A bHLH transcription factor,CsSPT,regulates high-temperature resistance in cucumber

High-temperature stress threatens the growth and yield of crops. Basic helix-loop-helix(bHLH) transcription factors(TFs) have been shown to play important roles in regulating high-temperature resistance in plants. However, the bHLH TFs responsible for high-temperature tolerance in cucumbers have not been identified. We used transcriptome profiling to screen the high temperature-responsive candidate bHLH TFs in cucumber. Here, we found that the expression of 75 CsbHLH genes was altered under high-temperature stress. The expression of the CsSPT gene was induced by high temperatures in TT(Thermotolerant) cucumber plants. However, the Csspt mutant plants obtained by the CRISPR-Cas9 system showed severe thermosensitive symptoms, including wilted leaves with brown margins and reduced root density and cell activity.The Csspt mutant plants also exhibited elevated H_(2)O_(2) levels and down-regulated photosystem-related genes under normal conditions.Furthermore, there were high relative electrolytic leakage(REC), malondialdehyde(MDA), glutathione(GSH), and superoxide radical(O_(2)^(·-)) levels in the Csspt mutant plants, with decreased Proline content after the high-temperature treatment. Transcriptome analysis showed that the photosystem and chloroplast activities in Csspt mutant plants were extremely disrupted by the high-temperature stress compared with wildtype(WT) plants. Moreover, the plant hormone signal transduction, as well as MAPK and calcium signaling pathways were activated in Csspt mutant plants under high-temperature stress. The HSF and HSP family genes shared the same upregulated expression patterns in Csspt and WT plants under high-temperature conditions. However, most bHLH, NAC, and bZIP family genes were significantly down-regulated by heat in Csspt mutant plants. Thus, these results demonstrated that CsSPT regulated the high-temperature response by recruiting photosynthesis components, signaling pathway molecules, and transcription factors. Our results provide important insights into the heat response mechanism of CsSPT in cucumber and its potential as a target for breeding heat-resistant crops.

Transcriptomic analysis on cucumber tendril reveals GLRs play important roles in thigmotropism and thigmomorphogenesis

Thigmotropism and thigmomorphogenesis are two related and pervasive processes that play crucial roles in plant adaptation to the environment.However,there have been few investigations into the molecular regulatory mechanisms of these phenomena.Cucumber(Cucumis sativus L.)tendrils are ideal material for studying thigmotropism and thigmomorphogenesis because they display a combination of the two processes.Here,we generated the transcriptome profiles of cucumber tendrils at the young,stretch,and coiling stages.Genes related to receptor proteins,transmembrane transport,and ion transport were significantly enriched among those differentially expressed between stages.Pharmacological assays illustrated that three GLUTAMATE RECEPTOR(GLR)genes might play a vital function in perceiving or transducing touch stimulation signals.Comparing the transcriptomes of tendrils and roots after touch stimulation,we found that genes related to extracellular stimulus and xyloglucan metabolism might have conserved functions in the regulation of thigmomorphogenesis.The transcriptome atlas of thigmotropism and thigmomorphogenesis of cucumber tendrils constructed in this study will help further elucidate the molecular mechanisms behind these processes.

The intrinsic developmental age signal defines an age-dependent climbing behavior in cucumber

The tendril is a climbing organ in cucurbits and functions in physical support and to avoid shading by neighboring vegetation.However,how cucurbits produce tendrils to obtain climbing ability is largely unknown.In this study,tendril phenotypes were investigated during different developmental stages.Our results revealed that tendril growth exhibited an age-dependent pattern in cucurbits.Tendril growth was inhibited,and the tendril was formed as a short tendril[nonfunctional tendril(nonF-tendril),approximately 0.1 cm]during the seedling stage.In contrast,enhanced cell proliferation and cell expansion led to rapid elongation of the tendril during the climbing stage,and the tendril formed as a functional tendril(F-tendril,approximately 30 cm)to obtain climbing ability.RT-qPCR detection showed that age-dependent tendril growth correlated negatively with the abundance of the conserved age regulator CsmiR156.Defoliation induced CsmiR156 to inhibit CsSPLs,and F-tendril formation and climbing ability were delayed in defoliated cucumbers,which confirmed the role of CsmiR156 in regulating tendril growth in vivo.Additionally,exogenous gibberellin(GA)treatment showed that GA positively regulated tendril growth,and RT-qPCR detection showed that the GA bio-synthetic genes and metabolic genes were affected by age pathway,suggesting that the age pathway depended on GA bio-synthetic and metabolic pathway to regulate cell expansion to determine tendril growth.In summary,our work reveals that change in tendril type is an important marker of phase transition in cucumber,and tendril growth is regulated by an intrinsic developmental age signal,ensuring that the cucumber obtains climbing ability at a suitable age.

