The determination of sex morphs and the optimization of fruit shape are key challenges in cucumber cultivation and breeding, with ethylene recognized as a crucial regulatory factor in both processes. Ethylene is synthesized through the coordinated actions of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO). While previous studies have highlighted the significant role of CsACSs in cucumber sex determination, investigations on CsACOs remain limited. In this study, we generated mutants of all members of the ACO gene family involved in ethylene synthesis during cucumber flower development and systematically analyzed their functions. Our results demonstrate that CsACO2 plays a dual role: it is involved in the early induction of carpel initiation during flower development and subsequently regulates ovary elongation. Mutations in CsACO2 affect ovary elongation, as evidenced by the shorter ovaries of female flowers in the cswip1/csaco2 double mutants. In developing female buds, after carpel initiation, we identified CsACO3 as the core regulatory gene responsible for stamen arrest, in cooperation with either CsACO2 or CsACO4. Notably, the cswip1/csaco2/csaco3 triple mutants produce hermaphrodite flowers with shorter ovaries. In contrast, the cswip1/csaco3/csaco4 triple mutants exhibit hermaphrodite flowers with normal ovaries and fruit shapes, and more importantly, their fruit-setting ability is comparable to that of the subgynoecious control. Our results enrich the ethylene regulatory network of cucumber female/hermaphrodite flower development and fruit shape, and provide an optional breeding pathway for high-yield varieties.

Sex determination underpins genesis of male and female flowers with particularly important implications in plant breeding. Auxin and ethylene regulate femaleness in cucurbits. In this work, we identified an auxin response factor 3 (CsARF3) that plays an essential role in carpel development in cucumber. Deletion of CsARF3 resulted in an androecious phenotype with only male flowers, whereas overexpression of CsARF3 led to an increased number of female flowers. CsARF3 promotes femaleness by directly stimulating the expression of the meristem maintenance gene CsSTM and repressing the activity of the gynoecious gene CsWIP1. Auxin and ethylene exhibit a reciprocal relationship during sex determination, in which ethylene promotes carpel formation through auxin at the early stage of flower development. The auxin signaling in carpels then enhances ethylene biosynthesis to inhibit stamen development.

WUSCHEL-related homeobox 1 (WOX1), categorized into the WUS clade, has been determined as the essential gene for leaf expansion as well as promoting leaf outgrowth along the mediolateral axis. The phenotypic characteristics of transgenic cucumber plants with CsWOX1 overexpression (CsWOX1-OE) and mutants (designated as mango fruit, mf) have been reported. Specifically, CsWOX1-OE cucumber plants exhibit leaves with a distinctive 'butterfly-shaped' appearance, while mf mutants show a rapid reduction in the area of their leaf tips. In this study, RNA-Seq was employed to sequence the transcriptome of CsWOX1-OE to reveal its differentially expressed genes (DEGs). Combined with the transcriptome sequencing data of the mf mutant, GO classification, and KEGG pathway enrichment analyses were performed, revealing new links between the WOX1 protein and many core plant regulatory pathways. The further joint analysis identified 80 common DEGs between CsWOX1-OE and mf, with a significant proportion of these common DEGs being annotated to the circadian pathway and the auxin polarity transport pathway. Six genes (CsGI, CsFKF1, CsRVE6, CsRVE8, CsABCB, and CsPin1At) were selected for Y1H and Dual-LUC interaction validation experiments. These experiments demonstrated that CsWOX1 directly targets the promoters of CsRVE6, which regulates leaf expansion, and CsPin1At, which is involved in auxin polarity transport, thereby promoting their transcription. In conclusion, these findings provide a foundation for exploring the potential regulatory mechanisms associated with CsWOX1, expecting to contribute to the construction of a more comprehensive gene network for leaf morphogenesis.

