Detailed analysis of the expression of an alpha-gliadin promoter and the deposition of alpha-gliadin protein during wheat grain development.
OBJECTIVE
Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development.


METHODS
A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences.


RESULTS
GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream.


CONCLUSIONS
The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.
Van Herpen
T W J M
TW
Plant Research International, Wageningen UR, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands.
Riley
M
M
Sparks
C
C
Jones
H D
HD
Gritsch
C
C
Dekking
E H
EH
Hamer
R J
RJ
Bosch
D
D
Salentijn
E M J
EM
Smulders
M J M
MJ
Shewry
P R
PR
Gilissen
L J W J
LJ
D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005903:Gliadin / Q000235:genetics* / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
D000939:Epitopes; D010940:Plant Proteins; D005903:Gliadin; D005966:Glucuronidase