Pan-genome Analysis of WOX Gene Family and Function Exploration of CsWOX9 in Cucumber.

Cucumber is an economically important vegetable crop, and the warts (composed of spines and Tubercules) of cucumber fruit are an important quality trait that influences its commercial value. WOX transcription factors are known to have pivotal roles in regulating various aspects of plant growth and development, but their studies in cucumber are limited. Here, genome-wide identification of cucumber WOX genes was performed using the pan-genome analysis of 12 cucumber varieties. Our findings revealed diverse CsWOX genes in different cucumber varieties, with variations observed in protein sequences and lengths, gene structure, and conserved protein domains, possibly resulting from the divergent evolution of CsWOX genes as they adapt to diverse cultivation and environmental conditions. Expression profiles of the CsWOX genes demonstrated that CsWOX9 was significantly expressed in unexpanded ovaries, especially in the epidermis. Additionally, analysis of the CsWOX9 promoter revealed two binding sites for the C2H2 zinc finger protein. We successfully executed a yeast one-hybrid assay (Y1H) and a dual-luciferase (LUC) transaction assay to demonstrate that CsWOX9 can be transcriptionally activated by the C2H2 zinc finger protein Tu, which is crucial for fruit Tubercule formation in cucumber. Overall, our results indicated that CsWOX9 is a key component of the molecular network that regulates wart formation in cucumber fruits, and provide further insight into the function of CsWOX genes in cucumber.

Comparison of Helicobacter pylori positive and negative gastric cancer via multi-omics analysis.

Helicobacter pylori (H. pylori) has been regarded as a definite carcinogenic bacterium for gastric cancer (GC). This multi-omics research was designed to investigate the genetic, microbial, and metabolic changes of GC patients when they are infected with H. pylori. We first mined The Cancer Genome Atlas Stomach Adenocarcinoma (STAD) data to identify the key genes and critical pathways in H. pylori-positive individuals with GC compared to H. pylori-negative individuals with GC. Then, fresh stool samples were collected from GC individuals screened for eligibility, and we analyzed the microbial changes and metabolite alterations between H. pylori-positive and H. pylori-negative GC individuals. Finally, we tried to explore the interaction between key gut flora and metabolite changes in GC patients infected with H. pylori. We identified three genes (GCG, APOA1, and IGFBP1) with significant relevance to H. pylori infection, and the survival monogram based on the three H. pylori-related genes showed good predictive ability for overall survival among GC individuals. 16S rRNA sequencing showed that the abundance of Escherichia-Shigella, Bacteroides, Enterococcus, and Lactobacillus was upregulated in GC cases with H. pylori at the level of genus. There exists a great difference in alpha and beta diversity between H. pylori group and non-H. pylori group. The untargeted metabolome analysis identified 295 significant fecal metabolites, and the levels of penitrem E, auberganol, stercobilinogen, and lys thr are upregulated in the H. pylori group. Finally, correlation analysis showed that there exists a significant correlation between the fecal metabolites and gut bacterial strains. This is the first clinical research to investigate the difference between GC patients with H. pylori and GC patients without H. pylori via multi-omics analysis. 16S rRNA sequencing along with untargeted metabolomics demonstrated decreased microbial diversity and metabolic dysregulation in gastric carcinoma individuals with H. pylori infection.IMPORTANCEThis is the first clinical research to systematically expound the difference between gastric cancer (GC) individuals with Helicobacter pylori and GC individuals without H. pylori from the perspective of multi-omics. This clinical study identified significant genes, microbes, and fecal metabolites, which exhibited nice power for differentiating GC individuals with H. pylori infection from GC individuals without H. pylori infection. This study provides a crucial basis for a better understanding of eradication therapy among the GC population.

Pan-Genome Analysis of TIFY Gene Family and Functional Analysis of CsTIFY Genes in Cucumber.

