Role And Mechanism Of Nitric Oxide In Regulating Photosynthetic Pigment Biosynthesis And Cold Tolerance In Tomato

Nitric oxide(NO)is an important signaling molecule in plants,which is widely present and plays a critical role in plant growth and stress responses.However,NO plays a dual role depending on the concentration in plants.High concentration of NO would be toxic to plant photosynthesis.On the contrary,an appropriate concentration of NO could act as a growth regulator to reduce the damage caused by abiotic stress on plants.In facility production,the application of pesticides and fertilizers,and the activity of soil microorganisms are potential pathways of NO production.Tomato(Solanum lycopersicum),an important facility vegetable,is a kind of photophilous and thermophilic plant.It becomes a key problem to improve its photosynthetic capacity and overcome cold stress in overwintering cultivation.Therefore,in actual production,rational use of NO produced in facilities to enhance plant photosynthetic capacity and cold tolerance has positive ecological and economic benefits.In this study,the key factors and mechanisms involved in the inhibition of photosynthesis and the response to cold tolerance by NO in tomato were studied by using plant physiology,genetics,molecular biology,RNA sequencing and other means.The main contents and conclusions are as follows:1.NO inhibits the biosynthesis of photosynthetic pigments and weaken the photosynthetic capacity of plants,and Long Hypocotyl 5(HY5)plays a leading role in this process.In NO-accumulated gsnor mutants and exogenous NO donors-treated plants,it was found that higher concentrations of NO inhibited the accumulation of chlorophyll and carotenoids,which were important photosynthetic pigments,and decreased plant photosynthetic capacity.At the same time,HY5 also played a key role in regulation of plant photosynthesis.The loss of HY5 also led to a significant decrease in both photosynthetic pigment accumulation and photosynthetic capacity.In addition,both HY5 gene expression and protein content were inhibited by NO signaling.These results suggested that NO weakens the photosynthetic capacity of tomato plants by inhibiting the accumulation of photosynthetic pigments,and HY5 plays a major role in the inhibition of tomato photosynthesis by NO.2.NO signaling accelerates the degradation of HY5 and reduces gene expression of PORC(Protochlorophyllide oxidoreductase C)and PSY2(Phytoene synthase 2),key genes encoding rate-limiting enzymes in chlorophyll and carotenoid biosynthesis pathway,by inhibiting the transactivation of HY5.The transcript levels of PORC and PSY2 were significantly decreased in gsnor,NO donor-treated plants,and hy5,indicating that the transcript level of PORC and PSY2 was regulated by both NO signaling and HY5.EMSA assay,dual-luciferase chemiluminescence assay,Y1 H assay and Ch IP assay were used to demonstrated that HY5 could directly bind to the G-box domain in promoters of PORC and PSY2 and directly activate their expression.In addition,in vitro cell-free protein degradation assay also demonstrated that NO signaling could reduce the stability of HY5.Furthermore,results of EMSA and dualluciferase chemiluminescence assay proved that NO signaling reduced the transcript level of PORC and PSY2 by stimulating the degradation of HY5 and further decelerate pigment accumulation and photosynthesis.3.The C2H2 zinc finger protein ZF14 acts as a positive regulator in NO-induced cold tolerance.By treating tomato plants by various concentrations of exogenous SNP(Sodium nitroprusside),the optimal concentration of SNP that can improve plant cold tolerance was confirmed.Then the C2H2 zinc finger proteins,ZF12,ZF13 and ZF14,of which the transcript levels were induced by both cold stress and NO signaling were found by RT-q PCR.VIGS technique was used to determine the function of ZF12,ZF13 and ZF14 in cold response.Results showed that ZF14 was important in the regulation of cold resistance in tomato.ZF14 overexpression and CRISPR/Cas9 transgenic plants were constructed by transgenic approaches and then treated with cold stress.The results confirmed that ZF14 could positively regulate the cold tolerance in tomato.Furthermore,ZF14 CRISPR/Cas9 plants were treated with SNP before exposure to cold stress.It was found that ZF14 was an important role downstream of NO signaling in cold response,and NO could induce the expression of ZF14 and improve plant cold tolerance.4.ZF14 activates the expression of COR47-like to response to cold stress in NOinduced cold tolerance.RT-q PCR results showed that ZF14 could directly induce the expression of COR47-like in response to cold stress.Furthermore,dual luciferase chemiluminescence assay and yeast one-hybrid assay were used to demonstrate that ZF14 could bind the promoter of COR47-like and transactivate the expression of COR47-like.The transcript level of COR47-like in ZF14 CRISPR/Cas9 plants treated with SNP under cold stress was determined,and results showed that ZF14 regulated the expression of COR47-like downstream of NO signaling in response to cold stress.Dualluciferase luminescence assay further demonstrated that NO could increase COR47-like gene expression by enhancing the transactivation ability of ZF14.On the other hand,it was detected in gsnor by biotin switch assay that NO increased the S-nitrosylation proteins in plants exposed to cold stress.In vitro experiments also proved that ZF14 could be S-nitrosylazed in the presence of GSNO,and Cys79 in the conserved zinc finger domain of ZF14 was found to be a potential target of S-nitrosylation.In conclusion,ZF14 could regulate the transcription of COR47-like in NO-mediated cold stress response pathway,and NO could enhance the transcriptional regulation of COR47-like by ZF14 and S-nitrosylated ZF14.In conclusion,this study proved that NO inhibits the biosynthesis of photosynthetic pigments and weakens the photosynthetic capacity of tomato plants by promoting the protein degradation of HY5 and inhibiting its transcriptional activity to key genes encoding rate-limiting enzymes of chlorophyll and carotenoids biosynthesis.For another,the C2H2 zinc finger protein ZF14 is a key factor in NO-induced cold tolerance.ZF14 improves plant cold tolerance by transcriptional regulation of CORs gene expression.S-nitrosylation of ZF14 by NO further enhances the transcriptional regulation activity of ZF14 and promotes the expression of CORs.NO and ZF14 jointly participate in the cold stress response of tomato plants.This study provides a further understanding of the dual role of NO in plant photosynthesis and cold stress response and provides a scientific basis for controlling the concentration of NO and rationally using genetic modification technology and plant growth regulators to improve crop yield,quality,and cold tolerance in agricultural production.