| Plants are constantly threatened by various pathogens in a challenging environment.Panax notoginseng(Bruk.)FH Chen is a valuable traditional herb in China.However,leaf diseases are a major factor limiting growth and production of Panax notoginseng.Panax notoginseng black spot disease(the pathogen is Alternaria panax Whetzel)is the main devastating disease in the production of Panax notoginseng,which brings huge losses to the production of Panax notoginseng every year.However,the molecular mechanism of Panax notoginseng immune response regulation is still unclear.The prevention and control of Panax notoginseng leaf diseases mainly relies on the application of a large number of chemical pesticides and the resulting excessive pesticide residues are undoubtedly the focus of society.Therefore,in order to dig deeper into the regulation mechanism of Panax notoginseng immune response,using biological methods to improve Panax notoginseng innate immunity and disease resistance is an effective strategy for the sustainable development of Panax notoginseng green agriculture.The research results of this study are as follows:1.By using transcriptome and proteomics techniques,this study analyzed the oxidative stress response of Panax notoginseng infected with Alternaria panax Whetzel(A.panax),and found lignin synthesis and melatonin synthesis pathways.Pathogen infection caused significant changes in the transcriptome and proteome of Panax notoginseng.Specifically,a total of 13,6100 transcripts and 4,468 proteins were identified.The integration of transcriptome and proteome profiles revealed many candidate transcripts/proteins that may be involved in lignin synthesis and melatonin synthesis during the activation of oxidative stress response in Panax notoginseng.In detail,the expression level and protein abundance of Pn COMT1,a key gene in the melatonin synthesis pathway,increased significantly,and the endogenous melatonin content also increased significantly.It shows that A.panax induces the accumulation of endogenous melatonin in Panax notoginseng,thereby activating the defense response of Panax notoginseng.2.In this study,the Pn COMT1 gene was cloned from Panax notoginseng and analyzed its function in pathogen resistance in transgenic Arabidopsis plants.Results showed that that subcellular localization of Arabidopsis protoplasts revealed that the encoded protein was localized in the cytoplasm.The transgenic arabidopsis expressing Pn COMT1(Pn COMT1-OE)contains higher concentrations of endogenous phytomelatonin than the wild type Col-0 plants.Compared with Col-0,Pn COMT1 OE plants showed higher stomatal sensitivity and MAPK activity after Pst DC3000 and A.panax infection.And overexpression of Pn COMT1 also increased the bacterial resistance of the transgenic arabidopsis plants.Under Pst DC3000 treatment,the expression of defense-related response genes in transgenic plants was higher than in wild-type plants.The transgenic plants show better growth and physiological performance under pathogen stress,such as higher antiperoxidase activity,indicating that Pn COMT1 positively regulates biotic stress resistance via enhancement of phytomelatonin synthesis.3.Here,we show that melatonin functions in stomatal immunity in Panax notoginseng and Arabidopsis(Arabidopsis thaliana).Biochemical analyses showed that melatonin-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv.tomato(Pst)DC3000 via activation of MAPK and NADPH oxidase-mediated reactive oxygen species production in P.notoginseng.The first putative phytomelatonin receptor 1(PMTR1)is a plasma membrane protein required for perceiving melatonin signaling in stomatal closure and activation of MAPK.Biochemical and genetic tests found PMTR1 is essential for flg22-and melatonin-induced MAPK activation in a heterotrimeric GTP-binding protein Gαsubunit GPA1-independent manner.GPA1 functions in the same genetic pathways of FLS2/BAK1-as well as PMTR1-mediated flg22 and melatonin signaling in stomatal closure.The stomata in pmtr1 are insensitive to melatonin and flg22,but the application of melatonin induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants,indicating that PMTR1 might be a downstream signaling component in FLS2-and BAK1-mediated stomatal immunity.4.We also investigated the role of melatonin in leaf disease resistance of Panax notoginseng in field conditions and analyzed saponin content to evaluate the applicability of melatonin in agricultural practice.Our results showed that exogenous application of melatonin promoted the endogenous phytomelatonin accumulation via upregulation of genes involved in its biosynthesis.The application of 10 μM melatonin decreased the incidence of leaf diseases(gray mold,round spot,and black spot)by about 40% compared with the solvent control,which might have been due to the increased expression of genes associated with immunity and disease resistance.Furthermore,concentrations of saponins and expression of their biosynthesis-related genes were significantly increased by melatonin.In summary,this study was firstly to analyze the transcriptome,proteome and metabolome data of Panax notoginseng leaves under the Alternaria panax Whetzel stress,and screen out a melatonin synthesis gene Pn COMT1 in Panax notoginseng.And through functional verification,it is found that Pn COMT1 may positively regulate the stress response and melatonin synthesis under the pathogen stress,and may become a potential candidate gene for future research on the disease resistance and defense mechanism of Panax notoginseng plants.Molecular genetic experiments have also confirmed that that phytomelatonin functions in priming of stomatal immunity and provide insights into the phytomelatonin signaling transduction pathway.Moreover,as a signaling molecule,melatonin increases the biosynthesis level of endogenous melatonin by regulating the expression of related genes,and stimulates the innate immunity and the biosynthesis of saponins in Panax notoginseng plants.These results indicate that melatonin can be used in the agricultural control of Panax notoginseng,and provide a new theoretical and practical basis for the green cultivation of Panax notoginseng. |
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