Physiological Responses And Molecular Mechanism Of Cucumber To Different Low Temperature And High Humidity

Cucumber（Cucumis sativus L.）is a thermophilic crop but often grow in greenhouse and plastic tunnel during off season throughout the world,where low temperatures（LT）and high humidity（HH）is one of the main restrictions.Although the effects of LT on growth of cucumbers are well known,there is very limited information about the effects of high humidity especially the combination of HH and LT on cucumber and other vegetable crops.Therefore,it is necessary to investigate the effects of different combinations of LT and HH stress on cucumber cultivars with different resistance,and the response of cucumber at different leaf stage.How about cucumber physiological responses to concurrent and contemporaneous effect of LT and HH on chlorophyll biosynthesis?How about the expression and performance of related genes and pathways against this stress to understand the underlying molecular mechanism?Whether melatonin has the potential to alleviate the detrimental effects of LT and HH?These problems need to be further explored.（1）Biochemical and Physiological Responses of Cucumber Cultivars to Different Combinations of Low Temperature and High Humidity StressCucumber growth and development are considerably affected by fluctuation in temperature and humidity thereby influencing its morphology,physiology,and biochemical properties.In this experiment two cucumber cultivars viz:Zhongnong37（ZN37:resistant）and Shuyanbailv（SYB:sensitive）were subjected to different LT and HH treatments（day/night:T1 15/10°C+95%RH,T2 12/8°C+95%RH,and T3 9/5°C+95%RH）along with a normal temperature and normal humidity（CK 25/18°C+80%RH）as control.The results revealed that LT and HH stress reduced morphological parameters,relative water content,net photosynthesis rate,stomatal conductance,transpiration rate,leaf pigments and increased electrolyte leakage,malondialdehyde（MDA）,hydrogen peroxide（H₂O₂）and intercellular concentration of carbon dioxide in cultivars cv.SYB than cv.ZN37 as compared to CK.Antioxidant enzymes such as,superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,ascorbate peroxidase（APX）,monodehydroascorbate reductase（MDHAR）,dehydroascorbate reductase（DHAR）,and glutathione reductase（GR）were decreased in cv.SYB under stress conditions as compared to cv.ZN37.Furthermore,LT and HH treatments showed an increase in proline and soluble protein content in cv.ZN37 as compared to cv.SYB.Abscisic acid（ABA）and jasmonic acid（JA）were augmented in cv.ZN37,while indole-3-acetic acid（IAA）,zeatin（ZT）,zeatin riboside（ZR）,and gibberellic acid（GA）was decreased but was higher than cv.SYB.Under T3 net photosynthesis,protoporphyrin,and ZT were extremely decreased by 71.3%,74.3%,and 82.4%,respectively,in cv.SYB compared to their control.Moreover,principal component analysis based on physiochemical traits confirmed that cv.ZN37 had the strongest correlation with antioxidant enzymes,proline,and soluble protein content than cv.SYB under LT and HH treatments.Our results suggest that a stress-tolerant cultivar might mitigates stress damage in cucumber by regulating photosynthetic efficiency,antioxidant capacity and hormonal profile when compared to a stress-sensitive cultivar.（2）Effect of LT and HH Stress on Physiology of Cucumber at Different Leaf StagesLT and HH are important environmental factors in green house and plastic tunnel during cold season that hamper plant growth and development.In this experiment,we studied the effect of different LT and HH treatments（day/night:CK 25/18°C+80%RH,LT+NH9/5°C+80%RH,NT+HH 25/18°C+95%RH,LT+HH 9/5°C+95%RH）on cucumber young plant at 2,4,or 6 leaf stages.The results showed that LT+HH treatment resulted in a decline in morphological parameters such as,shoot,root and total fresh and dry weights and decreased net photosynthesis rate（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）,photochemical efficiency（Fv/Fm）,photochemical quenching（q P）,electron transport rate（ETR）,and chlorophyll pigments parameters,and increased MDA,H₂O₂,O₂^-,non-photochemical quenching coefficient（NPQ）,intercellular CO₂ as compared to its control（CK）at the 2-leaf stage.Antioxidant defense like SOD,POD,CAT,APX and GR activities were upregulated under LT+HH as compared to the plants grown in control condition at the 6-leaf stage.ABA and JA increased under LT+HH stress as compared to its control（CK）at the 6-leaf stage,while IAA and GA decreased under LT+HH compared to its control（CK）at the 2-leaf stage.Our results show that LT+HH stress affects young cucumber plant photosynthetic efficiency,PSII activity,antioxidant defense system,ROS and hormone profile.MDA,H₂O₂,and O₂^-were elevated sharply by 140%,136%and 279%,respectively,at 2-leaf stage compared to its control（CK）.Plants at the 6-leaf stage were more tolerant than at the 2 leaf stages under stress conditions when compared to its control（CK）.