Abstract
Ten 16-year-old trees were used as test materials to investigate the effect of foliar calcium fertilizer on the sugar content of ‘Feizixiao’ litchi (Litchi chinensis Sonn.) pulp. The experiment began 35 days after anthesis (DAA) in 2020 and 2021, and the treatment was a foliar spray application of 0.3% CaCl2 aqueous solution, whereas the control was a foliar spray application of water. The sugar content, sucrose-metabolizing enzymes, and ATP-dependent phosphofructokinase (PFK) activities in pulp were measured in 2020 and 2021. Transcriptome sequencing (RNA-seq) was performed on RNA samples from treatment and control fruit pulps at 35, 63, and 69 DAA (full mature stage) in 2020, and 10 genes were chosen for confirmation by real-time polymerase chain reaction (PCR) in 2020 and 2021. At full maturity, the soluble sugar content in the calcium-treated group was extremely significantly or significantly higher than that in the control group. After 63 DAA, the net sucrose-metabolizing enzyme activity in the calcium-treated group was significantly higher than that in the control group. Furthermore, at full maturity, the calcium-treated group had significantly higher sucrose synthase cleavage activity and significantly lower PFK activity than the control group. Fifty-four highly expressed genes in the glycolytic pathway (EMP) were screened from transcriptome data, including hexokinase, PFK, and pyruvate kinase genes; 87% of these genes were downregulated in the treatment group compared with the control group at 69 DAA in 2020. The linear regression between RNA-seq and real-time PCR results was significant in 2020 (r = 0.9292) and 2021 (r = 0.8889). When the fruit is fully ripe, calcium treatment increases net sucrose-metabolizing enzyme activity by increasing sucrose synthase cleavage activity, promoting the accumulation of reducing sugars, and it downregulates phosphofructokinase gene expression in EMP, promoting sugar accumulation.
Litchi (Litchi chinensis Sonn.) is a well-known Lingnan fruit native to China (Menzel 2001). It has thousands of years of cultivation history in China (Li et al. 2013) and is cultivated in more than 20 countries worldwide (Hu et al. 2022). Litchi has become one of the most appealing tropical or subtropical fruits on the international market because of its excellent flavor and high nutrient content (Noh et al. 2011; Wall 2006). ‘Feizixiao’ litchi is a high-quality cultivar that ripens medium to early. It has a sweet taste and strong aroma, is juicy and refreshing, and is primarily distributed in Guangdong, Guangxi, Sichuan, Hainan, Taiwan, and other Chinese regions (Feng et al. 2015), with high economic value.
Sugar is an important flavor substance in fruit and a component of the cellular respiration metabolic substrate (Guo et al. 2022). It can also be used as a signal to regulate fruit growth and senescence as well as related gene expression (Batista-Silva et al. 2018; Gupta and Kaur 2005). Different cultivars of litchi accumulate different types of sugars. ‘Feizixiao’ litchi accumulates more reducing sugars than sucrose (Wang et al. 2003). Sugar accumulation is related to sucrose-metabolizing enzymes, including invertase (INV), sucrose synthase (SS), and sucrose phosphate synthase (SPS) (Qazi et al. 2012). Invertases include acid invertase (AI) and neutral invertase (NI), which catalyze the decomposition of sucrose into fructose and glucose; SS catalyzes the reversible reactions between fructose, uridine diphosphate glucose (UDPG) and sucrose; and SPS catalyzes the synthesis of sucrose. A change in fruit sugar content is not simply the result of the action of a single enzyme. Some studies have shown that changes in the activities of some enzymes are not consistent with changes in pulp sugar accumulation (Yang et al. 2013). Therefore, a change in the fruit sugar content should be the result of the combined action of these sucrose-metabolizing enzymes, that is, the net activity of sucrose-metabolizing enzymes. Studies have shown that the accumulation of sucrose in muskmelon (Cucumis melo L.) depends on the difference between the activities of INV, SS, and SPS (Hubbard et al. 1989).
Energy metabolism is based on the glycolytic pathway (EMP) in plants, and phosphofructokinase is a key enzyme in EMP. There are two types of phosphofructokinase proteins that phosphorylate fructose-6-phosphate: pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP) and ATP-dependent phosphofructokinase (PFK). PFK catalyzes the interconversion of fructose-1,6-bisphosphate and fructose-6-phosphate, and PFP can use pyrophosphate instead of ATP as a phosphoryl donor to catalyze the reversible phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate (Lü et al. 2019). PFP catalyzes the reaction and reacts near equilibrium in both directions, whereas PFK is virtually irreversible in vivo (Siebers and Schönheit 2005), so PFK may be more critical in the glycolytic pathway.
