BAHD Superfamily Research Articles

Identification and characterization of HCT genes in blackberry: Overexpression of RuHCT1 enhances anthocyanin contents

Emerging as a new generation of economic fruit, blackberries are rich in flavonoids and anthocyanins. Acylation can change the pharmacological activity and structural stability of anthocyanins and is of great significance to plant growth and secondary metabolism. In this study, based on the transcriptome data of the blackberry fruit, five blackberry hydroxycinnamoyl transferase (HCT) genes were screened and identified, all of which belong to the BAHD superfamily. Among them, RuHCT1, RuHCT2 and RuHCT3 have similar tertiary structures and functions. RuHCT1, the gene with the largest difference in expression between immature and mature blackberry fruits, was selected for overexpression research. The results showed that the content of anthocyanin in the leaves of transgenic plants was significantly increased, while the content of secondary metabolites such as lignan and chlorogenic acid was decreased. Moreover, the expression levels of the NtCCOMT, NtCHS and NtCYP98A genes in transgenic tobacco were lower than those in nontransgenic plants. In conclusion, RuHCT1 may be an important structural gene regulating anthocyanin synthesis in blackberry. RuHCT1 may also inhibit lignan synthesis and affect the expression of other key structural genes. This study provides a theoretical reference regarding the biosynthesis and regulatory mechanisms of anthocyanins in blackberry, contributing to the breeding of new blackberry cultivars with high anthocyanin content.

Identification and analysis of BAHD superfamily related to malonyl ginsenoside biosynthesis in Panax ginseng.

The BAHD (benzylalcohol O-acetyl transferase, anthocyanin O-hydroxycinnamoyl transferase, N-hydroxycinnamoyl anthranilate benzoyl transferase and deacetylvindoline 4-O-acetyltransferase), has various biological functions in plants, including catalyzing the biosynthesis of terpenes, phenolics and esters, participating in plant stress response, affecting cell stability, and regulating fruit quality. Bioinformatics methods, real-time fluorescence quantitative PCR technology, and ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer were used to explore the relationship between the BAHD gene family and malonyl ginsenosides in Panax ginseng. In this study, 103 BAHD genes were identified in P. ginseng, mainly distributed in three major clades. Most PgBAHDs contain cis-acting elements associated with abiotic stress response and plant hormone response. Among the 103 genes, 68 PgBAHDs are WGD (whole-genome duplication) genes. The significance of malonylation in biosynthesis has garnered considerable attention in the study of malonyltransferases. The phylogenetic tree results showed 34 PgBAHDs were clustered with genes that have malonyl characterization. Among them, seven PgBAHDs (PgBAHD4, 45, 65, 74, 90, 97, and 99) showed correlations > 0.9 with crucial enzyme genes involved in ginsenoside biosynthesis and > 0.8 with malonyl ginsenosides. These seven genes were considered potential candidates involved in the biosynthesis of malonyl ginsenosides. These results help elucidate the structure, evolution, and functions of the P. ginseng BAHD gene family, and establish the foundation for further research on the mechanism of BAHD genes in ginsenoside biosynthesis.

Roles of the MYB94/FUSED LEAVES1 (ZmFDL1) and GLOSSY2 (ZmGL2) genes in cuticle biosynthesis and potential impacts on Fusarium verticillioides growth on maize silks.

Maize silks, the stigmatic portions of the female flowers, have an important role in reproductive development. Silks also provide entry points for pathogens into host tissues since fungal hyphae move along the surface of the silks to reach the site of infection, i.e., the developing kernel. The outer extracellular surface of the silk is covered by a protective hydrophobic cuticle, comprised of a complex array of long-chain hydrocarbons and small amounts of very long chain fatty acids and fatty alcohols. This work illustrates that two previously characterized cuticle-related genes separately exert roles on maize silk cuticle deposition and function. ZmMYB94/FUSED LEAVES 1 (ZmFDL1) MYB transcription factor is a key regulator of cuticle deposition in maize seedlings. The ZmGLOSSY2 (ZmGL2) gene, a putative member of the BAHD superfamily of acyltransferases with close sequence similarity to the Arabidopsis AtCER2 gene, is involved in the elongation of the fatty acid chains that serve as precursors of the waxes on young leaves. In silks, lack of ZmFDL1 action generates a decrease in the accumulation of a wide number of compounds, including alkanes and alkenes of 20 carbons or greater and affects the expression of cuticle-related genes. These results suggest that ZmFDL1 retains a regulatory role in silks, which might be exerted across the entire wax biosynthesis pathway. Separately, a comparison between gl2-ref and wild-type silks reveals differences in the abundance of specific cuticular wax constituents, particularly those of longer unsaturated carbon chain lengths. The inferred role of ZmGL2 is to control the chain lengths of unsaturated hydrocarbons. The treatment of maize silks with Fusarium verticillioides conidia suspension results in altered transcript levels of ZmFDL1 and ZmGL2 genes. In addition, an increase in fungal growth was observed on gl2-ref mutant silks 72 hours after Fusarium infection. These findings suggest that the silk cuticle plays an active role in the response to F. verticillioides infection.

