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Dihydroflavonol-4-reductase (DFR) plays a crucial role in anthocyanin biosynthesis. In cultivated soybean [Glycine max (L.) Merr.], the W3 and W4 loci encode DFR1 and DFR2, respectively and are epistatic to each other. In this study, we discovered a new flower color variant of Glycine soja (L.) Merr., CW13133, which has pinkish-white flowers and investigated the genetic and molecular basis of the variation in flower color. We found that the W4 locus encoding DFR2 was responsible for the variation in flower color. Sequence analysis of the DFR2 gene of CW13133 revealed three single-nucleotide polymorphisms (SNPs). Among these, an SNP (T to C) at nucleotide position 2856 was noteworthy, since it led to a change in an amino acid residue (leucine to proline) at position 326 of DFR2. This amino acid substitution was located in a region that is highly conserved among different plant species. The new allele was designated w4-s1. A cleaved amplified polymorphic sequence analysis showed cosegregation of the w4-s1 allele with pinkish-white flowers. Interestingly, DFR1 was not expressed in the w4-s1 mutant, indicating that the W3 locus has no association with flower color variation. Based on a randomized comparative analysis using 10 different purple-flowered accessions of G. soja, we provide evidence for the nonassociation of DFR1 expression with total anthocyanin contents and flower color determination in G. soja. In conclusion, we found that the W3 locus in G. soja does not contribute to flower color determination and consequently there is no epistatic interaction between the W3 and W4 loci, as shown in G. max. [ABSTRACT FROM AUTHOR]

Photoreceptors perceive different wavelengths of light and transduce light signals downstream via a range of proteins. COP1, an E3 ubiquitin ligase, regulates light signaling by mediating the ubiquitination and subsequent proteasomal degradation of photoreceptors such as phytochromes and cryptochromes, as well as various development-related proteins including other light-responsive proteins. COP1 is itself regulated by direct interactions with several signaling molecules that modulate its activity. The control of photomorphogenesis by COP1 is also regulated by its localization to the cytoplasm in response to light. COP1 thus acts as a tightly regulated switch that determines whether development is skotomorphogenic or photomorphogenic. In this review, we discuss the effects of COP1 on the abundance and activity of various development-related proteins, including photoreceptors, and summarize the regulatory mechanisms that influence COP1 activity and stability in plants. [ABSTRACT FROM AUTHOR]

Vivipary, germination of seeds on the maternal plant, is observed in nature and provides ecological advantages in certain wild species, such as mangroves. However, precocious seed germination in agricultural species, such as preharvest sprouting (PHS) in cereals, is a serious issue for food security. PHS reduces grain quality and causes economical losses to farmers. PHS can be prevented by translating the basic knowledge of hormone biology in seeds into technologies. Biosynthesis of abscisic acid (ABA), which is an essential hormone for seed dormancy, can be engineered to enhance dormancy and prevent PHS. Enhancing nine-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting enzyme of ABA biosynthesis, through a chemically induced gene expression system, has successfully been used to suppress germination of Arabidopsis seeds. The more advanced system NCED positive-feedback system, which amplifies ABA biosynthesis in a seed-specific manner without chemical induction, has also been developed. The proofs of concept established in the model species are now ready to be applied to crops. A potential problem is recovery of germination from hyperdormant crop grains. Hyperdormancy induced by the NCED systems can be reversed by inducing counteracting genes, such as NCED RNA interference or gibberellin (GA) biosynthesis genes. Alternatively, seed sensitivity to ABA can be modified to rescue germination using the knowledge of chemical biology. ABA antagonists, which were developed recently, have great potential to recover germination from the hyperdormant seeds. Combination of the dormancy-imposing and -releasing approaches will establish a comprehensive technology for PHS prevention and germination recovery. [ABSTRACT FROM AUTHOR]

Flowering Locus C (FLC) is a key floral repressor that precisely controls flowering time. The role of FLC has been extensively studied at the transcriptional level using molecular biological and epigenetic approaches. However, how FLC functions and how its stability is controlled at the post-translational level are only beginning to be understood. Recent studies show that various post-translational modifications (PTMs) control the stability and activity of FLC. In this review, we focus on three types of PTMs that regulate FLC function: phosphorylation, ubiquitination, and sumoylation. This report should serve as a model to guide post-translational studies of other important floral regulators. [ABSTRACT FROM AUTHOR]

Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening. However, their germination rates increased to wild-type levels after cold stratification or long periods of ripening. In addition, exogenous gibberellin (GA) application improved the germination rates of the siz1 mutants to the wild-type level. In transgenic plants, suppression of AtSIZ1 caused rapid post-translational decay of SLEEPY1 (SLY1), a positive regulator of GA signaling, during germination, and inducible AtSIZ1 overexpression led to increased SLY1 levels. In addition, overexpressing wild-type SLY1 in transgenic sly1 mutants increased their germination ratios to wild-type levels, whereas the germination ratio of transgenic sly1 mutants overexpressing mSLY1 was similar to that of sly1. The germination ratios of siz1 mutant seeds in immature developing siliques were much lower than those of the wild-type. Moreover, SLY1 and DELAY OF GERMINATION 1 (DOG1) transcript levels were reduced in the siz1 mutants, whereas the transcript levels of DELLA and ABSCISIC ACID INSENSITIVE 3 (ABI3) were higher than those of the wild-type. Taken together, these results indicate that the reduced germination of the siz1 mutants results from impaired GA signaling due to low SLY1 levels and activity, as well as hyperdormancy due to high levels of expression of dormancy-related genes including DOG1. [ABSTRACT FROM AUTHOR]

Bacterial leaf blight is one of the major diseases in rice and affects yields. Thus, various methods have been applied to protect rice from this disease. Here, we show systemic translocation of the human drug niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)- 2-hydroxybenzamide) in rice and its long-term effect on prevention of rice leaf blight. The development of Xanthomonas oryzae pv. oryzae-induced rice leaf blight was effectively inhibited in untreated systemic leaves as in niclosamide-treated leaves, although its effect gradually decreased in a time-dependent manner. Time-course examination after niclosamide treatment showed that the niclosamide level was highest after 3 h in non-treated distal leaves, suggesting fast systemic movement of niclosamide from the treated local site to untreated distal regions. Our data indicate that niclosamide controls rice leaf blight by its rapid systemic movement and that its effect is maintained for a long time. [ABSTRACT FROM AUTHOR]

An ethyl methanesulfonate-induced mutant (PE1033) with near-white flowers was isolated from the soybean [Glycine max (L.) Merr.] cultivar Pungsannamul with purple flowers that had the W1w3W4 genotype. In the present study, we aimed to investigate the genetic and molecular bases of the flower color variation displayed by the PE1033 mutant. Analyses of genomic sequences and reverse transcription-polymerase chain reaction (RT-PCR) revealed that the color variation of PE1033 flowers originated from a recessive mutation in the W4 locus that encodes dihydroflavonol 4-reductase (DFR) 2. The new mutant allele, named w4-nw, harbored a single-nucleotide substitution (G to A) at the 5' spliceosome recognition site in the first intron of DFR2, resulting in the failure of splicing and producing two different abnormal transcripts: w4-nw-a and w4-nw-b. The former encoded a truncated DFR2 protein because of the occurrence of a premature stop codon in the first intron, which was retained in the transcript; the latter resulted from alternative splicing of the first intron and encoded a DFR2 protein that lacked 11 amino acid residues in the N-terminus. Derived cleaved amplified polymorphism sequence (dCAPS) analysis revealed a tight cosegregation pattern between the w4-nw mutant allele and near-white color phenotype. In conclusion, the novel mutation in the W4 locus, w4-nw, caused the production of near-white flowers under the w3 allelic background of PE1033. [ABSTRACT FROM AUTHOR]

Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation. [ABSTRACT FROM AUTHOR]

Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)SLY1 complex, are modulated at the posttranslational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than inwild-type plants, but theDELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing amutatedmSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1- 13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants overexpressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 andmSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation. [ABSTRACT FROM AUTHOR]

Crop production and productivity must be increased to provide a balanced diet for the global population. The entire genome sequences of crop species allow the elucidation of genes that regulate important traits related to the final crop seed yield, which frequently depends mainly on seed size. Seed size is a major factor that controls seed quantity and it is strongly affected by various biotic, abiotic and genetic factors. Epigenetic marks in the genome and phytohormones are also important factors affecting seed growth and development. Several genes are known to be involved in the control of seed size, but their interaction and functional characterization have yet to be resolved. In this review, we discuss the different factors that govern seed size in cereal crops and Arabidopsis. [ABSTRACT FROM AUTHOR]