When the fruit of ‘Feizixiao’ litchi matures, the soluble sugar content peaks, and the flavor and taste are at their best. Then, the soluble sugar content decreases, and the phenomenon of “sugar receding” occurs at the fully mature stage (Wang et al. 2017). The drop in sugar content seriously affects fruit quality and reduces its commercial value.
Calcium is considered to be an important mineral element that plays a key role in the growth and development of plants. Numerous studies have shown that calcium can delay ripening and inhibit fruit senescence (Sinha et al. 2019; Zhou et al. 2000), mainly by reducing fruit respiration (Recasens et al. 2004). An increase in the calcium content of sweet cherry (Prunus avium L.) fruit was accompanied by a decrease in the fruit respiration rate and an increase in peel firmness (Wang et al. 2014). Calcium also inhibits fungal spore germination and reduces fruit disease (Biggs 1999). In general, fruit undergo changes in membrane composition and structure during senescence (Jincy et al. 2017), and studies have shown that calcium maintains cell wall structure (Balic et al. 2014) and plasma membrane integrity, reducing fruit spoilage (Khaliq et al. 2015; Wang and Long 2015). In addition, calcium plays an important role in the prevention of physiological disorders in plant cells (Conway et al. 1992).
There have been many studies on the effect of calcium on the quality of various fruit, but little work has been done on the effect of calcium on the sugar content of litchi fruit. In ‘Feizixiao’ litchi fruit, the interval between when the best flavor and taste is present and the phenomenon of “sugar receding” is short. Therefore, when fruit are ripe, a large amount of labor is required to harvest them in a short period of time, which will increase production costs and reduce farmers’ income. CaCl2, the main inorganic calcium salt, is beneficial for postharvest preservation and storage of fruit due to its high efficiency (Youryon et al. 2018). For these reasons, this study aimed to explore the effect of foliar spraying of a 0.3% CaCl2 aqueous solution on the sugar content of ‘Feizixiao’ litchi fruit and to provide theoretical support for the artificial regulation of the sugar content of ‘Feizixiao’ litchi fruit.
Materials and Methods
Plant materials.
Ten 16-year-old ‘Feizixiao’ litchi trees with basically the same growth vigor and no bad performance in the Team 5 Litchi Garden, Jinpai Farm, Chengmai County, Hainan Province, were used as the test materials. Foliar spray application of 0.3% CaCl2 aqueous solution was used as the treatment, and foliar spray application of water was used as the control. There were five replicates in the treatment and control groups. In the garden, the fruit began to ripen after 63 d after anthesis (DAA) in 2020 and 2021 and ripened completely at 69 DAA in 2020 and 70 DAA in 2021 (Liao et al. 2022). Treatment was started at the end of the physiological fruit drop period (35 DAA), and three treatments were applied in total. The treatment times were 35, 42, and 50 DAA in 2020 and 35, 42, and 49 DAA in 2021. Sampling occurred during the fruit expansion period and fruit ripening period, starting at 35 DAA (19 Apr 2020, 18 Apr 2021), and five fruit with the same average size in the middle and periphery of each tree were selected. The size and color of the five fruit were used as a reference for subsequent sampling, with highly similar fruit selected as the test materials at 30 fruit per tree. The sampling times were 35, 42, 50, 56, 63, and 69 DAA in 2020 and 35, 42, 49, 56, 63, and 70 DAA in 2021. After collection, the samples were placed in liquid nitrogen for quick freezing and returned to the laboratory for storage in an ultralow temperature freezer (−80 °C).
Extraction and determination of glucose, fructose, and sucrose.
The method described by Wang et al. (2006) was used with some modifications. First, 0.5 g of pulp was weighed into a mortar and heated in a microwave oven for 30 s; then, 5 mL of 90% ethanol was added, and the sample was ground thoroughly and centrifuged at 10,000 gn for 15 min. The supernatant was collected, and 5 mL of 90% ethanol was added for a second extraction. The two supernatants were combined and evaporated to dryness in a 90 °C water bath, followed by the addition of deionized water to 10 mL. A small amount of the supernatant was then aspirated with a syringe and filtered through a 0.45-μm membrane for testing. The sugar content was determined by high-performance liquid chromatography with an evaporative light-scattering detector (model 2695; Waters Corp., Milford, MA, USA) and an amino column (Boston Analytics, Watertown, MA, USA). The mobile phase was 8 acetonitrile:2 water, the flow rate was 1 mL·min−1, the column temperature was 35 °C, and the injection volume was 10 μL. High-purity glucose, fructose, and sucrose (Beijing Tanmo Quality Inspection Technology Co., Ltd., Beijing, China) were used as the standards, and high-purity acetonitrile (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was used as the mobile phase. Sucrose, fructose, and glucose have been identified as the main sugars in litchi (Yang et al. 2013), so the sum of the fructose, glucose, and sucrose contents was used as the soluble sugar content, and the sum of the fructose and glucose contents was used as the reducing sugar content.
