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Growth-regulating factors (GRFs) are plant-specific transcription factors, and their activities are regulated by miR396 and the GRF-GIF interaction. The miR396-GRFs/GIFs module determines organ size by regulating cell proliferation. However, it is largely unknown in cucumber. In this study, the CsmiR396-CsGRFs/CsGIFs module was investigated in cucumber. Five CsMIR396 loci (CsMIR396A-E), eight CsGRFs and two CsGIFs were identified. CsMIR396A-E was distributed within two clusters and coded three different mature CsmiR396, and all CsGRFs acted as the target of CsmiR396. Bioinformatic analyses showed that miR396s were classified into five types, while GRFs were classified into six groups in plants. The GRFs from group Ⅰ exhibited high diversity and harbored specific characteristics (truncated C-terminus or two WRC domains). qRT-PCR results showed that CsMIR396s (CsMIR396A, CsMIR396B and CsMIR396D) and mature CsmiR396 increased, whereas CsGRFs declined as leaf age increased. In contrast, CsMIR396E was highly expressed in young leaves and shoot tissue, and it was expressed in an age-independent pattern. Yeast two-hybrid assays showed that CsGRF3 strongly interacted with CsGIFs, while CsGRF5 weakly interacted with CsGIFs. Overexpression of CsGRF3 resulted in an enlarged organ size; in contrast, overexpression of CsGRF5, which belonged to group Ⅰ and harbored two WRC domains, resulted in a reduced organ size in Arabidopsis. Section analysis showed that cell proliferation was increased in CsGRF3OE plants, whereas it was decreased in CsGRF5OE plants. In summary, our results reveal the diversity of the CsmiR396-CsGRFs/CsGIFs module in cucumber, and that CsGRF3 and CsGRF5 play an opposite role in regulating cell proliferation. |
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