Investigation On Molecular Mechanisms Of CsPIF4 Mediated Red Light-induced Carotenoid Accumulation In Citrus Fruit

Carotenoids,a class of secondary metabolites widely distributed in nature,play an essential role in plant development and human nutrition.Citrus fruits are rich in carotenoids,making them an ideal material for investigating carotenoid metabolism.However,even though the carotenoid metabolic pathway has been established,the transcriptional regulation mechanism remains unclear.Phytochrome interacting factors(PIFs),which are b HLH family transcription factors that interact with phytochromes,combined with other factors,participate in plant growth and development,abiotic(light,temperature,and circadian rhythm)and biotic(defense response)responses,as well as other signaling pathways.Our previous studies have found that red light can significantly promote carotenoid accumulation in citrus fruits.Integrating transcriptome analysis,we speculate that CsPIF4 may mediate this process.In this study,based on bioinformatics analysis,gene expression analysis,and phenotypic correlation analysis,we bagged ’Molo’blood orange fruit(with silenced CsPIF4)and ’Micro-Tom’ tomato fruit(which overexpressed CsPIF4)with white and red light transmittance bagging.After that,compositions and contents of carotenoids were detected by LC-MS/MS to parse the function of CsPIF4 in carotenoid metabolism.Meanwhile,we used q RT-PCR to explore candidate genes involved in the CsPIF4-regulated carotenoids metabolism under red light.Based on the correlation analysis and Y1 H experiment,the mechanisms of CsPIF4 mediated red light-induced carotenoid accumulation in citrus fruit were carried out preliminarily,providing theoretical and practical values for the regulation of citrus fruit color and nutritional quality.Main results are as follows:1.CsPIF4 is distributed on chromosome 7 according to the sweet orange genome,which contains numerous light responsive elements in its promoter region.CsPIF4 and At PIF4(AT2G43010)were shown to be clustered together through phylogenetic analysis,implying that CsPIF4 may be took part in light-mediated biological processes.Based on temporal and spatial expression analysis in citrus,CsPIF4 expression was much higher in leaves than in stems,flowers,and fruits at different developmental stages.As fruit ripening,CsPIF4 decreased and showed a significant negative correlation with carotenoid accumulation.The transient overexpressed CsPIF4 citrus fruit were acquired by Agrobacterium-mediated transient transformation.Lower total carotenoids content was found in CsPIF4 overexpressed citrus fruits.Red light transmittance treatment significantly inhibited CsPIF4 expression,suggesting that CsPIF4 may be involved in the red light-regulated carotenoids metabolism in citrus fruits.2.Via Agrobacterium-mediated temporary transformation,bacterial solution containing the recombinant PHB-CsPIF4-GFP plasmid was injected into tobacco leaves.Observed by laser scanning confocal microscope,CsPIF4 is a nuclear-localized protein.Based on the yeast GAL4 two-hybrid system,the recombinant p GBKT7-CsPIF4 and p GADT7 were co-introduced into Y2 H Gold strain.After screening with defective medium,CsPIF4 was found to have transcriptional activating activity and its transcriptional activation domain was identified between 70 and 235 aa.3.The blood orange fruit with transient silenced CsPIF4 were obtained through virus-induced gene silencing(VIGS)technology and Agrobacterium-mediated transient gene expression system of citrus fruits.Red light transmittance treatment could enhance the accumulation of total carotenoids both in VIGS-CsPIF4 and VIGS-Control blood orange fruit,especially in VIGS-CsPIF4 fruit.Moreover,the contents of phytoene,β-cryptoxanthin,zeaxanthin,and β-citraurin in silenced CsPIF4 fruit was significantly higher than that in VIGS-Control(p < 0.05).Expect for zeaxanthin,the contents of the other three carotenoids in each treatment were consistent with the expression level of CsPIF4,indicating that silencing CsPIF4 may increase the accumulation of total carotenoids in fruits by promoting the biosynthesis of phytoene,β-cryptoxanthin,and β-citraurin.4.Tomato plants overexpressing CsPIF4 were obtained by Agrobacterium-mediated plant transformation system.As increasing of CsPIF4 expression in the transgenic lines,total carotenoids content of tomato fruits decreased significantly,which were significantly lower than that of the wild type(WT)(p < 0.05).Red light transmittance treatment could alleviate the inhibition of CsPIF4 on carotenoids accumulation,particularly in lycopene synthesis.Thus,it is demonstrated that CsPIF4 negatively regulate carotenoid accumulation in fruits.5.Further q RT-PCR analysis of transient transformed citrus fruit showed that silencing CsPIF4 greatly increased the expression levels of CsZ-ISO,CsCRTISO,CsLCYB,and CsNSY,while dramatically inhibiting the expression of CsLCYE,CsCHYB,CsNCED2,and CsNCED3.In CsPIF4-VIGS fruits,CsCCD4 b expression could be remarkably induced by red light transmittance treatment.In CsPIF4-overexpression tomato fruit,the expression levels of Sl PSY1,Sl CRTISO,and Sl CHYB1 were significantly down-regulated,on the contrary,Sl LCYE expression was notably up-regulated.Sl PSY1 and Sl CHYB1 expression achieved the same level as wild type after red light transmittance treatment.Apparently,red light negatively impacted the expression of Sl LCYE.Consequently,we deduced that CsPIF4 response to red light promoted carotenoid accumulation in citrus fruits by negatively regulating CsCRTISO,CsLCYB,and CsCCD4 b expression,positively regulating the expression of LCYE.6.According to the correlation analysis,the correlation between structural genes involved in carotenoids metabolism and CsPIF4 were different in citrus and tomato.However,the expression levels of LCYE presented a significant positive correlation with CsPIF4 in both species(p < 0.05).As a particular gene in citrus,CsCCD4 b,showed a strong negative correlation with CsPIF4(p < 0.05).Results from yeast one-hybrid(Y1H)and dual luciferase(Dual-LUC)assays indicated that CsPIF4 may directly bind to the promoters of CsLCYE and CsCCD4 b,by activating CsLCYE transcription to increase the synthesis of β-ε branch carotenoids and reduce the accumulation of β-citraurin by inhibiting CsCCD4 b expression.