CsBPC2 is essential for cucumber survival under cold stress.

Cold stress affects the growth and development of cucumbers. Whether the BPC2 transcription factor participates in cold tolerance and its regulatory mechanism in plants have not been reported. Here, we used wild-type (WT) cucumber seedlings and two mutant Csbpc2 lines as materials. The underlying mechanisms were studied by determining the phenotype, physiological and biochemical indicators, and transcriptome after cold stress. The results showed that CsBPC2 knockout reduced cucumber cold tolerance by increasing the chilling injury index, relative electrical conductivity and malondialdehyde (MDA) content and decreasing antioxidant enzyme activity. We then conducted RNA sequencing (RNA-seq) to explore transcript-level changes in Csbpc2 mutants. A large number of differentially expressed genes (1032) were identified and found to be unique in Csbpc2 mutants. However, only 489 down-regulated genes related to the synthesis and transport of amino acids and vitamins were found to be enriched through GO analysis. Moreover, both RNA-seq and qPT-PCR techniques revealed that CsBPC2 knockout also decreased the expression of some key cold-responsive genes, such as CsICE1, CsCOR413IM2, CsBZR1 and CsBZR2. These results strongly suggested that CsBPC2 knockout not only affected cold function genes but also decreased the levels of some key metabolites under cold stress. In conclusion, this study reveals for the first time that CsBPC2 is essential for cold tolerance in cucumber and provides a reference for research on the biological function of BPC2 in other plants.

CsBPC2 is a key regulator of root growth and development.

BASIC PENTACYSTEINE (BPCs) transcription factors are important regulators of plant growth and development. However, the regulatory mechanism of BPC2 in roots remains unclear. In our previous study, we created Csbpc2 cucumber mutants by the CRISPR/Cas9 system, and our studies on the phenotype of Csbpc2 mutants showed that the root growth was inhibited compared with wide-type (WT). Moreover, the surface area, volume and number of roots decreased significantly, with root system architecture changing from dichotomous branching to herringbone branching. Compared with WT, the leaf growth of the Csbpc2 mutants was not affected. However, the palisade and spongy tissue were significantly thinner, which was not beneficial for photosynthesis. The metabolome of root exudates showed that compared with WT, amino acids and their derivatives were significantly decreased, and the enriched pathways were mainly regulated by amino acids and their derivatives, indicating that knockout of CsBPC2 mainly affected the amino acid content in root exudates. Importantly, transcriptome analysis showed that knockout of CsBPC2 mainly affected root gene expression. Knockout of CsBPC2 significantly reduced the gene expression of gibberellins synthesis. However, the expression of genes related to amino acid synthesis, nitrogen fixation and PSII-related photosynthesis increased significantly, which may be due to the effect of knocking out CsBPC2 on gibberellins synthesis, resulting in the inhibition of seedling growth, thus forming negative feedback regulation. Generally, we showed for the first time that BPC2 is a key regulator gene of root growth and development, laying the foundation for future mechanisms of BPC2 regulation in roots.

Multifaceted regulatory functions of CsBPC2 in cucumber under salt stress conditions.

BASIC PENTACYSTEINE (BPC) transcription factors are essential regulators of plant growth and development. However, BPC functions and the related molecular mechanisms during cucumber (Cucumis sativus L.) responses to abiotic stresses, especially salt stress, remain unknown. We previously determined that salt stress induces CsBPC expression in cucumber. In this study, Csbpc2 transgene-free cucumber plants were created using a CRISPR/Cas9-mediated editing system to explore CsBPC functions associated with the salt stress response. The Csbpc2 mutants had a hypersensitive phenotype, with increased leaf chlorosis, decreased biomass, and increased malondialdehyde and electrolytic leakage levels under salt stress conditions. Additionally, a mutated CsBPC2 resulted in decreased proline and soluble sugar contents and antioxidant enzyme activities, which led to the accumulation of hydrogen peroxide and superoxide radicals. Furthermore, the mutation to CsBPC2 inhibited salinity-induced PM-H+-ATPase and V-H+-ATPase activities, resulting in decreased Na+ efflux and increased K+ efflux. These findings suggest that CsBPC2 may mediate plant salt stress resistance through its effects on osmoregulation, reactive oxygen species scavenging, and ion homeostasis-related regulatory pathways. However, CsBPC2 also affected ABA signaling. The mutation to CsBPC2 adversely affected salt-induced ABA biosynthesis and the expression of ABA signaling-related genes. Our results indicate that CsBPC2 may enhance the cucumber response to salt stress. It may also function as an important regulator of ABA biosynthesis and signal transduction. These findings will enrich our understanding of the biological functions of BPCs, especially their roles in abiotic stress responses, thereby providing the theoretical basis for improving crop salt tolerance.

