Fig. 4

Validation and expression analysis of miR156-MaSPL module in banana peel under cold stress. a Alignments of miR156 with their targeted MaSPLs validated in banana. Scissors indicate the cleavage sites of the targeted MaSPL4/5/42/44, as detected by RLM-5’-RACE assay and the factions next to the scissors show the numbers of clones with an identified 5’ end detected in the total sequenced clones. b In vivo targeting validation of miR156-targeted MaSPLs. Four overexpression vectors were constructed for the transient expression assay in tobacco. a: empty vector; b: vector overexpressing pre-miR156c; c: vector overexpressing GFP fused with miR156 target sites of MaSPL4/5/42/44; d: vector overexpressing GFP fused with miR156 modified target sites of MaSPL4/5/42/44. Co-infiltrated leaves and control leaves were photographed on the 3rd day after infiltration under bright light and UV light. Combinations of vectors used in the assay were shown on the leaves. c A time-course phenotype of banana fruit under cold stress. d Detection of miR156c and miR528 levels in banana peel during cold stress. U6 was probed as a loading control. Numbers below the bands are the mean gray values of the samples relative to those of U6, calculated by the Image J software. The control (23℃) sample at 6 h was set as 1. e Gene expression pattern of miR156-targeted MaSPLs in banana peel during cold stress. The RPS2 gene was used as an internal reference. Data are presented as the means ± SD of three biological replicates (n = 3) and significant differences are indicated with asterisks, as determined by two-tailed unpaired Student’s t-test (*, P < 0.05; **, P < 0.01, ns, not significant)