Genome-wide expression quantitative trait locus studies facilitate isolation of causal genes controlling panicle structure.

The morphology of rice (Oryza sativa L.) panicles is an important determinant of grain yield, and elucidation of the genetic control of panicle structure is very important for fulfilling the demand for high yield in breeding programs. In a quantitative trait locus (QTL) study using 82 backcross inbred lines (BILs) derived from Koshihikari and Habataki, 68 QTLs for 25 panicle morphological traits were identified. Gene expression profiling from inflorescence meristems of BILs was obtained. A combination of phenotypic QTL (pQTL) and expression QTL (eQTL) analysis revealed co-localization between pQTLs and eQTLs, consistent with significant correlations between phenotypic traits and gene expression levels. By combining pQTL and eQTL data, two genes were identified as controlling panicle structure: OsMADS18 modulates the average length of the primary rachis and OsFTL1 has pleiotropic effects on the total number of secondary rachides, number of grains per panicle, plant height and the length of flag leaves. Phenotypes were confirmed in RNA interference knocked-down plants and overexpressor lines. The combination of pQTL and eQTL analysis could facilitate identification of genes involved in rice panicle formation.

SMALL ORGAN SIZE 1 and SMALL ORGAN SIZE 2/DWARF AND LOW-TILLERING Form a Complex to Integrate Auxin and Brassinosteroid Signaling in Rice.

Although auxin and brassinosteroid (BR) synergistically control various plant responses, the molecular mechanism underlying the auxin-BR crosstalk is not well understood. We previously identified SMOS1, an auxin-regulated APETALA2-type transcription factor, as the causal gene of the small organ size 1 (smos1) mutant that is characterized by a decreased final size of various organs in rice. In this study, we identified another smos mutant, smos2, which shows the phenotype indistinguishable from smos1. SMOS2 was identical to the previously reported DWARF AND LOW-TILLERING (DLT), which encodes a GRAS protein involved in BR signaling. SMOS1 and SMOS2/DLT physically interact to cooperatively enhance transcriptional transactivation activity in yeast and in rice nuclei. Consistently, the expression of OsPHI-1, a direct target of SMOS1, is upregulated only when SMOS1 and SMOS2/DLT proteins are both present in rice cells. Taken together, our results suggest that SMOS1 and SMOS2/DLT form a keystone complex on auxin-BR signaling crosstalk in rice.

Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice.

A genome-wide association study (GWAS) can be a powerful tool for the identification of genes associated with agronomic traits in crop species, but it is often hindered by population structure and the large extent of linkage disequilibrium. In this study, we identified agronomically important genes in rice using GWAS based on whole-genome sequencing, followed by the screening of candidate genes based on the estimated effect of nucleotide polymorphisms. Using this approach, we identified four new genes associated with agronomic traits. Some genes were undetectable by standard SNP analysis, but we detected them using gene-based association analysis. This study provides fundamental insights relevant to the rapid identification of genes associated with agronomic traits using GWAS and will accelerate future efforts aimed at crop improvement.

Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism.

Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait locus (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.

Plant Omics Data Center: an integrated web repository for interspecies gene expression networks with NLP-based curation.

Comprehensive integration of large-scale omics resources such as genomes, transcriptomes and metabolomes will provide deeper insights into broader aspects of molecular biology. For better understanding of plant biology, we aim to construct a next-generation sequencing (NGS)-derived gene expression network (GEN) repository for a broad range of plant species. So far we have incorporated information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max) from the public short read archive, digitally profiled the entire set of gene expression profiles, and drawn GENs by using correspondence analysis (CA) to take advantage of gene expression similarities. In order to understand the evolutionary significance of the GENs from multiple species, they were linked according to the orthology of each node (gene) among species. In addition to other gene expression information, functional annotation of the genes will facilitate biological comprehension. Currently we are improving the given gene annotations with natural language processing (NLP) techniques and manual curation. Here we introduce the current status of our analyses and the web database, PODC (Plant Omics Data Center; http://bioinf.mind.meiji.ac.jp/podc/), now open to the public, providing GENs, functional annotations and additional comprehensive omics resources.

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.

De novo transcriptome assembly of a fern, Lygodium japonicum, and a web resource database, Ljtrans DB.