Evolution of p-coumaroylated lignin in eudicots provides new tools for cell wall engineering.

Ester-linked p-coumarate (pCA) is a hallmark feature of the secondary cell walls in commelinid monocot plants. It has been shown that pCA groups arise during lignin polymerisation from the participation of monolignol conjugates assembled by p-coumaroyl-CoA:monolignol transferase (PMT) enzymes, members of the BAHD superfamily of acyltransferases. Herein, we report that a eudicot species, kenaf (Hibiscus cannabinus), naturally contains p-coumaroylated lignin in the core tissues of the stems but not in the bast fibres. Moreover, we identified a novel acyltransferase, HcPMT, that shares <30% amino acid identity with known monocot PMT sequences. Recombinant HcPMT showed a preference in enzyme assays for p-coumaroyl-CoA and benzoyl-CoA as acyl donor substrates and sinapyl alcohol as an acyl acceptor. Heterologous expression of HcPMT in hybrid poplar trees led to the incorporation of pCA in lignin, but no improvement in the saccharification potential of the wood. This work illustrates the value in mining diverse plant taxa for new monolignol acyltransferases. Furthermore, the occurrence of pCA outside monocot lineages may represent another example of convergent evolution in lignin structure. This discovery expands textbook views on cell wall biochemistry and provides a new molecular tool for engineering the lignin of biomass feedstock plants.

Genome-Wide Identification of BAHD Superfamily and Functional Characterization of Bornyl Acetyltransferases Involved in the Bornyl Acetate Biosynthesis in Wurfbainia villosa.

Bornyl acetate (BA) is known as a natural aromatic monoterpene ester with a wide range of pharmacological and biological activities. Borneol acetyltransferase (BAT), catalyzing borneol and acetyl-CoA to synthesize BA, is alcohol acetyltransferase, which belongs to the BAHD super acyltransferase family, however, BAT, responsible for the biosynthesis of BA, has not yet been characterized. The seeds of Wurfbainia villosa (homotypic synonym: Amomum villosum) are rich in BA. Here we identified 64 members of the BAHD gene family from the genome of W. villosa using both PF02458 (transferase) and PF07247 (AATase) as Hidden Markov Model (HMM) to screen the BAHD genes. A total of sixty-four WvBAHDs are distributed on 14 chromosomes and nine unanchored contigs, clustering into six clades; three WvBAHDs with PF07247 have formed a separated and novel clade: clade VI. Twelve candidate genes belonging to clade I-a, I-b, and VI were selected to clone and characterize in vitro, among which eight genes have been identified to encode BATs acetylating at least one type of borneol to synthesize BA. All eight WvBATs can utilize (−)-borneol as substrates, but only five WvBATs can catalyze (+)-borneol, which is the endogenous borneol substrate in the seeds of W. villosa; WvBAT3 and WvBAT4 present the better catalytic efficiency on (+)-borneol than the others. The temporal and spatial expression patterns of WvBATs indicate that WvBAT3 and WvBAT4 are seed-specific expression genes, and their expression levels are correlated with the accumulation of BA, suggesting WvBAT3 and WvBAT4 might be the two key BATs for BA synthesis in the seeds of W. villosa. This is the first report on BAT responsible for the last biosynthetic step of BA, which will contribute to further studies on BA biosynthesis and metabolism engineering of BA in other plants or heterologous hosts.

Systematic identification and expression profiles of the BAHD superfamily acyltransferases in barley (Hordeum vulgare)

BAHD superfamily acyltransferases play an important role in catalyzing and regulating secondary metabolism in plants. Despite this, there is relatively little information regarding the BAHD superfamily in barley. In this study, we identified 116 HvBAHD acyltransferases from the barley genome. Based on phylogenetic analysis and classification in model monocotyledonous and dicotyledonous plants, we divided the genes into eight groups, I-a, I-b, II, III-a, III-b, IV, V-a and V-b. The Clade IV genes, including Agmatine Coumarol Transferase (ACT) that is associated with resistance of plants to Gibberella fungi, were absent in Arabidopsis. Cis-regulatory element analysis of the HvBAHDs showed that the genes respond positively to GA3 treatment. In-silico expression and qPCR analysis showed the HvBAHD genes are expressed in a range of tissues and developmental stages, and highly enriched in the seedling stage, consistent with diverse roles. Single nucleotide polymorphism (SNP) scanning analysis revealed that the natural variation in the coding regions of the HvBAHDs is low and the sequences have been conserved during barley domestication. Our results reveal the complexity of the HvBAHDs and will help facilitate their analysis in further studies.

