Carbohydrates And Organic Acids Composition Of Different Apple Genotypes & The Role Of Malate Transporter

Apple(Malus domestica Boukh.)is one of the most popular fruits in the world. It is of great value to understand the developmental changes of carbohydrates and organic acids and malate metabolism profile in different genotypes of apple fruit for evaluation of apple germplasm and apple breeding. Here we determined carbohydrates and organic acids in different genotypes of apple during fruit development by GC-MS and investigated the relative expression levels of genes involved in malate metabolism with real time quantitative PCR. We cloned the genes of apple tonoplast malate transporter (MdtDT1) and apple malate channel 1/2 (MdMC1/2) then transformed MdtDT1 into arabidopsis AttDT mutant for complement experiment. The main results were as follows:1. Developmental changes of carbohydrates and organic compounds are similar in GoldRush,Red Delicious,Dabinett,Chisel Jersey,McIntosh which have different acidity levels.Fructose and sorbitol are the primary sugars in apple fruit during early development, followed by glucose, sucrose and galactose. As fruit developing, the percent of sucrose increased all the way to fruit harvest; the percent of fructose increased between 20 and 110 days after bloom (DAB) then decreased to fruit harvest; the percent of glucose peaked at 44 DAB then decreased to fruit harvest; the percent of sobitol decreased to fruit harvest; the percent of galactose remained almost unchanged before 110 days then decreased to fruit harvest. Fructose, sucrose and glucose are the main soluble sugars in mature apple fruit.On a fresh weight (FW) basis, the concentration of sucrose increased to fruit harvest and the rate of increase was higher during mature period. The concentrations of fructose and glucose increased with fluctuations. The concentration of sobitol decreased, and the concentration of galactose fluctuated during fruit development.Quinic acid and malic acid are the primary organic acids in apple fruit during early development. Along with fruit development, the percent of quinic acid decreased exponentially; the percent of malic acid increased all the way to fruit harvest; the percent of ascorbic acid and citric acid showed fluctuating increases; maleic acid increased between 20 and 110 DAB then decreased rapidly to fruit harvest. Malic acid was the main organic acid in mature apple fruit.On a fresh weight (FW) basis, the concentration of malic acid peaked at 44 DAB then decreased to fruit harvest, the concentration of citric acid and quinic acid decreased, whereas the concentration of ascorbic acid fluctuated during fruit development.On a fresh weight (FW) basis, the total sugar concentration increased before 71 DAB then decreased between 71 and 110 DAB and increased rapidly to fruit harvest while the total organic acid concentration decreased during fruit development. Along with fruit development, the ratio of total sugars to organic acid increased to fruit harvest, and the ratio of other varieties increased slower than that of Dabinett and Chisel, both of which are low acidity varieties.2. In mature apple fruit of GoldRush, Red Delicious, Dabinett, Chisel Jersey, McIntosh, Honeycrisp, NY 752, Line1, apple 214, apple 118, apple 109, apple 056, apple 017, Crabapple 1, Crabapple 2 and Crabapple 3, malic acid accounted for 88% ~ 94% of the organic acid and fructose, sucrose and glucose together accounted for more than 90% of the total soluble sugars, except for crabapple 1,crabapple 2 and crabapple3 in which sorbitol was another major sugar (alcohol).The ratio of total sugars to organic acids of different apple fruit at harvest ranged from 2.7 to 56.2, which corresponded roughly with that of genotypes from high acidity to low acidity. The ratio of total sugars to organic acids was an important determinant of fruit acidity level and the best predictor of acid taste.3. The tonoplast malate transporter (MdtDT1,GenBank Accession No. HM641021,HM641022) and malate channel (MdMC1/2,GenBank Accession No. HM641021/HM641022) gene we cloned from apple, which had high similarity with genes involved in malate/ dicaboxylate/ citrate metabolism in other species.The short and slow-growing Arabidopsis AttDT mutant was restored to regular growth after we transformed MdtDT1 into it. The concentrations of malic acid and citric acid in leaves were increased from 37% and 22% to 134% and 201% respectively compared with wild type. Other acods in the tricarboxylic acid cycle were also increased. The complemented lines also accumulated maleic acid and trehalose.4. Along with apple fruit development, mRNA expression level of vacuolar H+-ATPase (MdVHA), vacuolar H+-pyrophosphatase (MdVPP), NAD-dependent malate dehydrogenase (MdMDT) and malate channel 2 (MdMC2) increased; mRNA expression level of phosphoenolpyruvate carboxylase (MdPEPC), NADP-dependent malic enzyme (MdME) and malate channel 1 (MdMC1) decreased then increased; mRNA expression level of tonoplast malate transporter (MdtDT1) increased exponentially in GoldRush and Chisel Jersey fruit whereas fluctuated in Mclntosh, Dabinett and Red Delicious fruit; mRNA expression level of malate channel 1 (MdMC1) was much higher than malate channel 1 (MdMC2) during fruit development.Our data showed a positive correlation between mRNA expression level of MdtDT1 /MdMC1 and the concentration of malic acid in different genotypes of apple fruit at harvest, The mRNA expression level of MdMC1 was noticeably higher in the fruit with more malic acid. MdMC1 and MdtDT1 play a significant role in malate accumulation and their expression levels can be taken as one important indicator for measuring the ability of apple fruit to accumulate malic acid and citric acid.