Calcium is an essential macronutrient for plant growth and development. In the soil, Ca²⁺ is transported to the root surface primarily through mass flow, diffusion, and root interception, before being transferred into the root xylem. From there, it is transported to the aerial parts of the plant via the transpiration stream [29,30]. Fruits, as terminal organs with relatively weak transpiration and independent physiology, receive nutrients from the plant body through the fruit stalk. After calcium is absorbed by the roots, it is transported through the transpiration via the xylem or phloem stream to branches, young leaves, flowers, and fruits. It exists in various calcium forms within these tissues [31,32]. The fruit stalk, serving as the conduit between the tree and the fruit, is the sole pathway through which calcium from the plant body enters the fruit [33,34,35].

The present study found that the changes in the content of different calcium forms in the fruits and fruit stalks of different Cerasus humilis types and under 2,4-D treatment followed similar patterns. Both exhibited an overall increase in water-soluble calcium content, while total calcium, active calcium, calcium pectin, calcium phosphate, and calcium oxalate showed general declines, with the decrease being more pronounced in the fruit than in the fruit stalks. It is likely that as the fruit develops and matures, the rapid expansion of fruit cells occurs, with vacuole enlargement being the primary factor driving cell expansion. As an osmotic regulator, calcium accumulates in the vacuoles, primarily in the form of water-soluble calcium. In contrast, the amount of calcium pectin required for cell wall growth is limited. Therefore, during fruit maturation, the accumulation of water-soluble calcium is significantly higher than that of other calcium forms. Compared to distilled water spraying, 2,4-D treatment significantly increased the content of all calcium forms in the fruit, while enhancing the increase in water-soluble calcium content and reducing the decline of calcium pectin, calcium phosphate, calcium oxalate, and total calcium. This effect is likely due to the regulation of calcium absorption, transport, and accumulation by plant hormones. Studies have shown that auxins promote the translocation of calcium from the plant body to the fruit, with IAA and NAA primarily enhancing the symplastic pathway of calcium transport into young fruits [36]. As an auxin analog, 2,4-D not only promotes fruit enlargement and increased fruit weight but also facilitates calcium uptake and accumulation.

During the development and maturation of Cerasus humilis, the proportions of different calcium forms in the fruits and fruit stalks exhibited fluctuating changes, indicating that calcium forms can interconvert during fruit maturation. Previous studies on apples and kiwifruits have demonstrated that calcium can interconvert within the fruit, and the results of this study are consistent with those findings [37,38]. The proportions of the four main calcium components were similar in the fruit stalks, while in the fruits, there were significant differences, with water-soluble calcium and calcium pectin being the dominant forms. At the fully ripe stage, the sum of water-soluble calcium and calcium pectin in the fruits of both Cerasus humilis types approached 70%, making them the most prevalent calcium forms in the fruit. The proportion of calcium pectin exhibited clear differences between the two types in the fruits and fruit stalks. In the MY-2 fruits, the proportion of calcium pectin was significantly higher than in the fruit stalks, indicating that calcium pectin is more readily transported from the fruit stalk to the fruit. Conversely, in the MY-9 fruits, the proportion of calcium pectin was lower than in the fruit stalks, suggesting that calcium pectin tends to accumulate in the fruit stalks. This difference could be the primary factor contributing to the variation in calcium content between the two types. The application of 2,4-D altered the distribution of the four major calcium components in the fruit stalks, increasing the proportions of water-soluble calcium and calcium pectin while reducing the proportions of calcium phosphate and calcium oxalate. Furthermore, 2,4-D treatment increased the proportion of calcium phosphate and calcium oxalate in the fruit, suggesting that 2,4-D may promote the transport of these forms from the fruit stalks to the fruits. This effect may be due to hormone regulation of cell expansion, cell wall modification, xylem development, and phloem sucrose unloading. Calcium is known to participate in signaling pathways involving gibberellins, auxins, and abscisic acid, which regulate processes such as fruit set, ripening initiation, cell proliferation, and fruit softening. These physiological changes likely influence calcium distribution within the fruit [39]. The proportions of calcium forms in the fruit stalks and fruits showed clear differences between the young fruit stage and the fully ripe stage, with the most significant changes occurring between the hard–ripe stage and the fully ripe stage. These two stages are critical for calcium changes, transport, and accumulation.

