Flowering time of rice (Oryza sativa) plays important roles in regulating grain yield, quality, and adaptability. O. sativa GIGANTEAN (OsGI), heading date 1 (Hd1), and O. sativa flavin-binding, Kelch repeat, and F-box 1 (OsFKF1) are key factors controlling flowering time in rice. However, their relationships are not quite clear. In this study, we found that OsFKF1, an F-box domain-containing protein, physically interacts with OsGI and Hd1. OsFKF1 targets OsGI for degradation through ubiquitination-mediated 26S proteasome. OsFKF1 is functionally conserved with FKF1 in Arabidopsis. Mutations of OsFKF1, OsGI, and Hd1 generated through CRISPR-Cas9 in the same genetic background confer late flowering phenotypes. The osfkf1 mutations suppress the expression of Ehd1, Hd1, and Ehd2, while activating Ghd7 and Ehd3, which further reduces the expression of florigen genes Hd3a and RFT1, leading to delayed flowering time. Mutations of OsFKF1 also affect the agronomic traits and enhance grain yield. These findings reveal the molecular mechanisms of OsFKF1 on regulating flowering time and provide valuable germplasm resources for rice breeding to improve growth period.

Cold stress (CS) significantly impacts rice (Oryza sativa L.) growth during seedling and heading stages. Based on two-year field observations, this study identified two rice lines, L9 (cold stress-sensitive) and LD18 (cold stress-tolerant), showing contrasting CS responses. L9 exhibited a 38% reduction in photosynthetic efficiency, whereas LD18 remained unchanged, correlating with seed rates. Transcriptome analysis identified differentially expressed genes (DEGs) with LD18 showing enriched pathways (carbon fixation, starch/sucrose metabolism, and glutathione metabolism). LD18 displayed dramatically enhanced expression of MAPK-related genes (LOC4342017, LOC9267741, and LOC4342267) and increased ABA signaling genes (LOC4333690, LOC4345611, and LOC4335640) compared with L9 exposed to CS. Results from qPCR confirmed the enhanced expression of the three MAPK-related genes in LD18 with a dramatic reduction in L9 under CS relative to that under CK. We also observed up to 66% reduction in expression levels of the three genes related to the ABA signaling pathway in L9 relative to LD18 under CS. Consistent with the results of photosynthetic efficiency, metabolic analysis suggests pyruvate metabolism, TCA cycle, and carbon metabolism enrichment in LD18 under CS. The study reveals reprogramming of the carbon assimilation metabolic pathways, emphasizing the critical roles of the key DEGs involved in ABA and MAPK signaling pathways in positive regulation of LD18 response to CS, offering the foundation toward cold tolerance breeding through targeted gene editing.

Introduction: The angle of tillers is crucial for shaping plant architecture, which in turn affects grain yield of rice. The formation of tiller angle is associated with the asymmetrical distribution and polar transport of auxin. However, the roles of auxin signaling in regulating the development of tiller angle in rice remain unclear. Objective: In this study, we discovered that Oryza sativa Auxin Response Factor 5 (OsARF5) acts as a key regulator of tiller angle development in rice. Methods: The bta3-1 mutant was obtained through using chemical mutagenesis. The BTA3 gene was isolated by using a map-based positional cloning method. The differentially expressed genes were identified and examined through quantitative RT-PCR and high-throughput mRNA sequencing. The protein-DNA interactions were analyzed by chromatin immunoprecipitation and dual-luciferase reporter assays. The protein-protein interactions were assessed using yeast two-hybrid and bimolecular fluorescence complementation methods. Results: The bta3-1 mutation enlarges the tiller angles, weakens shoot gravitropism, and diminishes the response to auxin in rice. After map-based cloning, we found that the BTA3 gene encodes OsARF5, which belongs to an ARF transcription factor superfamily. OsARF5 binds to the cis-acting elements in the promoters of genes related to tiller angle development, including LA1, OsAGPL1, HASF2d and OsPIN1a, thereby activating their expression. Genome-wide studies identify thousands of differentially expressed genes (DEGs), including auxin response genes, between wild-type and osarf5-1. The number of DEGs in osarf5-1 decreases upon gravistimulation, indicating the involvement of OsARF5 in shoot gravitropism. The OsARF5 physically interacts with three rice Indole Acetic Acid (OsIAA) repressors, forming complexes that facilitate their functions. Mutations in OsIAAs lead to a more compact plant architecture, and the expression of OsARF5-target genes is elevated in the osiaa mutants, suggesting that the OsIAAs counteract OsARF5′s effects on the tiller angle formation. Conclusion: OsARF5 is physically associated with three OsIAAs to bind to the promoter of the target genes, thereby regulating the traget gene expression to manage shoot gravitropism and tiller angle in rice. These findings offer new insights into the principles governing tiller angle development in rice.

