DWARF AND LESS TILLERS ON CHROMOSOME 3 Promotes Tillering in Rice by Sustaining FLORAL ORGAN NUMBER 1 Expression.

Three key factors determine yield in rice (Oryza sativa): panicle number, grain number, and grain weight. Panicle number is strongly associated with tiller number. Although many genes regulating tillering have been identified, whether Dof proteins are involved in controlling plant architecture remains unknown. The dwarf and less tillers on chromosome 3 (dlt3) rice mutant produces fewer tillers than the wild type. We cloned DLT3, which encodes a Dof protein that interacts with MONOCULM 3 (MOC3) in vivo and in vitro and recruits MOC1, forming a DLT3-MOC3-MOC1 complex. DLT3 binds to the promoter of FLORAL ORGAN NUMBER 1 (FON1) to activate its transcription and positively regulate tiller number. The overexpression of MOC1, MOC3, or FON1 in the dlt3 mutant increased tiller number. Collectively, these results suggest a model in which DLT3 regulates tiller number by maintaining the expression of MOC1, MOC3, and FON1. We discovered that DLT3 underwent directional selection in the Xian/indica and Geng/japonica populations during rice domestication. To provide genetic resources for breeding varieties with optimal panicle numbers, we performed large-scale diversity sequencing of the 1080-bp DLT3 coding region of 531 accessions from different countries and regions. Haplotype analysis showed that the superior haplotype, DLT3H1, produced the most tillers, while haplotype DLT3H6 produced the fewest tillers. Our study provides important germplasm resources for breeding super high-yielding rice varieties with combinations of superior haplotypes in different target genes, which will help overcome the challenge of food and nutritional security in the future.

OsCOL5 suppresses heading through modulation of Ghd7 and Ehd2, enhancing rice yield.

KEY MESSAGE: OsCOL5, an ortholog of Arabidopsis COL5, is involved in photoperiodic flowering and enhances rice yield through modulation of Ghd7 and Ehd2 and interactions with OsELF3-1 and OsELF3-2. Heading date, also known as flowering time, plays a crucial role in determining the adaptability and yield potential of rice (Oryza sativa L.). CONSTANS (CO)-like is one of the most critical flowering-associated gene families, members of which are evolutionarily conserved. Here, we report the molecular functional characterization of OsCOL5, an ortholog of Arabidopsis COL5, which is involved in photoperiodic flowering and influences rice yield. Structural analysis revealed that OsCOL5 is a typical member of CO-like family, containing two B-box domains and one CCT domain. Rice plants overexpressing OsCOL5 showed delayed heading and increases in plant height, main spike number, total grain number per plant, and yield per plant under both long-day (LD) and short-day (SD) conditions. Gene expression analysis indicated that OsCOL5 was primarily expressed in the leaves and stems with a diurnal rhythm expression pattern. RT-qPCR analysis of heading date genes showed that OsCOL5 suppressed flowering by up-regulating Ghd7 and down-regulating Ehd2, consequently reducing the expression of Ehd1, Hd3a, RFT1, OsMADS14, and OsMADS15. Yeast two-hybrid experiments showed direct interactions of OsCOL5 with OsELF3-1 and OsELF3-2. Further verification showed specific interactions between the zinc finger/B-box domain of OsCOL5 and the middle region of OsELF3-1 and OsELF3-2. Yeast one-hybrid assays revealed that OsCOL5 may bind to the CCACA motif. The results suggest that OsCOL5 functions as a floral repressor, playing a vital role in rice's photoperiodic flowering regulation. This gene shows potential in breeding programs aimed at improving rice yield by influencing the timing of flowering, which directly impacts crop productivity.

Mutation of DEFECTIVE EMBRYO SAC1 results in a low seed-setting rate in rice by regulating embryo sac development.

