Comparison of ascorbic acid metabolism during the development of two jujube varieties

Seven-year-old trees Z. jujuba Mill. cv. 'Zhanshanmizao 1' (ZM1) and 7-year-old grafted trees Z. jujuba Mill. cv. 'Zhanshanpingguozao' (ZP) (the grafting rootstock is ZM1) with similar vigour and load (based on trunk cross-sectional area), which were grown in Zhanshan Village, Santai County, Mianyang City, Sichuan Province, were used for this study. The orchard and growing conditions are hilly terraces with yellowish reddish purple clay soil. The climate is subtropical monsoon, with an average annual temperature of 16.8°C, an average annual precipitation of 828 mm and a frost-free period of 291 days. Petals were collected during the first bloom period. Initial leaf sampling was conducted on 5-day-old foliage (from the fifth to seventh nodes of fruitbearing shoots). Subsequent collections were performed at 20-day intervals, covering foliage ages of 25, 45, 65 and 85 days. Fruits were collected at 10, 30, 50, 70 and 90 days after flowering (DAF). In total, 50 leaves and 30 fruits of uniform size were collected from different orientations at 10 a.m. Three replicates were included for each sampling. Samples were packed with ice and were transported to the laboratory immediately. First, the packed samples were counted and weighed. Next, they were frozen in liquid nitrogen. Then, they were stored at -80°C for further use. Three technical replicates were included for each analysis.

The AsA content was determined using HPLC method according to Huang et al. (2014), with some modifications. Exactly 0.5 g of sample was ground in 5 mL of 0.2% metaphosphoric acid solution. The mixture was then centrifuged at 8000 × g for 20 min at 4°C. The supernatant was diluted to a final volume of 10 mL, filtered through a 0.22 μm membrane and used for AsA detection. However, unlike AsA quantification, total ascorbic acid (T-AsA) quantification required the addition of 10 μL of fresh DTT (200 mM) solution to the supernatant. After being kept at room temperature for 4 h in the dark, the mixture was used for T-AsA quantification; a 15% methanol solution was used as the mobile phase. The separation was achieved by an Agilent 1260 MP-C18 column (250 mm × 4.6 mm, 5 μm). The flow rate was set to 0.5 mL ∙ min⁻¹, and the column temperature was maintained at 25°C. The AsA and T-AsA contents were quantified based on the detected peak areas at 243 nm by comparing with external standards. Dehydroascorbic acid (DHA) content was calculated by subtracting the AsA content from the T-AsA content.

The GalDH enzyme activity was assayed according to the method of Gatzek et al. (2002). Two grams of flesh was homogenised with 5 mL of 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 mM phenylmethanesulfonyl fluoride, 0.5% (v/v) Triton X-100, 0.2% (v/v) 2-mercaptoethanol and 2% (w/v) PVP, and then centrifuged at 12000 × g at 2°C for 20 min. The supernatants were collected to assay GalDH. One unit of GalDH activity was defined as the amount of enzyme causing an increase of 0.01 in the absorbance per min at 340 nm at 25°C.

The GalLDH enzyme activity was assayed according to the method of Gatzek et al. (2002), with some modifications. Exactly 0.5 g sample was ground in 8 mL of 0.1 mM phosphate buffer (containing 0.4M sucrose, 10% glycerin, 50 mM β-mercaptoethanol, 1 mM EDTA and 2% PVP, pH 7.4). The mixture was then centrifuged at 5000 × g at 4°C. After centrifugation, the supernatant was collected and further centrifuged at 12000 × g at 4°C to obtain the pellet. The pellet was subsequently suspended in Tris-HCl buffer (containing 10% glycerin and 5 mM GSH). The reaction system contained 2 mL of 50 mM phosphate buffer (containing 1 mg/mL cytochrome c, pH 8.0), 0.4 mL of galactono-1,4-lactone and 0.1 mL of enzyme solution. The reaction mixture was pre-incubated at 27°C for 1 min. One unit of enzyme activity was defined as the amount of enzyme required to increase the absorbance by 0.01 per min at 550 nm.

