Study On The Metabolism Of Carbonhydrates And Phenolics In Apple Fruit&the Effects Of MdSPS Interference On Carbon Partitioning In Leaf

Apple is a temperate-zone fruit, but consumed worldwide for its unique characteristics,such as color, flavor and aroma, freshness, crispness, especially its nutritional value. Itsprimary and secondary metabolites such as carbohydrates, organic acids, amino acids andphenolic compounds are responsible for its high acceptance and reputation. The levels ofthese metabolites vary according to developmental stage, genotype and tissue type, as well asenvironmental conditions and human involvement. Here, we identified members of thevarious gene families that encode key enzymes or transporters involved in sugar metabolismand accumulation using homology analysis based on Malus genome and EST sequences andcomparison of expression patterns in different tissues, and analyzed the relationship of theirrelative transcript abundance and activities of enzymes with sugar accumulation during applefruit development; In addition, the expression of SPSC was decreased via RNA interference todetermine its role in sucrose synthesis and plant growth in apple plants. Furthermore, weconducted a systematic examination of the effects of canopy position, fruit bagging andturning on the concentrations of the main primary and secondary metabolites in apple peeland flesh of different apple cultivars.The main results are as followes:1. At the early stage of fruit development, the transcript levels of sorbitol dehydrogenase,cell wall invertase, neutral invertase, sucrose synthase, fructokinase and hexokinase arerelatively high, and the resulting high enzyme activities are responsible for the rapidutilization of the imported sorbitol and sucrose for fruit growth, with low levels of sugaraccumulation. As the fruit continues to grow due to cell expansion, the transcript levels andactivities of these enzymes are down-regulated, with concomitant accumulation of fructoseand elevated transcript levels of tonoplast monosaccharide transporters (TMTs), MdTMT1andMdTMT2; the excess carbon is converted into starch. At the late stage of fruit development,sucrose accumulation is enhanced, consistent with the elevated expression ofsucrose-phosphate synthase (SPS), MdSPS5and MdSPS6, and an increase in its total activity. Our data indicate that sugar metabolism and accumulation in apple fruit is developmentallyregulated. The expression charactors of MdSDHs、MdCWINVs、MdNINVs、MdvAINVs、MdSUSYs、MdFKs、MdHKs、MdSPSs、MdSOTs、MdSUTs、MdTMTs�'�MdvGTs varied indifferent tissues indicated that they play different roles in sugar metabolism and accumulation.2. Six isoforms of SPS genes, MdSPS1/2/3, MdSPS4and MdSPS5/6, were expressed inapple leaves, which were grouped into three families, MdSPSA, MdSPSB and MdSPSC,respectively. MdSPSC accounted for approximately97%of all the SPS transcripts in appleleave. To determine the role of MdSPSC in sucrose synthesis in apple leaves, RNAinterference targeting MdSPS5/6was used to suppress the expression of MdSPS5/6. Relativeto the untransformed control, MdSPSC expression level in the transgenic lines decreased toaround42~61%, but total SPS activity was decreased slightly. Leaf sucrose, glucose andfructose concentrations at midday were decreased to approximately26~33%,51%~62%and40%of the untransformed control, respectively, whereas starch content almost doubled in thetransgenic lines, with sorbitol concentration unchanged. Plant vegetative growth was notsignificantly affected. These data indicate that type C SPS genes play a key role in sucrosesynthesis in mature apple leaves. The unaltered vegetative growth of the transgenic plants ismost likely related to the presence of compensatory mechanisms in end-product synthesis andexport in apple leaves.3. For ‘McIntosh’,‘Gala’ and ‘Mutsu’, peel of outer-canopy had higher concentration offructose than that of inner-canopy fruit. Both flesh and peel of outer-canopy fruit had higherconcentrations of glucose, sucrose, galactose, xylose, ribose, raffinose, rhamnose, sorbitol,maltitol and xylitol. Outer-canopy fruit had less starch than inner-canopy fruit. Canopyposition did not significantly affect malic acid concentrations except in the peel of ‘McIntosh’and the flesh of ‘Mutsu’. Although levels of ascorbic and succinic acids were higher in thepeels collected from the outer canopy, the pattern of distribution for other organic acids withinthe canopy depended upon tissue type and cultivar. Compared with the inner-canopy fruit,outer-canopy fruits had lower levels of amino acids, but higher concentrations of phenoliccompounds.4. Compared with the control, bagged ‘Jonagold’ fruit were yellowish, and had higherscores for lightness and hue angle, lower values for chroma, and the lowest concentration ofanthocyanins. Also, bagged fruit had more starch, glucose, and galactose, but less of the othersugars and sugar alcohols. The concentrations of fructose and total soluble carbohydrateswere not obviously affected by bagging. Although the malic acid concentration was notinfluenced by bagging, other acids were either increased (quinic, shikimic, and citric) ordecreased (ascorbic). The concentration of total organic acids did not show large difference among three treatments except that it was the lowest in the bagged fruit at14d after bagremoval. Phenolic compounds, especially flavonols in the peel, were severely inhibited, buttheir concentrations were enhanced by re-exposure to sunlight. The de-bagging treatment ledto up-regulation of the expression of MYB10and seven structural genes (MdPAL, MdCHS,MdCHI, MdF3H, MdDFR1, MdLDOX and MdUFGT) in anthocyanin biosynthesis. Alltranscripts were induced rapidly at6h after bag removal, peaking at Hour30before declining,with concomitant accumulation of anthocysnins.5. For the unturned (control) fruit, the sun-exposed peel had higher expression levels ofMdMYB10and seven structural genes in anthocyanin synthesis (MdPAL, MdCHS, MdCHI,MdF3H, MdDFR1, MdLDOX and MdUFGT), and higher levels of anthocyanins and favonolsthan in the shaded peel for both ‘Fortune’ and ‘Mutsu’ varieties. Exposure of the shaded peelto full sun caused marked up-regulation of the expression of MdMYB10and all sevenstructural genes, which peaked between6h and30h after fruit turning, consequently leadingto higher levels of anthocysnins, flavonols and total phenolics than in the sun-exposed peel ofcontrol fruit at174h. Interestingly, the levels of flavonols were higher in the shaded peel ofturned fruit (original sunny peel) than in the sun-exposed peel of both control and turned fruitin both varieties.