Proteome and phosphoproteome analysis of 2,4-epibrassinolide-mediated cold stress response in cucumber seedlings.

The 2, 4-epibrassinolide (EBR) significantly increased plants cold tolerance. However, mechanisms of EBR in regulating cold tolerance in phosphoproteome and proteome levels have not been reported. The mechanism of EBR regulating cold response in cucumber was studied by multiple omics analysis. In this study, phosphoproteome analysis showed that cucumber responded to cold stress through multi-site serine phosphorylation, while EBR further upregulated single-site phosphorylation for most of cold-responsive phosphoproteins. Association analysis of the proteome and phosphoproteome revealed that EBR reprogrammed proteins in response to cold stress by negatively regulating protein phosphorylation and protein content, and phosphorylation negatively regulated protein content in cucumber. Further functional enrichment analysis of proteome and phosphoproteome showed that cucumber mainly upregulated phosphoproteins related to spliceosome, nucleotide binding and photosynthetic pathways in response to cold stress. However, different from the EBR regulation in omics level, hypergeometric analysis showed that EBR further upregulated 16 cold-up-responsive phosphoproteins participated photosynthetic and nucleotide binding pathways in response to cold stress, suggested their important function in cold tolerance. Analysis of cold-responsive transcription factors (TFs) by correlation between proteome and phosphoproteome showed that cucumber regulated eight class TFs may through protein phosphorylation under cold stress. Further combined with cold-related transcriptome found that cucumber phosphorylated eight class TFs, and mainly through targeting major hormone signal genes by bZIP TFs in response to cold stress, while EBR further increased these bZIP TFs (CsABI5.2 and CsABI5.5) phosphorylation level. In conclusion, the EBR mediated schematic of molecule response mechanisms in cucumber under cold stress was proposed.

PBAT/PLA humic acid biodegradable film applied on solar greenhouse tomato plants increased lycopene and decreased total acid contents.

This work aims to resolve residual film pollution in farmlands and improve tomato quality. The mechanical properties and degradation of PBAT/PLA lignin (MZS) and PBAT/PLA humic acid (FZS) composite biodegradable film were analyzed, and its effect on soil temperature and humidity, soil microorganisms, soil physical and chemical properties, tomato yield, and quality was studied. Polyethylene film (PE) was used as a control. The results demonstrate a higher degradation degree of FZS film than of MZS film. The degradation degree of FZS and MZS films reached level 2 and level 1, respectively, after 131 days of film covering. The weight loss rate of FZS and MZS films reached 52.74 % and 57.82 %, respectively, when buried for 160 days. Compared to the coverings of PE and MZS films, FZS film could significantly increase the soil's electric conductivity and organic matter content (p < 0.05). The relative abundance of soil fungi Chaetomium also increased. The yield, soluble solids, vitamin C (Vc), soluble sugar, and lycopene of tomato plants covered with FZS film significantly increased by 6.74 %, 8.75 %, 15.41 %, 8.30 %, and 27.27 % compared to plants covered with PE film, and the total acid and hardness significantly decreased by 24.95 % and 8.46 %, respectively (p < 0.05). Using 10 µm PBAT/PLA humic acid biodegradable film for tomato cultivation in autumn and winter increased the lycopene and decreased the total acid content by changing the soil's physical and chemical characteristics and increasing the content of Chaetomium soil.

Photosynthesis Mediated by RBOH-Dependent Signaling Is Essential for Cold Stress Memory.