The ascorbate oxidase (AO) enzyme activity was assayed according to the method of Esaka et al. (1990). The ascorbate peroxidase (APX) enzyme activity was assayed according to the method of De Pinto et al. (1999). One unit of AO and APX activities was defined as the amount of enzyme causing an increase of 0.01 in the absorbance per min at 265 nm and 290 nm at 25°C, respectively.

Total RNA was isolated from the petals, leaves and fruits of ZM1 and ZP at different developmental stages using the improved CTAB method. RNA quality was assessed by electrophoresis on a 1% agarose gel, and RNA concentration was determined. First-strand cDNA was synthesised using a PrimeScript strand synthesis Kit (Takara RR047A, China). The expression of genes related to AsA metabolism in ZM1 and ZP was analysed using qPCR at different developmental stages of petals, leaves and fruits. Quantitative real-time PCR (qRT-PCR) was performed using a SYBR Premix Ex Taq Kit on a Bio-Rad CFX96 instrument. ZjH3 was the reference gene. The PCR protocol comprised initial heating at 95°C for 3 min, followed by 39 cycles at 95°C for 5 s, 55°C for 30 s and 72°C for 10 s. Primer specificity was determined using qRT-PCR and melting-curve analysis. The 25 μL reaction system contained 2 × SYBR Premix ExTaq (12.5 μL), 10 μmol/L primers (0.5 μL of each), H₂O (10.5 μL) and diluted cDNA (1 μL). Gene expression levels were calculated using the formula 2^−ΔΔCT. The primers used for qPCR analysis are listed in Table S1 in Supplementary Materials.

The correlation analysis was conducted using Origin 2022 software. All results are expressed as mean ± standard deviation (n = 3), with differences considered significant at p < 0.05 and highly significant at p < 0.01, as determined by Student’s t-tests. At the same time, a one-way analysis of variance was performed on the fruits and leaves of the two varieties at different stages of development, with significant differences (Duncan’s multiple range test) assessed at the 5% confidence level.

The growth dynamics of ZM1 and ZP are shown in Figure 2. Overall, the results showed that the change patterns of AsA and T-AsA content (Figure 3A) during the development of ZM1 and ZP fruits were consistent, showing an initial increase followed by a decrease. A sharp increase was observed from 10 DAF to 30 DAF, with AsA and T-AsA content in ZP being higher than that in ZM1. Both reached their maximum values at 50 DAF. However, the maximum content in ZM1 was significantly higher than that of ZP by 18.35% and 16.96%, and thereafter, a sharp decrease was observed in both from 50 DAF to 70 DAF. Although the DHA content (Figure 3A) generally increased during fruit development, the pattern of increase differed between ZM1 and ZP. Both ZM1 and ZP showed the same increasing trend from 10 DAF to 50 DAF. Nevertheless, the turning point occurred at 50 DAF, with a slight increase observed in ZP from 50 DAF to 90 DAF, increasing by 22.20%. In ZM1, the trend showed a sharp decrease followed by an increase. AsA/DHA (Figure 3A) reflects the degree of redox of AsA; the higher the ratio, the more favourable the accumulation of AsA. At the 10 DAF, the ratio in ZM1 was higher than that in ZP by 196.77%. However, at 30 DAF, the ratio was exactly the opposite. After 50 DAF, the ratio in ZM1 was higher than that in ZP by 13.73%.

Figure 2.

Fruit developmental stages of two jujube varieties. DAF, days after flowering.

Figure 3.

Changes in AsA, DHA, T-AsA contents and AsA/DHA ratio during the development of different tissues in two jujube varieties. (A) Changes in AsA, DHA, T-AsA contents and AsA/DHA ratio during fruit development of two jujube varieties. (B) Changes in AsA, DHA, T-AsA contents and AsA/DHA ratio during leaf development of two jujube varieties. (C) Comparison of AsA, DHA, T-AsA contents and AsA/DHA ratio in the petals of two jujube varieties. Note: * and ** indicate independent samples t-test, which analyse the differences between ZM1 and ZP in direct quantitative data at the same developmental stage. * indicates a significant difference (0.01 < p* < 0.05), ** indicates a highly significant difference (**p < 0.01); a, b, c, etc. represent one-way analysis of variance between different developmental stages of two varieties, with significant differences (Duncan’s multiple range test) assessed at the 5% confidence level. AsA, ascorbic acid; DHA, dehydroascorbic acid; T-AsA, total ascorbic acid; ZM1, 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

