Members of a low-copy number gene family encoding glutamyl-tRNA reductase are differentially expressed in barley

Abstract

In plants tetrapyrrole synthesis is initially light regulated on the level of 5-aminolevulinate (ALA) synthesis. ALA is formed from glutamate in three enzymatic steps. Glutamyl tRNA reductase (GluTR) catalyses the NADPH-dependent reduction of glutamyl tRNA to glutamate 1-semialdehyde. GluTR is encoded by a low-copy gene family consisting of three to four genes. Three different cDNA clones are presented. Full-length clones BHA1 and 87 differ in the length of the 3' untranslated region and code for a 58.5 kDa protein. The sequence of the partial clone, BHA13, contains at least 87 base mismatches in the coding region for the mature GluTR resulting in 11 amino acid substitutions. Synthesis of a recombinant mature and a truncated GluTR lacking the first 19 amino terminal amino acids in Escherichia coli lead only in the latter case to complementation of an E. coli hemA mutant. Steady-state level of BHA1- and BHA13-specific mRNA encoding GluTR were analysed by Northern blot hybridization using cDNA-specific oligo nucleotides and quantitative reverse transcriptase-polymerase chain reaction. Accumulation of the two RNA species is light induced in greening barley and controlled during cellular development. In contrast to BHA13, BHA1 transcripts are present in roots and are elevated after cytokinin treatment of dark-grown seedlings. Furthermore, BHA1 mRNA shows oscillation under circadian growth conditions. GluTR transcript levels correlate with the capacity for ALA synthesis indicating that the rate-limiting substrate flux through the ALA synthesizing pathway can be at least partially attributed to GluTR expression. Consequences of the initial control of the chlorophyll metabolic pathway on the level of ALA formation are discussed.