Identification and validation of an ECERIFERUM2- LIKE gene controlling cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata L.).

A single nucleotide mutation of BoCER2 is the primary cause of the wax deficiency in cabbage. An effective allele-specific KASP marker was developed for marker-assisted selection of glossiness. TL28-1 is a novel spontaneous wax-deficient mutant with a glossy phenotype identified from cabbage. In this study, the genetic analysis suggested that the wax-deficient trait of TL28-1 was controlled by a single recessive gene. All wax monomers longer than 28 carbons were significantly decreased in TL28-1. Fine-mapping results showed that the wax-deficient locus wdtl28 was located at an 80-kb interval between BOL01-20 and BOL01-24 markers on chromosome 1. According to the genome annotation of B. oleracea, the ECERIFERUM2- LIKE (CER2-LIKE) gene, BoCER2, was identified as the candidate gene. Phylogenetic analysis showed that BoCER2 and other CER2-LIKEs from vascular plants formed a clade within the BAHD superfamily of acyltransferases. The BoCER2 transcript was detected in various tissues, including stem, leaf, flower, and silique, but not in the cabbage roots. Subcellular localization indicated that BoCER2 protein functions in the endoplasmic reticulum. Further sequence analysis showed that a single nucleotide mutation (G to A) is present in the BoCER2 coding sequence in TL28-1, leading to a stop codon (TGA), hence premature translation termination. Linkage analysis showed that the homozygotic mutational BoCER2 co-segregated with wax deficiency. Moreover, the complementation test suggested that BoCER2 from wild type can rescue the wax deficiency of TL28-1. These results indicate that BoCER2 mutation hinders the elongation of very-long-chain fatty acid precursors in TL28-1, leading to wax deficiency. The allele-specific KASP marker designed in this study could be effective for marker-assisted selection of glossiness.

Molecular and structural characterization of agmatine coumaroyltransferase in Triticeae, the key regulator of hydroxycinnamic acid amide accumulation

Hydroxycinnamic acid amides (HCAAs) are involved in stress-induced defense in many plant species. Barley accumulates high concentrations of HCAAs irrespective of exogenous stressors, while other major cereals such as wheat and rice accumulate relatively low levels of HCAAs in intact tissues. The primary HCAA species in barley are biosynthesized by agmatine p-coumaroyltransferase (ACT), an N-acyltransferase of the BAHD superfamily. However, the molecular basis underlying barley's uniquely high HCAA accumulation has not been elucidated, and information regarding the structural details of BAHD N-acyltransferases is limited. Hence, we aimed to investigate the ACTs of family Poaceae. We isolated ACT (-like) genes, including those previously undescribed, and investigated their enzymatic and genetic features. All the identified enzymes belonged to clade IVa of the BAHD superfamily. The barley and wheat ACTs were further categorized, based on catalytic properties and primary structures, into ACT1 and ACT2 groups, the encoding loci of which are neighbors on the same chromosome. While all ACTs exhibited similar Km values for CoA-thioesters (acyl-group donors), members of the ACT1 group showed a distinctly higher affinity for agmatine (acyl-acceptor). Among the ACTs tested, an ACT isozyme in barley (HvACT1-1) showed the highest catalytic efficiency and transcript level, indicating that ACT regulates high-level HCAA accumulation in barley. For further enzymatic characterization of the ACTs, we crystalized wheat ACT2 (TaACT2) and determined its structure at 2.3 Å resolution. Structural alignment of TaACT2 and HvACT1-1 showed that the architectures of the substrate binding pockets were well conserved. However, the structure of a loop located at the entrance to acyl-acceptor binding site may be more flexible in TaACT2, which could be responsible for the lower affinity of TaACT2 to agmatine. Mutations of HvACT1-1 at Glu372 and Asp374 within one of the clade-IV specific motifs facing the deduced acyl-acceptor binding pocket caused significant catalytic deterioration toward agmatine both in Km and kcat, suggesting their key roles in acyl acceptor binding by the clade-IV enzymes. This study elucidated the molecular basis of how plants accumulate defensive specialized metabolites and provided insights into developing efficient and eco-friendly agricultural methods.