Tillering is an important agronomic trait of rice (Oryza sativa) that affects the number of effective panicles, thereby affecting yields. The phytohormone auxin plays a key role in tillering. Here we identified the high tillering and semi-dwarf 1 (htsd1) mutant with auxin-deficiency root characteristics, such as shortened lateral roots, reduced lateral root density, and enlarged root angles. htsd1 showed reduced sensitivity to auxin, but the external application of indole-3-acetic acid (IAA) inhibited its tillering. We identified the mutated gene in htsd1 as AUXIN1 (OsAUX1, LOC_Os01g63770), which encodes an auxin influx transporter. The promoter sequence of OsAUX1 contains many SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) binding sites, and we demonstrated that SPL7 binds to the OsAUX1 promoter. TEOSINTE BRANCHED1 (OsTB1), a key gene that negatively regulates tillering, was significantly downregulated in htsd1. Tillering was enhanced in the OsTB1 knockout mutant, and the external application of IAA inhibited tiller elongation in this mutant. Overexpressing OsTB1 restored the multi-tiller phenotype of htsd1. These results suggest that SPL7 directly binds to the OsAUX1 promoter and regulates tillering in rice by altering OsTB1 expression to modulate auxin signaling.

Grain weight, grain number per panicle, and the number of panicles are the three factors that determine rice (Oryza sativa L.) yield. Of these, grain weight, which not only directly determines rice yield but also influences appearance and quality, is often considered the most important for rice production. Here, we describe OsNF-YC1, a member of the NF-Y transcription factor family that regulates rice grain size. OsNF-YC1 knockout plants (osnf-yc1), obtained using CRISPR-Cas9 technology, showed reduced grain weight due to reduced width and thickness, with no change in grain length, leading to a slenderer grain shape. Downregulation of OsNF-YC1 using RNA interference resulted in similar grain phenotypes as osnf-yc1. OsNF-YC1 affects grain formation by regulating both cell proliferation and cell expansion. OsNF-YC1 localizes in both the nucleus and cytoplasm, has transcriptional activation activity at both the N-terminus and C-terminus, and is highly expressed in young panicles. OsNF-YC1 interacts with OsMADS1 both in vivo and in vitro. Further analysis showed that the histone-like structural CBFD-NFYB-HMF domain of OsNF-YC1 conserved in the OsNF-YC transcription factor family can directly interact with the MADS-box domain of OsMADS1 to enhance its transcriptional activation activity. This interaction positively regulates the expression of OsMADS55, the direct downstream target of OsMADS1. Therefore, this paper reveals a potential grain size regulation pathway controlled by an OsNF-YC1-OsMADS1-OsMADS55 module in rice.

The prevalence of soluble aluminum (Al) ions is one of the major limitations to crop production worldwide on acid soils. Therefore, understanding the Al tolerance mechanism of rice and applying Al tolerance functional genes in sensitive plants can significantly improve Al stress resistance. In this study, transcriptomics and metabolomics analyses were performed to reveal the mechanism of Al tolerance differences between two rice landraces (Al-tolerant genotype Shibanzhan (KR) and Al-sensitive genotype Hekedanuo (MR) with different Al tolerance. The results showed that DEG related to phenylpropanoid biosynthesis was highly enriched in KR and MR after Al stress, indicating that phenylpropanoid biosynthesis may be closely related to Al tolerance. E1.11.1.7 (peroxidase) was the most significant enzyme of phenylpropanoid biosynthesis in KR and MR under Al stress and is regulated by multiple genes. We further identified that two candidate genes Os02g0770800 and Os06g0521900 may be involved in the regulation of Al tolerance in rice. Our results not only reveal the resistance mechanism of rice to Al stress to some extent, but also provide a useful reference for the molecular mechanism of different effects of Al poisoning on plants.

