Properties of the Poplar Controlled by the Peroxidase Gene

Peroxidase is a final step enzyme in the pathway of lignin biosynthesis. This enzymatic step has no other bypass and is a rate limiting step for the dehydropolymerization of lignin. Therefore, we thought that it would be possible to inhibit lignin biosynthesis effectively using the peroxidase gene for the control of lignin content.

A vector having the CaMV 35S promoter, followed by a reversed prxA1, was constructed and introduced into the hybrid poplar. Transformants obtained with POX1 have died because the preoxidase enzyme activity of the POX1 transformants was strongly inhibited in all parts of the transformants. This fact shows that the CaMV 35S promoter, which though it can express everywhere in plants, yet cannot be used for inhibition by antisense RNA in the case of the peroxidase gene.

Therefore, we constructed an antisense vector having the original promoter of prxA3a (Fig. 26.4) and transformants were formed. These transformants have grown similarly to the control without death. Some transformants have changed characteristics, for example, more branches and delayed root growth. Figure 26.5 shows the peroxidase activity of the transformants. Obtained results indicate that peroxidase activity in the stem of transformants decreases 75-90% compared with control, and in the leaf decreases 20-80%. This result suggests that the prxA3a gene specifically expresses in the stem. From the results of isoelectric focusing analysis, we found that the transformants lack an acidic peroxidase band, pi 3.8, which mainly expresses in the wild stem.

in order to analyze the lignin chemical structure of transformants in detail, the samples are subjected to potassium permanganate oxi-dation7 and thioacidolysis analyses.8 These results show that the lignin content of transformants decreases 20-60% in comparison with the control (Fig. 26.6) and the characteristic chemical structure of transformants is a decrease of p-hydroxy, guaiacyl and syringyl units evenly. From this result, it seems that there is no effect for the process of polymerization among aromatic units when the peroxidse is inhibited by antisense RNA. As the amount of uncondensed units is almost similar between transformants and control, it is concluded that the the main effect of inhibition of peroxidase in transformants is to decrease the lignin content in the plant.

To clarify the amount and quality of polysaccharides in transformed aspens, the composition of monosaccharide degraded by the alditol acetate method9 was analyzed. This result indicates an increasing tendency for glucose, which forms cellulose, and a decreasing tendency for xylose that forms the hemicellu-lose. This result might mean that transformed aspens increase in cellulose content and decrease hemicellulose fractions in comparison with the wild.

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