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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 1,080-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., Competing Interests: Conflict of interest statement. None declared., (© Crown copyright 2024.)

The flowering period of rice significantly impacts variety adaptability and yield formation. Properly shortening the reproductive period of rice varieties can expand their ecological range without significant yield reduction. Targeted genome editing, like CRISPR/Cas9, is an ideal tool to fine-tune rice growth stages and boost yield synergistically. In this study, we developed a CRISPR/Cas9-mediated multiplex genome-editing vector containing five genes related to three traits, Hd2 , Ghd7 , and DTH8 (flowering-stage genes), along with the recessive rice blast resistance gene Pi21 and the aromatic gene BADH2 . This vector was introduced into the high-quality japonica rice variety in Zhejiang province, Jiahe212 (JH212), resulting in 34 T 0 plants with various effective mutations. Among the 17 mutant T 1 lines, several displayed diverse flowering dates, but most exhibited undesirable agronomic traits. Notably, three homozygous mutant lines (JH-C15, JH-C18, and JH-C31) showed slightly earlier flowering dates without significant differences in yield-related traits compared to JH212. Through special Hyg and Cas marker selection of T 2 plants, we identified seven, six, and two fragrant glutinous plants devoid of transgenic components. These single plants will serve as sib lines of JH212 and potential resources for breeding applications, including maintenance lines for indica - japonica interspecific three-line hybrid rice. In summary, our research lays the foundation for the creation of short-growth-period CMS (cytoplasmic male sterility, CMS) lines, and also provides materials and a theoretical basis for indica - japonica interspecific hybrid rice breeding with wider adaptability.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 H 2 O 2 , 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 H 2 O 2 . Overexpression of the phosphomimetic proteins OsCATA S11D and OsCATC S11D in oscpk12-cr reduced the level of H 2 O 2 accumulation. Moreover, OsCATA S11D and OsCATC S11D 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 H 2 O 2 homeostasis, thereby mediating oxidative stress tolerance in rice., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

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/β 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Chloroplast is an important organelle for photosynthesis and numerous essential metabolic processes, thus ensuring plant fitness or survival. Although many genes involved in chloroplast development have been identified, mechanisms underlying such development are not fully understood. Here, we isolated and characterized the stripe3 (st3) mutant which exhibited white-striped leaves with reduced chlorophyll content and abnormal chloroplast development during the seedling stage, but gradually produced nearly normal green leaves as it developed. Map-based cloning and transgenic tests demonstrated that a splicing mutation in ST3, encoding a human deoxynucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 homolog, was responsible for st3 phenotypes. ST3 is highly expressed in the third leaf at three-leaf stage and expressed constitutively in root, stem, leaf, sheath, and panicle, and the encoded protein, OsSAMHD1, is localized to the cytoplasm. The st3 mutant showed more severe albino leaf phenotype under exogenous 1-mM dATP/dA, dCTP/dC, and dGTP/dG treatments compared with the control conditions, indicating that ST3 is involved in dNTP metabolism. This study reveals a gene associated with dNTP catabolism, and propose a model in which chloroplast development in rice is regulated by the dNTP pool, providing a potential application of these results to hybrid rice breeding., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 ( qHD7a and qHD7b ) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs ( qHD7b ) using 2997 BC 5 F 2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs ( qHD2a , qHD4a , qHD4b , qHD5a , qHD6a , qHD6b , qHD7b , qHD7c , qHD8a , qHD10a , qHD10b , qHD11a , qHD12a , and qHD12b ). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC 5 F 2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 ( Os07g0261200 ) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.

Lesion mimic mutants (LMMs) are ideal materials for studying cell death and resistance mechanisms. Here, we identified and mapped a novel rice LMM, g380 . The g380 exhibits a spontaneous hypersensitive response-like cell death phenotype accompanied by excessive accumulation of reactive oxygen species (ROS) and upregulated expression of pathogenesis-related genes, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae ( Xoo ). Using a map-based cloning strategy, a 184,916 bp deletion on chromosome 2 that overlaps with the diterpenoid biosynthetic gene cluster was identified in g380 . Accordingly, the content of diterpenoids decreased in g380 . In addition, lignin, one of the physical lines of plant defense, was increased in g380 . RNA-seq analysis showed 590 significantly differentially expressed genes (DEG) between the wild-type 9311 and g380 , 585 of which were upregulated in g380 . Upregulated genes in g380 were mainly enriched in the monolignol biosynthesis branches of the phenylpropanoid biosynthesis pathway, the plant-pathogen interaction pathway and the phytoalexin-specialized diterpenoid biosynthesis pathway. Taken together, our results indicate that the diterpenoid biosynthetic gene cluster on chromosome 2 is involved in immune reprogramming, which in turn regulates cell death 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 'loose tiller angle (tillering stage)-compact (heading stage)-loose curved stem (maturing stage)' under natural long-day (NLD) conditions, and 'loose tiller angle (tillering and heading stages)-loose tiller 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.

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 H 2 O 2 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.

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1 , having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1 . Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Khan, Yu, Sun, Abbas, Shah, Xiang, Wang, Sohail, Zhang, Liu, Cheng and Cao.)

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.

Background: Plant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood., Results: Necrosis appeared in leaf tips at the tillering stage, finally resulting in 3-5 cm of dark brown necrotic tissue. ltn-212 showed obvious cell death and accumulation of H 2 O 2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212., Conclusion: These results demonstrate that OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.

