Leaves represent an important organ in plant photosynthesis, and moderately rolled leaves would be beneficial in establishing an ideal plant architecture and thereby increasing rice yields. In this study, a stable inherited rolled leaf mutant was obtained via ethyl methanesulfonate (EMS) mutagenesis from japonica variety WYJ27, which was named rll2 (rolling leaf 2). rll2 showed a leaf-rolling phenotype at the seedling stage, which increased with growth. Compared with the wild type, the leaves at all levels of rll2 were significantly shorter and narrower, and the leaf-rolling index gradually decreased from the highest leaf to the third-highest leaf. Semi-thin sections showed that the bulliform cells of rll2 were significantly larger than those of the wild type, and the number of cells was significantly higher than that of the wild type. Genetic analysis showed that rll2 is controlled by a pair of recessive nuclear genes. Map-based cloning revealed that RLL2 encodes a conserved and plant-specific calpain-like cysteine proteinase. RLL2 was mainly expressed in young roots, shoots, spikelets, and panicles. Transcriptome sequencing showed that a total of 104 genes were differentially expressed in the wild type and rll2. Moreover, several transcription factor genes were significantly altered in the rll2 mutant. Taken together, our findings indicate that RLL2 plays an important role in leaf rolling by regulating bulliform cells, which may be useful in breeding rice with an ideal plant architecture.

The tiller angle, which is an important agronomic trait, determines plant architecture and greatly influences the grain yield of rice. In this study, a population of chromosome segment substitution lines derived from a cross between a japonica variety with a compact plant architecture—Koshihikari—and an indica variety with a spread-out plant architecture—Nona Bokra—was used to investigate the genetic basis of the tiller angle. Five quantitative trait loci (qTA1, qTA5, qTA9-1, qTA9-2, and qTA11) for the tiller angle were detected on chromosomes 1, 5, 9, 9, and 11 in two different environments. The phenotypic variation in these QTLs ranged from 3.78% to 8.22%. Two pairs of digenic epistatic QTLs were detected in Lingshui. The epistatic interaction explained 15.19% and 13.60% of the phenotypic variance, respectively. Among the five QTLs, qTA9-2 was detected in both environments. An F2 mapping population containing the qTA9-2 QTL was established. The location of qTA9-2 was narrowed down to a 187 kb region between InDel markers M9 and M10 on chromosome 9. Thirty open reading frames (ORFs), including TAC1, a gene known to regulate the tiller angle, were identified in this region. The gene sequencing results suggested that a base substitution from G to A at position 1557 in the 3′-untranslated region led to a difference in the expression of qTA9-2 in Koshihikari and Nona Bokra. These findings provide a potential gene resource for the improvement of rice plant architecture.

BACKGROUND:R2R3-MYB transcription factors belong to one of the largest gene subfamilies in plants, and they are involved in diverse biological processes. However, the role of R2R3-MYB transcription factor subfamily genes in the response of rice (Oryza sativa L.) to salt stress has been rarely reported. RESULTS:In this study, we performed a genome-wide characterization and expression identification of rice R2R3-MYB transcription factor subfamily genes. We identified a total of 117 R2R3-MYB genes in rice and characterized their gene structure, chromosomal location, and cis-regulatory elements. According to the phylogenetic relationships and amino acid sequence homologies, the R2R3-MYB genes were divided into four groups. qRT-PCR of the R2R3-MYB genes showed that the expression levels of 10 genes significantly increased after 3 days of 0.8% NaCl treatment. We selected a high expression gene OsMYB2-115 for further analysis. OsMYB2-115 was highly expressed in the roots, stem, leaf, and leaf sheath. OsMYB2-115 was found to be localized in the nucleus, and the yeast hybrid assay showed that OsMYB2-115 has transcriptional activation activity. CONCLUSION:This result provides important information for the functional analyses of rice R2R3-MYB transcription factor subfamily genes related to the salt stress response and reveals that OsMYB2-115 may be an important gene associated with salt tolerance in rice.

