3.2.1 Exogenous application of methanol stimulates AsA biosynthesis in Oncidium PLB cultures
To study the effect of the MeOH dosage on AsA biosynthesis in Oncidium, 10-500 mM MeOH was applied exogenously to Oncidium PLB culture. The endogenous AsA level in tissues was measured at 6, 12, 24 and 30 h after MeOH application. As shown in Fig. 11, application of MeOH resulted in varied AsA levels in the PLB cultures. In general, the AsA level preferentially decreased during the first 6 h of incubation then showed an irreversible response to various concentrations of MeOH. Notably, the PLB culture was lethally affected by 500 mM MeOH (Fig. 12), and the AsA level was markedly decreased. Upon treatment with 50 mM MeOH, the AsA level of the Oncidium PLB culture increased following 24 h of inoculation. Thus, a 50 mM MeOH concentration was concluded to be appropriate for signaling AsA biosynthesis in Oncidium.
3.2.2 Characterization of AsA induction by MeOH stimulation
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To unravel the mechanism of AsA induction after 50 mM MeOH stimulation, several AsA-inducing compounds (Davey et al., 1999), such as D-galacturonate (D-GalUA) and L-galactose (L-Gal), were applied to the PLB culture and their effects were compared (Fig. 13, 14). The AsA levels in the PLB culture increased by MeOH (50 mM), D-GalUA (50 mM), and L-Gal (50 mM) treatment (Fig. 13); however, treatment of MeOH alone significantly decreased the AsA level during the first 6 h of
inoculation. Interestingly, assays of the AsA redox state (reduced form AsA / oxidized form AsA) in the PLB culture showed a similar pattern to that of AsA level (Fig. 14).
The distinct variation of the AsA profile with MeOH application suggests that MeOH is deleterious to Oncidium cells. The level of H2O2 significantly increased from 27.8 to 39.1 µM with MeOH application during the first 6 h of treatment (Table 1), whereas no significant effects were observed by the other chemicals, such as L-Gal and D-GalUA. Detoxification of MeOH during the first 6 h of treatment is important for the up-regulation of AsA-related genes. Moreover, MeOH has a distinct effect of H2O2 generation when applying to Oncidium culture.
3.2.3 Methanol enhances AsA levels by up-regulating AsA-biosynthesis and defense genes
Since the application of 50 mM MeOH to Oncidium PLB culture was effective in elevating the AsA level (Fig. 13), we investigated the effect of 50 mM MeOH on the expression level of AsA-biosynthetic genes in the GalUA pathway, such as polygalacturonase (OgPG), pectin methylesterase (OgPME) and galacturonate reductase (OgGalUAR; EC 1.1.1.19), as well as those in the Smirnoff-Wheeler pathway, such as GDP-D-mannose pyrophosphorylase (OgGMP) and galactose dehydrogenase (OgGalDH; EC:1.1.1.122). The RT-PCR data showed that OgPG and OgPME, which are involved in pectin degradation, were both up-regulated after 6 h of
MeOH treatment. However, the expression of OgPME was decreased at 24 h. In contrast, no further changes in the expression level of OgGalUAR by MeOH treatment were observed (Fig. 15). On the other hand, both OgGMP and OgGalDH of the Smirnoff-Wheeler pathway were up-regulated during the first 6 h of MeOH treatment, effects lasting for 30 h (Fig. 15). This is similar to the effect by L-Gal
routes (Fig. 15; Davey et al., 1999). Finally, the expression of galactono-1, 4-lactone dehydrogenase (OgGalLDH), an integrator of the AsA biosynthetic pathway, displayed an enhanced level upon MeOH treatment (Fig. 15). In addition, the levels of defense genes, including ascorbate peroxidase (OgAPX) and monodehydroascorbate reductase (OgMDHAR; EC: 1.6.5.4), were also increased at 6-24 h after MeOH treatment (Fig. 15). Taken together, a 50 mM MeOH treatment was effective to enhance the expression level of most AsA-related genes in the GalUA pathway, Smirnoff-Wheeler pathway and defense system.
