The OsNAC24-OsNAP protein complex activates OsGBSSI and OsSBEI expression to fine-tune starch biosynthesis in rice endosperm.

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.

Evaluation of oilseed proteins as precursors of antimicrobial peptides using bioinformatics method.

In this study, the potential of oilseed proteins from soybean, peanut, sesame, sunflower seed and flaxseed as antimicrobial peptide (AMP) precursors was assessed using the bioinformatics method. Thirty-four novel potential AMPs were obtained by in silico hydrolysis of 12 oilseed protein sequences, and 11 of them were positive in all four algorithm tests in CAMPR3. Among the six proteases analyzed, trypsin cleaved soybean, peanut, sesame and sunflower seed proteins most effectively to generate AMPs, with three, four, two and two AMPs obtained, respectively. Subtilisin was most effective for flaxseed AMPs release, obtaining three AMPs. More than 85% of AMPs were predicted to be cationic peptides, and some AMPs were hydrophobic. These potential AMPs were classified as non-toxic peptides, and 15 peptides were non-allergenic. All the AMPs were unstable to digestive enzymes according to in silico simulated digestion. The results of this study provide a theoretical basis for further development of AMPs using oilseed proteins.

OsNAC129 Regulates Seed Development and Plant Growth and Participates in the Brassinosteroid Signaling Pathway.

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.

Integrated Multi-Omics Perspective to Strengthen the Understanding of Salt Tolerance in Rice.

Salt stress is one of the major constraints to rice cultivation worldwide. Thus, the development of salt-tolerant rice cultivars becomes a hotspot of current rice breeding. Achieving this goal depends in part on understanding how rice responds to salt stress and uncovering the molecular mechanism underlying this trait. Over the past decade, great efforts have been made to understand the mechanism of salt tolerance in rice through genomics, transcriptomics, proteomics, metabolomics, and epigenetics. However, there are few reviews on this aspect. Therefore, we review the research progress of omics related to salt tolerance in rice and discuss how these advances will promote the innovations of salt-tolerant rice breeding. In the future, we expect that the integration of multi-omics salt tolerance data can accelerate the solution of the response mechanism of rice to salt stress, and lay a molecular foundation for precise breeding of salt tolerance.

High-resolution quantitative trait locus mapping for rice grain quality traits using genotyping by sequencing.

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 F2 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 F2 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.

Advances in Sensing, Response and Regulation Mechanism of Salt Tolerance in Rice.

Soil salinity is a serious menace in rice production threatening global food security. Rice responses to salt stress involve a series of biological processes, including antioxidation, osmoregulation or osmoprotection, and ion homeostasis, which are regulated by different genes. Understanding these adaptive mechanisms and the key genes involved are crucial in developing highly salt-tolerant cultivars. In this review, we discuss the molecular mechanisms of salt tolerance in rice-from sensing to transcriptional regulation of key genes-based on the current knowledge. Furthermore, we highlight the functionally validated salt-responsive genes in rice.

Genome-Wide Association Study of Grain Size Traits in Indica Rice Multiparent Advanced Generation Intercross (MAGIC) Population.

Rice grain size plays a crucial role in determining grain quality and yield. In this study, two multiparent advanced generation intercross (MAGIC) populations, DC1 and BIM, were evaluated for grain size across three environments and genotyped with 55K array-based SNP detection and genotype-by-sequencing (GBS), respectively, to identify QTLs and SNPs associated with grain length, grain width, grain length-width ratio, grain thickness, and thousand grain weight. A total of 18 QTLs were identified for the five grain size-related traits and explained 6.43-63.35% of the total phenotypic variance. Twelve of these QTLs colocalized with the cloned genes, GS3, GW5/qSW5, GW7/GL7/SLG7, and GW8/OsSPL16, of which the first two genes showed the strongest effect for grain length and grain width, respectively. Four potential new genes were also identified from the QTLs, which exhibited both genetic background independency and environment stability and could be validated in future studies. Moreover, the significant SNP markers identified are valuable for direct utilization in marker-assisted breeding to improve rice grain size.

Using Heading date 1 preponderant alleles from indica cultivars to breed high-yield, high-quality japonica rice varieties for cultivation in south China.

