A 2-bp deletion in the protein kinase domain region of the ERECTA-like receptor kinase gene in cucumber results in short internode phenotype.

Cucumber varieties with shortend internodes require less space than regular vining varieties, thus have great significance for germplasm improvement. Here, we found a novel spontaneous cucumber mutant si107 that exhibited short intenodes (si), smaller leaves, fruits, and seeds. The decrease in longitudinal cell length led to the shortened internodes of si107. The genetic analysis revealed a single recessive gene si-2 that was responsible for the mutation. Through multiple lines of evidence, we demonstrated that CsSI is the possible candidate gene for si-2, which encodes an ERECTA leucine-rich repeat receptor-like kinase. The shortened internode in si107 is attributed to a 2-bp deletion in the protein kinase domain region of this gene. The expression of CsSI was higher in the internodes, petioles, and fruit peels of si107 than in the wild type (WT). The transcriptome analysis between the si107 mutant and WT indicated that differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway, in which auxin signal genes comprised the largest group, and all were downregulated in si107. Phytohormone quantitation confirmed that endogenous auxin levels in the stems of si107 were decreased. Our results provide new insights into the molecular mechanisms underlying the internode length control in cucumber.

Comparative Biochemical and Transcriptomic Analyses Provide New Insights into Phytoplasma Infection Responses in Cucumber.

Flat stem and witches' broom phytoplasma-like symptoms in the cucumber inbred line C17 were observed in a greenhouse at Yangzhou University, China for three consecutive planting seasons; these symptoms resulted in a decreased yield. To better understand the cause of these symptoms, 16S rRNA PCR, plant hormones, mineral elements, and RNA-seq profiling were performed using symptomatic and normal stem samples. The results showed that the causal agent was classified as the Candidatus phytoplasma asteris strain, a plant pathogenic prokaryote that could not be cultured in vitro. Measurement of plant hormones showed that auxin, salicylic acid, and jasmonic acid contents were significantly increased, whereas that of ethylene's immediate biosynthetic precursor, 1-aminocyclopropane-1-carboxylic acid, was decreased in the phytoplasma-infected stems compared with the healthy stems. Furthermore, measurement of mineral element composition showed that magnesium, calcium, sodium, iron, and zinc concentrations significantly changed in the phytoplasma-infected cucumber stems compared with the uninfected stems. Comparative RNA-seq identified 253 differentially expressed genes, including 179 upregulated and 74 downregulated genes. Further analyses suggested that genes related to phenylpropanoid biosynthesis, phenylalanine metabolism, and plant hormone signal transduction contributed to phytoplasma infection. Taken together, this study presents the first in-depth assessment of disease symptoms and biochemical content of cucumber stems known to be infected with phytoplasma.

Characterization of Differences in the Composition and Content of Volatile Compounds in Cucumber Fruit.

The cucumber is characterized by the presence of a wide range of volatile organic compounds (VOCs), which are recognized as the main responsible for its unique flavor. However, research on the types and contents of VOCs in different cucumber cultivars remains fragmentary. Here, using an automatic headspace solid-phase microextraction coupled with the gas chromatography-mass spectrometry method, the VOCs were analyzed in three representative cucumber cultivars, including YX, KX, and GX, with the best, middle, and worst flavor quality, respectively, which were selected from 30 cultivars after flavor quality evaluation. Principal component analysis revealed that the six biological replicates were grouped, indicating high reliability of the data. A total of 163 VOCs were detected. There were 28 differential VOCs in YX compared to GX, 33 differential VOCs in YX compared to KX, and 10 differential VOCs in KX compared to GX. Furthermore, K-means clustering analysis showed that 38 of the 43 no-overlapping differential VOCs were represented by the most abundant compounds detected in YX. The prevailing VOCs in YX included: hydrocarbons, aldehydes, and ketones. The data obtained in the present study extend our understanding the impact of cultivars on VOCs in cucumber and will help facilitate targeted breeding.

Proteomic Analysis of Fusarium oxysporum-Induced Mechanism in Grafted Watermelon Seedlings.

