Functional Analysis Of Tomato Pectate Lyase And S-adenosylhomocysteine Hydrolase Genes In Fruit Ripening

Tomato(Solanum lycopersicum L.)is one of the most important horticultural crops throughout the world,which is rich in nutrition,helpful in health care and effective in defending cancer.Also,it is a model plant for studying fruit ripening and softening.In this thesis,we used tomato of Micro-Tom as research background and focused on the role of two enzyme(pectate lyase and S-adenosine cysteine hydrolase)-encoding genes in tomato fruit ripening.Although there was no direct relationship between these two genes,they were both functional genes which were different from transcription factors,and also,their expression were increased in tomato fruit ripening and responded strongly to ethylene.These data supplied important information in defining their gene functions and improving tomato varietal characteristic by genetic engineering.Pectate lyase(PL,E.C.4.2.2.2)is one of pectin-related enzymes.It degrades the esterified pectin into a short chain of pectin molecule and a 4,5-unsaturated uronic acid with beta elimination mechanism to make pectin breakdown and fruit softening.In strawberry,one of the non-climacteric fruits,PL can act as a candidate gene to control fruit softening.Therefore,we conducted study of the corresponding gene in tomato,one of the climacteric fruits.Firstly,22 individual PL genes in tomato were identified,and one ripening related pectate lyase gene SlPL was further investigated from physiological level,cell level,molecular level and postharvest level.The expression of SlPL was enhanced in fruit at breaker stage and could be strongly induced by ripening related hormones especially by ethylene.RNA interference of SlPL resulted in enhanced fruit firmness,smaller and more epicarp cells,denser mesocarp cells and more compact arrangement of cells from exocarp to endocarp.SlPL-RNAi fruit demonstrated greater anti-rotting and pathogen-resisting ability.It had higher levels of cellulose and hemicellulose,whereas the level of water-soluble pectin was lower.Consistent with this,the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)were higher in SlPL-RNAi fruit,and the malondialdehyde(MDA)concentration was lower.Evaluation for agronomic characteristics indicated that suppression of SlPL brought no negative effect to most properties such as fruit yield.Real-time PCR results revealed that a lot of genes related to cell wall degradation and signaling pathway were down-regulated.More over,DEGs selected by RNA-seq data also supported above phenotype in molecular levels.The selected DEGs involved in hormone transduction pathway,transcription factor,cell wall metabolism,plant-pathogen interaction and so on.Collectively,these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance.This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit.S-adenosylhomocysteine hydrolase(SAHH,EC 3.3.1.1)is a widespread enzyme in cells.It catalyzes the reversible hydrolysis of S-adenosylhomocysteine(SAH)to adenosine(Ado)and homocysteine(Hcy),and is considered to be essential in regulating the intracellular methylation cycle.SAHH plays important roles in decreasing the replication ability of virus,regulating plant growth and defending abiotic stresses,but no information is available on the role of SAHH in fruit ripening.In tomato,SAHH is encoded with a gene family containing three members,although their expression in hormones treated seedlings and abiotic stresses treated plants hinted partial functional redundancy,their expression patterns in WT different tissues were all not the same,SlSAHH2 was highly accumulated during fruit ripening and strongly responded to ethylene treatment.Over-expression of SlSAHH2 enhanced SAHH enzymatic activity in tomato fruit development and ripening stages and resulted in a major phenotypic change of reduced ripening time from anthesis to breaker.Consistent with this,the content of lycopene was higher in SlSAHH2 over-expression(SlSAHH2-OE)lines than in WT at the same developmental stage.Compared to WT,real-time PCR results revealed that the expression of ethylene biosynthesis and inducible genes turned from down-regulation to up-regulation accompanied by fruit development and ripening in SlSAHH2-OE lines.Their expressions were enhanced significantly in SlSAHH2-OE breaker fruit with much more ethylene production than WT.Expressions of several ripening regulators were regulated,it was speculated that the influence of SlSAHH2 on ethylene production was downstream of the regulation of SlSAHH2 on these ripening regulator genes.SlSAHH2-OE lines displayed higher sensitivity to ethylene in both fruit and seedlings.1-amino cyclopropane carboxylic acid(ACC)treatment accelerated ripening faster in SlSAHH2-OE fruit than in WT.Additionally,seedlings of transgenic lines displayed shorter hypocotyls and roots in ethylene triple response assay.SAHH was an important hydrolase influencing methylation cycle,in our results several genes encoding DNA methyltransferases were enhanced in SlSAHH2-OE fruit at breaker stage,which suggesting that SlSAHH2 may also affect fruit ripening through methylation cycle.In conclusion,this report extended the previous study,confirming that SlSAHH2 also played an important role in tomato fruit ripening.