Analysis of free amino acids accumulation during fruit development in three different types of watermelons

Abstract: 【Objective】Watermelon belongs to the genus Watermelon in the Cucurbitaceae family. It has 7 species, and there are many varieties within the genus. Among them, egusi watermelon (Citrullus mucosospermus), seed edible watermelon (C. lanatus var. megalospermus) and common watermelon (C. lanatus var. vulgaris) have been cultivated at commercial scales. Common watermelon is a sweetflesh watermelon for daily consumption. Egusi watermelon is an important economic crop in West African countries, and the seed edible watermelon is the main economic crop in the arid area of northwest China. Free amino acids are not only important nutrients in watermelon fruit, but also an important nitrogen source. They participate in fruit development and perform their respective functions, which are connected to each other. Studies have shown that there are significant differences in the accumulation and content of some free amino acids such as citrulline in different varieties of watermelons. The differences in metabolites can be used for distinguishing varieties. This study determined amino acids in fruits at four development stages (10, 18, 26, 34 d) in three watermelon varieties, PI532726 (egusi watermelon), Ningxia Red Seed (seed edible watermelon) and Bing Tang Cui (common watermelon) withHPLC. The accumulation and metabolism of 24 kinds of free amino acids was analyzed.【Methods】According to the development characteristics of different varieties, four watermelon fruit with the same growth and good development status were sampled for each variety and quickly frozen with liquid nitrogen. The watermelon pulp was completely freeze-dried in a freeze dryer and then ground into fine powder. A sample of 1.00 g of freeze- dried pulp was taken and suspended in 90 mL boiling water, and bathed in 95 ℃ water for 10 minutes after full mixed. The suspension was forced through a 0.22 μm water-phase filter, and the filtrate was cooled to room temperature and set to a volume of 125 mL. 10 μL of the filtrate was pipetted into a glass-lined tube, added with 20 μL of derivatization reagent and 70 μL of derivatization buffer reagent, vortexed for 10 s, oven heated at 55 ℃ for 10 min, and used for HPLC analysis, which was repeated for three times for each measurement. The standard curves of 24 free amino acids were constructed with four concentration gradients for HPLC determination, so as to quantify amino acids in the watermelon sample filtrate.【Results】It was found that the contents of citrulline, glycine and glutamine were among the highest in the three watermelon varieties. For example, citrulline was 0.11 g·kg-1 in PI532726, 0.12 g·kg^-1 in Ningxia Red Seed, and 0.39 g·kg-1 in Bing Tang Cui. The content of arginine (0.1-0.2 g·kg^-1 ) in Bing Tang Cui (0.12 g·kg^-1 ) was higher than in PI532726 and Ningxia Red Seed (0.018 g·kg-1 and 0.068 g·kg^-1 , respectively). The contents of γ-aminobutyric acid, glutamic acid, valine, isoleucine, asparagine and phenylalanine in the three watermelons ranged from 0.01 to 0.06 g · kg^{- 1} . The contents of theanine, hydroxyproline, tyrosine, sarcosine, alanine, leucine, ornithine and cysteine were all lower than 0.01 g·kg^-1 . The contents of the other free amino acids differed significantly in the three watermelon varieties. For example, the contents of proline and methionine in Bing Tang Cui were 0.028 g · kg^{- 1} and 0.013 g · kg^{- 1} , respectively, while those in PI532726 and Ningxia Red Seed were all lower than 0.01 g·kg^-1 . The content of serine in Ningxia Red Seed and Bing Tang Cui was 0.011 g·kg^-1 and 0.015 g·kg^-1 , respectively, while that in PI532726 was lower than 0.01 g·kg^-1 . Principal component analysis was carried out on the 24 amino acids in four developmental stages of the three watermelon varieties after standardization, and two principal components with eigenvalue＞1 were extracted. The eigenvalues of the two principal components for PI532726 were 14.503 and 5.511; those for Ningxia Red Seed were 19.006 and 3.368; and for Bing Tang Cui were 10.73 and 9.79. The cumulative variance contribution rates were all greater than 83%, showing strong information representability. PCA loading plot can be used to find the differential variables. The greater the distance from the origin, the greater the contribution rate to the samples. From the loading plot, it could