The Gametic Non-Lethal Gene Gal on Chromosome 5 Is Indispensable for the Transmission of the Co-Induced Semidwarfing Gene d60 in Rice.

The gametic lethal gene gal in combination with the semidwarfing gene d60 causes complementary lethality in rice. Here, we attempted to ascertain the existence of gal and clarify male gamete abortion caused by d60 and gal. Through the F2 to F4 generations derived from the cross between D60gal-homozygous and d60Gal-homozygous, progenies of the partial sterile plants (D60d60Galgal) were segregated in a ratio of 1 semidwarf (1 d60d60GalGal):2 tall and quarter sterile (2 D60d60Galgal):6 tall (2 D60d60GalGal:1 D60D60GalGal:2 D60D60Galgal:1 D60D60galgal), which is skewed from the Mendelian ratio of 1 semidwarf:3 tall. However, the F4 generation was derived from fertile and tall heterozygous F2 plants (D60d60GalGal), which were segregated in the Mendelian ratio of 1[semidwarf (d60d60GalGal)]:2[1 semidwarf:3 tall (D60d60GalGal)]:1[tall (D60D60GalGal)]. The backcrossing of D60Gal-homozygous tall F4 plants with Hokuriku 100 resulted in fertile BCF1 and BCF2 segregated in a ratio of 1 semidwarf:3 tall, proving that d60 is inherited as a single recessive gene in the D60d60GalGal genetic background (i.e., in the absence of gal). Further, gal was localized on chromosome 5, which is evident from the deviated segregation of d1 as 1:8 and linkage with simple sequence repeat (SSR) markers. Next-generation sequencing identified the candidate SNP responsible for Gal. In F1 and sterile F2, at the binucleate stage, partial pollen discontinued development. Degraded pollen lost vegetative nuclei, but second pollen mitosis raising two generative nuclei was observed. Thus, our study describes a novel genetic model for a reproductive barrier. This is the first report on such a complementary lethal gene, whose mutation allows the transmission of a co-induced valuable semidwarfing gene d60.

Allelic Differentiation at the E1/Ghd7 Locus Has Allowed Expansion of Rice Cultivation Area.

The photoperiod-insensitivity allele e1 is known to be essential for the extremely low photoperiod sensitivity of rice, and thereby enabled rice cultivation in high latitudes (42-53° north (N)). The E1 locus regulating photoperiod-sensitivity was identified on chromosome 7 using a cross between T65 and its near-isogenic line T65w. Sequence analyses confirmed that the E1 and the Ghd7 are the same locus, and haplotype analysis showed that the e1/ghd7-0a is a pioneer allele that enabled rice production in Hokkaido (42-45° N). Further, we detected two novel alleles, e1-ret/ghd7-0ret and E1-r/Ghd7-r, each harboring mutations in the promoter region. These mutant alleles alter the respective expression profiles, leading to marked alteration of flowering time. Moreover, e1-ret/ghd7-0ret, as well as e1/ghd7-0a, was found to have contributed to the establishment of Hokkaido varieties through the marked reduction effect on photoperiod sensitivity, whereas E1-r/Ghd7-r showed a higher expression than the E1/Ghd7 due to the nucleotide substitutions in the cis elements. The haplotype analysis showed that two photoperiod-insensitivity alleles e1/ghd7-0a and e1-ret/ghd7-0ret, originated independently from two sources. These results indicate that naturally occurring allelic variation at the E1/Ghd7 locus allowed expansion of the rice cultivation area through diversification and fine-tuning of flowering time.

Barley NARROW LEAFED DWARF1 encoding a WUSCHEL-RELATED HOMEOBOX 3 (WOX3) regulates the marginal development of lateral organs.

Barley (Hordeum vulgare L.) is the fourth most-produced cereal in the world and is mainly utilized as animal feed and malts. Recently barley attracts considerable attentions as healthy food rich in dietary fiber. However, limited knowledge is available about developmental aspects of barley leaves. In the present study, we investigated barley narrow leafed dwarf1 (nld1) mutants, which exhibit thin leaves accompanied by short stature. Detailed histological analysis revealed that leaf marginal tissues, such as sawtooth hairs and sclerenchymatous cells, were lacked in nld1, suggesting that narrowed leaf of nld1 was attributable to the defective development of the marginal regions in the leaves. The defective marginal developments were also appeared in internodes and glumes in spikelets. Map-based cloning revealed that NLD1 encodes a WUSCHEL-RELATED HOMEOBOX 3 (WOX3), an ortholog of the maize NARROW SHEATH genes. In situ hybridization showed that NLD1 transcripts were localized in the marginal edges of leaf primordia from the initiating stage. From these results, we concluded that NLD1 plays pivotal role in the increase of organ width and in the development of marginal tissues in lateral organs in barley.

