SLIPT4 Gene Regulates Leaf Senescence And Lycopene Biosynthesis During Fruit Ripening In Tomato

Cytokinins?CKs?are important phytohormones involved in a wide range of developmental and physiological processes in plants,including cell division,apical dominance,leaf senescence,root formation and development,fruit set and development,as well as roles in response to stress tolerance.However,their roles in fruit ripening are complete unknown.The isopentenyltransferases?IPTs?catalyze the initial rate-limiting step of cytokinins biosynthesis,and different IPT genes may regulate different CKs biosynthesis.Threfore,studying the biological function of IPT genes is important for revealing the regulatory mechanism of CKs biosynthesis.Lycopene is an important carotenoid pigment in red fruits and vegetables,and its accumulation,leading tomato fruit turn into red,is an important feature of tomato fruit ripening.Lycopene biosynthesis and catabolism are regulated by multiple factors including phytohormones,little is known about their regulatory mechanism.In this study,the expression leavels of SlIPT4 and its response to different hormone treatment and stress were analyzed by RT-PCR.At the same time its biological functions was analyzed through overexpression and silencing strategy.The main results are as follows:?1?Expression profiles of SlIPT4 were analyzed by RT-PCR,the results showed that SlIPT4 had a higher expression in the young leaves of tomato and decreased gradually with leaves senescence process;SlIPT4 had abundant accumulation at the early stage of fruit development,maintained a low expression level in green ripe fruit,and then increased gradually during fruit ripening process.This indicated that SlIPT4 may play important roles in the process of leaves senescence,early fruit development and fruit ripenning process?especially the biosynthesis of lycopene?.?2?The response analysis of SlIPT4 to different hormones and adversity treatment showed that auxin?IAA?,ethylene biosynthesis precursor ACC and ethylene synthesis inhibitor AgNO₃ treatment had almost no effect on the expression of SlIPT4,while auxin inhibitor NPA enhanced the expression of SlIPT4 gene;abscisic acid?ABA?could significantly inhibit the expression of SlIPT4;drought stress,salt stress and water stress could inhibit the expression of SlIPT4.These results indicated that SlIPT4 may be involved in the adaptation process of tomato under abiotic stress condition and regulated by ABA.?3?Overexpression and silencing vectors of SlIPT4 were constructed,and then these vectors were transformed into tomato by agrobacterium mediated transformation to obtain transgenic tomato lines.Phenotypic analysis showed that there was no significant difference between SlIPT4 overexpressing lines and wild-type plants,while SlIPT4silencing lines showed obvious phenotypic changes.?4?Silencing of SlIPT4 resulted in accelerated senescence of tomato leaves,decreased chlorophyll content in leaves;and down-regulated the expression of GLK2,a positive regulator of chlorophyll synthesis;up-regulated the expression of SGR,a positive regulator of chlorophyll degradation;and down-regulated the expression of Rbcs3A a positive regulator of photosynthesis.These results indicated that SlIPT4 may participate in the process of leaves senescence by regulating chlorophyll biosynthesis and metabolism of in tomato leaves.?5?Silencing of SlIPT4 leaded to the never-red orange fruits.Both total carotenoids and lycopene were dramatically decreased in SlIPT4-RNAi fruits.RT-PCR analysis showed that the expression of ZISO was significantly downregulated by more than 80%in SlIPT4-RNAi fruits compared with that in wild-type control fruits,and ZDS was mildly reduced in SlIPT4-RNAi lines,while other carotenoids biosynthesis genes?PSY1,PDS,CRTISOs?were up-regulated.These results indicate that SlIPT4 may regulate lycopene biosynthesis by regulating the expression of ZISO.?6?ABA is one of the major products of lycopene degradation,playing important roles in the process of plant adaptation to stress.Decreased ABA content was observed in SlIPT4-RNAi fruits,suggesting that the decrease of lycopene in the silencing fruits was not due to the enhancement of its degradation process,but the inhibition of its biosynthesis.Morever,the ABA biosynthesis related genes?ZEP,NCED1 and NCED2?were up-regulated in SlIPT4-RNAi fruits,consistented with the increased expression of PSY1 and PDS,suggesting the negative feedback regulation between the biosynthesis of ABA and carotenoids?lycopene?.In summary,this study demonstrated that SlIPT4 plays important roles in the regulation of leaves senescence and the network of lycopene biosynthesis,having significance to reveal the regulatory mechanism of CKs in the regulation of leaves senescence and lycopene biosynthesis.