Project description:Cucumber fruit wart composed of spine and tubercule is an important appearance quality trait, which affects product classification and market value of cucumber fruit. Although several key genes for initiation and development of spine and tubercule have been cloned, their underlying mechanisms and relationships have not been well studied. Here, we identified a cucumber basic Helix-Loop-Helix (bHLH) gene CsHEC2 that was strongly expressed in spines and tubercules of cucumber peel. Knockout lines obtained using CRISPR/Cas9 technology were used to explore the biological function of CsHEC2. Compared with the wild type, the Cshec2 mutants resulted in reduced density of wart, and decreased cytokinin accumulation in fruit peel compared to wild type. To comprehensively analyze the regulatory network, RNA sequencing (RNA-seq) experiments were conducted on female buds at 7 days before anthesis (DBA). Transcriptomic data analysis showed that 293 and 1295 genes were up- and down-regulated in Cshec2 mutants relative to WT, respectively. Several sets of genes for cytokinin biosynthesis and metabolism were expressed differently, which explained the decrease of cytokinin in Cshec2 mutants. Our results suggested that CsHEC2 is very likely to regulate the initiation of fruit wart by affecting cytokinin pathway.
Project description:Cucumber (Cucumis sativus L.) is an important vegetable crop bearing fleshy pepo fruits that harvested immature. The fruit length is one of the most important agricultural traits that directly determine the fruit yield and affects fruit quality, but the regulatory mechanism of fruit length variation remains elusive. Here we found a FRUITFULL-like MADS-box gene CsFUL1 functions as a key repressor for fruit length regulation in cucumber. The expression of CsFUL1 is highly enriched in male flowers and fruits, and negatively correlated with fruit length in different cucumber lines. Notably, a key SNP in CsFUL1 was selected during cucumber domestication for long fruit. Ectopic expression of CsFUL1 was unable to rescue the indehiscent fruit phenotype of ful-1 in Arabidopsis. Overexpression of CsFUL1 resulted in increased floral organs and reduced fruit length, whereas knockdown of CsFUL1 led to elongated fruit in cucumber. Transcriptome and biochemical analyses showed that CsFUL1 regulates fruit length through two pathways: one by inhibiting the PIN-FORWED (PIN1/7)-mediated auxin transport and thus downregulates auxin related genes in the fruit, and the other by forming a tetramer with other MADS-box genes to repress the CsSUP-mediated cell division and cell expansion. In addition, we found that CsFUL1 promotes locule number variation through the classical CsWUS-CsCLV pathway. Our findings uncover the regulatory commonality and specificity during development of different fruit types, and provide an important candidate gene to customize fruit length during cucumber breeding.
Project description:To evaluate the role of seeds in fruit quality, we induced parthenocarpy in tomato by regulating ovule-specific auxin synthesis or responsiveness using the INO promoter from A. thaliana, which is expressed in the outer layer of the integuments during early stages of ovule development. We compared these to fruit where the same coding regions were expressed from the DeFH9 promoter which is expressed in carpel tissues during early stages of ovule development. Expression of auxin synthesis or responsiveness genes by both of these promoters produced seedless parthenocarpic tomato fruit. We compared fruit samples using the Affymetrix tomato GeneChip (GPL4741) to determine how gene regulation and expression differed between wild-type and transgenic fruit. Keywords: genetic modification
Project description:Lateral organ development is important for cucumber yield, while the molecular mechanism controlling leaf and floral organ development in cucumber remain elusive. In this report, a novel EMS-mutaginized mutant, round leaf (rl) was distinguished with remarkable round leaf shape, abnormal floral organ and inhibited tendril outgrowth in early development phase. Moreover, the ovule organogenesis disrupted completely in parthenocarpy fruit of rl. MutMap+ analysis revealed that RL encodes a protein kinase PINOID (CsPID, Csa1G537400). A non-synoymous SNP in the second exon of CsPID resulted in an amino-acid substitution from Arg in the wild type to Lys in the rl mutant. CsPID was down-regulated in rl mutant and preferentially expressed in young leaf, and flower buds. IAA quantification showed that rl plants exhibited a lower IAA content than wild type in ovary and blade edge. IAA immunolocalization results confirmed the IAA content alteration in rl plants. Transcriptome profile analysis further suggested IAA biosynthesis, polar transport and signal transduction genes participated in the leaf and floral development process by CsPID. Biochemical analyses showed that CsPID may regulate leaf shape by interacting with CsREV. In conclusion, this study revealed that the extensive genetic architecture of lateral organ organogenesis and development via CsPID regulating auxin polar transport action in cucumber.
Project description:To evaluate the role of seeds in fruit quality, we induced parthenocarpy in tomato by regulating ovule-specific auxin synthesis or responsiveness using the INO promoter from A. thaliana, which is expressed in the outer layer of the integuments during early stages of ovule development. We compared these to fruit where the same coding regions were expressed from the DeFH9 promoter which is expressed in carpel tissues during early stages of ovule development. Expression of auxin synthesis or responsiveness genes by both of these promoters produced seedless parthenocarpic tomato fruit. We compared fruit samples using the Affymetrix tomato GeneChip (GPL4741) to determine how gene regulation and expression differed between wild-type and transgenic fruit. Experiment Overall Design: Wild-type fruit with seeds was compared with transgenic lines INO-IaaM, DefH9-IaaM, INO-RolB, and DefH9-RolB. To find genes with seed-specific expression, we also compared the control with wild-type fruit from which seeds had been manually removed. We had three biological replicates for each treatment and control except DefH9-RolB, for which only two replicates were available. Each CEL file from the microarray represents one plant from each line.