Cold tolerance is improved by cold stress acclimation (CS-ACC), and the cold tolerance level is 'remembered' by plants. However, the underlying signaling mechanisms remain largely unknown. Here, the CS memory mechanism was studied by bioinformation, plant physiological and photosynthetic parameters, and gene expression. We found that CS-ACC induced the acquisition of CS memory and enhanced the maintenance of acquired cold tolerance (MACT) in cucumber seedlings. The H2O2 content and NADPH oxidase activity encoded by CsRBOH was maintained at higher levels during recovery after CS-ACC and inhibition of RBOH-dependent signaling after CS-ACC resulted in a decrease in the H2O2 content, NADPH oxidase activity, and MACT. CsRBOH2, 3, 4, and 5 showed high expression during recovery after CS-ACC. Many BZR-binding sites were identified in memory-responsive CsRBOHs promoters, and CsBZR1 and 3 showed high expression during recovery after CS-ACC. Inhibition of RBOH-dependent signaling or brassinosteroids affected the maintenance of the expression of these memory-responsive CsRBOHs and CsBZRs. The photosynthetic efficiency (PE) decreased but then increased with the prolonged recovery after CS-ACC, and was higher than the control at 48 h of recovery; however, inhibition of RBOH-dependent signaling resulted in a lower PE. Further etiolated seedlings experiments showed that a photosynthetic capacity was necessary for CS memory. Therefore, photosynthesis mediated by RBOH-dependent signaling is essential for CS memory.

Effects of biodegradable films on melon quality and substrate environment in solar greenhouse.

With the increase in plastic pollution of farmland substrate, biodegradable mulch film research has become a hotspot. However, the degradation rate of biodegradable plastic film over the entire crop growth period is still unclear, as well as its impact on crop growth and product quality. Here, several properties of two kinds of composite biodegradable mulch films, PBAT/PLA-[S1] and PBAT/lignin-[S2], are studied with polyethylene-[PE] and uncovered substrate (CK) as controls. We tested the differences in morphological characterization, physical properties, and weight loss rate of the plastic films, as well as the effects of the different plastic films on melon yield and quality, substrate temperature and humidity, physical and chemical properties of the substrate, and substrate fungal species composition. Compared to PE, biodegradable plastic films S1 and S2 increased substrate temperature and the net photosynthetic rate of leaves. The results of substrate 18 s rDNA assay of CK, PE, S1, and S2 after 80 days of treatment and pre-treatment showed that a total of 12 fungal phylum, with 317 fungal genera were found, in which Ascomycota as the main phyla and Penicillium as the main genera. Compared with PE, the S2 treatment significantly increased the single fruit weight, central sugar content and soluble sugar of melon by 225.35 g, 1.26%, and 0.68%, respectively (p < 0.05). When buried for 240 d, the weight loss rate of S2 was significantly increased by 86.08% compared with PE (p < 0.05). From these results, we extrapolated that covering the substrate with the most biodegradable film, PBAT/lignin composite (10 µm), improved the yield and fruit quality of the melon in winter greenhouse production.

Physiological and Molecular Mechanisms of ABA and CaCl2 Regulating Chilling Tolerance of Cucumber Seedlings.

Cold stress is a limiting factor to the growth and development of cucumber in the temperate regions; hence, improving the crop's tolerance to low temperature is highly pertinent. The regulation of low-temperature tolerance with exogenous ABA and CaCl2 was investigated in the cucumber variety Zhongnong 26. Under low-temperature conditions (day/night 12/12 h at 5 °C), seedlings were sprayed with a single application of ABA, CaCl2, or a combination of both. Our analysis included a calculated chilling injury index, malondialdehyde (MDA) content, relative electrical conductivity, antioxidant enzyme activities (SOD, CAT, and APX), leaf tissue structure, and expression of cold-related genes by transcriptome sequencing. Compared with the water control treatment, the combined ABA + CaCl2 treatment significantly improved the superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) of the seedlings by 34.47%, 59.66%, and 118.80%, respectively (p < 0.05), and significantly reduced the chilling injury index, relative electrical conductivity, and MDA content, by 89.47%, 62.17%, and 44.55%, respectively (p < 0.05). Transcriptome analysis showed that compared with the water control treatment, 3442 genes were differentially expressed for the combined treatment, 3921 for the ABA treatment, and 1333 for the CaCl2 treatment. KEGG enrichment analysis for both the ABA and combined ABA + CaCl2 treatments (as compared to the water control) showed that it mainly involves genes of the photosynthesis pathway and metabolic pathways. Differentially expressed genes following the CaCl2 treatment were mainly involved in plant hormone signal transduction, plant-pathogen interaction, MAPK signaling pathway-plant, phenylpropanoid biosynthesis, and circadian rhythm-plant. qRT-PCR analysis and RNA-seq results showed a consistent trend in variation of differential gene expression. Overall, this study demonstrated that although all three treatments provided some protection, the combined treatment of ABA (35 mg/L) with CaCl2 (500 mg/L) afforded the best results. A combined ABA + CaCl2 treatment can effectively alleviate cold-stress damage to cucumber seedlings by inducing physiological changes in photosynthesis and metabolism, and provides a theoretical basis and technical support for the application of exogenous ABA and CaCl2 for low-temperature protection of cucumber seedlings.