As shown in Figure 3B, the DHA and T-AsA contents in ZM1 were significantly higher than those in ZP from 5 days to 65 days, and the AsA contents in ZM1 were significantly higher than those in ZP by 560.77%. At 85 days, the AsA, DHA and T-AsA contents in ZP were higher than those in ZM1. Although there was an overall increasing trend in AsA content during leaf development, the pattern of increase differed between ZM1 and ZP. From 5 days to 45 days, a gradual increase occurred in ZM1 and ZP. However, a sharp increase was observed from 45 days to 85 days, followed by a sharp decrease in ZM1. On the contrary, the AsA content in ZP decreased from 45 days to 85 days, followed by a sharp decrease thereafter.

According to the results (Figure 3C), petals also contain AsA, but the content was significantly lower than that in the leaves and fruits. The AsA content in ZP was higher than that in ZM1 by 311.86%. The DHA content in ZM1 was higher than that in ZP by 64.40%, but there was no significant difference between the two in T-AsA content. The noteworthy point was that the AsA/DHA ratio in ZP was much higher than that in ZM1 by 588.95%.

Changes in the expression of critical AsA biosynthetic genes involved in the L-galactose pathway, as well as the activities of corresponding enzymes during fruit development, were analysed in two varieties with significantly different AsA content (Figure 4A). In this pathway, an overall increase in GalLDH activity was observed, but a decrease occurred at 70 days in both species, decreasing by 244.97% and 45.94% respectively, compared with 50 DAF. The AsA content in the fruits of both varieties decreased sharply from 50 days to 70 days, and the changes in AsA content corresponded to the changes in enzyme activity. GalDH activity exhibited different changes in different varieties. In ZM1, it increased from 10 DAF to 50 DAF, representing a significant increase of 1056.59%, and then decreased from 50 DAF to 90 DAF, while in ZP, it increased from 10 DAF to 70 DAF, showing a sharp increase from 50 DAF to 70 DAF, followed by a relatively stable level in the later stage. The expression levels of GME and GME1 demonstrated similar trends in both varieties (Figure 5). The expression levels of GMP and GMP1 displayed similar trends (Figure 5). In both varieties, the expression levels of GGP, GalDH and GalLDH showed a significant increase during fruit development (Figure 5). The expression levels of the ZM1 gene increased by 15768.26%, 16214.28% and 46688.17%, respectively, while the expression levels of the ZP gene increased by 14350.74%, 75644.64% and 137379.42%, respectively. GPP showed a relatively stable level from 10 DAF to 70 DAF, followed by a sharp increase and subsequent decrease in the later stages in ZM1. By contrast, the expression levels of GPP demonstrated a gradual increase from 10 DAF to 50 DAF and then gradually decreased from 50 DAF to 90 DAF in ZM1.

Figure 4.

Changes in the activities of AsA-related enzymes during the development of different tissues in two jujube varieties. (A) Changes in the activities of AsA-related enzymes during fruit development of two jujube varieties. (B) Changes in the activities of AsA-related enzymes during leaf development of two jujube varieties. (C) Comparison of AsA-related enzyme activities in the petals of two jujube varieties. Note: * and ** indicate independent samples t-test, which analyse the differences between ZM1 and ZP in direct quantitative data at the same developmental stage. * indicates a significant difference (**p < 0.01), ** indicates a highly significant difference (0.01 < p* < 0.05); a, b, c, etc. represent oneway analysis of variance between different developmental stages of two varieties, with significant differences (Duncan’s multiple range test) assessed at the 5% confidence level. AO, ascorbate oxidase; APX, ascorbate peroxidase; AsA, ascorbic acid; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; ZM1, 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

Figure 5.