Rice ( Oryza sativa L.) occupies a very salient and indispensable status among cereal crops, as its vast production is used to feed nearly half of the world's population. Male sterile plants are the fundamental breeding materials needed for specific propagation in order to meet the elevated current food demands. The development of the rice varieties with desired traits has become the ultimate need of the time. Genic male sterility is a predominant system that is vastly deployed and exploited for crop improvement. Hence, the identification of new genetic elements and the cognizance of the underlying regulatory networks affecting male sterility in rice are crucial to harness heterosis and ensure global food security. Over the years, a variety of genomics studies have uncovered numerous mechanisms regulating male sterility in rice, which provided a deeper and wider understanding on the complex molecular basis of anther and pollen development. The recent advances in genomics and the emergence of multiple biotechnological methods have revolutionized the field of rice breeding. In this review, we have briefly documented the recent evolution, exploration, and exploitation of genic male sterility to the improvement of rice crop production. Furthermore, this review describes future perspectives with focus on state-of-the-art developments in the engineering of male sterility to overcome issues associated with male sterility-mediated rice breeding to address the current challenges. Finally, we provide our perspectives on diversified studies regarding the identification and characterization of genic male sterility genes, the development of new biotechnology-based male sterility systems, and their integrated applications for hybrid rice breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abbas, Yu, Sun, Yang, Chen, Cheng and Cao.)

KEY MESSAGE: qRN5a, a novel QTL for increasing root number under low K in rice, was fine mapped to a 48.8-kb region on chromosome 5, and LOC_Os05g27980 is the most likely candidate gene. Potassium (K) is a mineral nutrient essential for plant growth and development, but the molecular mechanism for low-K (LK) tolerance in rice remains poorly understood. In our previous study, the quantitative trait locus (QTL) qRN5a for root number (RN) under LK was identified in the chromosome segment substitution line CSSL35 carrying segments from XieqingzaoB in the genetic background of Zhonghui9308 (ZH9308). CSSL35 developed more roots than ZH9308 under LK at the seedling stage, and qRN5a was initially located within a 1,023-kb genomic region. In this study, to understand the molecular basis of qRN5a, a large F 2:3 (BC 5 F 2:3 ) population obtained from crossing CSSL35 and ZH9308 was constructed for fine mapping. High-resolution linkage analysis narrowed down qRN5a to a 48.8-kb interval flanked by markers A99 and A139. Seven putative candidate genes were annotated in the delimited region, and three genes (Os05g0346700, LOC_Os05g27980, and LOC_Os05g28000) had nonsynonymous single-nucleotide polymorphisms in the coding sequence between the two parents. Expression analysis suggests that LOC_Os05g27980, which encodes a LATERAL ORGAN BOUNDARIES domain-containing protein, is a positive regulator of RN under LK and is the most likely candidate gene for qRN5a. Moreover, we found that qRN5a promotes expression of OsIAA23 and represses OsHAK5 expression in root tissues to promote root initiation in CSSL35 under LK conditions. Additional investigations on OsHAK5 in rice are needed to elucidate the basis of changing root architecture under different K + concentrations. qRN5a is useful for marker-assisted selection to develop an ideotype with improved root architecture in rice under K deficiency.

Plant lesion mimic mutants have been used as ideal materials for studying pathogen defense mechanisms due to their spontaneous activation of defense responses in plants. Here, we report the identification and characterization of a rice lesion mimic mutant, oshpl3. The oshpl3 mutant initially displayed white spots on leaves of 7-day-old seedlings, and the white spots gradually turned into large brown spots during plant development, accompanied by poor metrics of major agronomic traits. Histochemical analysis showed that spontaneous cell death and H 2 O 2 hyperaccumulation occurred in oshpl3. Defense responses were induced in the oshpl3 mutant, such as enhanced ROS signaling activated by recognition of pathogen-associated molecular patterns, and also upregulated expression of genes involved in pathogenesis and JA metabolism. These defense responses enhanced resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae. The mutated gene was identified as OsHPL3 (LOC_Os02g02000) by map-based cloning. A G1006A mutation occurred in OsHPL3, causing a G-to-D mutation of the 295th amino acid in the transmembrane region of OsHPL3. OsHPL3 localized to the chloroplast, cytoplasm, and another unknown organelle, while the mutated protein OsHPL3 G295D was not obviously observed in the chloroplast, suggesting that the G295D mutation affected its chloroplast localization. Based on our findings, the G295D mutation in OsHPL3 is most likely responsible for the phenotypes of the oshpl3 mutant. Our results provide new clues for studying the function of the OsHPL3 protein., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Tiller number is a crucial agronomic trait that directly affects the number of effective panicles and yield formation in rice. Here, we report a semi-dwarf and low tillering mutant Osdlt10 (dwarf and low tillering 10) that exhibited reduced tiller number, semi-dwarfism, increased grain width, low seed-setting rate, curled leaf tip and a series of abnormalities of agronomic traits. Phenotypic observations showed that Osdlt10 mutants had defects in tiller bud formation and grew slowly at the tillering stage. Map-based cloning revealed that LOC_Os10g41310 was the responsible gene for OsDLT10, which was subsequently demonstrated using the CRISPR/Cas9 system and a complementary experiment. Expression pattern analysis indicated that OsDLT10 was primarily expressed in the stem node, the basic part of axillary bud and leaf sheath, pulvinus. The hormone treatment investigation indicated that extremely high of exogenous auxin concentrations can inhibit the expression of OsDLT10. Endogenous auxin content decreased significantly at the base of stem node and axillary bud in Osdlt10 mutants. The results showed that OsDLT10 was related to auxin. qPCR analysis results further showed that the expression levels of auxin transport genes (PINs) and early response genes (IAAs) were significantly increased. The expression levels of WUS-like and FON1 were substantially decreased in the Osdlt10 mutants. These results revealed that OsDLT10 played a critical role in influencing tiller number, likely in association with hormone signals and the WUS-CLV pathway, to regulate axillary bud development in rice., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

