The Mechanism Of SlCAS1 Involved In Tomato Fruit Maturation And Senescence And Leaves Disease Resistance

Tomato(Solanum Lycopersicum)is one of the most important economic crops which is widely cultivated in the North and South of China.Because of their high nutritional content,tomato fruits are deeply loved by consumers.Meanwhile,as a model plant species,tomato has been widely used for studying fruit maturation and senescence.Significant changes in color,flavor and texture are the characteristics of fruit maturation and senescence,and this series of physiological changes are regulated by many factors.Cyanoalanine synthase(CAS)plays a key role in cyanide detoxification of plants.Previous studies have shown that the expression pattern of CAS is similar to ethylene synthesis-related genes during fruit maturation and senescence.But whether CAS participates in the process of fruit maturation and senescence is unclear.In this study,SlCAS1/2 was used as the research object to explore the regulatory mechanism of SlCAS in tomato fruit maturation and senescence.Bioinformatics analysis identified 32 CAS genes in 16 plant species.The results showed that tomato CAS has the highest homology with Solanum tuberosum.The expression pattrens of SlCAS1/2 gene in wild-type tomato different tissues was analyzed by RT-qPCR.The expression levels of SlCAS1/2 were higher in mature fruit.Tomato SlCAS1/2 gene silencing plants were constructed by virus-induced gene silencing(VIGS).Gene silencing of SlCAS1/2 resulted in delayed fruit maturation and senescence,but the effect of SlCAS1 was more significant than SlCAS2.Furthermore,CRISPR/Cas9 gene editing and gene overexpression technology were used to construct gene editing T2 generation homozygous mutant plants(ΔSlCAS1)and gene overexpression plants(SlCAS1-OE).Compared with WT,the maturation and senescence process of SlCAS1OE tomato fruit was 7 days earlier,while ΔSlCAS1 tomato fruit was 5 days later.The pigment metabolism of WT,ΔSlCAS1 and SlCAS1-OE tomato fruits at 20 DPA(days post anthesis,DPA),30 DPA and 37 DPA was investigated.It was found that the SlCAS1OE tomato fruit had accelerated chlorophyll degradation and accumulated a large amount of carotenoid,lycopene and anthocyanin.At 30 DPA,the carotenoids content of SlCAS1OE tomato fruit was significantly higher than WT(P<0.01),which was about twice of WT.The trend of ΔSlCAS1 tomato fruit was opposite to SlCAS1-OE.ΔSlCAS1 tomato fruit accumulated a large amount of chlorophyll and the content of carotenoids,lycopene and anthocyanins were significantly reduced during the maturation and senescence process.The expression levels of chlorophyll degradation related genes(SlNYC1,SlSGR1,SlPPH and SlPAO),carotenoid synthesis related genes(SlPSY1,SlPDS and SlZDS),anthocyanin synthesis-related genes(SlDFR and SlANS)and ethylene metabolism pathway related genes(SlACO1,SlACO3,SlACS2,SlACS4,SlRIN and SlNOR)in WT,ΔSlCAS1 and SlCAS1-OE tomato fruits were analyzed by RT-qPCR.The expression levels of the above genes in WT,ΔSlCAS1 and SlCAS1-OE tomato fruits increased with fruit maturation and senescence.However,in the same period,the expression levels of genes related to pigment metabolism and ethylene metabolism in SlCAS1-OE fruit was the highest,while that in ΔSlCAS1 fruit was the lowest.The results indicated that SlCAS1 could participate in fruit maturation and senescence in pigment metabolism and ethylene synthesis.The upstream regulatory factors of SlCAS1 were screened by transcriptome data and correlation analysis.SlERF-H6,SlERF109-like and SlWRKY71 were positively correlated with SlCAS1 in tomato fruits.Dual luciferase reporter assay found that SlERF-H6 had the most significant activation effect on the promoter region of SlCAS1 gene.To verify the role of SlERF-H6 in fruit maturation and senescence,SlERF-H6 gene silencing plants were constructed by VIGS.Compared with the control,the maturation and senescence process of SlERF-H6-vigs tomato fruit was 4 days later.Compared to the control,the SlERF-H6-vigs tomato fruits expression levels of SlCAS1 was significantly downregulated.On the 7th day,the relative expression of SlCAS1 gene in the control was about 23 times higher than SlERF-H6-vigs.The yeast one-hybrid(Y1H)assay was used to analyze the interaction between SlERF-H6 and SlCAS1.The results showed that SlERFH6 can bind to SlCAS1-1/3,indicating that SlERF-H6 regulated the expression of SlCAS1 gene by binding to the promoter of SlCAS1.To preliminarily explore whether SlCAS1 gene is involved in the senescence and cell death of tomato leaves under biotic stress,Pseudomonas syringae pv.Tomato(Pst)DC3000 was inoculated in WT,ΔSlCAS1 and SlCAS1-OE tomato leaves.The necrosis symptoms in ΔSlCAS1 leaves inoculated with Pst DC3000 was the most obvious.Trypan blue staining was used to identify dead cells in leaves,the results showed that the number of dead cells in SlCAS1-OE tomato leaves was the least.Furthermore,bacterial number counting assay demonstrated that the number of Pst DC3000 in ΔSlCAS1 tomato leaves was the largest,and Pst DC3000 could grow better in ΔSlCAS1 tomato leaves.The content of hydrogen peroxide(H2O2)in tomato leaves inoculated with Pst DC3000 was identified by DAB staining.The content of malondialdehyde(MDA)in tomato leaves inoculated with Pst DC3000 was determined to identify the membrane lipid peroxidation.Compared with WT and SlCAS1-OE,ΔSlCAS1 tomato leaves accumulated large amounts of H2O2 and had increased peroxidation of cell membranes Furthermore,the expression levels of antioxidant-related genes(SlPOD and SlSOD)and pathogenesis-related genes(SlNPR1 and SlPR1)in leaves inoculated with Pst DC3000 were analyzed by RT-qPCR.In WT,ΔSlCAS1 and SlCAS1-OE,the expression level of SlSOD was highest in ΔSlCAS1 and lowest in SlCAS1-OE.However,in WT,ΔSlCAS1 and SlCAS1-OE,the expression level of SlPOD was highest in SlCAS1-OE and lowest in ΔSlCAS1.The results showed that the overexpression of SlCAS1 reduced the production of H2O2 and promoted the decomposition of H2O2.Compared with WT and ΔSlCAS1,the expression levels of SlNPR1 and SlPR1 in SlCAS1-OE tomato leaves was significantly up-regulated.Overexpression of SlCAS1 gene promoted the expression levels of disease resistance related genes and slowed down senescence and death of plant cells under Pst DC3000 stress.In conclusion,tomato SlCAS1 regulates fruit maturation and senescence through chlorophyll degradation,pigment accumulation and ethylene synthesis.SlERF-H6 activates SlCAS1 gene expression by binding to the SlCAS1 promoter region,thereby promoting tomato fruit maturation and senescence.Further studies showed that SlCAS1 delayed the senescence and death of plant cells under biotic stress.