A High-temperature-induced miR2871a Affects Rice (Oryza sativa) Grain Thickness by Regulating Aleurone Layer Development
XING Yuan-Hang1,*, JIANG Wen-Jing1,*, ZHU Xin-Feng1, XIE Yu-Jun1, NI Shen2, LUO Qiu-Hong1,**, YU Jin-Sheng1,**
1 College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, China; 2 State Key Laboratory of Rice Biology and Breeding, China National Rice Research Institute, Hangzhou 311400, China
Abstract：Grain shape is a core agronomic trait determining rice (Oryza sativa) yield and quality. As a key component of grain shape, grain thickness directly influences the thousand-grain weight and milling quality of rice. As important post-transcriptional regulators, microRNAs (miRNAs) play key roles in plant growth and development. In this study, taking grain shape regulation as the starting point, loss-of-function mutants of rice miR2871a were successfully generated using CRISPR/Cas9 technology. Systematic phenotypic analysis revealed that the mutants exhibited significantly reduced grain thickness and thousand-grain weight for both paddy and brown rice across multiple environments (P<0.05), accompanied by a significant increase in the chalky grain rate of milled rice (P<0.05). Histological observations demonstrated severe impairment in the development of the aleurone layer in mutant's caryopses, characterized by reduced cell layers, thinner thickness, and delayed developmental progression. Multi-element analysis revealed that defects in aleurone layer structure directly led to a significant decrease in the accumulation of key mineral elements, such as zinc, manganese, and calcium, in brown rice (P<0.05). Transcriptome sequencing (RNA-seq) analysis showed that differentially expressed genes were significantly enriched in thiamine metabolism and other biosynthetic pathways closely related to aleurone layer function. This study reveals the key regulatory role of miR2871a in the development of rice grain thickness and aleurone layer, and provides novel and important genetic resources for the synergistic genetic improvement of rice grain shape and nutritional quality.
[1] 姜平, 吴娜娜, 翟小童, 等. 2021. 米糠生物活性物质及其应用研究的进展[J]. 中国粮油学报, 36(10): 188-195. (Jiang P, Wu N N, Zhai X T, et al.2021. Research progress on bioactive substances from rice bran and their applications[J]. Journal of the Chinese Cereals and Oils Association, 36(10): 188-195.) [2] 李文冰, 张奇, 张振都, 等. 2020. 电感耦合等离子体质谱法分析不同产地大米中多种矿物元素[J]. 东北农业科学, 45(06): 129-134. (Li W B, Zhang Q, Zhang Z D, et al.2020. Analysis of multiple mineral elements in rice from different producing areas by inductively coupled plasma mass spectrometry[J]. Journal of Northeast Agricultural Sciences, 45(06): 129-134.) [3] 杨福, 聂小兰, 宋慧, 等. 2005. 2个不同垩白度粳稻胚乳养分输导组织解剖结构的比较研究[J]. 吉林农业大学学报,(01): 11-14+18. [4] 张帆涛, 谢建坤, 杨婉铃, 等. 2023. 一种调控植物耐冷性和耐盐性的miRNA及其应用. 中国, 202310290142.5[P]. (Zhang F T, Xie J K, Yang W L, et al. 2023. A miRNA regulating cold tolerance and salt tolerance in plants and its application. China, 202310290142.5[P].) [5] Alam I, Batool K, Huang Y Y, et al.2022. Developing genetic engineering techniques for control of seed size and yield[J]. International Journal of Molecular Sciences, 23(21): 13256. [6] Choi B S, Kim Y J, Markkandan K, et al.2018. GW2 functions as an E3 ubiquitin ligase for rice expansin-like 1[J]. International Journal of Molecular Sciences, 19(7): 1904. [7] Duan P G, Ni S, Wang J M, et al.2015. Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice[J]. Nature Plants, 2: 15203. [8] Fan C C, Xing Y Z, Mao H L, et al.2006. GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein[J]. Theoretical and Applied Genetics, 112(6): 1164-1171. [9] Garg R, Jhanwar S, Tyagi A K, et al.2010. Genome-wide survey and expression analysis suggest diverse roles of glutaredoxin gene family members during development and response to various stimuli in rice[J]. DNA Research, 17(6): 353-367. [10] Guo T, Lu Z Q, Shan J X, et al.2020. ERECTA1 acts upstream of the OsMKKK10-OsMKK4-OsMPK6 cascade to control spikelet number by regulating cytokinin metabolism in rice[J]. Plant Cell, 32(9): 2763-2779. [11] Hamm H E.1998. The many faces of G protein signaling[J]. The Journal of Biological Chemistry, 273(2): 669-672. [12] Hao J Q, Wang D K, Wu Y B, et al.2021. The GW2-WG1-OsbZIP47 pathway controls grain size and weight in rice[J]. Molecular Plant, 14(8): 1266-1280. [13] Hsieh P H, Chung Y H, Lee K T, et al.2021. The rice PALE1 homolog is involved in the biosynthesis of vitamin B1[J]. Plant Biotechnology Journal, 19(2): 218-220. [14] Li D Q, Wu X B, Wang H F, et al.2021. Defective mitochondrial function by mutation in THICK ALEURONE 1 encoding a mitochondrion-targeted single-stranded DNA-binding protein leads to increased aleurone cell layers and improved nutrition in rice[J]. Molecular Plant, 14(8): 1343-1361. [15] Li Y, He Y Z, Qin T, et al.2023. Functional conservation and divergence of miR156 and miR529 during rice development[J]. The Crop Journal, 11(3): 692-703. [16] Liu D P, Zhang X X, Li Q L, et al.2023. The U-box ubiquitin ligase TUD1 promotes brassinosteroid-induced GSK2 degradation in rice[J]. Plant Communications, 4(2): 100450. [17] Liu L C, Tong H N, Xiao Y H, et al.2015. Activation of Big Grain1 significantly improves grain size by regulating auxin transport in rice[J]. Proceedings of The National Academy of Sciences of The USA, 112(35): 11102-11107. [18] Mao H L, Sun S Y, Yao J L, et al.2010. Linking differential domain functions of the GS3 protein to natural variation of grain size in rice[J]. Proceedings of The National Academy of Sciences of The USA, 107(45): 19579-19584. [19] Miao C B, Wang D, He R, et al.2020. Mutations in MIR396e and MIR396f increase grain size and modulate shoot architecture in rice[J]. Plant Biotechnology Journal, 18(2): 491-501. [20] Miao C B, Wang Z, Zhang L, et al.2019. The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice[J]. Nature Communications, 10(1): 3822. [21] Moon J, Parry G, Estelle M.2004. The ubiquitin-proteasome pathway and plant development[J]. Plant Cell, 16(12): 3181-3195. [22] Qiao S L, Sun S Y, Wang L L, et al.2017. The RLA1/SMOS1 transcription factor functions with OsBZR1 to regulate brassinosteroid signaling and rice architecture[J]. Plant Cell, 29(2): 292-309. [23] Ren D Y, Ding C Q, Qian Q.2023. Molecular bases of rice grain size and quality for optimized productivity[J]. Science Bulletin, 68(3): 314-350. [24] Shi C L, Rao Y L, Liu L L, et al.2019. Ubiquitin specific protease 15 has an important role in regulating grain width and size in rice[J]. Plant Physiology, 180(1): 381-391. [25] Song X J, Huang W, Shi M, et al.2007. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J]. Nature Genetics, 39(5): 623-630. [26] Xu R, Duan P G, Yu H Y, et al.2018. Control of grain size and weight by the OsMKKK10-OsMKK4-OsMAPK6 signaling pathway in rice[J]. Molecular Plant, 11(6): 860-873. [27] Tong H N, Liu L C, Jin Y, et al.2012. DWARF AND LOW-TILLERING acts as a direct downstream target of a GSK3/SHAGGY-like kinase to mediate brassinosteroid responses in rice[J]. Plant Cell, 24(6): 2562-2577. [28] Yang B J, Wendrich J, De-Rybel B, et al.2020. Rice microtubule-associated protein IQ67-DOMAIN14 regulates grain shape by modulating microtubule cytoskeleton dynamics[J]. Plant Biotechnology Journal, 18(5): 1141-1152. [29] Yang W S, Wu K, Wang B, et al.2021a. The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice[J]. Molecular Plant, 14(10): 1699-1713. [30] Yang X F, Zhao X L, Dai Z Y, et al.2021b. OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408[J]. Plant Physiology, 186(1): 519-533. [31] Zhao Y F, Peng T, Sun H Z, et al.2019. miR1432-OsACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice[J]. Plant Biotechnology Journal, 17(4): 712-723. [32] Zhao Y F, Zhang X F, Cheng Y, et al.2023. The miR167-OsARF12 module regulates rice grain filling and grain size downstream of miR159[J]. Plant Communications, 4(5): 100604. [33] Zhu D P, Wan Y, Shao S X, et al.2025. Suppression of miR2871 enhances rice resistance to bacterial blight by modulating cell wall biosynthesis[J]. New Phytologist, 248(6): 3132-3150.