The seed-setting rate has a significant effect on grain yield in rice (Oryza sativa L.). Embryo sac development is essential for seed setting; however, the molecular mechanism underlying this process remains unclear. Here, we isolated defective embryo sac1 (des1), a rice mutant with a low seed-setting rate. Cytological examination showed degenerated embryo sacs and reduced fertilization capacity in des1. Map-based cloning revealed a nonsense mutation in OsDES1, a gene that encodes a putative nuclear envelope membrane protein (NEMP)-domain-containing protein that is preferentially expressed in pistils. The OsDES1 mutation disrupts the normal formation of functional megaspores, which ultimately results in a degenerated embryo sac in des1. Reciprocal crosses showed that fertilization is abnormal and that the female reproductive organ is defective in des1. OsDES1 interacts with LONELY GUY (LOG), a cytokinin-activating enzyme that acts in the final step of cytokinin synthesis; mutation of LOG led to defective female reproductive organ development. These results demonstrate that OsDES1 functions in determining the rice seed-setting rate by regulating embryo sac development and fertilization. Our study sheds light on the function of NEMP-type proteins in rice reproductive development.

Photoperiod and gravistimulation-associated Tiller Angle Control 1 modulates dynamic changes in rice plant architecture.

KEY MESSAGE: TAC1 is involved in photoperiodic and gravitropic responses to modulate rice dynamic plant architecture likely by affecting endogenous auxin distribution, which could explain TAC1 widespread distribution in indica rice. Plants experience a changing environment throughout their growth, which requires dynamic adjustments of plant architecture in response to these environmental cues. Our previous study demonstrated that Tiller Angle Control 1 (TAC1) modulates dynamic changes in plant architecture in rice; however, the underlying regulatory mechanisms remain largely unknown. In this study, we show that TAC1 regulates plant architecture in an expression dose-dependent manner, is highly expressed in stems, and exhibits dynamic expression in tiller bases during the growth period. Photoperiodic treatments revealed that TAC1 expression shows circadian rhythm and is more abundant during the dark period than during the light period and under short-day conditions than under long-day conditions. Therefore, it contributes to dynamic plant architecture under long-day conditions and loose plant architecture under short-day conditions. Gravity treatments showed that TAC1 is induced by gravistimulation and negatively regulates shoot gravitropism, likely by affecting auxin distribution. Notably, the tested indica rice containing TAC1 displayed dynamic plant architecture under natural long-day conditions, likely explaining the widespread distribution of TAC1 in indica rice. Our results provide new insights into TAC1-mediated regulatory mechanisms for dynamic changes in rice plant architecture.

Disruption of OsPHD1, Encoding a UDP-Glucose Epimerase, Causes JA Accumulation and Enhanced Bacterial Blight Resistance in Rice.

Lesion mimic mutants (LMMs) have been widely used in experiments in recent years for studying plant physiological mechanisms underlying programmed cell death (PCD) and defense responses. Here, we identified a lesion mimic mutant, lm212-1, which cloned the causal gene by a map-based cloning strategy, and verified this by complementation. The causal gene, OsPHD1, encodes a UDP-glucose epimerase (UGE), and the OsPHD1 was located in the chloroplast. OsPHD1 was constitutively expressed in all organs, with higher expression in leaves and other green tissues. lm212-1 exhibited decreased chlorophyll content, and the chloroplast structure was destroyed. Histochemistry results indicated that H2O2 is highly accumulated and cell death is occurred around the lesions in lm212-1. Compared to the wild type, expression levels of defense-related genes were up-regulated, and resistance to bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) was enhanced, indicating that the defense response was activated in lm212-1, ROS production was induced by flg22, and chitin treatment also showed the same result. Jasmonic acid (JA) and methyl jasmonate (MeJA) increased, and the JA signaling pathways appeared to be disordered in lm212-1. Additionally, the overexpression lines showed the same phenotype as the wild type. Overall, our findings demonstrate that OsPHD1 is involved in the regulation of PCD and defense response in rice.

Tiller Angle Control 1 Is Essential for the Dynamic Changes in Plant Architecture in Rice.