Starch accounts for up to 90% of the dry weight of rice endosperm and is a key determinant of grain quality. Although starch biosynthesis enzymes have been comprehensively studied, transcriptional regulation of starch-synthesis enzyme-coding genes (SECGs) is largely unknown. In this study, we explored the role of a NAC transcription factor, OsNAC24, in regulating starch biosynthesis in rice. OsNAC24 is highly expressed in developing endosperm. The endosperm of osnac24 mutants is normal in appearance as is starch granule morphology, while total starch content, amylose content, chain length distribution of amylopectin and the physicochemical properties of the starch are changed. In addition, the expression of several SECGs was altered in osnac24 mutant plants. OsNAC24 is a transcriptional activator that targets the promoters of six SECGs; OsGBSSI, OsSBEI, OsAGPS2, OsSSI, OsSSIIIa and OsSSIVb. Since both the mRNA and protein abundances of OsGBSSI and OsSBEI were decreased in the mutants, OsNAC24 functions to regulate starch synthesis mainly through OsGBSSI and OsSBEI. Furthermore, OsNAC24 binds to the newly identified motifs TTGACAA, AGAAGA and ACAAGA as well as the core NAC-binding motif CACG. Another NAC family member, OsNAP, interacts with OsNAC24 and coactivates target gene expression. Loss-of-function of OsNAP led to altered expression in all tested SECGs and reduced the starch content. These results demonstrate that the OsNAC24-OsNAP complex plays key roles in fine-tuning starch synthesis in rice endosperm and further suggest that manipulating the OsNAC24-OsNAP complex regulatory network could be a potential strategy for breeding rice cultivars with improved cooking and eating quality.

Rice is a major food crop that sustains approximately half of the world population. Recent worldwide improvements in the standard of living have increased the demand for high-quality rice. Accurate identification of quantitative trait loci (QTLs) for rice grain quality traits will facilitate rice quality breeding and improvement. In the present study, we performed high-resolution QTL mapping for rice grain quality traits using a genotyping-by-sequencing approach. An F-2 population derived from a cross between an elite japonica variety, Koshihikari, and an indica variety, Nona Bokra, was used to construct a high-density genetic map. A total of 3,830 single nucleotide polymorphism markers were mapped to 12 linkage groups spanning a total length of 2,456.4 cM, with an average genetic distance of 0.82 cM. Seven grain quality traits-the percentage of whole grain, percentage of head rice, percentage of area of head rice, transparency, percentage of chalky rice, percentage of chalkiness area, and degree of chalkiness-of the F-2 population were investigated. In total, 15 QTLs with logarithm of the odds (LOD) scores >4 were identified, which mapped to chromosomes 6, 7, and 9. These loci include four QTLs for transparency, four for percentage of chalky rice, four for percentage of chalkiness area, and three for degree of chalkiness, accounting for 0.01%-61.64% of the total phenotypic variation. Of these QTLs, only one overlapped with previously reported QTLs, and the others were novel. By comparing the major QTL regions in the rice genome, several key candidate genes reported to play crucial roles in grain quality traits were identified. These findings will expedite the fine mapping of these QTLs and QTL pyramiding, which will facilitate the genetic improvement of rice grain quality.

Brown rice traits are critical to both grain yield and quality. In the present study, the chromosome segment substitution lines (CSSLs) population derived from a cross between japonica Koshihikari and indica Nona Bokra was used to analyze the brown rice length (BRL), brown rice width (BRW), length–width ratio of brown rice (BLWR), brown rice thickness (BRT), brown rice perimeter (BRP), brown rice area (BRA), thousand-grain weight of brown rice (BRGW), brown rice ratio (BRR), taste value of brown rice (BTV), and water content of brown rice (BWC). Correlations analysis showed that most of the brown rice traits had significant correlations with each other, except for BRR, BTV, and BWC. A total of sixty-one QTLs for these traits were identified under three environments, which mapped to chromosomes 1, 2, 3, 5, 6, 7, 8, 10, 11, and 12, with the LOD ranging from 2.52 to 16.68 and accounting for 2.60 to 25.38% of the total phenotypic variations. Moreover, thirty pairs of epistatic interactions for BRL, BRW, BLWR, BRP, and BRA were estimated and distributed on all chromosomes except 10. These findings will provide a further understanding of the genetic basis of brown rice traits and facilitate the genetic improvement of rice yield and quality through breeding.