Relative expression levels of genes involved in AsA metabolism during fruit development of two jujube varieties. Note: * and ** indicate independent samples t-test, which analyse the differences between ZM1 and ZP in direct quantitative data at the same developmental stage. * indicates a significant difference (0.01 < p* < 0.05), ** indicates a highly significant difference (**p < 0.01); a, b, c, etc. represent one-way analysis of variance between different developmental stages of two varieties, with significant differences (Duncan’s multiple range test) assessed at the 5% confidence level. AO, ascorbate oxidase; APX, ascorbate peroxidase; AsA, ascorbic acid; DHAR, dehydroascorbate reductase; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GGP, GDP-L-galactose phosphorylase; GME, GDP-D-mannose 3',5'-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1-P phosphatase; MDHAR, monodehydroascorbate reductase; ZM1, 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

APX and AO are important degrading enzymes of plant AsA. As shown in Figure 4, AO activity displayed similar changes in ZM1 and ZP. It was highest in the first stage, sharply decreased at 30 DAF and then decreased significantly by 147.64% and 762.01%, respectively. Then it gradually maintained during the latter stage. In addition, APX activity exhibited similar changes in different varieties. It increased in the first stage, increased from 30 DAF to 70 DAF and then decreased significantly during the latter stage by 389.63% and 415.47%. AO showed the same trend in ZM1 and ZP, respectively (Figure 5). It reached the lowest level at 10 DAF, gradually increased from 10 DAF to 50 DAF, significantly increased by 857.98% and 2342.01%, respectively, and then sharply decreased. Eventually, it increased sharply from 70 DAF to 90 DAF and increased significantly by 287.72% and 1863.02%, respectively. The expression of APX showed a pattern of continuous increase during the ZM1 fruit development, significantly increased by 78044.47%, while it showed a different trend in ZP (Figure 5). In ZP, APX expression increased during the former stage, with a significant increase of 15658.65%, decreased from 50 DAF to 70 DAF and then sharply increased during the latter stage by 909.10%, peaking at 90 DAF. The expression of MDHAR in ZM1 and ZP showed a gradual increase and increased significantly by 8061.03% and 7983.09%, respectively. But the expression of DHAR exhibited different changes in ZM1 and ZP (Figure 5). In ZM1, the expression of DHAR generally increased from 10 DAF to 30 DAF, while a decrease was observed at 50 DAF, followed by an increase during the latter stage. In addition, the expression of DHAR generally increased from 10 DAF to 70 DAF, and a sharp increase occurred from 70 DAF to 90 DAF by 1608.90% in ZP.

As shown in Figure 4B, the activity of GalLDH showed less variability during the leaf growth in ZM1 and ZP, but GalDH activity exhibited different changes in different varieties. In ZM1, it peaked at 25 days and decreased continuously, while in ZP, it decreased sharply, then increased and finally decreased during the latter stage. GalDH activity decreased by 1660.31% at 85 days compared with 10 days. GalDH activity was not consistent with changes in ZM1 and ZP AsA content, respectively. The expression levels of GME, GME1, GMP, GMP1, GGP, GPP, GalDH and GalLDH demonstrated similar changes in ZP (Figure 6). They showed low expression levels during the former stage, increased sharply at 45 days, then decreased and finally increased sharply at 85 days by 1842.71%, 334.69%, 3839.66%, 2428.13%, 1456.25%, 408.65%, 2297.29% and 8286.52%. The expression levels were not in agreement with the changes in AsA content. GME, GME1, GMP, and GGP showed low expression levels during ZM1 leaf growth. The expression of GGP peaked at 25 days, then decreased sharply. They were almost not expressed at 85 days. The expression of GMP1 and GalLDH peaked at 65 days, in contrast to the changes in GalDH expression, but in agreement with the changes in AsA content during leaf growth, suggesting that these genes might be the key limiting factors involved in AsA biosynthesis in jujube.

Figure 6.