The bHLH transcription factor CsPIF4 positively regulates high temperature-induced hypocotyl elongation in cucumber

High temperature-induced hypocotyl elongation is a typical thermomorphogenesis trait that may significantly affect early seedling growth and subsequent crop yield.The ambient temperature and endogenous auxin are two critical factors that regulate hypocotyl growth.However,the mechanism of temperature and auxin integration in horticultural plants remains poorly understood.In this study,the roles of the basic helix-loop-helix transcription factor CsPIF4 in regulating auxin biosynthesis genes and the auxin content in the hypocotyl of cucumber(Cucumis sativus L.)seedlings under high temperature were investigated.qRT-PCR and in situ hybridization analysis revealed that expression of CsPIF4 was enhanced in the epidermis and vascular bundles in the hypocotyl of cucumber seedlings in response to high temperature.qRT-PCR and HPLC analysis showed that CsPIF4 positively regulated transcription of the auxin biosynthesis gene CsYUC8 and the auxin content in the hypocotyl under high temperature(35℃).The CRISPR/Cas9-mediated knockout of CsPIF4 resulted in a shorter hypocotyl compared with that of the wild type,together with decreased expression of CsYUC8 and lower auxin content in response to high temperature.Furthermore,biochemical assays showed that CsPIF4 could bind directly to the G-box motif of the CsYUC8 promoter and thereby activate CsYUC8 expression.These findings provide insight into the molecular mechanism of high temperature-mediated hypocotyl elongation in cucumber.

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis,photosynthesis mechanism and chloroplast development.Herein,we identified a spontaneous mutant,yc412,in cultivated cucumber that exhibited yellow cotyledons.The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure,and reduced photosynthetic capacity and pigment content.Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping,we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3.By resequencing and molecular cloning,we showed that Csyc is a potential candidate gene,which encodes a yellow stripe-like (YSL) transporter.The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412.Compared to YZU027A (WT),the expression of Csyc was significantly downregulated in the cotyledons of yc412.Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves,mainly by suppressing the chlorophyll content.Furthermore,a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons.Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

Sea Cucumber Fishery in Aurora, Philippines

This study was conducted to provide a preliminary assessment of the sea cucumber fishery in Aurora, Philippines. This was conducted from February to June 2013. All sea cucumber gatherers and traders, obtained through snowball sampling, in each identified sea cucumber fishery area in six coastal municipalities (Baler, Dipaculao, Dinalungan, Casiguran, Dilasag and Dingalan) were interviewed using a structured interview schedule and focused group discussions (FGD). Results revealed that gathering sea cucumbers were accomplished by handpicking, skin diving or compressor diving. A total of 15 commercial species were regularly gathered. Out of this, three species are of very high economic value (≥Php 4000/kg) (Holothuria nobilis, Holothuria scabra, and Actinopyga lecanora). The average number of kilograms harvested per hour per trip ranges from 0.21 (Holothuria fuscopunctata) to 2.71 (Holothuria scabra), while the average size of commonly gathered sea cucumber varies among species with Holothuria lecanora showed an average length (29.26 cm) greater than recorded 24 cm. Processing mainly involves washing, slicing, boiling, drying and packing. Processed sea cucumbers or trepan were sold to middlemen or direct buyers who usually offered higher prices (36.36% up to 69.20%) than the former. Problems encountered by sea cucumber gatherers and traders include a decline in total catch, a shift in area of collection from intertidal zones to deeper waters, an increase in the number of hours spent in finding and gathering sea cucumbers, the use of compressors in gathering, the decline of larger species and gathering of smaller sizes in huge amount, the absence of conservation program for sea cucumber, and the lenient implementation of government policies with regards to coastal resources management.

A novel mutation in ACS11 leads to androecy in cucumber

Sex determination in plants gives rise to unisexual flowers. A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis. Androecious cucumber(Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production. The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding. Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate(EMS)-mutagenized library of the cucumber inbred line ‘406’. Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11(ACS11) conferred androecy. The mutation caused an amino acid change from serine(Ser) to phenylalanine(Phe) at position 301(S301F). In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity. This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.