The effects of phytochrome-mediated light signals on the developmental acquisition of photoperiod sensitivity in rice.

Plants commonly rely on photoperiodism to control flowering time. Rice development before floral initiation is divided into two successive phases: the basic vegetative growth phase (BVP, photoperiod-insensitive phase) and the photoperiod-sensitive phase (PSP). The mechanism responsible for the transition of rice plants into their photoperiod-sensitive state remains elusive. Here, we show that se13, a mutation detected in the extremely early flowering mutant X61 is a nonsense mutant gene of OsHY2, which encodes phytochromobilin (PΦB) synthase, as evidenced by spectrometric and photomorphogenic analyses. We demonstrated that some flowering time and circadian clock genes harbor different expression profiles in BVP as opposed to PSP, and that this phenomenon is chiefly caused by different phytochrome-mediated light signal requirements: in BVP, phytochrome-mediated light signals directly suppress Ehd2, while in PSP, phytochrome-mediated light signals activate Hd1 and Ghd7 expression through the circadian clock genes' expression. These findings indicate that light receptivity through the phytochromes is different between two distinct developmental phases corresponding to the BVP and PSP in the rice flowering process. Our results suggest that these differences might be involved in the acquisition of photoperiod sensitivity in rice.

Early embryogenesis-specific expression of the rice transposon Ping enhances amplification of the MITE mPing.

Miniature inverted-repeat transposable elements (MITEs) are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu), mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP), and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP). Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.

Se14, encoding a JmjC domain-containing protein, plays key roles in long-day suppression of rice flowering through the demethylation of H3K4me3 of RFT1.

Floral transition from the vegetative to the reproductive growth phase is a major change in the plant life cycle and a key factor in reproductive success. In rice (Oryza sativa L.), a facultative short-day plant, numerous flowering time and flower formation genes that control floral transition have been identified and their physiological effects and biochemical functions have been clarified. In the present study, we used a Se14-deficient mutant line (HS112) and other flowering mutant lines to investigate the photoperiodic response, chromosomal location and function in the photoperiod sensitivity of the Se14 gene. We also studied the interactive effects of this locus with other crucial flowering time genes. We found that Se14 is independent of the known photoperiod-sensitive genes, such as Hd1 and Ghd7, and is identical to Os03g0151300, which encodes a Jumonji C (JmjC) domain-containing protein. Expression analysis revealed that the expressions of RFT1, a floral initiator known as a "florigen-like gene", and Ehd1 were up-regulated in HS112, whereas this up-regulation was not observed in the original variety of 'Gimbozu'. ChIP assays of the methylation states of histone H3 at lysine 4 (H3K4) revealed that the trimethylated H3K4 in the promoter region of the RFT1 chromatin was significantly increased in HS112. We conclude that Se14 is a novel photoperiod-sensitivity gene that has a suppressive effect on floral transition (flowering time) under long day-length conditions through the modification of chromatin structure by H3K4me3 demethylation in the promoter region of RFT1.

The effects of the photoperiod-insensitive alleles, se13, hd1 and ghd7, on yield components in rice.

Flowering time is closely associated with grain yield in rice (Oryza sativa L.). In temperate regions, seasonal changes in day length (known as the photoperiod) are an important environmental cue for floral initiation. The timing of flowering is important not only for successful reproduction, but also for determining the ideal balance between vegetative growth and reproductive growth duration. Recent molecular genetics studies have revealed key flowering time genes responsible for photoperiod sensitivity. In this study, we investigated the effect of three recessive photoperiod-insensitive alleles, se13, hd1 and ghd7, on yield components in rice under Ehd1-deficient genetic background conditions to ensure vegetative growth of each line. We found that se13-bearing plants had fewer panicles, hd1-bearing plants showed decreased grain-filling percentage, and ghd7-bearing plants appeared to have fewer grains per panicle and fewer secondary branches. Our results indicate that the pleiotropic effects of photoperiod-insensitive genes on yield components are independent of short vegetative growth. This will provide critical information which can be used to create photoperiod-insensitive varieties that can be adapted to a wide range of latitudes.

