AsA and GSH concentrations in two tomato cultivars
Fully ripe tomato fruits have only ‘moderate’ totAsA-AsA concentrations
 compared to the fruits of other species such as blackcurrant
 and kiwifruit
. Under the standardised hydroponic greenhouse conditions used here, fruit AsA concentrations of ‘Santorini’ and ‘Ailsa Craig’ at the red ripe stage were 14.6 and 8.69 mg/100gFW, respectively, representing a 1.7-fold difference between the cultivars. This is identical to the differences observed between the same cultivars when grown under field conditions in Greece
, indicating that there is a strong and stable genetic basis for the regulation of the AsA pool size in tomato. However, the field-grown tomatoes had about 1.5-fold higher fruit AsA levels compared to greenhouse-grown tomatoes, presumably due to the higher levels of irradiance (and temperature) experienced in the field
. The differences in totAsA concentrations of red ripe fruits however were not as great (21.2 and 17.7 nmol/gFW in ‘Santorini’ and ‘Ailsa Craig’, respectively) due to the fact that the AsA pool in ‘Ailsa Craig’ contained a higher % DHA (i.e. it was more oxidised). This is possibly due to the lower activities of the enzymes involved in AsA recycling at this stage (Figure
On average, totAsA, AsA, and GSH concentrations were 1.8-, 2.4-, and 1.4-fold higher in ‘Santorini’ fruit than ‘Ailsa Craig’ throughout ripening. However, the concentrations of totGSH-GSH did not vary significantly during ripening. Since the time to ripening was similar in both cultivars and there were also no significant differences in fruit water contents it is unlikely that the changes are related to a metabolite ‘dilution’ effect (Additional file
1: Table S1). These differences therefore represent clear genetic differences in the capacity to accumulate fruit totAsA-AsA.
AsA and GSH accumulation in tomato fruits during ripening
The accumulation of totAsA during fruit ripening has been well studied in several plant species, and reports show that patterns vary according to the species and the cultivar studied, as well as growth conditions. For example in fruits of different tomato cultivars, totAsA concentrations were shown to both increase
[11, 18, 33, 34], or to remain essentially unchanged
 during ripening. The two cultivars used here also showed different patterns of fruit totAsA-AsA accumulation even under identical growth conditions. Nevertheless, one feature common to both cultivars was a characteristic spike in totAsA-AsA concentrations at the breaker stage, which proved useful to help understand the factors involved in regulating AsA concentrations during ripening. The breaker stage in tomatoes (when fruit colour first begins to change from green to red) marks the onset of ripening linked to the induction of ethylene–related metabolic pathways, and leads to a major shift in fruit metabolism. The biological basis for this spike in AsA (and GSH) concentrations is unclear, but an increase in H2O2 accumulation at the breaker stage has previously been reported in tomatoes
. While H2O2 levels were not measured here, the enhancement of the size of the totAsA-AsA pool could be indicative of an adaptive response to such an increase in H2O2 concentrations.
In ‘Ailsa Craig’, the breaker stage was associated with a 15% decrease in % DHA and a 48% decrease in % GSSG, due to an increase in both AsA and GSH concentrations. Taken together with the 56% increase in fruit MDHAR activity in ‘Ailsa Craig’ fruits relative to the B-1 stage, these results suggests that the enhanced capacity for AsA recycling could be linked to the observed increases in AsA and GSH around these stages. Unfortunately we do not have data on changes in the AsA biosynthetic capacity (substrate incubations) at this specific time point as there were insufficient fruit to carry out all incubations. However, similar results were observed in ‘Santorini’, where a 65% increase in MDHAR activity was correlated with an increase in the AsA pool at the breaker stage. In fact, MDHAR was the only enzyme whose activity was associated with the increase in fruit totAsA-AsA levels of both cultivars at the breaker stage, and although activity results represent the mean activities of all subcellular isoforms, the results are supported by changes in the expression of SlMDHARs in ‘Ailsa Craig’ – see discussion here under.
Previous work on GSH levels in tomato fruits has shown that concentrations increase during the later stages of ripening, but that changes in GSH are not correlated with changes in AsA
. Our results support these conclusions, and we see that both totGSH and GSH concentration profiles differ from the totAsA-AsA profiles during ripening in both cultivars, suggesting that these antioxidant pools are differently regulated.
