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Root development is a fundamental process that supports plant survival and crop productivity. One of the essential factors to consider when developing biotechnology crops is the selection of a promoter that can optimize the spatial-temporal expression of introduced genes. However, there are insufficient cases of suitable promoters in crop plants, including rice.This study aimed to verify the usefulness of a new rice root-preferred promoter to optimize the function of a target gene with root-preferred expression in rice.osrns1 mutant had defects in root development based on T-DNA insertional mutant screening and CRISPR technology. To optimize the function of OsRNS1, we generated OsRNS1-overexpression plants under two different promoters: a whole-plant expression promoter and a novel root-preferred expression promoter. Root growth, yield-related agronomic traits, RNA-seq, and reactive oxygen species (ROS) accumulation were analyzed for comparison.OsRNS1 was found to be involved in root development through T-DNA insertional mutant analysis and gene editing mutant analysis. To understand the gain of function of OsRNS1, pUbi1::OsRNS1 was generated for the whole-plant expression, and both root growth defects and overall growth defects were found. To overcome this problem, a root-preferential overexpression line using Os1-CysPrxB promoter (Per) was generated and showed an increase in root length, plant height, and grain yield compared to wild-type (WT). RNA-seq analysis revealed that the response to oxidative stress-related genes was significantly up-regulated in both overexpression lines but was more obvious in pPer::OsRNS1. Furthermore, ROS levels in the roots were drastically decreased in pPer::OsRNS1 but were increased in the osrns1 mutants compared to WT.The results demonstrated that the use of a root-preferred promoter effectively optimizes the function of OsRNS1 and is a useful strategy for improving root-related agronomic traits as well as ROS regulation.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

Summary Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild‐type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10–16 clumped nuclei while wild‐type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild‐type nuclei were evenly distributed by microtubules while the enl2‐2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

The tapetum plays a crucial role in pollen development by nursing and releasing the microspore. The tapetum undergoes programmed cell death (PCD), and appropriate timing of PCD is essential for microsporogenesis. Undeveloped Tapetum1 (UDT1) is known to function in anther development, but the regulation mechanism of tapetum differentiation and degeneration by UDT1 is largely unknown. Through comparative transcriptome analysis, we selected 100 genes as udt1 downregulated genes. The biological process related to the negative regulation of translation is the most enriched in udt1 downregulated genes. It is attributed by ribosome inactivating proteins (RIPs), but the role of RIPs is not well defined, and they are assumed to participate in tapetal PCD. Lipid transport is another overrepresented Gene Ontology (GO) term in udt1 downregulated genes, indicating the functional loss of the tapetum as the nursing cell. Using comparative analysis and real-time PCR, we infer that UDT1 can trigger tapetal PCD by controlling the expression of RIPs and three aspartyl proteases (AP) grouped with OsAP37, which is well known as being involved in tapetum degeneration.

Rice is an important food staple that is consumed by half of the human population. Therefore, understanding the regulatory mechanism of male fertility in rice can improve production by enhancing the efficiency of hybrid seed production. However, information on the control mechanism of male fertility by anther dehiscence or wall development in rice is very limited. To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther walls. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our result could provide useful insight for future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

Root hairs are tubular shaped protuberances of root epidermal cells and are found in nearly all vascular plants. Co-ordinate expression of a number of root hair morphogenesis genes involved in cytoskeleton reorganization, changes in homeostasis and distribution of ion gradients, and cell wall reassembly are required during root hair cell elongation. In this report, we have characterized a root hairspecific putative cellulase gene in rice, OsRHC. OsRHC is specifically expressed in elongating root hairs and OsRHC is targeted to the plasma membrane. The mutation of the OsRHC gene by a T-DNA knock-out and CRISPR-Cas9 system causes a severe reduction in root hair length. Bimolecular fluorescence complementation analysis demonstrated that the OsRHC protein interacts with a root hair-specific cellulose synthase protein (OsCSLD1) in the plasma membrane. Furthermore, we observed a moderate reduction of cellulose content in the osrhc mutant. Our results suggest that the plasma membrane-localized OsRHC plays a critical role in cell wall remodeling during root hair extension.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Promoters are key components for the application of biotechnological techniques in crop plants. Reporter genes such as GUS or GFP have been used to test the activity of promoters for diverse applications. A huge number of T-DNAs carrying promoterless GUS near their right borders have been inserted into the rice genome, and 105,739 flanking sequence tags from rice lines with this T-DNA insertion have been identified, establishing potential promoter trap lines for 20,899 out of 55,986 genes in the rice genome. Anatomical meta-expression data and information on abiotic stress related to these promoter trap lines enable us to quickly identify new promoters associated with various expression patterns. In the present report, we introduce a strategy to identify new promoters in a very short period of time using a combination of meta-expression analysis and promoter trap lines.

