Abstract
Key message
Our results show SPL13 plays a crucial role in regulating vegetative and reproductive development in Medicago sativa L. (alfalfa), and that MYB112 is targeted and downregulated by SPL13 in alfalfa.
Abstract
We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156) show a bushy phenotype, reduced internodal length, delayed flowering time, and enhanced biomass yield. In alfalfa, transcripts of seven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL13, are targeted for cleavage by miR156. Thus, association of each target SPL gene to a trait or set of traits is essential for developing molecular markers for alfalfa breeding. In this study, we investigated SPL13 function using SPL13 overexpression and silenced alfalfa plants. Severe growth retardation, distorted branches and up-curled leaves were observed in miR156-impervious 35S::SPL13m over-expression plants. In contrast, more lateral branches and delayed flowering time were observed in SPL13 silenced plants. SPL13 transcripts were predominantly present in the plant meristems, indicating that SPL13 is involved in regulating shoot branch development. Accordingly, the shoot branching-related CAROTENOID CLEAVAGE DIOXYGENASE 8 gene was found to be significantly downregulated in SPL13 RNAi silencing plants. A R2R3-MYB gene MYB112 was also identified as being directly silenced by SPL13 based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays, suggesting that MYB112 may be involved in regulating alfalfa vegetative growth.
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Acknowledgements
This project was funded by grants from Forage Genetics International (J-001068) and Agriculture and Agri-Food Canada (J-000260) to AH. We would like to thank Mr. Biruk Feyissa and the staff at the Biotron Department, University of Western Ontario for their assistance with in situ hybridization. We also would like to thank Professor Hao YU from National University of Singapore for providing us the pGreen-GFP vector. RG was the recipient of a NSERC Visiting Fellowship to a Canadian Government Laboratory.
Funding
This project was funded by grants from Forage Genetics International (J-001068) and Agriculture and Agri-Food Canada (J-000260) to AH.
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RG, MYG and AH conceived of and designed the work. RG and LA conducted the experiments. RG, MYG, LA and AH analyzed the data. RG and LA drafted the manuscript. MYG and AH revised and approved the final manuscript.
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Margaret Y. Gruber—retired from Agriculture and Agri-Food Canada.
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11103_2017_683_MOESM3_ESM.tif
Fig. S3 Nuclear localization of SPL13. (A), Bipartite nuclear localization signal in SPL13. (B), Detection of nuclear localization signal of SPL13-GFP fusion protein in N. benthamiana leaves by transient expression analysis. DAPI-stained nuclei (blue-color foci) were superimposed onto the differential interference contrast (DIC) image to form a merged imager. Free GFP represents agro-infiltration with vector lacking of inserted gene. The bar=5µm (TIF 3383 KB)
11103_2017_683_MOESM4_ESM.tif
Fig. S4 A proposed model for miR156/SPL13/MYB112 regulation of alfalfa plant development. Solid black and red arrows indicate the direct activation and regulation effect. Blunted line shows direct repression. (TIF 701 KB)
11103_2017_683_MOESM14_ESM.docx
Dataset S1. Genomic DNA sequences (CDS and its upstream 4000 bp promoter regions) of genes in Medicargo sativa (DOCX 13 KB)
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Gao, R., Gruber, M.Y., Amyot, L. et al. SPL13 regulates shoot branching and flowering time in Medicago sativa . Plant Mol Biol 96, 119–133 (2018). https://doi.org/10.1007/s11103-017-0683-8
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DOI: https://doi.org/10.1007/s11103-017-0683-8