Ki-Hong Jung

Rice Undeveloped Tapetum1 Is a Major Regulator of Early Tapetum Development

The tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis.

Wax-deficient anther1 Is Involved in Cuticle and Wax Production in Rice Anther Walls and Is Required for Pollen Development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.

The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors.

We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

Towards a better bowl of rice: assigning function to tens of thousands of rice genes.

Rice, one of the most important food crops for humans, is the first crop plant to have its genome sequenced. Rice whole-genome microarrays, genome tiling arrays and genome-wide gene-indexed mutant collections have recently been generated. With the availability of these resources, discovering the function of the estimated 41,000 rice genes is now within reach. Such discoveries have broad practical implications for understanding the biological processes of rice and other economically important grasses such as cereals and bioenergy crops.

Towards Establishment of a Rice Stress Response Interactome

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%–60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein–protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance.

The Rice Oligonucleotide Array Database: an atlas of rice gene expression

BackgroundMicroarray technologies facilitate high-throughput gene expression analysis. However, the diversity of platforms for rice gene expression analysis hinders efficient analysis. Tools to broadly integrate microarray data from different platforms are needed.ResultsIn this study, we developed the Rice Oligonucleotide Array Database (ROAD,http://www.ricearray.org) to explore gene expression across 1,867 publicly available rice microarray hybridizations. The ROAD’s user-friendly web interface and variety of visualization tools facilitate the extraction of gene expression profiles using gene and microarray element identifications. The ROAD supports meta-analysis of genes expressed in different tissues and at developmental stages. Co-expression analysis tool provides information on co-regulation between genes under general, abiotic and biotic stress conditions. Additionally, functional analysis tools, such as Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology, are embedded in the ROAD. These tools facilitate the identification of meaningful biological patterns in a list of query genes.ConclusionsThe Rice Oligonucleotide Array Database provides comprehensive gene expression profiles for all rice genes, and will be a useful resource for researchers of rice and other grass species.

Reverse genetic approaches for functional genomics of rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.

Isolation and characterization of a rice cysteine protease gene, OsCP1 using T-DNA gene-trap system

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cyteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.

Production of transgenic rice plants showing reduced heading date and plant height by ectopic expression of rice MADS-box genes

Plant architecture is an important factor that controls several characteristics, such as light interception, harvest index, and lodging. Plant architecture is controlled by a group of regulatory genes. It was previously found that ectopic expression of some MADS-box genes resulted in early flowering and a dwarf habit in Arabidopsis and tobacco (Nicotianatabacum) plants. To explore ways to alter heading time and heading height, we expressed ectopically several OsMADS (Oryza sativa MADS-box) genes in rice plants. The OsMADS cDNA clones were connected to the maize ubiquitin promoter in the sense orientation, and the constructs were introduced into rice plants by the Agrobacterium-mediated transformation. Ectopic expression of the rice MADS-box genes hastened flowering to varying extents. RNA-blot analyses revealed that the phenotypes of early flowering and dwarfism were correlated with the transcript level of the transgene. In addition, weak expression of OsMADS1 using the nopaline synthase (nos) promoter, shortened the heading time by a few days and resulted in a mild reduction of heading height without any pleiotropic effects. The present study suggests that the rice MADS box genes could be used as sources of early-flowering and dwarfing traits in monocot species.

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6−phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.