Abstract
The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156—TC70253, AU089191, and TC57859—were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-014-0931-4) contains supplementary material, which is available to authorized users.
Highlights
MicroRNAs have emerged as a new tool to improve plant traits; from biotic and abiotic stress tolerance to grain and biomass yield (Zhou and Luo 2013)
Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes and in leafy vegetables to positively impact economically valuable crop species
Because it had the highest homology to AtmiR156a, we designated it as pre-LjmiR156a (Supplementary Fig. S1)
Summary
MicroRNAs (miRNAs) have emerged as a new tool to improve plant traits; from biotic and abiotic stress tolerance to grain and biomass yield (Zhou and Luo 2013). MiRNAs are generally 21–24 nucleotides long noncoding RNA molecules. These are derived from singlestranded RNA precursors that are transcribed by RNA. The precursor is further processed by Dicer-like proteins into mature miRNAs which are incorporated into an RNA-induced silencing complex where they negatively regulate target gene expression at the post-transcriptional level by basepairing to complementary targets (Dugas and Bartel 2004; Kidner and Martienssen 2005). MiRNAs can silence genes at the transcriptional level by affecting chromatin methylation (Brodersen and Voinnet 2009). Temporal and spatial accumulations of a few highly conserved miRNAs are crucial for maintaining proper plant development. The most conserved miRNAs tend to be the most highly abundant in organisms
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