An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.
OBJECTIVE
Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence.


METHODS
Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques.


RESULTS
Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels.


CONCLUSIONS
There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.
Kis
Mihaly
M
Institute of Bioengineering and Agroecology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
Burbridge
Emma
E
Brock
Ian W
IW
Heggie
Laura
L
Dix
Philip J
PJ
Kavanagh
Tony A
TA
D000595:Amino Acid Sequence; D031215:Armoracia / Q000201:enzymology* / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009195:Peroxidase / Q000235:genetics* / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D009195:Peroxidase