Cucumbers are frequently affected by gray mold pathogen Botrytis cinerea, a pathogen that causes inhibited growth and reduced yield. Jasmonic acid (JA) plays a primary role in plant responses to biotic stresses, and the jasmonate-ZIM-Domain (JAZ) proteins are key regulators of the JA signaling pathway. In this study, we used the pan-genome of twelve cucumber varieties to identify cucumber TIFY genes. Our findings revealed that two CsTIFY genes were present in all twelve cucumber varieties and showed no differences in protein sequence, gene structure, and motif composition. This suggests their evolutionary conservation across different cucumber varieties and implies that they may play a crucial role in cucumber growth. On the other hand, the other fourteen CsTIFY genes exhibited variations in protein sequence and gene structure or conserved motifs, which could be the result of divergent evolution, as these genes adapt to different cultivation and environmental conditions. Analysis of the expression profiles of the CsTIFY genes showed differential regulation by B. cinerea. Transient transfection plants overexpressing CsJAZ2, CsJAZ6, or CsZML2 were found to be more susceptible to B. cinerea infection compared to control plants. Furthermore, these plants infected by the pathogen showed lower levels of the enzymatic activities of POD, SOD and CAT. Importantly, after B. cinerea infection, the content of JA was upregulated in the plants, and cucumber cotyledons pretreated with exogenous MeJA displayed increased resistance to B. cinerea infection compared to those pretreated with water. Therefore, this study explored key TIFY genes in the regulation of cucumber growth and adaptability to different cultivation environments based on bioinformatics analysis and demonstrated that CsJAZs negatively regulate cucumber disease resistance to gray mold via multiple signaling pathways.

Ectopic Expression of CsSUN in Tomato Results in Elongated Fruit Shape via Regulation of Longitudinal Cell Division.

Fruit shape, an important agronomic trait of cucumber (Cucumis sativus L.), is tightly controlled by a series of genes such as CsSUN, a homologue of SlSUN that is responsible for the tomato (Solanum lycopersicum) fruit shape via the modulation of cell division. However, the direct genetic evidence about the CsSUN-mediated regulation of fruit shape is still scarce, limiting our mechanistic understanding of the biological functions of CsSUN. Here, we introduced CsSUN into the round-fruited tomato inbred line 'SN1' (wild type, WT) via the Agrobacterium tumefaciens-mediated method. The high and constitutive expression of CsSUN was revealed by real-time PCR in all the tested tissues of the transgenic plants, especially in the fruits and ovaries. Phenotypic analyses showed that the ectopic expression of CsSUN increased fruit length while it decreased fruit diameter, thus leading to the enhanced fruit shape index in the transgenic tomato lines relative to the WT. Additionally, the reduction in the seed size and seed-setting rate and the stimulation of seed germination were observed in the CsSUN-expressed tomato. A histological survey demonstrated that the elongated fruits were mainly derived from the significant increasing of the longitudinal cell number, which compensated for the negative effects of decreased cell area in the central columellae. These observations are different from action mode of SlSUN, thus shedding new insights into the SUN-mediated regulation of fruit shape.

Morphological Characterization and Integrated Transcriptome and Proteome Analysis of Organ Development Defective 1 (odd1) Mutant in Cucumis sativus L.

Cucumber (Cucumis sativus L.) is an economically important vegetable crop with the unique growth habit and typical trailing shoot architecture of Cucurbitaceae. Elucidating the regulatory mechanisms of growth and development is significant for improving quality and productivity in cucumber. Here we isolated a spontaneous cucumber mutant organ development defective 1 (odd1) with multiple morphological changes including root, plant stature, stem, leaf, male and female flowers, as well as fruit. Anatomical and cytological analyses demonstrated that both cell size and number decreased, and the shoot apical meristem (SAM) was smaller in odd1 compared with WT. Pollen vigor and germination assays and cross tests revealed that odd1 is female sterile, which may be caused by the absence of ovules. Genetic analysis showed that odd1 is a recessive single gene mutant. Using the MutMap strategy, the odd1 gene was found to be located on chromosome 5. Integrated profiling of transcriptome and proteome indicated that the different expression genes related to hormones and SAM maintenance might be the reason for the phenotypic changes of odd1. These results expanded the insight into the molecular regulation of organ growth and development and provided a comprehensive reference map for further studies in cucumber.

Gibberellins regulate lateral root development that is associated with auxin and cell wall metabolisms in cucumber.