（3）Effect of LT and HH Stress on Leaf Pigments and Chlorophyll Biosynthesis of CucumberLT and HH stress evidently inhibited chlorophyll biosynthesis in cucumber seedlings during greening.The chlorophyll（Chl）biosynthesis inhibitory mechanism under LT and HH stress is still widely unclear.To elucidate the underlying mechanism of chlorophyll biosynthesis cucumber seedlings at two true leaves stage were exposed to LT and HH treatments（CK 25/18℃+80%RH,NT+HH 25/18℃+95%RH,LT+NH 9/5℃+80%RH,LT+HH 9/5℃+95%RH）.Experimental results revealed that LT+HH stress reduced chlorophyll pigments and carotenoid content as compared to CK.In addition,chlorophyll biosynthesis precursors such as amino-levulinic acid,porphobilinogen,urogen III,coprogen III,protoporphyrin IX,Mg-protoporphyrin,Mg-protoporphyrin monomethyl ester and chlorophyllide content were significantly inhibited under LT and HH stress and extreme inhibition was detected under LT+HH treated seedlings.Meanwhile,the activities of Chl biosynthesis related enzymes such as GSA-AT,ALAD,Mg-chelatase,and POR were also decreased under LT+HH stress conditions.Chl biosynthesis related genes like,CAB,CHl H,PBG and POR were upregulated under the NT+HH condition,while significantly downregulated under LT+HH stress treatment as compared to their CK treated plants.Histochemical observation showed increased O₂^-and H₂O₂content in cucumber leaves during the LT+HH stress periods.Taken together,the results revealed that LT+HH treatment seriously reduced total chlorophyll（14.2%）,Chl biosynthesis intermediates such as ALA（84.1%）,and Pchlide content（88.2%）,which insight our understanding that LT+HH stress impair chlorophyll biosynthesis and induce oxidative injury in cucumber at seedling stage.（4）Transcriptomic Profiling of Cucumber Reveals Multiple Genes and Complex Hormonal-Mediated Interaction During LT and HH StressDue to increased environmental constraints as a result of global climate change,LT+HH（day/night 9/5°C+95%RH）stress limit agriculture productivity.Several researches performed on LT responses to plant,however the combination of LT+HH stress responses to cucumber remain widely unclear.Therefore,it is necessary to increase the effort to explain the physiological and molecular mechanisms of LT+HH stress tolerance in cucumber.In the present investigation,cucumber seedlings were treated with LT+HH taking CK（day/night25/18°C+80%RH）as the control,and samples were taken at 24,48,and 72 h after treatment,respectively,for RNA-seq analysis.To understand the molecular mechanism of this stress effect,we performed RNA-seq analysis.A total of 10459 differentially expressed genes（DEGs）were found to have pathway annotation from RNA-seq analysis.These pathways were involved in plant hormone signal transduction,protein processing endoplasmic reticulum,MAPK signaling pathway,carbon fixation in photosynthetic organisms,and glycerolipid metabolism.In addition,123 DEGs were identified as associated with hormone signaling pathways,and their responses under LT+HH stress were deeply discussed.We also performed RT-q PCR analysis of LT and HH related genes and hormone signaling to validate the result of RNA-seq analysis.The RT-q PCR result showed that hormone signaling related genes were significantly influenced by LT+HH treatment and upregulated genes showed highest expression at each time points.（5）Melatonin Improved Photosynthesis and Robust Defense Mechanism of Cucumber under LT and HH StressEven though low temperature and high humidity are dominant environmental factors that restricts plant growth and development.To investigate whether exogenous melatonin（MT）has the potential to mitigate the damaging effects of LT and HH stress.In this study,cucumber seedlings at fully developed two true leaf stage were treated with 200μM MT and LT+HH treatments（CK 25/18℃+80%RH,CK+MT 25/18℃+80%RH+MT,LT+HH15/10℃+95%RH,LT+HH+MT 15/10℃+95%RH+MT）to examine the effect of MT on seedling tolerance under the employed treatment in cucumber seedlings,and explored the underlying mechanism.The results showed that LT+HH stress inhibited growth and biomass accumulation,produced leaf chlorosis,led to oxidative stress,lowered chlorophyll a,chlorophyll b and carotenoid contents,reduced photosynthetic and photosystem II（PSII）activities,and increased the level of intercellular carbon dioxide and the non-photochemical quenching of PSI and PSII.However,foliar application of MT significantly improved the morphological indices and photosynthetic efficiency of cucumber seedlings,which entailed the elevation of electrolyte leakage,lipid peroxidation,and reactive oxygen species accumulation by boosting the antioxidant enzyme defense system under LT+HH conditions.Additionally,measurement of nitrogen,magnesium,and iron contents in roots and leaves showed that MT significantly augmented the nutrient uptake of cucumber seedlings exposed to LT+HH stress.Furthermore,MT application increased the transcripts levels of genes encoding antioxidant enzymes（Cs SOD,Cs POD,Cs CAT,Cs APX,Cs MDHAR,Cs DHAR and Cs GR）under LT+HH conditions,whereas treatment with LT+HH suppressed these genes,suggesting that MT application increases the LT+HH tolerance of cucumber seedlings.Overall,the results showed that MT application strikingly activating the main endogenous antioxidant enzymes like,superoxide dismutase（201.3%）,peroxidase（213.9%）,catalase（477.6%）,and ascorbate peroxidase（205.5%）at 4 DAT vs.CK,suggest that MT application increases the tolerance of cucumber seedlings to LT+HH stress.Thus,the exogenous application of MT could be potentially employed as a strategy to improve the LT and HH tolerance of cucumber production.