Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

Dwarf and High Tillering1 represses rice tillering through mediating the splicing of D14 pre-mRNA.

Strigolactones (SLs) constitute a class of plant hormones that regulate many aspects of plant development, including repressing tillering in rice (Oryza sativa). However, how SL pathways are regulated is still poorly understood. Here, we describe a rice mutant dwarf and high tillering1 (dht1), which exhibits pleiotropic phenotypes (such as dwarfism and increased tiller numbers) similar to those of mutants defective in SL signaling. We show that DHT1 encodes a monocotyledon-specific hnRNP-like protein that acts as a previously unrecognized intron splicing factor for many precursor mRNAs (pre-mRNAs), including for the SL receptor gene D14. We find that the dht1 (DHT1I232F) mutant protein is impaired in its stability and RNA binding activity, causing defective splicing of D14 pre-mRNA and reduced D14 expression, and consequently leading to the SL signaling-defective phenotypes. Overall, our findings deepen our understanding of the functional diversification of hnRNP-like proteins and establish a connection between posttranscriptional splicing and SL signaling in the regulation of plant development.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

KEY MESSAGE: We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

Rice STOMATAL CYTOKINESIS DEFECTIVE2 regulates cell expansion by affecting vesicular trafficking in rice.

Vesicular trafficking plays critical roles in cell expansion in yeast and mammals, but information linking vesicular trafficking and cell expansion in plants is limited. Here, we isolated and characterized a rice (Oryza sativa) mutant, decreased plant height 1-1 (dph1-1), which exhibited a wide spectrum of developmental phenotypes, including reduced plant height and smaller panicles and grains. Cytological analysis revealed that limited cell expansion was responsible for the dph1-1 mutant phenotype compared to the wild-type. Map-based cloning revealed that DPH1 encodes a plant-specific protein, OsSCD2, which is homologous to Arabidopsis (Arabidopsis thaliana) STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). Subcellular localization revealed that OsSCD2 is associated with clathrin. Confocal microscopy showed that the dph1-1 mutant has defective endocytosis and post-Golgi trafficking. Biochemical and confocal data indicated that OsSCD2 physically interacts with OsSCD1 and that they are associated with intracellular structures that colocalize with microtubules. Furthermore, we found that cellulose synthesis was affected in the dph1-1 mutant, evidenced by reduced cellulose synthase gene accumulation at the transcript and protein levels, most likely resulting from an impaired localization pattern. Our results suggest that OsSCD2 is involved in clathrin-related vesicular trafficking with an important role in maintaining plant growth in rice.

The APC/CTE E3 Ubiquitin Ligase Complex Mediates the Antagonistic Regulation of Root Growth and Tillering by ABA and GA.

The antagonistic regulation of seed germination by the phytohormones abscisic acid (ABA) and gibberellic acid (GA) has been well-established. However, how these phytohormones antagonistically regulate root growth and branching (tillering in rice, Oryza sativa) remains obscure. Rice TILLER ENHANCER (TE) encodes an activator of the APC/CTE E3 ubiquitin ligase complex that represses tillering but promotes seed germination. In this study, we identified a dual role of GA and APC/CTE in regulating root growth. High GA levels can activate APC/CTE to promote the degradation of rice SHORT-ROOT1 (OsSHR1, a key factor promoting root growth) in the root meristem (RM) or MONOCULM1 (MOC1, a key factor promoting tillering) in the axillary meristem (AM), leading to restricted root growth and tillering, while low GA levels can activate the role of APC/CTE in stimulating RM cell division to promote root growth. In addition, moderate enhancement of ABA signaling helps maintain the RM and AM size, sustaining root growth and tillering by antagonizing the GA-promoted degradation of OsSHR1 and MOC1 through the SnRK2-APC/CTE regulatory module. We conclude that APC/CTE plays a key role in regulating plant architecture by mediating the crosstalk between ABA and GA signaling pathways.

GPA5 Encodes a Rab5a Effector Required for Post-Golgi Trafficking of Rice Storage Proteins.

Dense vesicles (DVs) are vesicular carriers, unique to plants, that mediate post-Golgi trafficking of storage proteins to protein storage vacuoles (PSVs) in seeds. However, the molecular mechanisms regulating the directional targeting of DVs to PSVs remain elusive. Here, we show that the rice (Oryza sativa) glutelin precursor accumulation5 (gpa5) mutant is defective in directional targeting of DVs to PSVs, resulting in discharge of its cargo proteins into the extracellular space. Molecular cloning revealed that GPA5 encodes a plant-unique phox-homology domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN. We show that GPA5 is a membrane-associated protein capable of forming homodimers and that it is specifically localized to DVs in developing endosperm. Colocalization, biochemical, and genetic evidence demonstrates that GPA5 acts in concert with Rab5a and VPS9a to regulate DV-mediated post-Golgi trafficking to PSVs. Furthermore, we demonstrated that GPA5 physically interacts with a class C core vacuole/endosome tethering complex and a seed plant-specific VAMP727-containing R-soluble N-ethylmaleimide sensitive factor attachment protein receptor complex. Collectively, our results suggest that GPA5 functions as a plant-specific effector of Rab5a required for mediating tethering and membrane fusion of DVs with PSVs in rice endosperm.