Agronomic Biofortification of Cayenne Pepper Cultivars with Plant Growth-Promoting Rhizobacteria and Chili Residue in a Chinese Solar Greenhouse.

Agronomic biofortification of horticultural crops using plant growth-promoting rhizobacteria (PGPR) under crop residue incorporation systems remains largely underexploited. Bacillus subtilis (B1), Bacillus laterosporus (B2), or Bacillus amyloliquefaciens (B3) was inoculated on soil containing chili residue, while chili residue without PGPR (NP) served as the control. Two hybrid long cayenne peppers, succeeding a leaf mustard crop were used in the intensive cultivation study. Net photosynthesis, leaf stomatal conductance, transpiration rate, photosynthetic water use efficiency, shoot and root biomass, and fruit yield were evaluated. Derivatives of folate, minerals, and nitrate contents in the pepper fruits were also assessed. B1 elicited higher net photosynthesis and photosynthetic water use efficiency, while B2 and B3 had higher transpiration rates than B1 and NP. B1 and B3 resulted in 27-36% increase in pepper fruit yield compared to other treatments, whereas B3 produced 24-27.5% and 21.9-27.2% higher 5-methyltetrahydrofolate and total folate contents, respectively, compared to B1 and NP. However, chili residue without PGPR inoculation improved fruit calcium, magnesium, and potassium contents than the inoculated treatments. 'Xin Xian La 8 F1' cultivar had higher yield and plant biomass, fruit potassium, total soluble solids, and total folate contents compared to 'La Gao F1.' Agronomic biofortification through the synergy of Bacillus amyloliquefaciens and chili residue produced better yield and folate contents with a trade-off in the mineral contents of the greenhouse-grown long cayenne pepper.

Adaptation of cucumber seedlings to low temperature stress by reducing nitrate to ammonium during it's transportation.

BACKGROUND: Low temperature severely depresses the uptake, translocation from the root to the shoot, and metabolism of nitrate and ammonium in thermophilic plants such as cucumber (Cucumis sativus). Plant growth is inhibited accordingly. However, the availability of information on the effects of low temperature on nitrogen transport remains limited. RESULTS: Using non-invasive micro-test technology, the net nitrate (NO3-) and ammonium (NH4+) fluxes in the root hair zone and vascular bundles of the primary root, stem, petiole, midrib, lateral vein, and shoot tip of cucumber seedlings under normal temperature (NT; 26 °C) and low temperature (LT; 8 °C) treatment were analyzed. Under LT treatment, the net NO3- flux rate in the root hair zone and vascular bundles of cucumber seedlings decreased, whereas the net NH4+ flux rate in vascular bundles of the midrib, lateral vein, and shoot tip increased. Accordingly, the relative expression of CsNRT1.4a in the petiole and midrib was down-regulated, whereas the expression of CsAMT1.2a-1.2c in the midrib was up-regulated. The results of 15N isotope tracing showed that NO3--N and NH4+-N uptake of the seedlings under LT treatment decreased significantly compared with that under NT treatment, and the concentration and proportion of both NO3--N and NH4+-N distributed in the shoot decreased. Under LT treatment, the actual nitrate reductase activity (NRAact) in the root did not change significantly, whereas NRAact in the stem and petiole increased by 113.2 and 96.2%, respectively. CONCLUSIONS: The higher net NH4+ flux rate in leaves and young tissues may reflect the higher NRAact in the stem and petiole, which may result in a higher proportion of NO3- being reduced to NH4+ during the upward transportation of NO3-. The results contribute to an improved understanding of the mechanism of changes in nitrate transportation in plants in response to low-temperature stress.

