CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Genome-wide identification and expression analysis of autophagy genes in cucumber

Background Autophagy is an evolutionarily conserved physiological and developmental process in eukaryotes. In this process, damaged proteins in cells are degraded and cytoplasmic materials recycled. When plants are exposure to stress conditions or their growth and development are blocked, autophagy is induced to maintain the cell homeostasis by degrading long-lived proteins in the cells and organelles that function abnormally due to aging or damage. Cell autophagy has multiple functions in plants, it involved in growth and development, senescence, and responses to biotic and abiotic stress. So far, thirty three autophagy genes (ATG) have been found in rice, and more than 30 autophagy-related genes have been found in Arabidopsis, tobacco and corn, respectively. Four autophagy genes induced by salicylic acid were found in cucumber, but a little still unknown about all of autophagy genes in cucumber genome. Our experiment fully explored the ATG gene family of cucumber genome based on bioinformatics methods and identified 20 CsATG genes. We systematically analyzed the structure, conserved motifs, expression and phylogeny relationship of these ATG genes, which lays the foundation for exploring the function of the genes. Results A total of 20 putative ATG genes were identified in the cucumber genome. Gene duplication analysis showed that both fragmented and tandem duplication played vital roles in the amplification of cucumber ATG gene family. Gene expression analysis showed that 16 CsATG genes were induced by the salicylic acid (SA) treatment, and 16 CsATG genes were down-regulated by Methyl jasmonate (MeJA) treatment. Under high salinity stress, 10 CsATG genes were induced in roots. Under drought stress, 16 CsATG genes were induced in roots. Under carbon starvation stress, all of 20 CsATG genes were induced to express in leaves, suggesting that cell autophagy has a potential role in nutritional starvation tolerance. Conclusion Our results clearly have deepened our understanding of the characteristics and functions of cucumber ATG gene, and also found some new gene resources that can be used for the future development of cucumber and other crop varieties, which can resist stress.

Chromosome-scale genome assembly of Cucumis hystrix—a wild species interspecifically cross-compatible with cultivated cucumber

Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in “defense response,” and C. hystrix harbored 104 nucleotide-binding site–encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber–C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

Genome-Wide Characterization of HSP90 Gene Family in Cucumber and Their Potential Roles in Response to Abiotic and Biotic Stresses

Heat shock protein 90 (HSP90) possesses critical functions in plant developmental control and defense reactions. The HSP90 gene family has been studied in various plant species. However, the HSP90 gene family in cucumber has not been characterized in detail. In this study, a total of six HSP90 genes were identified from the cucumber genome, which were distributed to five chromosomes. Phylogenetic analysis divided the cucumber HSP90 genes into two groups. The structural characteristics of cucumber HSP90 members in the same group were similar but varied among different groups. Synteny analysis showed that only one cucumber HSP90 gene, Csa1G569290, was conservative, which was not collinear with any HSP90 gene in Arabidopsis and rice. The other five cucumber HSP90 genes were collinear with five Arabidopsis HSP90 genes and six rice HSP90 genes. Only one pair of paralogous genes in the cucumber HSP90 gene family, namely one pair of tandem duplication genes (Csa1G569270/Csa1G569290), was detected. The promoter analysis showed that the promoters of cucumber HSP90 genes contained hormone, stress, and development-related cis-elements. Tissue-specific expression analysis revealed that only one cucumber HSP90 gene Csa3G183950 was highly expressed in tendril but low or not expressed in other tissues, while the other five HSP90 genes were expressed in all tissues. Furthermore, the expression levels of cucumber HSP90 genes were differentially induced by temperature and photoperiod, gibberellin (GA), downy mildew, and powdery mildew stimuli. Two cucumber HSP90 genes, Csa1G569270 and Csa1G569290, were both differentially expressed in response to abiotic and biotic stresses, which means that these two HSP90 genes play important roles in the process of cucumber growth and development. These findings improve our understanding of cucumber HSP90 family genes and provide preliminary information for further studies of cucumber HSP90 gene functions in plant growth and development.

Characteristics and expression patterns of six α-galactosidases in cucumber (Cucumis sativus L.)

