The leaf is a major organ for photosynthesis, and its shape plays an important role in plant development and yield determination in rice (Oryza sativa L.). In this study, an adaxial curled leaf mutant, termed curly leaf 1-1 (cul1-1), was obtained by chemical mutagenesis. The leaf rolling index of the cul1-1 mutant was higher than that of the wild-type, which was caused by the abnormal development of bulliform cells (BCs). We cloned the CUL1 gene by map-based cloning. A nonsense mutation was present in the cul1-1 mutant, converting a tryptophan codon into a stop codon. The CUL1 gene encodes a chromodomain, helicase/ATPase and DNA-binding domain containing protein. Genes related to leaf rolling and BC development, such as ADL1, REL1 and ROC5, were activated by the cul1-1 mutation. The trimethylation of lysine 27 in histone 3 (H3K27me3), but not H3K4me3, at the ADL1, REL1 and ROC5 loci, was reduced in the cul1-1 mutant. High-throughput mRNA sequencing indicated that the cul1-1 mutation caused genome-wide differential gene expression. The differentially expressed genes were classified into a few gene ontology terms and Kyoto encyclopedia of genes and genomes pathways. In the natural population, 22 missense genomic variations in the CUL1 locus were identified, which composed of 7 haplotypes. A haplotype network was also built with haplotype II as the ancestor. The findings revealed that CUL1 is essential for normal leaf development and regulates this process by inhibiting the expression of genes involved in leaf rolling and BC development. (c) 2025 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

水稻是我国主要粮食作物之一,在我国种植面积不会发生重大变化的社会环境下,通过增加水稻的亩产便成为我国粮食每年保持增产的根本途径和确保国家粮食安全的有力保障。而氮素是限制水稻生长、发育和影响水稻亩产的最重要因素之一,因此水稻氮素利用相关性状的研究对水稻遗传改良具有重要意义。本研究利用一套以粳稻品种日本晴为供体亲本和籼稻品种广陆矮4号为轮回亲本构建的染色体单片段代换系（Chromosome Single Segment Substituted Lines,CSSLs）为材料,以株高（PH）、根干质量（RDW）、苗干质量（SDW）、总干质量（PDW）、氮素生理利用效率（NUE）以及氮素积累总量（NA）等6个性状作为高低氮两种条件下研究氮素利用相关QTL的检测指标并对其进行了 QTL鉴定,主要研究结果如下:1、染色体片段代换系群体和亲本的株高（PH）、根干质量（RDW）、苗干质量（SDW）、总干质量（PDW）、氮素生理利用效率（NUE）以及氮素积累总量（NA）等6个性状的表型分析表明（表1）,双亲（粳稻品种日本晴和籼稻品种广陆矮4号）和染色体片段代换系群体在低氮（LN）条件下的株高（PH）、苗干质量（SDW）、总干质量（PDW）、氮素积累总量（NA）均比高氮（HN）条件下有不同程度地降低,而根干质量（RDW）增加。因此表明水稻幼苗在低氮（LN）条件下能诱导根系抑制地上部茎叶的生长,并且广陆矮和日本晴两亲本对低氮（LN）条件的反应是一致的。从染色体片段代换系群体来看,各性状值均表现连续正态分布且呈双向超亲分离现象,表现出数量性状的特点（图1）。2、高氮（HN）和低氮（LN）条件下水稻苗期各性状的相关性分析表明（表2）,水稻苗期高低氮两种情况下的株高（PH）、根干质量（RDW）、苗干质量（SDW）和氮素积累总量（NA）均表现出极显著的正相关性,说明水稻苗期的生长发育是水稻地下部根系和水稻地上部茎叶协同作用的结果。其中低氮（LN）条件下株高（PH）与高氮（HN）条件下根干质量（RDW）之间的相关系数最小（0.211）,低氮条件（LN）下的根干质量（RDW）和高氮（HN）条件下的根干质量（RDW）之间的相关系数最大（0.924）。3、本实验共检测到高低氮两种条件下株高（PH）、根干质量（RDW）、苗干质量（SDW）、总干质量（PDW）、氮素生理利用效率（NUE）和氮素积累总量（NA）及其相对性状的35个QTL,其中高氮条件下检测到13个QTL,低氮条件下检测到10个QTL,高低氮相对性状检测到12个QTL。检测到2个聚集了不同氮水平及其相对性状的包含多个QTL的区间,称其为QTL热点区,第1染色体的长臂靠近末端附近检测到的QTL热点区和前人已经报道的QTL热点区在同一染色体区域附近,而在第1染色体短臂末端检测到的QTL热点区目前还未发现报道。以上这些结果将为利用分子标记辅助选育水稻苗期氮高效利用品种提供依据。