This study applies parallel reaction monitoring (PRM) proteomics and CRISPR-Cas9 mutagenesis to identify relationships between cell metabolism, cell death, and disease resistance. In oscul3a ( oscullin3a ) mutants, OsCUL3a-associated molecular switches are responsible for disrupted cell metabolism that leads to increased total lipid content in rice grain, a late accumulation of H 2 O 2 in leaves, enhanced Xanthomonas oryzae pv. oryzae disease resistance, and suppressed panicle and first internode growth. In oscul3a mutants, PRM-confirmed upregulated molecular switch proteins include lipoxygenases (CM-LOX1 and CM-LOX2), suggesting a novel connection between ferroptosis and rice lesion mimic formation. Rice immunity-associated proteins OsNPR1 and OsNPR3 were shown to interact with each other and have opposing regulatory effects based on the cell death phenotype of osnpr1 / oscul3a and osnpr3 / oscul3a double mutants. Together, these results describe a network that regulates plant growth, disease resistance, and grain quality that includes the E3 ligase OsCUL3a, cell metabolism-associated molecular switches, and immunity switches OsNPR1 and OsNPR3.

Leaf senescence, which affects plant growth and yield in rice, is an ideal target for crop improvement and remarkable advances have been made to identify the mechanism underlying this process. We have characterized an early senile mutant es5 (early leaf senescence 5) in rice exhibiting leaf yellowing phenotype after the 4-leaf stage. This phenotype was confirmed by the higher accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), the disintegration of chloroplasts, reduction in chlorophyll content and photosynthetic rate and up-regulation of senescence-associated genes (SAGs) like Osh36, OsI57, and OsI85. Positional cloning revealed that the es5 phenotype is the result of one base substitution in ES5, encoding phosphatidylserine synthase (PSS) family protein, which is involved in the base-exchange type reaction to synthesize the minor membrane phospholipid phosphatidylserine. Functional complementation of ES5 in the es5 plants completely restored the wild-type phenotype. Ultra-high-performance liquid chromatography (UHPLC) analysis showed that es5 plants had increased levels of phosphatidylserine (PS) and decreased level of phosphatidylcholine (PC). These results provide evidence about the role of PS in rice leaf senescence.

Purpose: The aim of this study was to determine the anatomical variations of the superior transverse scapular ligament (STSL) for better understanding the possible predisposing factors for suprascapular nerve entrapment., Methods: The study was using fifty 10% formalin solution-fixed human cadaveric shoulders. After dissection of the suprascapular region, the length, medial width, lateral width and middle width of the suprascapular opening were measured for each STSL., Results: The STSL displayed six types as: (1) band-shaped in 11 cases; (2) fan-shaped in 27 cases; (3) triangular-shaped in 5 cases; (4) linear type in 2 cases; (5) bifid in 1 case; (6) absent in 1 case. The ossified type of STSL was found in 3 cases. There were statistically significant differences in the length (P = 0.009), medial width (P = 0.001), lateral width (P = 0.029) of the three types of fan-shaped, band-shaped and triangular-shaped. However, there was no statistical difference in the middle width of the suprascapular opening of the three types (P = 0.340)., Conclusion: Knowing the morphological features and variations of the STSL is important for better understanding the anatomical conditions, which could be taken into consideration during open suprascapular operations or arthroscopic decompressions.

Lesion mimic mutants are ideal genetic materials to study programmed cell death and defense signaling in plants. However, the molecular basis of lesion mimic formation remains largely unknown. Here, we first used a proteomic approach to identify differentially expressed proteins during dynamic lesion mimic formation in the rice oscul3a mutant, then electron microscope observation and physiological assays were used to analyze the mutant. The oscul3a mutant had disrupted cell metabolism balance, and the identified differentially expressed proteins were mainly located in the chloroplast and cytoplasm, which caused enhanced lipid metabolism, but suppressed carbon/nitrogen metabolism with reduced growth and grain quality. The oscul3a mutant had higher salicylic acid (SA) concentration in leaves, and H 2 O 2 was shown to accumulate late in the formation of lesions. The secondary metabolite coumarin induced reactive oxygen species (ROS) and had rice blast resistance activity. Moreover, the cell death initiated lesion mimic formation of oscul3a mutant was light-sensitive, which might be associated with metabolite biosynthesis and accumulation. This study sheds light on the metabolic transition associated with cell death and defense response, which is under tight regulation by OsCUL3a and metabolism-related proteins, and the newly identified chemicals in the secondary metabolic pathway can potentially be used to control disease in crop plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

