MsmiR171 targets MsSCL6 to mediate selenium-regulated chlorophyll biosynthesis in alfalfa

Abstract

Alfalfa (Medicago sativa L.) is a globally pivotal legume forage. Selenium (Se), an essential trace element for humans and animals, can significantly enhance the growth and development of alfalfa. Chlorophyll is the central pigment of plant photosynthesis. Previous research on chlorophyll synthesis in alfalfa has mainly focused on transcriptional regulation, environmental factors (light, nutrient availability), and phytohormone signaling, while fewer studies have been conducted at the post-transcriptional level. Through whole transcriptome sequencing analysis, microRNAs (miRNAs) were identified as positively responsive to Se. This study focused on the regulation of chlorophyll synthesis by the miR171-SCL6 module in alfalfa. β-glucuronidase staining and dual-luciferase assays revealed that MsmiR171 negatively regulated the transcript levels of the SCARECROW-LIKE 6 transcription factor MsSCL6. Subcellular localization analysis revealed that MsSCL6 was mainly in the cell nucleus. Functional analyses demonstrated that MsmiR171 promoted chlorophyll synthesis and photosynthesis in alfalfa, while MsSCL6 negatively regulated chlorophyll synthesis. Notably, Se treatment upregulated MsmiR171 expression, downregulated MsSCL6 expression, and enhanced chlorophyll accumulation. qRT-PCR analysis revealed differential expression of MsPOR in MsmiR171 and MsSCL6 overexpression or silencing plants. Combined yeast one-hybrid and dual-luciferase assays demonstrated that MsSCL6 transcriptionally represses MsPOR through direct promoter binding, suppressing chlorophyll accumulation. In summary, this study for the first time revealed the mechanism of the MsmiR171-MsSCL6-MsPOR module mediating Se-regulated chlorophyll biosynthesis in alfalfa. These findings provide a theoretical foundation and technical guidance for alfalfa breeding and the production of Se-enriched forage.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 51508518) and the Special Fund for Agro-scientific Research in the Public Interest (grant no. 201503134). We thank Professor Yule Liu (Tsinghua University) and Tao Zhou (China Agricultural University) for providing the silencing vector TRV2.

Authors contributions

Q.W. conceived and designed the experiments. S.S. wrote the paper and conducted the experiment. Y.S., M.X., and B.T. helped to revise the manuscript and provided valuable opinions. Y.M. and Y.S. took part in the experimental design, project administration and funding acquisition. All authors have read and approved the final manuscript.

Data availability

All data supporting this study are included in the article and its supplementary materials.

Conflicts of interest statement

All authors declare no conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Tangjaidee P, Swedlund P, Xiang J. et al. Selenium-enriched plant foods: selenium accumulation, speciation, and health functionality. Front Nutr. 2022; 9:962312

[2] Hossain A, Skalicky M, Brestic M. et al. Selenium biofortifica-tion: roles, mechanisms, responses and prospects. Molecules. 2021; 26:881

[3] Rayman MP. Selenium intake, status, and health: a complex relationship. Hormones. 2019; 19:9-14

[4] Abdullah, Wani KI, Hayat K. et al. Multifaceted role of selenium in plant physiology and stress resilience: a review. Plant Sci. 2025; 355:112456

[5] Hu H, Hu J, Wang Q. et al. Transcriptome analysis revealed accumulation-assimilation of selenium and physio-biochemical changes in alfalfa ( Medicago sativa L.) leaves. J Sci Food Agric. 2022; 102:4577-88

[6] Chen J, Chen H, Wang H. et al. Selenium treatment promotes anthocyanin accumulation in radish sprouts (Raphanus sativus L.) by its regulation of photosynthesis and sucrose transport. Food Res Int. 2023; 165:112551

[7] Zhang M, Tang S, Huang X. et al. Selenium uptake, dynamic changes in selenium content and its influence on photosyn-thesis and chlorophyll fluorescence in rice (Oryza sativa L.). Environ Exp Bot. 2014; 107:39-45

[8] Borbély P, Molnár Á, Valyon E. et al. The effect of foliar selenium (Se) treatment on growth, photosynthesis, and oxidative-nitrosative signalling of Stevia rebaudiana leaves. Antioxidants. 2021; 10:72

[9] Ameres SL, Zamore PD. Diversifying microRNA sequence and function. Nat Rev Mol Cell Biol. 2013; 14:475-88

[10] Zhan J, Meyers BC. Plant small RNAs: their biogenesis, regula-tory roles, and functions. Annu Rev Plant Biol. 2023; 74:21-51

