Naoki Sentoku

Cloning and functional analysis of two gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice

We have cloned two gibberellin (GA) 3β-hydroxylase genes, OsGA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obtained by PCR using degenerate primers. We have used full-scan GC-MS and Kovats retention indices to show function for the two encoded recombinant fusion proteins. Both proteins show 3β-hydroxylase activity for the steps GA20 to GA1, GA5 to GA3, GA44 to GA38, and GA9 to GA4. In addition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-desaturase activity, which catalyzes the steps GA9 to 2,3-dehydro-GA9 and GA20 to GA5 (2,3-dehydro GA20), and 2β-hydroxylase activity, which catalyzes the steps GA1 to GA8 and GA4 to GA34. Molecular and linkage analysis maps the OsGA3ox1 gene to the distal end of the short arm of chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm of chromosome 1 that corresponds to the D18 locus. The association of the OsGA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with the OxGA3ox2 gene results in transgenic plants with a normal phenotype. Although both genes show transient expression, the highest level for OsGA3ox1 is from unopened flower. The highest level for OsGA3ox2 is from elongating leaves.

Loss‐of‐function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants

The rice homeobox gene OSH15 (Oryza sativa homeobox) is a member of the knotted1‐type homeobox gene family. We report here on the identification and characterization of a loss‐of‐function mutation in OSH15 from a library of retrotransposon‐tagged lines of rice. Based on the phenotype and map position, we have identified three independent deletion alleles of the locus among conventional morphological mutants. All of these recessive mutations, which are considered to be null alleles, exhibit defects in internode elongation. Introduction of a 14 kbp genomic DNA fragment that includes all exons, introns and 5′‐ and 3′‐ flanking sequences of OSH15 complemented the defects in internode elongation, confirming that they were caused by the loss‐of‐function of OSH15. Internodes of the mutants had abnormal‐shaped epidermal and hypodermal cells and showed an unusual arrangement of small vascular bundles. These mutations demonstrate a role for OSH15 in the development of rice internodes. This is the first evidence that the knotted1‐type homeobox genes have roles other than shoot apical meristem formation and/or maintenance in plant development.

Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development.

We report the isolation, sequence, and pattern of gene expression of members of the KNOTTED1 (KN1)-type class 1 homeobox gene family from rice. Phylogenetic analysis and mapping of the rice genome revealed that all of the rice homeobox genes that we have isolated have one or two direct homologs in maize. Of the homeobox genes that we tested, all exhibited expression in a restricted region of the embryo that defines the position at which the shoot apical meristem (SAM) would eventually develop, prior to visible organ formation. Several distinct spatial and temporal expression patterns were observed for the different genes in this region. After shoot formation, the expression patterns of these homeobox genes were variable in the region of the SAM. These results suggest that the rice KN1-type class 1 homeobox genes function cooperatively to establish the SAM before shoot formation and that after shoot formation, their functions differ.

OsMADS22 , an STMADS11 -like MADS-box gene of rice, is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy

We report the cDNA sequence and gene expression patterns of OsMADS22, a novel member of the STMADS11-like family of MADS-box genes, from rice. In contrast to previously reported STMADS11-like genes, whose expression is detected in vegetative tissues, OsMADS22 is mainly expressed during embryogenesis and flower development. In situ hybridization analysis revealed that OsMADS22 expression is localized in the L1 layer of embryos and in developing stamen primordia. Ectopic expression of OsMADS22 in transgenic rice plants resulted in aberrant floral morphogenesis, characterized by a disorganized palea, an elongated glume, and a two-floret spikelet. The results are discussed in terms of rice spikelet development and a novel non-vegetative role for a STMADS11-like gene.

The COP1 Ortholog PPS Regulates the Juvenile–Adult and Vegetative–Reproductive Phase Changes in Rice

Rice pps is a heterochronic mutant that shows a prolonged juvenile phase by repressing GA biosynthetic genes and altering expression patterns of microRNA genes but is early flowering. Positional cloning revealed that PPS is the ortholog of Arabidopsis COP1. Although PPS has a role in photomorphogenesis as COP1 does, PPS has additional roles in vegetative phase change and flowering time regulation. Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA3 content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots.

Isolation and characterization of a rice homebox gene, OSH15.

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.