Mitogen-activated protein kinase (MAPK) cascades play central roles in response to biotic and abiotic stresses. However, the mechanisms by which various MAPK members regulate the plant immune response in rice remain elusive. In this article, to characterize the mechanisms, the knock-out and overexpression mutants of OsMPK15 were constructed and the disease resistance was investigated under the various fungal and bacterial inoculations. The knock-out mutant of OsMPK15 resulted in the constitutive expression of pathogenesis-related ( PR ) genes, increased accumulation of reactive oxygen species (ROS) triggered by the pathogen-associated molecular pattern (PAMP) elicitor chitin, and significantly enhanced the disease resistance to different races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae ( Xoo ), which cause the rice blast and bacterial blight diseases, respectively. On contrary, the expression of PR genes and ROS were down-regulated in the OsMPK15 -overexpressing (OsMPK15-OE) lines. Meanwhile, phytohormones such as salicylic acid (SA) and jasmonic acid (JA) were accumulated in the mpk15 mutant lines but decreased in the OsMPK15-OE lines. The expression of SA- and JA-pathway associated genes were significantly upregulated in the mpk15 mutant, whereas it was down regulated in the OsMPK15-OE lines. We conclude that OsMPK15 may negatively regulate the disease resistance through modulating SA- and JA-mediated signaling pathway.

Chlorophyll plays a vital role in harvesting light and turning it into chemical energy. In this study, we isolated and characterized a chlorophyll-deficient mutant, which we named cs3 (chlorotic seedling 3). The cs3 mutant seedlings exhibit a yellowish phenotype at germination, and they do not survive at the seedling stage. In addition, brown necrotic spots appear on the surface of the leaves and leaf sheaths during development. DAB staining and H 2 O 2 content measurement showed that there was excessive H 2 O 2 accumulation in the cs3 mutant leaf. Accompanying the chlorophyll deficiency, the chloroplasts in cs3 leaf cells were abnormal. Using a map-based cloning strategy, we mapped the CS3 gene, which encodes a Ycf54 domain-containing protein, to a locus on chromosome 3. CS3 is mainly expressed in green tissues and the S136 F would influence CS3 interacting with YGL8 and its chloroplast localization. qRT-PCR analysis revealed the changes in the expression of genes involved in chlorophyll biosynthesis and degradation, chloroplast development, senescence, and photosynthesis in the cs3 mutant. In addition, our study also supports the notion that the mutation in the CS3/Ycf54 gene arrests chlorophyll biosynthesis by negatively affecting the activity of magnesium protoporphyrin IX monomethylester cyclase (MgPME-cyclase)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Seed setting rate is one of the major components that determine rice (Oryza sativa L.) yield. Successful fertilization is necessary for normal seed setting. However, little is known about the molecular mechanisms governing this process. In this study, we report a novel rice gene, LOW SEED SETTING RATE1 (LSSR1), which regulates the seed setting rate by facilitating rice fertilization. LSSR1 encodes a putative GH5 cellulase, which is highly conserved in plants. LSSR1 is predominantly expressed in anthers during the microsporogenesis stage, and its encoded protein contains a signal peptide at the N-terminal, which may be a secretory protein that stores in pollen grains and functions during rice fertilization. To explore the physiological function of LSSR1 in rice, loss-of-function mutants of LSSR1 were created through the CRISPR-Cas9 system, which showed a significant decrease in rice seed setting rate. However, the morphology of the vegetative and reproductive organs appears normal in lssr1 mutant lines. In addition, lssr1 pollen grains could be normally stained by I 2 -KI solution. Cytological results demonstrate that the blockage of fertilization mostly accounted for the low seed setting rate in lssr1 mutant lines, which was most likely caused by abnormal pollen grain germination, failed pollen tube penetration, and retarded pollen tube elongation. Together, our results suggest that LSSR1 plays an important role in rice fertilization, which in turn is vital for maintaining rice seed setting rate.

Premature leaf senescence affects plant yield and quality, and numerous researches about it have been conducted until now. In this study, we identified an early senescent mutant es4 in rice ( Oryza sativa L.); early senescence appeared approximately at 60 dps and became increasingly senescent with the growth of es4 mutant. We detected that content of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as activity of superoxide dismutase (SOD) were elevated, while chlorophyll content, soluble protein content, activity of catalase (CAT), activity of peroxidase (POD) and photosynthetic rate were reduced in the es4 mutant leaves. We mapped es4 in a 33.5 Kb physical distance on chromosome 4 by map-based cloning. Sequencing analysis in target interval indicated there was an eight bases deletion mutation in OsCPK12 which encoded a calcium-dependent protein kinase. Functional complementation of OsCPK12 in es4 completely restored the normal phenotype. We used CRISPR/Cas9 for targeted disruption of OsCPK12 in ZH8015 and all the mutants exhibited the premature senescence. All the results indicated that the phenotype of es4 was caused by the mutation of OsCPK12 . Overexpression of OsCPK12 in ZH8015 enhanced the net photosynthetic rate ( P n ) and chlorophyll content. OsCPK12 was mainly expressed in green organs. The results of qRT-PCR analysis showed that the expression levels of some key genes involved in senescence, chlorophyll biosynthesis, and photosynthesis were significantly altered in the es4 mutant. Our results demonstrate that the mutant of OsCPK12 triggers the premature leaf senescence; however, the overexpression of OsCPK12 may delay its growth period and provide the potentially positive effect on productivity in rice.