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1-CCZ1.

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1-CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

OsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in Rice.

Tillering and panicle branching are important determinants of plant architecture and yield potential in rice (Oryza sativa). IDEAL PLANT ARCHITECTURE1 (IPA1) encodesSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14, which acts as a key transcription factor regulating tiller outgrowth and panicle branching by directly activating the expression of O. sativa TEOSINTE BRANCHED1 (OsTB1) and O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), thereby influencing grain yield in rice. Here, we report the identification of a rice mutant named shi1 that is characterized by dramatically reduced tiller number, enhanced culm strength, and increased panicle branch number. Map-based cloning revealed that O. sativa SHORT INTERNODES1 (OsSHI1) encodes a plant-specific transcription factor of the SHI family with a characteristic family-specific IGGH domain and a conserved zinc-finger DNA binding domain. Consistent with the mutant phenotype, OsSHI1 is predominantly expressed in axillary buds and young panicle, and its encoded protein is exclusively targeted to the nucleus. We show that OsSHI1 physically interacts with IPA1 both in vitro and in vivo. Moreover, OsSHI1 could bind directly to the promoter regions of both OsTB1 and OsDEP1 through a previously unrecognized cis-element (T/GCTCTAC motif). OsSHI1 repressed the transcriptional activation activity of IPA1 by affecting its DNA binding activity toward the promoters of both OsTB1 and OsDEP1, resulting in increased tiller number and diminished panicle size. Taken together, our results demonstrate that OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 and provide insight into the establishment of plant architecture in rice.

Genetic characterization and fine mapping of qHMS4 responsible for pollen sterility in hybrids between Oryza sativa L. and Oryza glaberrima Steud.

African cultivated rice (Oryza glaberrima Steud) contains many favorable genes for tolerance to biotic and abiotic stresses and F1 hybrids between Asian cultivated rice (Oryza sativa L.) show strong heterosis. However, the hybrids of two species often exhibit hybrid sterility. Here, we identified a male sterility locus qHMS4 on chromosome 4 (Chr.4), which induces pollen semi-sterility in F1 hybrids of japonica rice variety Dianjingyou1 (DJY1) and a near-isogenic line (NIL) carrying a Chr.4 segment from Oryza glaberrima accession IRGC101854. Cytological observations indicated that non-functional pollen grains produced by the hybrids and lacking starch accumulation abort at the late bicellular stage. Molecular genetic analysis revealed distorted segregation in male gametogenesis carrying qHMS4 allele from DJY1. Fine-mapping of qHMS4 using an F2 population of 22,500 plants delimited qHMS4 to a region of 110-kb on the short arm of Chr.4. Sequence analysis showed that the corresponding sequence region in DJY1 and Oryza glaberrima were 114-kb and 323-kb, respectively, and that the sequence homology was very poor. Gene prediction analysis identified 16 and 46 open reading frames (ORFs) based on the sequences of DJY1 and O. glaberrima, respectively, among which 3 ORFs were shared by both. Future map-based cloning of qHMS4 will help to understand the underlying molecular mechanism of hybrid sterility between the two cultivated rice species. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01306-8.

Ectopic Expression of Executor Gene Xa23 Enhances Resistance to Both Bacterial and Fungal Diseases in Rice.

Bacterial blight (BB) and bacterial leaf streak (BLS), caused by phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are the most serious bacterial diseases of rice, while blast, caused by Magnaporthe oryzae (M. oryzae), is the most devastating fungal disease in rice. Generating broad-spectrum resistance to these diseases is one of the key approaches for the sustainable production of rice. Executor (E) genes are a unique type of plant resistance (R) genes, which can specifically trap transcription activator-like effectors (TALEs) of pathogens and trigger an intense defense reaction characterized by a hypersensitive response in the host. This strong resistance is a result of programed cell death induced by the E gene expression that is only activated upon the binding of a TALE to the effector-binding element (EBE) located in the E gene promoter during the pathogen infection. Our previous studies revealed that the E gene Xa23 has the broadest and highest resistance to BB. To investigate whether the Xa23-mediated resistance is efficient against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of BLS, we generated a new version of Xa23, designated as Xa23p1.0, to specifically trap the conserved TALEs from multiple Xoc strains. The results showed that the Xa23p1.0 confers broad resistance against both BB and BLS in rice. Moreover, our further experiment on the Xa23p1.0 transgenic plants firstly demonstrated that the E-gene-mediated defensive reaction is also effective against M. oryzae, the causal agent of the most devastating fungal disease in rice. Our current work provides a new strategy to exploit the full potential of the E-gene-mediated disease resistance in rice.

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression.

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B-type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A-box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus-localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1-interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1-dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine-tuning heading date to improve rice regional adaptability.

OsALMT7 Maintains Panicle Size and Grain Yield in Rice by Mediating Malate Transport.