International audience; There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Rice (Oryza sativa L.) is a staple crop with agricultural traits that have been intensively investigated. However, despite the variety of mutant population and multi-omics data that have been generated, rice functional genomic research has been bottlenecked due to the functional redundancy in the genome. This phenomenon has masked the phenotypes of knockout mutants by functional compensation and redundancy. Here, we present an intuitive tool, CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice (CAFRI-Rice; cafri-rice.khu.ac.kr). To create this tool, we generated a phylogenetic heatmap that can estimate the similarity between protein sequences and expression patterns, based on 2,617 phylogenetic trees and eight tissue RNA-sequencing datasets. In this study, 33,483 genes were sorted into 2,617 families, and about 24,980 genes were tested for functional redundancy using a phylogenetic heatmap approach. It was predicted that 7,075 genes would have functional redundancy, according to the threshold value validated by an analysis of 111 known genes functionally characterized using knockout mutants and 5,170 duplicated genes. In addition, our analysis demonstrated that an anther/pollen-preferred gene cluster has more functional redundancy than other clusters. Finally, we showed the usefulness of the CAFRI-Rice-based approach by overcoming the functional redundancy between two root-preferred genes via loss-of-function analyses as well as confirming the functional dominancy of three genes through a literature search. This CAFRI-Rice-based target selection for CRISPR/Cas9-mediated mutagenesis will not only accelerate functional genomic studies in rice but can also be straightforwardly expanded to other plant species.

Background Root hairs are valuable in taking up nutrients and water from the rhizosphere and serving as sites of interactions with soil microorganisms. By increasing the external surface area of the roots or interacting with rhizobacteria, root hairs directly and indirectly promote plant growth and yield. Transcriptome data can be used to understand root-hair development in rice. Result We performed Agilent 44 K microarray experiments with enriched root-hair samples and identified 409 root hair-preferential genes in rice. The expression patterns of six genes were confirmed using a GUS reporter system and quantitative RT-PCR analysis. Gene Ontology (GO) analysis demonstrated that 13 GO terms, including oxygen transport and cell wall generation, were highly over-represented in those genes. Although comparative analysis between rice and Arabidopsis revealed a large proportion of orthologous pairs, their spatial expression patterns were not conserved. To investigate the molecular network associated with root hair-preferential genes in rice, we analyzed the PPI network as well as coexpression data. Subsequently, we developed a refined network consisting of 24 interactions between 10 genes and 18 of their interactors. Conclusion Identification of root hair-preferential genes and in depth analysis of those genes will be a useful reference to accelerate the understanding of root-hair development in rice. Electronic supplementary material The online version of this article (10.1186/s12284-018-0241-2) contains supplementary material, which is available to authorized users.

Background Understanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development. Results We performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer. Conclusion Through the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis and storage reserves of pollen. In addition, these candidates might be useful targets for future examinations of late pollen development, and will be a valuable resource for accelerating the understanding of molecular mechanisms for pollen maturation and germination processes in rice. Electronic supplementary material The online version of this article (10.1186/s12284-018-0219-0) contains supplementary material, which is available to authorized users.

