Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.
Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.
Harholt
Jesper
J
Department of Plant Biology and Biotechnology, Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Bach
Inga C
IC
Lind-Bouquin
Solveig
S
Nunan
Kylie J
KJ
Madrid
Susan M
SM
Brinch-Pedersen
Henrik
H
Holm
Preben B
PB
Scheller
Henrik V
HV
D001089:Arabinose / Q000032:analysis; D001234:Aspergillus niger / Q000201:enzymology; D001412:Bacillus subtilis / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D003373:Coumaric Acids / Q000032:analysis; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D056625:Endosperm / Q000201:enzymology*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D014170:Transformation, Genetic; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D014990:Xylans / Q000032:analysis; D014994:Xylose / Q000032:analysis
D003373:Coumaric Acids; D014990:Xylans; C085118:arabinoxylan; D014994:Xylose; C004999:ferulic acid; D001089:Arabinose; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D043364:Endo-1,4-beta Xylanases