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

Plant J. 2001 Jul;27(2):139-48. doi: 10.1046/j.1365-313x.2001.01074.x.

Abstract

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.

MeSH terms

  • Ascorbic Acid / metabolism*
  • Cell Division
  • Cell Line
  • DNA, Complementary
  • Oxidoreductases / genetics*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Plants, Genetically Modified / cytology
  • Plants, Genetically Modified / enzymology
  • Plants, Genetically Modified / genetics*
  • Plants, Genetically Modified / metabolism
  • Plants, Toxic*
  • RNA, Antisense / genetics*
  • Tobacco / cytology
  • Tobacco / enzymology
  • Tobacco / genetics*
  • Tobacco / metabolism

Substances

  • DNA, Complementary
  • RNA, Antisense
  • Oxidoreductases
  • Oxidoreductases Acting on CH-CH Group Donors
  • galactonolactone dehydrogenase
  • Ascorbic Acid