Plant root systems play essential roles in developmental processes, such as the absorption of water and inorganic nutrients, and structural support. Gene expression is affected by growth conditions and the genetic background of plants. To identify highly conserved root-preferred genes in rice across diverse growth conditions and varieties, we used two independent meta-anatomical expression profiles based on a large collection of Affymetrix and Agilent 44 K microarray data sets available for public use. We then identified 684 loci with root-preferred expression, which were validated with in silico analysis using both meta-expression profiles. The expression patterns of four candidate genes were confirmed in vivo by monitoring expression of β-glucuronidase under control of the candidate-gene promoters, providing new tools to manipulate agronomic traits associated with roots. We also utilized real-time PCR to examine the root-preferential expression of 14 genes across four rice varieties, including japonica and indica cultivars. Using a database of rice genes with known functions, we identified the reported functions of 39 out of the 684 candidate genes. Sixteen genes are directly involved in root development, while the remaining are involved in processes indirectly related to root development (i.e., soil-stress tolerance or growth retardation). This indicates the importance of our candidate genes for studies on root development and function. Gene ontology enrichment analysis in the ‘biological processes’ category revealed that root-preferred genes in rice are closely associated with nutrient transport-related genes, indicating that the primary role of roots is the uptake of nutrients from soil. In addition, predicted protein–protein interaction analysis suggested a molecular network for root development composed of 215 interactions associated with 44 root-preferred or root development-related genes. Taken together, our data provide an important foundation for future research on root development in rice.

This work suggests 2020 potential candidates in rice for the functional annotation of unannotated genes using meta-analysis of anatomical samples derived from microarray and RNA-seq technologies and this information will be useful to identify novel morphological agronomic traits. Although the genome of rice (Oryza sativa) has been sequenced, 14,365 genes are considered unannotated because they lack putative annotation information. According to the Rice Genome Annotation Project Database ( http://rice.plantbiology.msu.edu/ ), the proportion of functionally characterized unannotated genes (0.35%) is quite limited when compared with the approximately 3.9% of annotated genes with assigned putative functions. Researchers require additional information to help them investigate the molecular mechanisms associated with those unannotated genes. To determine which of them might regulate morphological or physiological traits in the rice genome, we conducted a meta-analysis of expression data that covered a wide range of tissue/organ samples. Overall, 2020 genes showed cultivar-, tissue-, or organ-preferential patterns of expression. Representative candidates from featured groups were validated by RT-PCR, and the GUS reporter system was used to validate the expression of genes that were clustered according to their leaf or root preference. Taking a molecular and genetics approach, we examined meta-expression data and found that 127 genes were differentially expressed between japonica and indica rice cultivars. This is potentially significant for future agronomic applications. We also used a T-DNA insertional mutant and performed a co-expression network analysis of Sword shape dwarf1 (SSD1), a gene that regulates cell division. This network was refined via RT-PCR analysis. Our results suggested that SSD1 represses the expression of four genes related to the processes of DNA replication or cell division and provides insight into possible molecular mechanisms. Together, these strategies present a valuable tool for in-depth characterization of currently unannotated genes.

Studying endosperm development in crop species enables us to understand the molecular mechanisms for producing and metabolizing carbohydrates as a main energy source. A significant accumulation of genome-wide transcriptome data can enhance the performance of metaexpression data for diverse applications. Using a gene search tool in Genevestigator (https://genevestigator.com/) for anatomical samples, we first conducted a meta-anatomical expression analysis based on 2566 Affymetrix array data in rice (Oryza sativa) and intended to identify 400 endospermpreferred probes. Tissue-preferred expression patterns were confirmed by performing an additional meta-analysis of anatomical expression data comprising 219 spatial or temporal Agilent 44K array data. We then identified 299 genes that showed strong endosperm-preferred expression. The functional significance of 24 previously characterized genes was evaluated, and tissue-specific expression patterns of two genes were validated using a GUS reporter system. This second approach demonstrated that new tools are available for delivering agronomic traits in the endosperm. MapMan (mapman.gabipd.org/) analysis revealed the Metabolism and Regulation overviews associated with the process of endosperm development. In particular, the starch metabolism pathway is very closely related to that process in rice. We then constructed a regulatory network using both KEGG (www. genome.jp/ kegg/) pathway information for our candidate genes and a predicted protein–protein interaction network tool. Examination of an osbzip58-1 mutant with defects in endosperm development was combined with global transcriptome data in the network. Our results indicated that osbzip58-1 regulates the starch and sucrose metabolism pathways as well as the production of seed storage protein precursors. This new fundamental information adds to our understanding about the molecular mechanism for endosperm development in rice, and the resulting data will contribute to future studies that work to enhance the agronomic trait(s) associated with the endosperm.

