Aromatic Metabolite Profiling And Functional Analysis For Monosaccharide Transporters In Fruit

Sugar and aroma compounds in fruit can directly affect its flavor quality and marketing traits. Fruit aroma characteristics rely on many factors, such as genotype,stresses, signal transduction. Fruit sugar transporters are important for sugar partitioning and metabolism, consequently crucial for plant growth and development. Aroma profiling and major sugar(fructose) transportation mechanism analysis can lead to a better understanding of the mechanisms underlying fruit flavor formation, which will be surely helpful for fruit flavor enhancement. Thus this study has great scientific and practical value.Volatile profiles of two local cultivars, ‘Xiang-Mian Li’(XML) and ‘Mu-Tou Su’(MTS), with substantial aroma grown in Anhui, China were compared with those of control cultivars ‘Dang-Shan Suli’(DSS)(Pyrus bretschneideri) and ‘Nan-Guo Li’(NGL)(P. ussuriensis). Headspace solid-phase microextraction(HS-SPME) and static headspace gas chromatography-mass spectrometry(GC-MS) were employed for volatiles detection and quantification. Odor active value(OAV) was employed to verify the contribution to the sensory traits of certain compound. In addition, orthogonal partial least-squares discriminant analysis(OPLS-DA) was done to uncover the contribution of the volatile compounds to cultivar clustering. The possible biosynthetic pathways were also discussed.Volatiles detected from the intact fruit of XML, MTS, NGL and DSS were 11, 14, 7and 25, respectively. These compounds were roughly divided into 4 groups: esters,terpenes, alcohols and hydrocarbons. The order of the total OAV(>1) was NGL(275.15) >XML(118.67) > MTS(98.91) >DSS(49.83). Esters, derived from lipoxygenase(LOX)and shikimate pathway, were the most abundant group and followed by terpenes produced from the precursors of MEP and MVA pathways. Characterization of the sensory traits revealed that the fruit of XML and MTS processed aroma notes like apple peel and grass,respectively. Since the total OAV of the two cultivars were mainly contributed by esters,they were defined as ‘Ester-Aroma Type’.The volatiles detected in pulp, peel and core of XML were 8, 9 and 7, while 10, 4 and8 in MTS. These volatiles were mainly alcohols, esters, hydrocarbons and others. The order of the total amount of the volatiles in pulp was XML(2240.74 μg kg-1 FW) > NGL(1882.39 μg kg-1 FW) > MTS(1307.76 μg kg-1 FW) > DSS(487.64 μg kg-1 FW), while in peer was NGL(2515.68 μg kg-1 FW) > XML(1583.68 μg kg-1 FW) > DSS(718.46μg kg-1FW) > MTS(225.48 μg kg-1 FW), and in core was MTS(1227.97 μg kg-1 FW) > NGL(1060.4 μg kg-1 FW) > XML(741.86 μg kg-1 FW) > DSS(45.63 μg kg-1 FW). The major volatiles in pulp, peel and core of XML and MTS were esters resulted from the ADH pathway.OPLS-DA analysis showed good reproducibility and separability. The volatile varieties and concentrations and OAV(>1) basis OPLS-DA clustering results showed that volatile varieties and concentrations were similar in XML, MTS and DSS. Moreover,VIP(variables of importance) analysis revealed that ethyl hexanoate was the most important contributor to the aroma variance in intact fruit and peel of the four pear cultivars. Our data also showed that XML, MTS and DSS were closely related but distant to NGL.Based on homology search in strawberry genome(http://www.strawberrygenome.org/), 13 possible fructose transporter protein genes homologous to Arabidopsis mono-carbohydrate transporter were found. q RT-PCR analysis of different stages of strawberry leaves, flowers and fruits revealed that g-31477 was positively correlated with fructose concentrations in the fruits, while g-20355 was highly expressed in all three organs. Thus, g-31477 and g-20355 were chosen as candidate genes.Full length of g-31477(CDS length was 2562 bp and encoded 853 amino acid residues) was obtained and subsequently used to make the plant expression construct35S::g-31477 in p CAMBIA2300 for plant transformation. Further characterization of transgenic plants are under way.Transient expression, yeast expression and genetic transformation of the way to verify the gene function, respectively. The results show that transient infection way improved 3.82% of fructose, increase 3.07% of xylose, improved 1.22% of glucose,increase 2.55% of galactose, improved 2.07% of mannose. By way of a yeast expression containing sugar plasmid transporter gene may be the medium of monosaccharides(fructose, glucose and mannose, galactose) is transported to a yeast to survive. These results fully demonstrated that gene g-31477 has a monosaccharide transporter features.