Cucumber is an economically important crop cultivated worldwide. Gibberellins (GAs) play important roles in the development of lateral roots (LRs), which are critical for plant stress tolerance and productivity. Therefore, it is of great importance for cucumber production to study the role of GAs in LR development. Here, the results showed that GAs regulated cucumber LR development in a concentration-dependent manner. Treatment with 1, 10, 50 and 100 µM GA3 significantly increased secondary root length, tertiary root number and length. Of these, 50 µM GA3 treatment had strong effects on increasing root dry weight and the root/shoot dry weight ratio. Pairwise comparisons identified 417 down-regulated genes enriched for GA metabolism-related processes and 447 up-regulated genes enriched for cell wall metabolism-related processes in GA3-treated roots. A total of 3523 non-redundant DEGs were identified in our RNA-Seq data through pairwise comparisons and linear factorial modeling. Of these, most of the genes involved in auxin and cell wall metabolisms were up-regulated in GA3-treated roots. Our findings not only shed light on LR regulation mediated by GA but also offer an important resource for functional studies of candidate genes putatively involved in the regulation of LR development in cucumber and other crops.

Camrelizumab plus apatinib as second-line treatment for advanced oesophageal squamous cell carcinoma (CAP 02): a single-arm, open-label, phase 2 trial.

BACKGROUND: Camrelizumab, an anti-PD-1 antibody, has shown moderate efficacy in oesophageal squamous cell carcinoma. Apatinib, a selective inhibitor of VEGFR2, has a synergistic effect with immunotherapy. We aimed to assess the combination of camrelizumab and apatinib as second-line treatment for advanced oesophageal squamous cell carcinoma. METHODS: This single-arm, open-label, phase 2 study was conducted at eight centres in China. Eligible patients were aged 18-75 years, with an Eastern Cooperative Oncology Group performance status of 0 or 1, who had unresectable locally advanced, locally recurrent, or metastatic oesophageal squamous cell carcinoma, and had progressed after or were intolerant to first-line chemotherapy. Patients received intravenous camrelizumab 200 mg once every 2 weeks plus oral apatinib 250 mg once daily for a 28-day cycle until disease progression, unacceptable adverse events, or withdrawal of consent. The primary endpoint was investigator-assessed confirmed objective response rate. Efficacy was analysed in patients who had received at least one dose of study drug, and safety was analysed in patients who received the study drug and had at least one post-baseline safety assessment. The study of this cohort is complete and this trial is registered with ClinicalTrials.gov, number NCT03736863. FINDINGS: Between Dec 5, 2019, and Feb 10, 2021, 52 patients were enrolled and included in analyses. At data cutoff (June 20, 2021), median follow-up was 7·5 months (IQR 4·0-11·2). 18 (34·6%, [95% CI 22·0-49·1]) of 52 patients had a confirmed objective response. 23 (44%) of 52 patients had grade 3 or worse treatment-related adverse events. The most common grade 3 or worse treatment-related adverse events were increased aspartate aminotransferase (10 [19%]), increased gamma-glutamyltransferase (10 [19%]), and increased alanine aminotransferase (five [10%]). No treatment-related deaths occurred. INTERPRETATION: Camrelizumab combined with apatinib showed promising activity and manageable toxicity, and might be a potential second-line treatment option for patients with advanced oesophageal squamous cell carcinoma. Another cohort of this study, enrolling patients previously treated with first-line immunotherapy, is ongoing. FUNDING: Jiangsu Hengrui Pharmaceuticals.

[Effect of circ-SFMBT2 on the biological behavior of non-small cell lung cancer cells by targeting the miR-7-5p/ADAM10 axis].

