The CsHEC1-CsOVATE module contributes to fruit neck length variation via modulating auxin biosynthesis in cucumber
Thursday, 2022/09/29 | 08:32:54
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Zhongyi Wang, Zhaoyang Zhou, Liming Wang, +12 , Shuangshuang Yan, Zhihua Cheng, Xiaofeng Liu, Lijie Han, Guangxin Chen, Shaoyun Wang, Weiyuan Song, Jiacai Chen, Liu Liu, Xiaofei Song, Liying Yan, Jianyu Zhao and Xiaolan Zhang. PNAS September 19, 2022; 119 (39) e2209717119 SignificanceShape variations of plant edible organs are the result of adaptive evolution and domestication. Fruit neck is an undesirable trait that affects fruit shape and commercial value in cucumber. Despite fruit neck length (FNL) varying greatly among cucumber germplasms, the molecular mechanisms controlling FNL remain unknown. Here, we demonstrate that HECATE1 (CsHEC1) positively regulates FNL by directly activating the auxin biosynthesis gene CsYUC4. CsOVATE, whose expression is negatively correlated with FNL, combats CsHEC1 to attenuate the CsHEC1-mediated CsYUC4 transcriptional activation. Our work not only paves a way to shorten FNL by manipulating the CsHEC1-CsOVATE module to decrease local auxin levels during cucumber breeding but also provides significant insights into fruit shape regulation in pepo fruits. AbstractFruit neck is the proximal portion of the fruit with undesirable taste that has detrimental effects on fruit shape and commercial value in cucumber. Despite the dramatic variations in fruit neck length of cucumber germplasms, the genes and regulatory mechanisms underlying fruit neck elongation remain mysterious. In this study, we found that Cucumis sativus HECATE1 (CsHEC1) was highly expressed in fruit neck. Knockout of CsHEC1 resulted in shortened fruit neck and decreased auxin accumulation, whereas overexpression of CsHEC1 displayed the opposite effects, suggesting that CsHEC1 positively regulated fruit neck length by modulating local auxin level. Further analysis showed that CsHEC1 directly bound to the promoter of the auxin biosynthesis gene YUCCA4 (CsYUC4) and activated its expression. Enhanced expression of CsYUC4 resulted in elongated fruit neck and elevated auxin content. Moreover, knockout of CsOVATE resulted in longer fruit neck and higher auxin. Genetic and biochemical data showed that CsOVATE physically interacted with CsHEC1 to antagonize its function by attenuating the CsHEC1-mediated CsYUC4 transcriptional activation. In cucumber germplasms, the expression of CsHEC1 and CsYUC4 positively correlated with fruit neck length, while that of CsOVATE showed a negative correlation. Together, our results revealed a CsHEC1-CsOVATE regulatory module that confers fruit neck length variation via CsYUC4-mediated auxin biosynthesis in cucumber.
See https://www.pnas.org/doi/10.1073/pnas.2209717119
Figure 5: CsHEC1 directly interacts with CsOVATE at the protein level. (A) CsOVATE colocalization with the CsHEC1 protein in N. benthamiana leaves. The yellow signal in the merged field represents the colocalization signal in the nucleus. (B) Interaction of CsHEC1 and CsOVATE in the yeast two-hybrid system. The combination of T-AD and 53-BD was used as a positive control. (C) CsHEC1 interacts with CsOVATE in vitro tested by a GST pull-down assay. The combination of GST and His-CsOVATE was used as a control. (D) Firefly luciferase complementation imaging analysis. CsHEC1-nLUC and cLUC-CsOVATE were transiently coexpressed in N. benthamiana, and the remaining combinations were used as controls. Representative pictures (Top) and the relative luciferase activity value are shown (Bottom). Values are means ± SD (n = 6). Two-tailed Student’s t test (**P < 0.01). (E) Co-IP assay showing that CsHEC1 interacted with CsOVATE in vivo. The total and precipitated proteins were detected by immunoblotting using anti-GFP antibody or anti-Flag antibody. The asterisks indicate nonspecific bands.
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