玉米（Zea mays L.）产量是一个复杂数量性状,受多基因控制。行粒数（kernel number per row,KNR）、穗行数（kernel row number,KRN）、穗长（ear length,EL）和穗重（ear weight,EW）是籽粒产量的组成因子。KERNEL NUMBER PER ROW6（KNR6）为本实验室前期克隆的一个控制穗长和行粒数的基因,深入解析KNR6调控行粒数的作用机制和遗传调控网络,将有助于人类认识产量性状形成的遗传学基础和调控机理,也有利于指导育种实践。本研究主要结果如下:1.KNR6与AGAP蛋白互作:采用免疫沉淀-质谱分析（immunoprecipitation mass spectrometry,IP-MS）共鉴定到135个KNR6互作蛋白。这些蛋白分别参与RNA翻译、生物合成与催化、能量代谢以及细胞内蛋白转运等重要生物过程。通过酵母双杂（yeast two hybrid,Y2H）、萤火虫荧光素酶互补技术（firefly luciferase complementation Imaging assay,LCI-assay）、双分子荧光互补（bimolecular fluorescence complementation,Bi FC）、蛋白质体外结合实验（pull-down assay）等实验,验证了KNR6与一个玉米Arf GTPase activating protein（AGAP）的物理互作。基因表达分析也揭示,KNR6与AGAP具有相似的表达模式和表达区域,暗示这两个基因的编码产物可能在相同的组织区域内富集,为这两个蛋白的互作提供了可能。2.KNR6具有蛋白激酶活性并能磷酸化AGAP:体外表达并纯化KNR6重组蛋白,自磷酸化和底磷酸化物活性检测证实,KNR6具有自磷酸化活性,但突变第74位或第172位氨基酸导致KNR6激酶活性丧失,表明第74位和第172位氨基酸是KNR6激酶活性所必需的。γ-[18O4]-ATP标记磷酸化分析共检测到包含AGAP在内的63个蛋白为KNR6的磷酸化底物,这些检测到的磷酸化底物蛋白主要参与DNA转录、蛋白结合、细胞转运和发育进程等生物过程。体外磷酸化证实AGAP和两个14-3-3蛋白为KNR6的磷酸化底物。酵母三杂分析证实KNR6、AGAP和14-3-3蛋白能互作形成三聚体结构,因此推测14-3-3蛋白可能作为小分子辅助因子参与到KNR6-AGAP的调控途径。3.AGAP在营养和生殖发育中具有多效性:通过CRISPR/Cas9系统,创制了导致AGAP编码提前终止的突变体材料（AGAP knock-out lines,agapko）。发现agapko1家系与野生型（non-transgenic sibling,AGAPNT）相比,花序分生组织（inflorescence meristem）变短、成熟果穗长度变短、果穗上每行籽粒数目减少;agapko2家系植株矮小、茎秆扭曲、叶片弯曲,特别是雌穗发育显著受到抑制且无法正常繁殖、雄穗呈现爪状、花粉管形态异常。因此认为AGAP具有多效性同时影响植株的营养和生殖发育。4.AGAP与KNR6遗传互作共同调控玉米穗长和行粒数:为证实AGAP与KNR6的互作,通过CRISPR/Cas9系统创制了KNR6突变体,相对野生型,KNR6突变体的花序分生组织长度变短、穗长变短、行粒数减少。将knr6ko1与agapko1杂交,在F2群体中分离出AGAP/KNR6、agapko1/KNR6、AGAP/knr6ko1和agapko1/knr6ko1基因型,表型分析发现两个单突变体（agapko1/KNR6和AGAP/knr6ko1）的穗长和行粒数比野生型AGAP/KNR6的短,而双突变体agapko1/knr6ko1的穗长比两个单突变体更短、行粒数更少,即2个基因突变能够增强单基因突变异常表型,表明AGAP与KNR6遗传互作共同调控穗长和行粒数。5.AGAP和Arf GTPase1互作参与囊泡运输:亚细胞定位结果表明AGAP与2个Arf GTPase1（ARF1）蛋白定位于高尔基体。LCI-assay和Bi FC分析发现2个ARF1亚家族成员能与AGAP蛋白物理互作。透射电镜观察发现,agapko细胞中的高尔基体片层变薄、结构弯曲,表明AGAP影响高尔基体形态。另外,agapko根表皮细胞中FM4-64标记的内吞体的内化延迟,BFA（brefeldin A）小体集聚减少,显示AGAP可能参与囊泡运输。推测AGAP与ARF1互作共同参与内吞作用和高尔基体/内质网网络的囊泡运输。KNR6磷酸化AGAP激活AGAP,调控AGAP与ARF1参与的细胞内的囊泡运输。KNR6参与的囊泡运输能维持雌穗花序中生长素含量的平衡,调控花序分生组织发育,影响穗长和行粒数。

Plant-specific WUSCHEL-related homeobox (Wox) transcription factors (TFs) are crucial for plant growth and development. However, the molecular mechanism of Wox-mediated regulation of thousand kernel weight (TKW) in crops remains elusive. In this research, we identified a major TKW-associated quantitative trait locus (QTL) on wheat chromosome 5DS by performing a genome-wide association study (GWAS) of a Chinese wheat mini-core collection (MCC) in four environments combined by bulked segregant analysis (BSA) and bulked segregant RNA-sequencing (BSR-seq) of wheat grains exhibiting a wide range of TKWs. The candidate TaWUS-like-5D was highly expressed in developing grains and was found to strongly negative influence grain TKW and wheat yield. Meanwhile, the RNAi lines, CRISPR/Cas9-edited single and double knockout mutants (AABBdd and AAbbdd), as well as the stop-gained aaBB Kronos mutants, exhibited a significant increase in grain size and TKW (P<0.05 or P<0.01) and a 10.0% increase in yield (P<0.01). Further analyses indicated that TaWUS-like-5D regulates TKW by inhibiting the transcription of sucrose, hormone and trehalose metabolism-related