OBJECTIVE: To explore the effect of circ-SFMBT2 on the biological behavior of non-small cell lung cancer (NSCLC) cells and its regulatory role on the miR-7-5p/ADAM10 axis. METHODS: qRT-PCR and Western blotting were used to determine the expression of circ-SFMBT2, miR-7-5p, and ADAM10 in NSCLC tissues and adjacent tissues. Pearson analysis was used to analyze the correlation between circ-SFMBT2 and miR-7-5p, and between miR-7-5p and ADAM10. In vitro cultured human bronchial epithelial-like cells (HBE) and lung cancer cell lines H1650, H460, A549, H1299. CCK-8 and EdU methods were used to assess the ability of cell proliferation. Plate experiment was used to detect the clone formation ability. Flow cytometry was used to detect the apoptosis rate. Transwell experiment was used to detect cell invasion ability. Dual luciferase reporter experiment detects the targeting relationship between circ-SFMBT2 and miR-7-5p, and between miR-7-5p and ADAM10. Transplanted tumor experiment in nude mice assessed the effect of knocking down circ-SFMBT2 on the growth of transplanted tumor. Immunohistochemical experiments were performed to detect the positive rates of ADAM10 and Ki67 proteins in transplanted tumor tissues. RESULTS: The expression levels of circ-SFMBT2 and ADAM10 were increased in NSCLC tissues and cell lines, while decreased the expression of miR-7-5p. circ-SFMBT2 was negatively correlated with miR-7-5p, while miR-7-5p was negatively correlated with ADAM10. Silencing the overexpression of circ-SFMBT2 and miR-7-5p could inhibit cell proliferation, clone formation and invasion, and also promote apoptosis. circ-SFMBT2 could target miR-7-5p, and ADAM10 was the target gene of miR-7-5p. The combined effect of silencing circ-SFMBT2 and inhibition of miR-7-5p, as well as miR-7-5p overexpression and ADAM10 overexpression could promote cell proliferation, clone formation and invasion, and also suppress cell apoptosis. Silencing circ-SFMBT2 could inhibit the growth of transplanted tumors. CONCLUSION: Silencing circ-SFMBT2 can suppress the proliferation, clone formation, invasion ability and induce apoptosis of NSCLC cells by regulating the miR-7-5p/ADAM10 axis.

CsMYB60 Confers Enhanced Resistance to Fusarium solani by Increasing Proanthocyanidin Biosynthesis in Cucumber.

Root rot caused by Fusarium solani is one of the most common fungal diseases in cucumber (Cucumis sativus). Proanthocyanidins (PAs) are known to play important roles in inhibiting the growth of phytopathogens. In addition, CsMYB60 is a known positive regulator of flavonol and PA biosynthesis in cucumber. However, it remains unclear that whether PAs can inhibit the growth of F. solani and whether CsMYB60 serves as a target gene for increasing resistance to phytopathogens in cucumber. In this study, we demonstrated that PAs (or their building block, catechin) could increase the resistance of cucumber seedlings to F. solani both in vitro and in vivo. The addition of catechins, or crude leaf extracts treated with different concentrations of catechins in culture medium, could significantly inhibit the hyphal growth of F. solani. On the other hand, cucumber seedlings treated with catechins showed higher resistance to F. solani than the seedlings of control group. Moreover, transgenic cucumber seedlings overexpressing CsMYB60, with the observed accumulation of PAs, were more resistant to F. solani than the nontransgenic siblings. Therefore, our results suggest that PAs (or catechin) can serve as a biological control agent to protect cucumber plants from the infection of F. solani. More importantly, CsMYB60 holds great promise as a target gene to confer disease resistance during the molecular breeding in cucumber.

Transcriptome profiling reveals response genes for downy mildew resistance in cucumber.

MAIN CONCLUSION: We discovered a potential defense pathway of cucumber to downy mildew. The signaling that activates the pathways of ROS and lignin accumulation may play an important role in the defense response. Many resistance genes were identified by transcriptome analysis. Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive diseases and causes severe yield losses of cucumber. However, the genes and pathways involved in regulating DM resistance were still poorly understood. In our study, we observed that the highly sensitive inbred line 53 (IL53) exhibited more severe disease symptoms than the highly resistant inbred line 51 (IL51) under P. cubensis infection. Furthermore, lignin, limiting the germination and extension of P. cubensis, and H2O2, as a signaling molecule during the resistant process, were both shown to increase, indicating that the signaling that activates these pathways might be responsible for the resistance divergence between IL51 and IL53. Transcriptome analysis, using the resistant and susceptible pools in F2 populations with IL51 and IL53 as parents, showed that a series of differentially expressed genes was involved in multiple functions of defense response: pathogen-associated molecular pattern recognition, signal transduction, reactive oxygen species and lignin accumulation, and transcription regulators. Combining physiological data with transcriptomes, we predicted a potential molecular mechanism of cucumber resistance to DM. Our research provided a foundation for further studies on the mechanism of cucumber resistance to DM.