Grafting can improve the resistance of watermelon to soil-borne diseases. However, the molecular mechanism of defense response is not completely understood. Herein, we used a proteomic approach to investigate the molecular basis involved in grafted watermelon leaf defense against Fusarium oxysporum f.sp. niveum (FON) infection. The bottle gourd rootstock-grafted (RG) watermelon seedlings were highly resistant to FON compared with self-grafted (SG) watermelon plants, with a disease incidence of 3.4 and 89%, respectively. Meanwhile, grafting significantly induced the activity of pathogenesis-related proteases under FON challenge. Proteins extracted from leaves of RG and SG under FON inoculation were analyzed using two-dimensional gel electrophoresis. Thirty-nine differentially accumulated proteins (DAPs) were identified and classified into 10 functional groups. Accordingly, protein biosynthetic and stress- and defense-related proteins play crucial roles in the enhancement of disease resistance of RG watermelon seedlings, compared with that of SG watermelon seedlings. Proteins involved in signal transduction positively regulated the defense process. Carbohydrate and energy metabolism and photosystem contributed to energy production in RG watermelon seedlings under FON infection. The disease resistance of RG watermelon seedlings may also be related to the improved scavenging capacity of reactive oxygen species (ROS). The expression profile of 10 randomly selected proteins was measured using quantitative real-time PCR, among which, 7 was consistent with the results of the proteomic analysis. The functional implications of these proteins in regulating grafted watermelon response against F. oxysporum are discussed.

Analysis of QTL DM4.1 for Downy Mildew Resistance in Cucumber Reveals Multiple subQTL: A Novel RLK as Candidate Gene for the Most Important subQTL.

One of the biggest problems in cucumber cultivation is cucurbit downy mildew (DM), caused by the obligate biotroph Pseudoperonospora cubensis. Whereas DM in cucumber was previously efficiently controlled by the dm-1 gene from Indian cucumber accession PI 197087, this resistance was broken by new DM strains, prompting the search for novel sources of resistance. A promising source of resistance is the wild cucumber accession PI 197088. It was previously shown that DM resistance in this genotype inherits polygenically. In this paper, we put the focus on one of the QTL, DM4.1 that is located on chromosome 4. QTL DM4.1 was shown to consist of three subQTL: DM4.1.1 affected pathogen-induced necrosis, DM4.1.2 was shown to have an additive effect on sporulation, and DM4.1.3 had a recessive effect on chlorosis as well as an effect on sporulation. Near-isogenic lines (NILs) were produced by introgressing the subQTLs into a susceptible cucumber line (HS279) with good horticultural traits. Transcriptomic analysis revealed that many genes in general, and defense pathway genes in particular, were differentially expressed in NIL DM4.1.1/.2 compared to NIL DM4.1.3 and the susceptible parent HS279. This indicates that the resistance from subQTL DM4.1.1 and/or subQTL DM4.1.2 likely involves defense signaling pathways, whereas resistance due to subQTL DM4.1.3 is more likely to be independent of known defense pathways. Based on fine-mapping data, we identified the RLK gene CsLRK10L2 as a likely candidate for subQTL DM4.1.2, as this gene was found to have a loss-of-function mutation in the susceptible parent HS279, and was strongly upregulated by P. cubensis inoculation in NIL DM4.1.1/.2. Heterologous expression of this gene triggered necrosis, providing further evidence that this gene is indeed causal for subQTL DM4.1.2.

Construction of a high-density genetic linkage map and identification of gene controlling resistance to cucumber mosaic virus in Luffa cylindrica (L.) Roem. based on specific length amplified fragment sequencing.

Luffa cylindrica L. is a cash crop which has important health, medicinal and industrial value, but no high saturation genetic map has been constructed owing to a lack of efficient markers. Furthermore, no genes were reportedly responsible for CMV resistance in Luffa spp. Specific length amplified fragment sequencing (SLAF-seq) is a valuable tool for large-scale discovery of markers and genetic mapping. The present study reported the construction of a high-density genetic map and the mapping of CMV resistant genes by using an F2 population of 130 individuals and their two inbred line parents. A total of 271.01 Mb pair-end reads were generated. 100,077 high-quality SLAFs were detected, and 7404 of them were polymorphic. Finally, 3701 of the polymorphic markers were selected for genetic map construction, and 13 linkage groups were generated. The map spanned 1518.56 cM with an average distance of 0.41 cM between adjacent markers. Based on the newly constructed high-density map, one gene located on chromosome 1 (100.072-100.457 cM) was identified to regulate CMV resistance in L. cylindrica. A gag-polypeptide of LTR copia-type retrotransposon was predicted as the candidate gene responsible for CMV resistance in L. cylindrica. The high-density genetic map and the CMV resistant gene mapped and predicted in this study will be useful in future research.

CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum.

KEY MESSAGE: CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.

Transcriptome analysis of Luffa cylindrica (L.) Roem response to infection with Cucumber mosaic virus (CMV).