Genome-wide analysis of the AINTEGUMENTA-like (AIL) transcription factor gene family in pumpkin (Cucurbita moschata Duch.) and CmoANT1.2 response in graft union healing.

AINTEGUMENTA-like (AIL) proteins are members of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain family of transcription factors involved in plant growth, development, and abiotic stress responses. However, the biological functions of AIL members in pumpkin (Cucurbita moschata Duch.) remain unknown. In this study, we identified 12 AIL genes in the pumpkin genome encoding proteins predicted to be localized in the nucleus. Phylogenetic analysis showed that the AIL gene family could be classified into six major subfamilies, with each member encoding two AP2/ERF domains separated by a linker region. CmoAIL genes were expressed at varying levels in the examined tissues, and CmoANT genes showed different expression patterns under auxin (IAA), 1-naphthylphthalamic acid (NPA), and abscisic acid (ABA) treatments. Ectopic overexpression of CmoANT1.2 in Arabidopsis increased organ size and promoted growth of grafted plants by accelerating graft union formation. However, there was no significant difference at the graft junction for WT/WT and WT/ANT under IAA or NPA treatments. Taken together, the results of this study provide critical information about CmoAIL genes and their encoded proteins, and suggest future work should investigate the functions of CmoANT1.2 in the grafting process in pumpkin.

Application of hollow fiber centrifugal ultrafiltrate purification as the pretreatment technology for traditional Chinese medicine: its application for analysis of honokiol and magnolol in TCM preparations containing Cortex Magnoliae Officinalis.

BACKGROUND: Traditional Chinese medicine (TCM) preparations are very complex mixtures, and the content of bioactive components is usually very low. Therefore, before final analysis, the preparation of an appropriate sample is necessary. Sample preparation is the most time-consuming and error-prone part of the analytical procedure, and the choice of purification technology greatly influences the reliability of the final analysis. METHODS: In the present study, we evaluated the feasibility of hollow fiber centrifugal ultrafiltrate (HFCF-UF) as a purification technology for the analysis of bioactive components in TCM preparations. The HFCF-UF technology was applied to analyze honokiol and magnolol in TCM preparations containing Cortex Magnoliae Officinalis (Hou Po in Chinese Pinyin). A mini centrifugal device based on hollow fiber was employed to remove the macromolecule components. A single step of simple centrifugation was required before the filtrate could be directly injected into an existing high performance liquid chromatography (HPLC) system without any further clean-up step or use of special columns. This greatly simplified the pretreatment steps, and improved the accuracy of analytic methods. The separation was achieved on a Diamonsil C18 column (i.d. 5 µm, 150 mm × 4.6 mm) with V (methanol):V (acetonitrile):V (0.5% acetic acid solution) =44:22:34 as the mobile phase at a flow rate of 1.0 mL/min. RESULTS: It had good linear relationship between the peak areas of honokiol and magnolol and their concentrations at 6.40-205 and 3.15-101 µg/mL (r=0.9999), respectively. The method recovery was over 92.6% with a relative standard deviation (RSD) of less than 3.0%. The average recovery of honokiol was 97.7% with an RSD of 3.0%, and that of magnolol was 96.8% with RSD of 2.8%. CONCLUSIONS: The application of HFCF-UF in TCM preparations could assist in making the quality control of TCM simple, rapid, and accurate. The HFCF-UF purification procedure can be used as an alternative means for analyzing bioactive components in TCM preparations.

The CsGPA1-CsAQPs module is essential for salt tolerance of cucumber seedlings.