Plant architecture is dynamic as plants develop. Although many genes associated with specific plant architecture components have been identified in rice, genes related to underlying dynamic changes in plant architecture remain largely unknown. Here, we identified two highly similar recombinant inbred lines (RILs) with different plant architecture: RIL-Dynamic (D) and RIL-Compact (C). The dynamic plant architecture of RIL-D is characterized by 'loosetiller angle (tillering stage)-compact (heading stage)-loosecurved stem (maturing stage)' under natural long-day (NLD) conditions, and 'loosetiller angle (tillering and heading stages)-loosetiller angle and curved stem (maturing stage)' under natural short-day (NSD) conditions, while RIL-C exhibits a compact plant architecture both under NLD and NSD conditions throughout growth. The candidate locus was mapped to the chromosome 9 tail via the rice 8K chip assay and map-based cloning. Sequencing, complementary tests, and gene knockout tests demonstrated that Tiller Angle Control 1 (TAC1) is responsible for dynamic plant architecture in RIL-D. Moreover, TAC1 positively regulates loose plant architecture, and high TAC1 expression cannot influence the expression of tested tiller-angle-related genes. Our results reveal that TAC1 is necessary for the dynamic changes in plant architecture, which can guide improvements in plant architecture during the modern super rice breeding.

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice.

KEY MESSAGE: The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

TDR INTERACTING PROTEIN 3, encoding a PHD-finger transcription factor, regulates Ubisch bodies and pollen wall formation in rice.

Male reproductive development involves a complex series of biological events and precise transcriptional regulation is essential for this biological process in flowering plants. Several transcriptional factors have been reported to regulate tapetum and pollen development, however the transcriptional mechanism underlying Ubisch bodies and pollen wall formation remains less understood. Here, we characterized and isolated a male sterility mutant of TDR INTERACTING PROTEIN 3 (TIP3) in rice. The tip3 mutant displayed smaller and pale yellow anthers without mature pollen grains, abnormal Ubisch body morphology, no pollen wall formation, as well as delayed tapetum degeneration. Map-based cloning demonstrated that TIP3 encodes a conserved PHD-finger protein and further study confirmed that TIP3 functioned as a transcription factor with transcriptional activation activity. TIP3 is preferentially expressed in the tapetum and microspores during anther development. Moreover, TIP3 can physically interact with TDR, which is a key component of the transcriptional cascade in regulating tapetum development and pollen wall formation. Furthermore, disruption of TIP3 changed the expression of several genes involved in tapetum development and degradation, biosynthesis and transport of lipid monomers of sporopollenin in tip3 mutant. Taken together, our results revealed an unprecedented role for TIP3 in regulating Ubisch bodies and pollen exine formation, and presents a potential tool to manipulate male fertility for hybrid rice breeding.

OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice.

Cullin3-based RING E3 ubiquitin ligases (CRL3), composed of Cullin3 (CUL3), RBX1, and BTB proteins, are involved in plant immunity, but the function of CUL3 in the process is largely unknown. Here, we show that rice (Oryza sativa) OsCUL3a is important for the regulation of cell death and immunity. The rice lesion mimic mutant oscul3a displays a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in pathogenesis-related gene expression as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. We cloned the OsCUL3a gene via a map-based strategy and found that the lesion mimic phenotype of oscul3a is associated with the early termination of OsCUL3a protein. Interaction assays showed that OsCUL3a interacts with both OsRBX1a and OsRBX1b to form a multisubunit CRL in rice. Strikingly, OsCUL3a interacts with and degrades OsNPR1, which acts as a positive regulator of cell death in rice. Accumulation of OsNPR1 protein is greater in the oscul3a mutant than in the wild type. Furthermore, the oscul3a osnpr1 double mutant does not exhibit the lesion mimic phenotype of the oscul3a mutant. Our data demonstrate that OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice.

Premature leaf senescence 3, encoding a methyltransferase, is required for melatonin biosynthesis in rice.

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H2 O2 ) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence-associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O-methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild-type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild-type rice. Moreover, overexpression of OsMTS1 in the wild-type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.