Determination of sucrose-metabolizing enzymes and PFK activities.
The sucrose synthase cleavage (SS-C) and synthesis (SS-S), AI, NI, SPS, and PFK activities in the pulp were measured with double antibody sandwich–enzyme-linked immunosorbent assay kits (Jiangsu Enzyme Immune Industrial Co., Ltd., Yancheng, Jiangsu, China) following the manufacturer’s protocol.
RNA extraction, library construction. and sequencing.
According to the changes in the total soluble sugar content in ‘Feizixiao’ litchi pulp in the control in 2020, RNA was extracted from the calcium-treated and control litchi pulp samples at 35, 63, and 69 DAA for transcriptome sequencing, with three biological replicates for each period. An RNA extraction kit (RNAprep Pure Plant Plus Kit, Tiangen Biochemical Technology Co., Ltd., Beijing, China) was used for RNA extraction following the manufacturer’s protocol. After the RNA was purified and qualified, a complementary DNA (cDNA) library was established (Wuhan Metwell Biotechnology Co., Ltd., Wuhan, China), and an Illumina HiSeq instrument (Illumina, San Diego, CA, USA) was used for transcriptome sequencing after the library was qualified.
Sequence assembly.
After sequencing, the raw reads were filtered to remove low-quality reads with adapters to obtain clean reads. Trinity software (Grabherr et al. 2011) was used to splice the clean reads. The obtained data were stored in FASTA format, and the longest transcript obtained after hierarchical clustering was considered a unigene.
Gene annotation and differentially expressed gene screening.
Unigenes were compared with the Kyoto Encyclopedia of Genes and Genomes [KEGG (Kanehisa et al. 2004)], National Center of Biotechnology Information (Bethesda, MD, USA) nonredundant (NR) database (Polashock et al. 2010), SwissProt protein sequence database (Sato et al. 2006), Gene Ontology [GO (Boyle et al. 2004)], cluster of orthologous groups of proteins and euKaryotic ortholog groups [COG/KOG (Li et al. 2003; Natale et al. 2000)], and TrEMB databases (O’Donovan et al. 2002) using BLAST software (Altschul et al. 1997), and the predicted amino acid sequences of the unigenes were compared with the protein family [Pfam (Bateman et al. 2002)] using HMMER software (Finn et al. 2011) to obtain annotation information. Fragments per kilobase of transcript per million fragments mapped (FPKM) was used to calculate the expression of the unigenes. DESeq2 software (Love et al. 2014; Varet et al. 2016) was used to identify differentially expressed genes (DEGs).
DEGs enrichment analysis.
DEGs were screened based on a false discovery rate (FDR) < 0.05 and a | log2Fold Change | ≥ 1. GO and KEGG enrichment analyses were performed for the DEGs, and significant GO terms and KEGG pathways were identified. A hypergeometric test was applied to find the pathways and GO terms that were significantly enriched for DEGs in the context of the whole genome.
Primer design and real-time PCR verification.
To further study the effect of calcium treatment on the sugar content of ‘Feizixiao’ litchi pulp, some genes in EMP with high expression were screened, and the FPKM values for these genes were horizontally normalized and drawn into a heatmap with TBtools (Chen et al. 2020). Ten genes were randomly selected from transcriptome sequencing (RNA-seq) data, and real-time PCR primers were designed with Primer3 (Rozen and Skaletsky 2000) and synthesized (Shanghai Bioengineering Co., Ltd., Shanghai, China). The extracted pulp RNA was reverse transcribed into cDNA using a cDNA synthesis kit (HiScript II first Strand cDNA Synthesis Kit; Novizan Biotechnology Co., Ltd., Nanjing, China) and a PCR instrument (T100TM Thermal Cycler; BIO-RAD Inc., Hercules, CA, USA). The procedure was performed according to the manufacturer’s instructions. Real-time PCR verification was performed using a real-time PCR instrument (qTOWER3; Analytik Jena Inc, Jena, Germany) with a real-time PCR kit (Taq Pro Universal SYBR qPCR Master Mix; Vazyme Inc., Nanjing, China). The relative expression of genes was calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001) with 35 DAA as the reference, and litchi actin was used as the internal reference gene (Jiang et al. 2017). The primers are shown in Table 1.
