Dominance complementation of parental heading date alleles of Hd1, Ghd7, DTH8, and PRR37 confers transgressive late maturation in hybrid rice.

Rice (Oryza sativa L.) is a short-day plant whose heading date is largely determined by photoperiod sensitivity (PS). Many parental lines used in hybrid rice breeding have weak PS, but their F1 progenies have strong PS and exhibit an undesirable transgressive late-maturing phenotype. However, the genetic basis for this phenomenon is unclear. Therefore, effective methods are needed for selecting parents to create F1 hybrid varieties with the desired PS. In this study, we used bulked segregant analysis with F1 Ningyou 1179 (strong PS) and its F2 population, and through analyzing both parental haplotypes and PS data for 918 hybrid rice varieties, to identify the genetic basis of transgressive late maturation which is dependent on dominance complementation effects of Hd1, Ghd7, DTH8, and PRR37 from both parents rather than from a single parental genotype. We designed a molecular marker-assisted selection system to identify the genotypes of Hd1, Ghd7, DTH8, and PRR37 in parental lines to predict PS in F1 plants prior to crossing. Furthermore, we used CRISPR/Cas9 technique to knock out Hd1 in Ning A (sterile line) and Ning B (maintainer line) and obtained an hd1-NY material with weak PS while retaining the elite agronomic traits of NY. Our findings clarified the genetic basis of transgressive late maturation in hybrid rice and developed effective methods for parental selection and gene editing to facilitate the breeding of hybrid varieties with the desired PS for improving their adaptability.

PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.

Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

Photoperiod and temperature synergistically regulate heading date and regional adaptation in rice.

Plants must accurately integrate external environmental signals with their own development to initiate flowering at the appropriate time for reproductive success. Photoperiod and temperature are key external signals that determine flowering time; both are cyclical and periodic, and they are closely related. In this review, we describe photoperiod-sensitive genes that simultaneously respond to temperature signals in rice (Oryza sativa). We introduce the mechanisms by which photoperiod and temperature synergistically regulate heading date and regional adaptation in rice. We also discuss the prospects for designing different combinations of heading date genes and other cold tolerance or thermo-tolerance genes to help rice better adapt to changes in light and temperature via molecular breeding to enhance yield in the future.

Characterization and fine-mapping of a new Asian rice selfish genetic locus S58 in Asian-African rice hybrids.

KEY MESSAGE: We identified and fine-mapped S58, a selfish genetic locus from Asian rice that confers hybrid male sterility in crosses between Asian and African cultivated rice, and found a natural neutral allele in Asian rice lines that will be useful for overcoming S58-mediated hybrid sterility. Hybrids between Asian cultivated rice (Oryza sativa L.) and African cultivated rice (Oryza glaberrima Steud) display severe hybrid sterility (HS), hindering the utilization of strong heterosis in hybrids between these species. Several African rice selfish loci causing HS in Asian-African cultivated rice hybrids have been identified, but few such Asian rice selfish loci have been found. In this study, we identified an Asian rice selfish locus, S58, which causes hybrid male sterility (HMS) in hybrids between the Asian rice variety 02428 and the African rice line CG14. Genetic analysis confirmed that S58 causes a transmission advantage for the Asian rice S58 allele in the hybrid offspring. Genetic mapping with near-isogenic lines and DNA markers delimited S58 to 186 kb and 131 kb regions of chromosome 1 in 02428 and CG14, respectively, and revealed complex genomic structural variation over these mapped regions. Gene annotation analysis and expression profiling analyses identified eight anther-expressed candidate genes potentially responsible for S58-mediated HMS. Comparative genomic analysis determined that some Asian cultivated rice varieties harbor a 140 kb fragment deletion in this region. Hybrid compatibility analysis showed that this large deletion allele in some Asian cultivated rice varieties can serve as a natural neutral allele, S58-n, that can overcome S58-mediated interspecific HMS. Our study demonstrates that this selfish genetic element from Asian rice is important for HMS between Asian and African cultivated rice, broadening our understanding of interspecific HS. This study also provides an effective strategy for overcoming HS in future interspecific rice breeding.

Effects of the core heading date genes Hd1, Ghd7, DTH8, and PRR37 on yield-related traits in rice.