Grain size and the endosperm starch content determine grain yield and quality in rice. Although these yield components have been intensively studied, their regulatory mechanisms are still largely unknown. In this study, we show that loss-of-function of OsNAC129, a member of the NAC transcription factor gene family that has its highest expression in the immature seed, greatly increased grain length, grain weight, apparent amylose content (AAC), and plant height. Overexpression of OsNAC129 had the opposite effect, significantly decreasing grain width, grain weight, AAC, and plant height. Cytological observation of the outer epidermal cells of the lemma using a scanning electron microscope (SEM) revealed that increased grain length in the osnac129 mutant was due to increased cell length compared with wild-type (WT) plants. The expression of OsPGL1 and OsPGL2, two positive grain-size regulators that control cell elongation, was consistently upregulated in osnac129 mutant plants but downregulated in OsNAC129 overexpression plants. Furthermore, we also found that several starch synthase-encoding genes, including OsGBSSI, were upregulated in the osnac129 mutant and downregulated in the overexpression plants compared with WT plants, implying a negative regulatory role for OsNAC129 both in grain size and starch biosynthesis. Additionally, we found that the expression of OsNAC129 was induced exclusively by abscisic acid (ABA) in seedlings, but OsNAC129-overexpressing plants displayed reduced sensitivity to exogenous brassinolide (BR). Therefore, the results of our study demonstrate that OsNAC129 negatively regulates seed development and plant growth, and further suggest that OsNAC129 participates in the BR signaling pathway.

Grain size traits are critical agronomic traits which directly determine grain yield, but the genetic bases of these traits are still not well understood. In this study, a total of 154 chromosome segment substitution lines (CSSLs) population derived from a cross between a japonica variety Koshihikari and an indica variety Nona Bokra was used to investigate grain length (GL), grain width (GW), length-width ratio (LWR), grain perimeter (GP), grain area (GA), and thousand grain weight (TGW) under four environments. QTL mapping analysis of six grain size traits was performed by QTL IciMapping 4.2 with an inclusive composite interval mapping (ICIM) model. A total of 64 QTLs were identified for these traits, which mapped to chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, and 12 and accounted for 1.6%-27.1% of the total phenotypic variations. Among these QTLs, thirty-six loci were novel and seven QTLs were identified under four environments. One locus containing the known grain size gene, qGL3/ GL3.1/OsPPKL1, also have been found. Moreover, five pairs of digenic epistatic interactions were identified except for GL and GP. These findings will facilitate fine mapping of the candidate gene and QTL pyramiding to genetically improve grain yield in rice.

Rice with low amylose content (AC) is popular due to the unique eating and cooking quality, but its transparency is poor. It is unclear why low AC rice has poor transparency until now. In this study, the transparency and microstructure of brown rice kernels were investigated in rices with different ACs. Chalkiness of endosperm resulted from the loosely packed starch granules, and existed in rice kernels with low and high moisture. However, dry kernels with low AC were opaque or dull due to the cavities in the center of starch granules, and could become transparent gradually with the increase of moisture. For rices with the same genetic background but different ACs, the cavity size was negatively correlated with AC, and the transparency of dry kernels was significantly correlated positively with AC and negatively with cavity size. The transparency of kernels was positively correlated with moisture of kernel, and the effect of moisture on transparency was significantly correlated positively with starch cavity size and negatively with kernel AC. The above results showed that the transparency of low AC rice was remarkably influenced by cavity size and AC of starch, and could be regulated through controlling the moisture of kernel.

