The role of OsCOM1 in homologous chromosome synapsis and recombination in rice meiosis

COM1/SAE2 is a highly conserved gene from yeast to higher eukaryotes. Its orthologs, known to cooperate with the MRX complex (Mre11/Rad50/Xrs2), are required for meiotic DNA double‐strand break (DSB) ends resection and specific mitotic DSB repair events. Here, the rice (Oryza sativa, 2n = 2x = 24) COM1/SAE2 homolog was identified through positional cloning, termed OsCOM1. Four independent mutants of OsCOM1 were isolated and characterized. In Oscom1 mutants, synaptonemal complex (SC) formation, homologous pairing and recombination were severely inhibited, whereas aberrant non‐homologous chromosome entanglements occurred constantly. Several key meiotic proteins, including ZEP1 and OsMER3, were not loaded normally onto chromosomes in Oscom1 mutants, whereas the localization of OsREC8, PAIR2 and PAIR3 seemed to be normal. Moreover, OsCOM1 was loaded normally onto meiotic chromosomes in Osrec8, zep1 and Osmer3 mutants, but could not be properly loaded in Osam1, pair2 and OsSPO11‐1^RNAi plants. These results provide direct evidence for the functions of OsCOM1 in promoting homologous synapsis and recombination in rice meiosis.     

The auxin response factor,OsARF19, controls rice leaf angles through positively regulating OsGH3‐5 and OsBRI1

Auxin and brassinosteroid (BR) are important phytohormones for controlling lamina inclination implicated in plant architecture and grain yield. But the molecular mechanism of auxin and BR crosstalk for regulating lamina inclination remains unknown. Auxin response factors (ARFs) control various aspects of plant growth and development. We here report that OsARF19‐overexpression rice lines show an enlarged lamina inclination due to increase of its adaxial cell division. OsARF19 is expressed in various organs including lamina joint and strongly induced by auxin and BR. Chromatin immunoprecipitation (ChIP) and yeast one‐hybrid assays demonstrate that OsARF19 binds to the promoter of OsGH3‐5 and brassinosteroid insensitive 1 (OsBRI1) directing their expression. OsGH3‐5‐overexpression lines show a similar phenotype as OsARF19‐O1. Free auxin contents in the lamina joint of OsGH3‐5‐O1 or OsARF19‐O1 are reduced. OsGH3‐5 is localized at the endoplasmic retieulum (ER) matching reduction of the free auxin contents in OsGH3‐5‐O1. osarf19‐TDNA and osgh3‐5‐Tos17 mutants without erected leaves show a function redundancy with other members of their gene family. OsARF19‐overexpression lines are sensitive to exogenous BR treatment and alter the expressions of genes related to BR signalling. These findings provide novel insights into auxin and BR signalling, and might have significant implications for improving plant architecture of monocot crops.     

Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.     

OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP ( http://rgp.dna.affrc.go.jp ). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3 ; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3 , which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation.     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.     

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

Two homologous regulatory genes, lin-12 and glp-1, have overlapping functions.

Two homologous genes, lin-12 and glp-1, encode transmembrane proteins required for regulatory cell interactions during C. elegans development. Based on their single mutant phenotypes, each gene has been thought to govern a distinct set of cell fates. We show here that lin-12 and glp-1 are functionally redundant during embryogenesis: Unlike either single mutant, the lin-12 glp-1 double mutant dies soon after hatching. Numerous cellular defects can be observed in these Lag (for lin-12 and glp-1) double mutants. Furthermore, we have identified two genes, lag-1 and lag-2, that appear to be required for both lin-12 and glp-1-mediated cell interactions. Strong loss-of-function lag mutants are phenotypically indistinguishable from the lin-12 glp-1 double; weak lag mutants have phenotypes typical of lin-12 and glp-1 single mutants. We speculate that the lin-12 and glp-1 proteins are biochemically interchangeable and that their divergent roles in development may rely largely on differences in gene expression.     

The role of aquaporin RWC3 in drought avoidance in rice

Although the discovery of aquaporins in plants has resulted in a paradigm shift in the understanding of plant water relations, the relationship between aquaporins and drought resistance still remains elusive. From an agronomic viewpoint, upland rice is traditionally considered as showing drought avoidance. In the investigation of different morphological and physiological responses of upland rice (Oryza sativa L. spp indica cv. Zhonghan 3) and lowland rice (O. sativa L. spp japonica cv. Xiushui 63) to water deficit, we observed young leaf rolling and the remarkable decline of cumulative transpiration in the upland rice. The expression of water channel protein RWC3 mRNA was increased in upland rice at the early response (up to 4 h) to the 20% polyethylene glycol (PEG) 6000 treatment, whereas there was no significant expression changes in lowland rice. Protein levels were increased in upland rice and decreased in lowland rice at 10 h after the water deficit. The up-regulation of RWC3 in upland rice fits well with the knowledge that upland rice adopts the mechanism of drought avoidance. The physiological significance of this RWC3 up-regulation was then explored with the over-expression of RWC3 in transgenic lowland rice (O. sativa L. spp japonica cv. Zhonghua 11) controlled by a stress-inducible SWPA2 promoter. Compared to the wild-type plant, the transgenic lowland rice exhibited higher root osmotic hydraulic conductivity (Lp), leaf water potential and relative cumulative transpiration at the end of 10 h PEG treatment. These results indicated that RWC3 probably played a role in drought avoidance in rice.     

