A Prenyltransferase Gene Confirmed To Be A Carotenogenic GGDS Gene From Sweetpotato

Sweetpotato ranks at the fifth most important food crop on a fresh-weight basis in developing countries after rice, wheat, maize and cassava. Sweetpotato cultivars have white, yellow, purple or orange flesh, but only orange-fleshed sweetpotato cultivars are the rich source of β-carotene that is the precursor of vitamin A. In fact, orange-fleshed sweetpotato is the main source providing β-carotene for the people in underdeveloped countries in Africa and Southeast Asia. Unfortunately, the β-carotene content in most sweetpotato cultivars is rather lower than the normal demand of human physiology. Therefore, it is desirable to develop sweetpotato cultivars rich in β-carotene.Metabolic engineering is a promising approach to develop sweetpotato cultivars with very high level of P-carotene and this is based on understanding sweetpotato carotenogenesis at molecular levels. Carotenogenesis has been extensively studied in fruits and flowers, but little is known about it in underground root organs. The20-carbon geranylgeranyl diphosphate (GGDP) is the general precursor for carotenoids.The prenyltransferases produce acyclic precursors of isoprenoids including carotenoids. In this study we cloned a prenyltransferase gene confirmed to be a carotenogenic GGDS gene from sweetpotato (IbGGDS, GenBank number:KF991091) by functional complementation, analyzed its subcellular localization, and established tissue profiles of carotenogenic genes and β-carotene. The full-length1409bp cDNA of IbGGDS contained a1092-bp open reading frame encoding a363-amino-acid polypeptide. The amino acid sequence of IbGGDS were similar with the reported large subunit of geranyl diphosphate synthase (GPPS) and geranylgeranyl diphosphate synthase (GGDS). There are a predicted63-amino-acid plastidial transit peptide (TP) at the N-terminus of IbGGDS. The189-bp corresponding sequence of IbGGDS plastidial transit peptide was fused with GFP and the fusion gene driven by35S promoter was transient expressed in transgenic tobacco protoplasts. The confocal microscopy of the fusion protein showed that the putative63-amino-acid TP of IbGGDS did direct the GFP into plastids. The plastidial localization of IbGGDS was consistent with the fact that plastids were the organelles to produce carotenoids. The truncated IbGGDS without transit peptide was used to reconstructed carotenoid biosynthesis in E. coli. Engineered E. coli strain became yellow given by carotenoids, in which IbGGDS was harboured. This suggested that IbGGDS acted as the function to produce geranylgeranyl diphosphate.Finally, the tissue expression profiles of IbGGDS and all the five downstream carotenogenic genes including PSY, PDS, ZDS, crtlSO and LYCB were analyzed by qPCR. All the six genes had the remarkably highest expression levels in underground tuberous roots. For IbGGDS, the relative expression level in tuberous roots was respectively18,25,18,4,5and14folds compared with that in fibrous roots, mature stems, mature petioles, mature leaves, young leaves and stem tips. For carotenogenic genes, the relative expression levels in tuberous roots were much higher in tuberous roots than those in other organs. Phytoene synthase (PSY) is the first committed-stem enzyme in regulating carotenoid biosynthesis and lycopene P-cyclase (LYCB) is the last enzyme involved in β-carotene biosynthesis in plant. The relative expression level of PSY in tuberous roots was respectively618,898,93,88,22and648folds and the relative expression level of LYCB was respectively167,45,42,15,8and49folds, compared with that in fibrous roots, mature stems, mature petioles, mature leaves, young leaves and stem tips. The three other carotenogenic genes including PDS, ZDS and crtlSO had the highly similar expression pattern with that of GGDS, PSY and LYCB in sweetpotato. The results showed that underground tuberous roots of sweetpotato were the main organs that biosynthesized P-carotene because both IbGGDS and all the five carotenogenic genes had much higher expression levels in underground tuberous roots than those in other organs such as fibrous roots, mature stems, mature petioles, mature leaves, young leaves and stem tips.The content of β-carotene in mature leaves (1.72±0.059mg.g-1FW) and young leaves (1.13±0.034mg.g-1FW) was respectively9.11times and5.98times compared with that in orange-fleshed tuberous root (0.19±0.020mg.g-1FW). Interestingly, it was found that the highest content of β-carotene was not in tuberous roots but in mature leaves and young leaves. This result suggested that the β-carotene transportation from underground tuberous roots to airborne leaves for storage. In summary, molecular characterization of GGDS from sweetpotato is helpful to understand and engineering carotenoid biosynthesis in sweetpotato.