Crystal structure of barley agmatine coumaroyltransferase, an N-acyltransferase from the BAHD superfamily.

The enzymes of the BAHD superfamily, a large group of acyl-CoA-dependent acyltransferases in plants, are involved in the biosynthesis of diverse secondary metabolites. While the structures of several O-acyltransferases from the BAHD superfamily, such as hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase, have been elucidated, no structural information on N-acyltransferases is available. Hordeum vulgare agmatine coumaroyltransferase (HvACT) is an N-acyltransferase from the BAHD superfamily and is one of the most important enzymes in the secondary metabolism of barley. Here, an apo-form structure of HvACT is reported as the first structure of an N-acyltransferase from the BAHD superfamily. HvACT crystals diffracted to 1.8 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 57.6, b = 59.5, c=73.6 Å, α = 90, β = 91.3 , γ = 90°. Like other known BAHD superfamily structures, HvACT contains two domains that adopt a two-layer αβ-sandwich architecture and a solvent-exposed channel that penetrates the enzyme core.

Molecular cloning, expression and in-silico characterization of rosmarinic acid synthase from Ocimum tenuiflorum L.

Rosmarinic acid synthase (RAS) is a control point at which metabolites derived from l-Phenylalanine and l-Tyrosine are channeled for the biosynthesis of rosmarinic acid (RA). Ocimum tenuiflorum or holy basil has been utilized as a source of RA. To acquire a better understanding of the biosynthesis of this metabolite, isolation, and cloning of O. tenuiflorum RAS (OtRAS) cDNA and its heterologous expression in bacterial cells was assessed. The full-length OtRAS (Accession No. MN542659) was 1278 bp in size encoding 425 amino acid long protein product. The subunit molecular mass of recombinant OtRAS was estimated to be ∼ 47.5 kDa with a theoretical pI of 6.35. Sequence analysis indicated OtRAS belongs to BAHD superfamily proteins. Phylogenetic analysis revealed close evolutionary relatedness of OtRAS with RAS proteins reported in other lamiaceae members. Gene expression analysis showed that callus derived from leaves accumulated more transcripts of OtRAS than leaf and stem tissues derived from field-grown plants. OtRAS was up-regulated under flood and cold stress whereas down-regulated under drought and salt conditions. Further, three-dimensional structure of OtRAS showed two pseudo symmetrical domains harboring an active site in between these two domains. OtRAS active site comprised of conserved His41, His152, Trp363, and Lys391 residues. Overall, our results indicated the OtRAS regulation is a mechanism that probably depended on a variety of abiotic stresses.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

Genome-wide comparative analysis of the BAHD superfamily in seven Rosaceae species and expression analysis in pear (Pyrus bretschneideri)

BackgroundThe BAHD acyltransferase superfamily exhibits various biological roles in plants, including regulating fruit quality, catalytic synthesizing of terpene, phenolics and esters, and improving stress resistance. However, the copy numbers, expression characteristics and associations with fruit aroma formation of the BAHD genes remain unclear.ResultsIn total, 717 BAHD genes were obtained from the genomes of seven Rosaceae, (Pyrus bretschneideri, Malus domestica, Prunus avium, Prunus persica, Fragaria vesca, Pyrus communis and Rubus occidentalis). Based on the detailed phylogenetic analysis and classifications in model plants, we divided the BAHD family genes into seven groups, I-a, I-b, II-a, II-b, III-a, IV and V. An inter-species synteny analysis revealed the ancient origin of BAHD superfamily with 78 syntenic gene pairs were detected among the seven Rosaceae species. Different types of gene duplication events jointly drive the expansion of BAHD superfamily, and purifying selection dominates the evolution of BAHD genes supported by the small Ka/Ks ratios. Based on the correlation analysis between the ester content and expression levels of BAHD genes at different developmental stages, four candidate genes were selected for verification as assessed by qRT-PCR. The result implied that Pbr020016.1, Pbr019034.1, Pbr014028.1 and Pbr029551.1 are important candidate genes involved in aroma formation during pear fruit development.ConclusionWe have thoroughly identified the BAHD superfamily genes and performed a comprehensive comparative analysis of their phylogenetic relationships, expansion patterns, and expression characteristics in seven Rosaceae species, and we also obtained four candidate genes involved in aroma synthesis in pear fruit. These results provide a theoretical basis for future studies of the specific biological functions of BAHD superfamily members and the improvement of pear fruit quality.

The Unique Role of the ECERIFERUM2-LIKE Clade of the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism.