Rice (Oryza sativa L.) is a cold-sensitive species that often faces cold stress, which adversely affects yield productivity and quality. However, the genetic basis for low-temperature adaptation in rice remains unclear. Here, we demonstrate that 2 functional polymorphisms in O. sativa SEC13 Homolog 1 (OsSEH1), encoding a WD40-repeat nucleoporin, between the 2 subspecies O. sativa japonica and O. sativa indica rice, may have facilitated cold adaptation in japonica rice. We show that OsSEH1 of the japonica variety expressed in OsSEH1MSD plants (transgenic line overexpressing the OsSEH1 allele from Mangshuidao [MSD], cold-tolerant landrace) has a higher affinity for O. sativa metallothionein 2b (OsMT2b) than that of OsSEH1 of indica. This high affinity of OsSEH1MSD for OsMT2b results in inhibition of OsMT2b degradation, with decreased accumulation of reactive oxygen species and increased cold tolerance. Transcriptome analysis indicates that OsSEH1 positively regulates the expression of the genes encoding dehydration-responsive element-binding transcription factors, i.e. OsDREB1 genes, and induces the expression of multiple cold-regulated genes to enhance cold tolerance. Our findings highlight a breeding resource for improving cold tolerance in rice.

The trichomes of rice leaves are formed by the differentiation and development of epidermal cells. Plant trichomes play an important role in stress resistance and protection against direct ultraviolet irradiation. However, the development of rice trichomes remains poorly understood. In this study, we conducted ethylmethane sulfonate (EMS)-mediated mutagenesis on the wild-type (WT) indica rice ‘Xida 1B’. Phenotypic analysis led to the screening of a mutant that is defective in trichome development, designated lhl1 (less hairy leaf 1). We performed map-based cloning and localized the mutated gene to the 70-kb interval between the molecular markers V-9 and V-10 on chromosome 2. The locus LOC_Os02g25230 was identified as the candidate gene by sequencing. We constructed RNA interference (LHL1-RNAi) and overexpression lines (LHL1-OE) to verity the candidate gene. The leaves of the LHL1-RNAi lines showed the same trichome developmental defects as the lhl1 mutant, whereas the trichome morphology on the leaf surface of the LHL1-OE lines was similar to that of the WT, although the number of trichomes was significantly higher. Quantitative real-time PCR (RT-qPCR) analysis revealed that the expression levels of auxin-related genes and positive regulators of trichome development in the lhl1 mutant were down-regulated compared with the WT. Hormone response analysis revealed that LHL1 expression was affected by auxin. The results indicate that the influence of LHL1 on trichome development in rice leaves may be associated with an auxin pathway.

Rice breeders are now developing new varieties with semi-high or even high plant height to further increase the grain yield, and the problem of lodging has re-appeared. We identified a major quantitative trait locus (QTL), qSCM4, for resistance to lodging by using an F2 segregant population and a recombinant self-incompatible line population from the cross between Shennong265 (SN265) and Lijiangxintuanheigu (LTH) after multiple years and multiple environments. Then, the residual heterozygous derived segregant population which consisted of 1781 individual plants, and the BC3F2 segregant population which consisted of 3216 individual plants, were used to shorten the physical interval of qSCM4 to 58.5 kb including 11 genes. DNA sequencing revealed the most likely candidate gene for qSCM4 was Os04g0615000, which encoded a functional protein with structural domains of serine and cysteine. There were 13 DNA sequence changes in LTH compared to SN265 in this gene, including a fragment deletion, two base changes in the 3′ UTR region, six base changes in the exons, and four base changes in the introns. A near-isogenic line carrying qSCM4 showed that it improved the lodging resistance through increasing stem thickness by 25.3% and increasing stem folding resistance by 20.3%. Furthermore, it was also discovered that qSCM4 enhanced the primary branch per panicle by 16.7%, secondary branch by per panicle 9.9%, and grain number per panicle by 14.7%. All the above results will give us a valuable genetic resource for concurrently boosting culm strength and lodging resistance, and they will also provide a basis for further research on the lodging resistance mechanism of rice.