KEY MESSAGE: CsGPA1 interacts with CsTIP1.1 (a member of CsAQPs) and suppression of CsGPA1 results the reverse expression of CsAQPs in leaves and roots, resulting in declining water content of cucumber seedlings under salt stress. Salt stress seriously affects cucumber growth and development. Whether the G-protein alpha subunit functions in cucumber during salt stress and its regulation mechanism remains unknown. We interrogated CsGPA1-RNAi lines to identify the role of CsGPA1 during salt stress. Phenotypically, compared with wild type, leaves were severely withered, and root cells showed signs of senescence under salt stress for RNAi lines. Compared with WT, SOD and CAT activity, soluble protein and proline contents all decreased in RNAi lines, while malondialdehyde and relative electrical conductivity increased. Through screening the yeast two-hybrid library and combined with yeast two-hybrid and GST pull-down, the interaction of CsGPA1 with CsTIP1.1 was found the first time in a plant. Then, the expression of aquaporin (AQP) family genes was detected. The expression of CsAQP genes in leaves and roots was primarily up-regulated in WT under salt stress. However, interference by CsGPA1 resulted in enhanced expression of CsAQPs except for CsTIP3.2 in leaves, but reduced expression of some CsAQPs in roots under salt stress. Furthermore, principal component analysis of CsAQP expression profiles and linear regression analysis between CsGPA1 and CsAQPs revealed that CsGPA1 reversely regulated the expression of CsAQPs in leaves and roots under salt stress. Moreover, the water content in leaves and roots of RNAi seedlings significantly decreased compared with WT under salt stress. Overall, CsGPA1 interacts with CsTIP1.1 and suppression of CsGPA1 results in opposite patterns of expression of CsAQPs in leaves and roots, resulting in declining water content of cucumber under salt stress.

Vasodilation activity of dipfluzine metabolites in isolated rat basilar arteries and their underlying mechanisms.

Identifying the metabolites of a drug has become an indispensable task in the development of new drugs. Dipfluzine (Dip) is a promising candidate for the treatment of cerebral vascular diseases and has 5 metabolites (M1∼M5) in rat urine and liver microsomes, but their biological activity is still unknown. Because selective cerebral vasodilation is a main role of Dip, we investigated the vasodilation of Dip and its 5 metabolites in isolated Sprague-Dawley (SD) male rat basilar arteries preconstricted with high-K+ or 5-HT. The results showed that only M1 possessed concentration-dependent inhibitory activity on the vasoconstriction of arteries with or without the endothelium, and M1 has a more potent vasodilatory effect than Dip on both contraction models. Like Dip, the vasodilatory mechanisms of M1 may be not only related to receptor-operated and voltage-dependent calcium ion channels of smooth muscle cells but also to the release of NO and EDHF from endothelial cells and the opening of Ca2+-activated K+ channels and ATP-sensitive potassium ion channels. Unlike Dip, the vasodilation mechanism of M1 is also related to the opening of voltage-sensitive K+ channel. Together with more selectivity to non-VDCC than Dip, this may partially explain why M1 has stronger vasodilatory effects than Dip. The mechanisms of vasodilation of Dip and M1 may result from the combined action of these or other factors, especially blocking non-endothelium dependent non-VDCC and endothelium dependent IKCa channels. These results point to the possibility that M1 provides synergism for the clinical use of Dip, which may inform the synthesis of new drugs.

Correction to: 24-Epibrassinolide promotes NO3- and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature.

In the original publication of this article [1], the author pointed out there is an error in Figs. 4 and 5.

Genome-Wide Identification and Characterization of Cucumber BPC Transcription Factors and Their Responses to Abiotic Stresses and Exogenous Phytohormones.

BASIC PENTACYSTEINE (BPC) is a small transcription factor family that functions in diverse growth and development processes in plants. However, the roles of BPCs in plants, especially cucumber (Cucumis sativus L.), in response to abiotic stress and exogenous phytohormones are still unclear. Here, we identified four BPC genes in the cucumber genome, and classified them into two groups according to phylogenetic analysis. We also investigated the gene structures and detected five conserved motifs in these CsBPCs. Tissue expression pattern analysis revealed that the four CsBPCs were expressed ubiquitously in both vegetative and reproductive organs. Additionally, the transcriptional levels of the four CsBPCs were induced by various abiotic stress and hormone treatments. Overexpression of CsBPC2 in tobacco (Nicotiana tabacum) inhibited seed germination under saline, polyethylene glycol, and abscisic acid (ABA) conditions. The results suggest that the CsBPC genes may play crucial roles in cucumber growth and development, as well as responses to abiotic stresses and plant hormones. CsBPC2 overexpression in tobacco negatively affected seed germination under hyperosmotic conditions. Additionally, CsBPC2 functioned in ABA-inhibited seed germination and hypersensitivity to ABA-mediated responses. Our results provide fundamental information for further research on the biological functions of BPCs in development and abiotic stress responses in cucumber and other plant species.