Plant height is a critical agronomic trait that affects crop yield, plant architecture, and environmental adaptability. Gibberellins (GAs) regulate plant height, with DELLA proteins acting as key repressors in the GA signaling pathway by inhibiting GA-induced growth. While DELLA phosphorylation is essential for regulating plant height, the precise mechanisms underlying this process remain incompletely understood. In this study, we identified a cucumber mutant with delayed growth, which exhibited reduced sensitivity to GA treatment. Through bulked segregant analysis (BSA-seq) combined with molecular marker linkage analysis, we successfully identified and cloned the gene responsible for the dwarf phenotype, CsIREH1 (INCOMPLETE ROOT HAIR ELONGATION 1), which encodes an AGC protein kinase. Further research revealed that CsIREH1 interacts with and phosphorylates DELLA proteins, specifically targeting CsGAIP and CsGAI2. We propose that IREH1-dependent phosphorylation of DELLA proteins prevents their excessive accumulation, thereby maintaining normal plant growth. Therefore, investigating the role of IREH1-mediated DELLA phosphorylation provides valuable insights and theoretical foundations for understanding how plants regulate growth mechanisms.

Cucumber (Cucumis sativus, Cs) tendrils are slender vegetative organs that typically require manual removal to ensure orderly growth during greenhouse cultivation. Here, we identified cucumber tendril-less (tl), a Tnt1 retrotransposon-induced insertion mutant lacking tendrils. Map-based cloning identified the mutated gene, CsaV3_3G003590, which we designated as CsTL, which is homologous to Arabidopsis thaliana LATERAL SUPPRESSOR (AtLAS). Knocking out CsTL repressed tendril formation but did not affect branch initiation, whereas overexpression (OE) of CsTL resulted in the formation of two or more tendrils in one leaf axil. Although expression of two cucumber genes regulating tendril formation, Tendril (CsTEN) and Unusual Floral Organs (CsUFO), was significantly decreased in CsTL knockout lines, these two genes were not direct downstream targets of CsTL. Instead, CsTL physically interacted with CsTEN, an interaction that further enhanced CsTEN-mediated expression of CsUFO. In Arabidopsis, the CsTL homolog AtLAS acts upstream of REVOLUTA (REV) to regulate branch initiation. Knocking out cucumber CsREV inhibited branch formation without affecting tendril initiation. Furthermore, genomic regions containing CsTL and AtLAS were not syntenic between the cucumber and Arabidopsis genomes, whereas REV orthologs were found on a shared syntenic block. Our results revealed not only that cucumber CsTL possesses a divergent function in promoting tendril formation but also that CsREV retains its conserved function in shoot branching.

Dwarf or semidwarf plant structures are well suited for intensive farming, maximizing yield, and minimizing labor costs. Watermelon (Citrullus lanatus) is classified as an annual vine plant with elongated internodes, yet the mechanism governing watermelon dwarfing remains unclear. In this study, a compact watermelon mutant dwarf, induced by the insertion of transferred DNA (T-DNA), was discovered. Through resequencing, a gene named domain of unknown function 21 (ClDUF21), located downstream of the T-DNA insertion site, was identified as the candidate gene for the dwarf mutant, and its functionality was subsequently confirmed. Watermelon mutants generated through CRISPR/Cas9-mediated knockout of ClDUF21 revealed that homozygous mutants displayed a pronounced dwarfing phenotype, and protein-protein interaction analysis confirmed the direct interaction between ClDUF21 and ClDWF1. Subsequently, we employed CRISPR/Cas9 technology to precisely modify the homologous gene CsDUF21 in cucumber (Cucumis sativus) and performed protein interaction validation between CsDUF21 and CsDWF1, thereby demonstrating that the CsDUF21 gene also exhibits analogous functionality in plant dwarfing. These findings demonstrate that ClDUF21 governs plant dwarfism by modulating the brassinosteroid synthesis pathway via ClDWF1.

