Biochemical characterization of 3-ketoacyl-acyl carrier protein synthase II from leek epidermis.

In order to define its possible involvement in production of stearic acid for wax biosynthesis, the presence of 3-ketoacyl acyl synthase II (KAS II) activity was investigated in different tissues of leek (Allium porrum L.) leaves. KAS II activity was identified in sheath and lamina epidermis, as well as in underlying parenchyma. In all three tissues, activity was inhibited by 50% on addition of 100 microM cerulenin, and showed an absolute requirement for acyl-ACP substrates. More interestingly, the different tissues did not display similar KAS II substrate specificities. Parenchyma and lamina epidermis tissues presented typical KAS II activities, since C(18:0)-ACP was the exclusive product. In contrast, in sheath epidermis, KAS II activity resulted in the synthesis of acyl-chains up to 22 carbons in length, suggesting the existence in this tissue of an unusual KAS II. This activity was sufficient to elongate all of the palmitoyl-ACP produced by the fatty acid synthase, suggesting that C(18:0) is the substrate of the microsomal elongases involved in wax biosynthesis.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana.

An Arabidopsis fatty acid elongase gene, KCS1, with a high degree of sequence identity to FAE1, encodes a 3-ketoacyl-CoA synthase which is involved in very long chain fatty acid synthesis in vegetative tissues, and which also plays a role in wax biosynthesis. Sequence analysis of KCS1 predicted that this synthase was anchored to a membrane by two adjacent N-terminal, membrane-spanning domains. Analysis of a T-DNA tagged kcs1-1 mutant demonstrated the involvement of the KCS1 in wax biosynthesis. Phenotypic changes in the kcs1-1 mutant included thinner stems and less resistance to low humidity stress at a young age. Complete loss of KCS1 expression resulted in decreases of up to 80% in the levels of C26 to C30 wax alcohols and aldehydes, but much smaller effects were observed on the major wax components, i.e. the C29 alkanes and C29 ketones on leaves, stems and siliques. In no case did the loss of KCS1 expression result in complete loss of any individual wax component or significantly decrease the total wax load. This indicated that there was redundancy in the elongase KCS activities involved in wax synthesis. Furthermore, since alcohol, aldehyde, alkane and ketone levels were affected to varying degrees, involvement of the KCS1 synthase in both the decarbonylation and acyl-reduction wax synthesis pathways was demonstrated.

Epicuticular wax accumulation and fatty acid elongation activities are induced during leaf development of leeks

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.

In vitro regeneration via caulogenesis and brassin-induced shoot conversion of dormant buds from plumular explants of peanut (Arachis hypogaea L. cv `Okrun').

Shoot buds were induced from plumular explants of peanut (Arachis hypogaea L., cv `Okrun') preconditioned on medium containing 2,4-dichlorophenoxyacetic acid and kinetin and then transferred to regeneration medium containing benzylaminopurine and ß-naphthoxyacetic acid. Buds differentiated 25 days following transfer to regeneration medium. Each explant produced 30 to 40 buds, but only 4 shoots. The remaining buds were dormant and did not produce shoots when maintained on regeneration medium. Shoots were regenerated continuously, however, when explants were subsequently transferred to shoot conversion medium containing 1 µM brassin, benzylaminopurine and ß-naphthoxyacetic acid, respectively. Approximately 5 shoots were harvested every 30 days after transfer to shoot conversion medium for up to 7 months. No further shoot production was observed from explants maintained on regeneration medium without brassin. Regenerated shoots could be rooted and produced viable seeds. This procedure provides an efficient and reliable system for regeneration and transformation studies using cv `Okrun'.

Molecular cloning of a cDNA encoding 3-ketoacyl-acyl carrier protein synthase III from leek.