Flowering time is one of the most important agronomic traits affecting the adaptation and yield of rice (Oryza sativa). Heading date 1 (Hd1) is a key factor in the photoperiodic control of flowering time. In this study, two basic helix-loop-helix (bHLH) transcription factors, Hd1 Binding Protein 1 (HBP1) and Partner of HBP1 (POH1) were identified as transcriptional regulators of Hd1. We generated knockout mutants of HBP1 and ectopically expressed transgenic lines of the two bHLH transcription factors and used these lines to investigate the roles of these two factors in regulating flowering time. HBP1 physically associated with POH1 forming homo- or heterodimers to perform their functions. Both HBP1 and POH1 bound directly to the cis-acting elements located in the promoter of Hd1 to activate its expression. CRISPR/Cas9-generated knockout mutations of HBP1, but not POH1 mutations, promoted earlier flowering time; conversely, HBP1 and POH1 overexpression delayed flowering time in rice under long-day and short-day conditions by activating the expression of Hd1 and suppressing the expression of Early heading date 1 (Ehd1), Heading date 3a (Hd3a), and Rice Flowering locus T 1 (RFT1), thus controlling flowering time in rice. Our findings revealed a mechanism for flowering time control through transcriptional regulation of Hd1 and laid theoretical and practical foundations for improving the growth period, adaptation, and yield of rice.

水稻种子快速萌发,可减短吸水膨胀至出土成苗时间,减少有害生物危害,进而有效提高种子成苗率,有利于培养强壮秧苗。为此,本研究对缙恢10号EMS （甲基磺酸乙酯）诱变体库进行了筛选,从中鉴定到一个种子萌发快且矮化多蘖的突变体rgs1,进一步分析发现矮化是由于各节间均缩短造成的,而多蘖则是分蘖芽发育较快引起的。遗传分析表明rgs1的突变性状受单隐性核基因调控,定位区间内叶绿体ζ-胡萝卜素异构酶编码基因LOC＿Os12g21710的第3个外显子发生了G至A的碱基替换,导致蛋白翻译提前终止,从而确定为目的基因。qPCR分析发现,与野生型相比,突变体中独角金内酯（SL）合成基因HTD1、D27、D10及信号传导相关基因D14、TB1的表达均极显著降低,SL信号途径抑制因子D53的表达则极显著升高,表明rgs1矮化多蘖性状与SL缺陷有关。在rgs1中,脱落酸（ABA）合成代谢关键酶基因OsNCED1几乎无表达,MOC3和FON1的表达极显著升高,表明rgs1种子的快速萌发与ABA合成缺陷有关。RGS1可能是水稻体内精准调控SL和ABA协同作用的一个关键基因,从而调控水稻种子萌发、株高、分蘖等的发育。

Grain filling influences grain size and quality in cereal crops. The molecular mechanisms that regulate grain endosperm development remain elusive. In this study, we characterized a filling-defective and grain width mutant, fgw1, whose mutation increased rice seed width mainly via cell division and expansion in grains. Sucrose contents were higher but starch contents lower in the fgw1 mutant during the grain-filling stage, resulting in inferior endosperm of opaque, white appearance with loosely packed starch granules. Map-based cloning revealed that FGW1 encoded a protein containing DUF630/DUF632 domains, localized in the plasma membrane with preferential expression in the panicle. RNA interference in FGW1 resulted in increased grain width and weight, whereas overexpression of FGW1 led to slightly narrower kernels and better grain filling. In a yeast two-hybrid assay, FGW1 interacted directly with the 14–3–3 protein GF14f, bimolecular fluorescence complementation verified that the site of interaction was the membrane, and the mutated FGW1 protein failed to interact with GF14f. The expression of GF14f was down-regulated in fgw1, and the activities of AGPase, StSase, and SuSase in the endosperm of fgw1 increased similarly to those of a reported GF14f-RNAi. Transcriptome analysis indicated that FGW1 also regulates cellular processes and carbohydrate metabolism. Thus, FGW1 regulated grain formation via the GF14f pathway.