The elongation of very-long-chain fatty acids is a conserved process used for the production of many metabolites, including plant cuticular waxes. The elongation of precursors of the most abundant cuticular wax components of some plants, however, is unique in requiring ECERIFERUM2-LIKE (CER2-LIKE) proteins. CER2-LIKEs are a clade within the BAHD superfamily of acyltransferases. They are known to be required for cuticular wax production in both Arabidopsis and maize based on mutant studies. Heterologous expression of Arabidopsis and rice CER2-LIKEs in Saccharomyces cerevisiae has demonstrated that they modify the chain-length specificity of elongation when paired with particular condensing enzymes. Despite sequence homology, CER2-LIKEs are distinct from the BAHD superfamily in that they do not appear to use acyl transfer activity to fulfill their biological function. Here, we review the discovery and characterization of CER2-LIKEs, propose several models to explain their function, and explore the importance of CER2-LIKE proteins for the evolution of plant cuticles.

Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L.

Lemon balm ( Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene ( MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli.

Identification and mapping of genes related to caffeoylquinic acid synthesis in Cynara cardunculus L.

The caffeoylquinic acid (CQA) biosynthesis pathway in plants is not fully understood, but the BAHD superfamily, a large class of acyl-CoA-dependent acyltransferases, may represent an interesting group for the isolation of enzymes playing a key role. Globe artichoke ( Cynara cardunculus var. scolymus) represents a relevant model species for studying CQA biosynthesis, because the plant accumulates high amounts and a diverse spectrum of CQAs in its leaves. An in silico scan of globe artichoke ESTs was based on a search string targeting the conserved motifs of BAHD proteins and complete BAHD sequences present in a range of species. EST hits were assembled into 32 unigenes and their phylogeny characterized. Among which were three putative BAHD acyltransferases showing a high level of similarity to globe artichoke HCT and HQT enzymes. The full-length cDNAs of these three proteins were isolated; two of these acyltransferases were able to synthesize both chlorogenic acid and p-coumaroylquinate, assessing their involvement in CQA biosynthesis. Upon exposure to UV-C radiation, which is known to induce CQA biosynthesis, expression of two of the enzyme transcripts was up-regulated while that of the third was unaffected. By analysing the globe artichoke unigene consensus sequences, further two genes (i.e. C4H, 4CL), related not only to flavonoids but also to chlorogenic acid (a mono-CQA) biosynthesis, were also identified and were genetically mapped along with the three acyltransferase genes. The research has improved the knowledge base surrounding globe artichoke BAHD members. The mapping of the genes related to the chlorogenic acid biosynthesis pathway may contribute to a greater understanding of the genetic basis of the synthesis of these compounds of nutraceutical and pharmaceutical value.

A hydroxycinnamoyltransferase responsible for synthesizing suberin aromatics in Arabidopsis

Suberin, a polyester polymer in the cell wall of terrestrial plants, controls the transport of water and nutrients and protects plant from pathogenic infections and environmental stresses. Structurally, suberin consists of aliphatic and aromatic domains; p-hydroxycinnamates, such as ferulate, p-coumarate, and/or sinapate, are the major phenolic constituents of the latter. By analyzing the "wall-bound" phenolics of mutant lines of Arabidopsis deficient in a family of acyl-CoA dependent acyltransferase (BAHD) genes, we discovered that the formation of aromatic suberin in Arabidopsis, primarily in seed and root tissues, depends on a member of the BAHD superfamily of enzymes encoded by At5g41040. This enzyme exhibits an omega-hydroxyacid hydroxycinnamoyltransferase activity with an in vitro kinetic preference for feruloyl-CoA and 16-hydroxypalmitic acid. Knocking down or knocking out the At5g41040 gene in Arabidopsis reduces specifically the quantity of ferulate in suberin, but does not affect the accumulation of p-coumarate or sinapate. The loss of the suberin phenolic differentially affects the aliphatic monomer loads and alters the permeability and sensitivity of seeds and roots to salt stress. This highlights the importance of suberin aromatics in the polymer's function.

BAHD superfamily of acyl-CoA dependent acyltransferases in Populus and Arabidopsis: bioinformatics and gene expression

Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by "in silico" northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members' evolutionary relationships and gene functions implicated in plant growth, development and metabolism.

Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases

Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.

Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina

Vinorine synthase (VS) is a central enzyme of the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure with significant sequence homology with VS is known. Crystals of VS and selenomethionyl-labelled VS from the medicinal plant Rauvolfia serpentina have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. VS crystals diffract to 2.8 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.3, b = 89.6, c = 136.2 A. The selenomethionyl VS crystal was nearly isomorphous with the VS crystal.

ThePun1gene for pungency in pepper encodes a putative acyltransferase

Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.