3-Ketoacyl-acyl carrier protein synthase III (KAS III) catalyzes the initial condensation of malonyl-acyl carrier protein (ACP) with acetyl-CoA in plant and bacterial fatty acid biosynthesis. The first cDNA clone encoding KAS III from a monocot is reported here. A cDNA clone was isolated from a leek epidermal cDNA library by screening with spinach and Arabidopsis heterologous probes from KAS III cDNA clones. When expressed in Escherichia coli, the cloned enzyme was able to catalyze the expected condensation reaction, was insensitive to cerulenin (100 microM) and cross-reacted with spinach KAS III antibody. The 1476-bp cDNA clone contained a 1206-bp open reading frame which encoded a 402-amino acid polypeptide. The deduced amino acid sequence showed significant similarities to other KAS IIIs although the leek sequence had some notable differences in regions otherwise completely conserved in dicots. Northern blot analyses indicated that KAS III transcript levels were similar in leaf epidermis and parenchyma, although developmental changes in transcript levels differed between these two tissues. In addition, leek KAS III was expressed in a manner comparable to leek oleoyl-ACP thioesterase (OTE), another enzyme of fatty acid biosynthesis, in both tissues.

Fatty Acid-Elongating Activity in Rapidly Expanding Leek Epidermis.

A microsomal fatty acid elongase activity measured in epidermis of rapidly expanding leek (Allium porrum L.) was 10-fold higher in specific activity than preparations from store-bought leek. These preparations elongated acyl chains effectively using endogenous or supplied primers. Elongation of C20:0 was specifically inhibited by 2 [mu]M cerulenin, and labeling experiments with [3H]cerulenin labeled two polypeptides (65 and 88 kD). ATP was required for maximal elongase activity in expanding leaves but was lost in nonexpanding tissues. Both [14C]stearoyl-coenzyme A (CoA) and [14C]stearate were maximally elongated in the presence of ATP. Addition of fully reduced CoA, however, inhibited [14C]stearate elongation, suggesting that stearoyl-CoA synthesis was not a prerequisite for elongation. Furthermore, microsomes preincubated with [14C]stearoyl-CoA plus ATP resulted in loss of radiolabel from the acyl-CoA pool without a corresponding loss in elongating activity. The lack of correlation between elongating activity and the label retained in the putative acyl-CoA substrate pool suggests that acyl-CoAs may not be the immediate precursors for elongation and that ATP plays a critical, yet undefined, role in the elongation process. We propose that an ATP-dependent elongating activity may generate the long-chain fatty acids required for wax biosynthesis.

Discovery of an epidermal stearoyl-acyl carrier protein thioesterase. Its potential role in wax biosynthesis.

Plant epicuticular, or surface, waxes are synthesized primarily, if not exclusively, by epidermal cells. The epicuticular wax constitutes almost 20% of the chloroform-extractable lipids in developing leek leaf and is derived predominantly from saturated fatty acids. The significant requirement for saturated fatty acids in epidermal tissues led us to investigate whether or not epidermal extracts have thioesterase activities that prefer saturated acyl-acyl carrier protein (ACP) substrates, rather than the 18:1-ACP more commonly hydrolyzed by total leaf extracts. Epidermal extracts from Brassica, pea, and leek exhibited higher activities toward saturated acyl-ACPs relative to 18:1-ACP when compared to total leaf or leaf parenchymal extracts. We identified and purified a stearoyl-ACP (18:0-ACP)-specific thioesterase from leek epidermal extracts which could be separated from 18:1-ACP thioesterase using hydroxyapatite chromatography. The stearoyl-ACP thioesterase exhibited a high preference for 18:0-ACP, having less than 10% of the 18:0-ACP hydrolyzing activity when presented with 18:1-ACP, 16:0-ACP, or 18:0-CoA substrates. The stearoyl-ACP thioesterase was predominantly, if not exclusively, expressed in epidermis and may play a role in generating the saturated fatty acid pool required for wax production.

Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase.

Expression of a plant lauroyl-acyl carrier protein (ACP) thioesterase in an Escherichia coli strain deficient in beta oxidation results in the accumulation of free fatty acids in the culture. Overall fatty acid production by the cultures is increased severalfold, particularly in the late log and stationary stages of growth. In control E. coli cells, malonyl-ACP levels and rates of fatty acid synthesis are highest during rapid logarithmic growth and decline to undetectable levels in stationary stage. In contrast, in cells expressing plant acyl-ACP thioesterase, malonyl-ACP levels remain high in late log and stationary stage in association with the continued fatty acid production. In addition, the biotin carboxyl carrier protein component of acetyl-CoA carboxylase is expressed at higher levels in cultures expressing the acyl-ACP thioesterase. The data presented indicate that removal of the acyl-ACP products of fatty acid synthesis results in increased production of both malonyl-ACP and fatty acids, which may in turn result from higher activity and/or expression of acetyl-CoA carboxylase.