The primer names and sequences used for real-time polymerase chain reaction validation of 10 genes selected in ‘Feizixiao’ litchi pulp.
Data analysis.
Data were summited to analysis of variance using statistical software (SAS version 9.4; SAS Institute Inc., Cary, NC, USA). Student’s t test was used to analyze significant differences between the calcium treatment and control at the same stage.
Results and Analysis
Chromatographic separation of standards.
The chromatogram of the standards is shown in Fig. 1. The peaks in the figure are fructose, glucose, and sucrose in sequence, which were all successfully separated before 14 min, indicating that the chromatographic conditions in this study could well separate the main sugar components of ‘Feizixiao’ litchi fruit.
High-performance liquid chromatography chromatograms of fructose, glucose, and sucrose in ‘Feizixiao’ litchi pulp.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
High-performance liquid chromatography chromatograms of fructose, glucose, and sucrose in ‘Feizixiao’ litchi pulp.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
High-performance liquid chromatography chromatograms of fructose, glucose, and sucrose in ‘Feizixiao’ litchi pulp.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Changes in the contents of fructose, glucose, and sucrose.
The dynamic changes in the fructose content are shown in Fig. 2A and B. In 2020 and 2021, the fructose content in the control continued to rise. In 2020, the fructose content in the calcium treatment continued to rise from 35 to 50 DAA, decreased at 56 DAA, and continued to rise after 56 DAA. The fructose content in the calcium treatment continued to rise in 2021. After 69 DAA in 2020 and 2021, the fructose content in the calcium treatment was significantly higher than that in the control, but there was no significant difference in the remaining stages.
Dynamic changes in the fructose, glucose, sucrose, soluble sugar, and fructose + glucose contents in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The fructose content in 2020 and 2021. (C, D) The glucose content in 2020 and 2021. (E, F) The sucrose content in 2020 and 2021. (G, H) The soluble sugar content in 2020 and 2021. (I, J) The fructose + glucose contents in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Dynamic changes in the fructose, glucose, sucrose, soluble sugar, and fructose + glucose contents in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The fructose content in 2020 and 2021. (C, D) The glucose content in 2020 and 2021. (E, F) The sucrose content in 2020 and 2021. (G, H) The soluble sugar content in 2020 and 2021. (I, J) The fructose + glucose contents in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Dynamic changes in the fructose, glucose, sucrose, soluble sugar, and fructose + glucose contents in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The fructose content in 2020 and 2021. (C, D) The glucose content in 2020 and 2021. (E, F) The sucrose content in 2020 and 2021. (G, H) The soluble sugar content in 2020 and 2021. (I, J) The fructose + glucose contents in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
The dynamic changes in glucose content are shown in Fig. 2C and D. In 2020, the glucose content in the control increased continuously from 35 to 56 DAA and first increased and then decreased from 56 to 69 DAA, whereas the glucose content in the control continued to rise in 2021. In 2020 and 2021, the change trend of the glucose content in the calcium treatment was the same, first continuously increasing, then declining and then continuously increasing. In 2020, the glucose content in the calcium treatment was significantly lower than that in the control at 56 DAA. In 2020 and 2021, the glucose content in the calcium treatment was significantly higher than that in the control at the fully mature stage, but there was no significant difference at the other stages.
The dynamic changes in the sucrose content are shown in Fig. 2E and F. The change trend in the calcium treatment in 2020 and 2021 was consistent with that in the control. In 2020, the sucrose content continued to rise before 50 DAA, after which it decreased and then rose and fell again from 50 to 69 DAA. In 2021, it continued to rise before 49 DAA, after which it decreased, then rose and then decreased again from 49 to 69 DAA. The sucrose content in the calcium treatment at 63 DAA in the 2 years was significantly or extremely significantly lower than that in the control, but there was no significant difference in the rest of the stages.
The dynamic changes in the soluble sugar content are shown in Fig. 2G and H. In 2020 and 2021, the soluble sugar content in the calcium treatment showed the same change trend, where it was significantly lower than that in the control after 56 DAA. “Sugar receding” appeared in the control after 63 DAA, and the soluble sugar content in the calcium treatment was lower than that in the control at 63 DAA. In contrast, it was higher than that in the control at the fully mature stage, showing a trend opposite to that in the control. The soluble sugar content in the calcium treatment was significantly higher than that in the control at 69 DAA in 2020, indicating that calcium treatment delayed the appearance of the soluble sugar content peak, thus playing a certain role in promoting the accumulation of soluble sugars at the fully mature stage.
