γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells.

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

Photocrosslinkable pMHC monomers stain T cells specifically and cause ligand-bound TCRs to be 'preferentially' transported to the cSMAC.

The binding of T cell antigen receptors (TCRs) to specific complexes of peptide and major histocompatibility complex (pMHC) is typically of very low affinity, which necessitates the use of multimeric pMHC complexes to label T lymphocytes stably. We report here the development of pMHC complexes able to be crosslinked by ultraviolet irradiation; even as monomers, these efficiently and specifically stained cognate T cells. We also used this reagent to probe T cell activation and found that a covalently bound pMHC was more stimulatory than an agonist pMHC on lipid bilayers. This finding suggested that serial engagement of TCRs is dispensable for activation when a substantial fraction of TCRs are stably engaged. Finally, pMHC-bound TCRs were 'preferentially' transported into the central supramolecular activation cluster after activation, which suggested that ligand engagement enabled linkage of the TCR and its associated CD3 signaling molecules to the cytoskeleton.

Targeting HLA-DR loss in hematologic malignancies with an inhibitory chimeric antigen receptor.

Chimeric antigen receptor natural killer (CAR-NK) cells have remarkable cytotoxicity against hematologic malignancies; however, they may also attack normal cells sharing the target antigen. Since human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in a substantial proportion of hematologic malignancies, presumably a mechanism to escape immune surveillance, we hypothesize that the anti-cancer specificity of CAR-NK cells can be enhanced by activating them against cancer antigens while inhibiting them against HLA-DR. Here, we report the development of an anti-HLA-DR inhibitory CAR (iCAR) that can effectively suppress NK cell activation against HLA-DR-expressing cells. We show that dual CAR-NK cells, which co-express the anti-CD19 or CD33 activating CAR and the anti-HLA-DR iCAR, can preferentially target HLA-DR-negative cells over HLA-DR-positive cells in vitro. We find that the HLA-DR-mediated inhibition is positively correlated with both iCAR and HLA-DR densities. We also find that HLA-DR-expressing surrounding cells do not affect the target selectivity of dual CAR-NK cells. Finally, we confirm that HLA-DR-positive cells are resistant to dual CAR-NK cell-mediated killing in a xenograft mouse model. Our approach holds great promise for enhancing CAR-NK and CAR-T cell specificity against malignancies with HLA-DR loss.

A New Temperature Correction Method for NaI(Tl) Detectors Based on Pulse Deconvolution.

To overcome the temperature effect of NaI(Tl) detectors for energy spectrometry without additional hardware, a new correction method was put forward based on pulse deconvolution, trapezoidal shaping and amplitude correction, named DTSAC. To verify this method, actual pulses from a NaI(Tl)-PMT detector were measured at various temperatures from -20 °C to 50 °C. Pulse processing and spectrum synthesis showed that the position drift of the 137Cs 662 keV peak was less than 3 keV, and the corresponding resolution at 662 keV of the sum spectra ranged from 6.91% to 10.60% with the trapezoidal width set from 1000 ns to 100 ns. The DTSAC method corrects the temperature effect via pulse processing, and needs no reference peak, reference spectrum or additional circuits. The method solves the problem of correction of pulse shape and pulse amplitude at the same time, and can be used even at a high counting rate.

Ground Radioactivity Distribution Reconstruction and Dose Rate Estimation Based on Spectrum Deconvolution.

Estimating the gamma dose rate at one meter above ground level and determining the distribution of radioactive pollution from aerial radiation monitoring data are the core technical issues of unmanned aerial vehicle nuclear radiation monitoring. In this paper, a reconstruction algorithm of the ground radioactivity distribution based on spectral deconvolution was proposed for the problem of regional surface source radioactivity distribution reconstruction and dose rate estimation. The algorithm estimates unknown radioactive nuclide types and their distributions using spectrum deconvolution and introduces energy windows to improve the accuracy of the deconvolution results, achieving accurate reconstruction of multiple continuous distribution radioactive nuclides and their distributions, as well as dose rate estimation of one meter above ground level. The feasibility and effectiveness of the method were verified through cases of single-nuclide (137Cs) and multi-nuclide (137Cs and 60Co) surface sources by modeling and solving them. The results showed that the cosine similarities between the estimated ground radioactivity distribution and dose rate distribution with the true value were 0.9950 and 0.9965, respectively, which could prove that the proposed reconstruction algorithm would effectively distinguish multiple radioactive nuclides and accurately restore their radioactivity distribution. Finally, the influences of statistical fluctuation levels and the number of energy windows on the deconvolution results were analyzed, showing that the lower the statistical fluctuation level and the more energy window divisions, the better the deconvolution results.