In most eukaryotes, a set of conserved proteins that are collectively termed ZMM proteins (named for molecular zipper 1 [ZIP1], ZIP2, ZIP3, and ZIP4, MutS homologue 4 [MSH4] and MSH5, meiotic recombination 3, and sporulation 16 [SPO16] in yeast [Saccharomyces cerevisiae]) are essential for the formation of the majority of meiotic crossovers (COs). Recent reports indicated that ZIP2 acts together with SPO16 and ZIP4 to control CO formation through recognizing and stabilizing early recombination intermediates in budding yeast. However, whether this mechanism is conserved in plants is not clear. Here, we characterized the functions of SHORTAGE OF CHIASMATA 1 (OsSHOC1; ZIP2 ortholog) and PARTING DANCERS (OsPTD; SPO16 ortholog) and their interactions with other ZMM proteins in rice (Oryza sativa). We demonstrated that disruption of OsSHOC1 caused a reduction of CO numbers to ∼83% of wild-type CO numbers, whereas synapsis and early meiotic recombination steps were not affected. Furthermore, OsSHOC1 interacts with OsPTD, which is responsible for the same set of CO formations as OsSHOC1. In addition, OsSHOC1 and OsPTD are required for the normal loading of other ZMM proteins, and conversely, the localizations of OsSHOC1 and OsPTD were also affected by the absence of OsZIP4 and human enhancer of invasion 10 in rice (OsHEI10). OsSHOC1 interacts with OsZIP4 and OsMSH5, and OsPTD interacts with OsHEI10. Furthermore, bimolecular fluorescence complementation and yeast-three hybrid assays demonstrated that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 were able to form various combinations of heterotrimers. Moreover, statistical and genetic analysis indicated that OsSHOC1 and OsPTD are epistatic to OsHEI10 and OsZIP4 in meiotic CO formation. Taken together, we propose that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 form multiple protein complexes that have conserved functions in promoting class I CO formation.

Transcription factors (TFs) are an important class of regulatory molecules. Despite their importance, only a small number of genes encoding TFs have been characterized in Oryza sativa (rice), often because gene duplication and functional redundancy complicate their analysis. To address this challenge, we developed a web-based tool called the Rice Transcription Factor Phylogenomics Database (RTFDB) and demonstrate its application for predicting TF function. The RTFDB hosts transcriptome and co-expression analyses. Sources include high-throughput data from oligonucleotide microarray (Affymetrix and Agilent) as well as RNA-Seq-based expression profiles. We used the RTFDB to identify tissue-specific and stress-related gene expression. Subsequently, 273 genes preferentially expressed in specific tissues or organs, 455 genes showing a differential expression pattern in response to 4 abiotic stresses, 179 genes responsive to infection of various pathogens and 512 genes showing differential accumulation in response to various hormone treatments were identified through the meta-expression analysis. Pairwise Pearson correlation coefficient analysis between paralogous genes in a phylogenetic tree was used to assess their expression collinearity and thereby provides a hint on their genetic redundancy. Integrating transcriptome with the gene evolutionary information reveals the possible functional redundancy or dominance played by paralog genes in a highly duplicated genome such as rice. With this method, we estimated a predominant role for 83.3% (65/78) of the TF or transcriptional regulator genes that had been characterized via loss-of-function studies. In this regard, the proposed method is applicable for functional studies of other plant species with annotated genome.

Rice microspore-promoters. Based on microarray data analyzed for developing anthers and pollen grains, we identified nine rice microspore-preferred (RMP) genes, designated RMP1 through RMP9. To extend their biotechnological applicability, we then investigated the activity of RMP promoters originating from monocotyledonous rice in a heterologous system of dicotyledonous Arabidopsis. Expression of GUS was significantly induced in transgenic plants from the microspore to the mature pollen stages and was driven by the RMP1, RMP3, RMP4, RMP5, and RMP9 promoters. We found it interesting that, whereas RMP2 and RMP6 directed GUS expression in microspore at the early unicellular and bicellular stages, RMP7 and RMP8 seemed to be expressed at the late tricellular and mature pollen stages. Moreover, GUS was expressed in seven promoters, RMP3 through RMP9, during the seedling stage, in immature leaves, cotyledons, and roots. To confirm microspore-specific expression, we used complementation analysis with an Arabidopsis male-specific gametophytic mutant, sidecar pollen-2 (scp-2), to verify the activity of three promoters. That mutant shows defects in microspore development prior to pollen mitosis I. These results provide strong evidence that the SIDECAR POLLEN gene, driven by RMP promoters, successfully complements the scp-2 mutation, and they strongly suggest that these promoters can potentially be applied for manipulating the expression of target genes at the microspore stage in various species.