The dynamic changes in the reducing sugar content are shown in Fig. 2I and J. In 2020, the reducing sugar content in the calcium treatment and control showed a rising trend. In 2021, it showed a trend of first increasing, then decreasing and then increasing again in the calcium treatment, and in the control, it increased and then stabilized. The reducing sugar content in the calcium treatment was significantly lower than that in the control at 56 DAA in 2020. At the fully mature stage in 2020 and 2021, the reducing sugar content was significantly higher in the calcium treatment than in the control, but there was no significant difference in the remaining stages.
In summary, calcium treatment can promote the accumulation of reducing sugars in pulp when the fruit is fully ripe and inhibit the accumulation of sucrose in pulp at 63 DAA. The soluble sugar content in the calcium treatment was lower than that in the control at 63 DAA, which was mainly caused by the sucrose content being lower than that in the control. When the fruit was fully ripe, the soluble sugar content in the calcium treatment was higher than that in the control because of the higher reducing sugar content.
Changes in the activities of sucrose-metabolizing enzymes and PFK.
The dynamic changes in AI activity are shown in Fig. 3A and B. In 2020, the AI activity in the calcium treatment was significantly higher than that in the control at 63 and 69 DAA. In 2021, the AI activity in the calcium treatment was significantly lower than that in the control at 63 and 70 DAA. The changes in the AI activity in response to calcium treatment were inconsistent between the 2 years, which may have been caused by interannual environmental differences, but in general, the AI activity was low.
Dynamic changes in acid invertase (AI), neutral invertase (NI), sucrose synthase cleavage (SS-C), sucrose synthase synthesis (SS-S), sucrose phosphate synthase (SPS), the net sucrose-metabolizing enzyme and ATP-dependent phosphofructokinase (PFK) activities in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The AI activity in 2020 and 2021. (C, D) The NI activity in 2020 and 2021. (E, F) The SS-C activity in 2020 and 2021. (G, H) The SS-S activity in 2020 and 2021. (I, J) The SPS activity in 2020 and 2021. (K, L) The net sucrose-metabolizing enzyme activity in 2020 and 2021. (M, N) The PFK activity in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Dynamic changes in acid invertase (AI), neutral invertase (NI), sucrose synthase cleavage (SS-C), sucrose synthase synthesis (SS-S), sucrose phosphate synthase (SPS), the net sucrose-metabolizing enzyme and ATP-dependent phosphofructokinase (PFK) activities in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The AI activity in 2020 and 2021. (C, D) The NI activity in 2020 and 2021. (E, F) The SS-C activity in 2020 and 2021. (G, H) The SS-S activity in 2020 and 2021. (I, J) The SPS activity in 2020 and 2021. (K, L) The net sucrose-metabolizing enzyme activity in 2020 and 2021. (M, N) The PFK activity in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Dynamic changes in acid invertase (AI), neutral invertase (NI), sucrose synthase cleavage (SS-C), sucrose synthase synthesis (SS-S), sucrose phosphate synthase (SPS), the net sucrose-metabolizing enzyme and ATP-dependent phosphofructokinase (PFK) activities in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water). (A, B) The AI activity in 2020 and 2021. (C, D) The NI activity in 2020 and 2021. (E, F) The SS-C activity in 2020 and 2021. (G, H) The SS-S activity in 2020 and 2021. (I, J) The SPS activity in 2020 and 2021. (K, L) The net sucrose-metabolizing enzyme activity in 2020 and 2021. (M, N) The PFK activity in 2020 and 2021. *, ** Significantly different at P ≤ 0.05, 0.01 during the same period. Error bars represent SEM.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
The dynamic changes in NI activity are shown in Fig. 3C and D. In 2020, the NI activity in the calcium treatment was significantly higher than that in the control at 63 DAA, but there was no significant difference from the control at 69 DAA. In 2021, the NI activity in the calcium treatment was significantly higher than that in the control at 63 and 70 DAA. This indicated that the NI activity showed an increasing trend in response to calcium treatment during fruit ripening.
The dynamic changes in SS-C activity are shown in Fig. 3E and F. In 2020, the SS-C activity in the calcium treatment was significantly lower than that in the control at 63 DAA and was significantly higher than that in the control at 69 DAA. In 2021, the SS-C activity in the calcium treatment was significantly higher than that in the control at 63 and 70 DAA. This indicated that calcium treatment increased the SS-C activity at the fully mature stage.