Heading date 1 (Hd1) is an important gene for the regulation of flowering in rice, but its variation in major cultivated rice varieties, and the effect of this variation on yield and quality, remains unknown. In this study, we selected 123 major rice varieties cultivated in China from 1936 to 2009 to analyse the relationship between the Hd1 alleles and yield-related traits. Among these varieties, 19 haplotypes were detected in Hd1, including two major haplotypes (H8 and H13) in the japonica group and three major haplotypes (H14, H15 and H16) in the indica group. Analysis of allele frequencies showed that the secondary branch number was the major aimed for Chinese indica breeding. In the five major haplotypes, SNP316 (C-T) was the only difference between the two major japonica haplotypes, and SNP495 (C-G) and SNP614 (G-A) are the major SNPs in the three indica haplotypes. Association analysis showed that H16 is the most preponderant allele in modern cultivated Chinese indica varieties. Backcrossing this allele into the japonica variety Chunjiang06 improved yield without decreasing grain quality. Therefore, our analysis offers a new strategy for utilizing these preponderant alleles to improve yield and quality of japonica varieties for cultivation in the southern areas of China.

Genetic Dissection of Leaf Senescence in Rice.

Leaf senescence, the final stage of leaf development, is a complex and highly regulated process that involves a series of coordinated actions at the cellular, tissue, organ, and organism levels under the control of a highly regulated genetic program. In the last decade, the use of mutants with different levels of leaf senescence phenotypes has led to the cloning and functional characterizations of a few genes, which has greatly improved the understanding of genetic mechanisms underlying leaf senescence. In this review, we summarize the recent achievements in the genetic mechanisms in rice leaf senescence.

Fine Mapping of a Novel defective glume 1 (dg1) Mutant, Which Affects Vegetative and Spikelet Development in Rice.

In cereal crops, vegetative and spikelet development play important roles in grain yield and quality, but the genetic mechanisms that control vegetative and spikelet development remain poorly understood in rice. Here, we identified a new rice mutant, defective glume 1 (dg1) mutant from cultivar Zhonghua11 after ethyl methanesulfonate treatment. The dg1 mutant displayed the dwarfism with small, rolled leaves, which resulted from smaller cells and more bulliform cells. The dg1 mutant also had an enlarged leaf angle and defects in brassinosteroid signaling. In the dg1 mutant, both the rudimentary glume and sterile lemma (glumes) were transformed into lemma-like organ and acquired the lemma identity. Additionally, the dg1 mutant produced slender grains. Further analysis revealed that DG1 affects grain size by regulating cell proliferation and expansion. We fine mapped the dg1 locus to a 31-kb region that includes eight open reading frames. We examined the DNA sequence and expression of these loci, but we were not able to identify the DG1 gene. Therefore, more work will be needed for cloning and functional analysis of DG1, which would contribute to our understanding of the molecular mechanisms behind whole-plant development in rice.

A Rice PECTATE LYASE-LIKE Gene Is Required for Plant Growth and Leaf Senescence.

To better understand the molecular mechanisms behind plant growth and leaf senescence in monocot plants, we identified a mutant exhibiting dwarfism and an early-senescence leaf phenotype, termed dwarf and early-senescence leaf1 (del1). Histological analysis showed that the abnormal growth was caused by a reduction in cell number. Further investigation revealed that the decline in cell number in del1 was affected by the cell cycle. Physiological analysis, transmission electron microscopy, and TUNEL assays showed that leaf senescence was triggered by the accumulation of reactive oxygen species. The DEL1 gene was cloned using a map-based approach. It was shown to encode a pectate lyase (PEL) precursor that contains a PelC domain. DEL1 contains all the conserved residues of PEL and has strong similarity with plant PelC. DEL1 is expressed in all tissues but predominantly in elongating tissues. Functional analysis revealed that mutation of DEL1 decreased the total PEL enzymatic activity, increased the degree of methylesterified homogalacturonan, and altered the cell wall composition and structure. In addition, transcriptome assay revealed that a set of cell wall function- and senescence-related gene expression was altered in del1 plants. Our research indicates that DEL1 is involved in both the maintenance of normal cell division and the induction of leaf senescence. These findings reveal a new molecular mechanism for plant growth and leaf senescence mediated by PECTATE LYASE-LIKE genes.

Rational design of high-yield and superior-quality rice.