Phosphopantethenylated Precursor Acyl Carrier Protein Is Imported into Spinach (Spinacia oleracea) Chloroplasts.

Acyl carrier protein (ACP) is an essential cofactor of fatty acid synthase. In plants, ACP is synthesized in the cytosol as a larger precursor protein and then is imported into the plastid where it is processed to a smaller mature form. The active form of ACP uses a covalently linked 4[prime]-phosphopantetheine prosthetic group derived from coenzyme A to covalently bind the acyl intermediates during fatty acid synthesis. The prosthetic group is added to ACP by holoACP synthase. This enzyme activity is associated with both the plastidial subcellular fraction and the soluble, or cytoplasmic, fraction. To gain further insight into potential in vivo pathways for the synthesis and maturation of ACP, in this study we examined whether precursor holoACP can be imported by isolated spinach (Spinacia oleracea) chloroplasts. Precursor holoACP containing a [35S]phosphopantetheine prosthetic group was prepared, and the radiolabel was used to demonstrate import of the phosphopantethenylated protein into isolated chloroplasts. In addition, timed chloroplast import assays indicated that in vitro import of the phosphopantethenylated protein is at least as efficient as import of the precursor apoprotein. Evidence was also obtained for a low level turnover of the prosthetic group among endogenous plastidial ACPs when coenzyme A was supplied exogenously.

Acetyl-acyl carrier protein is not a major intermediate in fatty acid biosynthesis in spinach.

The extent to which acetyl-acyl carrier protein (acetyl-ACP) is an intermediate in fatty acid biosynthesis was examined. Acetyl-ACP was the least effective primer of fatty acid synthesis by spinach extracts when compared to acetyl-CoA, butyryl-ACP or hexanoyl-ACP. Furthermore, the rate of acetyl-ACP-primed fatty acid synthesis was inhibited significantly by cerulenin, indicating that the slow utilization of acetyl-ACP was predominantly by 3-oxoacyl-ACP synthase I. In light-incubated isolated chloroplasts with high rates of fatty acid synthesis (greater than 800 nmol.h-1.mg chlorophyll-1), the rate of acetyl-ACP metabolism was at least 10-30-fold slower than the rate of butyryl-ACP metabolism. The relatively slow metabolism of acetyl-ACP provided in situ evidence that (a) butyryl-ACP was formed principally from condensation of malonyl-ACP with acetyl-CoA and (b) acetyl-ACP was a minor participant in fatty acid biosynthesis.

Is Acetylcarnitine a Substrate for Fatty Acid Synthesis in Plants?

Long-chain fatty acid synthesis from [1-14C]acetylcarnitine by chloroplasts isolated from spinach (Spinacia oleracea), pea (Pisum sativum), amaranthus (Amaranthus lividus), or maize (Zea mays) occurred at less than 2% of the rate of fatty acid synthesis from [1-14C]acetate irrespective of the maturity of the leaves or whether the plastids were purified using sucrose or Percoll medium. [1-14C]-Acetylcarnitine was not significantly utilized by highly active chloroplasts rapidly prepared from pea and spinach using methods not involving density gradient centrifugation. [1-14C]Acetylcarnitine was recovered quantitatively from chloroplast incubations following 10 min in the light. Unlabeled acetyl-L-carnitine (0.4 mM) did not compete with [1-14C]acetate (0.2 mM) as a substrate for fatty acid synthesis by any of the more than 70 chloroplast preparations tested in this study. Carnitine acetyltransferase activity was not detected in any chloroplast preparation and was present in whole leaf homogenates at about 0.1% of the level of acetyl-coenzyme A synthetase activity. When supplied to detached pea shoots and detached spinach, amaranthus, and maize leaves via the transpiration stream, 1 to 4% of the [1-14C]acetylcarnitine and 47 to 57% of the [1-14C]acetate taken up was incorporated into lipids. Most (78-82%) of the [1-14C]acetylcarnitine taken up was recovered intact. It is concluded that acetylcarnitine is not a major precursor for fatty acid synthesis in plants.

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts.

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 mum) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 mum in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.

Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis.

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hlousek-Radojcic, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.

In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis.

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.