Insights into flavonoid biosynthesis during cucumber fruit peel coloration based on metabolite profiling and transcriptome analyses

The fruit peel color is a crucial trait of cucumber.To better understand the molecular mechanisms underlying cucumber peel coloration,we compared the UPLC-ESI-MS/MS-based flavonoid metabolomic and RNA sequencing-based transcriptomic profiling of the brown peeled cucumber line‘PW’at six developmental stages.A total of 210 flavonoid metabolites were identified.Of which,117 flavonoid metabolites were differentially accumulated.In this study,weighted gene co-expression network analysis combined with Kyoto Encyclopedia of Gene and Genomes enrichment analysis revealed key genes coding for seven enzymes and eight transcription factors(TFs)associated with flavonoid biosynthesis.Among them,the R2R3MYB CsaV3_4G001130 is the best candidate gene that is responsible for controlling mature fruit colors in cucumber.Sanger sequencing revealed one nonsynonymous SNP in the exon of CsaV3_4G001130 among the selected 11 cucumber lines,which introduced a premature stop codon,generating a truncated protein in pale yellow or creamy peeled fruits.Yeast two-hybrid assays showed a direct interaction of CsaV3_4G001130 with the bHLH TF CsaV3_1G002260 and the WD40 protein CsaV3_5G001800.However,the interactions were influenced by the nonsynonymous SNP we identified.Our finding revealed that the integrated transcriptome and metabolome analysis further demonstrated that the abundance of some pigmented flavonoids(especially anthocyanins and chalcones)contributed to the coloration of‘PW’fruits.These findings pave the way for elucidation of flavonoid biosynthesis and improvement of cucumber peel color in the future.

Genome-wide association study reveals candidate genes for gummy stem blight resistance in cucumber

Gummy stem blight(GSB),caused by Didymella bryoniae,is a serious fungal disease that leads to decline in cucumber yield and quality.The molecular mechanism of GSB resistance in cucumber remains unclear.Here,we investigated the GSB resistance of cucumber core germplasms from four geographic groups at the seedling and adult stages.A total of 9 SNPs related to GSB resistance at the seedling stage and 26 SNPs at the adult stage were identified,of which some are co-localized to previously mapped Quantitative trait loci(QTLs)for GSB resistance(gsb3.2/gsb3.3,gsb5.1,and gsb-s6.2).Based on haplotype analysis and expression levels after inoculation,four candidate genes were identified within the region identified by both Genome-wide association study(GWAS)and previous identified QTL mapping,including Csa3G129470 for gsb3.2/gsb3.3,Csa5G606820 and Csa5G606850 for gsb5.1,and Csa6G079730 for gsb-s6.2.The novel GSB resistant accessions,significant SNPs,and candidate genes facilitate the breeding of GSB resistant cucumber cultivars and provide a novel idea for understanding GSB resistance mechanism in cucumber.

An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

Genetics,resistance mechanism,and breeding of powdery mildew resistance in cucumbers(Cucumis sativus L.)

Cucumber is an important vegetable worldwide,and powdery mildew(PM)is a common and serious disease of cucumbers.Breeding disease-resistant cucumber varieties is the most advantageous strategy to control this disease.In recent years,exploration and identification of cucumber PM resistance genes have achieved great advancement,and many genes have been cloned and verified using different methods.However,the resistance mechanism of cucumber PM is still unclear,and many ambiguities need to be elucidated urgently.In this review,we summarized the research advances in PM resistance in cucumbers,including genetic analysis,quantitative trait locus mapping,map-based cloning,transcriptomics,mlo-mediated PM resistance,and mining of noncoding RNAs involved in resistance.Finally,the research directions and the problems that need to be solved in the future were discussed.

Comparative Transcriptome Analyses Reveal Genes Related to Spine Development in Cucumber (Cucumis sativus)

Fruit spine is an important quality trait of cucumber.To better understand the molecular basis of cucumber spine development and function,RNA-Seq was performed to identify differentially expressed genes(DEGs)in fruit spines of different development stages,namely,8 days before anthesis(SpBA8),anthesis(SpA)and 8 days after anthesis(SpAA8).Stage-wise comparisons obtained 2,259(SpBA8 vs.SpA),4,551(SpA vs.SpAA8),and 5,290(SpBA8 vs.SpAA8)DEGs.All the DEGs were classified into eight expression clusters by trend analysis.Among these DEGs,in addition to the Mict,Tril,CsTTG1,CsMYB6,NS,and Tu genes that have been reported to regulate fruit spine formation,we found that the CsHDG11,CsSCL8,CsSPL8,CsZFP6 and CsZFP8 may also be involved in spine development in cucumber.Our study provides a theoretical basis for further research on molecular mechanisms of spine development in cucumber.

