Abstract
SQUAMOSA Promoter-binding protein-Like (SPL) genes affect a broad range of plant biological processes and show potential application in crop improvement by genetic modification. As the most widely planted forage crop in the world, biomass and abiotic stresses tolerance are important breeding targets for alfalfa (Medicago sativa L.). Nevertheless, the systematic analysis of SPL genes in alfalfa genome remains lacking. In the present study, we characterized 22 putative non-redundant SPL genes in alfalfa genome and uncovered the abundant structural variation among MsSPL genes. The phylogenetic analysis of plant SPL proteins separated them into 10 clades and clade J was an alfalfa-specific clade, suggesting SPL genes in alfalfa might have experienced gene duplication and functional differentiation within the genome. Meanwhile, 11 MsSPL genes with perfect matches to miRNA response elements (MREs) could be degraded by miR156, and the cleavage sites were gene specific. In addition, we investigated the temporal and spatial expression patterns of MsSPL genes and their expression patterns in response to multiple treatments, characterizing candidate SPL genes in alfalfa development and abiotic stress tolerant regulation. More importantly, overexpression of the alfalfa-specific SPL gene (MsSPL20) showed stable delayed flowering time, as well as increased biomass. Further studies indicated that MsSPL20 delayed flowering time by regulating the expression of genes involved in floret development, including HD3A, FTIP1, TEM1, and HST1. Together, our findings provide valuable information for future research and utilization of SPL genes in alfalfa and elucidate a possibly alfalfa-specific flowering time regulation, thereby supplying candidate genes for alfalfa molecular-assisted breeding.
Highlights
Over the past two decades, SQUAMOSA Promoter-binding protein-Like (SPL) genes, which encode a class of plantspecific transcription factors, have been shown to affect a broad range of plant biological processes (Chen et al, 2010)
After confirming the SQUAMOSA-PROMOTER BINDING PROTEIN (SBP) domain in SMART, 72 of them contained SBP domain and distributed in alfalfa chromosomes except Chr1.4, 4.3, 5.2, 6.1, 6.2, 6.3, and 6.4 (Supplementary Figure 1). We subsequently clustered these 72 putative SPL genes and obtained 22 non-redundant MsSPL genes, including their CDSs and genomic sequences in alfalfa (Supplementary Figure 2). These 22 MsSPL genes were randomly distributed on 7 chromosomal groups and were designated as MsSPL1–MsSPL22 on the basis of their chromosomal locations (Figure 1)
The molecular weight of these 22 MsSPL proteins ranged from 16.58 kD (MsSPL8) to 130.58 kD (MsSPL7), and their isoelectric points varied from 5.66 (MsSPL3) to 9.23 (MsSPL17) (Supplementary Table 2)
Summary
Over the past two decades, SQUAMOSA Promoter-binding protein-Like (SPL) genes, which encode a class of plantspecific transcription factors, have been shown to affect a broad range of plant biological processes (Chen et al, 2010). These processes include the timing of vegetative phase changes and floral induction, the rate of leaf initiation, shoot regeneration and branching, anthocyanin and trichome production, stress responses, carotenoid biosynthesis, and lateral root development (Gou et al, 2011; Jung et al, 2012; Cui et al, 2015; Zhang et al, 2015; Ioannidi et al, 2016; Preston et al, 2016). These findings indicate the vital roles of SPL genes in plants, as positive Darwinian selection reflects adaptation to novel ecological conditions
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