Genome-wide identification and characterization of WOX genes in Cucumis sativus.

WUSCHEL-related homeobox (WOX) proteins are plant-specific transcription factors that are profoundly involved in regulation of plant development and stress responses. In this study, we totally identified 11 WOX transcription factor family members in cucumber (Cucumis sativus, CsWOX) genome and classified them into three clades with nine subclades based on phylogenetic analysis results. Alignment of amino acid sequences revealed that all WOX members in cucumber contained the typical homeodomain, which consists of 60-66 amino acids and is folded into a helix-turn-helix structure. Gene duplication event analysis indicated that CsWOX1a and CsWOX1b were a segment duplication pair, which might affect the number of WOX members in cucumber genome. The expression profiles of CsWOX genes in different tissues demonstrated that the members sorted into the ancient clade (CsWOX13a and CsWOX13b) were constitutively expressed at higher levels in comparison to the others. Cis-element analysis in promoter regions suggested that the expression of CsWOX genes was associated with phytohormone pathways and stress responses, which was further supported by RNA-seq data. Taken together, our results provide new insights into the evolution of cucumber WOX genes and improve our understanding about the biological functions of the CsWOX gene family.

Overexpression of SlMYB75 enhances resistance to Botrytis cinerea and prolongs fruit storage life in tomato.

KEY MESSAGE: SlMYB75 increased the accumulation of JA and improved the scavenging of excess H2O2 to resist B. cinerea. Overexpression of SlMYB75 greatly prolongs tomato fruit storage life. Botrytis cinerea (B. cinerea) is a major threat to the production and storage life of tomato (Solanum lycopersicum) fruit around the world. SlMYB75 is an R2R3MYB transcription factor associated with the biosynthesis of anthocyanidin, but little is known about its function in the resistance of tomato to B. cinerea. In this study, we found that the overexpression of SlMYB75 regulated the accumulation of jasmonic acid (JA) and promoted the JA-mediated signaling pathway to resist B. cinerea infection. Moreover, the activities of peroxidase and superoxide dismutase, which were activated to scavenge hydrogen peroxide produced as a result of the B. cinerea infection, were enhanced in the transgenic tomato plants. Scanning electron microscopy images showed that the wax on the fruit skin surface was significantly decreased in the transgenic tomatoes compared with the wild type. However, SlMYB75 prolonged fruit storage life by both enhancing resistance to B. cinerea and directly downregulating the fruit shelf life-related gene SlFSR. Collectively, this study provides a good candidate gene for breeding high-quality tomatoes with a long storage life and high disease resistance.

CsWRKY10 mediates defence responses to Botrytis cinerea infection in Cucumis sativus.

Cucumber (Cucumis sativus) is one of the most widely cultivated vegetable crops in the world, and its yield is often reduced due to the infection of Botrytis cinerea (B. cinerea), which causes a serious disease. However, few genes involved in the response to B. cinerea have been identified in cucumber. In this study, we identified that CsWRKY10 plays a key role in the cucumber resistance to B. cinerea because that the overexpression of CsWRKY10 significantly increased the susceptibility to B. cinerea in cucumber. After the pathogen infection, the enzyme activities of catalase, superoxide dismutase and peroxidase in transgenic plants were affected, resulting in the decrease in reactive oxygen species (ROS) contents. In addition, the light microscopic images showed that overexpression of CsWRKY10 promoted the spore germination and mycelia elongation of B. cinerea in cucumber. Importantly, after B. cinerea infection, the contents of jasmonic acid (JA) are decreased, and the expression levels of JA- and salicylic acid- related defence genes significantly changed in transgenic plants. In contrast, overexpression of CsWRKY10 enhanced resistance to Corynespora cassiicola in cucumber. Collectively, this study indicated that CsWRKY10 negatively regulates the resistance of cucumber to B. cinerea by reducing the ROS contents and inhibiting the JA-mediated resistance signalling pathway, but strengthens resistance to Corynespora cassiicola.

