Benzylalcohol Acetyltransferase Research Articles

Increased substrate availability reveals the potential of scentless lisianthus flowers in producing fragrant benzenoid-phenylpropanoids.

Lisianthus (Eustoma grandiflorum), a leading plant in the cut flower industry, is scentless. Here we show that lisianthus flowers have potential to produce several fragrant benzenoid-phenylpropanoids when substrate availability is not limited. To enable hyperaccumulation of substrates for the production of volatile benzenoid-phenylpropanoids, lisianthus commercial hybrid "Excalibur Pink" was transformed via floral dipping with a feedback-insensitive Escherichia coli DAHP synthase (AroG*) and Clarkia breweri benzyl alcohol acetyltransferase (BEAT), under constitutive promoters. The T1 progeny of "Excalibur Pink" plants segregated into four visual phenotypes, with pink or white colored petals and multiple or single petal layers. Interestingly, transformation with AroG* and BEAT caused no significant effect in the pigment composition among phenotypes, but did increase the levels of down-stream fragrant volatile benzenoids. All the transgenic lines exclusively accumulated methyl benzoate, a fragrant benzenoid, either in their petals or leaves. Furthermore, feeding with benzyl alcohol resulted in the accumulation of two novel benzenoids, benzyl acetate (the product of BEAT) and benzoate, as well as a dramatic increase in the concentrations of additional benzenoid-phenylpropanoid volatiles. Presumably, the degree of benzaldehyde overproduction after benzyl alcohol feeding in both leaves and flowers revealed their reverse conversion in lisianthus plants. These findings demonstrate the concealed capability of lisianthus plants to produce a wide array of fragrant benzenoid-phenylpropanoids, given high substrate concentrations, which could in turn open opportunities for future scent engineering.

Expansion of PmBEAT genes in the Prunus mume genome induces characteristic floral scent production

Prunus mume is the only plant in the genus Prunus of the Rosaceae family with a characteristic floral scent, and the main component of this scent is benzyl acetate. By contrast, benzyl acetate is not synthesized in Prunus persica flowers. Here, we searched for benzyl alcohol acetyltransferase (BEAT) genes based on genomic data from P. mume and P. persica and found 44 unique PmBEATs in P. mume. These genes, which were mainly detected in clusters on chromosomes, originated from gene duplication events during the species evolution of P. mume, and retroduplication and tandem duplication were the two dominant duplication patterns. The genes PmBEAT34, PmBEAT36 and PmBEAT37, which were generated by tandem duplication, were highly expressed in flowers, and their highest levels were detected during the blooming stage. In vitro, PmBEAT34, PmBEAT3, and PmBEAT37 all had benzyl alcohol acetyltransferase activity that was localized in the cytoplasm. Overexpression of the PmBEAT36 or PmBEAT37 genes increased benzyl acetate production in the petal protoplasts of P. mume, and interference in the expression of these genes slightly decreased the benzyl acetate content. In addition, light and temperature regulated the expression of the PmBEAT34, PmBEAT36 and PmBEAT37 genes. According to these results, we hypothesize that the expansion of the PmBEAT genes in the genome induce the characteristic floral scent of P. mume.

Transcriptomic and proteomic approaches to explore the differences in monoterpene and benzenoid biosynthesis between scented and unscented genotypes of wintersweet.

Wintersweet (Chimonanthus praecox L.) is an important ornamental plant in China with a pleasant floral scent. To explore the potential mechanisms underlying differences in the fragrances among genotypes of this plant, we analyzed floral volatile organic compounds (VOCs) from two different genotypes: SW001, which has little to no fragrance, and the scented genotype H29. The major VOCs in H29 were linalool, trans-β-ocimene, benzyl acetate, methyl salicylate, benzyl alcohol (BAlc) and methyl benzoate. The most important aroma-active compound in H29, linalool, was emitted at a low concentration in SW001, which had markedly higher levels of trans-β-ocimene than H29. Next, to investigate scent biosynthesis, we analyzed the transcriptome and proteome of fully open flowers of the two genotypes. A total of 14 443 differentially expressed unigenes and 196 differentially expressed proteins were identified. Further analyses indicated that 56 differentially expressed genes involved in the terpenoid and benzenoid biosynthesis pathways might play critical roles in regulating floral fragrance difference. Disequilibrium expression of four terpene synthase genes resulted in diverse emission of linalool and trans-β-ocimene in both genotypes. In addition, the expressions of two CpMYC2 transcription factors were both upregulated in H29, implying that they may regulate linalool production. Notably, 16 of 20 genes in the benzenoid biosynthesis pathway were downregulated, corresponding to the relatively low level of benzenoid production in SW001. The lack of benzyl acetate might indicate that SW001 may lack substrate BAlc or functional acetyl-CoA:benzylalcohol acetyltransferase.

Successful floral-dipping transformation of post-anthesis lisianthus (Eustoma grandiflorum) flowers.

The adaptation of the Agrobacterium-mediated floral-dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback-insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin-resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral-dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4days pre-anthesis or 3-5days post-anthesis with 1.5 and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral-dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel.