Panicle size is a critical determinant of grain yield in rice (Oryza sativa) and other grain crops. During rice growth and development, spikelet abortion often occurs at either the top or the basal part of the panicle under unfavorable conditions, causing a reduction in fertile spikelet number and thus grain yield. In this study, we report the isolation and functional characterization of a panicle abortion mutant named panicle apical abortion1-1 (paab1-1). paab1-1 exhibits degeneration of spikelets on the apical portion of panicles during late stage of panicle development. Cellular and physiological analyses revealed that the apical spikelets in the paab1-1 mutant undergo programmed cell death, accompanied by nuclear DNA fragmentation and accumulation of higher levels of H2O2 and malondialdehyde. Molecular cloning revealed that paab1-1 harbors a mutation in OsALMT7, which encodes a putative aluminum-activated malate transporter (OsALMT7) localized to the plasma membrane, and is preferentially expressed in the vascular tissues of developing panicles. Consistent with a function for OsALMT7 as a malate transporter, the panicle of the paab1-1 mutant contained less malate than the wild type, particularly at the apical portions, and injection of malate into the paab1-1 panicle could alleviate the spikelet degeneration phenotype. Together, these results suggest that OsALMT7-mediated transport of malate into the apical portion of panicle is required for normal panicle development, thus highlighting a key role of malate in maintaining the sink size and grain yield in rice and probably other grain crops.

Rice FLOURY ENDOSPERM 18 encodes a pentatricopeptide repeat protein required for 5' processing of mitochondrial nad5 messenger RNA and endosperm development.

Pentatricopeptide repeat (PPR) proteins, composing one of the largest protein families in plants, are involved in RNA binding and regulation of organelle RNA metabolism at the post-transcriptional level. Although several PPR proteins have been implicated in endosperm development in rice (Oryza sativa), the molecular functions of many PPRs remain obscure. Here, we identified a rice endosperm mutant named floury endosperm 18 (flo18) with pleiotropic defects in both reproductive and vegetative development. Map-based cloning and complementation tests showed that FLO18 encodes a mitochondrion-targeted P-type PPR protein with 15 PPR motifs. Mitochondrial function was disrupted in the flo18 mutant, as evidenced by decreased assembly of Complex I in the mitochondrial electron transport chain and altered mitochondrial morphology. Loss of FLO18 function resulted in defective 5'-end processing of mitochondrial nad5 transcripts encoding subunit 5 of nicotinamide adenine dinucleotide hydrogenase. These results suggested that FLO18 is involved in 5'-end processing of nad5 messenger RNA and plays an important role in mitochondrial function and endosperm development.

Ubiquitin Specific Protease 15 Has an Important Role in Regulating Grain Width and Size in Rice.

Ubiquitination and deubiquitination are reversible processes that play crucial roles in regulating organ size in plants. However, information linking deubiquitination and seed size in rice (Oryza sativa) is limited. Here, we characterized a dominant large-grain mutant, large grain1-D (lg1-D), with a 30.8% increase in seed width and a 34.5% increase in 1,000-grain weight relative to the wild type. The lg1-D mutant had more cells oriented in the lateral direction of the spikelet hull compared with the wild type. Map-based cloning showed that LG1 encodes a constitutively expressed ubiquitin-specific protease15 (OsUBP15) that possesses deubiquitination activity in vitro. Loss-of-function and down-regulated expression of OsUBP15 produced narrower and smaller grains than the control. A set of in vivo experiments indicated that the mutant Osubp15 had enhanced protein stability relative to wild-type OsUBP15. Further experiments verified that OsDA1 directly interacted with OsUBP15. Genetic data indicated that OsUBP15 and GRAIN WIDTH 2 (GW2) were not independent in regulating grain width and size. In summary, we identified OsUBP15 as a positive regulator of grain width and size in rice and provide a promising strategy for improvement of grain yield by pyramiding OsUBP15 and gw2.

DELAYED HEADING DATE1 interacts with OsHAP5C/D, delays flowering time and enhances yield in rice.

Heading date is an important agronomic trait affecting crop yield. The GRAS protein family is a plant-specific super family extensively involved in plant growth and signal transduction. However, GRAS proteins are rarely reported have a role in regulating rice heading date. Here, we report a GRAS protein DHD1 (Delayed Heading Date1) delays heading and enhances yield in rice. Biochemical assays showed DHD1 physically interacts with OsHAP5C/D both in vitro and in vivo. DHD1 and OsHAP5C/D located in the nucleus and showed that rhythmic expression. Both DHD1 and OsHAP5C/D affect heading date by regulating expression of Ehd1. We propose that DHD1 interacts with OsHAP5C/D to delay heading date by inhibiting expression of Ehd1.

Disruption of gene SPL35, encoding a novel CUE domain-containing protein, leads to cell death and enhanced disease response in rice.

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)-like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL-PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H2 O2 , up-regulated expression of defence-related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain-containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta-COP1 and Delta-COP2 through the CUE domain, and down-regulation of these interacting proteins also cause development of HR-like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.