Plastid‐localised seed acyl‐carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family

Abstract

Acyl-carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N-terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 5 1 -amino-acid N-terminal extension.Immunocytochemical studies showed ACP to be localised solely within the plastids of B. nupus seed tissue and it would therefore appear that the N-terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.De novo synthesis of fatty acids is catalysed by fatty acid synthetase which consists of seven or eight catalytic domains. In animals [l] and yeast [2] the domains are present on one or two multifunctional polypeptide chains (type I fatty acid synthetase), which are localised within the cytoplasm. In contrast, in plants [3] the fatty acid synthetase domains exist as discreet, monofunctional activities (type 11) which are organellar in location.Some insight into the genetic regulation of type I1 fatty acid synthetase systems has recently been obtained through cloning of genes from both yeast [4] and mammals [5]. Our interest lies in understanding the genetic control of fatty acid biosynthesis in plants, in particular within developing oil seeds. As a first step towards that objective we report here the molecular cloning of cDNA encoding seed-expressed acylcarrier protein (ACP) from Brassica napus (oil seed rape).ACP is a key component of the plant Fatty acid biosynthetic machinery, serving both as a component of fatty acid synthetase and also as an acyl donor in desaturation and acyltransfer reactions [6]. Recent studies have shown two major ACP isoforms to be expressed in leaf tissue [7, 81, but apparently only one major isoform in seeds [8]. To date, characterisation of plant ACP has been largely confined to the leafexpressed forms. Thus, spinach [S] and barley [7] isoforms have been purified and N-terminal analysis suggests that, in both species, the isoforms are products of distinct genes. Using ACP as a representative marker protein, the site of fatty acid biosynthesis in leaves has been identified as the chloroplast [9]. In developing soybean seeds, ACP levels increase in close correlation with storage lipid synthesis [lo] suggestive of a regulatory role for ACP in this process. Despite this important role, ACP has not previously been localised within, or purified from, a seed source.This paper provides the first insight into the origin, structure and expression of gene...

Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

“…4B). We note that a highly expressed ACP isoform from B. napus seeds (17) was used for all experiments. The specificity of inhibition to 18:1 acyl moieties is consistent with 18:1-containing Tweens causing maximum feedback.…”

Expression of Constitutive and Tissue-Specific Acyl Carrier Protein Isoforms in Arabidopsis

“…In B. napus, the seed-expressed ACP multigene family has been estimated to have 34 to 36 genes (29). Although B. napus is an allotetraploid, it is possible that the large size of the ACP gene family is characteristic of the Brassicaceae.…”

“…In spinach, a major isoform (ACP-I) is expressed only in leaves, whereas ACP-II, a minor isoform in leaves, is apparently "constitutive" as it is also found in roots and developing seeds (21,31). ACP cDNA clones have been isolated from spinach leaves (30) and roots (31), barley leaves (13), and Brassica campestris (28) and B. napus (29) developing seeds. In addition, ACP genomic clones have been characterized from Arabidopsis (18,25), B. napus (32), and barley (14).…”

Expression of Lauroyl–Acyl Carrier Protein Thioesterase in Brassica napus Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation

“…RNA gel blots were prehybridized for 4 to 8 hr at 42ЊC, and hybridizations were performed at 42ЊC for 12 to 24 hr using a random-primed 32 P-radiolabeled DNA probe, as described previously (Bao et al, 1997). 32 P-radiolabeled random DNA probes were synthesized using cDNA templates for oilseed rape ACP (EcoRI and HindIII fragments from pBN45 [Safford et al, 1988] and oilseed rape BCCP [gift of Q. Sun, Monsanto, St. Louis, MO]) and Arabidopsis biotin carboxylase (Bao et al, 1997).…”