Nuclear factor-Y (NF-Y) transcription factors NF-YC2/-YC9 targeting CsTIC21 involves in light-induced chloroplast photomorphogenesis in cucumber. Light signals promote photomorphogenesis and photosynthesis, allowing plants to establish photoautotrophic growth. Chloroplasts are organelles responsible for photosynthesis in which light energy is converted into chemical energy and stored as organic matter. However, how light regulates chloroplast photomorphogenesis remains unclear. Here, we isolated a cucumber (Cucumis sativus L.) mutant albino seedling (as) from an ethyl methane sulfonate mutagenesis library with an albino phenotype. Map-based cloning revealed that the mutation occurred in a component of cucumber translocon at the inner membrane of chloroplasts (CsTIC21). Subsequently, virus-induced gene silencing and CRISPR/Cas9 analyses confirmed the association between the mutant gene and the as phenotype. Loss-of-function of CsTIC21 induces malformation of chloroplast formation, leading to albinism and death in cucumber. Notably, CsTIC21 transcription was very low in etiolated seedlings grown in the dark and was upregulated by light, with expression patterns similar to those of Nuclear factor-YC (NF-YC) genes. Here, 7 cucumber NF-YC family genes (CsNF-YC) were identified, among which the expression of 4 genes (CsNF-YC1, -YC2, -YC9, and -YC13) responded to light. Gene silencing of all CsNF-YC genes in cucumber indicated that CsNF-YC2, -YC9, -YC11-1, and -YC11-2 induced distinct etiolated growth and decreased chlorophyll content. Interaction studies verified that CsNF-YC2 and CsNF-YC9 target the CsTIC21 promoter directly and promote gene transcription. These findings provide mechanistic insights on the role of the NF-YCs-TIC21 module in chloroplast photomorphogenesis promoted by light in cucumber.

The lateral organs of watermelon (Citrullus lanatus), including lobed leaves, branches, flowers, and tendrils, together determine plant architecture and yield. However, the genetic controls underlying lateral organ initiation and morphogenesis remain unclear. Here, we found that knocking out the homologous gene of shoot branching regulator LATERAL SUPPRESSOR in watermelon (ClLs) repressed the initiation of branches, flowers, and tendrils and led to developing round leaves, indicating that ClLs undergoes functional expansion compared with its homologs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum). Using ClLs as the bait to screen against the cDNA library of watermelon, we identified several ClLs-interacting candidate proteins, including TENDRIL (ClTEN), PINOID (ClPID), and APETALA1 (ClAP1). Protein-protein interaction assays further demonstrated that ClLs could directly interact with ClTEN, ClPID, and ClAP1. The mRNA in situ hybridization assay revealed that the transcriptional patterns of ClLs overlapped with those of ClTEN, ClPID, and ClAP1 in the axillary meristems and leaf primordia. Mutants of ClTEN, ClPID, and ClAP1 generated by the CRISPR/Cas9 gene editing system lacked tendrils, developed round leaves, and displayed floral diapause, respectively, and all these phenotypes could be observed in ClLs knockout lines. Our findings indicate that ClLs acts as lateral organ identity protein by forming complexes with ClTEN, ClPID, and ClAP1, providing several gene targets for transforming the architecture of watermelon.

The presence or absence of spines is an important economic trait of cucumber fruit. Spines are believed to be a type of specialized trichome on the fruit surface, and all the identified cucumber trichome-less mutants lack fruit spines. However, genes that specifically regulate fruit spine initiation remain to be identified. Here, we found that knocking out cucumber TARGET OF EAT3 homolog (CsTOE3), belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, affected flower development and, more interestingly, inhibited cucumber fruit spine initiation. On analyzing expression patterns by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization assay, CsTOE3 was found to be highly expressed in male and female flowers, and its mRNA accumulated in the tips of sepal and petal primordia and in the cells of fruit spines and peels. Biochemical analyses indicated that CsTOE3 directly interacts with GLABRA1 (CsGL1) and TRANSPARENT TESTA GLABRA1 (CsTTG1), which are positive regulators of trichome formation. In addition, RNA-seq showed that the transcription levels of eight ERFs were significantly upregulated in CsTOE3 knockout lines. Phytohormone content analysis also revealed a significant increase in the amount of ethylene released by CsTOE3 knockout line, and treatment with the ethylene synthesis inhibitor aminoethoxyvinyl-glycine partly restored the spineless phenotype. Our results suggest that CsTOE3 specifically regulates fruit spine initiation but does not affect the formation of trichomes on other organs in cucumber. Our findings may have a far-reaching significance for cucumber germplasm improvement and quality breeding using fruit spines as the target trait.