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.

CsMYB60 directly and indirectly activates structural genes to promote the biosynthesis of flavonols and proanthocyanidins in cucumber.

Flavonols and proanthocyanidins (PAs) are the main pigments in the black spines of cucumber (Cucumis sativus) fruit, and CsMYB60 is a key regulator of the biosynthesis of flavonols and PAs. However, in cucumber, the tissue distribution pattern of flavonols and PAs and the mechanism of their biosynthesis regulated by CsMYB60 remain unclear. In this study, we clarified the tissue-specific distribution of flavonoids and the unique transcriptional regulation of flavonoid biosynthesis in cucumber. CsMYB60 activated CsFLS and CsLAR by binding to their promoters and directly or indirectly promoted the expression of CsbHLH42, CsMYC1, CsWD40, and CsTATA-box binding protein, resulting in the formation of complexes of these four proteins to increase the expression of Cs4CL and interact with CsTATA-box binding protein to regulate the expression of CsCHS, thereby regulating the biosynthesis of flavonols and PAs in cucumber. Our data provide new insights into the molecular mechanism of flavonoid biosynthesis, which will facilitate molecular breeding to improve fruit quality in cucumber.

Localization of quantitative trait loci for cucumber fruit shape by a population of chromosome segment substitution lines.

Cucumber fruit shape, a significant agronomic trait, is controlled by quantitative trait loci (QTLs). Feasibility of chromosome segment substitution lines (CSSLs) is well demonstrated to map QTLs, especially the minor-effect ones. To detect and identify QTLs with CSSLs can provide new insights into the underlying mechanisms regarding cucumber fruit shape. In the present study, 71 CSSLs were built from a population of backcross progeny (BC4F2) by using RNS7 (a round-fruit cucumber) as the recurrent parent and CNS21 (a long-stick-fruit cucumber) as the donor parent in order to globally detect QTLs for cucumber fruit shape. With the aid of 114 InDel markers covering the whole cucumber genome, 21 QTLs were detected for fruit shape-related traits including ovary length, ovary diameter, ovary shape index, immature fruit length, immature fruit diameter, immature fruit shape index, mature fruit length, mature fruit diameter and mature fruit shape index, and 4 QTLs for other traits including fruit ground and flesh color, and seed size were detected as well. Together our results provide important resources for the subsequent theoretical and applied researches on cucumber fruit shape and other traits.

Genome-wide identification and characterization of cucumber bHLH family genes and the functional characterization of CsbHLH041 in NaCl and ABA tolerance in Arabidopsis and cucumber.

BACKGROUND: The basic/helix-loop-helix (bHLH) transcription factor family exists in all three eukaryotic kingdoms as important participants in biological growth and development. To date, the comprehensive genomic and functional analyses of bHLH genes has not been reported in cucumber (Cucumis sativus L.). RESULTS: Here, a total of 142 bHLH genes were identified and classified into 32 subfamilies according to the conserved motifs, phylogenetic analysis and gene structures in cucumber. The sequences of CsbHLH proteins were highly conserved based on the results of multiple sequence alignment analyses. The chromosomal distribution, synteny analysis, and gene duplications of these 142 CsbHLHs were further analysed. Many elements related to stress responsiveness and plant hormones were present in the promoter regions of CsbHLH genes based on a cis-element analysis. By comparing the phylogeny of cucumber and Arabidopsis bHLH proteins, we found that cucumber bHLH proteins were clustered into different functional clades of Arabidopsis bHLH proteins. The expression analysis of selected CsbHLHs under abiotic stresses (NaCl, ABA and low-temperature treatments) identified five CsbHLH genes that could simultaneously respond to the three abiotic stresses. Tissue-specific expression profiles of these five genes were also analysed. In addition, 35S:CsbHLH041 enhanced the tolerance to salt and ABA in transgenic Arabidopsis and in cucumber seedlings, suggesting CsbHLH041 is an important regulator in response to abiotic stresses. Lastly, the functional interoperability network among the CsbHLH proteins was analysed. CONCLUSION: This study provided a good foundation for further research into the functions and regulatory mechanisms of CsbHLH proteins and identified candidate genes for stress resistance in cucumber.