Cucumber mosaic virus (CMV) can cause serious losses in Luffa cylindrica (L.) Roem. Chemical application to control CMV is ineffective and environmentally unfriendly. The development of resistant hybrids is the best way to control CMV disease. Elucidating the virus-host interaction of CMV and molecular basis underlying Luffa spp. resistance against CMV would undoubtedly facilitate breeding for resistance against CMV disease. Transcriptome sequencing was used to analyze differentially expressed genes (DEGs) caused by CMV infection. A total of 138,336 unigenes were assembled, and 74,525 unigenes were annotated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the three major enrichment pathways (according to the p-values) were flavonoid biosynthesis, sulfur metabolism, and photosynthesis. Genes involved in basal defenses, probably R genes, were determined to be related to CMV resistance. Using quantitative real-time PCR, we validated the differential expression of 8 genes. A number of genes associated with CMV resistance were found in this study. This study provides transcriptomic information regarding CMV-Luffa spp. interactions and will shed light on our understanding of host-virus interactions.

Identification of browning-related microRNAs and their targets reveals complex miRNA-mediated browning regulatory networks in Luffa cylindrica.

As a non-coding and endogenous small RNA, MicroRNA (miRNA) takes a vital regulatory role in plant growth and development. Long-term storage and processing of many fruits and vegetables, including Luffa, are subject to influences from browning, a common post-harvest problem that adversely affects flavor, aroma, and nutritional value. The browning regulatory networks mediated by miRNA, however, remain largely unexplored. For a systematic identification of browning-responsive miRNAs and the targets, we built two RNA libraries from Luffa pulps of near-isogenic line, with resistant and sensitive browning characteristics respectively, and then sequenced them using Solexa high-throughput technology. We consequently identified 179 known miRNAs that represent 17 non-conserved miRNA families and 24 conserved families, as well as 84 potential novel miRNAs, among which 16 miRNAs (eight known and eight novel miRNAs) were found to exhibit significant differential expressions and were thus identified as browning-related miRNAs. We then studied those browning-responsive miRNAs and the corresponding targets with RT-qPCR and finally validated their expression patterns. The results revealed that the expression patterns are specific to plant development stages and the miRNAs are identified with 39 target transcripts, which involve in plant development, defense response, transcriptional regulation, and signal transduction. After characterizing these miRNAs and their targets, we propose a browning regulatory network model of miRNA-mediatation in this paper. The findings of the work are helpful for the understanding of miRNA-mediated regulatory mechanisms of browning in Luffa, and will facilitate genetic improvement of pulp characteristics in Luffa.

Impact of salicylic acid on the antioxidant enzyme system and hydrogen peroxide production in Cucumis sativus under chilling stress.

Salicylic acid (SA) is a naturally produced compound and has been implicated to play important roles in defense of plants against diverse biotic and abiotic stresses. To understand how SA functions in the tolerance of cucumber (Cucumis sativus) to chilling stress, endogenous SA levels in two different cultivars with opposite chilling responsiveness were quantified. Membrane integrity, including malondialdehyde (MDA) content and leakage of electrolyte, was also examined in SA-pretreated cucumber plants under chilling conditions. In addition, activities of the two antioxidant enzymes peroxidase (POD) and catalase (CAT) were quantified, and hydrogen peroxide (H2O2) production was investigated histochemically in SA-treated leaves under chilling temperature. Chilling stress resulted in greater induction of SA levels in the chilling-tolerant cultivar Changchun mici in both leaves and seeds compared to the chilling-sensitive one Beijing jietou, while the former one contained higher levels of SA than the latter one in the seeds under normal conditions. Pretreatment with SA diminished the increased electrolyte leakage and MDA content caused by chilling in the leaves of both cultivars, while much less MDA and electrolyte leakage were produced in Changchun mici compared to Beijing jietou. Moreover, exogenous application of SA increased significantly the POD and CAT activities and soluble protein content. Most importantly, exogenous SA treatment could eliminate the accumulation of H202 in leaves and cotyledons of both cultivars caused by chilling stress. The data clearly demonstrated that the chilling-tolerant cultivar displays a higher SA level than the chilling-sensitive one, and that exogenous SA can enhance the chilling tolerance ability, which might be achieved through modulating the antioxidant system in cucumber.

[Inheritance of parthenocarpy in monoecious cucumber (Cucumis sativus L.) under different eco-environments].

By using the mixed major gene plus polygene inheritance model of quantitative traits, a joint analysis of four-generations obtained after cross between two monoecious inbred lines was made to study the inheritance of parthenocarpy in monoecious cucumber in Jiangning (Nanjing) and Changli (Hebei). The interactions between genotype and environment were detected, and the inheritance of parthenocarpy in the monoecious cucumber was fitted in E-1-1 model and controlled by two additive-dominant-epistatic major genes and additive-dominant polygene under different eco-environments. The F1 tendency and genetic parameters of the parthenocarpy were different, and the major gene heritability of F2 ranged from 42.1% to 97.5%, which was mainly due to the differences in sunlight intensity and air temperature during fruit-setting period at the two locations. It was suggested that the parents should be highly parthenocarpic in breeding program, and the characterization of parthenocarpy should be conducted under different eco-environments.