Jialing Yao

Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice

Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22–34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.

Linking differential domain functions of the GS3 protein to natural variation of grain size in rice

Grain yield in many cereal crops is largely determined by grain size. Here we report the genetic and molecular characterization of GS3, a major quantitative trait locus for grain size. It functions as a negative regulator of grain size and organ size. The wild-type isoform is composed of four putative domains: a plant-specific organ size regulation (OSR) domain in the N terminus, a transmembrane domain, a tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family cysteine-rich domain, and a von Willebrand factor type C (VWFC) in the C terminus. These domains function differentially in grain size regulation. The OSR domain is both necessary and sufficient for functioning as a negative regulator. The wild-type allele corresponds to medium grain. Loss of function of OSR results in long grain. The C-terminal TNFR/NGFR and VWFC domains show an inhibitory effect on the OSR function; loss-of-function mutations of these domains produced very short grain. This study linked the functional domains of the GS3 protein to natural variation of grain size in rice.

A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice

Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day–specific male-fertility–associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding.

A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice

Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica–japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene.

FLEXIBLE CULM 1 encoding a cinnamyl-alcohol dehydrogenase controls culm mechanical strength in rice

Culm mechanical strength is an important agronomic trait in crop breeding. To understand the molecular mechanisms that control culm mechanical strength, we identified a flexible culm1 (fc1) mutant by screening a rice T-DNA insertion mutant library. This mutant exhibited an abnormal development phenotype, including late heading time, semi-dwarf habit, and flexible culm. In this study, we cloned the FLEXIBLE CULM1 (FC1) gene in rice using a T-DNA tagging approach. FC1 encodes a cinnamyl-alcohol dehydrogenase and is mainly expressed in the sclerenchyma cells of the secondary cell wall and vascular bundle region. In these types of cells, a deficiency of FC1 in the fc1 mutant caused a reduction in cell wall thickness, as well as a decrease in lignin. Extracts from the first internodes and panicles of the fc1 plants exhibited drastically reduced cinnamyl-alcohol dehydrogenase activity. Further histological and biochemical analyses revealed that the p-hydroxyphenyl and guaiacyl monomers in fc1 cell wall were reduced greatly. Our results indicated that FC1 plays an important role in the biosynthesis of lignin and the control of culm strength in rice.

PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants.

A MADS-box gene was isolated using the suppressive subtractive hybridization library between early-flowering mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.). This gene is highly homologous with ArabidopsisSHORT VEGETATIVE PHASE (SVP). Based on real-time PCR and in situ hybridization during bud differentiation, PtSVP was expressed intensively in dormant tissue and vegetative meristems. PtSVP transcripts were detected in apical meristems before floral transition, then down-regulated during the transition. PtSVP expression was higher in differentiated (flower primordium) than in undifferentiated cells (apical meristems). The PtSVP expression pattern during apical meristem determination suggested that its function is not to depress flower initiation but to maintain meristem development. Transcription of PtSVP in Arabidopsis svp-41 showed partially rescued SVP function. Ectopic overexpression of PtSVP in wild-type Arabidopsis induced late flowering similar to the phenotypes induced by other SVP/StMADS-11-like genes, but transformants produced additional trichomes and floral defects, such as flower-like structures instead of carpels. Ectopic expression of PtSVP in tobacco also caused additional florets. Overexpression of PtSVP in tobacco inhibited early transition of the coflorescence and prolonged coflorescence development, thus causing additional florets at the later stage. A yeast two-hybrid assay indicated that PtSVP significantly interacted with PtAP1, a homolog of Arabidopsis APETALA1 (AP1). These findings suggest that citrus SVP homolog genes are involved in flowering time regulation and may influence inflorescence meristem identity in some conditions or genetic backgrounds. SVP homologs might have evolved among plant species, but the protein functions are conserved between Arabidopsis and citrus.

Genome-wide analysis of endosperm-specific genes in rice.