be seen that the free amino acids that contributed more to PI532726 were glycine and glutamine. The free amino acids that contributed more to Ningxia Red Seed were glycine, citrulline and glutamine, and those to Ningxia Red Seed were glycine, citrulline, arginine and glutamine. Clustering heat map analysis showed significant differences in the amounts and patterns of glycine, citrulline, arginine and glutamine among the three watermelon varieties, and the free amino acid evolutionary tree also showed differences among the three varieties. The metabolic characteristics of free amino acids in PI532726 and Ningxia Red Seed were more similar. In conclusion, there were significant difference in contents of free amino acids in the three watermelon varieties, which may be used as the signature differential metabolites to distinguish watermelon varieties. It was found that glycine (26%-34%), glutamine (12%-24%), citrulline (8%-23%) and γ- aminobutyric acid (4%- 9% ) were dominant in the fruit of PI532726. In Ningxia Red Seed, glycine (20%-35%), glutamine (12%-28%), citrulline (9%-15%), glutamic acid (4%-12%) and arginine (4%- 9%) were the major amino acids. Citrulline (23%-45%), glutamine (21%-35%), glycine (4%-19%) and arginine (5%-11%) were dominant in Bing Tang Cui. In addition, there were significant differences inthe proportion of various free amino acids among varieties and developmental stages. The results showed that there were always a large proportion of free amino acids in fruits of the three varieties, and there were significant differences. The presence of these free amino acids in fruit development may be related to genotype of watermelon. The changes in the total 24 detected free amino acids in PI532726 were in a parabolic (up-down) pattern. In Ningxia Red seed, the change pattern was an ascending type, while that in Bing Tang Cui watermelon showed an“N”pattern (up-down-up). The total free amino acid content in Bing Tang Cui was higher than that in PI532726 and Ningxia Red Seed at the four developmental stages. The overlap of total free amino acid accumulation curves of PI532726 and Ningxia Red Seed was high. The change patterns of each free amino acid were analyzed respectively. It was found that citrulline, γ- aminobutyric acid, aspartic acid and ornithine in PI532726 were ascending; cysteine was in a descending pattern, and the change patterns of glutamine, glycine, arginine, glutamic acid, phenylalanine, aspartic acid and isoleucine were parabolic. The contents of alanine and cysteine in Ningxia Red Seed descended constantly; glutamic acid showed a“V”pattern (down-up); glycine, leucine, serine and tyrosine showed a parabolic pattern; citrulline, glutamine, arginine, phenylalanine, γ-aminobutyric acid, aspartic acid, asparagine, lysine, valine, methionine, ornithine and theanine showed ascended constantly. In Bing Tang Cui, glycine and alanine were constantly decreasing; citrulline and ornithine were increasing; aspartic acid and γ-aminobutyric acid were parabolic; and glutamine, arginine, phenylalanine, glutamic acid, leucine, isoleucine, lysine, proline, serine, valine and methionine changed in an “N”pattern (up-down-up). Threonine displayed an inverted“N”pattern (down-up-down). The change curves in PI532726 and Ningxia Red seed were similar in various free amino acids, such as glycine, leucine, asparagine, threonine, isoleucine, serine, γ- aminobutyric acid and tyrosine, while the change curves of free amino acids in PI532726 and Bing Tang Cui had high similarities. The coincidence degree of cysteine was high only in Ningxia Red Seed and Bing Tang Cui. PCA analysis of free amino acid data of all samples showed that principal component 1 clustered PI532726 and Ningxia Red Seed together, and Bing Tang Cui was grouped into a single class. The results indicate that the free amino acid metabolic network and metabolic pattern in the fruits of PI532726 and Ningxia Red Seed may be more similar.【Conclusion】The contents of citrulline, glycine and glutamine were high in fruits of the three watermelon varieties, and the difference was significant. The content of arginine was high only in Bing Tang Cui. The contents and change patterns of free amino acids in PI532726 and Ningxia Red Seed were similar, and significantly different from Bing Tang Cui. The combination analysis of the contents and change patterns of various free amino acids may be used in the classification of watermelon germplasm.