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

Internode elongation is one of the key agronomic traits determining a plant&rsquo, s height and biomass. However, our understanding of the molecular mechanisms controlling internode elongation is still limited in crop plant species. Here, we report the functional identification of an atypical basic helix-loop-helix transcription factor (OsbHLH073) through gain-of-function studies using overexpression (OsbHLH073-OX) and activation tagging (osbhlh073-D) lines of rice. The expression of OsbHLH073 was significantly increased in the osbhlh073-D line. The phenotype of osbhlh073-D showed semi-dwarfism due to deficient elongation of the first internode and poor panicle exsertion. Transgenic lines overexpressing OsbHLH073 confirmed the phenotype of the osbhlh073-D line. Exogenous gibberellic acid (GA3) treatment recovered the semi-dwarf phenotype of osbhlh073-D plants at the seedling stage. In addition, quantitative expression analysis of genes involving in GA biosynthetic and signaling pathway revealed that the transcripts of rice ent-kaurene oxidases 1 and 2 (OsKO1 and OsKO2) encoding the GA biosynthetic enzyme were significantly downregulated in osbhlh073-D and OsbHLH073-OX lines. Yeast two-hybrid and localization assays showed that the OsbHLH073 protein is a nuclear localized-transcriptional activator. We report that OsbHLH073 participates in regulating plant height, internode elongation, and panicle exsertion by regulating GA biosynthesis associated with the OsKO1 and OsKO2 genes.

Figure S1. Expression graph after KMC analysis of anatomical meta-expression data including root hairs. Genes in Cluster 7 (marked with red box) were selected as group showing root hair-preferential pattern of expression. Figure S2. GUS expression pattern of 63 promoter trap candidates. Among them, five lines showed GUS expression (blue and red boxes), and 3 exhibited root hair-preferential pattern (red box). Figure S3. Co-segregation between genotyping and GUS expression, as checked for 3 promoter trap lines: LOC_Os05g45900 (a), LOC_Os10g42750 (b), and LOC_Os12g02240 (c). GUS-positive and GUS-negative are represented by + and -, respectively. Figure S4. Expression profiles of root hair-preferential genes. Analysis of expression patterns via real-time PCR for 6 genes: LOC_Os05g45900 (a), LOC_Os10g42750 (b), LOC_Os12g02240 (c), LOC_Os02g42820 (d), LOC_Os10g38340 (e), and LOC_Os12g38010 (f). Y-axis, gene expression relative to rice OsUbi5 transcript level. Figure S5. New motifs discovered in 409 root hair-preferential genes, based on MEME analysis. Figure S6. Expression graph after KMC analysis of anatomical meta-expression data in Arabidopsis. Clusters 2, 4, and 6 (in red boxes) show root hair-preferential expression patterns. Figure S7. Heatmap for expression profiles of root hair-preferential genes in Arabidopsis. Yellow, high expression; dark-blue, low expression. Figure S8. Functional gene network associated with root hair-preferential genes in rice, as indicated by large circles with red boundaries. Six functionally characterized genes are named. Interactions between nodes with PCC values >â 0.5 are represented by red lines. (DOCX 2500 kb)

Figure S1. a. Order of developmental stages for anatomical samples. ACF, formation of archesporial cells; BG, bi-cellular gametophyte; Fl, flowering; Me, meiosis; Me1, meiotic leptotene; Me2, meiotic zygotene-pachytene; Me3, meiotic diplotene-tetrad; MP, mature pollen; PMe, pre-meiosis; GP, germinating pollen; TG, tri-cellular pollen; UG, uni-cellular gametophyte. Red bar, sample containing late pollen. b. Expression graph of 36 clusters after KMC analysis with 57,382 probes. Clusters 2 and 35 exhibited mature pollen-preferential patterns of expression and are marked with red boxes. Figure S2. Schematic representation of 3 promoter trap lines for T-DNA insertions. a. T-DNA was inserted into 17th intron of SacI homology domain-containing protein (LOC_Os11g20384) in Line 1A-13,819 (mtd1â 1). b. Line 3A-05916 has T-DNA insertion in B12D protein (LOC_Os07g17310). BL, left T-DNA border; RB, right T-DNA border; Gray boxes, exons; lines, introns. Figure S3. Expression graph after KMC analysis of meta-expression data from Arabidopsis. Clusters marked with red box showed late pollen-preferred patterns. Figure S4. Heatmap for expression profiles of late pollen-preferred genes in Arabidopsis. Figure S5. Heatmap of genes involved in GA biosynthesis and signaling. CPS, GA3ox1, KAO, GA20ox3, and KO2 showed late pollen-preferred expression patterns and are outlined with red boxes. (DOCX 227 kb)

