PACLOBUTRAZOL-RESISTANCE4 positively regulates cell expansion to promote tendril elongation in cucumber.

As a climbing organ, the tendril undergoes rapid elongation to increase its length to locate support within a short growth time. However, the molecular mechanism underlying this observation is poorly understood. Here, tendril development was divided into 4 stages in cucumber (Cucumis sativus L.) along with its growth. Phenotypic observations and section analyses showed that the rapid elongation of tendril primarily happened during stage 3 and was mainly due to cell expansion. RNA-seq analysis showed that PACLOBUTRAZOL-RESISTANCE4 (CsPRE4) was highly expressed in the tendril. Our RNAi studies in cucumber and transgenic overexpression in Arabidopsis (Arabidopsis thaliana) suggested that CsPRE4 functions as a conserved activator of cell expansion to promote cell expansion and tendril elongation. Through a triantagonistic HLH (helix-loop-helix)-HLH-bHLH (basic helix-loop-helix) cascade, CsPRE4-CsPAR1 (PHYTOCHROME RAPIDLY REGULATED1)-CsBEE1 (BR-ENHANCED EXPRESSION 1), CsPRE4 released the transcription factor CsBEE1, which activated expansin A12 (CsEXPA12) to loosen the cell wall structure in tendrils. Gibberellin (GA) promoted tendril elongation by modulating cell expansion, and CsPRE4 expression was induced by exogenous GA treatment, suggesting that CsPRE4 acts downstream of GA in regulating tendril elongation. In summary, our work suggested a CsPRE4-CsPAR1-CsBEE1-CsEXPA12 pathway in regulating cell expansion in cucumber tendrils, which might enable rapid tendril elongation to quickly locate support.

Tendril morphogenesis is regulated by a CsaTEN-CsaUFO module in cucumber.

Tendril is a morphological innovation during plant evolution, which provides the plants to obtain climbing ability. However, the tendril morphogenesis is poorly understood. A novel tendril morphogenesis defective mutant (tmd1) was identified in cucumber. The apical part of tendril was replaced by a leaf blade in tmd1 mutant, and it lost the climbing ability. Map-based cloning, qPCR detection, bioinformatic analysis, yeast one-hybrid assay, electrophoretic mobility shift assay, and luciferase assay were used to explore the molecular mechanism of CsaTMD1 in regulating tendril morphogenesis. CsaUFO was the candidate causal gene, and a fragment deletion within promoter impaired CsaUFO expression in tmd1 mutant. A conserved motif 1, which harbored two putative TCP transcription factor binding sites, was located within this deleted fragment. CsaTEN directly bound the motif 1 and positively regulated CsaUFO, and mutation in motif 1 removed this regulation. Our work shows a CsaTEN-CsaUFO module in regulating tendril morphogenesis, indicating that evolution of tendril in cucumber due to simply drive of CsaUFO by CsaTEN in tendril. Additionally, the conserved motif 1 provides a strategy for engineering tendril-less Cucurbitaceae crops.

Modified photoperiod response of CsFT promotes day neutrality and early flowering in cultivated cucumber.

KEY MESSAGE: Map-based cloning and photoperiod response detection suggested that CsFT is the critical gene for cucumber photoperiod domestication. Photoperiod sensitivity is important for sensing seasonal changes and local adaptation. However, day-length sensitivity limits crop geographical adaptation and it should be modified during domestication. Cucumber was domesticated in southern Asia and is currently cultivated worldwide across a wide range of latitudes, but its photoperiod sensitivity and its change during cucumber domestication are unknown. Here, we confirmed wild cucumber (Hardwickii) was a short-day plant, and its flowering depends on short-day (SD) conditions, while the cultivated cucumber (9930) is a day-neutral plant that flowers independently of day length. A photoperiod sensitivity locus (ps-1) was identified by the 9930 × Hardwickii F2 segregating populations, which span a ~ 970 kb region and contain 60 predicted genes. RNA-seq analysis showed that the critical photoperiod pathway gene FLOWERING LOCUS T (CsFT) within the ps-1 locus exhibits differential expression between 9930 and Hardwickii, which was confirmed by qRT-PCR detection. CsFT in Hardwickii was sensitive to day length and could be significantly induced by SD conditions, whereas CsFT was highly expressed in 9930 and was insensitive to day length. Moreover, the role of CsFT in promoting flowering was verified by overexpression of CsFT in Arabidopsis. We also identified the genetic variations existing in the promoter of CsFT among the different geographic cucumbers and suggest they have possible roles in photoperiod domestication. The results of this study suggest that a variation in photoperiod sensitivity of CsFT is associated with day neutrality and early flowering in cultivated cucumber and could contribute to cucumber cultivation in diverse regions throughout the world.

Systematic analysis of the CsmiR396-CsGRFs/CsGIFs module and the opposite role of CsGRF3 and CsGRF5 in regulating cell proliferation in cucumber.

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.