During plant evolution, ferns originally evolved as a major vascular plant with a distinctive life cycle in which the haploid and diploid generations are completely separated. However, the low level of genetic resources has limited studies of their physiological events, as well as hindering research on the evolutionary history of land plants. In this study, to identify a comprehensive catalog of transcripts and characterize their expression traits in the fern Lygodium japonicum, nine different RNA samples isolated from prothalli, trophophylls, rhizomes and sporophylls were sequenced using Roche 454 GS-FLX and Illumina HiSeq sequencers. The hybrid assembly of the high-quality 454 GS-FLX and Illumina HiSeq reads generated a set of 37,830 isoforms with an average length of 1,444 bp. Using four open reading frame (ORF) predictors, 38,142 representative ORFs were identified from a total of 37,830 transcript isoforms and 95 contigs, which were annotated by searching against several public databases. Furthermore, an orthoMCL analysis using the protein sequences of L. japonicum and five model plants revealed various sets of lineage-specific genes, including those detected among land plant lineages and those detected in only L. japonicum. We have also examined the expression patterns of all contigs/isoforms, along with the life cycle of L. japonicum, and identified the tissue-specific transcripts using statistical expression analyses. Finally, we developed a public web resource, the L. japonicum transcriptome database at http://bioinf.mind.meiji.ac.jp/kanikusa/, which provides important opportunities to accelerate molecular research in ferns.

Isolation of a novel lodging resistance QTL gene involved in strigolactone signaling and its pyramiding with a QTL gene involved in another mechanism.

Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait loci (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.

Antheridiogen determines sex in ferns via a spatiotemporally split gibberellin synthesis pathway.

Some ferns possess the ability to control their sex ratio to maintain genetic variation in their colony with the aid of antheridiogen pheromones, antheridium (male organ)-inducing compounds that are related to gibberellin. We determined that ferns have evolved an antheridiogen-mediated communication system to produce males by modifying the gibberellin biosynthetic pathway, which is split between two individuals of different developmental stages in the colony. Antheridiogen acts as a bridge between them because it is more readily taken up by prothalli than bioactive gibberellin. The pathway initiates in early-maturing prothalli (gametophytes) within a colony, which produce antheridiogens and secrete them into the environment. After the secreted antheridiogen is absorbed by neighboring late-maturing prothalli, it is modified in to bioactive gibberellin to trigger male organ formation.

Reduction of gibberellin by low temperature disrupts pollen development in rice.

Microsporogenesis in rice (Oryza sativa) plants is susceptible to moderate low temperature (LT; approximately 19°C) that disrupts pollen development and causes severe reductions in grain yields. Although considerable research has been invested in the study of cool-temperature injury, a full understanding of the molecular mechanism has not been achieved. Here, we show that endogenous levels of the bioactive gibberellins (GAs) GA4 and GA7, and expression levels of the GA biosynthesis genes GA20ox3 and GA3ox1, decrease in the developing anthers by exposure to LT. By contrast, the levels of precursor GA12 were higher in response to LT. In addition, the expression of the dehydration-responsive element-binding protein DREB2B and SLENDER RICE1 (SLR1)/DELLA was up-regulated in response to LT. Mutants involved in GA biosynthetic and response pathways were hypersensitive to LT stress, including the semidwarf mutants sd1 and d35, the gain-of-function mutant slr1-d, and gibberellin insensitive dwarf1. The reduction in the number of sporogenous cells and the abnormal enlargement of tapetal cells occurred most severely in the GA-insensitive mutant. Application of exogenous GA significantly reversed the male sterility caused by LT, and simultaneous application of exogenous GA with sucrose substantially improved the extent of normal pollen development. Modern rice varieties carrying the sd1 mutation are widely cultivated, and the sd1 mutation is considered one of the greatest achievements of the Green Revolution. The protective strategy achieved by our work may help sustain steady yields of rice under global climate change.

A novel AP2-type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway.

The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants.

Identification of transcription factors involved in rice secondary cell wall formation.

Using co-expression network analysis, we identified 123 transcription factors (TFs) as candidate secondary cell wall regulators in rice. To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to the MYB, NAC or homeodomain-containing TF families were overexpressed or downregulated 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 secondary cell wall formation in rice. Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, the former being a TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and the latter being 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 was transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential secondary cell wall TFs in rice by the use of rice co-expression network analysis.

Survey of genes involved in rice secondary cell wall formation through a co-expression network.

The plant secondary cell wall is the major source of lignocellulosic biomass, a renewable energy resource that 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.

Genes offering the potential for designing yield-related traits in rice.

Breeding of high-yielding rice is crucial for meeting the food demand of the increasing world population. New technologies have facilitated identification of genes involved in quantitative traits, and many genes underpinning quantitative trait loci involved in rice crop yield have been isolated. Meanwhile, various kinds of mutants have been intensively studied, leading to characterization of many genes related to yield traits. A combination of quantitative trait locus analysis and studies of such mutants has made it possible to compile a list of genes available for breeding rice with higher yield.

OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice.

