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

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.