Abstract
In order to examine potential regulatory steps in plant fatty acid biosynthesis, we have developed procedures for the analysis of the major acyl-acyl carrier protein (ACP) intermediates of this pathway. These techniques have been used to separate and identify acyl-ACPs with chain configurations ranging from 2:0 to 18:1 and to determine the relative in vivo concentrations of acyl-ACPs in spinach leaf and developing seed. In both leaf and seed as much as 60% of the total ACPs were nonesterified (free), with the remaining proportion consisting of acyl-ACP intermediates leading to the formation of palmitate, stearate, and oleate. In spinach leaf the proportions of the various acyl groups esterified to each ACP isoform were indistinguishable, indicating that these isoforms are utilized similarly in de novo fatty acid biosynthesis in vivo. However, the acyl group distribution pattern of seed ACP-II differed significantly from that of leaf ACP-II. The malonyl-ACP levels were less than the 4:0-ACP and 6:0-ACP levels in leaf, and in contrast, the malonyl-ACP-II levels in seed were approximately 3-fold higher than the 4:0-ACP-II and 6:0-ACP-II levels. In addition, the ratio of oleoyl-ACP-II (18:1) to stearoyl-ACP-II (18:0) was higher in seed than in leaf. These data suggest that the differences in acyl-ACP patterns reflect a tissue/organ-specific difference rather than an isoform-specific difference. In extracts prepared from leaf samples collected in the dark, the levels of acetyl-ACPs were approximately 5-fold higher compared to samples collected in the light. The levels of free ACPs showed an inverse response, increasing in the light and decreasing in the dark. Notably there was no concomitant increase in the malonyl-ACP levels. The most likely explanation for the major increase in acetyl-ACP levels in the dark is that light/dark control over the rate of fatty acid biosynthesis occurs at the reaction catalyzed by acetyl-CoA carboxylase.
Highlights
From the $Departmentof Botany and Plant Pathology, Michigan State University, East Lansing,Michigan 48824-1312 and the TDepartment of Chemistry, Miami UniversityO, xford, Ohio 45056
During de nouo fatty acid biosynthesis, the condensation of malonate with acetate or longer chain acyl groups, and the and 6:O-ACP levels in leaf, and in contrast, the ma- subsequent dehydration and reduction steps take place with lonyl-ACP-I1levels in seed were approximately%fold the acyl groups esterified to thecofactor, acyl carrier protein higher than the 4:O-ACP-I1 and 6:O-ACP-I1 levels
In this study we have developed methods which allow estimation of the steady state concentrations of plant fatty acid biosynthetic intermediates and which can thereby provide an indication of the in uiuo activities of the pathway enzymes
Summary
ACP adduct, a glutathione-ACP-I1adduct standard was included in the immunoblotanalysis. A band which migrated just below the free ACP was tentatively identified as acetyl-ACP-I1based on its comigration with the acetyl-ACP-I1 standard in gels with different urea concentrations. Extracts prepared from added alone.The shortchain acyl-ACPs (4:O and 6:O) shown developing spinach seed wereseparated on 5 M urea gels andthe in Fig. 6 were identified by their co-migrationwith standards on several gel systems with varied urea concentrations. The levels of the short chain acyl-ACPs were only 3-10% of the total ACP, they were readily identified in developing spinach seed using 1 M urea gels. To obtain information on longer chain acyl-ACPs, seed extracts were prepared and separated on gels with higher concentrations of urea (Fig. 7). In leaf, the levels of malonyl-ACPs (both I and 11)were less than or equal to the levels of 40-ACP or chains with 8or more carbonsco-migrate
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