Xin Wang

Programmed cell death and immunity in plants are finely orchestrated to promote antimicrobial defense while preventing autoimmunity. However, the molecular mechanisms involved are not fully understood. Here, we isolated a rice mutant ecdr1 (enhanced cell death and resistance 1) that displayed an autoimmunity phenotype and enhanced resistance to rice blast and bacterial blight, and we identified ECDR1 as a new shared component in phosphoinositide 3-kinase (PI3K) and PI4K complexes, linking the hyccin-containing protein with plant defense responses. ECDR1 was expressed at all developmental stages and in all tissues examined. The ECDR1 protein was highly conserved in function across monocots and dicots. The 113 bp deletion in the ECDR1 promoter reduced its expression, leading to cell death and enhanced disease resistance. The ECDR1 protein was localized in plasma membrane and interacted with tetratricopeptide repeat protein 1 (TPR1) and TPR2, which in turn interacted with both PI4K1 and PI3K1, suggesting that ECDR1 could function as a component associated with not only PI4K complexes but also PI3K complexes to help catalyze PI into phosphatidylinositol 3-phosphate (PI3P) and PI4P. The lethality of all homozygous ecdr1, pi3k1, tpr1 and tpr2 mutants indicated the crucial roles of these genes in plant normal growth, which restricted our understanding of PI3K and PI4K functions. Alternatively, exogenous application of PI3K and PI4K inhibitors could substantially exacerbate the cell death and enhance disease resistance, implying their roles in plant immunity. Our findings provide novel insights into the regulatory mechanisms of ECDR1 in cell death and defense pathways, will aid in understanding the functions of PI3K and PI4K in plant immunity.

The endoplasmic reticulum (ER) is the largest intracellular membrane-bound organelle in eukaryotic cells, comprising an interconnected network of tubules and sheets. Lunapark (LNP) functions as an E3 ubiquitin ligase that targets RHD3, a dynamin-like GTPase essential for homotypic fusion of ER tubules, for proteasomal degradation, thereby modulating ER tubule stability. However, the regulatory mechanism governing LNP activity remains largely unknown. Here, we report the characterization of the rice oskish mutant, which exhibits aberrant aggregation of ER tubules and defective ER exit of seed storage proteins. OsKish is an ER-localized small protein that physically interacts with both OsLNPs and the RHD3-like (RHD3L) protein. OsKish stabilizes OsLNPs by suppressing their auto-ubiquitination; concomitantly, OsKish attenuates the membrane fusion activity of RHD3L, possibly by inhibiting its oligomerization. Altogether, our studies propose an OsKish–OsLNPs–RHD3L framework for fine-tuning homotypic ER tubule fusion in rice, providing mechanistic insights into the maintenance of ER architecture in plants.

Appropriate heading date is an important safeguard for stabilizing rice yield and quality. Heading integrator Ehd1 and florigen Hd3a are crucial for rice heading. However, the mechanism by which Ehd1 transmits heading signals to Hd3a and the accurate regulation of Hd3a expression remains unclear. Here, we found that OsWOX13 functions as a signal transducer from Ehd1 to Hd3a for the accurate regulation of rice heading date. Ehd1 can directly bind to the OsWOX13 promoter, OsWOX13, in turn, binds to the Hd3a promoter to activate its expression. OsWOX13 interacts with Hd3a to inhibit OsWOX13-mediated activation of Hd3a expression, which precisely controls Hd3a levels. Meanwhile, late-heading phenotypes were observed in the ehd1, Oswox13, Oswox13 ehd1, hd3a, and Oswox13 hd3a, demonstrating that Ehd1, OsWOX13, and Hd3a coordinately regulate rice heading date. Furthermore, OsWOX13 can be divided into 2 haplotypes, Hap-T and Hap-C. Ehd1 elevated OsWOX13 expression in Hap-T compared with Hap-C. Longjing 31T-C (carrying Hap-C) exhibited later heading dates than Longjing 31 (carrying Hap-T). Rice varieties carrying Hap-T are predominantly cultivated in high-latitude regions. This study provides genetic resources and a theoretical foundation for improving the regional and climatic adaptability of rice.

Seed storage proteins (SSPs), which accumulate specifically during seed development, constitute a major source of plant-derived protein in the human diet. Despite their critical role in determining crop quality, the molecular mechanisms underlying the intracellular trafficking of SSPs remain poorly understood. Here, we characterize the rice glutelin precursor accumulation16 (gpa16) mutant, which exhibits defective dense vesicle (DV)-mediated post-Golgi trafficking of proglutelins, resulting in their overaccumulation in the apoplast. GPA16 encodes OsOSCA2.4, a member of the reduced hyperosmolality-induced Ca2+ increase channel (OSCA) family. OsOSCA2.4 localizes to post-Golgi compartments including the trans-Golgi network (TGN) and prevacuolar compartment (PVC) in vegetative tissue, and DVs in the endosperm. The OsOSCA2.4(P397L) variant disrupts the formation of the Rab5a molecular module, a process influenced by intracellular Ca2+ homeostasis. Ca2+ imaging assays indicate that OsOSCA2.4 functions as a regulator of Ca2+ homeostasis of PVCs/DVs, the primary sites of Rab5a module activity. Genetic analyses reveal functional redundancy between OsOSCA2.4 and OsOSCA4.1 in modulating post-Golgi trafficking of proglutelins. Collectively, our findings identify OSCA-mediated Ca2+ homeostasis as a previously unrecognized regulatory layer governing post-Golgi trafficking of SSPs and uncover an unexpected role for OSCA proteins in vesicular trafficking in eukaryotes.