Background: Nitrogen (N) is a major input cost in rice production, in addition to causing severe pollution to agricultural and ecological environments. Root dry weight has been considered the most important component related to crop yields than the other root traits. Therefore, development of rice varieties/lines with low input of N fertilizer and higher root traits are essential for sustainable rice production., Results: In this context, a main effect quantitative trait locus qRDWN6 XB on the long arm of chromosome 6 which positively confers tolerance to N deficiency in the Indica rice variety XieqingzaoB, was identified using a chromosomal segment substitution line (CSSL) population. qRDWN6 XB was determined to be located near marker InD90 on chromosome 6 based on association analysis of phenotype data from three N levels and 120 polymorphic molecular markers. The target chromosomal segment substitution line CSSL45, which has the higher root dry weight (RDW) than indica cultivar Zhonghui9308 and carry qRDWN6 XB , was selected for further study. A BC 5 F 2:3 population derived from a cross between CSSL45 and Zhonghui9308 was constructed. To fine-map qRDWN6 XB , we used the homozygous recombinant plants and ultimately this locus was narrowed to a 52.3-kb between markers ND-4 and RM19771, which contains nine candidate genes in this region. One of these genes, LOC_Os06g15910 as a potassium transporter was considered a strong candidate gene for the RDWN6 XB locus., Conclusions: The identification of qRDWN6 XB provides a new genetic resource for breeding rice varieties and a starting point to improve grain yield despite the decreased input of N fertilizers. The newly developed and tightly linked InDel marker ND-4 will be useful to improve the root system architecture under low N by marker-assisted selection (MAS) in rice breeding programs.

Key Message: OsMS1 functions as a transcriptional activator and interacts with known tapetal regulatory factors through its plant homeodomain (PHD) regulating tapetal programmed cell death (PCD) and pollen exine formation in rice. The tapetum, a hallmark tissue in the stamen, undergoes degradation triggered by PCD during post-meiotic anther development. This degradation process is indispensable for anther cuticle and pollen exine formation. Previous study has shown that PTC1 plays a critical role in the regulation of tapetal PCD. However, it remained unclear how this occurs. To further investigate the role of this gene in rice, we used CRISPR/Cas9 system to generate the homozygous mutant named as osms1, which showed complete male sterility with slightly yellow and small anthers, as well as invisible pollen grains. In addition, cytological observation revealed delayed tapetal PCD, defective pollen exine formation and a lack of DNA fragmentation according to a TUNEL analysis in the anthers of osms1 mutant. OsMS1, which encodes a PHD finger protein, was located in the nucleus of rice protoplasts and functioned as a transcription factor with transcriptional activation activity. Y2H and BiFC assays demonstrated that OsMS1 can interact with OsMADS15 and TDR INTERACTING PROTEIN2 (TIP2). It has been reported that TIP2 coordinated with TDR to modulate the expression of EAT1 and further regulated tapetal PCD in rice. Results of qPCR suggested that the expression of the genes associated with tapetal PCD and pollen wall biosynthesis, such as EAT1, AP37, AP25, OsC6 and OsC4, were significantly reduced in osms1 mutant. Taken together, our results demonstrate that the interaction of OsMS1 with known tapetal regulatory factors through its PHD finger regulates tapetal PCD and pollen exine formation in rice.

In flowering plants, ideal male reproductive development requires the systematic coordination of various processes, in which timely differentiation and degradation of the anther wall, especially the tapetum, is essential for both pollen formation and anther dehiscence. Here, we show that OsGPAT3 , a conserved glycerol-3-phosphate acyltransferase gene, plays a critical role in regulating anther wall degradation and pollen exine formation. The gpat3-2 mutant had defective synthesis of Ubisch bodies, delayed programmed cell death (PCD) of the inner three anther layers, and abnormal degradation of micropores/pollen grains, resulting in failure of pollen maturation and complete male sterility. Complementation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) experiments demonstrated that OsGPAT3 is responsible for the male sterility phenotype. Furthermore, the expression level of tapetal PCD-related and nutrient metabolism-related genes changed significantly in the gpat3-2 anthers. Based on these genetic and cytological analyses, OsGPAT3 is proposed to coordinate the differentiation and degradation of the anther wall and pollen grains in addition to regulating lipid biosynthesis. This study provides insights for understanding the function of GPATs in regulating rice male reproductive development, and also lays a theoretical basis for hybrid rice breeding.

Most Quantitative Trait Loci (QTL) and gene isolation approaches, such as positional- or map-based cloning, are time-consuming and low-throughput methods. Understanding and detecting the genetic material that controls a phenotype is a key means to functionally analyzing genes as well as to enhance crop agronomic traits. In this regard, high-throughput technologies have great prospects for changing the paradigms of DNA marker revealing, genotyping, and for discovering crop genetics and genomic study. Bulk segregant analysis, based on whole genome resequencing approaches, permits the rapid isolation of the genes or QTL responsible for the causative mutation of the phenotypes. MutMap, MutMap Gap, MutMap+, modified MutMap, and QTL-seq methods are among those approaches that have been confirmed to be fruitful gene mapping approaches for crop plants, such as rice, irrespective of whether the characters are determined by polygenes. As a result, in the present study we reviewed the progress made by all these methods to identify QTL or genes in rice.

