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MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa

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MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.

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The authors are grateful for the technical support of Ms. Lisa Amyot, Mr. Tim McDowell and Mr. Brian Weselowski. Research was conducted through a grant (J-000260) from Agriculture and Agri-Food Canada to AH.

Author’s Contributions

BA carried out the molecular genetic studies, statistically analysed the data, and drafted the manuscript. RG carried out bioinformatics analysis of NGS data and revised the manuscript. Z-CY and MS conducted the nitrogen fixation experiment, analysed data and reviewed manuscript. MG helped design experiments and edited the manuscript. AH conceived of the study, and participated in its design and coordination and helped to revise the manuscript. All authors read and approved the final manuscript.

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Corresponding author

Correspondence to Abdelali Hannoufa.

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Conflict of interests

The authors declare that they have no conflict of interests.

Additional information

Margaret Y. Gruber–Retired from Agriculture and Agri-Food Canada, 106 Science Place, Saskatoon, SK, S7N 0X2, Canada

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Table S1

Primers used in this study. (DOC 97 kb)

Table S2

Effect of miR156 on nodulation and shoot biomass in transgenic alfalfa. (DOC 37 kb)

Table S3

Differential gene expression in 1-month-old roots of MsMIR156OE transgenic genotype A17 relative to wild type (WT) parental control. (PDF 855 kb)

Table S4

Reduce visualization analysis of gene ontology for differentially expressed genes in transgenic alfalfa roots. (XLS 55 kb)

Fig. S1

Calibration curve for ethylene quantification by GC-FID. The curve was generated using ethylene gas and GC-FID with a gas-tight syringe (Hamilton, Reno, NV, USA) as described in the Methods. (TIFF 337 kb)

Fig. S2

Expression of phytohormone-related genes in young roots of six MsMIR156OE alfalfa genotypes growing without N fertilizer after inoculation with S. Elliot. Expression profiles of (a) AUX, (b) ARF; (c) GRF, (d) GR, (e) AAR2, (f) CRH, (g) COD, (h) AOC, (i) ERT, and (j) ERF genes. Expression was analyzed using three biological replicates and two technical (machine) replicates. One or two asterisks indicate significant differences of the means (n = 3) from wild type control P < 0.05 or 0.01 (t test). Abbreviations are AUX: Auxin-responsive AUX/IAA family protein, ARF: Auxin response factor, GRF: Gibberellin-regulated family protein,GR: Gibberellin receptor GID1, ERT: Ethylene-responsive transcription factor 1B, ERF: Ethylene response factor, AOC: Allene oxide cyclase (Jasomic acid biosynthesis), COD: Cytokinin oxidase/dehydrogenase-like protein, CRH: Cytokinin receptor histidine kinase, and AAR2: Abscisic acid receptor 2. (TIFF 787 kb)

Fig. S3

Effect of MsmiR156 overexpression on nodulation-related gene expression measured by qRT-PCR. Gene expression profiles (a, c, e, g, i, k) in 12-day-old entire stem cutting and (b, d, f, h, j, l) in roots at 3 weeks after inoculation (5 weeks old). Transcript levels of (a, b) CLE, (c, d) ENOD93, (e, f) LysM, (g, h) NR, (i, j) NTR, and (k, l) cyclops. Expression was analyzed using three biological replicates and two technical (machine) replicates. Acetyl CoA carboxylase 1 and acetyl CoA carboxylase 2 genes were used as the reference for normalization. One, two or three asterisks indicate significant differences from wild type control or non-inoculated plant at P < 0.1, 0.05 or 0.01, respectively (t test). WT: wild type control. (TIFF 1219 kb)

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Aung, B., Gao, R., Gruber, M.Y. et al. MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa. Transgenic Res 26, 541–557 (2017).

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