Heading date is a key agronomic trait that affects crop yield and regional adaptability. In this study, we identified a rice (Oryza sativa) early heading mutant and cloned the causal heading inhibitor gene EARLY HEADING AT LONG DAY 4 (ELD4) using the MutMap method. The eld4 CRISPR mutants and ELD4 RNAi plants flowered earlier than the wild type under natural long-day (NLD) conditions. ELD4 is a zinc finger transcription factor that localizes in the nucleus. Biochemical and genetic evidence suggests that ELD4 physically interacts with the pseudo-response regulator (PRR) protein OsPRR95. The Osprr95 mutant also exhibits an earlier heading phenotype under NLD. ELD4 and OsPRR95 co-regulate heading date by directly binding to the promoter and the first intron of OsMADS51, which promotes heading date. Moreover, electrophoretic mobility shift and luciferase complementation assays showed that ELD4 enhances OsPRR95 binding to the first intron of OsMADS51. These results suggest that the ELD4-OsPRR95 module directly represses OsMADS51 in regulating flowering time. Haplotype analysis reveals that OsPRR95 haplotype 3, which is geographically distributed in the north, exhibits a shorter heading date than haplotype 1 found in the south. This indicates that selection at the OsPRR95 locus has enhanced the regional adaptability of rice.

Hybrid sterility is a major barrier in exploiting hybrid vigor in rice grains produced by crossing distantly related parents. While genetic mechanisms such as the killer-protector system have been extensively studied, novel systems underlying hybrid sterility remain poorly characterized. Here, a novel hybrid sterility system governed by two tightly pseudolinked loci SGA1 (on chromosome 1) and SGA2 (on chromosome 2) is reported, which induces semi-sterility in male and female gametes during hybridization between the indica and japonica subspecies. Chromosomal translocations were proposed as the basis for pseudolinkage and unbiased segregation, supported by cytological evidence of meiotic quadrivalent configurations and translocation breakpoint sequences. Gametophytic sterility was identified as the primary driver of dual male–female semi-sterility in translocated heterozygotes. Furthermore, large-segment chromosomal translocations are found to be widespread in rice. Analysis of 120 pangenomic rice accessions revealed that chromosomal translocations are prevalent among cultivars, with one-third exhibiting large translocations (>500 kb). Translocation breakpoints were mainly localized in intergenic and intronic regions, and the disrupted genes were identified as predominantly transposons and retrotransposons. Besides, large translocations were validated through sequence analysis and phenotypic assays. Overall, this study establishes chromosomal translocations as a critical driver of hybrid sterility and provides new insights into heterosis constraints.

G-protein signaling and brassinosteroid (BR) phytohormones play important roles in regulating rice (Oryza sativa) yield-related plant architecture, such as leaf inclination and grain size. However, the relationship between G-proteins and BR signaling has not been fully elucidated in rice. The present study indicates that the G-protein Gγ subunit DENSE AND ERECT PANICLE 1 (DEP1) positively regulates BR signaling in rice and that BRs promote DEP1 nuclear entry through GRAIN NUMBER ASSOCIATED (GNA). Additionally, DEP1 interacts with and acts upstream of OsMYB86, an R2R3-MYB family transcription factor that positively regulates BR signaling by directly binding to the promoter of its downstream gene BRASSINOSTEROID UPREGULATED 1 (BU1), activating its expression in rice. In the nucleus, DEP1 interacts with OsMYB86 and GNA, significantly enhancing OsMYB86-mediated activation of BU1 expression. Furthermore, BU1 interacts with another HLH protein, INCREASED LEAF INCLINATION1 (ILI1), and a bHLH protein, ILI1 BINDING bHLH (IBH1). Interaction between ILI1 and BU1 facilitates translocation of BU1 from the cytoplasm to the nucleus, where they impede IBH1 binding to the promoter of the AUXIN RESPONSE FACTOR 11 (OsARF11) gene, which is involved in crosstalk between BR and auxin, thus effectively relieving the IBH1-repressed transcription of OsARF11. These findings reveal a DEP1-mediated signaling pathway that links G-proteins to the traditional BR signaling pathway, ensuring the efficient activation of BR responses in rice.

The phytohormone cytokinin (CK) positively regulates the activity of the inflorescence meristem (IM). Cytokinin oxidase 2/Grain number 1a (OsCKX2/Gn1a)-mediated degradation of CK in rice (Oryza sativa L.) negatively regulates panicle grain number, whereas DENSE AND ERECT PANICLE 1 (DEP1) positively regulates grain number per panicle (GNP). However, the detailed regulatory mechanism between DEP1 and OsCKX2 remains elusive. Here, we report the GRAS (GIBBERELLIN ACID INSENSITIVE, REPRESSOR OF GA1, and SCARECROW) transcription factor GRAIN NUMBER ASSOCIATED (GNA), previously thought to be involved in the Brassinosteroids (BRs) signaling pathway, directly inhibits OsCKX2 expression in the IM through a DEP1-GNA regulatory module. Overexpressing GNA leads to increased CK levels and consequently higher branch number, GNP, and yield. Both DEP1 and dep1 enhance the inhibitory effect of GNA on OsCKX2 expression through interacting with GNA. GNA promotes the translocation of DEP1 to the nucleus, while the gain-of-function mutant dep1 translocates into the nucleus in the absence of GNA. Our findings provide insight into the regulatory mechanism underlying OsCKX2 and a strategy to improve rice yield.

Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome, also named the vacuole in plants. However, the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear. Here, we report the functional characterization of a rice (Oryza sativa) mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells. Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles. The mutant harbors a loss‐of‐function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1 (MON1). Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY‐RELATED 8a in maintaining normal growth and development. In addition, the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles. Furthermore, quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport, thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles. Together, our findings establish a molecular link between autophagy and vacuolar protein transport, and offer insights into the dual functions of the MON1–CCZ1 (CAFFEINE ZINC SENSITIVITY1) complex in plants.