Waterlogging-Stress-Responsive LncRNAs,Their Regulatory Relationships with miRNAs and Target Genes in Cucumber (Cucumis sativus L.)

Low oxygen level is a phenomenon often occurring during the cucumber cultivation period. Genes involved in adaptations to stress can be regulated by non-coding RNA. The aim was the identification of long non-coding RNAs (lncRNAs) involved in the response to long-term waterlogging stress in two cucumber haploid lines, i.e., DH2 (waterlogging tolerant—WL-T) and DH4 (waterlogging sensitive—WL-S). Plants, at the juvenile stage, were waterlogged for 7 days (non-primed, 1xH), and after a 14-day recovery period, plants were stressed again for another 7 days (primed, 2xH). Roots were collected for high-throughput RNA sequencing. Implementation of the bioinformatic pipeline made it possible to determine specific lncRNAs for non-primed and primed plants of both accessions, highlighting differential responses to hypoxia stress. In total, 3738 lncRNA molecules were identified. The highest number (1476) of unique lncRNAs was determined for non-primed WL-S plants. Seventy-one lncRNAs were depicted as potentially being involved in acquiring tolerance to hypoxia in cucumber. Understanding the mechanism of gene regulation under long-term waterlogging by lncRNAs and their interactions with miRNAs provides sufficient information in terms of adaptation to the oxygen deprivation in cucumber. To the best of our knowledge, this is the first report concerning the role of lncRNAs in the regulation of long-term waterlogging tolerance by priming application in cucumber.     

Genome-Wide Identification and Characterization of the CC-NBS-LRR Gene Family in Cucumber (Cucumis sativus L.)

The NBS-LRR (NLR) gene family plays a pivotal role in regulating disease defense response in plants. Cucumber is one of the most important vegetable crops in the world, and various plant diseases, including powdery mildew (PM), cause severe losses in both cucumber productivity and quality annually. To characterize and understand the role of the CC-NBS-LRR(CNL) family of genes in disease defense response in cucumber plants, we performed bioinformatical analysis to characterize these genes systematically. We identified 33 members of the CNL gene family in cucumber plants, and they are distributed on each chromosome with chromosome 4 harboring the largest cluster of five different genes. The corresponding CNL family member varies in the number of amino acids and exons, molecular weight, theoretical isoelectric point (pI) and subcellular localization. Cis-acting element analysis of the CNL genes reveals the presence of multiple phytohormone, abiotic and biotic responsive elements in their promoters, suggesting that these genes might be responsive to plant hormones and stress. Phylogenetic and synteny analysis indicated that the CNL proteins are conserved evolutionarily in different plant species, and they can be divided into four subfamilies based on their conserved domains. MEME analysis and multiple sequence alignment showed that conserved motifs exist in the sequence of CNLs. Further DNA sequence analysis suggests that CsCNL genes might be subject to the regulation of different miRNAs upon PM infection. By mining available RNA-seq data followed by real-time quantitative PCR (qRT-PCR) analysis, we characterized expression patterns of the CNL genes, and found that those genes exhibit a temporospatial expression pattern, and their expression is also responsive to PM infection, ethylene, salicylic acid, and methyl jasmonate treatment in cucumber plants. Finally, the CNL genes targeted by miRNAs were predicted in cucumber plants. Our results in this study provided some basic information for further study of the functions of the CNL gene family in cucumber plants.     

The Formation of Hollow Trait in Cucumber (Cucumis sativus L.) Fruit Is Controlled by CsALMT2

The hollow trait is crucial for commercial quality of cucumber (Cucumis sativus L.) fruit, and its molecular regulatory mechanism is poorly understood due to its environmental sensitivity. In the previous research, we obtained the hollow and the non-hollow materials of ecotype cucumbers of South China, which were not easily affected by the external environment through a systematic breeding method. In this study, first, we proposed to use the percentage of the hollow area as the criterion to compare the hollow characteristics between two materials, and to analyze the formation mechanism of early hollow trait from the perspective of cytology. The results showed that the hollow trait occurred in the early stage of fruit development, and formed with the opening of carpel ventral zipped bi-cell layer, which formed rapidly from 2 to 4 days, and then slowed to a constant rate from 14 to 16 days. Meanwhile, the different genetic populations were constructed using these materials, and fine mapping was performed by bulked segregant analysis (BSA) and kompetitive allele specific PCR (KASP) method. The Csa1G630860 (CsALMT2), encoding protein ALMT2, was determined as a candidate gene for regulating the hollow trait in fruit. Furthermore, the expression profile of CsALMT2 was analyzed by qRT-PCR and fluorescence in situ hybridization. The expression of CsALMT2 had obvious tissue specificity, and it was abundantly expressed in the ovule development zone inside the fruit. In the hollow material of cucumber fruit, the expression of CsALMT2 was significantly downregulated. The subcellular localization in tobacco leaves indicated that CsALMT2 was distributed on the plasma membrane. In conclusion, in this study, for the first time, we found the regulatory gene of hollow trait in cucumber fruit, which laid the foundation for subsequent research on the molecular mechanism of hollow trait formation in cucumber fruit, and made it possible to apply this gene in cucumber breeding.     

