Biosynthesis,Regulation And Transportation Of Bitterness In Cucumber

Cucumber is one of the important vegetables in the world,which has a pivotal position in global vegetable supply.However,the fruits often result in bitterness due to genetic factor and unreasonable management which seriously affects economic benefits.Cucurbitacins are highly oxidized triterpenoids that confer a bitter taste in cucurbits such as cucumber,melon,watermelon,squash and pumpkin.These compounds discourage most pests on the plant and have also been shown to have antitumor properties.By applying the integrated strategies,including genome,variatome,transcriptome,metabolome,molecular biology and biochemistry,we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C,including one oxidosqualene cyclase(OSC)(Bi),seven cytochrome P450s and one acetyltransferase(ACT),five of which constitute a gene cluster on chromosome 6.These nine genes all co-expressed in leave or wild fruits of cucumber,which were consistent to the distribution of bitterness.Four catalytic steps were elucidated.Bi encodes a cucurbitadienol synthase that catalyzes the cyclization of 2,3-oxidosqualene into the tetracyclic cucurbitane skeleton,the first committed step of cucurbitacin C biosynthesis.The two P450 genes Csa3G903540 and Csa6G088160 catalyze the second step and third step biosynthesis of cucurbitacin C,respectively.The ACT gene Csa6G088700 is the enzyme involved in the final step in the cucurbitacin C biosynthetic pathway.Meanwhile,we discovered two basic helix-loop-helix(bHLH)transcription factors Bl(Bitter leaf)and Bt(Bitter fruit)that regulated this pathway in leaves and fruits,respectively.Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of non-bitter cucurbits from their bitter ancestors.Two mutations SV-2195 and SNP-1601 that localized in the promoter region of Bt are the key factors in the domestication of non-bitterness fruits.Furthermore,we present the discovery of a tonoplast localized ATP-binding cassette(ABC)transporter,CsABC1,that export cucurbitacin C from cytosol to the vacuole by H~+-antiport mechanism.The function of CsABC1 was identified by yeast microsome vesicle assay and Xenopus lavis oocytes assay.This is the first time to discover triterpenoid transporter in plant.These new discoveries highlights the role that not only guide breeding workers to perform molecular marker breeding but also open a door for biological manufacturing and engineering of these triterpenoids as antitumor drugs in a manner similar to the biosynthesis of artemisinic acid,the antimalarial drug precursor.