To date, the genetic transformation system of watermelon has remained inefficient. In this study, the genetic transformation system of watermelon mediated by Agrobacterium tumefaciens was optimized, including different seedling ages, strains of Agrobacterium tumefaciens, concentrations of acetosyringone infected solution, co-culture time, selection pressure of antibiotics timentin and glufosinate, and concentrations of hormones 6-benzylaminopurine, indoleacetic acid and naphthylacetic acid in the corresponding culture medium. Our results suggested that cotyledons would be used as explants, disinfected with 6% sodium hypochlorite for 12 min, cultured for 3 d, and then infected with Agrobacterium inoculum (Agrobacterium EHA105) containing 200 µM acetosyringone and 0.02 of final OD600. The explants differentiated into adventitious shoots in the medium with 1.5 mg/L 6-benzylaminopurine and 200 µM timentin. Positive adventitious shoots were obtained through further screening by 1.4 mg/L herbicide glufosinate-ammonium, and were induced by 0.1 mg/L naphthalene acetic acid into independent plants. Our system improves the genetic transformation efficiency of watermelon and provides a technical basis for continuous acquisition of watermelon transgenic plants.

Wax gourd is one of the most important winter vegetables of the Cucurbitaceae family. There are only limited markers available for this crop and the draft genome of wax gourd provides a powerful tool for simple sequence repeats (SSR) marker development. In this study, we developed genome-wide SSR markers from wax gourd genome and characterized their distribution and frequency of different motifs and repeats. A total of 52,431 microsatellites from wax gourd genome were identified, of which 39,319 SSR markers were developed. The 1152 non-wax gourd SSR markers were selected from cucumber, melon, watermelon and pumpkin to test their transferability in wax gourd. The 580 SSR markers could be transferable in wax gourd, and 42 of them were detected with polymorphic in 11 tested accessions of wax gourd. In addition, 11 good polymorphic transferrable SSR markers and 21 SSR markers of wax gourd were selected to investigate the genetic diversity and population structure of 129 wax gourd accessions. One hundred twelve alleles were detected by these 32 SSR markers. The result of population structure showed that the 129 wax gourd accessions were divided into two main populations, and the genetic diversity analysis separated them into two clusters. The large number of wax gourd SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in wax gourd.

Cucumber (Cucumis sativus L.) has been a model for the study of sex differentiation over the last two decades. Cucumber sex differentiation is mainly under genetic control, but plant growth regulators can also influence or even change it. However, the effect of exogenous auxin application on cucumber sex differentiation is not well understood at the physiological level. In this study, we explored the effects of different exogenous auxin concentrations on cucumber varieties with different mutant sex-controlling genotypes and found that there was a dosage effect of exogenous indole-3-acetic acid (IAA) on the enhancement of cucumber femaleness. Several ACC synthetase (ACS) family members responded directly to exogenous IAA, increasing endogenous ethylene synthesis, and this process appeared to be independent of the previously identified sex-related ACC oxidase CsACO2. We further demonstrated that ENHANCER OF SHOOT REGENERATION 2 (ESR2) responded to exogenous auxin induction by binding to ERE cis-acting element regions in the CsACS2 promoter, directly activating CsACS2 expression and thus increasing endogenous ethylene content, which may induce femaleness. These findings reveal that exogenous auxin increases cucumber femaleness by inducing a sex-controlling gene and promoting ethylene synthesis.