AsA uptake, biosynthesis and recycling in tomato fruits
Earlier studies in foliar cell suspension cultures indicate that the preferred uptake form of AsA is as the oxidized form, DHA
[13, 36]. Our results with young tomato fruits of both cultivars support this, but by the end of ripening at the red stage there was a clear preference for AsA, which increased the size of the totAsA pool 3.3-fold more than incubation with DHA in ‘Santorini’, and 1.4-fold more in ‘Ailsa Craig’. These different responses of the cultivars may be related to the less oxidized redox status of the totAsA pool in ‘Santorini’ ripe fruits, or differences in the energetic requirements and capacity for transport of external AsA/DHA as reported in bean seedlings
The ability of fruit tissues to synthesize AsA de novo has been demonstrated by substrate feeding experiments in several fruit species, including courgette
[28, 39], and blackcurrant
. Here, the highest accumulation of intercellular totAsA-AsA in both tomato cultivars took place following incubations of fruit discs with L-Gal or L-GaL. This is to be expected as these are the substrates for the last two enzymatic steps of the L-galactose pathway (Figure
1). The results also show that when supplied with an external carbon source, substrates of the L-Gal pathway are more effective than precursors of alternative biosynthetic routes at increasing intercellular totAsA-AsA concentrations. Nonetheless, incubations with L-GuL or MI were also able to increase totAsA levels of MG and red fruits, respectively, but only in the low-AsA cultivar ‘Ailsa Craig’. Incubations with L-GuL have previously been reported to effectively stimulate AsA levels in Arabidopsis leaf suspension cultures
 and in apple fruits
, while overexpressing myo-inositol oxygenase in Arabidopsis, the gene encoding the enzyme responsible for the conversion of MI to D-glucuronate (Figure
1), supports a role for MI as a potential precursor of AsA, at least in foliar tissues
. In agreement with previous reports in apple
, incubations with D-glucuronolactone (D-GlcUL) did not increase fruit totAsA levels at any ripening stage in either cultivar, however little is known about the efficiency of uptake or feedback inhibition effects of this substrate. Therefore, the existence or functionality of alternative AsA biosynthetic pathways seems to be cultivar and/or developmental stage-specific.
Young fruits (IG, MG) of both cultivars displayed higher AsA biosynthetic capacities than mature fruits, presumably to help support higher rates of cellular metabolism during the stages of fruit cell division and expansion. Despite this, red ripe fruits had higher AsA concentrations (Figure
2B). Similarly, despite having lower totAsA-AsA concentrations, ‘Ailsa Craig’ fruits had a higher AsA biosynthetic capacity than ‘Santorini’. Therefore biosynthetic capacity is not related to totAsA-AsA concentrations. The lower biosynthetic capacities of the high versus the low-AsA cultivar may represent the results of a feedback inhibition of AsA on AsA biosynthesis in ‘Santorini’ fruits, or could be due to a lower demand for AsA in these ‘Santorini’ fruits, or differences in uptake capacities. Feedback regulation of AsA pool has been previously observed in spinach
, as well as in pea seedlings
, where the activity of GLDH, the last enzyme in the L-galactose biosynthetic pathway, was competitively inhibited by increased AsA levels.
Since differences in the rate of AsA biosynthesis do not correlate with cultivar-specific differences in fruit totAsA-AsA concentrations, or the changes observed during ripening, the accumulation of totAsA-AsA is possibly due to differences in the rate of AsA turnover and AsA recycling. Indeed, our results indicate that both of these mechanisms significantly influence the size of the fruit totAsA pool. For example, the % turnover of the AsA pool was significantly higher (2-fold) in young (low AsA) compared to the red (high AsA) fruits in ‘Ailsa Craig’, and 1.7-fold lower in ‘Santorini’ young fruits compared to ‘Ailsa Craig’ fruits at the same developmental stage. Nevertheless, the actual turnover rate was higher in ‘Santorini’ fruits, again indicating a possible link between the size and the rate of turnover of the AsA pool
. On the other hand, totAsA pool is smaller and more oxidized in ‘Ailsa Craig’ (Figure
2C) which is also the cultivar with a lower capacity for AsA recycling (Figure
5). Interestingly, of all the enzymes measured, it is the enhanced MDHAR activity that is associated with the increased totAsA–AsA concentrations measured at the breaker stage in both cultivars.