Project description:To identify the differentially expressed genes(DEGs) in the ventral and dorsal of cucumber bending fruits,cucumber bending fruit mRNA profiles of 2-day-old ventral and dorsal were generated by deep sequencing. After removing the low quality reads, the total number of clean reads in two library were 27.07 million and16.52 million, accounted for 85.21% and 80.71% of total reads .We identified a total of 4313 sequences differentially expressed in ventral and dorsal of bending fruit with a 2-fold or greater change and P < 0.01 , the Dorsal (D2) served as a control, in which 2351 up-regulated genes and 1962 down-regulated genes.For up-regulated genes, protein kinase activity, ethylene mediated signaling pathway, regulation of cell shape, auxin polar transport were significantly enriched, whereas down-regulated genes, the functional classes photosynthesis, oxidation reduction, response to auxin stimulus were significantly enriched. Moreover, among DEGs related to ethylene, we identified an ERF/AP2 gene CsERF025 that was significant difference in the dorsal and ventral of cucumber. up-regulation of CsERF025 in cucumber promoted fruit bendng and Increased the fruit bending angle, suggesting that CsERF025 plays an important role in cucumber bending fruit.
Project description:Fruit length is a key domestication trait that affects crop yield and appearance quality. Cucumber fruits vary from 5~60 cm in length. Despite multiple fruit length QTLs have been identified, the underlying genes and regulatory mechanisms are poorly understood. Map-based cloning identified a nonsynonymous SNP (G to A) in CRABS CLAW (CsCRC) confers the major effect fruit length QTL FS5.2. CsCRCA is a rare allele only exist in Xishuangbanna cucumber with round fruits. Construction of near-isogenic line (NIL) of CsCRCA led to 34~39% reduction in fruit length. Introduction of CsCRCG into the NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in reduced fruit length and decreased cell size. RNA-seq results showed that an auxin responsive protein CsARP1 expressed decreased in CsCRC-RNAi lines. Further, an auxin responsive protein Further, CsARP1 is the downstream target gene of CsCRCG, instead of CsCRCA. Knockout of CsARP1 produced decreased fruit length with smaller cells. Hence, our work suggested that CsCRCG positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhanced cell expansion. Utilization of CsCRC alleles provides a new strategy to manipulate fruit length in cucumber breeding.
Project description:Cucumber (Cucumis sativus L.) fruit is a type of fleshy fruit that is harvested immaturely. Early fruit development directly determines the final fruit length and diameter, and consequently the fruit yield and quality. Different cucumber varieties display huge variations of fruit length, but how fruit length is determined at the molecular level remains poorly understood. To understand the genes and gene networks that regulate fruit length in cucumber, high throughout RNA-seq data were used to compare the transcriptomes of early fruit from two near isogenic lines with different fruit lengths. 3955 genes were found to be differentially expressed, among which 2368 genes were significantly up-regulated and 1587 down-regulated in the line with long fruit. Microtubule and cell cycle related genes were dramatically activated in the long fruit, and transcription factors were implicated in the fruit length regulation in cucumber. Thus, our results built a foundation to dissect the molecular mechanism of fruit length control in cucumber, a key agricultural trait of significant economic importance.
Project description:The carpel number (CN) is an important fruit trait affecting fruit shape, size, and internal quality in cucumber. CsCLAVATA3 (CsCLV3) was previously showed to be the simply inherited gene responsible for carpel number variation in cucumber, but the molecular mechanism of CsCLV3 regulating carpel number remains elusive. Here, we found that the expression of CsCLV3 was negatively correlated with carpel number variation in different cucumber lines. Knock down of CsCLV3 by RNAi led to increased number of petals and carpels, suggesting that CsCLV3 functions as a negative regulator for floral organ number in cucumber. WUSCHEL (WUS) has been well characterized to promote CLV3-expressing stem cell activity in a non-cell autonomous manner to regulate meristem maintenance and floral organ number. However, here we found the expression region of CsCLV3 overlaps with CsWUS in the basal domain of meristem, and CsCLV3 interact with CsWUS at the protein level through binding to the WUS-box motif. Overexpression of CsFUL1, a FRUITFULL-like MADS-box gene involved in fruit length regulation, resulted in increased number of floral organs in cucumber. Biochemical analyses indicated that CsFUL1 can directly bind to CsWUS promoter to stimulate its expression. Further, we found that auxin participates in carpel number variation in cucumber through physical interaction of AUXIN RESPONSE FACTOR 14 (CsARF14) and CsWUS. Therefore, CsFUL1 and CsARF14 are two new players in the WUS-CLV pathway in determining carpel number variation in cucumber.
Project description:Cucumber (Cucumis sativus L.) fruit is a type of fleshy fruit that is harvested immaturely. Early fruit development directly determines the final fruit length and diameter, and consequently the fruit yield and quality. Different cucumber varieties display huge variations of fruit length, but how fruit length is determined at the molecular level remains poorly understood. To understand the genes and gene networks that regulate fruit length in cucumber, high throughout RNA-seq data were used to compare the transcriptomes of early fruit from two near isogenic lines with different fruit lengths. 3955 genes were found to be differentially expressed, among which 2368 genes were significantly up-regulated and 1587 down-regulated in the line with long fruit. Microtubule and cell cycle related genes were dramatically activated in the long fruit, and transcription factors were implicated in the fruit length regulation in cucumber. Thus, our results built a foundation to dissect the molecular mechanism of fruit length control in cucumber, a key agricultural trait of significant economic importance. Comparative analysis of fruit from two near-isogenic lines, 408 (long fruit) and 409 (short fruit), was employed to discover genes and networks that regulate the fruit length. Two biological replicates were used from each line.