Stigma exsertion is a key determinant to increase the efficiency of commercial hybrid rice seed production. The major quantitative trait locus (QTL) qSE7 for stigma exsertion rate was previously detected on the chromosome 7 using 75 Chromosome Segment Substitution Lines (CSSLs) derived from a cross between the high stigma exsertion indica maintainer XieqingzaoB (XQZB) and low stigma exsertion indica restorer Zhonghui9308 (ZH9308). The C51 line, a CSSL population with an introgression from XQZB, was backcrossed with ZH9308 to produce the secondary F 2 (BC 5 F 2 ) and F 2:3 (BC 5 F 2:3 ) populations. As a result, the Near Isogenic Line (NIL qSE7 XB ) was developed. Analysis indicated qSE7 acted as a single Mendelian factor and decreased the stigma exsertion. We hypothesized qSE7 regulates single, dual, and total stigma exsertion rate, provided experimental support. qSE7 was mapped and localized between RM5436 and RM5499 markers, within a physical distance of 1000-kb. With use of new insertion-deletion (InDel) markers and analysis of the heterozygous and phenotypic data, it was ultimately dissected to a 322.9-kb region between InDel SER4-1 and RM5436. The results are useful for additional identification and isolation of this candidate gene controlling stigma exsertion rate, and provide a basis for further fine mapping, gene cloning, and Marker Assisted Selection (MAS) breeding later.

Rice is a pivotal cereal crop that provides the staple food for more than half of the world's population. Along with improvements in the standard of living, people not only pay attention to the grain yield but also to the grain quality. Chalkiness is one of the most important index of grain quality. In this study, qPCG1 , a QTL for percentage of chalky grain, was mapped in an interval with a physical distance about 139 kb on chromosome 1 by residual heterozygous line (RHL) method. qPCG1 was incomplete dominant and the additive effect plays a major role and explained 6.8-21.9% of phenotypic variance within the heterogeneous region on chromosome 1. The effect of allele from Zhonghui9308 was decreasing the percentage of chalky grains (PCG). Microscope observation results indicated that there are great differences in the shape, structure and arrangement of starch granule between the chalky part and transparent part. Analysis of starch physicochemical properties showed that the total starch content, amylose content and chain length distribution of amylopectin changed while the protein contents were not apparently affected with the changed chalkiness. qPCG1 had little influence on main agronomic traits and it might be useful in rice breeding for it did not bring negative effect on grain yield while reducing the chalkiness.

Abnormally developed endosperm strongly affects rice ( Oryza sativa ) appearance quality and grain weight. Endosperm formation is a complex process, and although many enzymes and related regulators have been identified, many other related factors remain largely unknown. Here, we report the isolation and characterization of a recessive mutation of White Belly 1 ( WB1 ), which regulates rice endosperm development, using a modified MutMap method in the rice mutant wb1 . The wb1 mutant develops a white-belly endosperm and abnormal starch granules in the inner portion of white grains. Representative of the white-belly phenotype, grains of wb1 showed a higher grain chalkiness rate and degree and a lower 1000-grain weight (decreased by ~34%), in comparison with that of Wild Type (WT). The contents of amylose and amylopectin in wb1 significantly decreased, and its physical properties were also altered. We adopted the modified MutMap method to identify 2.52 Mb candidate regions with a high specificity, where we detected 275 SNPs in chromosome 4. Finally, we identified 19 SNPs at 12 candidate genes. Transcript levels analysis of all candidate genes showed that WB1 ( Os04t0413500 ), encoding a cell-wall invertase, was the most probable cause of white-belly endosperm phenotype. Switching off WB1 with the CRISPR/cas9 system in Japonica cv. Nipponbare demonstrates that WB1 regulates endosperm development and that different mutations of WB1 disrupt its biological function. All of these results taken together suggest that the wb1 mutant is controlled by the mutation of WB1 , and that the modified MutMap method is feasible to identify mutant genes, and could promote genetic improvement 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 (H 2 O 2 ) 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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Phosphorus (P) is the essential macro-element supporting rice productivity. Quantitative trait loci (QTL) underlying related traits at the seedling stage under two different phosphorus levels was investigated in rice using a population of 76 Chromosomal Sequence Substitution Lines (CSSLs) derived from a cross between the maintainer variety XieqingzaoB (P stress tolerant) and the restorer variety Zhonghui9308 (P stress sensitive); the parents of super hybrid rice Xieyou9308. A genetic linkage map with 120 DNA marker loci was constructed. At logarithmic odd (LOD) value of 2.0, a total of seven QTLs were detected for studied traits under two P levels and their relative ratio. The LOD values ranged from 2.00 to 3.32 and explaining 10.82% to 18.46% of phenotypic variation. Three QTLs were detected under low phosphorus (P - ), one under normal (P⁺) and three under their relative ratio (P - /P⁺) on the rice chromosomes 3, 5, 6, 8 and 10. No significant QTLs were found for shoot dry weight (SDW) and total dry weight (TDW). The pleiotropic QTLs influencing root number ( qRN5 ) and root dry weight ( qRDW5 ) as novel QTLs under P - level were detected near marker RM3638 on chromosome 5, which considered to directly contributing to phosphorus deficiency tolerance in rice. These QTLs need further analysis, including the fine mapping and cloning, which may use in molecular marker assisted breeding., Competing Interests: The authors declare no conflict of interest.