Exogenous Zeaxanthin Alleviates Low Temperature Combined with Low Light Induced Photosynthesis Inhibition and Oxidative Stress in Pepper (Capsicum annuum L.) Plants.

Low temperature combined with low light (LL) affects crop production, especially the yield and quality of peppers, in northwest China during the winter and spring seasons. Zeaxanthin (Z) is a known lipid protectant and active oxygen scavenger. However, whether exogenous Z can mitigate LL-induced inhibition of photosynthesis and oxidative stress in peppers remains unclear. In this study, we investigated the effects of exogenous Z on photosynthesis and the antioxidant machinery of pepper seedlings subject to LL stress. The results showed that the growth and photosynthesis of pepper seedlings were significantly inhibited by LL stress. In addition, the antioxidant machinery was disturbed by the uneven production and elimination of reactive oxygen species (ROS), which resulted in damage to the pepper. For example, membrane lipid peroxidation increased ROS content, and so on. However, exogenous application of Z before LL stress significantly increased the plant height, stem diameter, net photosynthetic rate (Pn), and stomata, which were obviously closed at LL. The activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), mono de-hydroascorbate reductase (MDHAR), de-hydroascorbate reductase (DHAR), ascorbate peroxidase (APX), and ascorbate oxidase (AAO) improved significantly due to the increased expression of CaSOD, CaCAT, CaAPX, CaMDHAR, and CaDHAR. The ascorbic (AsA) and glutathione (GSH) contents and ascorbic/dehydroascorbate (AsA/DHA) and glutathione/oxidized glutathione (GSH/GSSG) ratios also increased significantly, resulting in the effective removal of hydrogen peroxide (H2O2) and superoxide anions (O2•-) caused by LL stress. Thus, pre-treatment with Z significantly reduced ROS accumulation in pepper seedlings under LL stress by enhancing the activity of antioxidant enzymes and accumulation of components of the ascorbate-glutathione (AsA-GSH) cycle and upregulated key genes in the AsA-GSH cycle.

Heme is involved in the exogenous ALA-promoted growth and antioxidant defense system of cucumber seedlings under salt stress.

A biosynthetic precursor of tetrapyrrol, 5-aminolevulinic acid (ALA), is widely used in agricultural production, as an exogenous regulatory substance that effectively regulates plant growth. Previous studies have shown that heme and chlorophyll accumulate in plants under salt stress, when treated with exogenous ALA. In this study, we explored the regulatory role of heme in plants, by spraying 25 mg L-1 ALA onto the leaves of cucumber seedlings treated with heme synthesis inhibitor (2,2'-dipyridyl, DPD) and heme scavenger (hemopexin, Hx), under 50 mmol L-1 NaCl stress. The results showed that NaCl alone and DPD + Hx treatments to cucumber seedlings subjected to salt stress adversely affected their growth, by decreasing biomass accumulation, root activity, and root morphology. In addition, these treatments induced an increase in membrane lipid oxidation, as well as enhancement of anti-oxidase activities, proline content, and glutamate betaine. However, exogenous ALA application increased the plant growth and root architecture indices under NaCl stress, owing to a lack of heme in the seedlings. In addition, cucumber seedlings treated with DPD and Hx showed inhibition of growth under salt stress, but exogenous ALA effectively improved cucumber seedling growth as well as the physiological characteristics; moreover, the regulation of ALA in plants was weakened when heme synthesis was inhibited. Heme biosynthesis and metabolism genes, HEMH and HO1, which are involved in the ALA metabolic pathway, were upregulated under salinity conditions, when ferrochelatase activity was inhibited. Application of exogenous ALA increased the heme content in the leaves. Thus, exogenous ALA may supplement the substrates for heme synthesis. These results indicated that heme plays a vital role in the response of plants to salinity stress. In conclusion, heme is involved in ALA-mediated alleviation of damage caused to cucumber seedlings and acts as a positive regulator of plant adaption.

A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells.

We have developed a single-molecule imaging technique that uses quantum-dot-labeled peptide-major histocompatibility complex (pMHC) ligands to study CD4(+) T cell functional sensitivity. We found that naive T cells, T cell blasts, and memory T cells could all be triggered by a single pMHC to secrete tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) cytokines with a rate of ∼1,000, ∼10,000, and ∼10,000 molecules/min, respectively, and that additional pMHCs did not augment secretion, indicating a digital response pattern. We also found that a single pMHC localized to the immunological synapse induced the slow formation of a long-lasting T cell receptor (TCR) cluster, consistent with a serial engagement mechanism. These data show that scaling up CD4(+) T cell cytokine responses involves increasingly efficient T cell recruitment rather than greater cytokine production per cell.

