Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase

In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA-deficient transgenic tobacco BY-2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY-2 cells using PCR. Two transgenic cell-lines, AS1-1 and AS2-2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1-1 and AS2-2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild-type cells. The calli of AS1-1 and AS2-2 appeared to be sticky and soft. Back extrusion method also showed that AsA-deficient BY-2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1-1 and AS2-2 cells were abnormally slender, suggesting a potential for a significant and a uni-axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.