome of rice leaf tissues was performed by microarray at three time points--post 10 and 60 min heat stress at 42 °C and 30 min recovery at 26 °C following 60 min 42 °C heat stress to investigate specifically the early events in the heat stress and recovery response. The transcriptome profile was significantly modulated within 10 min of heat stress. Strikingly, the number of up-regulated genes was higher than the number of down-regulated genes in 10 min of heat stress. The enrichment of GO terms protein kinase activity/protein serine threonine kinase activity, response to heat and reactive oxygen species in up-regulated genes after 10 min signifies the role of signal transduction events and reactive oxygen species during early heat stress. The enrichment of transcription factor (TF) binding sites for heat shock factors, bZIPs and DREBs coupled with up-regulation of TFs of different families suggests that the heat stress response in rice involves integration of various regulatory networks. The interpretation of microarray data in the context of coexpression network analysis identified several functionally correlated genes consisting of previously documented heat upregulated genes as well as new genes that can be implicated in heat stress. Based on the findings on parallel analysis of growth of seedlings, associated changes in transcripts of selected Hsps, genome-wide microarray profiling and the coexpression network analysis, this study is a step forward in understanding heat response of rice, the world's most important food crop.

A Multi-Layered Screening Method to Identify Plant Regulatory Genes.

We used a seven-step process to identify genes involved in glucosinolate biosynthesis and metabolism in the Chinese cabbage (Brassica rapa). We constructed an annotated data set with 34,570 unigenes from B. rapa and predicted 11,526 glucosinolate-related candidate genes using expression profiles generated across nine stages of development on a 47k-gene microarray. Using our multi-layered screening method, we screened 392 transcription factors, 843 pathway genes, and 4,162 ortholog genes associated with glucosinolate-related biosynthesis. Finally, we identified five genes by comparison of the pathway-network genes including the transcription-factor genes and the ortholog-ontology genes. The five genes were anchored to the chromosomes of B. rapa to characterize their genetic-map positions, and phylogenetic reconstruction with homologous genes was performed. These anchored genes were verified by reverse-transcription polymerase chain reaction. While the five genes identified by our multi-layered screen require further characterization and validation, our study demonstrates the power of multi-layered screening after initial identification of genes on microarrays.

Transcriptome analysis of leaf and root of rice seedling to acute dehydration.

BACKGROUND: Water deficiency is one of the most serious worldwide problems for agriculture. Recently, it has become more serious and outspread, which urgently requires the production of drought-tolerant plants. Microarray experiments using mRNA from air-dried leaves and roots of rice were performed in an attempt to study genes involved in acute dehydration response. RESULTS: Set of 10,537 rice genes was significantly up- or down-regulated in leaves or roots under the treatment. Gene Ontology analysis highlighted gene expression during acute dehydration response depending on organ types and the duration of stress. Rice responded by down-regulating many processes which are mainly involved in inhibiting growth and development. On the other hand, phytohormones (ABA, cytokinin, brassinosteroid) and protective molecules were induced to answer to multiple stresses. Leaves induced more genes than roots but those genes were scattered in various processes, most significantly were productions of osmoprotectants and precursors for important pathways in roots. Roots up-regulated fewer genes and focused on inducing antioxidants and enhancing photosynthesis. Myb, zf-C3HC4, and NAM were most strongly affected transcription factors with the dominance of leaf over root. CONCLUSIONS: Leaf and root tissues shared some common gene expression during stress, with the purpose of enhancing protective systems. However, these two tissues appeared to act differently in response to the different level of dehydration they experience. Besides, they can affect each other via the signaling and transportation system.

Comprehensive analysis of genic male sterility-related genes in Brassica rapa using a newly developed Br300K oligomeric chip.

To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage (Brassicarapa ssp. pekinensis), floral bud transcriptome analysis was carried out using a B. rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B. rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.

Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities.

The expression of the six rice ASR genes is differentially regulated in a tissue-dependent manner according to environmental conditions and reproductive stages. OsASR1 and OsASR3 are the most abundant and are found in most tissues; they are enriched in the leaves and roots, respectively. Coexpression analysis of OsASR1 and OsASR3 and a comparison of the cis-acting elements upstream of OsASR1 and OsASR3 suggested that their expression is regulated in common by abiotic stresses but differently regulated by hormone and sugar signals. The results of quantitative real-time PCR analyses of OsASR1 and OsASR3 expression under various conditions further support this model. The expression of both OsASR1 and OsASR3 was induced by drought stress, which is a major regulator of the expression of all ASR genes in rice. In contrast, ABA is not a common regulator of the expression of these genes. OsASR1 transcription was highly induced by ABA, whereas OsASR3 transcription was strongly induced by GA. In addition, OsASR1 and OsASR3 expression was significantly induced by sucrose and sucrose/glucose treatments, respectively. The induction of gene expression in response to these specific hormone and sugar signals was primarily observed in the major target tissues of these genes (i.e., OsASR1 in leaves and OsASR3 in roots). Our data also showed that the overexpression of either OsASR1 or OsASR3 in transgenic rice plants increased their tolerance to drought and cold stress. Taken together, our results revealed that the transcriptional control of different rice ASR genes exhibit different tissue-dependent sugar and hormone-sensitivities.

A transcriptional repressor of the ERF family confers drought tolerance to rice and regulates genes preferentially located on chromosome 11.

Plant-specific ethylene response factors (ERFs) play important roles in abiotic and biotic stress responses in plants. Using a transgenic approach, we identified two rice ERF genes, OsERF4a and OsERF10a, which conferred drought stress tolerance. In particular, OsERF4a contains a conserved ERF-associated amphiphilic repression (EAR) motif in its C-terminal region that has been shown to function as a transcriptional repression domain. Expression profiling of transgenic rice plants over-expressing OsERF4a using either a constitutively active or an ABA-inducible promoter identified 45 down-regulated and 79 up-regulated genes in common. The increased stress tolerance by over-expression of the EAR domain-containing protein OsERF4a could result from suppression of a repressor of the defense response. Expression of the putative silent information regulator 2 (Sir2) repressor protein was repressed, and expression of several stress-response genes were induced by OsERF4a over-expression. The Sir2 and 7 out of 9 genes that were down-regulated by OsERF4a over-expression were induced by high salinity and drought treatments in non-transgenic control plants. Genes that were down- and up-regulated by OsERF4a over-expression were highly biased toward chromosome 11. Rice chromosome 11 has several large clusters of disease-resistance and defense-response genes. Taken together, our results suggest that OsERF4a is a positive regulator of shoot growth and water-stress tolerance in rice during early growth stages. We propose that OsERF4a could work by suppressing a repressor of the defense responses and/or by controlling the expression of a large number of genes located on chromosome 11.