Seong-Ryong Kim

leafy hull sterile1 Is a Homeotic Mutation in a Rice MADS Box Gene Affecting Rice Flower Development

Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 (lhs1). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1-conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene.

Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice

We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.

Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene

Recent studies with dicot plants reveal that floral organ development is controlled by a group of regulatory factors containing the MADS domain. In this study, we have isolated and characterized a cDNA clone from rice, OsMADS1, which encodes a MADS-domain-containing protein. The OsMADS1 amino acid sequence shows 56.2% identity to AGL2 and 44,4% identity to AP1. The MADS box region was the most homologous to other MADS-domain-containing proteins. Northern blot analysis indicated that the rice MADS gene was preferentially expressed in floral organs. In situ localization studies showed that the transcript was uniformly present in young flower primordia and later became localized in palea, lemma, and ovary. Ectopic expression of OsMADS1 with the CaMV 35S promoter in transgenic tobacco plants dramatically alters development, resulting in short, bushy, early-flowering plants with reduced apical dominance. These results suggest that the OsMADS1 gene is involved in flower induction and that it may be used for genetic manipulation of certain plant species.

T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses

T-DNA-tagged rice plants were screened under cold- or salt-stress conditions to determine the genes involved in the molecular mechanism for their abiotic-stress response. Line 0-165-65 was identified as a salt-responsive line. The gene responsible for this GUS-positive phenotype was revealed by inverse PCR as OsGSK1 (Oryza sativaglycogen synthase kinase3-like gene 1), a member of the plant GSK3/SHAGGY-like protein kinase genes and an orthologue of the Arabidopsisbrassinosteroid insensitive 2 (BIN2), AtSK21. Northern blot analysis showed that OsGSK1 was most highly detected in the developing panicles, suggesting that its expression is developmental stage specific. Knockout (KO) mutants of OsGSK1 showed enhanced tolerance to cold, heat, salt, and drought stresses when compared with non-transgenic segregants (NT). Overexpression of the full-length OsGSK1 led to a stunted growth phenotype similar to the one observed with the gain-of-function BIN/AtSK21 mutant. This suggests that OsGSK1 might be a functional rice orthologue that serves as a negative regulator of brassinosteroid (BR)-signaling. Therefore, we propose that stress-responsive OsGSK1 may have physiological roles in stress signal-transduction pathways and floral developmental processes.

Characterization of two rice MADS box genes homologous to GLOBOSA

Abstract A group of regulatory factors containing the MADS box domain is playing an important role in controlling floral organ induction and development. In this study, we have isolated and characterized two cDNA clones from rice, OsMADS2 and OsMADS4, which encode MADS-domain containing proteins. The OsMADS2 amino acid sequence shows more than 50% identity to GLO of Antirrhinum majus, PI of Arabidopsis, and GLO PI homologs of petunia and tobacco. Also, these two rice proteins contain, in addition to the conserved MADS box sequence, two other conserved sequences in the same locations as are found specifically in the GLO PI family. RNA blot analysis showed that the rice MADS box containing genes were expressed throughtout flower development. While OsMADS2 expression increased strongly as the flower matured, OsMADS4 expression was at a higher level from early stages of flower development. RNA blot hybridization with total RNA from each floral organ showed that the expression of these genes was restricted to anther and carpel. In situ hybridization studies indicated that the transcripts were present mainly in pollen, tapetum and also in stigma. Since rice flowers consist of a single perianth which resembles sepal, GLO PI homologs in rice would be expected to be expressed predominantly in reproductive organs. These results suggest that the rice OsMADS genes may be members of the GLO PI family.

Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin

Anthocyanin is the major color pigment in plants. In order to understand anthocyanin biosynthesis mechanism in apple, the cDNAs encoding flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were isolated from cDNA libraries prepared from the skin tissues of apple (Malus domestica Borkh. cv. Fuji). Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Each gene is a member of a multigene family. The mRNAs of anthocyanin biosynthetic genes were detected preferentially in the skin tissue and expression of the genes was coordinately induced by light. The transcripts were detected abundantly in the skins of cultivars with red skin, but rarely in that of a cultivar carrying non-red fruit, suggesting that these genes have major roles in determination of apple skin color.

