SlCOP1 Mediated SlJAF13 Ubiquitination Regulates Light-induced Anthocyanin Accumulation In Aft Tomato Fruit

Anthocyanins are naturally occurring plant pigments that are widely distributed in fruits,vegetables,and flowers.Their roles in plant life are manifold,spanning from attracting pollinators for pollination to offering defense against ultraviolet radiation.In humans,anthocyanins possess potent antioxidant effects that can help combat oxidative stress,reduce cellular damage,and lower the risk of several chronic diseases,including cardiovascular disease and cancer.Although tomatoes are one of the most widely consumed fruits and vegetables worldwide,the fruit of cultivated tomato varieties typically accumulates lycopene but not anthocyanins.However,certain tomato strains,such as Aft(Anthocyanin fruit),can accumulate high levels of anthocyanins in the fruit epidermis.Notably,studies have found that the accumulation of anthocyanins in the fruit skin of purple tomato varieties is dependent on light exposure,but the exact molecular regulatory mechanism remains elusive.Previous research in our laboratory have demonstrated that the bHLH transcription factor SlJAF13 positively regulates the synthesis of anthocyanins in the tomato fruit epidermis.A mutation in the SlJAF13 gene leads to an inability for the Aft tomatoes to accumulate anthocyanins.Further analysis revealed that SlJAF13 activates the expression of genes associated with anthocyanin biosynthesis by forming MBW complexes with SlAN2-like and SlAN11,thus contributing to the accumulation of anthocyanins in the Aft tomato fruit epidermis.Nevertheless,it remains unclear whether SlJAF13 is involved in the lightinduced anthocyanin accumulation process in tomato fruit and which light signal transduction factors could affect the expression level of SlJAF13.In this study,the Aft wild-type,sljaf13 mutant,SlJAF13-Com transgenic functional complementary line,and SlJAF13p: GUS transgenic line tomato fruits were used as materials.The expression level of SlJAF13 at the transcriptional and protein levels under light and darkness was investigated.We also analyzed the interaction between the light signal transduction factor SlCOP1 and SlJAF13 using yeast two-hybrid,luciferase complementation,and immunoprecipitation assays.Additionally,we analyze the ubiquitination and degradation effect of SlCOP1 on SlJAF13 through in vitro ubiquitination and MG132 treatment experiments,and assess the influence of elevated SlCOP1 expression levels on SlJAF13 expression levels and the accumulation of anthocyanins in fruits through transgenic experiments.The main results of this study are as follows:(1)Light promotes the stability of SlJAF13 protein and the accumulation of anthocyanins in Aft tomato fruits.Four different types of Aft tomatoes were subjected to both light and dark conditions as experimental materials.The accumulation of anthocyanin was observed to be dependent on light exposure,with its synthesis being inhibited in the absence of light.Analysis of these tomatoes using RT-qPCR,GUS staining,and immunoblotting showed that the transcription level of the SlJAF13 gene remained consistent under both light and dark conditions.However,a significant difference was found in the abundance of the SlJAF13 protein: its levels were markedly higher under light treatment compared to dark conditions.(2)SlCOP1 interacts with the SlJAF13 protein,leading to its ubiquitination and degradation.Yeast two-hybrid,luciferase complementation,and immunoprecipitation assays confirmed that SlJAF13 could interact with the E3 ubiquitin ligase,SlCOP1.In vitro ubiquitination analysis indicated that SlCOP1 could ubiquitinate SlJAF13.Co-transfection experiments revealed that the protein abundance of SlJAF13 decreases as the abundance of SlCOP1 protein increases,while the addition of the 26 S proteasome inhibitor,MG132,blocked the inhibitory effect of SlCOP1 on SlJAF13.This suggests that,SlCOP1 can interact with and ubiquitinate SlJAF13,leading to its degradation by the 26 S proteasome..(3)Overexpression of SlCOP1 inhibits anthocyanin accumulation in tomato fruits.A 35S:SlCOP1 overexpression plasmid was constructed and introduced into SlJAF13-Com tomato plants via genetic transformation.Subsequent phenotypic analyses revealed a decrease in anthocyanin levels in the fruits of the SlCOP1-overexpressing lines,indicating that SlCOP1 plays a important role in regulating anthocyanin accumulation in tomato fruits.Western blot analysis confirmed a decrease in the abundance of SlJAF13 protein in SlCOP1-overexpressing tomatoes,demonstrating that SlCOP1 suppresses anthocyanin accumulation by degrading SlJAF13 via the ubiquitin pathway.In summary,the accumulation of anthocyanins in Aft tomato fruits is light-dependent,promoting the stabilization and accumulation of SlJAF13 protein under light conditions,which in turn activates anthocyanin synthesis.Conversely,in darkness,SlCOP1 degrades SlJAF13 through ubiquitination,inhibiting anthocyanin synthesis.This study elucidates the molecular mechanism of light-induced anthocyanin synthesis in Aft tomato fruits,providing scientific rationale for the theory of light-induced anthocyanin synthesis.