Transformation and Functional Expression of the rFCA-RRM2 Gene in Rice

The primary aim of the present study was to investigate the overexpression of the rice (Oryza sativa L. subsp, japonica var. Zhonghua 11) flowering control gene (rFCA-RRM2) in monocotyledonous model rice. Constitutive expression of rFCA-RRM2 from the Actl-5 rice promoter caused late flowering in transgenic rice and increased grain weight that was more than 50% higher than that of control plants, which is the first demonstration of rFCA-RRM2 being able to increase rice production. Late flowering was accompanied by strong phenotype and some morphological modifications. These observations suggest that rFCA-RRM2 is a useful tool for phenotype improvement and yield enhancement in cereal crops.     

Functional conservation and divergence of FVE genes that control flowering time and cold response in rice and Arabidopsis

Recent molecular and genetic studies in rice, a short-day plant, have elucidated both conservation and divergence of photoperiod pathway genes and their regulators. However, the biological roles of rice genes that act within the autonomous pathway are still largely unknown. In order to better understand the function of the autonomous pathway genes in rice, we conducted molecular genetic analyses of OsFVE, a rice gene homologous to Arabidopsis FVE. OsFVE was found to be ubiquitously expressed in vegetative and reproductive organs. Overexpression of OsFVE could rescue the flowering time phenotype of the Arabidopsis fve mutants by up-regulating expression of the SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and down-regulating FLOWERING LOCUS C (FLC) expression. These results suggest that there may be a conserved function between OsFVE and FVE in the control of flowering time. However, OsFVE overexpression in the fve mutants did not rescue the flowering time phenotype in in relation to the response to intermittent cold treatment.     

Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana

CONSTANS (CO) is an important floral regulator in the photoperiod pathway, integrating the circadian clock and light signal into a control for flowering time. It is known that CO promotes flowering in Arabidopsis under long-day conditions. CONSTANS-LIKE 9 (COL9) is a member of the CONSTANS-LIKE gene family, encoding a nuclear protein. The expression of COL9 is regulated by the circadian clock in the photoperiod pathway and is detected in various organs. Unexpectedly, overexpression of COL9 in transgenic Arabidopsis resulted in delayed flowering, while co-suppression lines and a transferred DNA (T-DNA) knockout line showed earlier flowering under long-day conditions. Overexpression of COL9 did not enhance the late-flowering phenotype in a co mutant background. Double overexpressors produced by overexpression of CO in COL9 transgenic lines showed an early flowering phenotype similar to single CO overexpressors. The pattern of oscillation of a number of circadian-associated genes remained unchanged in the COL9 transgenic lines. Compared with wild-type plants, the abundance of CO and FLOWERING LOCUS T (FT) mRNA was reduced in the COL9 overexpression lines. Our results indicate that COL9 is involved in regulation of flowering time by repressing the expression of CO, concomitantly reducing the expression of FT and delaying floral transition.     

Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant)

The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.     

The differential expression of HvCO9, a member of the CONSTANS-like gene family, contributes to the control of flowering under short-day conditions in barley

HvCO9 was characterized to elucidate the barley flowering control mechanisms and to investigate the functional diversification of the barley CONSTANS-like (CO-like) genes in flowering. HvCO9 was located on the same chromosome, 1HL, as Ppd-H2 (HvFT3), which is a positive regulator of short-day (SD) flowering. A phylogenetic analysis showed that HvCO9 was located on the same branch of the CO-like gene tree as rice Ghd7 and the barley and wheat VRN2 genes, which are all negative regulators of flowering. High level HvCO9 expressions were observed under SD conditions, whereas its expression levels were quite low under long-day (LD) conditions. HvCO9 expression correlated with HvFT1 and HvFT2 expression under SD conditions, although no clear effect of HvCO9 on HvFT3 expression, or vice versa, under SD conditions was observed. The over-expression of HvCO9 in rice plants produced a remarkable delay in flowering. In transgenic rice, the expression levels of the flowering-related Ehd1 gene, which is a target gene of Ghd7, and its downstream genes were suppressed, causing a delay in flowering. These results suggest that HvCO9 may act as a negative regulator of flowering under non-inductive SD conditions in barley; this activity is similar to that of rice Ghd7 under non-inductive LD conditions, but the functional targets of these genes may be different. Our results indicate that barley has developed its own pathways to control flowering by using homologous genes with modifications for the timing of expression. Further, it is hypothesized that each pathway may target different genes after gene duplication or species diversification.     