A Large-Scale Genomic Association Analysis Identifies the Candidate Genes Regulating Salt Tolerance in Cucumber (Cucumis sativus L.) Seedlings

Salt stress seriously restricts plant growth and development, affects yield and quality, and thus becomes an urgent problem to be solved in cucumber stress resistance breeding. Mining salt tolerance genes and exploring the molecular mechanism of salt tolerance could accelerate the breeding of cucumber germplasm with excellent salt stress tolerance. In this study, 220 cucumber core accessions were used for Genome-Wide Association Studies (GWAS) and the identification of salt tolerance genes. The salinity injury index that was collected in two years showed significant differences among the core germplasm. A total of seven loci that were associated with salt tolerance in cucumber seedlings were repeatedly detected, which were located on Chr.2 (gST2.1), Chr.3 (gST3.1 and gST3.2), Chr.4 (gST4.1 and gST4.2), Chr.5 (gST5.1), and Chr.6 (gST6.1). Within these loci, 62 genes were analyzed, and 5 candidate genes (CsaV3_2G035120, CsaV3_3G023710, CsaV3_4G033150, CsaV3_5G023530, and CsaV3_6G009810) were predicted via the functional annotation of Arabidopsis homologous genes, haplotype of extreme salt-tolerant accessions, and qRT-PCR. These results provide a guide for further research on salt tolerance genes and molecular mechanisms of cucumber seedlings.     

Relationship between Key Environmental Factors and the Architecture of Fruit Shape and Size in Near-Isogenic Lines of Cucumber (Cucumis sativus L.)

Fruit shape and size are complex traits influenced by numerous factors, especially genetics and environment factors. To explore the mechanism of fruit shape and size development in cucumber, a pair of near-isogenic lines (NIL) Ln35 and Ln37 were used. The fruit length and diameter, cell length and diameter, and related gene expression were measured. Both the fruit length, diameter, and cell length and diameter showed sigmate curves in the two lines. The cell length and diameter were significantly positively correlated with fruit length and diameter both in two lines. The expression of CsACS2 and CsLNG showed significant positive correlations with fruit length and diameter increment in Ln35, and there was no correlation in Ln37. Furthermore, there were significant positive correlations between fruit size and thermal effectiveness (TE), as well as between fruit size and photosynthetic active radiation (PAR), both in two lines. Two models using logistic regression were formulated to assess the relationships among fruit length and diameter in Ln35 and Ln37, respectively, based on thermal effectiveness and photosynthetic active radiation (TEP). The coefficient R² values of the models were 0.977 and 0.976 in Ln35, and 0.987 and 0.981 in Ln37, respectively. The root mean square error (RMSE) was 12.012 mm and 4.338 mm in Ln35, and 5.17 mm and 7.082 mm in Ln37, respectively, which illustrated the accurate and efficient of these models. These biologically interpreted parameters will provide precision management for monitoring fruit growth and forecasting the time of harvesting under different temperatures and light conditions.     

Role of cucurbitacin C in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis sativus L.)

Cucurbitacins are bitter triterpenoid compounds that are toxic to most organisms and occur widely in wild and cultivated Cucurbitaceae. The only cucurbitacin identified in Cucumis sativus is cucurbitacin C. The bitter taste of cucumber has been correlated with resistance to the spider mite Tetranychus urticae, but a quantitative relationship has not been established. We determined the spider mite resistance and cucurbitacin C content in the dihaploid progeny derived from the F₁ generation of a cross between a bitter, spider-mite-resistant cucumber line and a bitter-free, spider-mite-susceptible line. The ratio of the number of bitter to bitter-free dihaploids conformed to the expected 1:1 ratio, based on a monogenic segregation pattern. Genetic analysis ascribed 69% of the variance of the difference in spider mite survival rate to the bitterness locus. Within the group of bitter dihaploids, cucurbitacin C content was significantly correlated with spider mite resistance. Thus, a quantitative relationship between cucurbitacin C content and spider mite resistance could be established.     

CsSWEET2, a Hexose Transporter from Cucumber (Cucumis sativus L.), Affects Sugar Metabolism and Improves Cold Tolerance in Arabidopsis

Sugars, which are critical osmotic compounds and signalling molecules in plants, and Sugars Will Eventually be Exported Transporters (SWEETs), which constitute a novel family of sugar transporters, play central roles in plant responses to multiple abiotic stresses. In the present study, a member of the SWEET gene family from cucumber (Cucumis sativus L.), CsSWEET2, was identified and characterized. Histochemical analysis of β-glucuronidase expression in transgenic Arabidopsis plants showed that CsSWEET2 is highly expressed in the leaves; subcellular localization indicated that CsSWEET2 proteins are localized in the plasma membrane and endoplasmic reticulum. Heterologous expression assays in yeast demonstrated that CsSWEET2 encodes an energy-independent hexose/H⁺ uniporter that can complement both glucose and fructose transport deficiencies. Compared with wild-type Arabidopsis plants, transgenic Arabidopsis plants overexpressing CsSWEET2 had much lower relative electrolyte leakage levels and were much more resistant to cold stress. Sugar content analysis showed that glucose and fructose levels in the transgenic Arabidopsis plants were significantly higher than those in the wild-type plants. Taken together, our results suggest that, by mediating sugar metabolism and compartmentation, CsSWEET2 plays a vital role in improving plant cold tolerance.