Responses of differential metabolites and pathways to high temperature in cucumber anther.

Cucumber is one of the most important vegetable crops, which is widely planted all over the world. Cucumber always suffers from high-temperature stress in South China in summer. In this study, liquid chromatography-mass spectrometry (LC-MS) analysis was used to study the differential metabolites of cucumber anther between high-temperature (HT) stress and normal condition (CK). After HT, the pollen fertility was significantly reduced, and abnormal anther structures were observed by the paraffin section. In addition, the metabolomics analysis results showed that a total of 125 differential metabolites were identified after HT, consisting of 99 significantly upregulated and 26 significantly downregulated metabolites. Among these differential metabolites, a total of 26 related metabolic pathways were found, and four pathways showed significant differences, namely, porphyrin and chlorophyll metabolism; plant hormone signal transduction; amino sugar and nucleotide sugar metabolism; and glycine, serine, and threonine metabolism. In addition, pollen fertility was decreased by altering the metabolites of plant hormone signal transduction and amino acid and sugar metabolism pathway under HT. These results provide a comprehensive understanding of the metabolic changes in cucumber anther under HT.

Cytological, genetic and transcriptomic characterization of a cucumber albino mutant.

Photosynthesis, a fundamental process for plant growth and development, is dependent on chloroplast formation and chlorophyll synthesis. Severe disruption of chloroplast structure results in albinism of higher plants. In the present study, we report a cucumber albino alc mutant that presented white cotyledons under normal light conditions and was unable to produce first true leaf. Meanwhile, alc mutant could grow creamy green cotyledons under dim light conditions but died after exposure to normal light irradiation. No chlorophyll and carotenoid were detected in the alc mutant grown under normal light conditions. Using transmission electron microscopy, impaired chloroplasts were observed in this mutant. The genetic analysis indicated that the albino phenotype was recessively controlled by a single locus. Comparative transcriptomic analysis between the alc mutant and wild type revealed that genes involved in chlorophyll metabolism and the methylerythritol 4-phosphate pathway were affected in the alc mutant. In addition, three genes involved in chloroplast development, including two FtsH genes and one PPR gene, were found to have negligible expression in this mutant. The quality of RNA sequencing results was further confirmed by real-time quantitative PCR analysis. We also examined 12 homologous genes from alc mutant in other plant species, but no genetic variation in the coding sequences of these genes was found between alc mutant and wild type. Taken together, we characterized a cucumber albino mutant with albinism phenotype caused by chloroplast development deficiency and this mutant can pave way for future studies on plastid development.

Phenotypic Characteristics and Transcriptome of Cucumber Male Flower Development Under Heat Stress.

Cucumber (Cucumis sativus L.) is an important vegetable crop, which is thermophilic not heat resistant. High-temperature stress always results in sterility at reproductive stage. In the present study, we evaluate the male flower developmental changes under normal (CK) and heat stress (HS) condition. After HS, the activities of peroxidase (POD) and superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) were increased. In addition, the pollen fertility was significantly decreased; and abnormal tapetum and microspore were observed by paraffin section. Transcriptome analysis results presented that total of 5828 differentially expressed genes (DEGs) were identified after HS. Among these DEGs, 20 DEGs were found at four stages, including DNA binding transcription factor, glycosyltransferase, and wound-responsive family protein. The gene ontology term of carbohydrate metabolic process was significantly enriched in all anther stages, and many saccharides and starch synthase-related genes, such as invertase, sucrose synthase, and starch branching enzyme, were significantly different expressed in HS compared with CK. Furthermore, co-expression network analysis showed a module (midnightblue) strongly consistent with HS, and two hub genes (CsaV3_6G004180 and CsaV3_5G034860) were found with a high degree of connectivity to other genes. Our results provide comprehensive understandings on male flower development in cucumber under HS.

Metabolome and transcriptome analyses reveal chlorophyll and anthocyanin metabolism pathway associated with cucumber fruit skin color.