genes, subsequently sharply decreasing starch synthesis in wheat grains. The results of this study provide a fundamental molecular basis for further elucidating the mechanism of Wox-mediated regulation of grain development in crops. © 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Grain size and weight are closely related traits determining yield in rice (Oryza sativa L.). Since indica and japonica rice varieties differ significantly in multiple traits, a high-generation recombinant inbred line (RIL) population derived from the crossing LH9 (indica) and RPY (japonica) was used to map grainrelated traits in six environments. Pyramiding of the quantitative trait loci (QTL) for thousand-grain weight showed that combinations of multiple QTL significantly increased the phenotypic effect. A novel gene named GSW3.1 controlling grain size and weight was discovered using the major QTL for the colocalization of grain width and thousand-grain weight on chromosome 3. Gene editing revealed that GSW3.1 (LOC_Os03g16850) was pleiotropic, positively regulating grain size and weight while affecting several other agronomic traits. Haplotype analysis indicated that some traits, including grain width and weight, were highly correlated with indica-japonica differentiation. (c) 2024 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

Heading date is one of the most important agronomic traits of rice, which critically affects rice ecogeographical adaptation, yield and quality. In this study, a late heading date 3 (lhd3) mutant was screened from the 60Co-& gamma; irradiation mutant library. The lhd3 delayed heading date in rice under both short day and long day conditions. Map-based cloning combined with Mutmap strategy was adopted to isolate the causal LHD3 gene. The LHD3 gene encodes a DNA_J domain protein, which was ubiquitously expressed in various plant organs, and dominant expressed in stems and leaves. Subcellular localization analysis showed that LHD3 was localized to nucleus, indicating that LHD3 may interact with other elements to regulate the expression of flowering genes. The transcriptions of the heading activators Ehd1, Hd3a and RFT1 significantly decreased in the lhd3 mutant, suggesting that LHD3 may control the heading date through the Ehd1-Hd3a/RFT1 photoperiodic flowering pathway. The variation and haplotype analyses of the genomic region of LHD3 showed that there were 7 haplotypes in the LHD3 region from 4 702 accessions. The haplotypes of LHD3 can be divided into two classes: class a and class b, and the heading dates of these two classes were significantly different. Further study showed that two single nucleotide polymorphisms (SNPs), SNP10 (G2100C) in Hap II and SNP3 (C861T) in Hap VII, may be the functional sites causing early and late heading in accessions. Nucleotide diversity analysis showed LHD3 had been selected in the indica population, rather than in the japonica population. Therefore, the present study sheds light on the regulation of LHD3 on heading date in rice and suggests that LHD3 is a novel promising new target for rice molecular design and breeding improvement.