The dynamic changes in SS-S activity are shown in Fig. 3G and H. In 2020 and 2021, the SS-S activity in the calcium treatment was significantly lower than that in the control at 63 DAA. The activity levels of the two were similar, and there was no significant difference at the fully mature stage, indicating that the effect of calcium treatment on SS-S activity was the same in the 2 years, with both showing a decreased activity level at 63 DAA.
The dynamic changes in SPS activity are shown in Fig. 3I and J. In 2020 and 2021, the SPS activity in the calcium treatment was significantly higher than that in the control during fruit ripening, indicating that calcium treatment increased the level of SPS activity during this developmental process.
The dynamic changes in the net activity of sucrose-metabolizing enzymes (the difference between the cleavage and synthesis activity of sucrose-metabolizing enzymes) are shown in Fig. 3K and L. In 2020 and 2021, the net activity of sucrose-metabolizing enzymes in the calcium treatment was significantly higher than that in the control during fruit ripening, indicating that calcium treatment increased the net activity of sucrose-metabolizing enzymes during this developmental process.
The dynamic changes in PFK activity are shown in Fig. 3M and N. In 2020 and 2021, the activity levels were comparable, and there was no significant difference between the calcium treatment and control at 63 DAA. When the fruit was fully ripe, the PFK activity in the calcium treatment was lower than that in the control, indicating that calcium treatment reduced the activity level of PFK at the fully mature stage.
Transcriptome sequencing quality assessment.
The transcriptome sequencing data are shown in Table 2. The total number of raw reads obtained from all samples was ≥ 43,294,196, and the total number of clean reads after filtering was ≥ 41,871,161. Ninety-seven percent of the bases had a mass value of 20 or greater, 94% of the bases had a mass value of 30 or greater, and the proportion of guanine and cytosine was more than 45%, indicating that the transcriptome sequencing quality was good for subsequent analysis.
Statistical results of transcriptome sequencing of ‘Feizixiao’ litchi pulp.
Statistics on the number of DEGs.
As shown in Fig. 4A–D, A1 vs. B1 yielded a total of 425 DEGs, of which 255 were downregulated and 170 were upregulated; A2 vs. B2 yielded a total of 212 DEGs, of which 60 were downregulated and 152 were upregulated; and B1 vs. B2 yielded a total of 393 DEGs, of which 193 were downregulated and 200 were upregulated. The numbers of upregulated, downregulated, and total DEGs between the calcium treatment and the control at 69 DAA were correspondingly lower than those at 63 DAA. The number of downregulated DEGs decreased the most, and the total number of DEGs decreased by half.
Volcano plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C). Bar plot of DEGs in A1 vs. B1, A2 vs. B2, B1 vs. B2 (D). A1 vs. B1 represents the comparison between Ca treatment and control at 63 d after anthesis. A2 vs. B2 represents the comparison between Ca treatment and control at 69 d after anthesis. B1 vs. B2 represents the comparison of Ca treatment between 63 d after anthesis and 69 d after anthesis.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Volcano plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C). Bar plot of DEGs in A1 vs. B1, A2 vs. B2, B1 vs. B2 (D). A1 vs. B1 represents the comparison between Ca treatment and control at 63 d after anthesis. A2 vs. B2 represents the comparison between Ca treatment and control at 69 d after anthesis. B1 vs. B2 represents the comparison of Ca treatment between 63 d after anthesis and 69 d after anthesis.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Volcano plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C). Bar plot of DEGs in A1 vs. B1, A2 vs. B2, B1 vs. B2 (D). A1 vs. B1 represents the comparison between Ca treatment and control at 63 d after anthesis. A2 vs. B2 represents the comparison between Ca treatment and control at 69 d after anthesis. B1 vs. B2 represents the comparison of Ca treatment between 63 d after anthesis and 69 d after anthesis.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
GO enrichment analysis.
DEGs in the A1 vs. B1, A2 vs. B2, and B1 vs. B2 comparisons were divided into three major categories: biological process, cellular composition, and molecular function. The 50 GO terms with the lowest q-values in the enrichment analysis results were selected, and a column chart of enrichment entries was drawn. The results are shown in Fig. 5A–C. Among the three groups, biological process was enriched for 1220, 641, and 1487 DEGs, of which 487, 429, and 448 were upregulated and 733, 212, and 1039 were downregulated, respectively; cellular composition was enriched for 209, 142, and 220 DEGs, of which 114, 93, and 75 were upregulated and 95, 49, and 145 were downregulated, respectively; and molecular function was enriched for 593, 205, and 469 DEGs, of which 227, 153, and 210 were upregulated and 366, 52, and 259 were downregulated, respectively. A further search for GO terms related to glucose metabolism revealed that seven DEGs were enriched in the fructose metabolism pathway in A1 vs. B1, indicating that calcium treatment may have a significant effect on fructose metabolism.