Relative expression levels of genes involved in AsA metabolism during leaf development of two jujube varieties. Note: * and ** indicate independent samples t-test, which analyse the differences between ZM1 and ZP in direct quantitative data at the same developmental stage. * indicates a significant difference (0.01 < p* < 0.05), ** indicates a highly significant difference (**p < 0.01); a, b, c, etc. represent one-way analysis of variance between different developmental stages of two varieties, with significant differences (Duncan’s multiple range test) assessed at the 5% confidence level. AO, ascorbate oxidase; APX, ascorbate peroxidase; AsA, ascorbic acid; DHAR, dehydroascorbate reductase; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GGP, GDP-L-galactose phosphorylase; GME, GDP-D-mannose 3',5'-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1-P phosphatase; MDHAR, monodehydroascorbate reductase; ZM1, 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

APX activity sharply decreased from 5 days to 25 days by 28.17% and 694.55%, then increased at 45 days and decreased during the later stages in ZM1 and ZP, with similar trends (Figure 4B). It peaked at 45 days in ZM1, while in ZP, it peaked at 5 days. Both AO and APX reached their lowest levels at 85 days (Figure 4B). This result indicated that jujube leaf senescence might be associated with APX activity.

The expression of AO showed different changes in ZM1 and ZP (Figure 6). In ZP, it increased sharply, with a significant increase of 1865.29% and peaked at 45 days, then decreased sharply at 65 days, and increased again during the later stage. By contrast, in ZM1, AO expression increased in the first stage, then decreased at 45 days, gradually increased afterwards and finally peaked at 85 days. A similar expression pattern of APX was observed during ZP leaf growth, while it was rarely detected in ZM1. In ZP, the expression of DHAR was higher at 45 days and 85 days, which was consistent with the AsA content. In ZM1, the expression of DHAR was higher at 65 days, also in agreement with AsA content. These results indicated that DHAR might be the key gene involved in the regeneration of AsA in both ZM1 and ZP. The expression of MDHAR was rarely detected in ZM1. However, in ZP, MDHAR expression was lower in the early stages until 25 days, then increased sharply and peaked at 70 days.

As shown in Figure 4C, the comparison of AsA enzyme activities in the petals of different varieties revealed that both GalDH and GalLDH activities were lower, which may explain the low AsA content in the petals. Compared with ZM1, the petals of ZP exhibited higher GalDH activity but lower GalLDH activity. GME, GME1, GMP and GMP1 showed lower expression levels in ZM1 and ZP, respectively (Figure 7), which corresponded to the lowest levels of their AsA. Notably, GalLDH had higher expression in both ZM1 and ZP (Figure 7). Additionally, GGP expression was detected at a high level in ZP but was rarely detected in ZM1.

Figure 7.

Comparison of the expression of AsA metabolic genes in petals of two jujube varieties. Note: * and ** indicate independent samples t-test, which analyse the differences between ZM1 and ZP in direct quantitative data at the same developmental stage. * indicates a significant difference (0.01 < p* < 0.05), ** indicates a highly significant difference (**p < 0.01). AO, ascorbate oxidase; APX, ascorbate peroxidase; AsA, ascorbic acid; DHAR, dehydroascorbate reductase; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GGP, GDP-L-galactose phosphorylase; GME, GDP-D-mannose 3',5'-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1-P phosphatase; MDHAR, monodehydroascorbate reductase; ZM1: 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

APX and AO showed higher activity in ZM1 and ZP, respectively (Figure 4C). Their activities were consistent with the highest T-AsA content. The expression level of AO was lower in jujube petals. Compared with ZM1, the petals of ZP exhibited higher expression of DHAR and MDHAR.

To deeply explore the mechanism of AsA accumulation in ZM1 and ZP, the correlation between AsA content, biosynthetic enzyme activities and gene expression was analysed in fruits of different varieties with contrasting AsA content. As shown in Figure 8A, in ZM1, the expression levels of AsA-related genes were not significantly correlated with the AsA pool, except for the GME, GME1 and GGP, which showed a positive correlation with the AsA pool, respectively. Similarly, GMP1 displayed a positive correlation with DHA. The activities of APX, GalDH and GalLDH were positively correlated with the AsA pool, whereas AO activity was negatively correlated with the AsA pool. On the contrary, in ZP, the expression levels of AsA-related genes were positively correlated with AsA, DHA and T-AsA, but not significantly correlated with AsA/DHA ratio, except for GPP, which was negatively correlated with the AsA pool. APX activity was negatively correlated with the AsA pool, and the activity of AO was negatively correlated with DHA and T-AsA. GalDH activity was significantly correlated with AsA, DHA and T-AsA. However, GalLDH activity was not significantly correlated with the AsA pool.