The development of soybean varieties that lack the β-conglycinin α′ subunit is an attractive goal because the β-conglycinin α′ subunit exerts a negative influence on nutrition and tofu gelation, and is also a major allergen. We sought to develop a co-dominant DNA marker for the β-conglycinin α′ subunit ( Cgy- 1) gene for use in marker-assisted selection (MAS). We identified a deleted sequence responsible for the null allele of Cgy- 1 in a soybean variety lacking the β-conglycinin α′ subunit known as 'Keburi'. The deletion spanned 12,998 bp and included Cgy- 1 and its incomplete tandem duplicate on chromosome 10. Based on the Cgy- 1 sequence from both the Williams 82 soybean reference sequence and the Keburi variety, a set of three allele-specific primers was designed for multiplex PCR assay. These primers enabled allelic discrimination by the sizes of PCR products. This amplified two distinct DNA fragments of 913 and 649 bp in Williams 82 and Keburi, respectively. The practicality of the developed co-dominant marker for Cgy- 1 was also confirmed by amplification in five other soybean varieties including three wild types and two mutants. The heterozygosity of the F plants at the Cgy- 1 locus was ascertained using our novel co-dominant marker. This PCR-based co-dominant marker is capable of detecting the presence or absence of β-conglycinin α′ subunit for soybean marker assisted breeding system. [ABSTRACT FROM AUTHOR]

Post-translational covalent modifications by small molecules or peptides remodel target proteins. One such modification, made by ubiquitin or small ubiquitin-related modifier (SUMO), is a rapidly expanding field in cell signaling pathways. Ubiquitin attachment controls the turnover and degradation of target proteins while SUMO conjugation regulates their activity and function. Recent studies report many examples of cross-talk between ubiquitin and SUMO pathways, indicating that the boundary is no longer clear. Here, we review recent progress concerning how ubiquitin and SUMO participate in new regulatory roles in plant cell, and how ubiquitination and sumoylation control plant growth and development. [ABSTRACT FROM AUTHOR]

The zebra-necrosis ( zn) mutant of rice ( Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness. [ABSTRACT FROM AUTHOR]

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3′-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 ( UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNA i construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T1 plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism. [ABSTRACT FROM AUTHOR]

Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the sfaygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase-impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana bentharniana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively. [ABSTRACT FROM AUTHOR]

Desensitization of activated receptors is an important mechanism for terminating signal transduction. Here we show that phytochrome (phy) A, a predominant photoreceptor for seedling deetiolation, colocalizes in nuclear bodies with CONSTITUTIVELY PHOTOMORPHOGENIC (COP) 1, a RING motif-containing E3 ligase. The phyA PAS domain interacts with the COP1 WD40 domain. Both the Pr and the Pfr forms of phyA, as well as the PHYA apoprotein, are ubiquitinated by COP1 in vitro. The phyA destruction rate is decreased in cop1 mutants and by expression of a COP1 RING motif mutant. Our results indicate that COP1 acts as an E3 ligase to regulate phyA signaling by targeting elimination of the phyA photoreceptor itself. [ABSTRACT FROM AUTHOR]

Reports on the significance of the expression of a bifunctional fusion of the Escherichia coli genes for trehalose–6–phosphate synthase and trehalose–6–phosphate phosphatase in transgenic rice plants. Role of trehalose in stress tolerance in plants; Change in the carbohydrate profiles in the seeds; Tolerance of rice to trehalose synthesis.

To study physiological roles of plant small GTP binding proteins, we isolated a cDNA clone (ORrab2) encoding the rab-related small GTP binding protein from rice (Oryza sativa L. IR-36) by using human cDNA rab2 as a probe. The deduced amino acid sequence of the ORrab2 gene shared all the conserved regions, important for GTPase/GTP binding activities, with those of other small GTP binding proteins. ORrab2 is a 1028 bp long cDNA, encodes a 23.2 kDa protein which shows 85.2 % similarity on the amino acid sequence level to the Hrab2 protein, and was used as a probe. Through Southern and Northern blot analyses, we found that ORrab2 is a single copy gene and actively expressed at the stages of cell division and elongation. We investigated GTP binding abilities by a filter assay procedure. Deletion of a binding motif, GDTGVGKS, within an ORrab2 protein showed a significant decrease of GTP binding affinity, suggesting its important role in nucleotide binding. [ABSTRACT FROM AUTHOR]

The ORrab2 gene encodes a GTP-binding. protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%}. Hrab4 (5i.9%}, Hrab1 (46.2%). YPT (40.7ck), Hrab3B (40.0%). Hrab3A (38.1%). SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatogra- phy. The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa. The guanine-nucleo- tide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [γ-35 S]GTP[S]/ tool of protein with a Ka value of 56.83 nM. The ratios koff GDP /koff GTP of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (kcat) of P1 release against [γ-32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [γ-32P]GTP (kGTPase) were calculated to be 432×10-4 ± 8×10-4 min-1 and 172×10-4 ±2 × 10-4 min-1, respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4. [ABSTRACT FROM AUTHOR]