Transcriptional repressor RST1 controls salt tolerance and grain yield in rice by regulating gene expression of asparagine synthetase.

Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH4+) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1Hap III decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.

Comparison of leaf transcriptome in response to Rhizoctonia solani infection between resistant and susceptible rice cultivars.

BACKGROUND: Sheath blight (SB), caused by Rhizoctonia solani, is a common rice disease worldwide. Currently, rice cultivars with robust resistance to R. solani are still lacking. To provide theoretic basis for molecular breeding of R. solani-resistant rice cultivars, the changes of transcriptome profiles in response to R. solani infection were compared between a moderate resistant cultivar (Yanhui-888, YH) and a susceptible cultivar (Jingang-30, JG). RESULTS: In the present study, 3085 differentially express genes (DEGs) were detected between the infected leaves and the control in JG, with 2853 DEGs in YH. A total of 4091 unigenes were significantly upregulated in YH than in JG before infection, while 3192 were significantly upregulated after infection. Further analysis revealed that YH and JG showed similar molecular responses to R. solani infection, but the responses were earlier in JG than in YH. Expression levels of trans-cinnamate 4-monooxygenase (C4H), ethylene-insensitive protein 2 (EIN2), transcriptome factor WRKY33 and the KEGG pathway plant-pathogen interaction were significantly affected by R. solani infection. More importantly, these components were all over-represented in YH cultivar than in JG cultivar before and/or after infection. CONCLUSIONS: These genes possibly contribute to the higher resistance of YH to R. solani than JG and were potential target genes to molecularly breed R. solani-resistant rice cultivar.

Young Seedling Stripe1 encodes a chloroplast nucleoid-associated protein required for chloroplast development in rice seedlings.

MAIN CONCLUSION: Young Seedling Stripe1 (YSS1) was characterized as an important regulator of plastid-encoded plastid RNA polymerase (PEP) activity essential for chloroplast development at rice seedling stage. Chloroplast development is coordinately regulated by plastid- and nuclear-encoding genes. Although a few regulators have been reported to be involved in chloroplast development, new factors remain to be identified, given the complexity of this process. Here, we report the characterization of a temperature-sensitive young seedling stripe1 (yss1) rice mutant, which develops striated leaves at the seedling stage, particularly in leaf 3, but produces wild-type leaves in leaf 5 and onwards. The chlorotic leaves have decreased chlorophyll (Chls) accumulation and impaired chloroplast structure. Positional cloning combined with sequencing demonstrated that aberrant splicing of the 8th intron in YSS1 gene, due to a single nucleotide deletion around splicing donor site, leads to decreased expression of YSS1 and accumulation of an 8th intron-retained yss1 transcript. Furthermore, complementation test revealed that downregulation of YSS1 but not accumulation of yss1 transcript confers yss1 mutant phenotype. YSS1 encodes a chloroplast nucleoid-localized protein belonging to the DUF3727 superfamily. Expression analysis showed that YSS1 gene is more expressed in newly expanded leaves, and distinctly up-regulated as temperatures increase and by light stimulus. PEP- and nuclear-encoded phage-type RNA polymerase (NEP)-dependent genes are separately down-regulated and up-regulated in yss1 mutant, indicating that PEP activity may be impaired. Furthermore, levels of chloroplast proteins are mostly reduced in yss1 seedlings. Together, our findings identify YSS1 as a novel regulator of PEP activity essential for chloroplast development at rice seedling stage.

FLOURY ENDOSPERM6 encodes a CBM48 domain-containing protein involved in compound granule formation and starch synthesis in rice endosperm.

Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDOSPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.

DEFORMED FLORAL ORGAN1 (DFO1) regulates floral organ identity by epigenetically repressing the expression of OsMADS58 in rice (Oryza sativa).

Floral organ identity in plants is controlled by floral homeotic A/B/C/D/E-class genes. In Arabidopsis thaliana, several epigenetic repressors that regulate these floral organ identity genes have been characterized. However, the roles of epigenetic factors in rice floral development have not been explored in detail. Here, we report the identification and functional characterization of a rice epigenetic repressor, DEFORMED FLORAL ORGAN1 (DFO1) gene, which causes abnormal floral morphology when mutated. We isolated dfo1 by mapping, and confirmed its function by rescue experiments, combined with genetic, cytological and molecular biological analysis. We showed that DFO1 is constitutively expressed and encodes a nuclear-localized protein. Mutation of DFO1 causes the ectopic expression of C-class genes in the dfo1-1 mutant, and overexpression of OsMADS58, a C-class gene, phenocopies the dfo1 mutants. In vitro and in vivo experiments demonstrated that DFO1 interacts with the rice polycomb group (PcG) proteins (OsMSI1 and OsiEZ1). Remarkably, trimethylation of histone H3 lysine 27, a mark of epigenetic repression, is significantly reduced on OsMADS58 chromatin in the dfo1-1 mutant. Our results suggest that DFO1 functions in maintaining rice floral organ identity by cooperating with PcG proteins to regulate the H3K27me3-mediated epigenetic repression on OsMADS58.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield.