An endogenous positively selecting peptide enhances mature T cell responses and becomes an autoantigen in the absence of microRNA miR-181a.

Thymic positive selection is based on the interactions of T cell antigen receptors (TCRs) with self peptide-major histocompatibility complex (MHC) ligands, but the identity of selecting peptides for MHC class II-restricted TCRs and the functional consequences of this peptide specificity are not clear. Here we identify several endogenous self peptides that positively selected the MHC class II-restricted 5C.C7 TCR. The most potent of these also enhanced mature T cell activation, which supports the hypothesis that one function of positive selection is to produce T cells that can use particular self peptide-MHC complexes for activation and/or homeostasis. We also show that inhibiting the microRNA miR-181a resulted in maturation of T cells that overtly reacted toward these erstwhile positively selecting peptides. Therefore, miR-181a helps to guarantee the clonal deletion of particular moderate-affinity clones by modulating the TCR signaling threshold of thymocytes.

Melatonin alleviates imidacloprid phytotoxicity to cucumber (Cucumis sativus L.) through modulating redox homeostasis in plants and promoting its metabolism by enhancing glutathione dependent detoxification.

Melatonin (Mel), a powerful antioxidant that has the ability to regulate physiological and biochemical processes in plants under abiotic stresses. However, its roles in pesticide detoxification is poorly understood. Herein, selecting leaf spraying insecticide imidacloprid (IMD) as the model, we demonstrated the detoxification mechanism underlying root pretreatment of Mel on IMD in cucumber. IMD treatment affected the primary light conversion efficiency of photosystem II (Fv/Fm), reduced the quantum yield, and increased hydrogen peroxide and superoxide anions contents as well as the levels of membrane lipid peroxidation, indicating that excessive IMD treatment induces oxidative stress. Nonetheless, by increasing the appropriate levels of exogenous Mel, the photosynthesis of cucumber under IMD treatment reached the control levels, effectively removing reactive oxygen species. Furthermore, the content and ratio of ascorbate (AsA) and glutathione (GSH) were decreased under IMD treatment; Mel treatment enhanced the AsA/DHA and GSH/GSSG ratios, as well as the activities of MDHAR, DHAR and GR, suggesting that Mel could alleviate oxidative stress of cucumber treated with IMD by regulating the ascorbic acid-glutathione cycle. Importantly, IMD degradation rate and glutathione S-transferase (GST) activity increased after Mel treatment. The levels of transcripts encoding antioxidant enzymes GPX and GST (GST1,2 and 3) were also increased, indicating that Mel accelerated IMD degradation. These results suggest that Mel plays an important role in the detoxification of IMD by promoting GST activity and transcription and the AsA-GSH cycle, thus providing an approach for plants to reduce IMD residue through the plant's own detoxification mechanism.

Exogenous silicon enhances the systemic defense of cucumber leaves and roots against CA-induced autotoxicity stress by regulating the ascorbate-glutathione cycle and photosystem II.

Cinnamic acid (CA), one of the main autotoxins secreted by cucumber roots during continuous cropping, inhibits plant growth and reduces yield. Silicon (Si) is an environmentally friendly element that alleviates abiotic stresses in plants, but the mechanism underlying its resistance to autotoxicity remain unclear. Here, we used 0.8 mmol L-1 CA to study the effects of Si application on the growth, chlorophyll fluorescence, and ascorbate-glutathione (AsA-GSH) cycle of cucumber seedlings under CA inducing conditions. Our results indicated that CA significantly induced photoinhibition and overaccumulation of reactive oxygen species (ROS), thereby inhibiting cucumber growth. Treatment with 1.0 mmol L-1 Si improved plant height, stem diameter and biomass accumulation, and protected the photosynthetic electron transport function of photosystem II in the presence of CA. Similarly, Si application maintained the ROS status by increasing ascorbate (AsA) and glutathione (GSH) production, as well as the ratios of AsA/DHA and GSH/GSSG in both leaves and roots during CA stress. In addition, Si application in CA-treated seedlings enhanced the activity of key enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and the transcription of several enzyme genes (CsAPX, CsMDHAR and CsGR） from the AsA-GSH cycle. These results suggest that exogenous Si enhances CA tolerance in cucumber seedlings by protecting photosystem II activity, upregulating AsA-GSH pathway, and reducing ROS levels.

