Acyl Carrier Protein Gene Research Articles

Fatty acid production and associated gene pathways are altered by increased salinity and dimethyl sulfoxide treatments during cryopreservation of Symbiodinium pilosum (Symbiodiniaceae)

The Symbiodinium genus is ancestral among other Symbiodiniaceae lineages with species that are both symbiotic and free living. Changes in marine ecosystems threaten their existence and crucial ecological roles. Cryopreservation offers an avenue for their long-term storage for future habitat restoration after coral bleaching. In our previous study we demonstrated that high salinity treatments of Symbiodiniaceae isolates led to changes in their fatty acid (FA) profiles and higher cell viabilities after cryopreservation. In this study, we investigated the role of increased salinity on FA production and the genes involved in FA biosynthesis and degradation pathways during the cryopreservation of Symbiodinium pilosum. Overall, there was a twofold increase in mass of FAs produced by S. pilosum after being cultured in medium with increased salinity (54 parts per thousand; ppt). Dimethyl sulfoxide (Me2SO) led to a ninefold increase of FAs in standard salinity (SS) treatment, compared to a fivefold increase in increased salinity (IS) treatments. The mass of the FA classes returned to baseline during recovery. Transcriptomic analyses showed an acyl carrier protein gene was significantly upregulated after Me2SO treatment in the SS cultures. Cytochrome P450 reductase genes were significantly down regulated after Me2SO addition in SS treatment preventing FA degradation. These changes in the expression of FA biosynthesis and degradation genes contributed to more FAs in SS treated isolates. Understanding how increased salinity changes FA production and the roles of specific genes in regulating FA pathways will help improve current freezing protocols for Symbiodiniaceae and other marine microalgae.

A Blue Light-Responsive Strong Synthetic Promoter Based on Rational Design in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (C. reinhardtii) is a single-cell green alga that can be easily genetically manipulated. With its favorable characteristics of rapid growth, low cost, non-toxicity, and the ability for post-translational protein modification, C. reinhardtii has emerged as an attractive option for the biosynthesis of various valuable products. To enhance the expression level of exogenous genes and overcome the silencing of foreign genes by C. reinhardtii, synthetic promoters such as the chimeric promoter AR have been constructed and evaluated. In this study, a synthetic promoter GA was constructed by hybridizing core fragments from the natural promoters of the acyl carrier protein gene (ACP2) and the glutamate dehydrogenase gene (GDH2). The GA promoter exhibited a significant increase (7 times) in expressing GUS, over the AR promoter as positive control. The GA promoter also displayed a strong responsiveness to blue light (BL), where the GUS expression was doubled compared to the white light (WL) condition. The ability of the GA promoter was further tested in the expression of another exogenous cadA gene, responsible for catalyzing the decarboxylation of lysine to produce cadaverine. The cadaverine yield driven by the GA promoter was increased by 1-2 times under WL and 2-3 times under BL as compared to the AR promoter. This study obtained, for the first time, a blue light-responsive GDH2 minimal fragment in C. reinhardtii, which delivered a doubling effect under BL when used alone or in hybrid. Together with the strong GA synthetic promoter, this study offered useful tools of synthetic biology to the algal biotechnology field.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates.

Peptide-Peptide Nucleic Acid (PNA) conjugates targeting essential bacterial genes have shown significant potential in developing novel antisense antimicrobials. The majority of efforts in this area are focused on identifying different PNA targets and the selection of peptides to deliver the peptide-PNA conjugates to Gram-negative bacteria. Notably, the selection of a linkage strategy to form peptide-PNA conjugate plays an important role in the effective delivery of PNAs. Recently, a unique Cysteine- 2-Cyanoisonicotinamide (Cys-CINA) click chemistry has been employed for the synthesis of cyclic peptides. Considering the high selectivity of this chemistry, we investigated the efficiency of Cys-CINA conjugation to synthesize novel antimicrobial peptide-PNA conjugates. The PNA targeting acyl carrier protein gene (acpP), when conjugated to the membrane-active antimicrobial peptides (polymyxin), showed improvement in antimicrobial activity against multidrug-resistant Gram-negative Acinetobacter baumannii. Thus, indicating that the Cys-CINA conjugation is an effective strategy to link the antisense oligonucleotides with antimicrobial peptides. Therefore, the Cys-CINA conjugation opens an exciting prospect for antimicrobial drug development.

Genome mining for drug discovery: cyclic lipopeptides related to daptomycin.

The cyclic lipopeptide antibiotics structurally related to daptomycin were first reported in the 1950s. Several have common lipopeptide initiation, elongation, and termination mechanisms. Initiation requires the use of a fatty acyl-AMP ligase (FAAL), a free-standing acyl carrier protein (ACP), and a specialized condensation (CIII) domain on the first NRPS elongation module to couple the long chain fatty acid to the first amino acid. Termination is carried out by a dimodular NRPS that contains a terminal thioesterase (Te) domain (CAT-CATTe). Lipopeptide BGCs also encode ABC transporters, apparently for export and resistance. The use of this mechanism of initiation, elongation, and termination, coupled with molecular target-agnostic resistance, has provided a unique basis for robust natural and experimental combinatorial biosynthesis to generate a large variety of structurally related compounds, some with altered or different antibacterial mechanisms of action. The FAAL, ACP, and dimodular NRPS genes were used as molecular beacons to identify phylogenetically related BGCs by BLASTp analysis of finished and draft genome sequences. These and other molecular beacons have identified: (i) known, but previously unsequenced lipopeptide BGCs in draft genomes; (ii) a new daptomycin family BGC in a draft genome of Streptomyces sedi; and (iii) novel lipopeptide BGCs in the finished genome of Streptomyces ambofaciens and the draft genome of Streptomyces zhaozhouensis.

