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.

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.

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.

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.

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.

Mapping and Validation of qHD7b: Major Heading-Date QTL Functions Mainly under Long-Day Conditions.

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 BC5F2:3 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 BC5F2:3 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.

qHD5 encodes an AP2 factor that suppresses rice heading by down-regulating Ehd2 expression.

Heading date is crucial for rice reproduction and the geographical expansion of cultivation. We fine-mapped qHD5 and identified LOC_Os05g03040, a gene that encodes an AP2 transcription factor, as the candidate gene of qHD5 in our previous study. In this article, using two near-isogenic lines NIL(BG1) and NIL(XLJ), which were derived from the progeny of the cross between BigGrain1 (BG1) and Xiaolijing (XLJ), we verified that LOC_Os05g03040 represses heading date in rice through genetic complementation and CRISPR/Cas9 gene-editing experiments. Complementary results showed that qHD5 is a semi-dominant gene and that the qHD5XLJ and qHD5BG1 alleles are both functional. The homozygous mutant line generated from knocking out qHD5XLJ in NIL(XLJ) headed earlier than NIL(XLJ) under both short-day and long-day conditions. In addition, the homozygous mutant line of qHD5BG1 in NIL(BG1) also headed slightly earlier than NIL(BG1). All of these results show that qHD5 represses the heading date in rice. Transient expression showed that the qHD5 protein localizes to the nucleus. Transactivation activity assays showed that the C-terminus is the critical site that affects self-activation in qHD5XLJ. qRT-PCR analysis revealed that qHD5 represses flowering by down-regulating Ehd2. qHD5 may have been selected during indica rice domestication.

Deletion of Diterpenoid Biosynthetic Genes CYP76M7 and CYP76M8 Induces Cell Death and Enhances Bacterial Blight Resistance in Indica Rice '9311'.

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.

STRIPE3, encoding a human dNTPase SAMHD1 homolog, regulates chloroplast development in rice.

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.

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.

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.

DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death 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.

OsPG1 Encodes a Polygalacturonase that Determines Cell Wall Architecture and Affects Resistance to Bacterial Blight Pathogen 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 H2O2 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.

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.

Exploiting Genic Male Sterility in Rice: From Molecular Dissection to Breeding Applications.

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.

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.

Dissection of Closely Linked Quantitative Trait Locis Controlling Grain Size in Rice.

Grain size is a key constituent of grain weight and appearance in rice. However, insufficient attention has been paid to the small-effect quantitative trait loci (QTLs) on the grain size. In the present study, residual heterozygous populations were developed for mapping two genetically linked small-effect QTLs for grain size. After the genotyping and the phenotyping of five successive generations, qGS7.1 was dissected into three QTLs and two were selected for further analysis. The qTGW7.2a was finally mapped into a 21.10 kb interval containing four annotated candidate genes. Transcript levels assay showed that the expression of the candidates LOC_Os07g39490 and the LOC_Os07g39500 were significantly reduced in the NIL-qTGW7.2aBG1 . The cytological observation indicated that qTGW7.2a regulated the grain width through controlling the cell expansion. Using the same strategy, qTGW7.2b was fine-mapped into a 52.71 kb interval containing eight annotated candidate genes, showing a significant effect on the grain length and width with opposite allelic directions, but little on the grain weight. Our study provides new genetic resources for yield improvement and for fine-tuning of grain size in rice.

Fine mapping and candidate gene analysis of qRN5a, a novel QTL promoting root number in rice under low potassium.

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 F2:3 (BC5F2:3) 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.

Rice dwarf and low tillering 10 (OsDLT10) regulates tiller number by monitoring auxin homeostasis.

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.