To further understand the proteins associated with the AsA-related genes under 50 mM MeOH stimulation, their enzymatic activities were assayed. As shown in Fig.
16, the activities of OgPG, OgMDHAR, OgAPX and OgSOD were specifically enhanced from 6 to 12 h upon MeOH treatment, whereas other enzymes, such as OgGalUAR, OgGMP, OgGalDH, OgGalLDH, were not significantly enhanced by MeOH stimulation, even though they were enhanced in RNA levels. These results indicated that mRNA levels of many of these genes are not correlated with enzymatic activities, which may be related to post-translational modifications.
The pectin content of the Oncidium PLB culture was decreased in 50 mM MeOH treatment, but not in L-Gal or D-GalUA treatment (Table 1). The degradation appeared to result mainly from the elevated activity of OgPG under MeOH stimulation (Fig.
16). In conclusion, the AsA level was elevated in Oncidium PLB culture by MeOH stimulation, primarily because of the enhanced expression level and enzymatic activity of OgPG. Although the mRNA levels of a number of AsA-biosynthetic genes were certainly induced and enhanced, their functional contribution in AsA biosynthesis is unclear due to the absence of increased enzymatic activity with MeOH treatment.
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3.2.4 Hydrogen peroxideproduction in Oncidium PLB cultures through the
activation of alcohol oxidase and NADPH oxidase under MeOH stimulation
Plant cells are able to convert MeOH to formaldehyde and H2O2 by alcohol oxidase (Gout et al., 2000). The H2O2 level was elevated from 27.8 to 39.1 μM in Oncidium PLB cultures in response to exogenous application of MeOH, but not
D-GalUA and L-Gal, during the first 6 h of treatment (Table 1). To unravel the source of H2O2 production, we applied hydroxylamine (1 mM) and DPI (5 mM), inhibitors of alcohol oxidase and NADPH oxidase respectively, with MeOH in PLB cultures, and monitored the H2O2 levels. In Oncidium PLB cultures incubated with 50 mM MeOH, an early H2O2 burst (~45 μM) was detected during the first 30 min, followed by a subsequent decrease in accumulation (~40 μM) that lasted for another 6 h (Fig. 17).
However, this H2O2 burst was attenuated by incubation with 50 mM MeOH combined with 1 mM hydroxylamine or 5 mM DPI. The DPI inhibitor was more effective in blocking H2O2 generation than the alcohol oxidase inhibitor. NADPH oxidase could play a more significant role in the systemic production of H2O2 than alcohol oxidase does. Therefore, the stimulation of the H2O2 level by MeOH in the Oncidium culture occurs directly, through MeOH metabolism (or detoxification) by alcohol oxidase activity, and indirectly, through the subsequent induction of NADPH oxidase activity to amplify H2O2 production. Moreover, the early oxidative peak of the H2O2 level in the Oncidium culture could be largely due to the conversion of MeOH by alcohol oxidase, and the later H2O2 burst could result primarily from NADPH oxidase activation (Fig. 17).
H2O2 signal transduction
To confirm the potential signaling effects of H2O2 on the expression of AsA-related genes, we investigated the expression of AsA-related genes under the application of the inhibitors alone or with MeOH. As shown in Fig. 18, the expression of AsA-related genes did not change after 6 h with 1 mM hydroxylamine or 5 mM DPI treatment. However, the expressional levels of AsA-related genes were lower with MeOH combined with hydroxylamine or DPI than with MeOH alone.
Hydroxylamine and DPI inhibited H2O2 production (Fig. 17), consequently reducing the MeOH effect on the expression of AsA-related genes in Oncidium PLB cultures (Fig. 18). The results suggest that H2O2 signaling is critical in up-regulating the expression of AsA-related genes.