Column chart of Gene Ontology (GO) classification of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The horizontal axis represents the secondary GO entries, and the vertical axis represents the number of DEGs. The labels to the right of the graph represent the categories to which the GO entries belong.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Column chart of Gene Ontology (GO) classification of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The horizontal axis represents the secondary GO entries, and the vertical axis represents the number of DEGs. The labels to the right of the graph represent the categories to which the GO entries belong.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Column chart of Gene Ontology (GO) classification of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The horizontal axis represents the secondary GO entries, and the vertical axis represents the number of DEGs. The labels to the right of the graph represent the categories to which the GO entries belong.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
KEGG enrichment analysis.
The 20 most significant pathways revealed by the KEGG enrichment analysis of DEGs in A1 vs. B1, A2 vs. B2, and B1 vs. B2 are shown in Fig. 6A–C, and 470, 136, and 330 DEGs were enriched in the three groups, respectively. Notably, pathways related to sugar metabolism, such as fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism, were found in A1 vs. B1, indicating that calcium treatment during this period may have a significant regulatory effect on these pathways.
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bubble plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The enrichment factor represents the ratio of the number of DEGs enriched by the pathway to the number of annotated genes. The color bar represents the significance test P value adjusted for multiple hypothesis testing. The number represents the number of DEGs enriched in the pathway.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bubble plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The enrichment factor represents the ratio of the number of DEGs enriched by the pathway to the number of annotated genes. The color bar represents the significance test P value adjusted for multiple hypothesis testing. The number represents the number of DEGs enriched in the pathway.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bubble plot of differentially expressed genes (DEGs) in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) at 63 d after anthesis (A), in ‘Feizixiao’ litchi pulp of Ca treatment and control at 69 d after anthesis (B) and in ‘Feizixiao’ litchi pulp of Ca treatment at 63 and 69 d after anthesis (C) from transcriptome sequencing results in 2020. The enrichment factor represents the ratio of the number of DEGs enriched by the pathway to the number of annotated genes. The color bar represents the significance test P value adjusted for multiple hypothesis testing. The number represents the number of DEGs enriched in the pathway.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Differential expression of key glycolysis enzyme genes.
Fifty-four key enzyme genes in EMP with high expression were selected, including seven hexokinase genes, 30 phosphofructokinase genes, and 17 pyruvate kinase genes. As shown in Fig. 7, in A1 vs. B1, 83% of the 54 genes were upregulated, including four hexokinase genes, 28 phosphofructokinase genes, and 13 pyruvate kinase genes. In A2 vs. B2, 87% of the 54 genes were downregulated, including seven hexokinase genes, 27 phosphofructokinase genes, and 13 pyruvate kinase genes.
Glycolytic pathway (EMP) map and heatmap of hexokinase (HK), ATP-dependent phosphofructokinase (PFK), and pyruvate kinase (PK) genes screened from transcriptome sequencing results in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) in 2020. A1 and B1 represent the stage of Ca treatment and control at 63 d after anthesis, respectively. A2 and B2 represent the stage of Ca treatment and control at 69 d after anthesis, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Glycolytic pathway (EMP) map and heatmap of hexokinase (HK), ATP-dependent phosphofructokinase (PFK), and pyruvate kinase (PK) genes screened from transcriptome sequencing results in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) in 2020. A1 and B1 represent the stage of Ca treatment and control at 63 d after anthesis, respectively. A2 and B2 represent the stage of Ca treatment and control at 69 d after anthesis, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Glycolytic pathway (EMP) map and heatmap of hexokinase (HK), ATP-dependent phosphofructokinase (PFK), and pyruvate kinase (PK) genes screened from transcriptome sequencing results in ‘Feizixiao’ litchi pulp of Ca treatment (foliar spray application of 0.3% CaCl2 aqueous solution) and control (foliar spray application of water) in 2020. A1 and B1 represent the stage of Ca treatment and control at 63 d after anthesis, respectively. A2 and B2 represent the stage of Ca treatment and control at 69 d after anthesis, respectively.
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Real-time PCR verification.
Using three samples of A2 as templates, 10 genes were randomly selected for real-time PCR verification, and the results are shown in Fig. 8A and B. The transcriptomic data had a significant linear relationship with the real-time PCR results in 2020 (r = 0.9292) and 2021 (r = 0.8889), demonstrating the reliability of the transcriptomic results.