To summarize this section therefore, AsA biosynthetic capacities are higher early in fruit development, even though concentrations of AsA (in both cultivars) and totAsA (only in ‘Ailsa Craig’) are higher in the later stages of ripening. The lack of accumulation of totAsA-AsA at IG fruits is due to higher rates of turnover of the AsA pool. The two cultivars also differ in net de novo biosynthetic capacities, with the ‘low-AsA’ ‘Ailsa Craig’ actually displaying a higher biosynthetic capacity. Again, this is compensated for by a higher % turnover of the AsA pool early in fruit development, and a lower rate of AsA recycling, as manifested by a more oxidized redox status compared to ‘Santorini’.
Candidate genes for AsA regulation throughout ripening
Similar to results in kiwi
 and tomato
, we found high relative expression of most of the biosynthetic genes from the L-galactose pathway early in fruit development (Figure
6, group 2 and 3). This correlates with the higher biosynthetic capacities measured and may reflect increased requirements of AsA for cell division/expansion
. In a previous study, the enhanced expression of GPP, as monitored by northern blot analysis, has been correlated with increased AsA concentrations during the later stages of the ripening process in ‘Ailsa Craig’ fruits
. However, no such correlation between GPP expression and AsA levels was observed by us during ‘Ailsa Craig’ fruit ripening. In fact of all genes examined, only the relative expression patterns of the ‘group 4’ genes (SlGGP1, SlMDHAR1 and SlMDHAR3) (Figure
6) were strongly correlated (P < 0.001) with the changes in fruit totAsA-AsA levels around the breaker stage, with a delay of one day (Additional file
6: Table S6). A similar delay between gene expression and changes in AsA concentrations was noticed in tomato leaves
. Therefore these three genes seem to represent good candidates for the regulation of fruit AsA concentrations. Significant correlations were also found between the expression of SlGMP2 and SlGGP2 and AsA concentrations around this stage (P < 0.05), but not with totAsA concentrations (Additional file
6: Table S6), indicating that these transcripts may also play a role in regulating the size of the reduced AsA pool.
GGP (VTC2) represents the first committed step of AsA biosynthesis, and has previously been suggested to be a key rate-limiting step in plants
[5, 9, 10, 43]. Recent results from our lab indicate that paralogues of apple GGP are key regulators of totAsA-AsA concentrations in fruits
, and others have shown that ectopic expression of the single kiwifruit GGP orthologue results in enhanced totAsA contents in both transgenic tomato (3- to 6-fold increase) and strawberry (2-fold increase)
. Therefore, our results here support a central role for GGP in the regulation of fruit AsA pool in fruits of several different plant species. In addition, although we cannot exclude the possibility that other genes may play a role in regulating fruit AsA pool size in the high AsA cultivar during ripening, differences in SlGGP1 expression could also help to explain the differences in fruit totAsA-AsA concentrations between cultivars, as expression levels were twice as high in red ripe fruits of ‘Santorini’ compared to the low-AsA cultivar, ‘Ailsa Craig’ (Table
3). These results should however be further validated by testing SlGGP1 expression in fruits of a larger collection of tomato cultivars with a wider range of totAsA-AsA concentrations.
On the basis of Quantitative Trait Loci (QTL) studies in several tomato populations, Stevens and co-authors have proposed a role for an orthologue of MDHAR, as a candidate gene for the genetic control of AsA in tomato fruits
[22, 23]. This candidate gene corresponds to SlMDHAR2 (SGN-U573751), but gene expression profiles here do not support this conclusion, and others have shown that overexpression of SlMDHAR2 in tomato can even lead to a moderate decrease in AsA levels of MG fruits
. However, MDHAR activity is well correlated with AsA concentration in ‘Santorini’ fruits during ripening (Additional file
4: Table S4), and is linked to the short-term changes in totAsA-AsA around the breaker stage in ‘Ailsa Craig’ fruits (Figure
5), while expression of the other two MDHAR orthologues, SlMDHAR1 (SGN-U583672) and SlMDHAR3 (SGN-U584073), do closely mirror changes in totAsA-AsA concentrations in ‘Ailsa Craig’ fruits around this stage (Figure
6). In contrast, expression levels of the two orthologues of the other main AsA-recycling gene DHAR, did not reflect changes in totAsA-AsA changes (Figure
Although the results presented here indicate a role for the SlMDHAR1 and SlMDHAR3 gene products in the control of AsA pool during ripening in ‘Ailsa Craig’, their relative expression levels did not differ significantly between the two cultivars at the red stage. Despite this, MDHAR enzyme activity was actually 1.6-fold higher in fruits of the high-AsA cultivar. This discrepancy between gene expression and enzyme activity is possibly related to the fact that activity results represent the mean activities of all subcellular isoforms, while expression results represent the expression of individual members of the gene family.