50 Cucumber ( Cucumis sativus L.) is a model for the study of sex differentiation in the last 51 two decades. In cucumber, sex differentiation is mainly controlled by genetic material, 52 but plant growth regulators can also influence or even change it. However, the effect of 53 exogenous auxin application on cucumber sex differentiation is mostly limited in 54 physiological level. In this study, we explored the effects of different exogenous auxin 55 concentrations on the varieties with different mutant sex-controlling genotypes and 56 found that there was a dosage effect of exogenous indole-3-acetic acid (IAA) on the 57 enhancement of cucumber femaleness. Several ACC synthetase (ACS) family members 58 could directly respond to the induction of exogenous IAA to improve endogenous 59 ethylene synthesis, and this process can be independent on the previously identified 60 sex-related ACC oxidase CsACO2. We further demonstrated that ENHANCER OF 61 SHOOT REGENERATION 2 (ESR2), responding to the induction of exogenous auxin, 62 could directly activate CsACS2 expression by combining the ERE cis -acting element 63 regions in the promoter, and then increase endogenous ethylene content, which may 64 induce femaleness. These findings reveal that exogenous auxin improves cucumber 65 femaleness via inducing sex-controlling gene and promoting ethylene synthesis.

In triploid watermelon (Citrullus lanatus), the homologous chromosomes of germ cells are disorder during meiosis, resulting in the failure of seeds formation and producing seedless fruit. Therefore, mutating the genes specifically functioning in meiosis may be an alternative way to achieve seedless watermelon. REC8, as a key component of the cohesin complex in meiosis, is dramatically essential for sister chromatid cohesion and chromosome segregation. However, the role of REC8 in meiosis has not yet been characterized in watermelon. Here, we identified ClREC8 as a member of RAD21/REC8 family with a high expression in male and female flowers of watermelon. In situ hybridization analysis showed that ClREC8 was highly expressed at the early stage of meiosis during pollen formation. Knocking out ClREC8 in watermelon led to decline of pollen vitality. After pollinating with foreign normal pollen, the ovaries of ClREC8 knockout lines could inflate normally but failed to form seeds. We further compared the meiosis chromosomes of pollen mother cells in different stages between the knockout lines and the corresponding wild type. The results indicated that ClREC8 was required for the monopolar orientation of the sister kinetochores in Meiosis I. Additionally, transcriptome sequencing (RNA-seq) analysis between WT and the knockout lines revealed that the disruption of ClREC8 caused the expression levels of mitosis-related genes and meiosis-related genes to decrease. Our results demonstrated ClREC8 has a specific role in Meiosis I of watermelon germ cells, and loss-of-function of the ClREC8 led to seedless fruit, which may provide an alternative strategy to breed cultivars with seedless watermelon.

Background: Wax gourd ( Benincasa hispida Cong., 2n=2x=24) is one of the most important winter vegetables of the Cucurbitaceae family. There are only limited markers available for this crop and the draft genome of wax gourd provides a powerful tool for SSR marker development. Results: In this study, we developed genome-wide SSR markers from wax gourd genome and characterized their distribution and frequency of different motifs and repeats. A total of 52,431 microsatellites from wax gourd genome were identified, of which 39,319 SSR markers were developed. 1,152 non-wax gourd SSR markers were selected from cucumber, melon, watermelon and pumpkin to test their transferability in wax gourd. 580 SSR markers could be transferable in wax gourd, and 42 of them were detected with polymorphic in 11 tested accessions of wax gourd. In addition, 11 good polymorphic transferrable SSR markers and 21 SSR markers of wax gourd were selected to investigate the genetic diversity and population structure of 129 wax gourd accessions. 112 alleles were detected by these 32 SSR markers. The result of population structure showed that the 129 wax gourd accessions were divided into two main populations, and the genetic diversity analysis separated them into two clusters. Conclusions: The large number of wax gourd SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in wax gourd.

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes, as plant-specific transcription factors, play vital roles in flower pattern, leaf development and plant architecture. Our recent study shows that the TCP gene BRANCHED1 (CsBRC1) specifically regulates shoot branching in cucumber. Here, we found CsBRC1 had a closely related paralogous gene CsBRC1-like. The synteny analysis revealed that these two genes originated from a segmental duplication. CsBRC1-like displayed different expression patterns in cucumber compared with CsBRC1, indicating that they may have functional differentiation. Ectopic expression of CsBRC1-like in Arabidopsis brc1 1 mutant resulted in reduced rosette branches and rosette leaves, whereas silencing CsBRC1-like in cucumber only led to a deformed true leaf of seedling rather than affecting the shoot branching. RNA-seq analysis of wild-type and CsBRC1-like-RNAi plants implicated that CsBRC1-like might regulate early leaf development through affecting the transcripts of auxin and cytokinin related genes in cucumber. Moreover, CsBRC1-like directly interacts with CsTCP10a and CsBRC1 in vivo. Our results demonstrated that CsBRC1-like has a specific role in regulating leaf development, and CsBRC1-like and CsBRC1 may have overlapping roles in shoot branching.

