Calcium Allocation to the Tree Canopy and the Edible Part of Sweet Cherry Fruit Is Hindered by Boron Soil Deficiency

Abstract

Calcium (Ca) and Boron (B) are structural components of the cell wall with limited phloem mobility. The absorption, movement, and distribution of these two nutrients have a greater effect on leaves than on fruits since their transport is dependent on transpiration flow. This research aimed to study the absorption and movement of ⁴⁵Ca applied to the soil and the fruit of sweet cherry trees under B-deficient and B-adequate soil conditions. In the first experiment, ⁴⁵Ca was applied to the soil surface before the occurrence of leaf senescence. Soil and tree components were sampled and analyzed 6 months after ⁴⁵Ca application. The second experiment involved a ⁴⁵Ca application to the surface of small fruits with 5 mm diameters, which were analyzed after 45 days. The tree Ca allocation in the B-deficient soil condition was significantly fewer in shoots and higher in roots, contrary to the B-adequate soil. On the other hand, the fruit evidenced significant differences in Ca levels in the edible portion of the fruit (i.e., the flesh and peel), which was higher in the B-adequate soil condition. Therefore, under B-deficient soil, Ca was ‘retained’ in the root system and in the fruit pit, suggesting a synergistic mechanism between Ca and B. This mechanism might indicate a survival ecological function where B triggers biological signals to restore Ca homeostasis.

1. Introduction

Calcium (Ca) is an essential macronutrient that performs different functions in plants. It is a structural component in plant cell walls and the middle lamella, and due to this characteristic, many physiological disorders in fruits have been related to Ca nutrition [1]. Ca uptake by plants through soil occurs via the roots, and its accumulation in fruit depends on its transportation through the xylem, as Ca has low phloem mobility [2].

The Ca content within the fruit is regulated by transpiration [3]. Therefore, its lower transpiration capacity accounts for the lower Ca concentrations observed in fruits than in leaves. Ca accumulation in fruit has been reported to be higher during the early stages of development and decreases through maturation, mainly due to the loss of xylem functionality [4]. Although Ca foliar applications can increase the Ca content within fruits [5,6], these applications are not always effective [7]. Conway et al. [8] have proposed that the major problem with foliar spraying is that it leads to insufficient absorption of Ca into the fruit. Other studies have reported that foliar applications of Ca significantly increased the mineral content and quality parameters of sweet cherry fruit [9].

Boron (B), on the other hand, is an essential micronutrient for higher plants as a constituent of the cell wall that is closely related to Ca [10], and the deprivation or excess of one element affects the nutritional status of the other [11]. Ca in the form of pectate contributes to cell wall rigidity [12], and B is involved in the biosynthesis of pectic acid [13]. Its deficiency is associated with reproductive processes affecting the fruit set, and net photosynthesis and transpiration rates are significantly lower in cherry trees with B deficiencies [14].

Ca and B are structural components of the cell wall, have limited phloem mobility, and are strongly dependent on transpiration flow. As a result, the absorption, movement, and distribution of these nutrients affect plant growth and development. Both elements play crucial roles in several developmental processes directly linked to yield and fruit quality, including pollen tube elongation, cell wall synthesis, and cell division [15]. Deficiencies in Ca and B can lead to flower bud abortion, poor fruit sets, and diminished fruit quality, resulting in soft fruit with a reduced shelf life [16]. Conversely, adequate soil B availability enhances Ca translocation and uptake by fruits, reducing physiological disorders and justifying foliar and soil applications, particularly in apple orchards [15,17]. In sweet cherry trees, there are primarily separate studies. For instance, preharvest Ca sprays in sweet cherry orchards have been reported to effectively reduce fruit cracking, although results have not always been consistent postharvest [18,19]. In contrast, preharvest B applications have shown limited effects on fruit quality [20]. Therefore, the interaction between these two elements has not been thoroughly explored.