Integrated Metabolome and Transcriptome Analysis Provide Insights into the Effects of Grafting on Fruit Flavor of Cucumber with Different Rootstocks.

Rootstocks frequently exert detrimental effects on the fruit quality of grafted cucumber (Cucumis sativus L.) plants. To understand and ultimately correct this deficiency, a transcriptomic and metabolomic comparative analysis was performed among cucumber fruits from non-grafted plants (NG), and fruits from plants grafted onto different rootstocks of No.96 and No.45 (Cucurbita moschata. Duch), known to confer a different aroma and taste. We found remarkable changes in the primary metabolites of sugars, organic acids, amino acids, and alcohols in the fruit of the grafted cucumber plants with different rootstocks, compared to the non-grafted ones, especially No.45. We identified 140, 131, and 244 differentially expressed genes (DEGs) in the comparisons of GNo.96 vs. NG, GNo.45 vs. NG, and GNo.45 vs. GNo.96. The identified DEGs have functions involved in many metabolic processes, such as starch and sucrose metabolism; the biosynthesis of diterpenoid, carotenoid, and zeatin compounds; and plant hormone signal transduction. Members of the HSF, AP2/ERF-ERF, HB-HD-ZIP, and MYB transcription factor families were triggered in the grafted cucumbers, especially in the cucumber grafted on No.96. Based on a correlation analysis of the relationships between the metabolites and genes, we screened 10 candidate genes likely to be involved in sugar metabolism (Fructose-6-phosphate and trehalose), linoleic acid, and amino-acid (isoleucine, proline, and valine) biosynthesis in grafted cucumbers, and then confirmed the gene expression patterns of these genes by qRT-PCR. The levels of TPS15 (Csa3G040850) were remarkably increased in cucumber fruit with No.96 rootstock compared with No.45, suggesting changes in the volatile chemical production. Together, the results of this study improve our understanding of flavor changes in grafted cucumbers, and identify the candidate genes involved in this process.

24-Epibrassinolide promotes NO3- and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature.

BACKGROUND: Suboptimal root zone temperature (RZT) causes a remarkable reduction in growth of horticultural crops during winter cultivation under greenhouse production. However, limited information is available on the effects of suboptimal RZT on nitrogen (N) metabolism in cucumber seedlings. The aim of this study is to investigate the effects of 24-Epibrassinolide (EBR) on nitrate and ammonium flux rate, N metabolism, and transcript levels of NRT1 family genes under suboptimal RZT in cucumber seedlings. RESULTS: Suboptimal RZT (LT) negatively affected on cucumber growth and proportionately decreased EBR contents, bleeding rate, root activity, enzyme activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), nitrate (NO3-) influx rate, ammonium (NH4+) efflux rate, and transcript levels of nitrate transporter (NRT1) encoding genes. However, exogenous EBR reduced the harmful effects of suboptimal RZT and increased endogenous EBR contents, bleeding rate, root activity, enzyme activities of NR, NiR, GS, and GOGAT, NH4+ and NO3- flux rates and contents, and N accumulation. EBR-treated seedlings also upregulated the transcript levels of nitrate transporters CsNRT1.1, CsNRT1.2A, CsNRT1.2B, CsNRT1.2C, CsNRT1.3, CsNRT1.4A, CsNRT1.5B, CsNRT1.5C, CsNRT1.9, and CsNRT1.10, and downregulated CsNRT1.5A and CsNRT1.8. LT treatment upregulated the expression level of CsNRT1.5A, while exogenous BZR application downregulated the expression level of NRT1 genes. CONCLUSION: These results indicate that exogenous application of EBR alleviated the harmful effects of suboptimal RZT through changes in N metabolism, NH4+ and NO3- flux rates, and NRT1 gene expression, leading to improved cucumber seedlings growth. Our study provides the first evidence of the role of EBR in the response to suboptimal RZT in cucumber, and can be used to improve vegetable production.

Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber-pumpkin grafted plants.

BACKGROUND: Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber-pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber-pumpkin grafted plants. METHODS: In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber-pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. RESULTS: The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber-pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber-pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. DISCUSSION: This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber-pumpkin grafted plants.