Table S1. Six series of microarray data comprising 64 slides (GPL2025) associated with anthers/pollen in rice. Table S2. Locus IDs and putative functions of late pollen-preferred genes from rice. Table S3. Locus IDs and promoter regions of genes used for promoter analysis with GUS reporter. Table S4. Classification of GO terms for biological processes associated with late pollen-preferred genes. Table S5. MapMan classification of late pollen-preferred genes. Table S6. Genes related to hormone metabolism term in MapMan. Table S7. Late pollen-preferred genes in Arabidopsis. Table S8. Assignment of rice orthologs to Arabidopsis late pollen-preferred genes. Locus numbers are shown in by red. Table S9. Assignment of Arabidopsis orthologs to rice late pollen-preferred genes. Locus numbers are shown in red. Table S10. MapMan terms related to cell wall organization and modifications in rice and Arabidopsis. Table S11. Primer sequences used in genotyping and real-time PCR. (DOCX 1980 kb)

Flag leaves in crops are one of the key organs determining grain yield, which significantly affects total yield. However, our understanding of the molecular and genetic regulation of flag leaves is very limited. To provide a genome-wide view of gene expression in flag leaves associated with grain yield, we compared the flag leaves of rice varieties with different yield potentials, such as Hwacheong (moderate yield), Milyang23 (high yield), Dasan (high yield), and IR64 (high yield), using an Agilent 8x60K microarray. As a result, we identified 245 genes that were up-regulated in high yield potential varieties compared to Hwacheong, along with 293 genes that were up-regulated in Hwacheong. GO enrichment analysis of the selected candidate genes revealed that the thiamin biosynthetic process and the sucrose metabolic process were the most enriched terms in flag leaves from the high yield potential varieties, while phosphate transport and the chitin catabolic process terms were the most significant in flag leaves of Hwacheong. In addition, MapMan analysis suggested that the biotic stress response and auxin signaling are important in Hwacheong, while the heat stress response, calcium and G-protein signaling are necessary in other high yield potential varieties. The functions of 11 of our candidate genes have been previously characterized in genetic and molecular biological studies and most of them are related to tolerance against environmental challenges or yield, thereby indicating the potential significance of our candidate genes in further applications.

Tissue-specific promoters are a very useful tool for manipulating gene expression in a target tissue or organ; however, their range of applications in other plant species has not been determined, to date. In this study, we identified two late pollen-specific rice promoters (ProOsLPS10 and ProOsLPS11) via meta-anatomical expression analysis. We then investigated the expression of both promoters in transgenic rice (a homologous system) and Arabidopsis (a heterologous system) using ProOsLPS10 or ProOsLPS11::GFP-GUS constructs. As predicted by microarray data, both promoters triggered strong GUS expression during the late stages of pollen development in rice, with no GUS signals detected in the examined microspores and sporophytic tissues. Interestingly, these promoters exhibited different GUS expression patterns in Arabidopsis. While in Arabidopsis, the OsLPS10 promoter conferred GUS expression at the uni- and bi-cellular pollen stages, as well as at the shoot apical region during the seedling stage, the OsLPS11 promoter was not active in the pollen at any stage, or in the examined sporophytic tissues. Furthermore, by performing a complementation analysis using a sidecar pollen (scp) mutant that displays developmental defects at the microspore stage, we found evidence that OsLPS10 promoter, which can be applied in Arabidopsis, is useful for directing gene expression in the early stages of pollen development. Our results indicate that the OsLPS10 and OsLPS11 promoters can drive the expression of target genes during the late stages of pollen development in rice, but not in Arabidopsis. Our results also emphasize the necessity of confirming the applicability of an established promoter to heterologous systems.