Biosynthesis of medium chain length alkanes for bio-aviation fuel by metabolic engineered Escherichia coli

The aim of this work was to study the synthesis of medium-chain length alkanes (MCLA), as bio-aviation product. To control the chain length of alkanes and increase the production of MCLA, Escherichia coli cells were engineered by incorporating (i) a chain length specific thioesterase from Umbellularia californica (UC), (ii) a plant origin acyl carrier protein (ACP) gene and (iii) the whole fatty acid synthesis system (FASs) from Jatropha curcas (JC). The genetic combination was designed to control the product spectrum towards optimum MCLA. Decanoic, lauric and myristic acid were produced at concentrations of 0.011, 0.093 and 1.657mg/g, respectively. The concentration of final products nonane, undecane and tridecane were 0.00062mg/g, 0.0052mg/g, and 0.249mg/g respectively. Thioesterase from UC controlled the fatty acid chain length in a range of 10–14 carbons and the ACP gene with whole FASs from JC significantly increased the production of MCLA.

Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium‐chain‐specific thioesterase and acyl carrier protein

SUMMARYBiofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil‐producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium‐chain‐specific thioesterase (TE) is expected to effectively alter algae to produce medium‐chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12‐specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14‐specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG‐containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0‐containing TAGs ranged from 1.25‐ to 1.58‐fold, indicating the ability of medium‐chain‐specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.

Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis.

It has been firmly established that FabB for UFA synthesis via type II fatty acid synthesis in FabA-containing bacteria such as E. coli is essential. However, S. oneidensis appears to be an exception. In this bacterium, FabF1, when sufficiently expressed, is able to fully complement the FabB loss. Importantly, such a capability can be obtained by spontaneous mutations, which lead to transcription read-through. Therefore, our data, by identifying the functional overlap between FabB and FabFs, provide new insights into the current understanding of KAS and help reveal novel ways to block UFA synthesis for therapeutic purposes.

Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3.

Isolation and molecular characterization of three acyl carrier protein genes in olive (Olea europaea L.)

In plants, acyl carrier proteins (ACPs) are generally encoded by multigene families. We have isolated from olive (Olea europaea L.) six ACP cDNAs, and their corresponding genomic sequences, coding for plastidial ACP isoforms. Three different OeACP loci have been identified, each with a pair of alleles. The transcript abundance of OeACP1 in olive tissues is extremely low in comparison to the other OeACP genes. The OeACP2 and OeACP3 transcripts are easily detected in all tissues. OeACP2 is the most abundant in fruit and leaf tissues. In addition, OeACP3 seems to be highly expressed in flower. At the protein level, ACP is predominantly found in fruit, and to a minor extent, in flowers and leaves. The analysis of the expression pattern of ACP genes during fruit development has revealed a good correlation between the level of transcripts and the amount of protein, which indicates that ACP expression in olive pulp is mainly regulated at the transcriptional level. We suggest that OeACP2 and OeACP3 proteins are involved in the synthesis of fatty acids for the accumulation of triacylglycerols in olive fruits.

Complete Chloroplast Genome Sequence of Freshwater Araphid Pennate Diatom Alga Synedra acus from Lake Baikal

Complete chloroplast genome of diatom alga Synedra acus possesses canonical quadripartite structure with two inverted repeats containing ribosomal RNA gene loci that separate small and large single-copy regions. Chloroplast genome maps as a circular molecule of 116 251 bp. It encodes 27 tRNAs, three rRNAs, two small RNA genes, and 128 protein-coding genes. Comparison of the genic features across diatom chloroplast genomes reveals the absence of an overlap between atpD and atpF gene coding sequences that is present in other plastid genomes of diatom origin. This feature is a clear synapomorphy of S. acus plastid genome that is likely a result of either relaxed constraints or extensive selection forces acting upon atpF gene. We also characterized nuclear-encoded acyl-carrier protein gene with chloroplastic targeting in S. acus . The transfer of acpp gene into the nuclear host genome is hypothesized to have occurred independently in several lineages of diatoms.

Detection and Characterization of cry1Ac Transgene Construct in Bt Cotton: Multiple Polymerase Chain Reaction Approach

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis

Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.