Approximately 30% of the world’s ice-free land and ~12% of the global area used for arable crops are acidic, with low levels of several nutrients, including B and Ca [21,22]. In addition, Ca and B have similar behaviors, and both are vital during critical stages in the development of fruit, affecting yield and quality. In this context, this work aimed to explore Ca absorption and distribution after Ca applications to the soil and the fruit of sweet cherry trees growing under boron-deficient and boron-adequate soil conditions. This study provides preliminary insights to help understand the interactions between Ca and B.

2. Materials and Methods

2.1. Plant Materials and Treatments

Two outdoor experiments with 3-year-old sweet cherry trees (Prunus avium L.) cv. Regina on a Gisela 6 rootstock growing in pots were conducted in central Chile (35°23′ S, 71°27′ W). The trees were grown in 20 L pots with drip irrigation. A year before this study, a basal nutrient solution with and without B (boric acid, Soquimich, Santiago, Chile) was prepared, according to Arredondo and Bonomelli [14]. One liter of the basal solution without B was applied every 15 days to six trees to obtain the B-deficient (0.04 mg B kg⁻¹) soil conditions, and one liter of the basal solution with B was applied to six trees to obtain adequate (1.1 mg B kg⁻¹) soil conditions, according to Mathew et al. [23].

In the first experiment, 2 mL of a ⁴⁵Ca solution was applied to the soil surface on April 5 in 2022, before the leaves started senescence (BBCH91) [24] to allow it to be absorbed by the roots, stored in the tree reserves, and subsequently utilized during the following growing season (Figure 1a). The 2 mL solutions were split into four aliquots making an equidistant cross with 0.5 mL each. A second experiment was carried out on 21 October in 2022, where 5 µL of ⁴⁵Ca-solution was applied with a micropipette to the surface of all small green fruit (BBCH72) on the tree (Figure 1b) to explore ⁴⁵Ca uptake and distribution in fruit tissues. The specific ⁴⁵Ca volume applied was determined based on preliminary studies to prevent leaching from soil or the dripping of the solution from the fruit’s surface, as appropriate.

In both experiments, the experimental setup consisted of a completely randomized design with two treatments and three replicates, with a single tree as the experimental unit. Treatments were assessed using ANOVA, and the means were separated by the least significant differences (LSD) test when the treatment effect was significant (p-value ≤ 0.05).

2.2. Isotopic ⁴⁵Ca Application

To trace the absorption and movement of Ca, a ⁴⁵CaCl₂ solution (42.8 mg mL⁻¹) was prepared with an activity of 85,281.3 Bq at the Plant and Soil Analysis Laboratory of the Chilean Nuclear Energy Commission (CCHEN). The ⁴⁵Ca was manufactured and calibrated at the Ionizing Radiation Metrology Laboratory at the Chilean Nuclear Energy Comission, Santiago, Chile.

2.3. Plant and Soil Samples for Analytical Determinations of Ca

In the first experiment, 200 days (~6.2 months) after Ca application, 2 kg of soil were sampled from each pot on October 21 in 2022 (BBCH72), homogenized, air dried (to a constant weight), and later sieved with a 2 mm mesh. In addition, the sampled trees were divided into different biomass components: roots, trunk, shoots, and fruits, and then analyzed separately.

In the second experiment, fruits were collected 46 days later, on 6 December 2022, at veraison or the beginning of the fruit’s coloring (BBCH81). Fruits were further partitioned into three sections: pedicel, pit, and flesh and peel (i.e., the edible portion). In both experiments, all plant tissues were oven-dried at 65 °C, ground, and homogenized separately. The activity of ⁴⁵Ca in the trees’ biomass components and soil was determined using the calcination method for isotope ratio analysis via liquid scintillation [25] and expressed as a percentage of the total activity.

3. Results and Discussion

3.1. Total Ca Recovery in the Trees

For the first experiment, the recovery of ⁴⁵Ca by the cherry trees was significantly affected by B soil levels (

Table 1. Recovery of isotopic ⁴⁵Ca by sweet cherry trees cv. Regina applied to the soil before leaf senescence under two B soil conditions.

