Identification of domains in an Arabidopsis acyl carrier protein gene promoter required for maximal organ-specific expression

Abstract

Deletions were made in the promoter of the acyl carrier protein (ACP) Acll.2 gene from Arabidopsis to investigate the nature of the cis-acting elements that direct its expression. These constructs, which included the untranslated leader region, were fused to a reporter gene coding for beta-glucuronidase (GUS) and transformed into tobacco. Quantitative fluorometric analysis of GUS activity in transgenic plants showed that expression in young leaves drops to a basal level when a 85 bp domain, from -320 to -236 relative to transcription initiation, is deleted. Maximum promoter activity in roots also depends on this domain, but two other regions are also important. In total, deletion of the sequences from -466 to -55 caused an ca. 80-fold reduction in Acl1.2 promoter activity in roots. The -320 to -236 domain forms a complex with a protein factor found in leaves and roots, which was not detectable in seeds. The formation of this protein-DNA complex was abolished by mutation of a bZIP core motif, ACGT, found within the context AAGACGTAG, which is dissimilar to the other bZIP-binding sites thus far characterized in plants. Previously we showed that Acl1.2 promoter activity is highest in seeds [2]. Here we find, in contrast to leaves and roots, that deletion to position -236 has no effect on GUS levels in seeds. However, nearly a 100-fold drop was observed when the -235 to -55 region was removed. Hence, this 180 bp domain contains all the cis-acting information necessary for Acl1.2 promoter activity in seeds. The same region is necessary for Acl1.2 activity in the receptacle, stigma, tapetum and pollen of the flower, as demonstrated by histochemical staining.

Regulation of Fatty Acid Synthesis

“…Surprisingly, although ACP is not an abundant protein, the activity of the ACP/GUS construct was comparable to that obtained from the strong 35S promoter. Several constructs of a promoter from another Arabidopsis ACP gene, Acl1.2, have been fused to GUS and examined after transformation into tobacco (4). Fluorometric analysis indicated strongest expression in developing seeds.…”

Solution Structure and Dynamics of Oxytetracycline Polyketide Synthase Acyl Carrier Protein from Streptomyces rimosus,

“…Acyl carrier proteins play an essential role in the formation of both fatty acid and polyketide products. In the type II synthases found in bacteria (10) and plants (11), ACPs are small acidic proteins of about 80 residues which are posttranslationally modified from the inactive apo protein to the active holo ACP by the transfer of a 4′-phosphopantetheine group from coenzyme A to a conserved serine on the ACP by holo ACP synthase (12). NMR studies have provided solution structures for the type II ACPs from the Streptomyces coelicolor actinorhodin (act) polyketide synthase (13), Bacillus subtilis FAS (14), Escherichia coli FAS (15), Mycobacterium tuberculosis FAS (16), and the expressed domain of the type I rat FAS (17).…”

The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs

“…4). 42, 43 The NMR studies described here show that the secondary structure of the rat apo-ACP domain comprises four α-helical regions (8-16 [1], 41-51 [2], 58-63 [3] and 66-74 [4]). The initial pattern of long-range NOEs observed for the ACP domain of rat Type I FAS shows strong similarities to the Type II FAS ACPs of E. coli, 48 spinach, 15 B. subtilis, 16 M. tuberculosis 44 and the S. coelicolor act PKS ACP 18 suggesting that it forms a small four-helix bundle, the archetypal fold for these proteins.…”

“…Acyl carrier proteins are small proteins, which play a key role in fatty acid biosynthesis. These proteins have been found in a diverse range of organisms including bacteria, 1 plants, 2 insects 3 and mammals 4 where their main role is to chaperone the extending fatty acyl chain and, on chain completion, to act as a donor of mature fatty acids to diverse biosynthetic processes. 5 In bacteria and plants each cycle of condensation, dehydration and further reduction is catalysed by a group of monofunctional polypeptides (the Type II FAS), 6 while large multifunctional enzymes (Type I synthases) found in fungi and animals perform similar biosynthetic cycles.…”