Characterization of the acyl carrier protein gene and the fab gene locus in Xanthomonas albilineans

A genomic region containing the fatty acid biosynthetic (fab) genes was isolated from the sugarcane leaf-scald pathogen Xanthomonas albilineans. The order and predicted products of fabG (β-ketoacyl reductase), acpP (acyl carrier protein), fabF (ketoacyl synthase II) and downstream genes in X. albilineans are very similar to those in Escherichia coli, with one exception. Sequence analysis, confirmed by insertional knockout and specific substrate feeding experiments, shows that the position occupied by pabC (encoding aminodeoxychorismate lyase) in other bacteria is occupied instead by pabB (encoding aminodeoxychorismate synthase component I) in X. albilineans. Downstream of pabB, X. albilineans resumes the arrangement common to characterized Gram-negative bacteria, with three transcriptionally coupled genes, encoding an ORF340 protein of undefined function, thymidylate kinase and δ′ subunit of DNA polymerase III holoenzyme (HolB). Different species may obtain a common advantage from coordinated regulation of the same biosynthetic pathways using different genes in this region.

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms.

To examine as randomly as possible the role of the beta-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KSalpha) and ACP genes specifying the formation of 16- to 24-carbon polyketides, two CLF (KSbeta) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KSbeta genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KSbeta gene substituted for the actI-ORF2 or tcmL KSbeta subunit genes, along with the respective actI-ORF1 or tcmK KSalpha, tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KSalpha gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.

Identification of a cotton fiber-specific acyl carrier protein cDNA by differential display

Transcripts from immature fibers and stripped ovules (fibers removed) of cotton ( Gossypium hirsutum L.) were compared by differential display to identify cDNA fragments that represent mRNAs that are expressed primarily in cotton fibers. Eight independent fiber-specific cDNA fragments were isolated. One of these cDNAs had strong sequence similarity with acyl carrier protein (ACP). A full-length cDNA for the cotton fiber-specific ACP was isolated using a PCR cDNA library screening technique. This 713 bp cDNA has an open reading frame that encodes a 136 amino acid polypeptide. Overall nucleotide and amino acid sequence identities with other plant ACP gene sequences averaged 66% and 60% respectively. A 19 amino acid sequence surrounding the prosthetic group attachment site is nearly identical to other plant ACP genes. Northern blot analyses showed that transcripts homologous to this fiber-specific ACP cDNA were predominantly expressed during the elongation stage of fiber development. Initial genomic Southern blot analysis indicated that a single copy of the fiber-specific ACP gene may be present in both the cotton A and D genomes, since diploid Gossypium species with A or D genomes gave identical bands. We speculate that this putative fiber-specific ACP may play an important role in rapidly elongating cotton fibers by contributing to the synthesis of membrane lipids. It is also apparent that during the evolution of cotton a member of the ACP gene family has been recruited for specific expression in cotton fibers.

An Isoleucine to Valine Substitution in Escherichia coli Acyl Carrier Protein Results in a Functional Protein of Decreased Molecular Radius at Elevated pH

Escherichia coli acyl carrier protein (ACP) has been reported to exist in at least two distinct conformers in solution. A novel form of ACP having an increased electrophoretic mobility on polyacrylamide gel electrophoresis was noted previously during work on beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants of E. coli (Jackowski, S., and Rock, C. O.(1987) J. Bacteriol. 169, 1469-1473). These workers reported that the increased electrophoretic mobility of the ACP from fabF strains occurred irrespective of prosthetic group attachment or the state of acylation of the prosthetic group. Since these workers were unable to detect a difference between the amino acid sequence of the ACP from the fabF mutants and that of wild type ACP, they suggested that the increased electrophoretic mobility was due to an unknown post-translational modification of the polypeptide chain. We have reinvestigated these mutants and report that the increased electrophoretic mobility is due to a mutation within the gene (acpP) that encodes ACP. This mutation results in substitution of isoleucine for valine 43 of ACP. Site-directed mutagenesis of a synthetic ACP gene demonstrated that the amino acid substitution at residue 43 is the cause of the increased electrophoretic mobility. Gel filtration experiments indicated that the increased electrophoretic mobility results from the more compact structure of V43I ACP at high pH. The altered residue lies within the ACP region of greatest conformational lability, and thus the V43I substitution may shift the equilibrium toward the more compact conformation(s). The disulfide-linked dimer of V43I ACP was readily formed and had an electrophoretic migration greater than the dimer of wild type ACP, suggesting that formation of ACP.ACP dimers does not require structural deformation of the protein.

Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5.

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, epsilon-rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity.

Molecular cloning and sequence analysis of a seed-expressed acyl carrier protein (ACP) gene from Brassica campestris (Agrani)

We describe here the nucleotide sequence of the Brassica campestris ACPSF1 gene which encodes a seed-expressed acyl carrier protein (ACP). The 3600 by sequence consists of 1740 by upstream of the translation start codon, 828 by spanning the coding region which is interrupted by three introns and 1032 by downstream of the stop codon. Using a ACPSF1 gene-specific probe, transcripts could be detected in developing seeds, but not in leaves. This gene is now the only known member that represents group I seed-expressed ACP multigene family of Brassica species. The 5′ flanking sequence of the ACPSF1 gene was examined for putative transcriptional regulatory elements. A sequence alignment of the 5′ flanking regions of the available seed-expressed ACP genes of Brassica species showed some conserved regions which might have some common regulatory significance.

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

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.