KEY MESSAGE: We clarify the influence of the genotypes of the heading date genes Hd1, Ghd7, DTH8, and PRR37 and their combinations on yield-related traits and the functional differences between different haplotypes. Heading date is a key agronomic trait in rice (Oryza sativa L.) that determines yield and adaptability to different latitudes. Heading date 1 (Hd1), Grain number, plant height, and heading date 7 (Ghd7), Days to heading on chromosome 8 (DTH8), and PSEUDO-RESPONSE REGULATOR 37 (PRR37) are core rice genes controlling photoperiod sensitivity, and these genes have many haplotypes in rice cultivars. However, the effects of different haplotypes at these genes on yield-related traits in diverse rice materials remain poorly characterized. In this study, we knocked out Hd1, Ghd7, DTH8, or PRR37, alone or together, in indica and japonica varieties and systematically investigated the agronomic traits of each knockout line. Ghd7 and PRR37 increased the number of spikelets and improved yield, and this effect was enhanced with the Ghd7 DTH8 or Ghd7 PRR37 combination, but Hd1 negatively affected yield. We also identified a new weak functional Ghd7 allele containing a mutation that interferes with splicing. Furthermore, we determined that the promotion or inhibition of heading date by different PRR37 haplotypes is related to PRR37 expression levels, day length, and the genetic background. For rice breeding, a combination of functional alleles of Ghd7 and DTH8 or Ghd7 and PRR37 in the hd1 background can be used to increase yield. Our study clarifies the effects of heading date genes on yield-related traits and the functional differences among their different haplotypes, providing valuable information to identify and exploit elite haplotypes for heading date genes to breed high-yielding rice varieties.

Improving yield-related traits by editing the promoter of the heading date gene Ehd1 in rice.

KEY MESSAGE: We developed an efficient promoter editing method to create different weak Ehd1 alleles in elite japonica rice variety ZJ8 with slightly delayed heading and improved yield for use in breeding. Heading date is an important agronomic trait of rice (Oryza sativa) that determines the planting areas and cultivation seasons of different varieties, thus affecting final yield. Early heading date 1 (Ehd1) is a major rice integrator gene in the regulatory network of heading date whose expression level is negatively correlated with heading date and grain yield. Some elite japonica varieties such as Zhongjia 8 (ZJ8) show very early heading with poor agronomic traits when planted in South China. This problem can be addressed by downregulating the expression of Ehd1. In this study, we analyzed the cis-regulatory elements in the Ehd1 promoter region. We then used CRISPR/Cas9-mediated editing to modify the Ehd1 promoter at multiple target sites in ZJ8. We rapidly identified homozygous allelic mutations in the T2 generation via long-read sequencing. We obtained several Ehd1 promoter mutants with different degrees of lower Ehd1 expression, delayed heading date, and improved yield-related traits. We developed an efficient promoter editing method to create different weak Ehd1 alleles for breeding selection. Using this method, a series of heading date materials from elite varieties can be created to expand the planting area of rice and improve grain yields.

Plasma-Membrane-Localized Transporter NREET1 is Responsible for Rare Earth Element Uptake in Hyperaccumulator Dicranopteris linearis.

Rare earth elements (REEs) are critical for numerous modern technologies, and demand is increasing globally; however, production steps are resource-intensive and environmentally damaging. Some plant species are able to hyperaccumulate REEs, and understanding the biology behind this phenomenon could play a pivotal role in developing more environmentally friendly REE recovery technologies. Here, we identified a REE transporter NRAMP REE Transporter 1 (NREET1) from the REE hyperaccumulator fern Dicranopteris linearis. Although NREET1 belongs to the natural resistance-associated macrophage protein (NRAMP) family, it shares a low similarity with other NRAMP members. When expressed in yeast, NREET1 exhibited REE transport capacity, but it could not transport divalent metals, such as zinc, nickel, manganese, or iron. NREET1 is mainly expressed in D. linearis roots and predominantly localized in the plasma membrane. Expression studies in Arabidopsis thaliana revealed that NREET1 functions as a transporter mediating REE uptake and transfer from root cell walls into the cytoplasm. Moreover, NREET1 has a higher affinity for transporting light REEs compared to heavy REEs, which is consistent to the preferential enrichment of light REEs in field-grown D. linearis. We therefore conclude that NREET1 may play an important role in the uptake and consequently hyperaccumulation of REEs in D. linearis. These findings lay the foundation for the use of synthetic biology techniques to design and produce sustainable, plant-based REE recovery systems.