Six putative α-galactosidase genes (α-Gals), three acid forms (CsGAL1, CsGAL2, CsGAL3) and three alkaline forms (CsAGA1, CsAGA2, CsAGAL3), were found in the cucumber genome. It is interesting to know the expression pattern and possible function of these α-Gals in the cucumber plant since it is a stachyose-translocating species. In this study, full-length cDNAs of six α-Gals were cloned and heterologously expressed. The result showed that all recombinant proteins revealed acid or alkaline α-Gal activities with different substrate specificities and pH or temperature responding curves, indicating their distinct roles in cucumber plants. Phylogenetic analysis of collected α-Gal amino acid sequences from different plants indicated that the ancestor of both acid and alkaline α-Gals existed before monocots and dicots separated. Generally, six α-Gal genes are universally expressed in different cucumber organs. CsGAL2 highly expressed in fasting-growing leaves, fruits and germinating seeds; CsGAL3 mainly distributes in vacuoles and significantly expressed in cucumber fruits, senescent leaves and seeds during late stage germination; The expression of CsAGA1 increased from leaf 1 to leaf 3 (sink leaves) and then declined from leaf 4 to leaf 7 (source leaves), and this isoform also highly expressed in male flowers and germinating seeds at early stage; CsAGA2 significantly expressed in cucumber leaves and female flowers; CsAGA3 is localized in plastids and also actively expressed in senescent leaves and germinating seeds; The role of CsGAL1 in cucumber plants is now unclear since its expression was relatively low in all organs. According to their expression patterns, subcellular localizations and previously reported functions of these isoforms in other plants, combining the data of soluble sugars contents in different tissues, the possible functions of these α-Gals were discussed.

Genome-Wide Characterization of Cucumber (Cucumis sativus L.) GRAS Genes and Their Response to Various Abiotic Stresses

The GRAS (gibberellic acid insensitive, repressor of GAI, and scarecrow) proteins are a family of plant-specific transcription factors that regulate plant growth, development, and stress response. Currently, the role of GRAS transcription factors in various abiotic stress responses has not been systematically studied in cucumber (Cucumis sativus L.), a popular vegetable crop. Here, we provide a comprehensive bioinformatics analysis of the 35 GRAS genes identified in the cucumber genome. In this study, cucumber genotypes, i.e., “CG104”, which is stress-tolerant, and genotype “CG37”, which is stress-sensitive, were examined to provide insight on potential differences in the GRAS-regulated abiotic stress pathways. Transcriptional analysis by RNA-seq or qRT-PCR of these two genotypes revealed common and divergent functions of CsGRAS genes regulated by low and high temperatures, salinity, and by exposure to the phytohormones gibberellin (GA) and abscisic acid (ABA). Notably, CsGRAS2 (DELLA) and CsGRAS26 (LISCL) were regulated by all abiotic stresses and hormone treatments, suggesting that they may function in the biological cross-talk between multiple signaling pathways. This study provides candidate genes for improving cucumber tolerance to various environmental stresses.

Genome-wide analysis of the WRKY gene family in the cucumber genome and transcriptome-wide identification of WRKY transcription factors that respond to biotic and abiotic stresses

Background Cucumber (Cucumis sativus L.) is an economically important vegetable crop species. However, it is susceptible to various abiotic and biotic stresses. WRKY transcription factors play important roles in plant growth and development, particularly in the plant response to biotic and abiotic stresses. However, little is known about the expression pattern of WRKY genes under different stresses in cucumber. Results In the present study, an analysis of the new assembly of the cucumber genome (v3.0) allowed the identification of 61 cucumber WRKY genes. Phylogenetic and synteny analyses were performed using related species to investigate the evolution of the cucumber WRKY genes. The 61 CsWRKYs were classified into three main groups, within which the gene structure and motif compositions were conserved. Tissue expression profiles of the WRKY genes demonstrated that 24 CsWRKY genes showed constitutive expression (FPKM > 1 in all samples), and some WRKY genes showed organ-specific expression, suggesting that these WRKYs might be important for plant growth and organ development in cucumber. Importantly, analysis of the CsWRKY gene expression patterns revealed that five CsWRKY genes strongly responded to both salt and heat stresses, 12 genes were observed to be expressed in response to infection from downy mildew and powdery mildew, and three CsWRKY genes simultaneously responded to all treatments analysed. Some CsWRKY genes were observed to be induced/repressed at different times after abiotic or biotic stress treatment, demonstrating that cucumber WRKY genes might play different roles during different stress responses and that their expression patterns vary in response to stresses. Conclusions Sixty-one WRKY genes were identified in cucumber, and insight into their classification, evolution, and expression patterns was gained in this study. Responses to different abiotic and biotic stresses in cucumber were also investigated. Our results provide a better understanding of the function of CsWRKY genes in improving abiotic and biotic stress resistance in cucumber.