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.     

Genome-wide DNA polymorphism and transcriptome analysis of an early-maturing rice mutant

In order to develop a rice population with improved important traits such as flowering time, we developed 2,911 M₂ targeting-induced local lesions in genomes (TILLING) lines by irradiating rice seeds with γ-rays. In all, 15 M₃ lines were obtained from 3 different M₂ lines that exhibited an early-maturing phenotype: these plants matured approximately 25 days faster than wild-type (WT) plants. To identify genome-wide DNA polymorphisms, we performed whole-genome resequencing of both the plant types, i.e., WT and early-maturing TILLING 1 (EMT1), and obtained mapped reads of 118,488,245 bp (99.53 %) and 128,489,860 bp (99.72 %), respectively; Nipponbare was used as the reference genome. We obtained 63,648 and 147,728 single nucleotide polymorphisms (SNPs) and 33,474 and 31,082 insertions and deletions (InDels) for the WT and EMT1, respectively. Interestingly, there was a higher number of SNPs (2.6-fold) and slightly lower number of InDels (0.9-fold) in EMT1 than in WT. The expression of at least 202 structurally altered genes was changed in EMT1, and functional enrichment analysis of these genes revealed that their molecular functions were related to flower development. These results might provide a critical insight into the regulatory pathways of rice flowering.     

Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.)

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.     

Ef7 encodes an ELF3-like protein and promotes rice flowering by negatively regulating the floral repressor gene Ghd7 under both short- and long-day conditions

Much progress has been made in our understanding of photoperiodic flowering of rice and the mechanisms underlying short-day (SD) promotion and long-day (LD) repression of floral induction. In this study, we identified and characterized the Ef7 gene, one of the rice orthologs of Arabidopsis EARLY FLOWERING 3 (ELF3). The ef7 mutant HS276, which was induced by γ-irradiation of the japonica rice cultivar 'Gimbozu', flowers late under both SD and LD conditions. Expression analyses of flowering time-related genes demonstrated that Ef7 negatively regulates the expression of Ghd7, which is a repressor of the photoperiodic control of rice flowering, and consequently up-regulates the expression of the downstream Ehd1 and FT-like genes under both SD and LD conditions. Genetic analyses with a non-functional Ghd7 allele provided further evidence that the delayed flowering of ef7 is mediated through the Ghd7 pathway. The analysis of light-induced expression of Ghd7 revealed that the ef7 mutant was more sensitive to red light than the wild-type plant, but the gate of Ghd7 expression was unchanged. Thus, our results show that Ef7 functions as a floral promoter by repressing Ghd7 expression under both SD and LD conditions.     

Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants

Five rice homeobox (OSH) genes were overexpressed under the control of the cauliflower mosaic virus 35S promoter or the rice actin gene promoter in transgenic rice plants. Almost all of the transgenic plants showed abnormal phenotypes, which could be classified into three types according to their severity. Plants with the most severe phenotype formed only green organs, with many shoot apices on their adaxial sides. Plants with an intermediate phenotype formed bladeless leaves with normally developed leaf sheaths. Plants with a mild phenotype formed normal leaf sheaths and blades, but lacked ligules and showed diffusion of the blade-sheath boundary. The leaf structure of this phenotype was similar to that of dominant maize mutants, such as Kn1, Rs1, Lg3, and Lg4. Based on these phenotypes, we suggest that ectopic expression of the rice OSH genes interferes with the development of leaf blades and maintains leaves in less differentiated states. These results are discussed in relation to the leaf maturation schedule hypothesis (M. Freeling et al., 1992, BioEssays 14, 227-236).     

Circadian-associated rice pseudo response regulators (OsPRRs): insight into the control of flowering time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.     

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.     