Double Mutation of Days to Heading 2 and CONSTANS 3 Improves Agronomic Performance of Japonica Rice under Short Daylight Conditions in Southern China.

Some progress has been made in understanding the pathways related to rice heading, but their applications to breeding japonica rice varieties adapted to grow in low-latitude areas ("indica to japonica") are limited. We edited eight adaptation-related genes via a lab-established CRISPR/Cas9 system in a japonica variety, Shennong265 (SN265). All T0 plants and their progeny bearing random mutation permutations were planted in southern China and screened for changes in heading date. We found that the double mutant of Days to heading 2 (DTH2) and CONSTANS 3 (OsCO3) (dth2-osco3), two CONSTANS-like (COL) genes, showed significantly delayed heading under both short-day (SD) and long-day (LD) conditions in Guangzhou and manifested great yield increase under SD conditions. We further demonstrated that the heading-related Hd3a-OsMADS14 pathway was down-regulated in the dth2-osco3 mutant lines. The editing of the COL genes DTH2 and OsCO3 greatly improves the agronomic performance of japonica rice in Southern China.

Effects of Biological Nitrogen Metabolism on Glufosinate-Susceptible and -Resistant Goosegrass (Eleusine indica L.).

Glufosinate is a broad-spectrum herbicide used to control most weeds in agriculture worldwide. Goosegrass (Eleusine indica L.) is one of the top ten malignant weeds across the world, showing high tolerance to glufosinate via different mechanisms that are not yet fully understood. This study revealed that nitrogen metabolism could be a target-resistant site, providing clues to finally clarify the mechanism of glufosinate resistance in resistant goosegrass populations. Compared to susceptible goosegrass (NX), the resistant goosegrass (AUS and CS) regarding the stress of glufosinate showed stronger resistance with lower ammonia contents, higher target enzyme GS (glutamine synthetase) activity, and lower GOGAT (glutamine 2-oxoglutarate aminotransferase) activity. The GDH (glutamate dehydrogenase) activity of another pathway increased, but its gene expression was downregulated in resistant goosegrass (AUS). Analyzing the transcriptome and proteome data of goosegrass under glufosinate stress at 36 h showed that the KEGG pathway of the nitrogen metabolism was enriched in glufosinate-susceptible goosegrass (NX), but not in glufosinate-resistant goosegrass (CS and AUS). Several putative target genes involved in glufosinate stress countermeasures were identified. This study provides specific insights into the nitrogen metabolism of resistant goosegrass, and gives a basis for future functional verification of glufosinate-tolerance genes in plants.

Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.).

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 µmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.

PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.

Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

Functional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time-consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15-nt 3' single-strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII-UNiE. Using TGSII-UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII-UNiE. In conclusion, UNiEDA is an efficient, convenient and low-cost method for DNA cloning and multigene stacking, and the TGSII-UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5'-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Blocking Rice Shoot Gravitropism by Altering One Amino Acid in LAZY1.

Tiller angle is an important trait that determines plant architecture and yield in cereal crops. Tiller angle is partially controlled during gravistimulation by the dynamic re-allocation of LAZY1 (LA1) protein between the nucleus and plasma membrane, but the underlying mechanism remains unclear. In this study, we identified and characterized a new allele of LA1 based on analysis of a rice (Oryza sativa L.) spreading-tiller mutant la1G74V, which harbors a non-synonymous mutation in the predicted transmembrane (TM) domain-encoding region of this gene. The mutation causes complete loss of shoot gravitropism, leading to prostrate growth of plants. Our results showed that LA1 localizes not only to the nucleus and plasma membrane but also to the endoplasmic reticulum. Removal of the TM domain in LA1 showed spreading-tiller phenotype of plants similar to la1G74V but did not affect the plasma membrane localization; thus, making it distinct from its ortholog ZmLA1 in Zea mays. Therefore, we propose that the TM domain is indispensable for the biological function of LA1, but this domain does not determine the localization of the protein to the plasma membrane. Our study provides new insights into the LA1-mediated regulation of shoot gravitropism.

A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves.

BACKGROUND: Plant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined. RESULTS: Here an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14. CONCLUSIONS: These data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

Rice MutLγ, the MLH1-MLH3 heterodimer, participates in the formation of type I crossovers and regulation of embryo sac fertility.

