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.
Tabata
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Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-8528 Japan.
Oba
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Suzuki
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Esaka
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D001205:Ascorbic Acid / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D018076:DNA, Complementary; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D030821:Plants, Genetically Modified / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D016372:RNA, Antisense / Q000235:genetics*; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D016372:RNA, Antisense; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D001205:Ascorbic Acid