B Soil Availability	Total Plant 45Ca Recovery (%)
Deficient	29.9
Adequate	54.5
p-value *	0.05

* p-value ≤ 0.05 indicates statistically significant differences between the two soil B conditions.

). Under B-deficient soil conditions, 70.1% of the ⁴⁵Ca remained in the soil and only 29.9% was absorbed by the plant, while under B-adequate soil conditions, the ⁴⁵Ca plant recovery was 54.5%, suggesting a limitation at the root system. In fact, one of the earliest effects of B deficiency is a cessation of growth at the growing tips, or meristems, reducing cell division and elongation, which also affects root growth and calcium uptake [26]. At the root level, under B deprivation there is a strong inhibition of root growth and a reduction in root meristem activity [27], leading to a balanced reduction of the shoot-to-root biomass.

3.2. Ca Recovery in Different Tree Tissues

Regarding the allocation of Ca through the different plant tissues, a B-deficient soil condition affected Ca’s transportation to aerial tissues (shoots), a preferential sink of xylem flow. As a result, the Ca mainly remained in the roots (

Table 2. ⁴⁵Ca allocation in different tissues of sweet cherry trees cv. Regina under two B soil conditions. ⁴⁵Ca was applied to the soil before leaf senescence and analyzed at green 5 mm fruit stage.

Boron Soil Availability	45Ca Allocation in Sweet Cherry Tree (%)
	Shoots	Fruits	Trunk	Roots
Deficient	31 b	11 a	13 a	45 a
Adequate	45 a	13 a	14 a	28 b
p-value *	0.03	0.76	0.81	0.02

* p-value ≤ 0.05 indicates statistically significant differences between the two soil B conditions. Different letters within the column indicate significant difference (p ≤ 0.05).

), possibly due to the loss of root and xylem functionality derived from the B deficiency, as proposed by Wimmer and Eichert [28]. Moreover, a Ca deficiency can increase cell wall degradation, which inhibits root development. Also, a B deficiency can stimulate the cell wall’s thickness and affect the capacity of the root system and, consequently, the absorption of nutrients [11]. Conversely, an adequate B soil availability may have a synergistic effect in the retranslocation of Ca within plants, as have been reported in apple trees, pepper and tomato plants, and other species [22,29,30].

The Ca content in the fruit is usually much lower than in other parts of the plant [31], which coincides with what was found in our study (Table 2). Ca enters the plant passively with water through the roots and is transported via xylem vessels [32]. Ca cannot be mobilized from older tissues and redistributed via the phloem, forcing the developing tissues to rely on the immediate supply of Ca in the xylem. Thus, Ca supply to the plant’s organs depends on transpiration, where fruits have a lower transpiration rate than leaves [31]. The xylem inflow resulting from fruit transpiration and growth is a crucial determinant for fruit to acquire Ca [1], and fruit transpiration is a function of vapor pressure deficit (VPD). Therefore, the Ca content in the fruit is influenced by climatic factors [3] and soil characteristics (texture–porosity) [33].

3.3. Ca Recovery in the Fruit

Regarding the second experiment,

Table 3. ⁴⁵Ca allocation in the fruit of sweet cherries cv. Regina under two B soil conditions. ⁴⁵Ca was applied at green 5 mm fruit stage and analyzed at veraison or beginning of fruit coloring.

B Soil Availability	45Ca Allocation in Sweet Cherry Fruit (%)
	Pedicel	Flesh + Peel	Pit
Deficient	22.6 a	21.3 b	56.1 a
Adequate	24.3 a	25.1 a	50.6 a
p-value *	0.78	0.04	0.53

* p-value ≤ 0.05 indicates statistically significant differences between the two soil B conditions. Different letters within the column indicate significant difference (p ≤ 0.05).

shows the Ca allocation in different tissues within the sweet cherry fruit. The B soil condition did not affect the cherry fruit’s ⁴⁵Ca allocation in the pedicel and pit; however, there was a significant difference in the flesh and peel, where the ⁴⁵Ca allocation was lower under the B-deficient soil conditions (Table 3).