Figure 8.

Correlation analysis of AsA accumulation with metabolic enzyme activities and gene expression in two jujube varieties. (A) Correlation analysis of AsA accumulation with metabolic enzyme activities and gene expression during fruit development in two jujube varieties. (B) Correlation analysis of AsA accumulation with metabolic enzyme activities and gene expression during leaf development in two jujube varieties. AO, Ascorbate oxidase; APX, ascorbate peroxidase; AsA, ascorbic acid; AsA, ascorbic acid; DHA, dehydroascorbic acid; DHAR, dehydroascorbate reductase; GalDH, L-galactose dehydrogenase; GalDH, L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GGP, GDP-L-galactose phosphorylase; GME, GDP-D-mannose 3',5'-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1-P phosphatase; MDHAR, monodehydroascorbate reductase; T-AsA, total ascorbic acid; ZM1, 'Zhanshanmizao 1'; ZP, 'Zhanshanpingguozao'.

During the leaf growth (Figure 8A), in ZM1, the expression levels of GME, GME1, GMP1, GalLDH and DHAR were positively correlated with the AsA pool, whereas the expression levels of GPP, GGP and APX were negatively correlated with the AsA pool. The expression levels of GMP, GalDH and MDHAR were not significantly correlated with the AsA pool. The activity of APX was negatively correlated with the AsA pool, whereas the activity of AO was positively correlated with the AsA pool. GalDH and GalLDH were not correlated with the AsA pool. Meanwhile, in ZP, the expression levels of all the genes were correlated with AsA, DHA and T-AsA, but none of them were significantly correlated with the AsA/DHA ratio. The activity of AO was not significantly correlated with the AsA pool. The activities of APX and GalDH were negatively correlated with AsA, DHA and T-AsA, whereas the activity of GalLDH was positively correlated with AsA, DHA and T-AsA. Apart from APX and AO, the activities of GalDH and GalLDH were not significantly correlated with the AsA/DHA ratio.

AsA content can vary significantly among species or cultivars, such as peaches (Imai et al., 2009), strawberries (Aragueez et al., 2013) and blackcurrants (Ioannidi et al., 2009). In this study, the AsA content in ZP fruit was higher than that in ZM1 fruit from 10 DAF to 30 DAF, while ZM1 fruit had higher AsA content than ZP fruit from 50 DAF to 90 DAF. Since the growing environment and harvesting times of ZM1 and ZP were similar, the differences in AsA content were likely genetically determined. The patterns of AsA accumulation vary among varieties. For example, during fruit development, the total AsA content remained unchanged or increased slightly in oranges (Alos et al., 2021), decreased in apples (Fang et al., 2017) and sweet cherry (Liang et al., 2017), and increased in grapes (Cruz-Rus et al., 2010) and chestnut rose (Huang et al., 2014). In this study, AsA content increased during the early stage of fruit development, peaked at the white mature stage and then gradually decreased with fruit ripening. This pattern suggests that rapid AsA accumulation during the early stage may promote fruit growth and that AsA accumulation mainly occurs during the fruit expansion stage. Our results were consistent with the findings for kiwifruit (Lin et al., 2022) and blueberry (Liu et al., 2015), but differed from those for northern fresh jujube (Chen, 2015).

