HECATE2 acts with GLABROUS3 and Tu to boost cytokinin biosynthesis and regulate cucumber fruit wart formation

Abstract

Warty fruit in cucumber (Cucumis sativus L.) is an important quality trait that greatly affects fruit appearance and market value. The cucumber wart consists of fruit trichomes (spines) and underlying tubercules, in which the existence of spines is prerequisite for tubercule formation. Although several regulators have been reported to mediate spine or tubercule formation, the direct link between spine and tubercule development remains unknown. Here, we found that the basic Helix-Loop-Helix (bHLH) gene HECATE2 (CsHEC2) was highly expressed in cucumber fruit peels including spines and tubercules. Knockout of CsHEC2 by the CRISPR/Cas9 system resulted in reduced wart density and decreased cytokinin accumulation in the fruit peel, whereas overexpression of CsHEC2 led to elevated wart density and cytokinin level. CsHEC2 directly bound to the promoter of the cytokinin hydroxylase-like1 gene (CsCHL1) that catalyzes cytokinin biosynthesis, and activated CsCHL1 expression. Moreover, CsHEC2 physically interacted with GLABROUS3 (CsGL3, a key spine regulator) and Tuberculate fruit (CsTu, a core tubercule formation factor), and such interactions further enhanced CsHEC2-mediated CsCHL1 expression. These data suggested that CsHEC2 promotes wart formation by acting as an important cofactor for CsGL3 and CsTu to directly stimulate cytokinin biosynthesis in cucumber. Thus, CsHEC2 can serve as a valuable target for molecular breeding of cucumber varieties with different wart density requirements.

A novel HD‐Zip I/C2H2‐ZFP/WD‐repeat complex regulates the size of spine base in cucumber

“…Previous studies indicated that the ethylene, auxin, cytokinin and gibberellin signaling pathways are involved in cucumber spine formation (Che & Zhang, 2019; Wang et al ., 2021; Zhang et al ., 2021). Thus, we measured the level of endogenous gibberellic acid (GA3), auxin (indole‐3‐acetic acid, IAA; indole‐3‐butytric acid, IBA), cytokinin (isopentenyl adenosine, IPA; trans‐zeatin riboside, TZR), and ethylene in the ovary of L‐SB and S‐SB.…”

“…To measure the level of endogenous gibberellic acid, auxin, and cytokinin, 1 g of ovary samples at 2 DBA from L‐SB and S‐SB were frozen in liquid nitrogen. The extraction and quantification of phytohormones were performed using high‐performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) as previously described (Wang et al ., 2021). Ethylene release rate of ovary samples at 5 DPP was measured as previously described (Zhang et al ., 2021).…”

“…Recently, Wang et al . (2021) identified a bHLH gene, CsHEC2 , that regulated fruit spine density through mediating cytokinin biosynthesis and metabolism in cucumber. However, most of the earlier genes regulate the development for both fruit spine and leaf trichrome.…”

“…In previous studies, we have revealed that the WD-repeat protein CsTTG1 and MIXTA-like protein CsMYB6 regulated fruit spines initiation and further differentiation in cucumber (Chen et al, 2016;Yang et al, 2018Yang et al, , 2019Guo et al, 2020;Zhao et al, 2020). Recently, Wang et al (2021) identified a bHLH gene, CsHEC2, that regulated fruit spine density through mediating cytokinin biosynthesis and metabolism in cucumber. However, most of the earlier genes regulate the development for both fruit spine and leaf trichrome.…”

NS encodes an auxin transporter that regulates the ‘numerous spines’ trait in cucumber (Cucumis sativus) fruit

“…In this study, one NS candidate gene, encoding an auxin transport protein, was identified by genome‐wide association study (GWAS). Recently, the powerful biotechnology CRISPR/Cas9 has been applied in the improvement of cucumber agronomic traits (Chandrasekaran et al., 2016; Hu et al., 2017; Wang et al., 2021; Xin et al., 2019; Yang et al., 2020; Zhang et al., 2020; Zhang, Li, et al., 2021). We here demonstrate that the knockout ns mutant generated by CRISPR/Cas9 display the numerous fruit spine phenotype.…”