Involvement of the miR156/SPL module in flooding response in Medicago sativa

Abstract

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes

“…A subset of miR156-targeted SPL genes in alfalfa have been found previously to lead to an increased ability to withstand abiotic stresses such as drought ( SPL13 , SPL9 ; SPL8; Arshad et al, 2017 ; Gou et al, 2018 ; Feyissa et al, 2019 ; Hanly et al, 2020 ), heat ( SPL13 ; Matthews et al, 2019 ), salinity ( SPL8 ; Gou et al, 2018 ) and/or flooding ( SPL13 ; Feyissa et al, 2021 ) when down-regulated. Correspondingly, both SPL8-gRNA1 genotypes assessed in the present study exhibited improvements in their resilience to water deficit, with lower volumetric soil contents at the first sign of wilting, an improved ability to maintain RWC and photosynthetic rate under drought treatment, and higher survival following extreme drought stress ( Figure 6 ).…”

“…Unlike MsSPL8 in alfalfa, Arabidopsis SPL8 appears to be mainly involved in the regulation of male and female fertility, as well as root growth, with very few apparent roles in aboveground vegetative growth ( Unte et al, 2003 ; Zhang et al, 2007 ; Xing et al, 2013 ). In addition, while SPL8 does not appear to be regulated by miR156 in Arabidopsis ( Xing et al, 2010 ), it is a target of this miRNA in alfalfa ( Feyissa et al, 2021 ), which suggests that some level of evolutionary divergence has occurred in this gene among plant species. However, despite these possible functional and regulatory disparities among species, both Arabidopsis and alfalfa SPL8 have been suggested to function, at least in part, through the transcriptional regulation of genes involved in phytohormone biosynthesis or signaling ( Zhang et al, 2007 ; Xing et al, 2013 ; Gou et al, 2018 ), although their precise functions remain to be unraveled in full.…”

“…In alfalfa, a total of sixteen SPL genes have been identified to date, ten of which are directly silenced by miR156 ( Gao et al, 2016 ; Feyissa et al, 2021 ). Intriguingly, while SPL8 does not appear to be a target of miR156 in Arabidopsis thaliana ( Xing et al, 2010 ), it is cleaved by miR156 in alfalfa ( Feyissa et al, 2021 ).…”

“…In alfalfa, a total of sixteen SPL genes have been identified to date, ten of which are directly silenced by miR156 ( Gao et al, 2016 ; Feyissa et al, 2021 ). Intriguingly, while SPL8 does not appear to be a target of miR156 in Arabidopsis thaliana ( Xing et al, 2010 ), it is cleaved by miR156 in alfalfa ( Feyissa et al, 2021 ). This, along with the fact that there is considerable functional disparity among SPL8 homologs in different plant species ( Unte et al, 2003 ; Zhang et al, 2007 ; Xing et al, 2013 ; Gou et al, 2018 , 2019 ), suggests that it may have evolved in a distinct manner at some point following the divergence of particular lineages.…”

Characterization of Squamosa-Promoter Binding Protein-Box Family Genes Reveals the Critical Role of MsSPL20 in Alfalfa Flowering Time Regulation

“…Alfalfa ( Medicago sativa L.) is the most important, widely grown forage plant in the world because of its high biomass, notable adaptability, exceptional nutritive value, and remarkable biological nitrogen fixation capacity ( Russelle et al, 2007 ; Gou et al, 2018 ). Given the critical roles of SPL genes in plants, several SPL genes in alfalfa have been reported to participate in the regulation of multiple developmental processes and abiotic stress tolerance ( Gao et al, 2018 ; Gou et al, 2018 ; Feyissa et al, 2019 , 2021 ; Lorenzo et al, 2019 ). For instance, transgenic alfalfa silencing MsSPL13 displays more lateral branches and delayed flowering time, and the shoot branching genes were significantly down-regulated in SPL13 RNAi plants ( Gao et al, 2018 ).…”