Transcriptome analysis of biological pathways associated with heterosis in Chinese cabbage.

Chinese cabbage is an important vegetable in Asia, and high-yielding hybrids are needed to cope with the growing demand. A comparative transcriptome profiling was conducted to reveal the differentially expressed genes (DEGs) associated with heterosis in two hybrids relative to their parents. Our data suggests that heterosis is underlined by a significant upregulation of gene expression. High expression of DEGs in glycolysis and photosynthesis pathways in hybrids depicted their relation with growth and hybrid vigor. Besides, DEGs related to auxin, abscisic acid, ethylene and gibberellin were identified, implying that these hormones may boost the mechanisms of growth and developmental processes in the hybrids. Furthermore, transcription factors, including bHLH, ERF, MYB and WRKY were predicted to regulate downstream genes linked to hybrid vigor. Collectively, the present study will be helpful for a better understanding of the regulation mechanisms of heterosis to aid cabbage yield improvement.

Hydrogen sulfide negatively regulates cd-induced cell death in cucumber (Cucumis sativus L) root tip cells.

BACKGROUND: Hydrogen sulfide (H2S) is a gas signal molecule involved in regulating plants tolerance to heavy metals stress. In this study, we investigated the role of H2S in cadmium-(Cd-) induced cell death of root tips of cucumber seedlings. RESULTS: The results showed that the application of 200 µM Cd caused cell death, increased the content of reactive oxygen species (ROS), chromatin condensation, the release of Cytochrome c (Cyt c) from mitochondria and activated caspase-3-like protease. Pretreatment of seedlings with 100 µM sodium hydrogen sulfide (NaHS, a H2S donor) effectively alleviated the growth inhibition and reduced cell death of root tips caused by Cd stress. Additionally, NaHS + Cd treatment could decrease the ROS level and enhanced antioxidant enzyme activity. Pretreatment with NaHS also inhibited the release of Cyt c from the mitochondria, the opening of the mitochondrial permeability transition pore (MPTP), and the activity of caspase-3-like protease in the root tips of cucumber seedling under Cd stress. CONCLUSION: H2S inhibited Cd-induced cell death in cucumber root tips by reducing ROS accumulation, activating the antioxidant system, inhibiting mitochondrial Cyt c release and reducing the opening of the MPTP. The results suggest that H2S is a negative regulator of Cd-induced cell death in the root tips of cucumber seedling.

Transcriptome Analysis Reveals the Different Response to Toxic Stress in Rootstock Grafted and Non-Grafted Cucumber Seedlings.

Autotoxicity of root exudates is one of the main reasons for consecutive monoculture problem (CMP) in cucumber under greenhouse cultivation. Rootstock grafting may improve the tolerance of cucumber plants to autotoxic stress. To verify the enhanced tolerance to autotoxic stress and illuminate relevant molecular mechanism, a transcriptomic comparative analysis was performed between rootstock grafted (RG) and non-grafted (NG) cucumber plants by a simulation of exogenous cinnamic acid (CA). The present study confirmed that relatively stable plant growth, biomass accumulation, chlorophyll content, and photosynthesis was observed in RG than NG under CA stress. We identified 3647 and 2691 differentially expressed genes (DEGs) in NG and RG cucumber plants when compared to respective control, and gene expression patterns of RNA-seq was confirmed by qRT-PCR. Functional annotations revealed that DEGs response to CA stress were enriched in pathways of plant hormone signal transduction, MAPK signaling pathway, phenylalanine metabolism, and plant-pathogen interaction. Interestingly, the significantly enriched pathway of photosynthesis-related, carbon and nitrogen metabolism only identified in NG, and most of DEGs were down-regulated. However, most of photosynthesis, Calvin cycle, glycolysis, TCA cycle, and nitrogen metabolism-related DEGs exhibited not or slightly down-regulated in RG. In addition, several stress-related transcription factor families of AP2/ERF, bHLH, bZIP, MYB. and NAC were uniquely triggered in the grafted cucumbers. Overall, the results of this study suggest that rootstock grafting improve the tolerance of cucumber plants to autotoxic stress by mediating down-regulation of photosynthesis, carbon, and nitrogen metabolism-related DEGs and activating the function of stress-related transcription factor. The transcriptome dataset provides an extensive sequence resource for further studies of autotoxic mechanism at molecular level.