Correlation analysis between real-time PCR and transcriptome sequencing data of 10 genes selected in ‘Feizixiao’ litchi pulp in 2020 (A) and 2021 (B).
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Correlation analysis between real-time PCR and transcriptome sequencing data of 10 genes selected in ‘Feizixiao’ litchi pulp in 2020 (A) and 2021 (B).
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Correlation analysis between real-time PCR and transcriptome sequencing data of 10 genes selected in ‘Feizixiao’ litchi pulp in 2020 (A) and 2021 (B).
Citation: Journal of the American Society for Horticultural Science 148, 1; 10.21273/JASHS05258-22
Discussion
Foliar calcium treatment inhibits EMP at the fully mature stage.
Calcium may reduce the catabolism of endogenous substrates by limiting the diffusion of substrates from the vacuole to the cytoplasm (Bangerth et al. 1972). As the main respiratory substrate, sugar is involved in ATP synthesis through EMP, the tricarboxylic acid (TCA) cycle, pentose phosphate, oxidative phosphorylation, and other pathways to provide energy for growth and development (Guo et al. 2022), and EMP-TCA is the main pathway of cellular respiration. The classic EMP is regulated by phosphofructokinase degrading glucose (Plaxton 1996). Previous studies have shown that upregulation of the expression of genes related to glycolysis, the TCA cycle, fermentation, and energy metabolism can reduce the content of soluble sugar in the pulp of ‘Shixia’ longan [Dimocarpus longan Lour. (Luo et al. 2021)]. In this study, in the treatment and control groups, it was found that the expression of the phosphofructokinase genes in EMP changed significantly. During the fruit ripening stage, the soluble sugar content of the control decreased, and most of the phosphofructokinase genes were upregulated, resulting in “sugar receding”; the soluble sugar content in the calcium treatment increased, and the expression of most of the phosphofructokinase genes showed a trend of first increasing and then decreasing compared with that in the control. At 63 DAA, the soluble sugar content in the calcium treatment was lower than that in the control, and the expression of most phosphofructokinase genes was higher than that in the control. The soluble sugar content in the calcium treatment was higher than that in the control, and the expression of most phosphofructokinase genes was lower than that in the control at the fully mature stage. The soluble sugar content and the expression of phosphofructokinase genes showed the opposite trend, such that the phosphofructokinase gene was the key gene affecting the soluble sugar content of ‘Feizixiao’ litchi pulp.
Foliar calcium fertilizer increases the net activity of sucrose-metabolizing enzymes and reduces the activity of phosphofructokinase.
Previous studies have found that calcium fertilizer can increase the activities of sucrose metabolism–related enzymes in peanut (Arachis hypogaea L.) leaves (Lin et al. 2020) and melon [C. melo (Li et al. 2011)], thereby improving their quality, and this study produced similar results. At 63 DAA, the sucrose content in the calcium treatment was significantly or extremely significantly lower than that in the control, and the net activity of sucrose-metabolizing enzymes was significantly higher than that in the control. In 2020, the INV activity in the calcium treatment was significantly higher than that in the control, and the SS-S activity was significantly lower than that in the control. In 2021, the NI and SS-C activities in the calcium treatment were significantly higher than those in the control, and the SS-S activity was significantly lower than that in the control.
The reducing sugar content in the calcium treatment was significantly higher than that in the control, the sucrose content was comparable, and the net activity of sucrose-metabolizing enzymes was significantly higher than that in the control at the fully mature stage. In 2020, the AI and SS-C activities in the calcium treatment were significantly higher than those in the control. In 2021, the NI and SS-C activities in the calcium treatment were significantly higher than those in the control.
From these results, it can be inferred that calcium treatment mainly increased the net activity of sucrose-metabolizing enzymes by increasing the NI activity and decreasing the SS-S activity at 63 DAA, thereby promoting the decomposition of sucrose, resulting in a lower content than in the control. When the fruit was fully ripe, calcium treatment mainly increased the net activity of sucrose-metabolizing enzymes by increasing SS-C activity and simultaneously reducing PFK activity to inhibit EMP, thereby promoting the decomposition of sucrose and the accumulation of reducing sugars and soluble sugars.
Conclusions
When the fruit was fully ripe, calcium treatment increased the net activity of sucrose-metabolizing enzymes by increasing SS-C activity, thereby promoting the accumulation of reducing sugars. At the same time, downregulating the expression of the phosphofructokinase genes in EMP reduced PFK activity, thereby reducing the consumption of respiratory substrates and promoting the accumulation of sugar. Finally, the pulp in the calcium treatment had a high soluble sugar content.
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