Understanding the genetic and epigenetic architecture in complex network of rice flowering pathways

Although the molecular basis of flowering time control is well dissected in the long day (LD) plant Arabidopsis, it is still largely unknown in the short day (SD) plant rice. Rice flowering time (heading date) is an important agronomic trait for season adaption and grain yield, which is affected by both genetic and environmental factors. During the last decade, as the nature of florigen was identified, notable progress has been made on exploration how florigen gene expression is genetically controlled. In Arabidopsis expression of certain key flowering integrators such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) are also epigenetically regulated by various chromatin modifications, however, very little is known in rice on this aspect until very recently. This review summarized the advances of both genetic networks and chromatin modifications in rice flowering time control, attempting to give a complete view of the genetic and epigenetic architecture in complex network of rice flowering pathways.     

水稻C2H2型锌指蛋白基因RZF71的克隆与表达分析

利用生物信息学和RT-PCR方法从水稻幼苗组织中分离了1个新的C2H2型锌指蛋白基因RZF71, 该基因编码一条250个氨基酸残基的多肽, 含有两个典型的C2H2型锌指结构。半定量RT-PCR分析表明: RZF71在根、茎、叶和幼穗中呈组成性表达, 在根中的表达丰度略高; 在高盐和PEG6000胁迫的水稻幼苗组织中, RZF71的表达显著增强, 但低温和ABA处理对该基因的表达量影响不大。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明: RZF71定位于细胞核内。讨论了RZF71可能作为一个转录调控因子在水稻耐高盐和渗透胁迫中的作用。     

OsELF3-1, an Ortholog of Arabidopsis EARLY FLOWERING 3, Regulates Rice Circadian Rhythm and Photoperiodic Flowering

Arabidopsis thaliana EARLY FLOWERING 3 (ELF3) as a zeitnehmer (time taker) is responsible for generation of circadian rhythm and regulation of photoperiodic flowering. There are two orthologs (OsELF3-1 and OsELF3-2) of ELF3 in rice (Oryza sativa), but their roles have not yet been fully identified. Here, we performed a functional characterization of OsELF3-1 and revealed it plays a more predominant role than OsELF3-2 in rice heading. Our results suggest OsELF3-1 can affect rice circadian systems via positive regulation of OsLHY expression and negative regulation of OsPRR1, OsPRR37, OsPRR73 and OsPRR95 expression. In addition, OsELF3-1 is involved in blue light signaling by activating EARLY HEADING DATE 1 (Ehd1) expression to promote rice flowering under short-day (SD) conditions. Moreover, OsELF3-1 suppresses a flowering repressor GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) to indirectly accelerate flowering under long-day (LD) conditions. Taken together, our results indicate OsELF3-1 is essential for circadian regulation and photoperiodic flowering in rice.     

Pollen germination and in vivo fertilization in response to high‐temperature during flowering in hybrid and inbred rice

High‐temperature during flowering in rice causes spikelet sterility and is a major threat to rice productivity in tropical and subtropical regions, where hybrid rice development is increasingly contributing to sustain food security. However, the sensitivity of hybrids to increasing temperature and physiological responses in terms of dynamic fertilization processes is unknown. To address these questions, several promising hybrids and inbreds were exposed to control temperature and high day‐time temperature (HDT) in Experiment 1, and hybrids having contrasting heat tolerance were selected for Experiment 2 for further physiological investigation under HDT and high‐night‐time‐temperature treatments. The day‐time temperature played a dominant role in determining spikelet fertility compared with the night‐time temperature. HDT significantly induced spikelet sterility in tested hybrids, and hybrids had higher heat susceptibility than the high‐yielding inbred varieties. Poor pollen germination was strongly associated with sterility under high‐temperature. Our novel observations capturing the series of dynamic fertilization processes demonstrated that pollen tubes not reaching the viable embryo sac was the major cause for spikelet sterility under heat exposure. Our findings highlight the urgent need to improve heat tolerance in hybrids and incorporating early‐morning flowering as a promising trait for mitigating HDT stress impact at flowering.     

Transformation of rice with the Arabidopsis floral regulator LEAFY causes early heading

Onset of flowering, or heading date, is an important agronomic trait of cereal crops such as rice and early-heading varieties are required for certain regions in which rice is cultivated. Since the floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in dictoyledonous plants, the usefulness of LEAFY for manipulating heading date in rice has been tested. Constitutive expression of LEAFY from the cauliflower mosaic virus 35S promoter caused early flowering in transgenic rice, with a heading date that was 26–34 days earlier than that of wild-type plants. Early flowering was accompanied by a small yield penalty and some panicle abnormality. These observations suggest that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops.