ANGUSTIFOLIA (AN), a plant homolog of CtBP/BARS, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. AN has a plant-specific long C-terminal region of about 200 aa. We isolated four homologous genes of AN from the moss Physcomitrella patens (PpAN). While two PpAN proteins (1-1 and 1-2) are homologous to AN, PpAN2-1 and 2-2 have no plant-specific C-terminus. Northern analysis showed as follows. (1) PpAN1-1 and 1-2 were expressed both in protonemata and gametophores. (2) Gene expression level for PpAN1-2 was lower than that forPpAN1-1. (3) Transcripts for PpAN2-1 and 2-2 were more abundant in gametophores than in protonemata. Gene knockout lines with the insertion of the drug-resistant genes were produced for each PpAN gene in addition to generation of the double knockout lines by a gene-targeting technique. Moreover, We are now making the PpAN1-1pro:GUS and PpAN2-1pro:GUS lines. With these results, we discussed functions of PpAN genes in moss.

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CRES-T system (Chimeric REpressor gene-Silencing Technology) is a reliable gene-silencing system using the chimeric transcriptional repressor. Transcription factors can be changed into transcriptional repressors by connecting the transcription repression domain (SRDX). The chimeric repressor dominantly suppresses the target genes. In some cases, it caused a growth defect and a decrease in fertilization. In this study, we developed a new system toward the improvement of genetically modified floricultural plants. The GAL4SRDX, which is an yeast transcription factor with the repression domain, was used to repress the over-expressed chimeric repressor under CaMV35S promoter::GAL4 UAS. The chimeric repressor of AGAMOUS (AGSRDX) which caused double-petaled flower, and the chimeric repressor of DUPLICATE (DPSRDX) which is a homolog of AG in Pharbitis was introduced to Arabidopsis and Pharbitis, respectively. The growth of regenerating shoots of Pharbitis transformed with CaMV35S::DPSRDX was defective. However, using our new system, GAL4SRDX could repress the DPSRDX during redifferentiation, and we succeeded to bloom double-petaled Pharbitis flower.

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Because many transcription factors participate in floral organ development, manipulation of such kind of factors can be a powerful tool for modification of floral traits of horticultural plants. Our recent study using transgenic torenias revealed that utilization of Arabidopsis transcription factors is effective for generation of new floral traits. However, it demands time and effort to find out useful genes by generating transgenic plants one by one. To facilitate this process, we transformed torenias by bulk introduction of 50 different chimeric repressors for Arabidopsis transcription factors which participate in regulation of organ development and/or cell differentiation. In the observations of 402 transgenic plants, we found many attractive phenotypes in petal color and/or shape, such as big corollas, enlarged blotches on ventral petals, curled or serrated petal margins and altered color patterns. These phenotypes seemed more valuable and variegated contrary to our expectations from the putative gene function, suggesting that they will provide novel information on the functions of transcription factors which could not be revealed by previous studies in Arabidopsis.

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While genetic information concerning floral development has been accumulated over the past decade, it seems still insufficient to make us control floral traits in various ornamental flowers. In this study, we analyzed functions of torenia class B genes, TfGLO and TfDEF, which are thought to play important roles in developments of petals and stamens.
TfGLO-overexpressing transgenic plants showed ectopic accumulation of anthocyanin pigments in sepals, while TfDEF-overexpressing plants showed no phenotypic exchange. TfGLO-repressed plants showed distinctive serrations in petal margins, and TfDEF-repressed plants showed partially-decolorized petals. We isolated putative downstream genes of the class B genes, TfXEG and TfCAB, according to a microarray analysis of Arabidopsis, and examined their expressions in the transgenic plants. Expression patterns of the TfXEG and TfCAB genes were similar between TfGLO and TfDEF plants, while the patterns were partly different from results reported in Arabidopsis. These results suggested that there is a functional divergence between TfGLO and TfDEF in floral development, and their functions are partly different from those in Arabidopsis.

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Alleles of rice Dense panicle 1 (Dn1) mutant, which is semi-dwarf and increases spikelet number per plant, were compared. Based on their nucleotide sequences, DN1-1 was presumed to be a truncated protein which lacked most of cysteine-rich region in the C-terminus, and Dn1-3 was predicted to encode only the coiled-coil domain and a NLS. The expression level in shoot apical meristem and/or root tip was two or three times higher in Dn1-1 plant, and more than ten times higher in Dn1-3 plant than wild type. Since expression of DN1, DN1-1, and DN1-3 was driven by the same endogenous promoter, the wild-type mRNA was implied to be more degraded than that of the mutants. In the Nipponbare background, both Dn1-1 and Dn1-3 were semi-dwarf, but only Dn1-1 increased spikelet number.
Using transgenics which overexpressed fusion protein of GFP and DN1, it was clarified that at least DN1-1 contains transmembrane domain and that ca. 4 kDa N-terminus fragment was cleaved and released into the cytoplasm in both wild type and Dn1-1 plants.
Introgression of Dn1-1 allele in different backgrounds by backcrossing revealed that the timing of the effect of this allele depended on the genetic background.

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Previously, we reported on isolation of rice Short grain 1 (Sg1) gene that encodes a novel protein by activation-tagging. SG1:OX rice shows semi-dwarf phenotype with erected leaves reminiscent of brassinosteroid (BR) deficient mutants, and shows BR-insensitive lamina joint phenotype. In this study, we found that SG1:OX rice showed short culm and dense panicle phenotype, in addition to short grain phenotype. Despite short organ phenotype, cellular sizes in organs of SG1:OX rice are similar to those in WT, implicating that reduction of organ size is mainly caused by reduction of cell number. In SG1:OX line, BR-content was comparable with WT, suggesting that BR signaling or response was suppressed. Spatial expression of SG1 was monitored using GUS reporter plants. GUS activity was detected in seedling roots, nodes of stem, and young panicles mainly at lower part of rachises andtheir branches. Considering on the expression pattern and OX phenotype, SG1 may repress elongation of rice panicle by suppression of cell division. To elucidate the role of SG1, we are currently making RNAi plants repressing SG1 and its homologue.

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The differences in grass inflorescence architecture have important implications for grain yield. Previously we demonstrated the amount of expression of APO1 is positively associated with the number of spikelets per panicle in rice. In this study, we performed chemical mutagenesis and screened for phenotypically aberrant panicle. We identified aberrant panicle organization 2 (apo2) mutant exhibiting a short panicle and resembling apo1 mutant. The axis of apo2 had a terminal flower in contrast to that of wild type and the total number of spikelets in apo2 was reduced to 15% of that in wild type. Main-axis meristem at initial stage was narrower than that of the wild type and produced reduced number of primary branch primordia. In addition to inflorescence, apo2 showed pleiotropic phenotypes during vegetative and floral development. By positional cloning, we identified APO2 gene that is mutated in Rice FLORICAULA (RFL). Our analyses indicated that APO1 and APO2 regulated the conversion from inflorescence to floral meristem identity in the same pathway.

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