Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRC^A is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRC^A exhibited a 34∼39% reduction in fruit length. Introducing CsCRC^G into this NIL rescued the short-fruit phenotype, and knockdown of CsCRC^G resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRC^G expression was positively correlated with fruit length. Further, CsCRC^G, but not CsCRC^A, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRC^G positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.

WUSCHEL-related homeobox (WOX) transcription factors are plant-specific members that characterized by the presence of a homeodomain. It has been shown that WOX members regulate several aspects of plant development, but the biological functions of this CsWOX gene family remain largely unknown in cucumber (Cucumis sativus L.). In this study, we identified and characterized 11 putative CsWOX genes in cucumber, which are also divided into three major clades (e.g., the Ancient clade, the Intermediate clade and the WUS clade). Expression pattern analysis revealed tissue-specific expression patterns of CsWOX genes, including that CsWOX9 is mainly expressed in developing fruit and also has lower expression in tip and axillary bud, which was further confirmed by in situ hybridization assay. Moreover, overexpression of CsWOX9 in Arabidopsis led to increased branches and rosette leaves, and shorter siliques. Together, these results indicated that CsWOX members may regulate cucumber growth and development.

Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16–CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.

Fruit glossiness is an important external fruit quality trait that greatly affects the marketability of fresh cucumber (Cucumis sativus) fruits. A few reports have suggested that the extent of cuticular wax loading influences the glossiness of the fruit surface. In the present study, we tested the wax contents of two inbred cucumber lines, comparing a line with waxy fruit (3401) and a line with glossy fruit (3413). Wax content analysis and dewaxing analysis demonstrate that fruit cuticular wax loads negatively correlate with fruit glossiness in cucumber. Identifying genes that were differentially expressed in fruit pericarps between 3401 and 3413 and genes induced by abscisic acid suggested that the wax biosynthesis gene CsCER6 (Cucumis sativus ECERIFERUM 6) and the regulatory gene CsCER7 may affect wax accumulation on cucumber fruit. Expression analysis via RT-qPCR, GUS-staining, and in situ hybridization revealed that CsCER6 and CsCER7 are abundantly expressed in the epidermis cells in cucumber fruits. Furthermore, the overexpression and RNAi lines of CsCER6 and CsCER7 showed dramatic effects on fruit cuticular wax contents, fruit glossiness, and cuticle permeability. Our results suggest that CsCER6 and CsCER7 positively regulate fruit cuticular wax accumulation and negatively influence fruit glossiness.

The leaf angle is an important factor determining plant shoot architecture that may boost crop yield by increasing photosynthetic efficiency and facilitating high-density planting. Auxin is an important phytohormone involved in leaf angle regulation. Here, we identified two Single-Nucleotide Polymorphisms (SNPs) in the Indoleacetic Acid (IAA) glucosyltransferase gene CsIAGLU in 80 re-sequenced cucumber lines, of which the CsIAGLU^717G,1234T is the dominant allele associated with a small leaf pedicle angle (LPA), whereas CsIAGLU^717C,1234A is linked with a large LPA. CsIAGLU was highly expressed in leaves and petioles. In natural cucumber populations, the expression of CsIAGLU was negatively correlated with the LPA. The mutation of CsIAGLU induced by the CRISPR-Cas9 system resulted in elevated free IAA levels and enlarged cell expansion on the adaxial side of the petiole base, thus producing a greater LPA. Consistently, exogenous IAA treatment led to increased LPA and cell size. Therefore, our findings suggest that CsIAGLU functions as a negative regulator of LPA development via auxin-mediated cell expansion in cucumber, providing a valuable strategy for cucumber breeding with small LPAs.