Transitory starch is an important carbon source in leaves, and its biosynthesis and metabolism are closely related to grain quality and yield. The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice (Oryza sativa) quality and yield remain unclear. Here, we show that OsLESV and OsESV1, the rice orthologs of AtLESV and AtESV1, are associated with transitory starch biosynthesis in rice. The total starch and amylose contents in leaves and endosperms are significantly reduced, and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants. Furthermore, we found that OsLESV and OsESV1 bind to starch, and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain. Importantly, OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis, granule-bound starch synthase I (GBSSI), GBSSII, and pyruvate orthophosphote dikiase (PPDKB), to maintain their protein stability and activity. OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules. Overexpression of GBSSI, GBSSII, and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants. Thus, we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice. These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield, providing useful information for the genetic improvement of rice grain quality and yield.

Young Leaf Chlorosis 1, a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice.

Chlorophyll (Chl) and lutein are the two most abundant and essential components in photosynthetic apparatus, and play critical roles in plant development. In this study, we characterized a rice mutant named young leaf chlorosis 1 (ylc1) from a 6°Co-irradiated population. Young leaves of the ylc1 mutant showed decreased levels of Chl and lutein compared to those of wild type, and transmission electron microscopy analysis revealed that the thylakoid lamellar structures were obviously loosely arranged. Whereas, the mutant turns green gradually and approaches normal green at the maximum tillering stage. The Young Leaf Chlorosis 1 (YLC1) gene was isolated via map-based cloning and identified to encode a protein of unknown function belonging to the DUF3353 superfamily. Complementation and RNA-interference tests confirmed the role of the YLC1 gene, which expressed in all tested rice tissues, especially in the leaves. Real-time PCR analyses showed that the expression levels of the genes associated with Chl biosynthesis and photosynthesis were affected in ylc1 mutant at different temperatures. In rice protoplasts, the YLC1 protein displayed a typical chloroplast location pattern. The N-terminal 50 amino acid residues were confirmed to be necessary and sufficient for chloroplast targeting. These data suggested that the YLC1 protein may be involved in Chl and lutein accumulation and chloroplast development at early leaf development in rice.

OPAQUE3, encoding a transmembrane bZIP transcription factor, regulates endosperm storage protein and starch biosynthesis in rice.

Starch and storage proteins are the main components of rice (Oryza sativa L.) grains. Despite their importance, the molecular regulatory mechanisms of storage protein and starch biosynthesis remain largely elusive. Here, we identified a rice opaque endosperm mutant, opaque3 (o3), that overaccumulates 57-kDa proglutelins and has significantly lower protein and starch contents than the wild type. The o3 mutant also has abnormal protein body structures and compound starch grains in its endosperm cells. OPAQUE3 (O3) encodes a transmembrane basic leucine zipper (bZIP) transcription factor (OsbZIP60) and is localized in the endoplasmic reticulum (ER) and the nucleus, but it is localized mostly in the nucleus under ER stress. We demonstrated that O3 could activate the expression of several starch synthesis-related genes (GBSSI, AGPL2, SBEI, and ISA2) and storage protein synthesis-related genes (OsGluA2, Prol14, and Glb1). O3 also plays an important role in protein processing and export in the ER by directly binding to the promoters and activating the expression of OsBIP1 and PDIL1-1, two major chaperones that assist with folding of immature secretory proteins in the ER of rice endosperm cells. High-temperature conditions aggravate ER stress and result in more abnormal grain development in o3 mutants. We also revealed that OsbZIP50 can assist O3 in response to ER stress, especially under high-temperature conditions. We thus demonstrate that O3 plays a central role in rice grain development by participating simultaneously in the regulation of storage protein and starch biosynthesis and the maintenance of ER homeostasis in endosperm cells.

OsVPS9A functions cooperatively with OsRAB5A to regulate post-Golgi dense vesicle-mediated storage protein trafficking to the protein storage vacuole in rice endosperm cells.

In the rice endosperm cells, glutelins are synthesized on rough endoplasmic reticulum as proglutelins and are sorted to the protein storage vacuoles (PSVs) called protein body IIs (PBIIs), where they are converted to the mature forms. Dense vesicle (DV)-mediated trafficking of proglutelins in rice seeds has been proposed, but the post-Golgi control of this process is largely unknown. Whether DV can fuse directly with PSV is another matter of debate. In this study, we propose a regulatory mechanism underlying DV-mediated, post-Golgi proglutelin trafficking to PBII (PSV). gpa2, a loss-of-function mutant of OsVPS9A, which encodes a GEF of OsRAB5A, accumulated uncleaved proglutelins. Proglutelins were mis-targeted to the paramural bodies and to the apoplast along the cell wall in the form of DVs, which led to a concomitant reduction in PBII size. Previously reported gpa1, mutated in OsRab5a, has a similar phenotype, while gpa1gpa2 double mutant exacerbated the conditions. In addition, OsVPS9A interacted with OsRAB5A in vitro and in vivo. We concluded that OsVPS9A and OsRAB5A may work together and play a regulatory role in DV-mediated post-Golgi proglutelin trafficking to PBII (PSV). The evidence that DVs might fuse directly to PBII (PSV) to deliver cargos is also presented.