Investigation On Molecular And Cellular Mechanisms Of Red Light-induced Citrus Fruit Colouration

The colouration of citrus fruit is a very important indicator for evaluating external quality.The yellow,orange and red colors in citrus fruits are attributed to the large accumulation of carotenoids.In citrus fruits,there are many phenomena that the flesh and pericarp are unable to synchronously mature.When fruits reach commercial harvesting standards,the pericarp cannot completely change colour,and even the flesh reaches the full maturity,the pericarp still cannot de-green normally,which seriously affects the fruit’s market period and commercial value.The mature stage of the citrus in China mostly occurs in November,leading to great pressure on the sales during the ripening period.Some citrus varieties of early or late-maturing are usually with poor colouration,which results in cheapness.This study introduced a method of uniformly colouring citrus peel through red light(RL)irradiation,and explored the mechanism of RL-regulated colouration by using ‘Huapi’ kumquat(Fortunella crassifolia Swingle)as the experimental material.Moreover,the study successfully established a citrus fruit transient expression system by using Agrobacterium-mediated transformation,creating a new platform for genetic improvement and gene function research of perennial woody plants.The main results were as follows:1.Transient transformation of citrus fruit for gene function verification and cell biology research.Kumquat fruits cv.‘Huapi’ and ‘Rongan’ were used for transient transformation of citrus fruits using an Agrobacterium-mediated method;as they exhibited higher transformation efficiency and prolonged expression window among all citrus fruit have been tested.Fruits were transformed with different fluorescent reporters,such as microfilament(GFP-Lifeact),endoplasmic reticulum(GFP-HDEL),Golgi(GFP-ST and RFP-ST),plastid(RFP-PT)and nucleus(H2B-GB),confocal microscopy and live cell imaging were used to study their localization and dynamics.Furthermore,transient overexpression of PSY significantly increased the carotenoid content in citrus fruits,indicating that this transient expression system can also be used to verify gene function.2.Red LED light promoted chlorophyll degradation and carotenoid accumulation to improve colouration of citrus fruits after harvest.Using kumquat fruits with uneven colour as materials,the effects of RL,ethephon and RL combined with ethephon on fruit degreening were evaluated.The results showed that all above treatments significantly promoted the degreening of the outer peel of citrus fruit.In comparison of the degreening effects,RL treatment was superior to the application of ethephon.RL significantly accelerated the colour change,accompanied by the conversion of chloroplasts to chromoplasts,the degradation of chlorophyll and the accumulation of carotenoids.When RL was applied to other citrus varieties,such as Satsuma mandarin(Citrus unshiu Marc.)and Tribute Citru(Citrus reticulata Blanco Var.Gonggan),and even tomato fruit,RL could also promote fruit colour change,indicating that it is a common phenomenon that RL promotes fruit degreening.3.Discover the key genes that promote fruit colour change in response to red light irradiation and analyze the mechanism of Fcr NAC22 regulating fruit colour change.In the process of RL-promoted degreening of kumquat fruits,the transcription factor Fcr NAC22 was identified to be significantly up-regulated through transcriptome data in a RL-specific manner,while blue light did not affect its expression.Subcellular localization study has shown that Fcr NAC22 localized to the nucleus,and its gene expression was up-regulated as fruits colour change.Fcr NAC22 overexpression accelerated the yellowing of tobacco leaves,promoted the colour change of tomatoes and citrus fruits,elevated carotenoid accumulation and plastid conversion.In the transgenic citrus callus,it was found that Fcr NAC22 overexpression promoted yellowing of the callus and increased carotenoid synthesis;Knock down of Fcr NAC22 expression in transient transformed citrus fruits attenuated fruit colouration induced by RL.Results from dual luciferase,yeast one-hybrid(Y1H)and electrophoretic mobility shift assays(EMSAs)indicated that Fcr NAC22 directly binds to the promoters of the carotenoid metabolism pathway LCYB1 and BCH2,as well as the promoter of NCED5 on the ABA synthesis pathway,and activates the transcriptional expression of these genes,leading to increased carotenoids level and ABA content in citrus fruit.The above results not only introduced a safe and effective method for improving the colouration of citrus fruits through RL irradiation,but also explored theoretical basis for regulating the external quality of citrus from multiple perspectives at the molecular and cellular level.In addition,successful transient transformation of citrus fruits provided new insights into fruit biology research and gene function study.