Overexpression of GCN2-type protein kinase in wheat has profound effects on free amino acid concentration and gene expression

Abstract

A key point of regulation of protein synthesis and amino acid homoeostasis in eukaryotes is the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by protein kinase general control nonderepressible (GCN)-2. In this study, a GCN2-type PCR product (TaGCN2) was amplified from wheat (Triticum aestivum) RNA, while a wheat eIF2α homologue was identified in wheat genome data and found to contain a conserved target site for phosphorylation by GCN2. TaGCN2 overexpression in transgenic wheat resulted in significant decreases in total free amino acid concentration in the grain, with free asparagine concentration in particular being much lower than in controls. There were significant increases in the expression of eIF2α and protein phosphatase PP2A, as well as a nitrate reductase gene and genes encoding phosphoserine phosphatase and dihydrodipicolinate synthase, while the expression of an asparagine synthetase (AS1) gene and genes encoding cystathionine gamma-synthase and sulphur-deficiency-induced-1 all decreased significantly. Sulphur deficiency-induced activation of these genes occurred in wild-type plants but not in TaGCN2 overexpressing lines. Under sulphur deprivation, the expression of genes encoding aspartate kinase/homoserine dehydrogenase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase was also lower than in controls. The study demonstrates that TaGCN2 plays an important role in the regulation of genes encoding enzymes of amino acid biosynthesis in wheat and is the first to implicate GCN2-type protein kinases so clearly in sulphur signalling in any organism. It shows that manipulation of TaGCN2 gene expression could be used to reduce free asparagine accumulation in wheat grain and the risk of acrylamide formation in wheat products.