BACKGROUND: Fruit skin color play important role in commercial value of cucumber, which is mainly determined by the content and composition of chlorophyll and anthocyanins. Therefore, understanding the related genes and metabolomics involved in composition of fruit skin color is essential for cucumber quality and commodity value. RESULTS: The results showed that chlorophyll a, chlorophyll b and carotenoid content in fruit skin were higher in Lv (dark green skin) than Bai (light green skin) on fruit skin. Cytological observation showed more chloroplast existed in fruit skin cells of Lv. A total of 162 significantly different metabolites were found between the fruit skin of the two genotypes by metabolome analysis, including 40 flavones, 9 flavanones, 8 flavonols, 6 anthocyanins, and other compounds. Crucial anthocyanins and flavonols for fruit skin color, were detected significantly decreased in fruit skin of Bai compared with Lv. By RNA-seq assay, 4516 differentially expressed genes (DEGs) were identified between two cultivars. Further analyses suggested that low expression level of chlorophyll biosynthetic genes, such as chlM, por and NOL caused less chlorophylls or chloroplast in fruit skin of Bai. Meanwhile, a predicted regulatory network of anthocyanin biosynthesis was established to illustrate involving many DEGs, especially 4CL, CHS and UFGT. CONCLUSIONS: This study uncovered significant differences between two cucumber genotypes with different fruit color using metabolome and RNA-seq analysis. We lay a foundation to understand molecular regulation mechanism on formation of cucumber skin color, by exploring valuable genes, which is helpful for cucumber breeding and improvement on fruit skin color.

Understanding the heat resistance of cucumber through leaf transcriptomics.

Heat stress is a major environmental factor limiting plant productivity and quality in agriculture. Cucumber, one of the most important vegetables among cucurbitaceae, prefers to grow in a warm environment. Until now the molecular knowledge of heat stress in cucumber remained unclear. In this study, we performed transcriptome analysis using two diverse genetic cucumber cultivars, L-9 and A-16 grown under normal and heat stress. L-9 displayed heat-tolerance phenotype with higher superoxide dismutase enzyme (SOD) enzyme activity and lower malondialdehyde (MDA) content than A-16 under heat stress. RNA-sequencing revealed that a total of 963 and 2778 genes are differentially expressed between L-9 and A-16 under normal and heat stress respectively. In addition, we found that differentially expressed genes (DEGs) associated with plant hormones signally pathway, transcription factors, and secondary metabolites showed significantly change in expression level after heat stress, which were confirmed by quantitative real-time PCR assay. Our results not only explored several crucial genes involved in cucumber heat resistance, but also provide a new insight into studying heat stress.

Transcriptome Analyses Provide Novel Insights into Heat Stress Responses in Chieh-Qua (Benincasa hispida Cogn. var. Chieh-Qua How).

Temperature rising caused by global warming has imposed significant negative effects on crop qualities and yields. To get the well-known molecular mechanism upon the higher temperature, we carefully analyzed the RNA sequencing-based transcriptomic responses of two contrasting chieh-qua genotypes: A39 (heat-tolerant) and H5 (heat-sensitive). In this study, twelve cDNA libraries generated from A39 and H5 were performed with a transcriptome assay under normal and heat stress conditions, respectively. A total of 8705 differentially expressed genes (DEGs) were detected under normal conditions (3676 up-regulated and 5029 down-regulated) and 1505 genes under heat stress (914 up-regulated and 591 down-regulated), respectively. A significant positive correlation between RNA-Seq data and qRT-PCR results was identified. DEGs related to heat shock proteins (HSPs), ubiquitin-protein ligase, transcriptional factors, and pentatricopeptide repeat-containing proteins were significantly changed after heat stress. Several genes, which encoded HSPs (CL2311.Contig3 and CL6612.Contig2), cytochrome P450 (CL4517.Contig4 and CL683.Contig7), and bHLH TFs (CL914.Contig2 and CL8321.Contig1) were specifically induced after four days of heat stress. DEGs detected in our study between these two contrasting cultivars would provide a novel basis for isolating useful candidate genes of heat stress responses in chieh-qua.

RNA-Seq analysis of gynoecious and weak female cucumber revealing the cell cycle pathway may regulate sex determination in cucumber.