[11] Shankar N, Nath U. Advantage looping: gene regulatory cir-cuits between microRNAs and their target transcription fac-tors in plants. Plant Physiol. 2024; 196:2304-19

[12] Bresso EG, Chorostecki U, Rodriguez RE. et al. Spatial control of gene expression by miR319-regulated TCP transcription factors in leaf development. Plant Physiol. 2018; 176:1694-708

[13] Li J, Cao Y, Zhang J. et al. The miR165/166-PHABULOSA mod-ule promotes thermotolerance by transcriptionally and post-translationally regulating HSFA1. Plant Cell. 2023; 35:2952-71

[14] Wei X, Wang C, Wang Y. et al. miR166e/ZmATHB14 module contributes to drought tolerance in maize root. Int J Biol Macromol. 2025; 297:139707

[15] Dong Y, Tang M, Huang Z. et al. The miR164a-NAM3 module confers cold tolerance by inducing ethylene production in tomato. Plant J. 2022; 111:440-56

[16] Zhu X, Leng X, Sun X. et al. Discovery of conservation and diversification of miR171 genes by phylogenetic analysis based on global genomes. Plant Genome. 2015;8:eplantgenome2014.10.0076

[17] Bolle C. The role of GRAS proteins in plant signal transduction and development. Planta. 2004; 218:683-92

[18] Wang L, Mai YX, Zhang YC. et al. MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis. Mol Plant. 2010; 3:794-806

[19] Xing JX, Zang QL, Ye ZL. et al. Overexpression of larch SCL6 inhibits transitions from vegetative meristem to inflorescence and flower meristem in Arabidopsis thaliana (L.) Heynh. Plants (Basel). 2024; 13:1232

[20] Li H, Yu J, Qin J. et al. Regulatory mechanisms of miR171d-SCL6 module in the rooting process of Acer rubrum L. Planta. 2024; 260:109

[21] Yan R, Song S, Li H. et al. Functional analysis of the eTM-miR171-SCL6 module regulating somatic embryogenesis in Lilium pumilum DC.Fisch. Hortic Res. 2022;9:uhac045

[22] Um T, Choi J, Park T. et al. Rice microRNA171f/SCL6 mod-ule enhances drought tolerance by regulation of flavonoid biosynthesis genes. Plant Direct. 2022; 6:e374

[23] Hu G, Ge X, Wang P. et al. The cotton miR171a-SCL6 module mediates plant resistance through regulating GhPR1 expres-sion. Plant Physiol Biochem. 2023; 202:107995

[24] Ma Z, Hu X, Cai W. et al. Arabidopsis miR171-targeted scarecrow-like proteins bind to GT cis-elements and medi-ate gibberellin-regulated chlorophyll biosynthesis under light conditions. PLoS Genet. 2014; 10:e1004519

[25] Li H, Zhang Q, Li L. et al. Ectopic overexpression of bol-miR171b increases chlorophyll content and results in sterility in broccoli (Brassica oleracea L var. italica). J Agric Food Chem. 2018; 66:9588-97

[26] Li X, Zhang W, Niu D. et al. Effects of abiotic stress on chloro-phyll metabolism. Plant Sci. 2024; 342:112030

[27] Nagata N, Tanaka R, Satoh S. et al. Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of prochlorococ-cus species. Plant Cell. 2005; 17:233-40

[28] Radovic J, Sokolovic D, Markovic J. Alfalfa-most important perennial forage legume in animal husbandry. Biotechnol Anim Husb. 2009; 25:465-75

[29] Ma J, Huangfu W, Yang X. et al. "King of the forage"-alfalfa sup-plementation improves growth, reproductive performance, health condition and meat quality of pigs. Front Vet Sci. 2022; 9:1025942

[30] Wang T, Zhang WH. Priorities for the development of alfalfa pasture in northern China. Fundam Res. 2023; 3:225-8

[31] Wang Q, Hu J, Hu H. et al. Integrated eco-physiological, bio-chemical, and molecular biological analyses of selenium for-tification mechanism in alfalfa. Planta. 2022; 256:114

[32] Tong A, Yuan Q, Wang S. et al. Altered accumulation of osa-miR171b contributes to rice stripe virus infection by regulat-ing disease symptoms. J Exp Bot. 2017; 68:4357-67

[33] Schulze S, Schäfer BN, Parizotto EA. et al. LOST MERISTEMS genes regulate cell differentiation of central zone descen-dants in Arabidopsis shoot meristems. Plant J. 2010; 64:668-78

