3. Results and discussion
We showed previously that (i) d-lactate and MG negatively affect A. thaliana seedling development in a concentration-dependent manner and that (ii) d-ldh mutants i.e., plants lacking an active d-LDH, show lower tolerance than wild-type plants, indicating an impaired detoxification of these substances caused by the loss of d-LDH activity [5]. We therefore hypothesized that d-LDH overexpressing plants would be more resistant to these substances. To test this, we produced A. thaliana plants constitutively overexpressing d-LDH (35S::d-LDH plants). Seeds from wild-type as well as from 35S::D-LDH plants were germinated on MS plates in the presence of either MG (0.1–1 mM) or d-lactate (5–20 mM). In accordance with our hypothesis, the constitutive overexpression of d-LDH mitigated the negative effects on growth of MG up to 0.1 mM and of d-lactate up to 10 mM (Fig. 2). 35S::d-LDH overexpressing lines grew better than the wild type in the presence of MG up to 0.5 mM. At higher MG concentrations similar toxic effects were found in all genotypes, probably because glyoxylase I and/or glyoxylase II may control of the flux through the pathway. On the other hand, 35S::D-LDH overexpressing lines grew better than the wild type in the presence of d-lactate in all tested concentration (Fig. 2). It is worth mentioning that at all d-lactate concentrations tested 35S::D-LDH plants showed cotyledons and true leaves at the same developmental stage as plants grown on media without d-lactate (Fig. 2). To illustrate the better performance of 35S::d-LDH plants over wild type in the presence of d-lactate, the effect of increasing concentrations of this compound on root elongation was analyzed at 4, 8 and 12 days after imbibition in two independent 35S::D-LDH lines. Fig. 3 shows a dose-dependent reduction of root growth in wild type and the D-LDH overexpressing lines. While the wild type showed a continuous reduction in root length with increasing concentrations of d-lactate, 35S::D-LDH lines showed this dose-dependent toxic effect only at very high concentrations (5–10 mM d-lactate). Moreover, the developmental differences between the wild type and the overexpressing lines could unambiguously be observed already at 8 days after imbibition ( Fig. 2, Fig. 3 and Fig. 4). Growth on 5–10 mM d-lactate seemed ideal to most clearly distinguish transformed from untransformed plants. At this early developmental stage as the wild-type seedlings remained in the early cotyledonary stage and were yellowish while the 35S::D-LDH developed normal true leaves and roots. If plants were allowed to further grow in this condition, untransformed (wild-type) plants also developed the first true leaves. However, the root elongation was completely blocked ( Fig. 4, 12 dai) and these plants died within the following week (Fig. 5, 16 dai). If even shorter selection times are desired higher d-lactate concentrations in the media could be used. For example, at 20 mM d-lactate untransformed (wild-type) plants did not develop further (Fig. 2). However, since primary root elongation of 35S::D-LDH was slightly affected by growth on d-lactate concentrations higher than 20 mM (Fig. 2) further selection was performed with 10 mM d-lactate.
3. ผล และการอภิปรายWe showed previously that (i) d-lactate and MG negatively affect A. thaliana seedling development in a concentration-dependent manner and that (ii) d-ldh mutants i.e., plants lacking an active d-LDH, show lower tolerance than wild-type plants, indicating an impaired detoxification of these substances caused by the loss of d-LDH activity [5]. We therefore hypothesized that d-LDH overexpressing plants would be more resistant to these substances. To test this, we produced A. thaliana plants constitutively overexpressing d-LDH (35S::d-LDH plants). Seeds from wild-type as well as from 35S::D-LDH plants were germinated on MS plates in the presence of either MG (0.1–1 mM) or d-lactate (5–20 mM). In accordance with our hypothesis, the constitutive overexpression of d-LDH mitigated the negative effects on growth of MG up to 0.1 mM and of d-lactate up to 10 mM (Fig. 2). 35S::d-LDH overexpressing lines grew better than the wild type in the presence of MG up to 0.5 mM. At higher MG concentrations similar toxic effects were found in all genotypes, probably because glyoxylase I and/or glyoxylase II may control of the flux through the pathway. On the other hand, 35S::D-LDH overexpressing lines grew better than the wild type in the presence of d-lactate in all tested concentration (Fig. 2). It is worth mentioning that at all d-lactate concentrations tested 35S::D-LDH plants showed cotyledons and true leaves at the same developmental stage as plants grown on media without d-lactate (Fig. 2). To illustrate the better performance of 35S::d-LDH plants over wild type in the presence of d-lactate, the effect of increasing concentrations of this compound on root elongation was analyzed at 4, 8 and 12 days after imbibition in two independent 35S::D-LDH lines. Fig. 3 shows a dose-dependent reduction of root growth in wild type and the D-LDH overexpressing lines. While the wild type showed a continuous reduction in root length with increasing concentrations of d-lactate, 35S::D-LDH lines showed this dose-dependent toxic effect only at very high concentrations (5–10 mM d-lactate). Moreover, the developmental differences between the wild type and the overexpressing lines could unambiguously be observed already at 8 days after imbibition ( Fig. 2, Fig. 3 and Fig. 4). Growth on 5–10 mM d-lactate seemed ideal to most clearly distinguish transformed from untransformed plants. At this early developmental stage as the wild-type seedlings remained in the early cotyledonary stage and were yellowish while the 35S::D-LDH developed normal true leaves and roots. If plants were allowed to further grow in this condition, untransformed (wild-type) plants also developed the first true leaves. However, the root elongation was completely blocked ( Fig. 4, 12 dai) and these plants died within the following week (Fig. 5, 16 dai). If even shorter selection times are desired higher d-lactate concentrations in the media could be used. For example, at 20 mM d-lactate untransformed (wild-type) plants did not develop further (Fig. 2). However, since primary root elongation of 35S::D-LDH was slightly affected by growth on d-lactate concentrations higher than 20 mM (Fig. 2) further selection was performed with 10 mM d-lactate.
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