This study aims to investigate key genes involved in molecular regulatory networks of cucumber sex determination. Genome-wide high-throughput RNA sequencing was performed for young apical buds of gynoecious and weak female cucumber at three growth stages (one-leaf one-bud, three-leaf one-bud, and five-leaf one-bud). Seven comparisons from the same cultivar at three different stages and at the same stage between the two cultivars were analyzed, and the results revealed that compared with differentially expressed genes (DEGs) in weak female cucumber, more genes were upregulated at the one-leaf one-bud stage and downregulated at the three-leaf one-bud stage in gynoecious cucumber. In addition, there were four kinds of gene expression trends (0, 1, 6, and 7), which were significantly enriched in gynoecious cucumber, while only two kinds of gene expression trends (5 and 6) were significantly enriched in weak female cucumber. Together with the data of the Gene Ontology (GO), pathway, gene expression trends and qRT-PCR, nine genes were identified and considered as candidate genes that may be involved in sex differentiation regulation in cucumber. These genes included Cs-MCM6, Cs-ACT3, Cs-XRCC4, Cs-MCM2, Cs-CDC45, Cs-Dpri, Cs-H2B, Cs-CDC20 and Cs-CNGC1. Among these genes, five genes (Cs-MCM6, Cs-MCM2, Cs-CDC45, Cs-Dpri, and Cs-CDC20) were involved in the cell cycle pathway, suggesting that the cell cycle pathway may play an important role in sex determination in cucumber.

Transcriptome Analyses in Different Cucumber Cultivars Provide Novel Insights into Drought Stress Responses.

Drought stress is one of the most serious threats to cucumber quality and yield. To gain a good understanding of the molecular mechanism upon water deficiency, we compared and analyzed the RNA sequencing-based transcriptomic responses of two contrasting cucumber genotypes, L-9 (drought-tolerant) and A-16 (drought-sensitive). In our present study, combining the analysis of phenotype, twelve samples of cucumber were carried out a transcriptomic profile by RNA-Seq under normal and water-deficiency conditions, respectively. A total of 1008 transcripts were differentially expressed under normal conditions (466 up-regulated and 542 down-regulated) and 2265 transcripts under drought stress (979 up-regulated and 1286 down-regulated). The significant positive correlation between RNA sequencing data and a qRT-PCR analysis supported the results found. Differentially expressed genes (DEGs) involved in metabolic pathway and biosynthesis of secondary metabolism were significantly changed after drought stress. Several genes, which were related to sucrose biosynthesis (Csa3G784370 and Csa3G149890) and abscisic acid (ABA) signal transduction (Csa4M361820 and Csa6M382950), were specifically induced after 4 days of drought stress. DEGs between the two contrasting cultivars identified in our study provide a novel insight into isolating helpful candidate genes for drought tolerance in cucumber.

High-density genetic map construction and gene mapping of pericarp color in wax gourd using specific-locus amplified fragment (SLAF) sequencing.

BACKGROUND: High-density map is a valuable tool for genetic and genomic analysis. Although wax gourd is a widely distributed vegetable of Cucurbitaceae and has important medicinal and health value, no genetic map has been constructed because of the lack of efficient markers. Specific-locus amplified fragment sequencing (SLAF-seq) is a newly developed high-throughput strategy for large-scale single nucleotide polymorphism (SNP) discovery and genotyping. RESULTS: In our present study, we constructed a high-density genetic map by using SLAF-seq and identified a locus controlling pericarp color in wax gourd. An F2 population of 140 individuals and their two parents were subjected to SLAF-seq. A total of 143.38 M pair-end reads were generated. The average sequencing depth was 26.51 in the maternal line (B214), 27.01 in the parental line (B227), and 5.11 in each F2 individual. When filtering low-depth SLAF tags, a total of 142,653 high-quality SLAFs were detected, and 22,151 of them were polymorphic, with a polymorphism rate of 15.42 %. And finally, 4,607 of the polymorphic markers were selected for genetic map construction, and 12 linkage groups (LGs) were generated. The map spanned 2,172.86 cM with an average distance between adjacent markers for 0.49 cM. The inheritance of pericarp color was also studied, which showed that the pericarp color was controlled by one single gene. And based on the newly constructed high-density map, a single locus locating on chromosome 5 was identified for controlling the pericarp color of wax gourd. CONCLUSIONS: This is the first report of high-density genetic map construction and gene mapping in wax gourd, which will be served as an invaluable tool for gene mapping, marker assisted breeding, map-based gene cloning, comparative mapping and draft genome assembling of wax gourd.

Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars.

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic (Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses.