**1. Introduction**

Litchi (Litchi chinensis *Sonn*.) is a widely cultivated specialty fruit tree in tropical and subtropical areas of China and has high economic value [1]. The tender, juicy, and delicious fruit of "Concubine Xiao" litchi makes it one of the most popular litchi varieties in China, occupying a large share in the litchi market. Therefore, good appearance and inner quality are fundamental guarantees for "smile of princess" litchi fruit's market competitiveness; however, "smile of princess" litchi fruit experience dehiscent problems during the process of growth and development, which seriously influence yield and quality [2], weaken market competitiveness, and damage growers' economic benefit. Many technical measures have been explored to solve the problem of litchi fruit cracking, among which the simplest one is foliar spraying of Ca fertilizer [3].

Foliar calcium fertilizer has a good effect that prevents the fruit cracking, not only on a variety of fruits, but it also improves the intrinsic quality at the same time. Foliar application of Ca fertilizer effectively reduces grape berry cracking [4], increases the content of sugar in pulp, and promotes the degradation of citric acid in ripe grape pulp [5]. Foliar application of Ca fertilizer reduces cracking [6] and sunburn damage of pomegranate fruits, increases the content of total sugar and chlorophyll, and effectively accelerates the vegetative growth process of the tree [5]. Foliar application of Ca fertilizer can increase

**Citation:** Shui, X.; Wang, W.; Ma, W.; Yang, C.; Zhou, K. Mechanism by Which High Foliar Calcium Contents Inhibit Sugar Accumulation in Feizixiao Lychee Pulp. *Horticulturae* **2022**, *8*, 1044. https://doi.org/ 10.3390/horticulturae8111044

Academic Editor: Rosario Paolo Mauro

Received: 6 October 2022 Accepted: 3 November 2022 Published: 7 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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the hardness of apple meat by acting on the cell wall [5]. Foliar application of Ca fertilizer improved fruit quality and reduced cracking, increased fruit Ca levels and total phenolic content, and reduced the percentage of dehydrated and rotten berries after storage [5]. Therefore, foliar spraying Ca to prevent fruit cracking has become an effective measure for many fruits.

In actual production, owing to the difficulty in accurate control of operation, the problem of spraying Ca fertilizer solution when the concentration is too high leads to the fruit being damaged by Ca fertilizer, resulting in the deterioration of fruit quality, ion toxicity, and salt stress reaction [7]. At the same time, we found that after spraying 0.6% calcium chloride to the canopy, the branches were unchanged but leaves and only a few fruits were covered with some necrotic spots, and the common abnormal performance was a decline in the soluble sugar content of the fruit. However, the physiological, biochemical, and molecular mechanisms of excessive application of Ca fertilizer to fruit trees are rarely studied. The excessive use of Ca fertilizer in pineapple causes a decrease in glucose, fructose, and β-carotene content [8]. High Ca treatment of grapes results in a decrease in sugar–acid ratio, an increase in tannin content, and a decrease in fruit taste quality [9]. In other plants, excessive calcium also affects the normal growth and development of plants: MICU gene inhibits root growth and reduces mRNA transcription levels in land plants under high Ca conditions [10]. Under high Ca stress, the root volume of wheat plants decreased, and the buds showed greening symptoms and shortened length [11]. Under the influence of high Ca, the germination rate of herbage seeds decreased [12]. High Ca is absorbed by cells and accumulated in mitochondria, which may cause cell death [10].

At present, in litchi production, foliar calcium injection is often used to prevent the fruit from cracking [13], but at the same time, it is also found that the litchi tree body is sensitive to calcin; even a little excessive use causes leaf necrosis and a decline in fruit sugar content, similar to the harm to the fruits and other plants described above. Therefore, it is necessary to explore the fertilizer damage caused by excessive Ca in litchi and its mechanism. This paper intends to start from the mechanism of high leaf calcium inhibiting sugar accumulation in "Feizixiao" litchi pulp, so as to provide a theoretical foundation for the development of comprehensive and effective measures for foliar calcium spraying to prevent litchi fruit from cracking.

#### **2. Materials and Methods**

## *2.1. Experimental Setup and Materials*

The lychee orchard at the Yongfa Scientific Research Demonstration Station of the Tropical Fruit Tree Research Institute of Hainan Academy of Agricultural Sciences served as the experimental site (latitude and longitude of 19.9◦ and 109.8◦, respectively). The site is located in the town of Yongfa in northern Chengmai County, Hainan Province. The terrain is a plateau and lies in the tropical monsoon climate area. The annual average temperature is 23.8 ◦C, the annual average number of sunshine hours is 2059 h, the annual average rainfall is 1786.1 mm, and there is no frost year-round. Rain and heat occur in the same season, and the soil is fertile and lateritic. At the time of this study, the soil organic matter content was 23.37 ± 0.87 g/kg, the content of available K was 125.63 ± 2.38 mg/kg, the content of available Ca was 600.51 ± 12.35 mg/kg, and the content of available Mg was 150.47 ± 3.86 mg/kg. Ten 20-year-old grafted Feizixiao lychee trees displaying the same growth trend with no signs of poor growth were selected for sampling. Feizixiao lychee plants mature quickly when grown in this area—nearly 3–4 weeks earlier than those grown in the Guangdong production area. The main phenological periods of lychee in this region are as follows: the fruit dropping period is in early April, the fruit expansion period is in late April, maturity and harvest occur in early May, and the autumn shoot growth period is from July to September.