Underwater Sea Cucumber Target Detection Based on Edge-Enhanced Scaling YOLOv4

Sea cucumber detection is widely recognized as the key to automatic culture.The underwater light environment is complex and easily obscured by mud,sand,reefs,and other underwater organisms.To date,research on sea cucumber detection has mostly concentrated on the distinction between prospective objects and the background.However,the key to proper distinction is the effective extraction of sea cucumber feature information.In this study,the edge-enhanced scaling You Only Look Once-v4(YOLOv4)(ESYv4)was proposed for sea cucumber detection.By emphasizing the target features in a way that reduced the impact of different hues and brightness values underwater on the misjudgment of sea cucumbers,a bidirectional cascade network(BDCN)was used to extract the overall edge greyscale image in the image and add up the original RGB image as the detected input.Meanwhile,the YOLOv4 model for backbone detection is scaled,and the number of parameters is reduced to 48%of the original number of parameters.Validation results of 783images indicated that the detection precision of positive sea cucumber samples reached 0.941.This improvement reflects that the algorithm is more effective to improve the edge feature information of the target.It thus contributes to the automatic multi-objective detection of underwater sea cucumbers.

Effects of Different Irrigation Amounts on Water Consumption and Water Use Efficiency of Greenhouse Cucumber

[Objective] This study was to investigate the effects of different irrigation amount on water consumption and water use efficiency of greenhouse cucumber.[Method]Under the condition of drip irrigation with different water amounts in sunlight greenhouse of the arid areas in Ningxia,the soil water was measured and the water consumption of crop was calculated.[Result]When irrigation amount was 563 mm,the water consumption as a whole gradually increased with the delay of growth period,reached peak during the thriving stage of fruit setting,and then gradually declined;in each treatment,the daily water consumption increased with the increasing of irrigation amount during each growth period.However,the consumption of soil moisture reduced with the significant increase of irrigation.563 mm of irrigation amount could meet the water requirements of cucumber and began to add water to soil,and water utilization efficiency could reach 33.4 kg/m3.[Conclusion]The research had provided theoretical basis for water management in the production process of greenhouse cucumber.

Virus Movement Protein Gene Mediated Resistance Against Cucumber Mosaic Virus Infection

Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.

Construction of Fusion Expression Vector of α-galactosidase-EGFP in Cucumber

[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.

ACC Synthase Gene (ACSG) as a Possible Molecular Marker for Female Lines in Cucumber

利用本实验室根据已知序列分离得到的ACC合酶基因 (ACSG)为探针对不同黄瓜 (CucumissativusL .)品种(系 )的基因组DNA进行Southern杂交 ,初步分析了该基因与黄瓜性别表型之间的相关性。发现在所检测的 10个不同品系中 ,ACSG与雌性系表型之间存在明显的相关关系 ,而且这种相关关系在不同的实验中具有良好的重复性。ACSG基因可能是鉴定黄瓜雌性系的一个分子标记。

Study on the Activity of Herbal Extract to Kill Root-knot Nematode of Cucumber Seedlings Stage

Present mature plants hydroponic technology was used,combined with some excellent characteristics,such as growth conditions was easy to control and process of root growth was easy to continuously observe,the nematicidal activity of 5 kinds of Chinese herbs extracts and the compound solution of Avermectin,with strong contact toxicity effect indoor,was systematically studied and investigated the affection on the root-knot nematode parasitized on the cucumber seeding stage. It is found that under the premise of no influence on root growth of cucumber,extracts from Picrorhiza scrophulariiflora and Punica granatum showed strong prevention and nematicidal activity,and had the similar efficacy of Avermectin; while the extracts from Cibotium barometz,Aucklandia lappa Decne and Fructus cnidii showed low nematicidal activity and various degrees inhibition effect on plant growth.

Evaluation of Seed Oil Content in Cucumber Germplasm

With 35 different ecological types of cucumber germplasm resources as test materials, the soxhlet extraction was used to extract the crude fat from the cu- cumber seeds to analyze the seed oil content, with the aim to lay foundations for the application and new variety breeding related with cucumber seed oil. The results showed that there were significant differences in seed oil content between different cucumber varieties with the amplitude of variation rangeing from 31.03% to 40.59%, among which the seed oil content of south China cucumber was the highest of 40.59%, while the seed oil content of north China cucumber was the lowest of 31.03%. Four va.rieties with oil content of greater than 38.50% and four varieties with oil content of less than 32.01% werescreened out.

Construction of Cucumber Powdery Mildew Early Warning System in Solar Greenhouse Based on Internet of Things

ln order to explore the design and construction of cucumber powdery mildew warning system in solar greenhouse, internet of things technology was used to conduct the real-time dynamic monitoring of the incidence of cucumber powdery mildew and cucumber growth environment in solar greenhouse. The growth environ-ment included temperature and humidity of air and soil. Logistic regression model was used to construct cucumber powdery mildew warning model. The results showed that humidity characteristic variable （maximum air humidity） and temperature characteristic variable （maximum air temperature） had significant effects on the inci-dence probability of cucumber powdery mildew in solar greenhouse. And it was fea-sible to construct cucumber powdery mildew warning system in solar greenhouse with internet of things.