The fruit trichomes of Cucurbitaceae are widely desired in many Asian countries and have been a key determinant of cucumber (Cucumis sativus L.) cultivar selection for commercial production and breeding. However, our understanding of the initiation and development of cucumber trichomes is still limited. Here, we found that the cucumber TINY BRANCHED HAIR (TBH) gene is preferentially expressed in multicellular trichomes. Overexpression of CsTBH in tbh mutants restored the trichome phenotype and increased the percentage of female flowers, whereas silencing of CsTBH in wild-type plants resulted in stunted trichomes with a lower rate of female flowers. Furthermore, we provide evidence that CsTBH can directly bind to the promoters of cucumber 1-Aminocyclopropane-1-Carboxylate Synthase (CsACS) genes and regulate their expression, which affects multicellular trichome development, ethylene accumulation, and sex expression. Two cucumber acs mutants with different trichome morphology and sex morphs compared with their near-isogenic line further support our findings. Collectively, our study provides new information on the molecular mechanism of CsTBH in regulating multicellular trichome development and sex expression through an ethylene pathway.

The WUSCHEL-CLAVATA3 pathway genes play an essential role in shoot apical meristem maintenance and floral organ development, and under intense selection during crop domestication. The carpel number is an important fruit trait that affects fruit shape, size and internal quality in cucumber, but the molecular mechanism remains elusive. Here, we found that CsCLV3 expression was negatively correlated with carpel number in cucumber cultivars. CsCLV3-RNAi led to increased number of petals and carpels, whereas overexpression of CsWUS resulted in more sepals, petals and carpels, suggesting that CsCLV3 and CsWUS function as a negative and a positive regulator for carpel number variation, respectively. Biochemical analyses indicated that CsWUS directly bound to the promoter of CsCLV3 and activated its expression. Overexpression of CsFUL1A , a FRUITFULL-like MADS-box gene, resulted in more petals and carpels. CsFUL1A can directly bind to the CsWUS promoter to stimulate its expression. Furthermore, we found that auxin participated in carpel number variation in cucumber through interaction of CsARF14 with CsWUS. Therefore, we have identified a gene regulatory pathway involving CsCLV3, CsWUS, CsFUL1A and CsARF14 in determining carpel number variation in an important vegetable crop - cucumber.

Cucumber is an important vegetable crop bearing fleshy pepo fruit harvested immature. Fruits left unpicked in time during summer production, as well as unfavorable environmental conditions during post-harvest shelf, will cause cucumber fruits to turn yellow and ripen, and thus impair the market value. Identification of maturity-related genes is of great agricultural and economic importance for cucumber production. Here, we isolated and characterized a MADS-box gene, Cucumis sativus SHATTERPROOF (CsSHP) in cucumber. Expression analysis indicated that CsSHP was specifically enriched in reproductive organs including stamens and carpels. Ectopic expression of CsSHP was unable to rescue the indehiscence silique phenotype of shp1 shp2 mutant plant in Arabidopsis. Instead, overexpression of CsSHP resulted in early flowering, precocious phenotypes and capelloid organs in wild-type Arabidopsis. Biochemical analysis indicated that CsSHP directly interacted with cucumber SEPALLATA (SEP) proteins. CsSHP expression increased significantly during the yellowing stage of cucumber ripening, and was induced by exogenous application of abscisic acid (ABA). Therefore, CsSHP may participate in fruit maturation through the ABA pathway and floral organ specification via interaction with CsSEPs to form protein complex in cucumber.

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family gene BRANCHED1 (BRC1) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 (CsBRC1) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED (CsPIN3) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.