Identification of Methyl jasmonate and Salicylic Acid Response Elements from the Nopaline Synthase (nos) Promoter

Transgenic tobacco plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (cat) were studied for their inducibility by salicylic acid (SA) or methyl jasmonate (MJ) treatments. Either chemical significantly increased CAT activity to a level much higher than that achieved by wounding. Northern blot analysis showed a corresponding increase in mRNA levels. After 20 h of induction of flowering plants, the response to MJ treatment was weaker in old leaves compared with young leaves, whereas the SA response was stronger in old leaves. Kinetic experiments showed that the SA response was much faster than the MJ response, suggesting that the induction mechanism of the nos promoter by these chemicals may differ. Deletion analysis showed that both SA and MJ responses require the DNA sequence between -119 and -112 from the transcription initiation site. This region contains the hexamer sequence (TGACGT) that has been found to be an important regulatory element for several promoters. The MJ response was also reduced by deletions of the CAAT box region or the sequence between -112 and -101, whereas the SA response was not significantly affected by these deletions. This suggests that the nos upstream region containing the hexamer motif is essential for the SA or MJ response and that the CAAT box region and the sequence immediately downstream from the hexamer motif are required for maximum induction by MJ.

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor.

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.

Insights into genomics of salt stress response in rice

Plants, as sessile organisms experience various abiotic stresses, which pose serious threat to crop production. Plants adapt to environmental stress by modulating their growth and development along with the various physiological and biochemical changes. This phenotypic plasticity is driven by the activation of specific genes encoding signal transduction, transcriptional regulation, ion transporters and metabolic pathways. Rice is an important staple food crop of nearly half of the world population and is well known to be a salt sensitive crop. The completion and enhanced annotations of rice genome sequence has provided the opportunity to study functional genomics of rice. Functional genomics aids in understanding the molecular and physiological basis to improve the salinity tolerance for sustainable rice production. Salt tolerant transgenic rice plants have been produced by incorporating various genes into rice. In this review we present the findings and investigations in the field of rice functional genomics that includes supporting genes and networks (ABA dependent and independent), osmoprotectants (proline, glycine betaine, trehalose, myo-inositol, and fructans), signaling molecules (Ca2+, abscisic acid, jasmonic acid, brassinosteroids) and transporters, regulating salt stress response in rice.

Developmental and environmental regulation of two ribosomal protein genes in tobacco.

Two cDNA clones, TSC29 and TSC40, were isolated from a cDNA library prepared from three-day-old tobacco cell suspension grown to early exponential stage. DNA sequence analyses and database searches revealed that the TSC29 transcript encodes a protein which is highly homologous to eukaryotic 60S ribosomal (r)-protein L25 and that the TSC40 product is homologous to rat 60S r-protein L34. Southern blot analysis showed that the putative r-protein genes are members of multigene families. Transcript levels of both genes were most abundant in three-day-old cell suspension and declined in older cultures. Transcripts were also present in plant vegetative and reproductive organs. However, for TSC40 in particular, the mRNA levels were lower in plant organs than in three-day-old cell suspension. Stems and roots exhibited higher expression than leaves and flowers, indicating that these clones are differentially regulated in various cell types. Both genes were expressed at low levels in mature seeds but transcript levels significantly increased after one day of germination, remained at a high level until day 4, and declined after day 5.in situ localization experiments with germinating seedlings revealed that the TSC29 transcript was preferentially localized in root tips, epidermis, and endosperm. Wounding increased the steady-state mRNA amounts of these r-protein genes, and 2,4-dichlorophenoxyacetic acid and benzyladenine further increased the transcript level.