Rice (Oryza sativa L.) is a staple food for more than half of the world's population. To meet the ever-increasing demand for food, because of population growth and improved living standards, world rice production needs to double by 20301. The development of new elite rice varieties with high yield and superior quality is challenging for traditional breeding approaches, and new strategies need to be developed. Here, we report the successful development of new elite varieties by pyramiding major genes that significantly contribute to grain quality and yield from three parents over five years. The new varieties exhibit higher yield potential and better grain quality than their parental varieties and the China's leading super-hybrid rice, Liang-you-pai-jiu (LYP9 or Pei-ai 64S/93-11). Our results demonstrate that rational design is a powerful strategy for meeting the challenges of future crop breeding, particularly in pyramiding multiple complex traits.

Regulatory Role of OsMADS34 in the Determination of Glumes Fate, Grain Yield, and Quality in Rice.

Grasses produce seeds on spikelets, a unique type of inflorescence. Despite the importance of grass crops for food, the genetic mechanisms that control spikelet development remain poorly understood. In this study, we used m34-z, a new mutant allele of the rice (Oryza sativa) E-class gene OsMADS34, to examine OsMADS34 function in determining the identities of glumes (rudimentary glume and sterile lemma) and grain size. In the m34-z mutant, both the rudimentary glume and sterile lemma were homeotically converted to the lemma-like organ and acquired the lemma identity, suggesting that OsMADS34 plays important roles in the development of glumes. In the m34-z mutant, most of the grains from the secondary panicle branches (spb) were decreased in size, compared with grains from wild-type, but no differences were observed in the grains from the primary panicle branches. The amylose content and gel consistency, and a seed-setting rate from the spb were reduced in the m34-z mutant. Interesting, transcriptional activity analysis revealed that OsMADS34 protein was a transcription repressor and it may influence grain yield by suppressing the expressions of BG1, GW8, GW2, and GL7 in the m34-z mutant. These findings revealed that OsMADS34 largely affects grain yield by affecting the size of grains from the secondary branches.

Fine Mapping Identifies a New QTL for Brown Rice Rate in Rice (Oryza Sativa L.).

BACKGROUND: High yield and quality determine the commercial potential of rice variety. Brown rice rate (BRR) is a key factor ensuring grain yield and quality in rice. So far, there were few reports about the genes that directly controlled the BRR in rice. Therefore, dissecting the genetic mechanism of the BRR genes can facilitate improving effective rice supply or edible grain yield. RESULTS: A double haploid population derived from the cross between Taichung Native 1 (TN1) (an indica variety) and Chunjiang 06 (CJ06) (a japonica variety) was used to investigate the genetic basis of grain milling and appearance traits affecting the BRR. By using a constructed molecular linkage map, four quantitative trait loci (QTLs) for the BRR were detected on chromosomes 1, 8, 9, and 10, respectively. In addition, three QTLs for appearance traits, including grain weight and grain length/width ratio, were detected on chromosomes 6, 9 and 10, respectively. Chromosome segment substitution lines (CSSLs) were established at the qBRR-10 locus. Finally, the qBRR-10 was narrowed to a 39.5 kb region on chromosome 10. In this region, two candidate genes, LOC_Os10g32124 and LOC_Os10g32190, showed significantly differential expression in TN1 and CSSL1-2 compared with CJ06. Histocytological analysis suggested that cell size and hull thickness may be important factors for the BRR. CONCLUSION: In the study, the qBRR-10 affected the BRR and was finally located to a region between two markers, P13 and P14. Two candidate genes were selected based on the expression difference between two parents, which facilitated the further cloning of the qBRR-10 gene and largely contributed to improve the grain yield and quality in rice.

The pleiotropic ABNORMAL FLOWER AND DWARF1 affects plant height, floral development and grain yield in rice.

Moderate plant height and successful establishment of reproductive organs play pivotal roles in rice grain production. The molecular mechanism that controls the two aspects remains unclear in rice. In the present study, we characterized a rice gene, ABNORMAL FLOWER AND DWARF1 (AFD1) that determined plant height, floral development and grain yield. The afd1 mutant showed variable defects including the dwarfism, long panicle, low seed setting and reduced grain yield. In addition, abnormal floral organs were also observed in the afd1 mutant including slender and thick hulls, and hull-like lodicules. AFD1 encoded a DUF640 domain protein and was expressed in all tested tissues and organs. Subcellular localization showed AFD1-green fluorescent fusion protein (GFP) was localized in the nucleus. Meantime, our results suggested that AFD1 regulated the expression of cell division and expansion related genes.