LONGIFOLIA1 and LONGIFOLIA2, two homologous genes, regulate longitudinal cell elongation in Arabidopsis.

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2,in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis.     

Expression pattern and putative function of EXL1 and homologous genes in Arabidopsis

The Arabidopsis EXORDIUM-LIKE1 (EXL1) gene (At1g35140) is required for adaptation to carbon (C)- and energy-limiting growth conditions. An exl1 loss of function mutant showed diminished biomass production in a low total irradiance growth regime, impaired survival during extended night, and impaired survival of anoxia stress. We show here additional expression data and discuss the putative roles of EXL1. We hypothesize that EXL1 suppresses brassinosteroid-dependent growth and controls C allocation in the cell. In-depth expression analysis of homologous genes suggests that the EXL2 (At5g64260) and EXL4 (At5g09440) genes play similar roles.     

OsHAP3 genes regulate chloroplast biogenesis in rice

We have isolated three genes that potentially encode a HAP3/nuclear factor-YB (NF-YB)/CCAAT binding factor-A (CBF-A) subunit of a CCAAT-box binding complex in rice (Oryza sativa), and named them OsHAP3A, OsHAP3B and OsHAP3C. These genes were expressed in various organs including leaves. In the transgenic rice plants with antisense or RNAi construct of OsHAP3A, reduced expression of not only OsHAP3A but also OsHAP3B and OsHAP3C was observed. These plants had pale green leaves, in which the amount of chlorophyll was reduced and chloroplasts were degenerated. Lamella was not well developed and accumulation of starch was not detected. The degenerated chloroplast formation was accompanied by reduced expression of nuclear-encoded photosynthesis genes such as RBCS and CAB, while expression of chloroplast-encoded genes was not affected or rather increased. These results suggest that one or more OsHAP3 genes regulate the expression of nuclear-encoded chloroplast-targeted genes and normal development of chloroplasts.     

Chloroplastic and mitochondrial GPX genes play a critical role in rice development

Plant glutathione peroxidases (GPX) catalyze the reduction of H₂O₂ or organic hydroperoxides to water, mitigating the toxicity of these compounds to cells. In rice plants, the GPX gene family is composed of five members that are distributed in a range of sub-cellular compartments including cytosol, mitochondria, chloroplasts, or endoplasmic reticulum. Of these, OsGPX1 and OsGPX4 are located in mitochondria and chloroplasts, respectively. To understand the role of these GPX in rice, the effect of knockdown of OsGPX1 and OsGPX4 in rice plants was evaluated. Our data show that OsGPX4 was essential for in vitro rice regeneration because no plants were obtained from calli carrying a hairpin construct against OsGPX4. Although the knockdown of OsGPX1 did not impair plant regeneration, the plants with silenced OsGPX1 (GPX1s plants) showed reduced shoot length and a reduced number of seeds compared to the non-transformed rice plants. These results indicate that OsGPX1 and OsGPX4 are essential for redox homeostasis which leads to normal growth and development of rice.     

Genetic interaction between 2 tillering genes, reduced culm number 1 (rcn1) and tillering dwarf gene d3, in rice

Mutant genes, reduced culm number 1 (rcn1) and bunketsuwaito tillering dwarf (d3), affect tiller number in rice (Oryza sativa L.) in opposite directions. The d3 mutant was reported to increase tiller number and reduce plant stature. Our objective was to compare the phenotype of the d3rcn1 double mutant with each single mutant and parental rice cultivar "Shiokari" and to clarify whether the Rcn1 gene interacted with the D3 gene. We recovered a new rcn1 mutant from Shiokari and developed d3rcn1 double mutant with Shiokari genetic background. A new rcn1 mutant, designated as "S-97-61" exhibited a reduction in tiller number and plant stature to about the same level as the previously reported original rcn1 mutant. Three near-isogenic lines, rcn1 mutant, d3 mutant, and d3rcn1 double mutant, were grown together with the parental Shiokari. The reduction in tillering by the rcn1 mutation was independent of the d3 genotype, and tillering number of d3rcn1 double mutant was between those of the d3 and rcn1 mutants. These results demonstrated that the Rcn1 gene was not involved in the D3-associated pathway in tillering control.     

A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans

The homeotic genes of the bithorax complex (BX-C) and the Antennapedia complex (ANT-C) of Drosophila appear to specify the developmental fate of segments or parts of segments of the fly. We have previously reported weak DNA sequence homology between 3' portions of the Antennapedia and fushi tarazu genes of the ANT-C and the Ultrabithorax gene of the BX-C. Here we show that this DNA homology (the homeo box) is due to a conserved protein-coding sequence present in these three pattern-formation genes. Thus the functional homology between these developmental controlling genes is reflected in a structural homology in their gene products. The homeo box sequence is also present in a few copies in the genomes of some other invertebrates, and is even conserved in vertebrate genomes, including the human genome. Apparently at least a part of these developmental switch genes from Drosophila is highly conserved during evolution, and might perform an analogous function in many metazoans .