#### *2.2. Field Experimental Design and Sampling Methods*

The treatments involved spraying a 54 μM anhydrous CaCl2 aqueous solution onto the leaf surfaces (high-Ca treatment hereafter; the CaCl2 used was chemically pure), and a water treatment served as the control (CK). Five replications of single-tree plots were established. The field experiment was initiated at the beginning of fruit expansion (the aril completely covered the seeds). The foliar fertilizer was sprayed once every 7 d for a total of 3 times. The high-Ca treatment was applied between 8 and 9 a.m. on 10, 18, and 25 April 2019 (corresponding to 35, 43, and 50 d after anthesis). The first sampling occurred on 18 April 2019, and subsequent sampling was conducted every 7 d, i.e., at 43, 50, 57, 64, 71, and 77 d after anthesis. As references, five fruits of moderate size and shape were taken from four sides of the tree around the middle of the crown. Thirty fruits similar to the reference fruits were collected from each tree at each collection time. The fruits were quickly frozen in liquid nitrogen and taken back to the laboratory. Then, they were put into a −80 ◦C ultralow-temperature freezer for storage.

#### *2.3. Measurement Methods of Observed Indicators*

The soluble sugar content was determined by anthrone colorimetry. Then, 0.1 g of the sample was ground in a boiling water bath, centrifuged, and placed in a second boiling water bath. After cooling, the anthrone reacted with free hexose or hexosyl, pentosyl, and hexuronic acid in the polysaccharide. After the reaction, the solution was blue-green and had maximum absorption at 620 nm, and the specific content was obtained by comparison with the standard curve [14]. The contents of sugars such as sucrose, fructose, and glucose were determined by the use of a high-performance liquid chromatograph. Here, 0.5 g of pulp was weighed, and 5 mL of 90% ethanol was added to fully grind the sample. Then, the sample was centrifuged, the supernatant collected, and ethanol used for secondary extraction. The two supernatants were combined, placed in a water bath, evaporated to dryness, and brought up to 10 mL with deionized water. The sample was filtered through a 0.45 μm filter membrane prior to testing. The sugar content was determined using a Waters 2695 high-performance liquid chromatograph manufactured in the United States and equipped with an evaporative light-scattering detector [15]. The fresh sample was weighed and then dried to an ashed sample. The ashed sample was allowed to cool and 10 drops of deionized water followed by 3–4 mL of nitric acid were added to the sample. Excess nitric acid was evaporated by placing the sample on a hot plate set at 100–120 ◦C. The sample was returned to the furnace and ashed for an additional 1 h and after being cooled, the ash was dissolved in 10 mL hydrochloric acid and transferred quantitatively to 50 mL volumetric acid. Analysis for calcium was carried out using atomic absorption spectroscopy set at different wavelengths for the optimum working conditions of the minerals.

Extraction of the acid invertase (AI), sucrose phosphate synthase (SPS), sucrose synthase II (SS-II), neutral invertase (NI), and sucrose synthase I (SS-I) enzymes was performed according to Nielsen's method [16], and the enzyme activity was determined according to the avidin biotin peroxidase complex enzyme-linked immunosorbent assay and ELISA reagent test kit (Catalogue No.: KT8013-A, KT50452-A, KT5045-A, KT8107-A, KT5044-A; Meimian, Yancheng, Jiangsu, China). For example: Take 2 g of pulp, add 2 mL of acetic acid buffer, grind into a paste with a mortar in an ice bath, 12 000 r/min for 10 min, and retain the supernatant for enzyme activity determination. Using 2 brace-plug graduated tubes, add 0.8 mL of acetic acid buffer, 0.2 mL of 0.5 mmol/L sucrose solution, and 1 mL of appropriately diluted enzyme solution to each tube, and use the same treatment but no enzyme solution as a blank control, and place it at room temperature for 10 min. Then add 1 mL of Nelson reagent to each tube and place the tubes in a boiling water bath for 20 min. Cool to room temperature, add 1 mL of arsenic–molybdic acid reagent to each tube, and after 5 min, add 7 mL of distilled water to each tube, colorimetric at 510 nm, and determine the optical density (OD) 510nm.

Several key time points were selected: 35 d after anthesis was the starting point and 64 or 71 d after anthesis was the end point. The samples were sent to Wuhan Metwell Biotechnology Co., Ltd., for transcriptome sequencing. Four unigene databases, namely, NCBI nonredundant (NR) protein, SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups of proteins, were used in conjunction with alignment via BLASTX software to obtain protein sequences with high similarity; thus, we obtained the protein functional annotation information corresponding to each unigene. To analyze the differentially expressed genes and their clustering, RSEM software was used for quantitative analysis of gene expression based on fragments per kilobase of exon per million mapped fragment (FPKM) values [17], and DESeq2 was used for screening differentially expressed genes [18] and for analyzing subsequent Gene Ontology (GO) and KEGG functional enrichment. Eight differentially expressed genes were randomly selected from the transcriptome results, and their expression was measured via real-time quantitative PCR (qRT–PCR) [19]; the 2−ΔΔCT value was calculated and used to evaluate whether the transcriptome sequencing results were reliable.

## *2.4. Statistical Analysis*

The online version of SAS software was used for statistical analysis of the data. ANOVA was used for variance analysis, and the Duncan method was used for multiple comparison analysis. T tests were used to analyze the significance of the differences between the high-Ca treatment and the CK treatment.