Abscisic acid (ABA) plays key roles in plant development and responses to abiotic stresses. A wide number of transcriptional and posttranslational regulatory mechanisms of ABA signaling are known; however, less is known about the regulatory roles of alternative splicing. In this work, we found that SKIP, a splicing factor, positively regulates ABA signaling. SKIP binds to the pre-mRNA of ABA signaling-related genes, such as PYL7, PYL8, ABI1, HAB1 and ABI5, to regulate their splicing. The precursor mRNA alternative splicing of several PYL receptors, PP2C phosphatases and ABF transcriptional factors is disrupted by the skip-1 mutation. The abnormal alternative splicing in skip-1 represses the expression of ABA-positive regulators, including PYLs and ABFs, and activates the expression of ABA-negative regulators, such as PP2Cs, which confers ABA hyposensitive phenotype of skip-1. We also found that ABA-mediated genome-wide alternative splicing and differential gene expression are changed by the skip-1 mutation. The number of the differential splicing events is increased by skip-1; however, the number of differential expressed genes in response to ABA is reduced by skip-1. Our results reveal a principle on how a splicing factor regulates ABA signaling and ABA-mediated genome-wide alternative splicing.

Sheath blight (ShB) caused by Rhizoctonia solani is a major disease of rice, seriously affecting yield; however, the molecular defense mechanism against ShB remains unclear. A previous transcriptome analysis of rice identified that R. solani inoculation significantly induced MDPK. Genetic studies using MDPK RNAi and overexpressing plants identified that MDPK positively regulates ShB resistance. This MDPK protein was found localized in the endoplasmic reticulum (ER) and Golgi apparatus. Yeast one-hybrid assay, electrophoresis mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) showed that the intermediate domain proteins IDD12, IDD13, and IDD14 bind to the MDPK promoter. Moreover, IDD14 was found to interact with IDD12 and IDD13 to form a transcription complex to activate MDPK expression. The three IDDs demonstrated an additive effect on MDPK activation. Further genetic studies showed that the IDD13 and IDD14 single mutants were more susceptible to ShB but not IDD12, while IDD12, IDD13, and IDD14 overexpressing plants were less susceptible than the wild-type plants. The IDD12, IDD13, and IDD14 mutants also proved the additive effect of the three IDDs on MDPK expression, which regulates ShB resistance in rice. Notably, MDPK overexpression maintained normal yield levels in rice. Thus, our study proves that IDD12, IDD13, and IDD14 activate MDPK to enhance ShB resistance in rice. These results improve our knowledge of rice defense mechanisms and provide a valuable marker for resistance breeding.

Contemporary climatic stress seriously affects rice production. Unfortunately, long-term domestication and improvement modified the phytohormones network to achieve the production needs of cultivated rice, thus leading to a decrease in adaptation. Here, we identify a 14-3-3 protein-coding gene OsGF14h in weedy rice that confers anaerobic germination and anaerobic seedling development tolerance. OsGF14h acts as a signal switch to balance ABA signaling and GA biosynthesis by interacting with the transcription factors OsHOX3 and OsVP1, thereby boosting the seeding rate from 13.5% to 60.5% for anaerobic sensitive variety under flooded direct-seeded conditions. Meanwhile, OsGF14h co-inheritance with the Rc (red pericarp gene) promotes divergence between temperate japonica cultivated rice and temperate japonica weedy rice through artificial and natural selection. Our study retrieves a superior allele that has been lost during modern japonica rice improvement and provides a fine-tuning tool to improve flood adaptation for elite rice varieties.

Tiller angle is a key factor determining rice plant architecture, planting density, light interception, photosynthetic efficiency, disease resistance and grain yield. However, the mechanisms underlying tiller angle control are far from clear. In this study, we identified a mutant, termed bta1-1, with an enlarged tiller angle throughout its life cycle. A detailed analysis reveals that BTA1 has multiple functions because tiller angle, shoot gravitropism and tolerance to drought stress are changed in bta1-1 plants. Moreover, BTA1 is a positive regulator of shoot gravitropism in rice. Shoot responses to gravistimulation are disrupted in bta1-1 under both light and dark conditions. Gene cloning reveals that bta1-1 is a novel mutant allele of LA1 renamed la1-SN. LA1 is able to rescue the tiller angle and shoot gravitropism defects observed in la1-SN. The nuclear localization signal of LA1 is disrupted by la1SN, causing changes in its subcellular localization. LA1 is required to regulate the expression of auxin transporters and signaling factors that control shoot gravitropism and tiller angle. High-throughput mRNA sequencing is performed to elucidate the molecular and cellular functions of LA1. The results show that LA1 may be involved in the nucleosome and chromatin assembly, and protein-DNA interactions to control gene expression, shoot gravitropism and tiller angle. Our results provide new insight into the mechanisms whereby LA1 controls shoot gravitropism and tiller angle in rice.