The presence of ⁴⁵Ca was detected in the sweet cherry fruits at the beginning of the fruit coloring period, indicating that Ca absorption occurred regardless of the two soil B conditions. The largest Ca partitioning was found in the pit or endocarp, followed by the pedicel, and the flesh and peel of the fruit. The pit is a specialized part of the pericarp, which becomes woody during development and protects the seed inside. The seeds comprise distinct tissues, including the seed coat, which distributes imported assimilates to the developing internal storage tissues [34]. During stage I of fruit development, the pericarp sections increase significantly. The endocarp and seed show a higher relative growth rate, but during stage II, when pit hardening occurs, the embryo and endosperm show the highest rate. This relative growth rate suggests that the strongest sink is the seed, an organ with a remarkable ability to import assimilates [35] and, therefore, is an indication of the need for boron, which must be present in the meristems. Another likely explanation for the highest Ca accumulation in the pit, compared to the edible part of the cherry (the flesh and peel), is the loss of xylem functionality from early stage III. In the second experiment, the cherries were harvested at the beginning of coloring or stage III, therefore, the xylem rupture of the vessels had already happened. The xylem rupture begins with minor and major veins of the middle bundle, which are all in the flesh and peel regions of the fruit, and then the rupture continues with the lateral bundles and veins close to the suture [36]. The order in which the xylem ruptures and how its inflow diminishes may explain the higher Ca content in the pit than in the flesh and peel. Likely, the pit is the last tissue where the xylem is still functional before its total rupture. It is noteworthy that no xylogenesis (the formation of new xylem vessels) occurs from stages II and III in the sweet cherry fruit, as occurs in grapes, leading to the complete shutdown of the xylematic vasculature [37]. Consequently, soil conditions deficient in B hinder the already limited physiological translocation of Ca, thereby affecting its availability in the fruit. Therefore, to obtain cherry fruits with adequate Ca nutrition, it is necessary to have adequate B nutrition in the tree.

Figure 2 shows a summary diagram of the results from both experiments. Arredondo and Bonomelli [14] indicated that plants subjected to a low B soil deficiency showed a higher proportion of white-root biomass than those with an adequate soil level, probably to address the lack of supply of this essential nutrient within the plant. A synergistic mechanism between Ca and B has been proposed. Under a B deficiency, Ca⁺² cytosolic levels are affected; in roots, the Ca signaling gene expression is upregulated and that of the Ca channel/transporters out of the root systems is repressed, according to a recent review by Vera-Maldonado et al. [22]. It is likely that a B deficiency triggers a responsive mechanism (signal transduction) to restore Ca homeostasis, providing relevant evidence of synergism (B–Ca interaction).

4. Conclusions

Ca absorption and distribution were explored under interaction with boron in different sweet cherry tissues. Notably, the mechanism underlying the interaction between B and Ca nutrition remains unclear. A synergistic relationship between Ca and B mediated by the upregulation of Ca signaling gene expression to restore Ca homeostasis under B deficiency has been proposed by other authors; however, further investigation is needed.

Sweet cherry tree roots effectively recovered the ⁴⁵Ca applied to the soil at the end of the season when the B soil availability was adequate. This Ca remained in tree reserves (probably in roots) during wintertime and was subsequently remobilized to various cherry tree tissues, reaching the next-season tree organs. However, Ca remobilization from roots to aerial organs was more efficient under adequate B conditions than in B-deficient soil conditions. Therefore, sweet cherry growers should ensure sufficient B levels through soil and foliar applications to improve the Ca nutritional status of the trees. Furthermore, the higher Ca allocation found in the fruit’s flesh from trees growing under soil B-adequate conditions could potentially enhance the postharvest fruit’s quality and reduce susceptibility to some Ca-related physiological disorders.