Analysis of changes and correlations between calciums and organic acids in fruits of Cerasus humilis during different development stages

Abstract:【Objective】Calcium is an important nutritional component in fruits, however, the regulatory mechanisms of calcium in fruits is still limited. This study investigated the changes of organic acids, related metabolic enzymes, and different forms of calcium during the development of the fruits of Cerasus humilis, and analyzed the correlations between the calcium and organic acids. The purpose is to reveal the relationship between calcium accumulation and organic acid metabolism, and provide a theoretical basis for investigating the regulation of calcium in fruits.【Methods】The high-calcium and low-calcium C. humilis fruits from Inner Mongolia were used as research materials. The samples were collected at different stages of fruit development, including the young fruit stage (S1), hard kernel stage (S2), coloring and enlargement stage (S3), hardening stage (S4), and fully ripe stage (S5). Each fruit sample was washed with distilled water and then rapidly frozen in liquid nitrogen, and stored at -80 ℃. The progressive extraction method was used to extract water-soluble calcium, pectin calcium, calcium phos-phate, oxalate calcium, and residual calcium in the fruits. The content of these forms of calcium was determined using flame atomic absorption spectrophotometry. The content of malic acid and citric acid in the C. humilis fruits were measured using high- performance liquid chromatography. Additionally, the activities of malic enzyme (NADP-ME) and malate dehydrogenase (NAD-MDH) were determined. The correlation between the organic acid metabolism and calcium accumulation was analyzed. The tests were repeated three times, with three biological repeats each time.【Results】(1) During the development and maturation of C. humilis fruits, the water-soluble calcium content in high-calcium and low-calcium C. humilis fruits showed an initial decrease followed by an increase, indicating an overall upward trend. In contrast, the total calcium, pectin calcium, calcium phosphate, and oxalate calcium contents exhibited an initial increase followed by a decrease, indicating an overall downward trend. The residual calcium content showed a continuous decrease throughout the process. At the fully ripe stage of the fruits, the low-calcium fruits exhibited higher levels of water-soluble calcium. However, the other calcium components and total calcium content were higher in the high-calcium fruits. Among these components, the proportion of active calcium was the highest, accounting for 70.29% in the high- calcium fruits and 68.30% in the low-calcium fruits. (2) During the development and maturation of C. humilis fruits, the patterns of NAD-MDH activity, malic acid content, and total organic acid content were consistent in high-calcium and low-calcium C. humilis fruits. They showed an initial increase followed by a decrease, reaching their peak values at the hard ripening stage, indicating a significant overall upward trend. Notably, the activity or content of these three factors was higher in the low-calcium fruits. The NADP-ME activity exhibited an overall decreasing trend. The citric acid content showed an initial increase followed by a decrease, reaching its highest value at the hard kernel stage, indicating an overall downward trend. The high-calcium fruits had higher content of citric acid. The activity of NAD-MDH was significantly and positively correlated with the content of malic acid, and was significantly and negatively correlated with the content of citric acid. The activity of the NADP-ME was significantly and negatively correlated with the content of malic acid and was significantly and positively correlated with the content of citric acid. There was a highly significantly negative correlation between the activities of the NADP-ME and NAD-MDH. (3) The correlation analysis revealed significantly positive correlations between the activity of the NAD-MDH, malic acid content, content of total organic acid and content of water- soluble calcium in high-calcium and low-calcium C. humilis fruits. They exhibited varying degrees of negative correlations with other forms of calcium and total calcium content. The activity of NADP-ME and the content of citric acid showed a highly significantly negative correlation with watersoluble calcium content. In addition, they exhibited significant or highly significantly positive correlations with other forms of calcium and total calcium content.【Conclusion】The accumulation of calciums in C. humilis fruits during the development and maturation was closely related to the organic acid metabolism. The malic acid is the predominant organic acid in C. humilis fruits. As the activity of NADMDH increases and the activity of NADP-ME decreases, the synthesis of malic acid would be enhanced while its degradation would be reduced, leading to an increase in malic acid accumulation. This increase in the malic acid contributed to the increase in the water- soluble calcium, which would inhibit the synthesis of the other calcium components. The increase of the citric acid content tended to promote the accumulation of the non-water-soluble calcium components such as pectin calcium, calcium phosphate, and oxalate calcium.