LMM24 Encodes Receptor-Like Cytoplasmic Kinase 109, Which Regulates Cell Death and Defense Responses in Rice.

Lesion mimic mutants are excellent models for research on molecular mechanisms of cell death and defense responses in rice. We identified a new rice lesion mimic mutant lmm24 from a mutant pool of indica rice cultivar "ZhongHui8015". The LMM24 gene was identified by MutMap, and LMM24 was confirmed as a receptor-like cytoplasmic kinase 109 by amino acid sequence analysis. The lmm24 mutant displayed dark brown lesions in leaves and growth retardation that were not observed in wild-type ZH8015. The results of histochemical staining and TUNEL assays showed enhanced ROS accumulation and cell death in lmm24. Chloroplast degradation was observed in lmm24 leaves, with decreased expression of photosynthesis-related genes and increased expression of the senescence-induced STAYGREEN (SGR) gene and other senescence-associated genes. Furthermore, lmm24 exhibited enhanced resistance to rice blast fungus Magnaporthe oryzae (M. oryzae) and up-regulation of defense response genes. Our data demonstrate that LMM24 regulates cell death and defense responses in rice.

The rice CONSTANS-like protein OsCOL15 suppresses flowering by promoting Ghd7 and repressing RID1.

The photoperiodic flowering pathway is one of the most important regulatory networks controlling flowering time in rice (Oryza sativa L.). Rice is a facultative short-day (SD) plant; flowering is promoted under inductive SD conditions and delayed under non-inductive long-day (LD) conditions. In rice, flowering inhibitor genes play an important role in maintaining the trade-off between reproduction and yield. In this study, we identified a novel floral inhibitor, OsCOL15, which encodes a CONSTANS-like transcription factor. Consistent with a function in transcriptional regulation, OsCOL15 localized to the nucleus. Moreover, OsCOL15 had transcriptional activation activity, and the central region of the protein between the B-box and CCT domains was required for this activity. We determined that OsCOL15 is most highly expressed in young organs and exhibits a diurnal expression pattern typical of other floral regulators. Overexpression of OsCOL15 resulted in a delayed flowering phenotype under both SD and LD conditions. Real-time quantitative RT-PCR analysis of flowering regulator gene expression suggested that OsCOL15 suppresses flowering by up-regulating the flowering repressor Grain number, plant height and heading date 7 (Ghd7) and down-regulating the flowering activator Rice Indeterminate 1 (RID1), thus leading to the down-regulation of the flowering activators Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T1. These results demonstrate that OsCOL15 is an important floral regulator acting upstream of Ghd7 and RID1 in the rice photoperiodic flowering-time regulatory network.

Fine mapping and candidate gene analysis of qHD5, a novel major QTL with pleiotropism for yield-related traits in rice (Oryza sativa L.).

KEY MESSAGE: A major QTL for heading date, qHD5, was fine-mapped to a 52.59-kb region on the short arm of rice chromosome 5. Heading date (HD) is one of the most important traits that enables rice to adapt to seasonal differences and specific growth conditions in diverse growing regions. In this study, a major-effect quantitative trait locus (QTL), qHD5, was resolved as a single Medelian factor that causes NIL(BG1) and NIL(XLJ) (two near-isogenic lines (NILs) used in our study) to have at a minimum of 10-day difference in HD under both long-day and short-day conditions in rice. qHD5 was initially mapped to a 309.52-kb genomic region in our previous study. Here, using an advanced BC4F3 population and map-based cloning, we further narrowed the location of qHD5 to a 52.59-kb region between the H71 and RD502 markers. Sequence analysis revealed that Os05g03040, which putatively encodes an AP2 (APETALA2) transcription factor, has six single nucleotide polymorphisms (SNPs) between NIL(BG1) and NIL(XLJ). On this basis, this gene was concluded to be the most probable candidate gene for qHD5. Our results also showed that Hd3a, RFT1, Hd1, Ehd1, and Ghd7 were differentially expressed in the two NILs. Moreover, qHD5 was found to affect yield-related traits such as flag leaf width, flag leaf length, branch number, and 1000-grain weight.

