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Ectopic expression of LjmiR156 delays flowering, enhances shoot branching, and improves forage quality in alfalfa

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Abstract

MicroRNA156 (miR156) is a regulator of flowering time and biomass production through regulation of members of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. To expand our investigations on the utility of miR156 in alfalfa, we generated transgenic alfalfa expressing a heterologous miR156 from Lotus japonicus (LjmiR156a). 5′ RACE and qRT-PCR analysis confirmed that the same three SPL genes (MsSPL6, MsSPL12, and MsSPL13) targeted by MsmiR156d are also targets of LjmiR156a in alfalfa. Ectopic expression of LjmiR156a down-regulated these SPL genes in transgenic alfalfa, coupled with reduced internode length, a more extreme delay in flowering time than occurred with MsmiR156d, enhanced shoot branching, and elevated biomass production. While root length was maintained, nodulation was reduced in some transgenic genotypes. Furthermore, heterologous expression of LjmiR156a enhanced the contents of starch, soluble sugars, and phenolics in all transgenic genotypes in contrast to the impact from MsmiR156d enhancement, even though the effects on lignin, cellulose, pectin, structural sugars, flavonoids, and carotenoids were variable among the new alfalfa genotypes. The variations among the traits/genotypes reflect the change in expression of alfalfa SPL genes targeted by LjmiR156a and show that LjmiR156a could be employed as a tool to improve quality and yield of alfalfa biomass.

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Acknowledgments

Technical assistance for isolation of LjmiR156a precursor from Ying Wang is greatly appreciated. We thank Alex Molnar for his help with preparing the figures and M. Udvari (Noble Foundation, Ardmore, OK) for a kind gift of S. meliloti SM1021. This project was funded by a Grant from the National Science and Engineering Research Council to AH and a Grant from Agriculture and Agri-Food Canada to MYG and AH.

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Authors and Affiliations

  1. Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V4T3, Canada

    Banyar Aung, Khaled Omari & Abdelali Hannoufa

  2. Biology Department, Western University, 1151 Richmond Street, London, ON, N6A5B7, Canada

    Banyar Aung, Lisa Amyot & Abdelali Hannoufa

  3. Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N0X2, Canada

    Margaret Y. Gruber

  4. Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec City, QC, G1V2J3, Canada

    Annick Bertrand

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  1. Banyar Aung
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  2. Margaret Y. Gruber
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Correspondence to Abdelali Hannoufa.

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Fig. S1

Validation of miR156 targets in alfalfa using 5′ RLM-RACE PCR. 1st refers to outer PCR products whereas 2nd represents inner PCR products from each SPL gene. Ladder; HyperLadder II DNA ladder (TIFF 388 kb)

Fig. S2

Analysis of mature miR156 using stem-loop qRT-PCR (TIFF 18 kb)

Fig. S3

Effects of LjmiR156a on shoot branching in 40-day-old alfalfa genotypes (TIFF 2840 kb)

Fig. S4

Effects of LjmiR156a on shoot branching in two-month-old alfalfa genotypes (TIFF 2346 kb)

Fig. S5

Effects of LjmiR156a on stem thickness in alfalfa. a phenotypes of alfalfa stems, b quantitative measurement of stem thickness measured at six months after propagation (TIFF 438 kb)

Table S1

Primers used in this work (DOCX 21 kb)

Table S2

Effects of LjmiR156a on root generating capacity (DOCX 13 kb)

Table S3

Effects of LjmiR156a on structural and non-structural carbohydrates contents in six-month-old transgenic alfalfa (DOCX 16 kb)

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Aung, B., Gruber, M.Y., Amyot, L. et al. Ectopic expression of LjmiR156 delays flowering, enhances shoot branching, and improves forage quality in alfalfa. Plant Biotechnol Rep 9, 379–393 (2015). https://doi.org/10.1007/s11816-015-0375-2

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