PGL, encoding chlorophyllide a oxygenase 1, impacts leaf senescence and indirectly affects grain yield and quality in rice.

Chlorophyll (Chl) b is a ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. This pigment is synthesized from Chl a by chlorophyllide a oxygenase and plays a key role in adaptation to various environments. This study characterizes a rice mutant, pale green leaf (pgl), and isolates the gene PGL by using a map-based cloning approach. PGL, encoding chlorophyllide a oxygenase 1, is mainly expressed in the chlorenchyma and activated in the light-dependent Chl synthesis process. Compared with wild-type plants, pgl exhibits a lower Chl content with a reduced and disorderly thylakoid ultrastructure, which decreases the photosynthesis rate and results in reduced grain yield and quality. In addition, pgl exhibits premature senescence in both natural and dark-induced conditions and more severe Chl degradation and reactive oxygen species accumulation than does the wild-type. Moreover, pgl is sensitive to heat stress.

EARLY SENESCENCE1 Encodes a SCAR-LIKE PROTEIN2 That Affects Water Loss in Rice.

The global problem of drought threatens agricultural production and constrains the development of sustainable agricultural practices. In plants, excessive water loss causes drought stress and induces early senescence. In this study, we isolated a rice (Oryza sativa) mutant, designated as early senescence1 (es1), which exhibits early leaf senescence. The es1-1 leaves undergo water loss at the seedling stage (as reflected by whitening of the leaf margin and wilting) and display early senescence at the three-leaf stage. We used map-based cloning to identify ES1, which encodes a SCAR-LIKE PROTEIN2, a component of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous complex involved in actin polymerization and function. The es1-1 mutants exhibited significantly higher stomatal density. This resulted in excessive water loss and accelerated water flow in es1-1, also enhancing the water absorption capacity of the roots and the water transport capacity of the stems as well as promoting the in vivo enrichment of metal ions cotransported with water. The expression of ES1 is higher in the leaves and leaf sheaths than in other tissues, consistent with its role in controlling water loss from leaves. GREEN FLUORESCENT PROTEIN-ES1 fusion proteins were ubiquitously distributed in the cytoplasm of plant cells. Collectively, our data suggest that ES1 is important for regulating water loss in rice.

Quantitative trait loci identification, fine mapping and gene expression profiling for ovicidal response to whitebacked planthopper (Sogatella furcifera Horvath) in rice (Oryza sativa L.).

BACKGROUND: The whitebacked planthopper (WBPH), Sogatella furcifera Horváth, is a serious rice pest in Asia. Ovicidal resistance is a natural rice defense mechanism against WBPH and is characterized by the formation of watery lesions (WLs) and increased egg mortality (EM) at the WBPH oviposition sites. RESULTS: This study aimed to understand the genetic and molecular basis of rice ovicidal resistance to WBPH by combining genetic and genomic analyses. First, the ovicidal trait in doubled haploid rice lines derived from a WBPH-resistant cultivar (CJ06) and a WBPH-susceptible cultivar (TN1) were phenotyped based on the necrotic symptoms of the leaf sheaths and EM. Using a constructed molecular linkage map, 19 quantitative trait loci (QTLs) associated with WLs and EM were identified on eight chromosomes. Of them, qWL6 was determined to be a major QTL for WL. Based on chromosome segment substitution lines and a residual heterozygous population, a high-resolution linkage analysis further defined the qWL6 locus to a 122-kb region on chromosome 6, which was annotated to encode 20 candidate genes. We then conducted an Affymetrix microarray analysis to determine the transcript abundance in the CJ06 and TN1 plants. Upon WBPH infestation, 432 genes in CJ06 and 257 genes in TN1 were significantly up-regulated, while 802 genes in CJ06 and 398 genes in TN1 were significantly down-regulated. This suggests that remarkable global changes in gene expression contribute to the ovicidal resistance of rice. Notably, four genes in the 122-kb region of the qWL6 locus were differentially regulated between CJ06 and TN1 in response to the WBPH infestation, suggesting they may be candidate resistance genes. CONCLUSIONS: The information obtained from the fine mapping of qWL6 and the microarray analyses will facilitate the isolation of this important resistance gene and its use in breeding WBPH-resistant rice.