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.

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.

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.

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.

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.

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.

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.

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.

Proteomic Analysis of Sea Cucumber (Holothuria leucospilota) from the South China Sea

[Objectives]Proteomics methods were used to analyze the total proteome of Holothuria leucospilota in the South China Sea,so as to provide a theoretical basis for elucidating the medicinal material basis of sea cucumber proteins and peptides.[Methods]H.leucospilota was collected from the South China Sea.Label-free proteomics methods were used to detect the protein profile of enzymatically hydrolyzed H.leucospilota total protein,and identify sea cucumber protein and peptide sequences,and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)bioinformatics analysis software were used to analyze significantly enriched proteins.The String database was used to construct a protein-protein interaction network of significantly enriched H.leucospilota proteins(PPI network).[Results]Proteomics identified 1200 sea cucumber proteins and 2967 peptides.GO analysis and KEGG analysis showed that the four significantly-enriched sea cucumber proteins hsc70,COX,CYTB and rbbp4 participated in 8 metabolic pathways,and COX1 and CYTB among them were significantly related to cardiac muscle contraction,oxidative phosphorylation and metabolic pathways.The PPI network analysis showed that the 13 significantly-enriched sea cucumber proteins had close protein-protein interaction relationships.[Conclusions]This study used proteomics to conduct preliminary identification and research on the proteins and peptides of H.leucospilota,providing a scientific basis for the application of H.leucospilota proteins and peptides in the fields of functional health food and drug research and development.

Yield and Quality Response of Cucumber to Irrigation and Nitrogen Fertilization Under Subsurface Drip Irrigation in Solar Greenhouse

The aims of this research were to compare subsurface drip irrigation scheduling and nitrogen fertilization rates in cucumber, and evaluate yield and quality of cucumber fruit, water （WUE）, irrigation water （IWUE）, and nitrogen use （NUE） efficiencies in the solar greenhouse in Southwest China. The irrigation water amounts were determined based on the 20 cm diameter pan （Ep） placed over the crop canopy, and cucumber plant was subjected to three irrigation water levels （I1, 0.6 Ep; I2, 0.8 Ep; and I3, 1.0 Ep） in interaction with three nitrogen fertilization levels （N1, 300 kg ha-1; N2, 450 kg ha-1; and N3, 600 kg ha-1）. The results showed that the cucumber fruit yield increased with the improvement of irrigation water. Irrigation water increased yields by increasing the mean weight of the fruits, and also by increasing fruit number. But the highest values of IWUE and WUE were obtained from I2 treatment. NUE significantly decreased with the improvement of N application, but increased by irrigating more water. The quality of cucumber fruit decreased with the improvement irrigation water and nitrogen fertilization. In conclusion, the optimum irrigation level and nitrogen fertilizer application level for cucunber under subsurface drip irrigation in the solar greenhouse in Southwest China were 0.8 Ep and 450 and 600 kg ha-1, respectively.

Effect of cypermethrin insecticide on the microbial community in cucumber phyllosphere

Cucumber (Cucumis sativus) is one of the most widely used vegetable in the world,and different pesticides have been extensively used for controlling the insects and disease pathogens of this plant.However,little is known about how the pesticides affect the microbial community in cucumber phyllosphere.This study was the first attempt to assess the impact of pyrethroid insecticide cyperemethrin on the microbial communities of cucumber phyllosphere using biochemical and genetic approaches.Phospholipid fatty ac...

Development strategies for the sea cucumber industry in China

The sea cucumber, Apostichopus japonicus, is an important marine aquaculture species in China. After nearly thirty years of development, the production of A. japonicus has become commercially lucrative and successful. In this report, current advances in sea cucumber industry are addressed in terms of the basic biology, culturing methods, and health care bene?ts. Next, the challenges restricting development of the sea cucumber industry are discussed, including weaknesses in the basic biological research, the problem of germplasm degradation, environmental stress caused by global climate change, and food safety problems. Finally, several strategies are presented that might contribute to sustainable development of the sea cucumber industry. These strategies include advances in genome studies, behavioral studies, selective breeding, ecological culture technologies, reforms in food safety management, and the development of health care functions based on contemporary medical practices. Thus, our work provides new insights into how to explore the sustainable development of the sea cucumber industry in the future.

Effects of phosphate solubilization and phytohormone production of Trichoderma asperellum Q1 on promoting cucumber growth under salt stress

Salinity is one of the major abiotic stresses limiting crop growth and yield.This study investigated the underlying mechanisms of Trichoderma asperellum Q1 in promoting cucumber growth under salt stress, including the abilities of the strain to solubilize phosphate and to produce phytohormone.The results showed that T.asperellum Q1 could solubilize inorganic or organic phosphate and the activities of phosphatases and phytase could be detected in the culture supernatant.In hydroponic experiments, the growth of cucumber seedlings was increased in the hydroponic system treated by culture filtrate of strain Q1 with tricalcium phosphate or calcium phytate under salt stress.This strain also exhibited the ability to produce indole acetic acid（IAA）, gibberellic acid（GA） and abscisic acid（ABA） in liquid medium without any inducers.The levels of those three phytohormones in cucumber seedling leaves also increased after inoculated with this strain, along with increased root growth and root activities of the plant.These results demonstrated the mechanisms of T.asperellum Q1 in alleviating the suppression effect of salt stress involving the change of phytohormone levels in cucumber plant and its ability of phosphate solubilization.