Anther cuticle and pollen exine are two elaborated lipid-soluble barriers protecting pollen grains from environmental and biological stresses. However, less is known about the mechanisms underlying the synthesis of these lipidic polymers. Here, we identified a no-pollen male-sterility mutant cyp703a3-3 from the indica restorer line Zhonghui 8015 (Zh8015) mutant library treated with 60 Coγ-ray radiation. Histological analysis indicated that cyp703a3-3 underwent abnormal tapetal cells development, produced few orbicules and secreted less sporopollenin precursors to anther locule, as well as cutin monomers on anther. Genetic analysis revealed that cyp703a3-3 was controlled by a single recessive gene. Map-based cloning was performed to narrow down the mutant gene to a 47.78-kb interval on the chromosome 8 between two markers S15-29 and S15-30. Sequence analysis detected three bases (GAA) deletion in the first exon of LOC_Os08g03682, annotated as CYP703A3 with homologous sequences related to male sterility in Arabidopsis, causing the Asparagine deletion in the mutant site. Moreover, we transformed genomic fragment of CYP703A3 into cyp703a3-3, which male-sterility phenotype was recovered. Both the wild-type and cyp703a3-3 mutant 3D structure of CYP703A3 protein were modeled. Results of qPCR suggested CYP703A3 mainly expressed in anthers with greatest abundance at microspore stage, and genes involved in sporopollenin precursors formation and transportation, such as GAMYB, TDR, CYP704B2, DPW2, OsABCG26 and OsABCG15, were significantly reduced in cyp703a3-3. Collectively, our results further elaborated CYP703A3 plays vital role in anther cuticle and pollen exine development in rice (Oryza sativa L.)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The rate of stigma exsertion (SE) is an important trait in rice breeding because the efficiency of hybrid rice seed production can be improved by increasing the percentage of stigmas that exsert. In this study, we developed a near isogenic line (NIL) from two parents, XieqingzaoB (XQZB) and Zhonghoi9308 (ZH9308), which have high and low SE rates in that order. In our previous study, we employed 75 chromosome segment substitution lines (CSSLs) and analyzed quantitative trait loci (QTLs) for their influence on SE rate. The single gene QTL (qSE11), which is located on chromosome 11, was responsible for this trait. In this study, we focused on one of the CSSLs (C65), namely, the NIL (qSE11XB). It contains an introgression segment of XQZB in the genetic background of ZH9308, and exhibits a significantly higher SE rate than that of the parents. We demonstrated that qSE11 regulated both the single and the dual SE rates. They both contribute to the total SE rate. Genetic analysis revealed that qSE11 acted as a single Mendelian factor and that the allele from XQZB increased the SE rate. The validity of our conclusions was established when C65 was used to develop secondary F2 (BC5F2) and F2:3 (BC5F2:3) populations by backcrossing to ZH9308, with subsequent selfing. We entered 3600 plants from the F 2 population and 3200 from the F 2:3 populations into a genetic dissection program and dissected the major QTL qSE11 to a 350.7-kb region located on chromosome 11. This study will contribute to the future isolation of candidate genes of SE and will play a vital role in future hybrid rice seed production programs.

Meiosis is crucial in reproduction of plants and ensuring genetic diversity. Although several genes involved in homologous recombination and DNA repair have been reported, their functions in rice ( Oryza sativa ) male meiosis remain poorly understood. Here, we isolated and characterized the rice OsFIGNL1 ( OsFidgetin-like 1 ) gene, encoding a conserved AAA-ATPase, and explored its function and importance in male meiosis and pollen formation. The rice Osfignl1 mutant exhibited normal vegetative growth, but failed to produce seeds and displayed pollen abortion phenotype. Phenotypic comparisons between the wild-type and Osfignl1 mutant demonstrated that OsFIGNL1 is required for anther development, and that the recessive mutation of this gene causes male sterility in rice. Complementation and CRISPR/Cas9 experiments demonstrated that wild-type OsFIGNL1 is responsible for the male sterility phenotype. Subcellular localization showed that OsFIGNL1-green fluorescent protein was exclusively localized in the nucleus of rice protoplasts. Male meiosis in the Osfignl1 mutant exhibited abnormal chromosome behavior, including chromosome bridges and multivalent chromosomes at diakinesis, lagging chromosomes, and chromosome fragments during meiosis. Yeast two-hybrid assays demonstrated OsFIGNL1 could interact with RAD51A1, RAD51A2, DMC1A, DMC1B, and these physical interactions were further confirmed by BiFC assay. Taken together, our results suggest that OsFIGNL1 plays an important role in regulation of male meiosis and anther development.

