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Melatonin is a crucial regulator of plant growth and development as well as stress tolerance. However, we only have a limited understanding of the functions of endogenous melatonin. Tryptophan decarboxylase (TDC) serves as the initial rate-limiting enzyme in the melatonin synthesis pathway. To date, no cucumber TDC gene has been cloned and characterized. In this study, we identified two TDC genes (CsTDC1 and CsTDC2) in the cucumber genome. The subcellular localization analysis indicated that CsTDC1 and CsTDC2 are predominantly localized in the cytoplasm and plasma membrane. Tissue-specific expression analyses revealed that CsTDC1 and CsTDC2 are expressed in both vegetative and reproductive organs. Many cis-elements related to stress, hormone, and light responses as well as development were identified in the CsTDC promoter regions. Furthermore, the expression of CsTDC1 and CsTDC2 was strongly induced by treatments with various abiotic stresses and exogenous hormones. The transient overexpression of CsTDC1 and CsTDC2 in tobacco leaves resulted in increases in the TDC activity and melatonin content, along with improved tolerance of tobacco leaves to salt, drought, and low-temperature stresses. Notably, the overexpression of CsTDC2 had a more pronounced effect than the overexpression of CsTDC1. Accordingly, both CsTDC genes, but especially CsTDC2, may be important for regulating cucumber growth, development, and stress tolerance. The study findings provide a theoretical and experimental basis for future functional analyses of endogenous melatonin in cucumber. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • TAG had obviously higher daily growth rate than HIG, SG during grafted 7 days. • Isolation layer, callus formation and vascular connection were all happened at graft junction using HIG, TAG, SG. • Vascular bridging first occurred between scion and rootstock using HIG or TAG at 5 DAG; this process was delayed with SG. • Higher level of POD, CAT activities and total phenolic compounds, and lower level of PPO activity and lignin content were maintained at graft union using TAG at 7DAG. Abstract To determine the effect of grafting method (hole insertion grafting, HIG; tongue approach grafting, TAG; and spliced grafting, SG) on graft union formation in cucumber grafted onto bottle gourd rootstock, an anatomical and physiological study was carried out during the healing stage. The results showed that the TAG method resulted in an obviously higher daily growth rate than the other grafting methods 7 days after grafting (DAG), with significantly higher values in the scion than in the rootstock. The isolation layer, callus formation, and vascular connection all occurred at the graft junction when using HIG, TAG, and SG. Vascular bridging first occurred between scion and rootstock using HIG or TAG at 5 DAG; this process was delayed with SG. The activities of superoxide dismutase (SOD), POD (peroxidase), CAT (catalase), PAL (Phenylalanine ammonia-lyase), and PPO (polyphenol oxidase) were significantly enhanced in the graft union during the healing process when different grafting methods were used. Compared with HIG and SG, the POD and CAT activities and total phenolic compounds were higher with TAG at 7 DAG; a reduction in PPO activity and lignin content were also overserved at the graft with TAG at 7 DAG. These results suggest that the association of TAG with a higher growth rate during the healing period is linked with the earlier improvement in vascular bundle connection that may be caused by increased antioxidant activities and lower lignin content at the graft junction. These morphological and physiological differences may provide valuable information for revealing the graft union healing process. [ABSTRACT FROM AUTHOR]

Background: Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress. Results: The CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16 days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16 days compared with that at 0 day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit. Conclusions: The plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phytohormone biosynthesis and accumulation are essential for plant growth and development and stress responses. Here, we investigated the effects of 24-epibrassinolide (EBR) on physiological and biochemical mechanisms in cucumber leaves under low-temperature stress. The cucumber seedlings were exposed to treatments as follows: NT (normal temperature, 26 °C/18 °C day/night), and three low-temperature (12 °C/8 °C day/night) treatments: CK (low-temperature stress); EBR (low-temperature and 0.1 μM EBR); and BZR (low-temperature and 4 μM BZR, a specific EBR biosynthesis inhibitor). The results indicated that low-temperature stress proportionately decreased cucumber seedling growth and the strong seedling index, chlorophyll (Chl) content, photosynthetic capacity, and antioxidant enzyme activities, while increasing reactive oxygen species (ROS) and malondialdehyde (MDA) contents, hormone levels, and EBR biosynthesis gene expression level. However, EBR treatments significantly enhanced cucumber seedling growth and the strong seedling index, chlorophyll content, photosynthetic capacity, activities of antioxidant enzymes, the cell membrane stability, and endogenous hormones, and upregulated EBR biosynthesis gene expression level, while decreasing ROS and the MDA content. Based on these results, it can be concluded that exogenous EBR regulates endogenous hormones by activating at the transcript level EBR biosynthetic genes, which increases antioxidant enzyme capacity levels and reduces the overproduction of ROS and MDA, protecting chlorophyll and photosynthetic machinery, thus improving cucumber seedling growth. [ABSTRACT FROM AUTHOR]