Improving osmotic stress tolerance is critical to help crops to thrive and maintain high yields in adverse environments. Here, we characterized a core subunit of the transport protein particle (TRAPP) complex, ZmBET5L1, in maize using knowledge-driven data mining and genome editing. We found that ZmBET5L1 can interact with TRAPP I complex subunits and act as a tethering factor to mediate vesicle aggregation and targeting from the endoplasmic reticulum to the Golgi apparatus. ZmBET5L1 knock-out increased the primary root elongation rate under 20% polyethylene glycol-simulated osmotic stress and the survival rate under drought stress compared to wild-type seedlings. In addition, we found that ZmBET5L1 moderates PIN1 polar localization and auxin flow to maintain normal root growth. ZmBET5L1 knock-out optimized auxin flow to the lateral side of the root and promoted its growth to generate a robust root, which may be related to improved osmotic stress tolerance. Together, these findings demonstrate that ZmBET5L1 inhibits primary root growth and decreases osmotic stress tolerance by regulating vesicle transport and auxin distribution. This study has improved our understanding of the role of tethering factors in response to abiotic stresses and identified desirable variants for breeding osmotic stress tolerance in maize. © 2022 John Wiley & Sons Ltd.

Indica(xian)-japonica(geng) hybrid rice has many heterosis traits that can improve rice yield. However, the traditional hybrid technology will struggle to meet future needs for the development of higher-yield rice. Available genomics resources can be used to efficiently understand the gene-trait association trait for rice breeding. Based on the previously constructed high-density genetic map of 272 high-generation recombinant inbred lines (RILs) originating from the cross of Luohui 9 (indica, as female) and RPY geng (japonica, as male) and high-quality genomes of parents, here, we further explore the genetic basis for an important complex trait: possible causes of grain number per panicle (GNPP). A total of 20 genes related to grains number per panicle (GNPP) with the differences of protein amino acid between LH9 and RPY were used to analyze genotype combinations, and PCA results showed a combination of PLY1, LAX1, DTH8 and OSH1 from the RPY geng with PYL4, SP1, DST and GNP1 from Luohui 9 increases GNPP. In addition, we also found that the combination of LAX1-T2 and GNP1-T3 had the most significant increase in GNPP. Notably, Molecular Breeding Knowledgebase (MBK) showed a few aggregated rice cultivars, LAX1-T2 and GNP1-T3, which may be a result of the natural geographic isolation between the two gene haplotypes. Therefore, we speculate that the pyramiding of japonica-type LAX-T2 with indica-type GNP1-T3 via hybridization can significantly improve rice yield by increasing GNPP.