The relationship between starch components and hydrolysis previously focused on starches with different amylose contents (ACs) and amylopectin structures. In this study, starches were isolated from six rice seeds with the same genetic background but different activities of granule-bound starch synthase I. The starch components and enzyme hydrolysis were measured and analysed to reveal their relationships. The results showed that the six rice starches had the same amylopectin structure but different ACs ranging from approximately 1%–15%. The starch component parameters measured by different methods were significantly correlated with each other. The hydrolysis parameters of starch by both α-amylase and amyloglucosidase were analysed using three methods including a non-linear fit to the degree of hydrolysis, a first-order kinetics fit to the degree of hydrolysis, and a first-order kinetics fit to the hydrolysis rate, and they had significant correlation each other. Waxy starch had a fast hydrolysis rate and showed a single hydrolysis phase during early hydrolysis, while starch with a high AC had a bi-phasic hydrolysis process. Starch component parameters including the maximum absorption wavelength, iodine blue, AC, and low molecular weight fraction were significantly negatively correlated to the hydrolysis rate of starch. The above results indicated that starches with different ratios of amylose to amylopectin but the same amylopectin structure had significantly different hydrolysis properties. This study could provide some information for rice quality breeding and starch applications through changing the activity of granule-bound starch synthase I.

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.

Starch composition and the amount in endosperm, both of which contribute dramatically to seed yield, cooking quality, and taste in cereals, are determined by a series of complex biochemical reactions. However, the mechanism regulating starch biosynthesis in cereal seeds is not well understood. This study showed that OsbZIP58, a bZIP transcription factor, is a key transcriptional regulator controlling starch synthesis in rice endosperm. OsbZIP58 was expressed mainly in endosperm during active starch synthesis. osbzip58 null mutants displayed abnormal seed morphology with altered starch accumulation in the white belly region and decreased amounts of total starch and amylose. Moreover, osbzip58 had a higher proportion of short chains and a lower proportion of intermediate chains of amylopectin. Furthermore, OsbZIP58 was shown to bind directly to the promoters of six starch-synthesizing genes, OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb, and ISA2, and to regulate their expression. These findings indicate that OsbZIP58 functions as a key regulator of starch synthesis in rice seeds and provide new insights into seed quality control.

Granule-bound starch synthase 1 (GBSS1) is responsible for amylose synthesis in cereals, and this enzyme is regulated at the transcriptional and post-transcriptional levels. In this study, we show that GBSS1 from Oryza sativa L. (OsGBSS1) can form oligomers in rice endosperm, and oligomerized OsGBSS1 exhibits much higher specific enzymatic activity than the monomer. A monomer–oligomer transition equilibrium for OsGBSS1 occurs in the endosperm during development. Redox potential is a key factor affecting the oligomer percentage as well as the enzymatic activity of OsGBSS1. Adenosine diphosphate glucose, the direct donor of glucose, also impacts OsGBSS1 oligomerization in a concentration-dependent manner. OsGBSS1 oligomerization is influenced by phosphorylation status, which was strongly enhanced by Mitogen-activated protein kinase (MAPK) and ATP treatment and was sharply weakened by protein phosphatase (PPase) treatment. The activity of OsGBSS1 affects the ratio of amylose to amylopectin and therefore the eating quality of rice. Understanding the regulation of OsGBSS1 activity may lead to the improvement of rice eating quality.

For macromolecular compounds(e.g.protein),it is necessary to form correct three-dimensional structure for performing functions in vivo.As the primary tool,crystallization coupled with X-ray diffraction,NMR and electron microscopy is widely utilized in data analysis of macromolecular three-dimensional structures.Along with the improvement of these technologies,co-crystallization of protein-ligand complexes has been accomplished.It’s a multi-step process for genetic information transduction from DNA(or RNA) to protein,which was catalyzed by enzymes or protein complexes.The solved structures of these enzymes have been promoting intensive study in this field.The application of crystallization technology in structure-based functional property analysis of the main enzymes or protein complexes which play roles during the genetic information transduction will be expounded.