Calcium (Ca2+), as a universal second messenger, plays an important role in various signal transduction pathway networks in plants. There are several components of Ca2+ mediating signaling pathways including calcium-dependent protein kinases, calmodulin and calcineurin B-like proteins (CBLs), and CBL-interacting protein kinases (CIPK). Here, we provide a systematic view of 33 rice CIPK (OsCIPK) family members, which are further classified into seven subgroups. The integration of various meta-expression data such as anatomy, diurnal regulation, and phylogenomic tree context of the OsCIPK family reveals that 11 of the family members show preferred expression in leaves during vegetative growth. In addition, 20 OsCIPK genes show peak expression during the day, while none of the genes peaked expression in the dark. Regarding abiotic stress, 16 OsCIPK genes were induced by drought stress in leaf organs; of these, 14 OsCIPK genes showed up-regulation and two OsCIPK genes were down-regulated based on quantitative RT-PCR analyses. We then developed a functional gene network that consists of 15 out of the 16 genes. This network proposes a useful hypothetical model to understand the molecular mechanism of drought response and circadian regulation associated with OsCIPK family genes.

The pollen wall consists of an exine and an intine. The mechanism underlying its formation is not well understood. Glycosyltransferases catalyze the modification of biological molecules by attaching a single or multiple sugars and play key roles in a wide range of biological processes. We examined the role of GLYCOSYLTRANSFERASE1 (OsGT1) in pollen wall development in rice (Oryza sativa). This gene is highly expressed in mature pollen, and plants containing alleles caused by transfer DNA insertion do not produce homozygous progeny. Reciprocal crosses between OsGT1/osgt1 and the wild type indicated that the mutation leads to a male gametophyte defect. Microscopic analyses revealed that osgt1 pollen developed normally to the pollen mitosis stage but failed to produce mature grains. In osgt1 pollen, intine structure was disrupted. In addition, starch and protein levels were much lower in the mutant grains. Recombinant OsGT1 transferred glucose from UDP-glucose to the third and seventh positions of quercetin, a universal substrate of glycosyltransferases. Consistent with the role of OsGT1, an OsGT1-green fluorescent protein fusion protein was localized to the Golgi apparatus. Taken together, our results suggest that OsGT1 is a Golgi-localized glycosyltransferase essential for intine construction and pollen maturation, providing new insight into male reproductive development.

Data are limited on the value of non-invasive diagnostic methods, such as the cytomegalovirus (CMV) antigenemia assay, and the clinical features of CMV pneumonia in patients who have undergone solid organ transplant (SOT) compared with those who have had hematopoietic stem cell transplant (HSCT). All adult patients with suspected CMV pneumonia, who had received SOT or HSCT in a tertiary hospital during a 5-year period, were retrospectively enrolled. CMV pneumonia was defined as clinical and radiographic evidence of pneumonia in association with the isolation of CMV in viral cultures of bronchoalveolar lavage or lung tissue specimens, or with the identification of CMV in lung tissue. In total, 36 patients with CMV pneumonia were identified. Of these, 29 (80%) had received SOT and 7 (20%) had received HSCT. The incidence of CMV pneumonia in the patients with SOT (3.0 per 1000 person-years [95% confidence interval {CI} 0.6–8.7]) was lower than in those with HSCT (17.0 per 1000 person-years [95% CI 9.9–27.2], P = 0.003) and CMV-related mortality showed a tendency to have lower mortality in patients with SOT (10% [3/29]) than with HSCT (43% [3/7], P = 0.07). The overall sensitivity of the CMV assay (≥ 1/200,000 leukocytes) in patients with CMV pneumonia was 69% (95% CI 52–84%). The CMV antigenemia test is of limited value in diagnosing CMV pneumonia, given the high cost of false-negative diagnoses of CMV pneumonia.

RNA editing is the alteration of RNA sequences via insertion, deletion and conversion of nucleotides. In flowering plants, specific cytidine residues of RNA transcribed from organellar genomes are converted into uridines. Approximately 35 editing sites are present in the chloroplasts of higher plants; six pentatricopeptide repeat genes involved in RNA editing have been identified in Arabidopsis. However, although approximately 500 editing sites are found in mitochondrial RNAs of flowering plants, only one gene in Arabidopsis has been reported to be involved in such editing. Here, we identified rice mutants that are defective in seven specific RNA editing sites on five mitochondrial transcripts. Their various phenotypes include delayed seed germination, retarded growth, dwarfism and sterility. Mutant seeds from heterozygous plants are opaque. This mutation, named opaque and growth retardation 1 (ogr1), was generated by T-DNA insertion into a gene that encodes a pentatricopeptide repeat protein containing the DYW motif. The OGR1-sGFP fusion protein is localized to mitochondria. Ectopic expression of OGR1 in the mutant complements the altered phenotypes. We conclude that OGR1 is essential for RNA editing in rice mitochondria and is required for normal growth and development.