Enzymatic production and emission of floral scent volatiles in Jasminum sambac

Floral scent composed of low molecular weight volatile organic compounds. The sweet fragrance of any evening blooming flower is dominated by benzenoid and terpenoid volatile compounds. Floral scent of Jasminum sambac (Oleaceae) includes three major benzenoid esters – benzylacetate, methylbenzoate, and methylsalicylate and three major terpene compounds viz. (E)-β-ocimene, linalool and α-farnesene. We analyzed concentrations and emission rates of benzenoids and terpenoids during the developmental stages of J. sambac flower. In addition to spatial emission from different floral parts, we studied the time-course mRNA accumulations of phenylalanine ammonia-lyase (PAL) and the two representative genes of terpenoid pathway, namely 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and terpene synthase (TPS). Further, in vitro activities of several enzymes of phenylpropanoid/benzenoid pathway viz., PAL and acetyl-coenzyme A: benzylalcohol acetyltransferase (BEAT), S-adenosyl-l-methionine: benzoic acid carboxyl methyl transferase (BAMT) and S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase (SAMT) were studied. All the above enzyme activities along with the in vitro activities of DXR and TPS were found to follow a certain rhythm as observed in the emission of different benzenoid and terpenoid compounds. Linalool emission peaked after petal opening and coincided with maximal expression of JsTPS gene as evidenced from RT-PCR analyses (semi-quantitative). The maximum transcript accumulation of this gene was observed in flower petals, indicating that the petals of J. sambac flower play an important role as a major contributor of volatile precursors. The transcripts accumulation of JsDXR and JsTPS in different developmental stages and in different floral part showed that emissions of terpenoid volatiles in J. sambac flower are partially regulated at transcription levels.

A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins.

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.

Post-harvest enhancement of aroma in transgenic lisianthus ( Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase ( BEAT) gene

Lisianthus ( Eustoma grandiflorum) is an ornamental plant with beautiful but scentless flowers. In an attempt to induce a fragrance in their flowers, lisianthus plants were transformed with the Clarkia breweri gene coding for benzyl alcohol acetyltransferase ( BEAT), catalyzing the synthesis of the volatile compound benzyl acetate under the regulation of the CaMV35S promoter. An external supply of benzyl alcohol induced five to seven times higher production of benzyl acetate in detached flowers and leaves of transgenic lisianthus plants, compared to non-transformed plants. No benzyl acetate was detected in tissues of both control and transgenic plants fed with water. When fed with additional alcoholic compounds, i.e. hexanol, benzyl alcohol, isoamyl alcohol, phenethyl alcohol, and cinnamyl alcohol, assumed to be used as substrates by BEAT, transgenic in vitro-grown lisianthus plantlets produced significantly higher levels of acetates than control plants. These results demonstrate the possibility of producing substrate-dependent acetates in transgenic lisianthus plants, which could lead to induction of new aromas.

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Identification and characterization of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers: an enzyme that is phylogenetically separated from other anthocyanin acyltransferases.

Anthocyanin acyltransferases (AATs) catalyze a regiospecific acyl transfer from acyl-CoA to the glycosyl moiety of anthocyanins, thus playing an important role in flower coloration. The known AATs are subfamily members of an acyltransferase family, the BAHD family, which play important roles in secondary metabolism in plants. Here, we describe the purification, characterization, and cDNA cloning of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (hereafter referred to as Ss5MaT2) is a monomeric 46-kDa protein that catalyzes the transfer of the malonyl group from malonyl-CoA to the 4"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Thus, it is a malonyl-CoA:anthocyanin 5-glucoside 4"'-O-malonyltransferase. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA that encodes an acyltransferase protein. The steady-state transcript level of the gene was the highest in recently opened, fully pigmented flowers and was also correlated with the trend observed for an AAT gene responsible for the first malonylation step during salvianin biosynthesis. Immunoprecipitation studies using antibodies against the recombinant acyltransferase protein corroborated the identity of this cDNA as that encoding Ss5MaT2. The deduced amino acid sequence of Ss5MaT2 showed a low similarity (22-24% identity) to those of AATs and lacked the AAT-specific signature sequence. A phylogenetic analysis suggested that Ss5MaT2 is more related to acetyl-CoA:benzylalcohol acetyltransferase (BEAT) rather than to AAT. This is another example in which enzymes with similar, although not identical, substrate evolved from different branches of the BAHD family.

MODIFYING LISIANTHUS TRAITS BY GENETIC ENGINEERING

The control of flowering time is an essential issue for most ornamentals. The molecular basis of this trait has been extensively studied in model plants, yielding useful information on the role of various genes which could potentially be used in heterologous systems. For instance, overexpression of the meristem-identity gene LEAFY (LFY) from Arabidopsis has previously been shown to be sufficient to reduce flowering time in plants as diverse as Arabidopsis itself, tobacco and aspen. We tested the effect of LFY overexpression on flower development in lisianthus plants transformed with LFY cDNA under constitutive control of the 35S CaMV promoter (35S::LFY, provided by Dr. Weigel). F1 progenies from two different transformed plants flowered in average one to two weeks earlier than control plants at two consecutive years. The number of nodes to the first flower as well as the number of flowers per inflorescence were reduced in the early-flowering plants, suggesting a faster flower development. Normal fertile flowers were obtained from all plants. These results indicate that flowering time can me manipulated by transgenic means. Lisianthus transformations with genes acting upstream to LFY are currently being made. The possibility of introducing a fragrance to lisianthus flowers by genetic engineering is being investigated using genes from the scented plant Clarkia breweri: Acetyl CoA benzyl alcohol acetyl transferase (BEAT) and S-linalool synthase (LIS). These two genes code for key enzymes in the synthesis of the aroma compounds benzyl acetate and S-linalool respectively. Aroma modification of the fruit of transgenic tomato plants carrying a LIS construct has already been demonstrated. The effect of BEAT and LIS transgenes on tobacco and lisianthus fragrance is under investigation.

Acetyl-CoA:benzylalcohol acetyltransferase--an enzyme involved in floral scent production in Clarkia breweri.

Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but a 35-residue region from position 135-163 has significant similarity (42-56% identity) to several proteins known or suspected to use an acyl-CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.

Floral Scent Production in Clarkia breweri1

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.