The endosperm of the cereal crop is an important nutrient source for humans. It also acts as a critical integrator of plant seed growth and development. Despite its importance, the comprehensive understanding in regulating of endosperm development in rice remains elusive. Here, we performed a genomic survey comprising the identification and functional characterization of the endosperm-specific genes (OsEnS) in rice using Affymetrix microarray data and Gene Ontology (GO) analysis. A total of 151 endosperm-specific genes were identified, and the expression patterns of 13 selected genes were confirmed by qRT-PCR analysis. Promoter regions of the endosperm-specific expression genes were analyzed by PLACE Signal Scan Search. The results indicated that some motifs were involved in endosperm-specific expression regulation, and some cis-elements were responsible for hormone regulation. The bootstrap analysis indicated that the RY repeat (CATGCA box) was over-represented in promoter regions of endosperm-specific expression genes. GO analysis indicated that these genes could be classified into 12 groups, namely, transcription factor, stress/defense, seed storage protein (SSP), carbohydrate and energy metabolism, seed maturation, protein metabolism, lipid metabolism, transport, cell wall related, hormone related, signal transduction, and one unclassified group. Taken together, our results provide informative clues for further functional characterization of the endosperm-specific genes, which facilitate the understanding of the molecular mechanism in rice endosperm development.

OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development.

Cell cycle regulators are crucial for normal endosperm development and seed size determination. However, how the cell cycle related genes regulate endosperm development remains unclear. In this study, we reported a rice Nuclear Factor Y (NF-Y) gene OsNF-YB1, which was also identified as an endosperm-specific gene. Transcriptional profiling and promoter analysis revealed that OsNF-YB1 was highly expressed at the early stages of rice endosperm development (5-7 DAP, days after pollination). Repression of OsNF-YB1 resulted in differential expression of the genes in cell cycle pathway, which caused abnormal seeds with defected embryo and endosperm. Basic cytological analysis demonstrated that the reduced endosperm cell numbers disintegrated with the development of those abnormal seeds in OsNF-YB1 RNAi plants. Taken together, these results suggested that the endosperm-specific gene OsNF-YB1 might be a cell cycle regulator and played a role in maintaining the endosperm cell proliferation.

The integrative expression and co-expression analysis of the AGO gene family in rice.

Argonautes (AGOs) play crucial roles in RNAi and related pathways in several species and regulate plant growth and development. However the investigation in rice argonautes (OsAGOs) remains elusive. Here we focused on the expression pattern and co-expression profiles of OsAGO genes. Microarray-based and qRT-PCR expression profiling of 19 OsAGO genes indicated that most OsAGOs expressed specifically and preferentially during stages of reproductive development, and exhibited preferential up-regulation in panicle stages. Six OsAGO genes showed specific up/down-regulation in response to Gibberellin A3 (GA3), Kinetin (KT), or 1-Naphthaleneacetic acid (NAA) treatments. And three OsAGOs presented specific up-regulation in response to light and dark treatments. Ten OsAGOs were co-expressed with Dicer-like (DCL), Double-stranded RNA Binding (DRB) and RNA-dependent RNA polymerase (RDR) genes, which were related with RNA processing including RNAi pathways. Twelve OsAGOs were correlated with 17 kinds of transcription factors involving diverse functions. Four OsAGOs RNAi plants were constructed, the expression level of co-expression genes, including DCL3, DRB2, RDR4 etc., were changed while OsAGOs were down-regulated in RNAi lines, providing experimental evidence for co-expression networks. The results provide new insights in understanding the biological pathways of OsAGO genes, as well as in selecting the candidate genes involved in RNA silencing mechanisms.

A lipid transfer protein, OsLTPL36, is essential for seed development and seed quality in rice

Storage lipid is a vital component for maintaining structure of seed storage substances and valuable for rice quality and food texture. However, the knowledge of lipid transporting related genes and their function in seed development have not been well elucidated yet. In this study, we identified OsLTPL36, a homolog of putative lipid transport protein, and showed specific expression in rice developing seed. Transcriptional profiling and in situ hybridization analysis confirmed that OsLTPL36 was exclusively expressed in developing seed coat and endosperm aleurone cells. Down-regulated expression of OsLTPL36 led to decreased seed setting rate and 1000-grain weight in transgenic plants. Further studies showed that suppressed expression of OsLTPL36 caused chalky endosperm and resulted in reduced fat acid content in RNAi lines as compared with wild type (WT). Histological analysis showed that the embryo development was delayed after down regulation of OsLTPL36. Moreover, impeded seed germination and puny seedling were also observed in the OsLTPL36 RNAi lines. The data demonstrated that OsLTPL36, a lipid transporter, was critical important not only for seed quality but also for seed development and germination in rice.