[34] Pei LL, Zhang LL, Liu X. et al. Role of microRNA miR171 in plant development. PeerJ. 2023; 11:e15632

[35] Xue XY, Zhao B, Chao LM. et al. Interaction between two tim-ing microRNAs controls trichome distribution in Arabidopsis. PLoS Genet. 2014; 10:e1004266

[36] Huang W, Peng S, Xian Z. et al. Overexpression of a tomato miR171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis. Plant Biotechnol J. 2017; 15:472-88

[37] Talesh Sasani S, Soltani BM, Mehrabi R. et al. Expression alter-ation of candidate rice miRNAs in response to sheath blight disease. Iran J Biotechnol. 2020; 18:e2451

[38] Wang K, Cai S, Xing Q. et al. Melatonin delays dark-induced leaf senescence by inducing miR171b expression in tomato. J Pineal Res. 2022; 72:e12792

[39] Zhao L, Gao L, Wang H. et al. The R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid biosynthesis. Funct Integr Genomics. 2013; 13:75-98

[40] Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell. 2009; 136:669-87

[41] Rogers K, Chen X. Biogenesis, turn,over, and mode of action of plant microRNAs. Plant Cell. 2013; 25:2383-99

[42] Curaba J, Talbot M, Li Z. et al. Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley. BMC Plant Biol. 2013; 13:6

[43] Li Y, Tong Y, He X. et al. The rice miR171b-SCL6-IIs module controls blast resistance, grain yield, and flowering. Crop J. 2022; 10:117-27

[44] Croft H, Chen JM, Luo X. et al. Leaf chlorophyll content as a proxy for leaf photosynthetic capacity. Glob Chang Biol. 2017; 23:3513-24

[45] Yuan Y, Xu X, Gong Z. et al. Auxin response factor 6A regulates photosynthesis, sugar accumulation, and fruit development in tomato. Hortic Res. 2019; 6:85

[46] Meng L, Fan Z, Zhang Q. et al. BEL1-LIKE HOMEODOMAIN 11 regulates chloroplast development and chlorophyll synthesis in tomato fruit. Plant J. 2018; 94:1126-40

[47] Cahoon AB, Timko MP. Yellow-in-the-dark mutants of Chlamy-domonas lack the CHLL subunit of light-independent pro-tochlorophyllide reductase. Plant Cell. 2000; 12:559-68

[48] Wu M, Xu X, Hu X. et al. SlMYB72 regulates the metabolism of chlorophylls, carotenoids, and flavonoids in tomato fruit. Plant Physiol. 2020; 183:854-68

[49] Dai X, Zhuang Z, Zhao PX. psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Res. 2018;46:W49-54

[50] Tamura K, Stecher G, Peterson D. et al. MEGA6: molecu-lar evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013; 30:2725-9

[51] Sparkes IA, Runions J, Kearns A. et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006; 1:2019-25

[52] Zhang B, Yang HJ, Qu D. et al. The MdBBX22-miR858-MdMYB9/11/12 module regulates proanthocyanidin biosynthesis in apple peel. Plant Biotechnol J. 2022; 20: 1683-700

[53] Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987; 6:3901-7

[54] Xia H, Shen Y, Hu R. et al. Methylation of MYBA1 is associated with the coloration in "manicure finger" grape skin. J Agric Food Chem. 2021; 69:15649-59

[55] Wu HJ, Wang ZM, Wang M. et al. Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants. Plant Physiol. 2013; 161:1875-84

[56] Liu J, Shi K, Wang S. et al. MsCYP71 is a positive regulator for drought resistance in alfalfa. Plant Physiol Biochem. 2023; 203: 107999

[57] Wijekoon C, Singer SD, Weselake RJ. et al. Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek. Can J Plant Sci. 2019; 99:917-26

[58] Li Y, Liang L, Li W. et al. ZnO nanoparticle-based seed priming modulates early growth and enhances physio-biochemical and metabolic profiles of fragrant rice against cadmium tox-icity. J Nanobiotechnology. 2021; 19:75

[59] Bradford MM. A rapid and sensitive method for the quan-titation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54

[60] Masuko T, Minami A, Iwasaki N. et al. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 2005; 339:69-72

[61] Hu X, Lu K, Mu L. et al. Interactions between graphene oxide and plant cells: regulation of cell morphology, uptake, organelle damage, oxidative effects and metabolic disorders. Carbon. 2014; 80:665-76