Wide genetic variation in phenolic compound content of seed coats among black soybean cultivars.

Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.

Transient dual-luciferase assay combined with a glucocorticoid-inducible system for rice protoplasts.

We describe a transient dual-luciferase assay combined with a glucocorticoid-inducible system for rice protoplasts. Luciferase genes were efficiently induced by adding 0.1 µM of dexamethasone to the protoplast suspension, the activity of the luciferases reaching a maximum 6 h after induction. This assay system is applicable to studying the translation efficiency of rice by using the luciferase gene harboring tandem copies of an interesting codon at the 5' end.

Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice.

Transposable elements (TEs) are DNA fragments that have the ability to move from one chromosomal location to another. The insertion of TEs into gene-rich regions often affects changes in the expression of neighboring genes. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome. It has been found to show exceptionally active transposition in a few japonica rice varieties, including Gimbozu, where mPing insertion rendered adjacent genes stress-inducible. In the Gimbozu population, it is highly possible that several genes with modified expression profiles are segregating due to the de novo mPing insertions. In our study, we utilized a screening system for detecting de novo mPing insertions in the upstream region of target genes and evaluated the effect of mPing on the stress response of the target genes. Screening for 17 targeted genes revealed five genes with the mPing insertion in their promoters. In most cases, the alteration of gene expression was observed under stress conditions, and there was no change in the expression levels of those five genes under normal conditions. These results indicate that the mPing insertion can be used as a genetic tool to modify an expression pattern of a target gene under stress conditions without changing the expression profiles of those under natural conditions.

Loss-of-function of a ubiquitin-related modifier promotes the mobilization of the active MITE mPing.

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

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.

Complete loss of photoperiodic response in the rice mutant line X61 is caused by deficiency of phytochrome chromophore biosynthesis gene.

In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35°01'N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis.

Long-culm mutations with dominant genes are induced by mPing transposon in rice.

Development of semidwarf rice cultivars contributed to the epoch of high yielding crops called the 'Green Revolution'. However, over-reliance on semidwarf rice also has intrinsic limitations to supply food for an ever expanding world population. As a solution to the food supply problem, we propose the development of 'tall dwarf' rice cultivars that are characterized by increased biomass with long culms or large grains. However, genetic studies on the elongation of rice culms have remained scarce. This study seeks to analyze mutant genes involved in culm elongation in long-culm mutants induced by the MITE transposon mPing, which has been shown to be active in the japonica cultivar Gimbozu. Through analysis of the experimental results, we have confirmed that the three mutant long-culm genes exhibit genetic dominance. These represent rare cases of artificially induced dominant mutations. It is very likely that the mPing transposons played an important role in inducing the dominant mutations and also play an evolutionary interesting role.

Gene cloning, bacterial expression, and purification of a novel rice (Oryza sativa L.) ubiquitin-related protein, RURM1.

A rice ubiquitin-related modifier-1 (Rurm1) gene was cloned and transformed in Escherichia coli. We successfully expressed the RURM1 protein as a glutathione S-transferase (GST)-fusion protein by cultivating the E. coli cells at 16 degrees C for 16 h. After cleavage of GST, we obtained a single protein of 12 kDa. This protein was identified as the RURM1 protein by western blot analysis.

Co-expression of alpha' and beta subunits of beta-conglycinin in rice seeds and its effect on the accumulation behavior of the expressed proteins.

A transgenic rice that produces both the alpha' and beta subunits of beta-conglycinin has been developed through the crossing of two types of transgenic rice. Although the accumulation level of the alpha' subunit in the alpha'beta-transgenic rice was slightly lower than that in the transgenic rice producing only the alpha' subunit, the accumulation level of the beta subunit in the alpha'beta-transgenic rice was about 60% higher than that in the transgenic rice producing only the beta subunit. Results from sequential extraction and gel-filtration experiments indicated that part of the beta subunit formed heterotrimers with the alpha' subunit in a similar manner as in soybean seeds and that the heterotrimers interacted with glutelin via cysteine residues. These results imply that the accumulation level of the beta subunit in the alpha'beta-transgenic rice increases by an indirect interaction with glutelin. Immunoelectron microscopy revealed that the alpha' and beta subunits are localized in a low electron-dense region of protein body-II (PB-II) and that alpha' homotrimers in the alpha'beta-transgenic rice seeds seem to accumulate outside of this low electron-dense region.