Flowering time is an important agronomic trait that coordinates the plant life cycle with regional adaptability and thereby impacts yield potentials for cereal crops. The CONSTANS (CO)-like gene family plays vital roles in the regulation of flowering time. CO-like proteins are typically divided into four phylogenetic groups in rice. Several genes from groups I, III, and IV have been functionally characterized, though little is known about the genes of group II in rice. We report the functional characterization in rice of a constitutive floral inhibitor, OsCOL16, encoding a group-II CO-like protein that delays flowering time and increases plant height and grain yield. Overexpression of OsCOL16 resulted in late heading under both long-day and short-day conditions. OsCOL16 expression exhibits a diurnal oscillation and serves as a transcription factor with transcriptional activation activity. We determined that OsCOL16 up-regulates the expression of the floral repressor Ghd7, leading to down-regulation of the expression of Ehd1, Hd3a, and RFT1. Moreover, genetic diversity and evolutionary analyses suggest that remarkable differences in flowering times correlate with two major alleles of OsCOL16. Our combined molecular biology and phylogeographic analyses revealed that OsCOL16 plays an important role in regulating rice photoperiodic flowering, allowing for environmental adaptation of rice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glutamate synthase (GOGAT) is a key enzyme for nitrogen metabolism and ammonium assimilation in plants. In this study, an early senescence 7 (es7) mutant was identified and characterized. The leaves of the es7 mutant begin to senesce at the tillering stage about 60day after sowing, and become increasingly senescent as the plants develop at the heading stage. When es7 plants are grown under photorespiration-suppressed conditions (high CO 2 ), the senescence phenotype and chlorophyll content are rescued. qRT-PCR analysis showed that senescence- associated genes were up-regulated significantly in es7. A map-based cloning strategy was used to identify ES7, which encodes a ferredoxin-dependent glutamate synthase (Fd-GOGAT). ES7 was expressed constitutively, and the ES7 protein was localized in chloroplast. qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7. Further, we also demonstrated that chlorophyll synthesis-associated genes were significantly down-regulated in es7. In addition, when seedlings are grown under increasing nitrogen concentrations (NH 4 NO 3 ) for 15days, the contents of chlorophyll a, chlorophyll b and total chlorophyll were significantly lower in es7. Our results demonstrated that ES7 is involved in nitrogen metabolism, effects chlorophyll synthesis, and may also associated with photorespiration, impacting leaf senescence in rice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

With the development and application of super rice breeding, elite rice hybrids with super high-yielding potential have been widely developed in last decades in China. Xieyou9308 is one of the most famous super hybrid rice varieties. To uncover the genetic mechanism of Xieyou9308's high yield potential, a recombinant inbred line (RIL) population derived from cross of XieqingzaoB and Zhonghui9308 was re-sequenced and investigated on the grain yield (GYD) and its three component traits, number of panicles per plant (NP), number of filled grains per panicle (NFGP), and grain weight (GW). Unconditional and conditional genome-wide association analysis, based on a linear mixed model with epistasis and gene-environment interaction effects, were conducted, using ~0.7 million identified SNPs. There were six, four, seven, and seven QTSs identified for GYD, NP, NFGP, and GW, respectively, with accumulated explanatory heritability varying from 43.06% to 48.36%; additive by environment interactions were detected for GYD, some minor epistases were detected for NP and NFGP. Further, conditional genetic mapping analysis for GYD given its three components revealed several novel QTSs associated with yield than that were suppressed in our unconditional mapping analysis.

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., (© 2017 American Society of Plant Biologists. All rights reserved.)

Xieyou9308 is a certified super hybrid rice cultivar with a high grain yield. To investigate its underlying genetic basis of high yield potential, a recombinant inbred line (RIL) population derived from the cross between the maintainer line XieqingzaoB (XQZB) and the restorer line Zhonghui9308 (ZH9308) was constructed for identification of quantitative trait SNPs (QTSs) associated with two important agronomic traits, plant height (PH) and heading date (HD). By re-sequencing of 138 recombinant inbred lines (RILs), a total of ~0.7 million SNPs were identified for the association studies on the PH and HD. Three association mapping strategies (including hypothesis-free genome-wide association and its two complementary hypothesis-engaged ones, QTL-based association and gene-based association) were adopted for data analysis. Using a saturated mixed linear model including epistasis and environmental interaction, we identified a total of 31 QTSs associated with either the PH or the HD. The total estimated heritability across three analyses ranged from 37.22% to 45.63% and from 37.53% to 55.96% for the PH and HD, respectively. In this study we examined the feasibility of association studies in an experimental population (RIL) and identified several common loci through multiple strategies which could be preferred candidates for further research.

Source size, sink size and heading date (HD) are three important classes of traits that determine the productivity of rice. In this study, a set of recombinant inbred lines (RILs) derived from the cross between an elite indica line Big Grain1 (BG1) and a japonica line Xiaolijing (XLJ) were used to map quantitative trait loci (QTLs) for source-sink size and heading date. Totally, thirty-one QTLs for source size, twenty-two for sink size, four for heading date and seven QTL clusters which included QTLs for multiple traits were identified in three environmental trials. Thirty QTLs could be consistently detected in at least two trials and generally located in the clusters. Using a set of BC4F2 lines, the QTL cluster in C5-1-C5-2 on chromosome 5 was validated to be a major QTL pleiotropically affecting heading date, source size (flag leaf area) and panicle type (neck length of panicle, primary branching number and the ratio of secondary branching number to primary branching number), and was narrowed down to a 309.52Kb region. QTL clusters described above have a large effect on source-sink size and/or heading date, therefore they should be good resources to improve the adaptability and high yield potential of cultivars genetically., (Copyright © 2015. Published by Elsevier B.V.)

Pseudomonas parafulva (formerly known as Pseudomonas fulva) is an antagonistic bacterium against several rice bacterial and fungal diseases. The total genome size of P. parafulva CRS01-1 is 5,087,619 bp with 4389 coding sequences (CDSs), 77 tRNAs, and 7 rRNAs. The annotated full genome sequence of the P. parafulva CRS01-1 strain might shed light on its role as an antagonistic bacterium., (Copyright © 2015. Published by Elsevier B.V.)