OsCPK12 phosphorylates OsCATA and OsCATC to regulate H2O2 homeostasis and improve oxidative stress tolerance in rice.

Calcium-dependent protein kinases (CPKs), the best-characterized calcium sensors in plants, regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. However, how CPKs regulate the antioxidant defense system remains largely unknown. We previously found that impaired function of OsCPK12 leads to oxidative stress in rice, with more H2O2, lower catalase (CAT) activity, and lower yield. Here, we explored the roles of OsCPK12 in oxidative stress tolerance in rice. Our results show that OsCPK12 interacts with and phosphorylates OsCATA and OsCATC at Ser11. Knockout of either OsCATA or OsCATC leads to an oxidative stress phenotype accompanied by higher accumulation of H2O2. Overexpression of the phosphomimetic proteins OsCATAS11D and OsCATCS11D in oscpk12-cr reduced the level of H2O2 accumulation. Moreover, OsCATAS11D and OsCATCS11D showed enhanced catalase activity in vivo and in vitro. OsCPK12-overexpressing plants exhibited higher CAT activity as well as higher tolerance to oxidative stress. Our findings demonstrate that OsCPK12 affects CAT enzyme activity by phosphorylating OsCATA and OsCATC at Ser11 to regulate H2O2 homeostasis, thereby mediating oxidative stress tolerance in rice.

Transcriptome analysis of rice root heterosis by RNA-Seq.

BACKGROUND: Heterosis is a phenomenon in which hybrids exhibit superior performance relative to parental phenotypes. In addition to the heterosis of above-ground agronomic traits on which most existing studies have focused, root heterosis is also an indispensable component of heterosis in the entire plant and of major importance to plant breeding. Consequently, systematic investigations of root heterosis, particularly in reproductive-stage rice, are needed. The recent advent of RNA sequencing technology (RNA-Seq) provides an opportunity to conduct in-depth transcript profiling for heterosis studies. RESULTS: Using the Illumina HiSeq 2000 platform, the root transcriptomes of the super-hybrid rice variety Xieyou 9308 and its parents were analyzed at tillering and heading stages. Approximately 391 million high-quality paired-end reads (100-bp in size) were generated and aligned against the Nipponbare reference genome. We found that 38,872 of 42,081 (92.4%) annotated transcripts were represented by at least one sequence read. A total of 829 and 4186 transcripts that were differentially expressed between the hybrid and its parents (DGHP) were identified at tillering and heading stages, respectively. Out of the DGHP, 66.59% were down-regulated at the tillering stage and 64.41% were up-regulated at the heading stage. At the heading stage, the DGHP were significantly enriched in pathways related to processes such as carbohydrate metabolism and plant hormone signal transduction, with most of the key genes that are involved in the two pathways being up-regulated in the hybrid. Several significant DGHP that could be mapped to quantitative trait loci (QTLs) for yield and root traits are also involved in carbohydrate metabolism and plant hormone signal transduction pathways. CONCLUSIONS: An extensive transcriptome dataset was obtained by RNA-Seq, giving a comprehensive overview of the root transcriptomes at tillering and heading stages in a heterotic rice cross and providing a useful resource for the rice research community. Using comparative transcriptome analysis, we detected DGHP and identified a group of potential candidate transcripts. The changes in the expression of the candidate transcripts may lay a foundation for future studies on molecular mechanisms underlying root heterosis.

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions.

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14-18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC3F3 recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC3F3 recombinants, the root lengths of their BC3F4 progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice.

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/ß Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1-1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7.

INTRODUCTION: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. OBJECTIVES: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s-OsELF3-1-OsLUX complex directly regulates flowering-related genes to mediate rice heading. METHODS: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s-OsELF3-1-OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s-OsELF3-1-OsLUX) complex. RESULTS: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s-OsELF3-1-OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. CONCLUSION: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s-OsELF3-1-OsLUX)-Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.