FT-INTERACTING PROTEIN (FTIP) gene family in rice are the members of multiple C2 domain and transmembrane region proteins (MCTPs). There are many homologs of OsFTIPs in plants; however, the bioinformatics of them remains unclear. In the studies, 13 OsFTIP genes are identified in rice. OsFTIPs are unevenly located in 12 chromosomes. The OsFTIPs are phylogenetically divided into three clades. Cis-elements respond to abiotic stress, light, and hormones are found in the promoter region of OsFTIPs which are induced by the stimuli. All OsFTIPs are expressed with different profiles. Syntenic analysis of 128 OsFTIPs and FTIP-like homologs reveals that various number of gene pairs are identified between rice and other species. The 128 FTIP-like homologs are divided into six groups which fall into three classes. Ten motifs are shared by most OsFTIPs and their homologs. The studies provide a theoretical basis for further elucidating the functions of OsFTIP gene family.

Background Similar to other eukaryotes, splicing is emerging as an important process affecting development and stress tolerance in plants. Ski-interacting protein (SKIP), a splicing factor, is essential for circadian clock function and abiotic stress tolerance; however, the mechanisms whereby it regulates flowering time are unknown. Results In this study, we found that SKIP is required for the splicing of serrated leaves and early flowering ( SEF) pre-messenger RNA (mRNA), which encodes a component of the ATP-dependent SWR1 chromatin remodeling complex (SWR1-C). Defects in the splicing of SEF pre-mRNA reduced H2A.Z enrichment at FLC , MAF4 , and MAF5 , suppressed the expression of these genes, and produced an early flowering phenotype in skip-1 plants. Conclusions Our findings indicate that SKIP regulates SWR1-C function via alternative splicing to control the floral transition in Arabidopsis thaliana .

Mesocotyl elongation in rice is essential for seedling emergence. Our previous screening identified two weedy rice accessions (Oryza sativa f. spontanea L.) (WR04-6 and WR04-43) were with unusually long mesocotyl. In this study, using rice cultivar (Nipponbare, Oryza sativa L., subspecies temperate japonica) as a control, we found that at the middle and end of mesocotyl elongation, the predominant cortical microtubules (CMTs) in weedy rice are transversely oriented that related to cell elongation, while, cultivated rice had oblique MT arrays. Interestingly, more extensive MT depolymerization was observed in the early stage of mesocotyl elongation in weedy rice accessions than that in cultivated rice. Moreover, MT-destabilizing agent oryzalin treatment (25 nM) led to an increase of rice mesocotyl length, which is correlated with mesocotyl cell elongation. On the contrary, MT-stabilizing agent taxol led to a full inhibition of weedy rice mesocotyl growth. Further, oryzalin treatments resulted in an endogenous gibberellin (GA) increase and the high expression of GA biosynthesis related gene-GA20ox2, GA3ox2, and GIBBERELLIN-INSENSITIVE DWARF1 (GID1), and taxol treatments had an opposite effect on the change of GA content and the GA biosynthesis gene expression. By contrast, the early CMT depolymerization in the weedy rice mesocotyl elongation was not affected by the change of GA content. Our findings suggested that an early depleted form of CMTs is a main reason of unusually long mesocotyls in weedy rice, and it plays an important role in rice mesocotyl growth, which has a close relationship with an increase of endogenous GAs.

The production of japonica rice in northern China plays an important role in food security for the world. Agrobacterium-mediated gene transfer is becoming a powerful approach to generate germplasm and develop varieties. However, the transgenic efficiency of the japonica rice varieties in northern China has been completely unknown, which obstructs the development of transgenic breeding and the exploration of gene functions. In this study, the transgenic efficiencies of six japonica rice varieties developed in northern China are evaluated. The rates of primary and secondary callus induction of the varieties are similar. However, transgenic efficiency and the regeneration ability of the varieties are greatly different. The results have established a platform for transformation of the rice varieties and proposed a suitable variety, SN9816, for gene transfer. SN9816 can be applied as an elite germplasm for transgenic breeding and basic research of molecular biology in northern China or an area in the same latitude.