In order to isolate genes and their promoters in rice,we created a promoter trap system containing the promoterless β-glucuronidase(GUS) reporter gene in the T-DNA region.113# was further analysisied,in which the GUS reporter gene was expressed constitutively.A single copy of the T-DNA was inserted into the plant genome,and the flanking sequence around T-DNA was isolated by inverse PCR.Sequencing and BLAST analyses suggested the T-DNA was inserted reversely in a candidate gene of rice chromosome 4.The promoter of the candidate gene directed GUS expression constitutively,analogous to the GUS expression pattern observed in 113#.The results confirmed that the candidate gene was trapped by T-DNA(GUS) structure.

OsRRMh, a homologue of OsRRM, encodes a Spen-like protein, and is composed of two N-terminal RNA recognition motifs (RRM) and one C-terminal Spen paralogue and an orthologue C-terminal domain (SPOC). The gene has been found to be constitutively expressed in the root, stem, leaf, spikelet, and immature seed, and alternative splicing patterns were confirmed in different tissues, which may indicate diverse functions for OsRRMh. The OsRRMh dsRNAi lines exhibited late-flowering and a larger panicle phenotype. When full-length OsRRMh and/or its SPOC domain were overexpressed, the fertility rate and number of spikelets per panicle were both markedly reduced. Also, overexpression of OsRRMh in the Arabidopsis fpa mutant did not restore the normal flowering time, and it delayed flowering in Col plants. Therefore, we propose that OsRRMh may confer one of its functions in the vegetative-to-reproductive transition in rice (Oryza sativa L. subsp. japonica cv. Zhonghua No. 11 (ZH11)).

A high performance ion exchange chromatographic method coupled with pulsed amperometric detection was established for the analysis of amylopectin chain-length distribution.The amylopectin was hydrolysised into unit chain under the function of isoamylase which can broke the α-1,6 chain of glucan.The determination method was optimized by comparing different chromatographic columns and elution conditions.Water,100 mmol/L NaOH and 1 mol/L NaAc+100 mmol/L NaOH aqueous solution were used as mobile phase,and the analysis of degree of polymerization(DP) more than 80 could be achieved by using CarboPac PA20 anion exchange column.Maize,cassava,potato and rice starch were selected as the example for the determination of chain-length distribution,which could be used to judge their molecular configurations.This method was proved to be simple,extensive and reproducible,and the RSD was less than 5% when the DP was under 65.The method could provide the basis for further research of physical and chemical properties of starch and breed improvement.

Starch is made up of amylose (linear α-1,4 polyglucans) and amylopectin (polyglucans linked together mainly by α-1,4 linkages but with 5%?6% of (α-1,6) bonds at the branch points). Amylose exists as a linear and random form but amylopectin is the main component of starch semi-crystal structure. Except for AG-Pase which charges for the synthesis of ADP-Glc (the glucose direct donor), the synthesis of amylose is under GBSSI’s control. But for amylopectin synthesis, more enzymes are involved in, such as SS, SBE, DBE, SP etc. In this paper, we reviewed the research development on enzymes involved in starch synthesis pathway, and what’s more, we give our view on the research prospect in this field.

Xieyou 57,an elite indica hybrid between the male-sterile line Xieqingzao A and the restorer line 057,owns high yield and wide adaptability.The high amylose content(AC) in the endosperm of both parents relatively results in poor cooking and eating quality of the hybrid.057(the restorer line,designated as 057(GG)) and XieqingzaoA(the male-sterile line,designated as XA(GG)) on Wx gene was improved.The AC eating quality and granule-bound starch synthase(GBSS) activity of all the four combinations were analyzed.The results revealed that the AC of the single parent improved hybrids,Xieqingzao A(GG) 057(TT) and Xieqingzao A(TT) 057(GG) were 19.9% and 19.3%,respectively.Both were lower than that of original Xieyou 57(GG,26.2%).Result indicated that the improved line of hybrid with homozygous TT-Wx-Xieqingzao A(TT)×057(TT),not only showed a decreased AC(12.5%),but also had better uniform AC for different seeds and good cooking and eating quality.The GBSS activity was tightly related to AC,and the three Wx genotype showed GGGTTT.