Unexpected consequences of a sudden and massive transposon amplification on rice gene expression.

High-copy-number transposable elements comprise the majority of eukaryotic genomes where they are major contributors to gene and genome evolution. However, it remains unclear how a host genome can survive a rapid burst of hundreds or thousands of insertions because such bursts are exceedingly rare in nature and therefore difficult to observe in real time. In a previous study we reported that in a few rice strains the DNA transposon mPing was increasing its copy number by approximately 40 per plant per generation. Here we exploit the completely sequenced rice genome to determine 1,664 insertion sites using high-throughput sequencing of 24 individual rice plants and assess the impact of insertion on the expression of 710 genes by comparative microarray analysis. We find that the vast majority of transposable element insertions either upregulate or have no detectable effect on gene transcription. This modest impact reflects a surprising avoidance of exon insertions by mPing and a preference for insertion into 5' flanking sequences of genes. Furthermore, we document the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible. As such, this study provides evidence for models first proposed previously for the involvement of transposable elements and other repetitive sequences in genome restructuring and gene regulation.

Identification of a novel gene ef7 conferring an extremely long basic vegetative growth phase in rice.

A late heading-time mutant line, HS276, which was induced by gamma-irradiation of seeds of the japonica rice (Oryza sativa L.) variety Gimbozu, exhibits an extremely long basic vegetative growth phase (BVP). A genetic analysis using the F(2) population from the cross between HS276 and Gimbozu revealed that the late heading of HS276 is governed by a single recessive mutant gene. The subsequent analysis on heading responses of HS276 and Gimbozu to four photoperiods (12, 13, 14, and 15 h) and to the photoperiodic transfer treatment from a short photoperiod to a long photoperiod revealed that the mutant gene confers an extremely long BVP and increases photoperiod sensitivity under long photoperiod (14 and 15 h). The BVP durations of HS276 and Gimbozu were estimated at 30.1 and 16.0 days, respectively; the mutant gene, compared with its wild type allele, elongates the duration of BVP by 14 days. Linkage analysis showed that the mutant gene is located in the 129 kb region between the two INDEL markers, INDELAP0399_6 and INDELAP3487_2, on the distal part of the short arm of chromosome 6. None of the other BVP genes are located in this region; therefore, we declared this a newly detected mutant gene and designated it ef7. A recently established program to breed rice suitable for low latitudes, where short photoperiodic conditions continue throughout the year, aims to develop varieties with extremely long BVPs and weak photoperiod sensitivities; the mutant gene ef7, therefore, will be quite useful in these programs because it confers an extremely long BVP and little enhances photoperiod sensitivity under short photoperiod.

Multiple alleles at Early flowering 1 locus making variation in the basic vegetative growth period in rice (Oryza sativa L.).

A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F(2) and F(3) populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes.

Identification and characterization of the erect-pose panicle gene EP conferring high grain yield in rice (Oryza sativa L.).

The breeding of japonica varieties with erect-pose panicle (EP) has recently progressed in the northern part of China, because these varieties exhibit a far higher grain yield than the varieties with normal-pose panicle (NP). A genetic analysis using the F(2) population from the cross between Liaojing5, the first japonica EP variety in China, and the Japanese japonica NP variety Toyonishiki revealed that EP is governed by a single dominant gene EP. Based on previous studies, map-based cloning of EP locus was conducted using Liaojing5, Toyonishiki, their F(2) population, and a pair of near-isogenic lines for EP locus (ZF14 and WF14) derived from the cross between the two varieties; consequently, the STS marker H90 was found to completely cosegregate with panicle pose. The H90 is located in the coding sequence AK101247 in the database, and the AK101247 of Liaojing5 has a 12 bp sequence in exon 5 replaced with a 637 bp sequence of its wild type allele. It was therefore considered that the AK101247 encodes the protein of the wild type allele at EP locus, and that the sequence substitution in exon 5 of Liaojing5 is crucial for expression of the EP phenotype. The effects of EP gene on agronomic traits were investigated using two pairs of near-isogenic lines (ZF6 vs. WF6 and ZF14 vs. WF14) derived from the cross between the two varieties. Experimental results showed that EP gene markedly enhanced grain yield, chiefly by increasing number of secondary branches and number of grains on the secondary branch. EP gene also produced a remarkable increase in grain density.