Analysis of the transgenic tobacco plants expressing Panicum miliaceum aspartate aminotransferase genes.

Abstract Expression of Panicum miliaceum L. (proso millet) mitochondrial and cytosolic aspartate aminotransferase (mAspAT and cAspAT, respectively) genes in transgenic tobacco plants (Nicotiana tabacum) and their influences on protein synthesis were examined. The mAspAT- or cAspAT-transformed plants had about threefold or 3.5-fold higher AspAT activity in the leaf than non-transformed plants, respectively. Interestingly, the leaves of both transformed plants had increased levels of phosphoenolpyruvate carboxylase (PEPC) and transformed plants with cAspAT also had increased levels of mAspAT in the leaf. These results suggest that the increased expression of Panicum cAspAT in transgenic tobacco enhances the expression of its endogenous mAspAT and PEPC, and the increased expression of Panicum mAspAT enhances the expression of its endogenous PEPC.

Change of shoot architecture during juvenile-to-adult phase transition in soybean

Juvenile-to-adult phase change is an indispensable event which guarantees a successful life cycle. Phase change has been studied in maize, Arabidopsis and rice, but is mostly unknown in other species. Soybean/Fabaceae plants undergo drastic changes of shoot architecture at the early vegetative stage including phyllotactic change and leaf type alteration from simple to compound. These characteristics make soybean/Fabaceae plants an interesting taxon for investigating vegetative phase change. Following the expansion of two cotyledons, two simple leaves simultaneously emerge in opposite phyllotaxy. The phyllotaxy of the third and fourth leaves is not fixed; both opposite and distichous phyllotaxis are observed within the same population. Leaves were compound from the third leaf. But the third leaf was rarely simple. Morphological and quantitative changes in early vegetative phase were recognized in leaf size, leaf shape, number of trichomes, stipule size and shape, and shoot meristem shape. Two microRNA genes, miR156 and miR172, are known to be associated with vegetative phase change. Examination of the expression level revealed that miR156 expression was high in the first two leaves and subsequently down-regulated, and that of miR172 showed the inverse expression pattern. These expression patterns coincided with the case of other species. Taken all data together, the first and second leaves represent juvenile phase, the fifth and upper leaves adult phase, and the third and fourth leaves intermediate stage. Further investigation of soybean phase change would give fruitful understandings on plant development.

The homeobox gene NTH23 of tobacco is expressed in the basal region of leaf primordia

We reported isolation and characterization of a homeobox gene from tobacco, NTH23. The homeodomain structure of NTH23 was highly homologous to the same regions of class 2 genes of the KN1-type homeobox (sharing more than 85% amino acid identity), but was less similar to class 1 genes of KN1-type. RNA gel blot analysis revealed that NTH23 was expressed in all organs we tested although the gene is primarily expressed in young leaves. To determine more precisely the spatial expression pattern of NTH23 in tobacco, a chimeric NTH23::GUS fusion gene was introduced into tobacco. The signal of GUS activity was observed at the basal part of leaf blade primordia in the NTH23::GUS transgenic tobacco plants. This observation suggests the possibility that NTH23 may be important for the lateral growth of leaf blades.

Expression patterns of cytoplasmic pyruvate, orthophosphate dikinase of rice (C3) and maize (C4) in a C3 plant, rice

Two types of mRNAs are transcribed from the C4-type pyruvate, orthophosphate dikinase gene (Pdk) with different sizes, which encode chloroplastic and cytoplasmic forms of the enzyme. The two transcripts are pro duced by two independent promoters and this unusual dual promoter system is also found in the C4-like Pdk gene of the C3 plant, rice. In order to elucidate the expression pattern of the cytoplasmic transcript from the maize C4-type and rice C4-like Pdk genes, we have produced chimeric constructs with the s-glucuronidase (GUS) reporter gene under the control of the cytoplasmic promoters and introduced the constructs into rice. Both cytoplasmic promoters directed GUS expression in non-photosynthetic organs, such as endosperm and roots, in transgenic rice plants, while expression was low in photosynthetic organs. These results indicate that the organ-specific localization of the cyto-plasmic enzyme is similar in C3 and C4 plants. The results also suggest the possibility that the cytoplasmic enzyme has a similar function(s) in non-photosynthetic organs both in C3 and C4 plants.