Seedling vigor is among the major determinants of stable stand establishment in direct-seeded rice (Oryza sativa L.) in temperate regions. Quantitative trait loci (QTL) for seedling vigor were identified using 250 recombinant inbred lines (RILs) derived from a cross between two japonica rice cultivars Kakehashi and Dunghan Shali. Seedling heights measured at 14 days after sowing were 20.3 and 29.4 cm for Kakehashi and Dunghan Shali, respectively. For the RILs, the height ranged from 14.1 to 31.7 cm. Four putative QTLs associated with seedling height were detected. qPHS3-2, the major QTL that was located on the long arm of chromosome 3, accounted for 26.2 % of the phenotypic variance. Using progeny of the near isogenic lines (NILs) produced by the backcross introduction of a chromosome segment carrying this major QTL into an elite cultivar Iwatekko, we fine-mapped qPHS3-2 to a 81-kb interval between two markers, ID_CAPS_01 and RM16227. Within this mapped region, we identified the gene OsGA20ox1, which is related to gibberellin (GA) biosynthesis. The relative expression levels of GA20ox1 in seedlings of Dunghan Shali and NILs were higher than that of Iwatekko. Concomitantly, the amount of endogenous active GA was higher in Dunghan Shali and the NILs compared to the level detected in Iwatekko. These results indicate that OsGA20ox1 is a strong candidate gene for major QTL controlling seedling vigor in rice.

Efficacy of microarray profiling data combined with QTL mapping for the identification of a QTL gene controlling the initial growth rate in rice.

Seedling vigor, which is controlled by many quantitative trait loci (QTLs), is one of several important agronomic traits for direct-seedling rice systems. However, isolating these QTL genes is laborious and expensive. Here, we combined QTL mapping and microarray profiling to identify QTL genes for seedling vigor. By performing QTL mapping using 82 backcross inbred lines (BILs) of the Koshihikari (japonica) and Habataki (indica) cultivars for the rice initial growth, we identified two QTLs, early-stage plant development1/2 (qEPD1 and qEPD2), whose Koshihikari alleles promote plant height and/or leaf sheath length. Phenotypic analysis of the two substituted lines carrying the Habataki qEPD1 or qEPD2 allele revealed that qEPD2 functioned more dominantly for the initial growth of rice. From the microarray experiment, 55 and 45 candidate genes were found in the qEPD1 and qEPD2 genomic regions, which are expressed differentially between each substitution line (SL) and Koshihikari. Gibberellin 20 oxidase-2 (OsGA20ox2), which is identical to Semi Dwarf1 (SD1), was included among the 55 candidate genes of qEPD1, whereas its paralog, OsGA20ox1, was included among the 45 candidate genes of qEPD2. Consistently, introduction of the Koshihikari OsGA20ox1 allele into SL(qEPD2) increaseed its plant height and leaf sheath length significantly relative to the introduction of the Habataki OsGA20ox1 allele. Therefore, microarray profiling could be useful for rapidly screening QTL candidate genes. We concluded that OsGA20ox1 and OsGA20ox2 (SD1) function during the initial growth of rice, but OsGA20ox1 plays a dominant role in increasing plant height and leaf sheath length at the initial growth stage.

The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity.

When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.

OsCAD2 is the major CAD gene responsible for monolignol biosynthesis in rice culm.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step of monolignol biosynthesis. The rice genome contains 12 CAD-like genes, and whereas the proteins encoded by OsCAD2 and OsCAD7 are known to function in monolignol biosynthesis, the degree to which these enzymes contribute to this process and the involvement of the enzymes encoded by the remaining ten genes is unclear. This paper investigates the role of OsCAD2 and the nine other OsCAD-like proteins in monolignol biosynthesis. Among the OsCAD genes analyzed, OsCAD2, an enzyme belonging to the bona fide CAD phylogenetic group, was the most abundantly expressed gene in the uppermost internode, and was expressed at levels that were more than seven times greater than those of the second most abundantly expressed gene, OsCAD1. Promoter-GUS analysis of OsCAD2 (pCAD::GUS) in the internode, sheath, and roots revealed that GUS expression was strong in tissues that accumulated high levels of lignin. Furthermore, expression always preceded lignin accumulation, showing the tight correlation between OsCAD2 expression and monolignol biosynthesis. Additionally, expression of pCAD::GUS was well synchronized with that of rice caffeic acid 3-O-methyltransferase (OsCOMT::GUS), suggesting that the two enzymes function cooperatively during monolignol biosynthesis. Co-expression network analysis of eight OsCAD genes further revealed that, among the OsCAD genes, expression of OsCAD2 was most tightly associated with the transcription of lignin biosynthesis-related genes. These results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monolignol biosynthesis.

Molecular determinants that convert hormone sensitive lipase into gibberellin receptor.

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.

Comprehensive network analysis of anther-expressed genes in rice by the combination of 33 laser microdissection and 143 spatiotemporal microarrays.

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.