The T-AsA content in the green photosynthetic tissues of species or cultivars is generally higher than that in fruits, such as apple (Li et al., 2010a), kiwifruit (Li et al., 2010b) and strawberry (Bulley et al., 2012). Nevertheless, in this study, the T-AsA content in the leaves of ZM1 and ZP was much lower than that in the fruit. The results were consistent with the findings for acerola (Badejo et al., 2007), Chinese jujube (Zhang et al., 2016) and Rosa roxburghii fruit (An et al., 2007; Yan et al., 2015). This result may be attributed to the transfer of AsA content from the leaves to the fruits. DHA content was higher than AsA content during the leaf growth, a finding consistent with studies in apple (Li et al., 2010a) and R. roxburghii fruit (Li et al., 2017). This result suggests that jujube leaves may contain high levels of oxidised substances. In R. roxburghii, AsA content remained at a high level with no significant change during the early stage of leaf development (Li et al., 2017). However, in this study, AsA content increased as the leaves matured and decreased when the leaves began to senesce, which is similar to the study of apple leaves (Li et al., 2010a). It suggests that the rate of AsA synthesis exceeds the rate of oxidation loss in young leaves, whereas oxidation loss exceeds biosynthesis in senescent leaves. Moreover, the change in AsA content in the leaves was more pronounced but significantly different from that in the fruits. The analysis of AsA and DHA content revealed that petals had the lowest of T-AsA levels, which were 120 times lower than the maximum levels detected in fruits. Interestingly, the AsA content in orange leaves differed from this study, with orange leaves showing the highest levels of T-AsA (Alos et al., 2021). The physiological reason for the accumulation of AsA in petals is not well understood. These findings suggest that each organ or tissue exhibits a distinct pattern of AsA accumulation during development.

The activities of the relevant metabolic enzymes showed different trends in ZM1 and ZP. In this study, GalDH activity correlated significantly with AsA content in ZM1 during fruit development, while in ZP, it was continuous throughout fruit development. The result indicated that GalDH plays a significant role in the AsA synthesis process. A study on ‘Jinsixiaozao’ (Wang et al., 2019) showed that GalLDH activity correlated with changes in AsA content. In this study, GalLDH activity was low during the early stage of fruit development and increased significantly with ripening in ZM1 and ZP, suggesting that GalLDH may play a role in synthesis at maturity. In the most widely accepted L-galactose pathway, GalDH and GalLDH are the key enzymes (Wheeler et al., 1998). In apple (Li et al., 2010a), changes in GalDH activity showed a strong correlation with AsA content in leaves of different ages. In this study, GalDH activity was negatively correlated with AsA content in ZP leaves, and AsA content remained low during leaf growth. Further investigation is necessary on the role of GalDH in controlling AsA synthesis via the L-galactose pathway. GalLDH activity was positively correlated with AsA in ZP leaves. As suggested by Ishikawa et al. (2007), GalLDH activity is strongly correlated with AsA content in developing tissues and cells. In addition, in ZM1, the activities of GalDH and GalLDH were not correlated with AsA content during leaf growth, suggesting that GalDH and GalLDH do not significantly affect AsA synthesis in ZM1 leaves. Young leaves exhibited much lower DHA content and much higher AO and APX activities compared with mature leaves, yet both had low AsA content. The results indicated that much of the AsA in young leaves may be oxidised and broken down, and mature leaves may contain higher levels of oxides. AO and APX activities were particularly low in aged leaves of ZP and ZM1, suggesting that leaf senescence may be associated with a decrease in APX and AO activities. GalDH and GalLDH activities were extremely low, while AO and APX activities were high in petals of ZP and ZM1. Meanwhile, ASA content was low, and DHA content was high. The results implied that AsA in petals is likely oxidised due to the high DHA/AO activity of ZP and ZM1.