Autophagy Plays a Potential Role in the Process of Sea Cucumber Body Wall “Melting” Induced by UV Irradiation

The changes of tissue appearances and structures in the process of UV-induced ＂melting＂ for sea cucumber （Stichopus japonicus） body wall were studied. And the localization and determination of acid phosphatase （ACP）, Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi＇s body and endoplasmic reticulure swelled, curled, and eventually double- or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions, It indicates that autophagy plays a potential role in the ＂melting＂ process for sea cucumber body wall induced by UV irradiation.

Gene Expression and Activities of SOD in Cucumber Seedlings Were Related with Concentrations of Mn^(2+),Cu^(2+),or Zn^(2+) Under Low Temperature Stress

Effects of increasing Mn^2＋, Cu^2＋, or Zn^2＋ on SOD expressions were studied in cucumber seedlings under low temperature stress. Both gene expressions and activities of Cu/Zn-SOD and Mn-SOD in cucumber seedling leaves were induced by increasing Mn^2＋, Cu^2＋, or Zn^2＋ under low temperature stress, especially 48 h afterwards. The activities of Cu/Zn-SOD and Mn-SOD at 0 and 48 h after treatment were in accordance with their gene expression levels, which implied that the transcriptional regulation plays key roles in regulating their activities at the early stage of low temperature stress. Gene expressions of Cu/Zn-SOD and Mn-SOD declined at 96 h, but Cu/Zn-SOD and Mn-SOD activities still remain high, which suggested that Cu/Zn-SOD and Mn-SOD activities might be regulated by other factors after transcription at the later stage of low temperature stress. Therefore, we concluded that the increasing Mn^2＋, Cu^2＋, or Zn^2＋ could increase the capacity of scavenging ROS in cucumber seedlings under low temperature stress by inducing gene expressions of Cu/ Zn-SOD and Mn-SOD, elevating activities of Cu/Zn-SOD, Mn-SOD, or regulating other factors after transcription.

Genome-wide identification, characterization, and expression analysis of the SWEET gene family in cucumber

SWEETs （sugars will eventually be exported transporters） are a novel class of recently identified sugar transporters that play important roles in diverse physiological processes. However, only a few species of the plant SWEETgene family have been functionally identified. Up till now, there has been no systematic analysis of the SWEETgene family in Cucurbitaceae crops. Here, a genome-wide characterization of this family was conducted in cucumber（Cucumis sativus L.）. A total of 17 CsSWEETgenes were identified, which are not evenly distributed over the seven cucumber chromosomes. Cucumber SWEET protein sequences possess seven conserved domains and two putative serine phosphorylation sites. The phylo- genetic tree of the SWEET genes in cucumber, Arabidopsis thaliana, and Oryza sativa was constructed, and all the SWEET genes were divided into four clades. In addition, a number of putative cis-elements were identified in the promoter regions of these CsSWEET genes： nine types involved in phytohormone responses and eight types involved in stress responses. Moreover, the transcript levels of CsSWEETgenes were analyzed in various tissues using quantitative real-time polymerase chain reaction. A majority （70.58%） of the CsSWEET genes were confined to reproductive tissue development. Finally, 18 putative watermelon ClaSWEETgenes and 18 melon CmSWEETgenes were identified that showed a high degree of similarity with CsSWEETgenes. The results from this study provided a basic understanding of the CsSWEETgenes and may also facilitate future research to elucidate the function of SWEET genes in cucumber and other Cucurbitaceae crops.

Effects of Exogenous Silicon on Photosynthetic Capacity and Antioxidant Enzyme Activities in Chloroplast of Cucumber Seedlings Under Excess Manganese

Effects of silicon on photosynthetic parameters and antioxidant enzymes of chloroplast in cucumber seedlings under excess Mn were studied. Compared with the control, excess Mn significantly inhibited net photosynthetic rate （Pn）, stomatal conductance, as well as the maximum yield of the photosystem II photochemical reactions （Fv/Fm） and the quantum yield of photosysytem II electron transport （Φ PSII）, application of Si reversed the negative effects of excess Mn. In the further investigation, it was obtained that application of Si significantly increased the activities of enzymes related with ascorbate-glutathione cycle including ascorbate peroxidase （APX）, dehydroascorbate reductase （DHAR） and glutathione reductase （GR） in cucumber chloroplast under excess Mn, this could be responsible for the lower accumulation of H2O2 and lower lipid peroxidation of chloroplast induced by Mn, and resulted in keeping higher photosynthesis.