The correlation analysis of calcium nutrition in the fruits and fruit stalks of Cerasus humilis reveals that the changes in the content of different calcium forms in the fruits are closely related to those in the fruit stalks. The fruit stalks, as the primary pathway for calcium transport to the fruit, play a crucial role in calcium transport. Studies by Huang et al. [40] found that the calcium concentration in the fruit stalks of lychee is significantly higher than in the fruit. Similarly, Song et al. [41] also found that the calcium content in lychee fruit is an order of magnitude lower than that in their fruit stalks, indicating a significant calcium concentration difference ratio between the fruit stalks and the fruits. Zhong Weiliang [42], using the ⁴⁵Ca isotope, injected calcium into lychee fruit stalks and found that less than 1% of the calcium was transported into the fruits. This study found that during the development and maturation of Cerasus humilis fruits, the calcium content in the fruit stalks was much higher than in the fruits, showing a clear concentration difference ratio, suggesting the existence of a ’bottleneck’ in calcium transport between the fruit stalks and the fruits. Calcium in the plant can be divided into two major categories: water-soluble calcium (free Ca²⁺) and non-water-soluble calcium, which includes calcium pectin, calcium phosphate, and calcium oxalate [43]. Calcium pectin, a calcium form associated with the cell wall, plays a crucial role in maintaining its integrity. During fruit growth and softening, calcium pectin undergoes enzymatic degradation, leading to the disintegration of pectin–calcium complexes. The calcium ions released from pectin may either remain as water-soluble calcium or combine with other components to form calcium oxalate and calcium phosphate [44]. Water-soluble calcium and calcium pectin are considered active forms of calcium. Due to the free mobility of water-soluble calcium and the ease with which calcium pectin can be converted, we hypothesize that active calcium (water-soluble calcium and calcium pectin) is more readily translocated and transported within the plant. In contrast, non-active calcium forms, such as calcium oxalate and calcium phosphate, once fixed, are less likely to be redistributed, making them more likely to be retained in the fruit stalks [43,45]. Studies have shown that most of the retained calcium in the fruit stalks is bound to the cell wall or precipitated as calcium oxalate. Electron probe microscopy has revealed large granular calcium oxalate crystals in the phloem of the fruit stalks [46]. Most studies focus on calcium oxalate as the primary cause of the calcium transport “bottleneck”. For instance, Yi Junwen [47] found that the calcium content in longan fruit stalks is higher than in the fruits, with large amounts of calcium oxalate crystals present in the fruit stalks. Zhang Xinsheng et al. [48] found that, in the early stages of apple development, the formation of calcium oxalate in the fruit stalks does not affect calcium influx. However, as the fruit develops, the accumulated calcium oxalate crystals gradually block vascular tissues, hindering later calcium influx. In contrast, Song Wenpei [17] suggested that, in citrus and other fruit trees, calcium retained in the fruit stalks is primarily in the form of calcium oxalate or structural calcium (calcium pectin and calcium phosphate), but the formation of calcium oxalate in the fruit stalks may not be the primary cause of the calcium transport ‘bottleneck’. In this study, different calcium forms exhibited varying characteristics in concentration differences and transport. The concentration difference of water-soluble calcium was the smallest and remained stable, with no significant fluctuations, allowing for easy transport from the fruit stalks to the fruits throughout development. The concentration differences in calcium oxalate and calcium phosphate were the largest, showing only a small difference in the early stages but a significantly larger difference in the later stages. These forms of calcium were more easily transported to the fruits in the early stages but became more likely to remain in the fruit stalks in the later stages, making their transport into the fruits more difficult. Throughout development, the MY-9 fruits exhibited a significantly higher concentration difference compared to MY-2, which may explain the differences in calcium content between the two types. The ’bottleneck’ effect in the MY-9 fruit stalks becomes more pronounced during the later stages of fruit development, with more calcium oxalate and calcium phosphate precipitating in the fruit stalks, leading to reduced calcium transport to the fruits. Further treatment with 2,4-D, an auxin analog, demonstrated that 2,4-D could significantly enhance the correlation between calcium phosphate, calcium oxalate, and the various calcium forms in the fruits. It also notably reduced the concentration differences in the calcium forms between the fruit stalks and fruits, indicating that 2,4-D promotes the transport of non-water-soluble calcium forms, such as calcium pectin, calcium phosphate, and calcium oxalate, from the fruit stalks to the fruits. The application of 2,4-D effectively alleviated the ’bottleneck’ in calcium transport in Cerasus humilis fruit. This effect is likely mediated by 2,4-D’s regulation of calcium transporters and channels, which are responsible for calcium uptake and translocation. 2,4-D may enhance the activity of these transporters, thereby increasing the movement of calcium from the fruit stalks to the fruits. However, the precise mechanisms underlying this regulation require further investigation.