Sunok Moon

Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses

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.

Rice GLYCOSYLTRANSFERASE1 Encodes a Glycosyltransferase Essential for Pollen Wall Formation

Summary: This study elucidates functional roles of Golgi-localized rice Glycosyltransferase1 that is essential for intine construction and pollen maturation. 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.

Defective Tapetum Cell Death 1 (DTC1) Regulates ROS Levels by Binding to Metallothionein during Tapetum Degeneration

Timely production of superoxides is essential for initiation of tapetum degeneration in rice. 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.

The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis

Tapetum development and meiosis play crucial roles in anther development. Here we identified a rice gene, DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), which controls the early stages of that development. This gene encodes for an endoplasmic reticulum (ER) membrane protein that is present only in cereals. Our T-DNA insertion mutations gave rise to abnormal tapetal formation. Cellular organelles, especially the ER, were underdeveloped, which led to hampered differentiation and degeneration of the tapetum. In addition, the development of pollen mother cells was arrested at the early stages of meiotic prophase I. RNA in-situ hybridization analyses showed that DTM1 transcripts were most abundant in tapetal cells at stages 6 and 7, and moderately in the pollen mother cells and meiocytes. Transcripts of UDT1, which functions in tapetum development during early meiosis, were reduced in dtm1 anthers, as were those of PAIR1, which is involved in chromosome pairing and synapsis during meiosis. However, expression of MSP1 and MEL1, which function in anther wall specification and germ cell division, respectively, was not altered in the dtm1 mutant. Moreover, transcripts of DTM1 were reduced in msp1 mutant anthers, but not in udt1 and pair1 mutants. These results, together with their mutant phenotypes, suggest that DTM1 plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1.

Evaluation of rice promoters conferring pollen-specific expression in a heterologous system, Arabidopsis

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.

Genome-wide identification and extensive analysis of rice-endosperm preferred genes using reference expression database

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.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage

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.

A systematic view of the rice calcineurin B-like protein interacting protein kinase family

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.

Genome-wide analyses of late pollen-preferred genes conserved in various rice cultivars and functional identification of a gene involved in the key processes of late pollen development

BackgroundUnderstanding 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.ResultsWe 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.ConclusionThrough 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.

Genome-wide transcriptome comparison of flag leaves among japonica and indica varieties

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.