AsA biosynthesis is recognised as the primary determinant of AsA accumulation in most plant varieties, though the predominant metabolic pathways vary among taxa. Current evidence indicates that in situ synthesis serves as the principal mechanism for AsA accumulation in numerous varieties, including blackcurrant (Ioannidi et al., 2009), kiwifruit (Li et al., 2010b) and strawberry (Duan et al., 2012). In this study of ZM1 and ZP, we identified eight cDNA sequences associated with the L-galactose pathway. The results of qRT-PCR showed that eight genes were expressed during the development of ZM1 and ZP. The expression patterns of GMP, GGP and GalLDH exhibited strong correlation with AsA accumulation patterns in both cultivars from 10 DAF to 50 DAF. The expression of GME and GME1 in ZM1 fruits showed ‘M’ type changes during development, but the general trend was first increasing and then decreasing, which was consistent with the changes. These findings align with previous studies demonstrating that GME1, GMP1, GGP, GPP and GalLDH function as key regulators of AsA biosynthesis during 'Junzao' fruit development, while GME2 and GMP2 appear particularly important during the ripening stages (Zhang et al., 2016). The expression of GMP1 in ZM1 fruits showed a general trend of increasing and then decreasing, but the expression of the gene increased from 50 days to 70 days. The reason for analysing may be that the GMP gene regulates the generation of mannose from D-mannose-1-phosphate, which is the substrate of the L-galactose pathway, and the elevated expression of the GMP1 gene synthesises more substrates of the L-galactose pathway, but the activity of the enzyme GalLDH decreased. Its gene expression is reduced, resulting in lower AsA content. Furthermore, GalLDH has been identified as the crucial gene controlling AsA production in 'Dongzao', 'Jinsixiaozao' and 'Zanhuangdazao' fruits (Chen, 2015). Collectively, these results strongly suggest that the L-galactose pathway represents the predominant route for AsA biosynthesis in southern jujube fruits, with GME1, GMP, GGP, GPP, GalDH and GalLDH serving as key regulatory genes. However, it is important to note that final AsA in fruits represents a complex balance between multiple processes, including not only biosynthesis but also catabolism, oxidation, reduction, and transport mechanisms (Ishikawa et al., 2007).

The results of this study showed that the expression of AO genes was low at the young fruit stage, whereas the results of the 'Junzao' study showed that the expression of most of the AO-type genes exhibited high expression during early fruit development. Similar studies have been documented in sweet orange (Alos et al., 2014), chilli pepper (Alos et al., 2013) and R. roxburghii Tratt (Huang et al., 2014). These discrepancies may reflect genotypic differences, environmental influences or the multifaceted roles of AO in physiological processes such as cell division and rapid fruit expansion (Sanmartin et al., 2007). Regarding the AsA regeneration pathway, our results demonstrated coordinated regulation by both MDHAR and DHAR in ZM1 and ZP fruits. In cherry (Eltelib et al., 2011), AsA accumulation was not correlated with DHAR, but in R. roxburghii Tratt (Huang et al., 2014), DHAR was the key gene for AsA accumulation, indicating that the key genes of the AsA regeneration pathway were different in different varieties. Genotypes and the strength of transcriptional regulation of key genes for AsA synthesis, degradation and regeneration vary by environment.

The key genes for AsA synthesis in leaves of different varieties differ. GPP is the key gene for AsA synthesis in apple leaves, overexpression of the GGP gene in kiwifruit led to a dramatic increase in AsA content, and co-expression of GGP and GME in Arabidopsis thaliana led to a seven-fold increase in AsA content. In this study, we found that the expression of GMP1 and GalLDH in ZM1 leaves was similar to the changes in their AsA content, suggesting that the L-galactose pathway plays an important role in the synthesis of AsA in southern fresh jujube leaves, and that GMP1 and GalLDH play a key role in the synthesis of AsA in ZM1 leaves. In ZP leaves, on the other hand, it seems that we could not find a single gene whose expression change was consistent with the change of its AsA content, which was similar to the study of R. roxburghii Tratt leaves (Huang et al., 2014), suggesting that some other pathways may be involved in the synthesis of AsA in jujube leaves, or post-transcriptional regulation may have occurred. In R. roxburghii Tratt leaves (Huang et al., 2014), MDHAR was the key gene of the cycling pathway, and APX was the main oxidase, and similar reports were also found in tomato (Stevens et al., 2008). The expression of AO in ZM1 leaves increased continuously, and the changes in the expression of DHAR were in line with the changes in the content of AsA, but the expression of APX and MDHAR was almost non-existent, which indicated that AO played a key role in the oxidation of AsA, and DHAR was the key gene of AsA regeneration in ZP leaves at 45 days and 85 days. These results suggest that the regeneration pathway plays an important role in the accumulation of AsA in fresh jujube leaves, but the key genes differed among genotypes.