Expression And Identification Of Recombinant Lycopene Cyclases And Carotenoid Metabolism In Citrus Fruits

Citrus fruits are rich in carotenoids and application of lycopene cyclase inhibitors is an effective way of studying on the carotenoid metabolism of citrus fruits. In the present work,’Newhall’ navel orange (Citrus sinensis Osbeck),’Valencia’orange (C. sinensis Osbeck),’Nichinan 1’ and ’Guoqing 1’satsuma mandarin (C. unshiu Marcow) are used as the materials. Effects of two lycopene cyclase inhibitors,2-(4-chlorophenylthio)-triethylammonium chloride (CPTA) and 2-(4-methylphenoxy)-triethylammonium chloride (MPTA), on citrus fruits etc., as well as effects of CPTA treatment on carotenoid composition (e.g. lycopene) and gene expression (e.g. phytoene synthase, PSY) in the flavedo of’Valencia’orange were investigated, and regulatory mechanism of CPTA on carotenogenesis was subsequently discussed. Proteins in the flavedo of ’Valencia’ orange that responded to CPTA treatment were identified via proteomic methods. Moreover, two lycopene cyclase genes, lycopene s-cyclase (LCYe) and lycopene (3-cyclase (LCYb), from citrus fruit were cloned, and their recombinant vectors for expression in pichia pastoris were constructed respectively. After the induced expression by methanol, recombinant proteins were purified and identified. Main results are listed as follows:(1) Effects of lycopene cyclase inhibitors CPTA and MPTA on citrus fruits etc.Daubing treatment on the fruits of’Valencia’orange with 0.1% to 5% CPTA resulted in massive lycopene accumulation and red coloration in the flavedo, but the coloration and regular qualities (e.g. soluble sugar), as well as the ultrastructure of rind-oil spots, were hardly affected. On the contrary, the peel coloration was not affected by the injection of 0.1% CPTA solution into the fruit center, while the juice sacs, the central axis and surrounding segment membrane and albedo tissues turned red. A relative stable red color formed in the peel of’Nichinan 1’satsuma mandarin at 5 d when treated with 0.1% CPTA after the turning stage; however, when treated with 0.5% CPTA before the turning stage, only the rind-oil spots turned red at 20 d, which indicating that the timing is very important to the treatment. Besides, white buds turned pink at 5 d after sprayed with 0.1% CPTA, while the leaves were not affected by 0.1% CPTA or MPTA, suggesting the coloration of organs and tissues with vigorous carotenoid metabolism, such as buds and peel, are more sensitive to CPTA induction.(2) Effects of CPTA treatment on carotenoid metabolism of ’Valencia’ orange.5% CPTA was applied to fruits of ’Valencia’ orange on the tree to investigate the carotenogenesis. HPLC results showed that quick and ever-increasing accumulation of lycopene together with the red color was the most apparent changes in the flavedo. Lycopene does not usually accumulate in the flavedo, while about 1.57μg/g FW (fresh weight) of lycopene was detected right at 2 d after CPTA treatment and then went up to 23.58μg/g FW at 30 d. Accumulation of carotenoids in both downstream branches were also affected, actually, as a whole, were suppressed to a similar degree. Real-time PCR analysis revealed that neither of the lycopene cyclase genes (LCYe and LCYb) was inhibited as once believed, while expression levels of those upstream genes, PSY, phytoene desaturase (PDS) andζ-carotene desaturase (ZDS), were all up-regulated during the first 8 days after CPTA treatment, which were significantly and positively correlated with the rapid accumulation of lycopene.(3) Effects of CPTA treatment on the flavedo proteome of ’Valencia’ orange.Totally 49 protein spots were considered to be differentially expressed between CPTA-treated and control samples via 2-DE gel analysis and 33 spots were further identified via MALDI-TOF/TOF MS analysis combined with MASCOT search, thereinto, 76% of the proteins matched belongs to citrus. However, neither of the lycopene cyclases was included. We thereby speculated that the targets of CPTA treatment might be the enzymatic activities of lycopene cyclases, instead of their gene expression transcriptionally or translationally. According to their biological functions, these identified proteins were categorized into eight groups, in which 76% of the proteins involved in carbohydrate/energy and nucleotide metabolisms. However, only carotenoid isomerase (CRTISO) was identified to be relevant to carotenoid biosynthesis, in spite of most carotenogenic genes were up- or down-regulated by CPTA treatment at the transcriptional levels.(4) Expression and identification of recombinant lycopene cyclases.Gene cloning and sequencing results showed that the open reading frame (ORF) of LCYe and LCYb was 1590 bp and 1515 bp, which encoding 529 and 504 amino acids, respectively. Subsequently, corresponding secretory recombinant vectors were constructed and transformed into pichia pastoris X-33 strains. After induced by methanol, expressed proteins were purified with His-tags and then identified by SDS-PAGE and Western blot analysis, none of the desired recombinant proteins was detected, which was probably due to their excessively low expression levels.