Genome-wide characterization of two-component system (TCS) genes in melon (Cucumis melo L.).

To better understand cytokinin signaling in melon (Cucumis melo L.), one of the most important fruit crops in the Cucurbitaceae family, we identified and characterized melon two-component system (TCS) genes in this study. The results showed that there were 51 genes encoding putative TCS proteins in melon, and these TCS genes were classified into 3 subgroups, with 17 HK(L)s (histidine kinase/histidine-kinase like; 9 HKs and 8 HKLs), 9 HPs (histidine phosphotransfer proteins; 6 authentic and 3 pseudo), and 25 RRs (response regulators; 8 Type-A, 11 Type-B and 6 pseudo). The identity values of these cytokinin signaling proteins were revealed by analyzing their conserved motifs, domains and amino acid sequences. By analyzing TCS genes in different plant species, we found that melon HK(L)s, HPs and RRs had closer phylogenetic relationships with cucumber genes than with the genes of other plants, and the expansion of melon cytokinin signaling genes might be attributed to segmental duplication events. Analysis of the putative promoter regions (2-kb upstream regions of the start codon) revealed the enrichment of stress- and hormone-response cis-elements. The involvement of these putative TCS genes in melon cytokinin signaling was further supported by qRT-PCR data.

SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato.

Salinity threatens the productivity of tomato (Solanum lycopersicum L.). R2R3-type MYB transcription factors are important regulators in response to environmental stress. Here, we analyzed the function of the tomato R2R3-type MYB gene SlMYB102. A transcriptional activation assay showed that SlMYB102 had transactivation activity in yeast. Promoter analysis showed that multiple stress-related elements were found in the promoter of SlMYB102. Furthermore, SlMYB102 was induced by osmotic stress, particularly by salt stress. The overexpression of SlMYB102 in tomato affected multiple parameters under salinity stress. Under long-term salt stress, the degree of growth inhibition was significantly reduced in the two overexpression (OE) lines. In addition, the two OE lines maintained a better K+/Na+ ratio, lower reactive oxygen species (ROS) generation (O2•- production rate and H2O2 content) and lower electrolytic leakage rates than the wild type (WT). The activity of ROS scavenging enzymes including superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and the accumulation of antioxidants (ascorbic acid and glutathione) and proline was higher in the two OE lines compared with WT. The qRT-PCR analysis confirmed that the transcript abundance of many salt stress-related genes (SlSOS1, SlSOS2, SlNHX3, SlNHX4, SlHAK5, SlCPK1 and SlCPK3) was upregulated in two OE lines under salt stress. Collectively, these results suggest that SlMYB102 participates in tomato tolerance through the regulation of a series of molecular and physiological processes.

Transcriptome profiling reveals genes involved in spine development during CsTTG1-regulated pathway in cucumber (Cucumis sativus L.).

The cucumber (Cucumis sativus L.), a type of fleshy fruit, is covered with spines (multicellular trichomes), which have a crucial impact on the economic value of the crop. Previous studies have found that CsTTG1 plays important roles in the initiation and further differentiation of cucumber spines, but how spine formation is regulated at the molecular level by CsTTG1 remains poorly understood. In this study, we characterized a cucumber 35S:CsTTG1 transgenic T2 line, OE-2, which bears relatively large and long spines compared with the small and short spines of the wild type (WT). Phenotypic measurements and histological analyses revealed that this phenotypic change was attributed to significant increases in cell number and size. Comparison of ovary epidermis transcriptomes between OE-2 and WT by DGE (Digital Gene Expression) analysis identified 1241 differentially expressed genes, among which 712 genes were dramatically upregulated and 529 downregulated in the ovary epidermis of OE-2. XTH23 and Cyclin family genes were significantly activated in OE-2, and transcription factors (TFs) were found to participate in spine size regulation in OE-2. Further analyses confirmed that GA was implicated in the regulation of fruit spine development in cucumber. Thus, our study provides a foundation for dissecting the molecular regulatory networks of fruit spine control in cucumber.