The development of embryo sacs is crucial for seed production in plants, but the genetic basis regulating the meiotic crossover formation in the macrospore and microspore mother cells remains largely unclear. Here, we report the characterization of a spontaneous rice female sterile variation 1 mutant (fsv1) that showed severe embryo sacs abortion with low seed-setting rate. Through map-based cloning and functional analyses, we isolated the causal gene of fsv1, OsMLH3 encoding a MutL-homolog 3 protein, an ortholog of HvMLH3 in barley and AtMLH3 in Arabidopsis. OsMLH3 and OsMLH1 (MutL-homolog 1) interact to form a heterodimer (MutLγ) to promote crossover formation in the macrospore and microspore mother cells and development of functional megaspore during meiosis, defective OsMLH3 or OsMLH1 in fsv1 and CRISPR/Cas9-based knockout lines results in reduced type I crossover and bivalent frequency. The fsv1 and OsMLH3-knockout lines are valuable germplasms for development of female sterile restorer lines for mechanized seed production of hybrid rice.

Strong photoperiod sensitivity is controlled by cooperation and competition among Hd1, Ghd7 and DTH8 in rice heading.

Rice (Oryza sativa) is a short-day (SD) plant originally having strong photoperiod sensitivity (PS), with SDs promoting and long days (LDs) suppressing flowering. Although the evolution of PS in rice has been extensively studied, there are few studies that combine the genetic effects and underlying mechanism of different PS gene combinations with variations in PS. We created a set of isogenic lines among the core PS-flowering genes Hd1, Ghd7 and DTH8 using CRISPR mutagenesis, to systematically dissect their genetic relationships under different day-lengths. We investigated their monogenic, digenic, and trigenic effects on target gene regulation and PS variation. We found that Hd1 and Ghd7 have the primary functions for promoting and repressing flowering, respectively, regardless of day-length. However, under LD conditions, Hd1 promotes Ghd7 expression and is recruited by Ghd7 and/or DTH8 to form repressive complexes that collaboratively suppress the Ehd1-Hd3a/RFT1 pathway to block heading, but under SD conditions Hd1 competes with the complexes to promote Hd3a/RFT1 expression, playing a tradeoff relationship with PS flowering. Natural allelic variations of Hd1, Ghd7 and DTH8 in rice populations have resulted in various PS performances. Our findings reveal that rice PS flowering is controlled by crosstalk of two modules - Hd1-Hd3a/RFT1 in SD conditions and (Hd1/Ghd7/DTH8)-Ehd1-Hd3a/RFT1 in LD conditions - and the divergences of these genes provide the basis for rice adaptation to broad regions.

CRISPR/Cas9 Guided Mutagenesis of Grain Size 3 Confers Increased Rice (Oryza sativa L.) Grain Length by Regulating Cysteine Proteinase Inhibitor and Ubiquitin-Related Proteins.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas9)-mediated genome editing has become an important way for molecular breeding in crop plants. To promote rice breeding, we edited the Grain Size 3 (GS3) gene for obtaining valuable and stable long-grain rice mutants. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteome-wide changes in the GS3 mutants compared with wild type (WT). Two target sites were designed to construct the vector, and the Agrobacterium-mediated method was used for rice transformation. Specific mutations were successfully introduced, and the grain length (GL) and 1000-grain weight (GWT) of the mutants were increased by 31.39% and 27.15%, respectively, compared with WT. The iTRAQ-based proteomic analysis revealed that a total of 31 proteins were differentially expressed in the GS3 mutants, including 20 up-regulated and 11 down-regulated proteins. Results showed that differentially expressed proteins (DEPs) were mainly related to cysteine synthase, cysteine proteinase inhibitor, vacuolar protein sorting-associated, ubiquitin, and DNA ligase. Furthermore, functional analysis revealed that DEPs were mostly enriched in cellular process, metabolic process, binding, transmembrane, structural, and catalytic activities. Pathway enrichment analysis revealed that DEPs were mainly involved in lipid metabolism and oxylipin biosynthesis. The protein-to-protein interaction (PPI) network found that proteins related to DNA damage-binding, ubiquitin-40S ribosomal, and cysteine proteinase inhibitor showed a higher degree of interaction. The homozygous mutant lines featured by stable inheritance and long-grain phenotype were obtained using the CRISPR/Cas9 system. This study provides a convenient and effective way of improving grain yield, which could significantly accelerate the breeding process of long-grain japonica parents and promote the development of high-yielding rice.