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity.

Regulating the transition of meristem identity is a critical step in reproductive development. After the shoot apical meristem (SAM) acquires inflorescence meristem identity, it goes through a sequential transition to second- and higher-order meristems that can eventually give rise to floral organs. Despite ample information on the molecular mechanisms that control the transition from SAM to inflorescence meristems, little is known about the mechanism for inflorescence development, especially in monocots. Here, we report the identification of the SUPERNUMERARY BRACT (SNB) gene controlling the transition from spikelet meristem to floral meristem and the floral organ development. This gene encodes a putative transcription factor carrying two AP2 domains. The SNB:GFP fusion protein is localized to the nucleus. SNB is expressed in all the examined tissues, but most strongly in the newly emerging spikelet meristems. In SNB knockout plants, the transition from spikelet meristems to floral meristems is delayed, resulting in the production of multiple rudimentary glumes in an alternative phyllotaxy. The development of additional bracts interferes with subsequent floral architecture. In some spikelets, the empty glumes and lodicules are transformed into lemma/palea-like organs. Occasionally, the number of stamens and carpels is altered and an ectopic floret occurs in the axil of the rachilla. These phenotypes suggest that snb is a heterochronic mutant, affecting the phase transition of spikelet meristems, the pattern formation of floral organs and spikelet meristem determinancy.

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.

Although the super family of ATP-binding cassette (ABC) proteins plays key roles in the physiology and development of plants, the functions of members of this interesting family mostly remain to be clarified, especially in crop plants. Thus, systematic analysis of this family in rice (Oryza sativa), a major model crop plant, will be helpful in the design of effective strategies for functional analysis. Phylogenomic analysis that integrates anatomy and stress meta-profiling data based on a large collection of rice Affymetrix array data into the phylogenic context provides useful clues into the functions for each of the ABC transporter family members in rice. Using anatomy data, we identified 17 root-preferred and 16-shoot preferred genes at the vegetative stage, and 3 pollen, 2 embryo, 2 ovary, 2 endosperm, and 1 anther-preferred gene at the reproductive stage. The stress data revealed significant up-regulation or down-regulation of 47 genes under heavy metal treatment, 16 genes under nutrient deficient conditions, and 51 genes under abiotic stress conditions. Of these, we confirmed the differential expression patterns of 14 genes in root samples exposed to drought stress using quantitative real-time PCR. Network analysis using RiceNet suggests a functional gene network involving nine rice ABC transporters that are differentially regulated by drought stress in root, further enhancing the prediction of biological function. Our analysis provides a molecular basis for the study of diverse biological phenomena mediated by the ABC family in rice and will contribute to the enhancement of crop yield and stress tolerance.

Promoters can direct gene expression specifically to targeted tissues or cells. Effective with both crop species and model plant systems, these tools can help researchers overcome the practical obstacles associated with transgenic protocols. Here, we identified promoters that allow one to target the manipulation of gene expression during pollen development. Utilizing published transcriptomic databases for rice, we investigated the promoter activity of selected genes in Arabidopsis. From various microarray datasets, including those for anthers and pollen grains at different developmental stages, we selected nine candidate genes that showed high levels of expression in the late stages of rice pollen development. We named these Oryza sativa late pollen-specific genes. Their promoter regions contained various cis-acting elements that could be responsible for anther-/pollen-specific expression. Promoter::GUS–GFP reporters were constructed and introduced into Arabidopsis plants. Histochemical GUS staining revealed that six of the nine rice promoters conferred strong GUS expression that was restricted to the anthers in Arabidopsis. Further analysis showed that although the GUS signals were not detected at the unicellular stage, they strengthened in the bicellular or tricellular stages, peaking at the mature pollen stage. This paralleled their transcriptomic profiles in rice. Based on our results, we proposed that these six rice promoters, which are active in the late stages of pollen formation in the dicot Arabidopsis, can aid molecular breeders in generating new varieties of a monocot plant, rice.