Functional Analysis Of Phytoene Synthase 1 Modulate Tomato Fruit Quality

Tomato(Solanum lycopersicum L.)is one of the most widely consumed vegetables worldwide.Thousands of structurally distinct metabolites have been characterized in tomato fruits,many of which make a substantial contribution to the human diet.Over the decade years,we have made great progress in tomato breeding,and tomato output has been enough for the consumer market.Howere,breeders and producers paid more attention to the tomato yield storage and transportation tolerance and resistance,ignoring fruit quality,the flavor and nutrition of tomato have decreased significantly.Therefore,improving the fruit quality of tomato is an important goal of tomato breeding.Tomato quality mainly includes flavor,nutritional and commodity,which are complex comprehensive character with multiple factors.The excavation and utilization of key gene sites have always been difficult points in tomato breeding.According to 33 flavor chemicals identified and 49 key sites that regulated the accumulation of flavor substances through tasting experiments and multi-omics analysis,our study further analyzed the key genes of MHO3.1(6-methyl-5-hepten-2-one,MHO)by a recombinant population and fine mapped it to 60.21 kb interval.After that,through CRISPR/Cas9 transgene verification,it was confirmed that Phytoene Synthases 1(PSY1)played a key role in regulating the accumulation of MHO in tomato fruits.Furthermore,the psy1-cr mutant and wild type tomato fruit were systematically analyzed by the metabolomes and transcriptome.with developed non-targeted metabolites detecting methods to analysis the metabolites changing in psy1-cr mutant.The results indicated that PSY1 regulated tomato quality including flavor,nutrition and puncture.The main results are as follows:(1)Through a map-based cloning approach and transgenic verification,we revealed that mho3.1 locus was fine mapped into 60.21 kb interval with recombination population which drived from TS-106 and TS-629,and PSY1 was responsible for the accumulation of an important flavor chemical MHO.(2)In order to further study the regulation effect of PSY1 gene on tomato quality,this study established non-targeted GC-MS detection methods.The detection results of non-targeted GC-MS show that 236 of the 1045 identified volatile substances are significantly different between wild type and psy1-cr mutant.It is found that these substances have different degrees of changes,among which 7 volatiles have significant changes,by analyzing the content of 27 volatiles that affect tomato flavor.This result shows that the change of these substances gives tomato new flavor.(3)We developed the tomato fruit carotenoid metabolome determined 89 kinds of carotenoids,among which 40 kinds of carotenoids decreased significantly in the mutant and 7kinds of carotenoids accumulated significantly.Among them,Prephytoene diphosphate(PPPP)significantly accumulated in psy1-cr mutant,while the content of various downstream carotenoids decreased significantly.This result indicates that PSY1 is a rate-limiting enzyme that catalyzes the conversion of PPPP to 15-cis-phytoene.(4)We performed a transcription analysis showed that genes synthesized by cutin and wax were enriched in psy1-cr mutant.Furthermorem,the cutin composition analysis on tomato peel showed that the content of 7 cutins in the mutant increased significantly,and paraffin sectioning and transmission electron microscope observed that the cuticle of pericarp thickened.The psy1-cr mutant showed higher puncture and long shelf life.These results indicated that PSY1 gene might regulate the puncture and shelf life of tomato fruit by affecting the synthesis of cutin wax.(5)1361 secondary metabolites in tomato fruits were detected,of which 217 were significantly different in MM and mutant by a Q Exactive Orbitrap/mass spectromerter coupled with U3000 UHPLC.Based on KEGG enrichment analysis of these differential substances and transcription group differential expression genes,it is found that the substances and key synthetic genes in flavonoid synthesis pathway are significantly enriched and improved to varying degrees,this indicates that the mutation of PSY1 gene leads to the truncation of the carotenoid pathway and promotes the synthesis of the flavonoid pathway.In order to further study the relationship between carotenoid pathway and flavonoid pathway,this study develops a Virus-induced gene silencing(Virus-induced gene silencing,VIGS)the key synthetic gene CHS1 in flavonoid pathway was silenced in tomato fruits.It was found that the accumulation of flavonoids decreased,while the content of carotenoids increased significantly.It indicates that the accumulation of flavonoids inhibits the synthesis of carotenoid,and there is direct metabolic regulation between the carotenoid pathway and the flavonoid pathway.In summary,by integrating a metabolite-based genome-wide association study with linkage mapping and transgenic verification,we revealed that Phytoene Synthase 1 contributes to the accumulation of MHO.Through generating and analyzing a dataset encompassing transcriptomes and metabolomes,we demonstrate a redirection of metabolic flux between the carotenoid and flavonoid pathways.Our data indicate that Sl PSY1 can modulate tomato flavor chemistry,color and firmness and shelf life through redirecting the metabolic flux between carotenoid and flavonoid pathways.