Downregulation of TNF-α/TNF-R1 Signals by AT-Lipoxin A4 May Be a Significant Mechanism of Attenuation in SAP-Associated Lung Injury.

Our previous studies verified the potent anti-inflammatory effects against severe acute pancreatitis (SAP) of AT-Lipoxin A4 and their analogues. However, the anti-inflammatory effects of AT-Lipoxin A4 on SAP-associated lung injury are not thoroughly known. We used western blot, polymerase chain reaction (PCR), and immunofluorescence to investigate the downregulation of TNF-α signals in cellular and animal models of SAP-associated lung injury following AT-Lipoxin A4 intervention. In vitro, we found that AT-Lipoxin A4 markedly suppressed protein expression in TNF-α signals in human pulmonary microvascular endothelial cell, such as tumor necrosis factor receptor-associated factor 2 (TRAF2), TNF-R1-associated death domain (TRADD), receptor-interacting protein (RIP), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Moreover, AT-Lipoxin A4 inhibited downstream signals activated by TNF-α, including NF-κB/p65, JNK/MAPK, and ERK/MAPK. In vivo, AT-Lipoxin A4 significantly decreased pathological scores of the pancreas and lungs and the serum levels of IL-6 and TNF-α. Immunofluorescence, western blotting, and real-time PCR assay showed that AT-Lipoxin A4 significantly attenuated the expression of TNF-R1, TRADD, TRAF2, and RIP in the lungs of SAP rats. In addition, the activation of NF-κB was also downregulated by AT-Lipoxin A4 administration as compared with SAP rats. AT-Lipoxin A4 could inhibit the production of proinflammatory mediators and activation of TNF-α downstream signals such as NF-κB and MAPK. Downregulation of TNF-α signals by AT-Lipoxin A4 may be a significant mechanism in the attenuation of SAP-associated lung injury.

Quantitative Trait Module-Based Genetic Analysis of Alzheimer's Disease.

The pathological features of Alzheimer's Disease (AD) first appear in the medial temporal lobe and then in other brain structures with the development of the disease. In this work, we investigated the association between genetic loci and subcortical structure volumes of AD on 393 samples in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Brain subcortical structures were clustered into modules using Pearson's correlation coefficient of volumes across all samples. Module volumes were used as quantitative traits to identify not only the main effect loci but also the interactive effect loci for each module. Thirty-five subcortical structures were clustered into five modules, each corresponding to a particular brain structure/area, including the limbic system (module I), the corpus callosum (module II), thalamus-cerebellum-brainstem-pallidum (module III), the basal ganglia neostriatum (module IV), and the ventricular system (module V). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicate that the gene annotations of the five modules were distinct, with few overlaps between different modules. We identified several main effect loci and interactive effect loci for each module. All these loci are related to the function of module structures and basic biological processes such as material transport and signal transduction.

Proteomic Investigation of S-Nitrosylated Proteins During NO-Induced Adventitious Rooting of Cucumber.

Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 µM S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.

The Involvement of Ethylene in Calcium-Induced Adventitious Root Formation in Cucumber under Salt Stress.

Calcium and ethylene are essential in plant growth and development. In this study, we investigated the effects of calcium and ethylene on adventitious root formation in cucumber explants under salt stress. The results revealed that 10 µM calcium chloride (CaCl2) or 0.1 µM ethrel (ethylene donor) treatment have a maximum biological effect on promoting the adventitious rooting in cucumber under salt stress. Meanwhile, we investigated that removal of ethylene suppressed calcium ion (Ca2+)-induced the formation of adventitious root under salt stress indicated that ethylene participates in this process. Moreover, the application of Ca2+ promoted the activities of 1-aminocyclopropane-l-carboxylic acid synthase (ACS) and ACC Oxidase (ACO), as well as the production of 1-aminocyclopropane-l-carboxylic acid (ACC) and ethylene under salt stress. Furthermore, we discovered that Ca2+ greatly up-regulated the expression level of CsACS3, CsACO1 and CsACO2 under salt stress. Meanwhile, Ca2+ significantly down-regulated CsETR1, CsETR2, CsERS, and CsCTR1, but positively up-regulated the expression of CsEIN2 and CsEIN3 under salt stress; however, the application of Ca2+ chelators or channel inhibitors could obviously reverse the effects of Ca2+ on the expression of the above genes. These results indicated that Ca2+ played a vital role in promoting the adventitious root development in cucumber under salt stress through regulating endogenous ethylene synthesis and activating the ethylene signal transduction pathway.