Dataset Information

Identification and Characterization of Five BAHD Acyltransferases Involved in Hydroxycinnamoyl Ester Metabolism in Chicory.

ABSTRACT: Chicory (Cichorium intybus) accumulates caffeic acid esters with important significance for human health. In this study, we aim at a better understanding of the biochemical pathway of these bioactive compounds. Detailed metabolic analysis reveals that C. intybus predominantly accumulates caftaric and chicoric acids in leaves, whereas isochlorogenic acid (3,5-diCQA) was almost exclusively accumulated in roots. Chlorogenic acid (3-CQA) was equally distributed in all organs. Interestingly, distribution of the four compounds was related to leaf age. Induction with methyljasmonate (MeJA) of root cell suspension cultures results in an increase of 3-CQA and 3,5-diCQA contents. Expressed sequence tag libraries were screened using members of the BAHD family identified in Arabidopsis and tobacco as baits. The full-length cDNAs of five genes were isolated. Predicted amino acid sequence analyses revealed typical features of BAHD family members. Biochemical characterization of the recombinant proteins expressed in Escherichia coli showed that two genes encode HCTs (hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferases, HCT1 and HCT2) whereas, three genes encode HQTs (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferases, HQT1, HQT2, and HQT3). These results totally agreed with the phylogenetic analysis done with the predicted amino acid sequences. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT3, HCT1, and HCT2 might be more directly associated with CQA accumulation in cell culture in response to MeJA elicitation. Transient expression of HCT1 and HQT1 in tobacco resulted in a higher production of 3-CQA. All together these data confirm the involvement of functionally redundant genes in 3-CQA and related compound synthesis in the Asteraceae family.

SUBMITTER: Legrand G

PROVIDER: S-EPMC4893494 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification and Characterization of Five BAHD Acyltransferases Involved in Hydroxycinnamoyl Ester Metabolism in Chicory.

Legrand Guillaume G Delporte Marianne M Khelifi Chahinez C Harant Adeline A Vuylsteker Christophe C Mörchen Monika M Hance Philippe P Hilbert Jean-Louis JL Gagneul David D

Frontiers in plant science 20160606

Chicory (Cichorium intybus) accumulates caffeic acid esters with important significance for human health. In this study, we aim at a better understanding of the biochemical pathway of these bioactive compounds. Detailed metabolic analysis reveals that C. intybus predominantly accumulates caftaric and chicoric acids in leaves, whereas isochlorogenic acid (3,5-diCQA) was almost exclusively accumulated in roots. Chlorogenic acid (3-CQA) was equally distributed in all organs. Interestingly, distribu ...[more]

PMID: 27375627

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Project description:Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1) have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato (Solanum lycopersicum cv. Micro-Tom) orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants, respectively. Expression of SlPSAT1 in the psat1-2 background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs) while, in contrast to the endoplasmic reticulum (ER) localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM). The possibility that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of SlPSAT1 and SlASAT1 genes in different tomato organs together with their moderate transcriptional response to several stresses suggests a dual role of SlPSAT1 and SlASAT1 in tomato plant and fruit development and the adaptive responses to stress. Overall, this study contributes to enlarge the current knowledge on plant sterol acyltransferases and set the basis for further studies aimed at understanding the role of SE metabolism in tomato plant growth and development.

Project description:Plant acyl-CoA dominated acyltransferases (named BAHD) comprise a large appointed protein superfamily and play varied roles in plant secondary metabolism like synthesis of modified anthocyanins, flavonoids, volatile esters, etc. Tea (Camellia sinensis) is an important non-alcoholic medicinal and fragrancy plant synthesizing different secondary metabolites, including flavonoids. In the tea (C.A sinensis) cultivar Longjing 43 (LJ43), eight samples were performed into three groups for transcriptome analysis under three biological replications. Among the BAHD acyltransferase genes in tea cultivars, the expression of TEA031065 was highest in buds and young leaves following the RNA sequencing data, which was coincident with the tissue rich in catechins and other flavonoids. We then transformed this gene into wild-type Arabidopsis as an over-expression (OX) line 1 and line 2 in ½ MS media to verify its function. In the wild types (WT), the primary root length, number of secondary roots, and total root weight were significantly higher at 24%, 15%, and 53.92%, respectively, compared to the transgenic lines (OX1 and OX2). By contrast, the leaves displayed larger rosettes (21.58%), with higher total leaf weight (32.64%) in the transgenic lines than in the wild type (WT). This result is consistent with DCR mutant At5g23940 gene in Arabidopsis thaliana. Here, anthocyanin content in transgenic lines was also increased (21.65%) as compared to WT. According to the RNA sequencing data, a total of 22 growth regulatory genes and 31 structural genes with TFs (transcription factors) that are correlative with plant growth and anthocyanin accumulation were identified to be differentially expressed in the transgenic lines. It was found that some key genes involved in IAA (Auxin) and GA (Gibberellin) biosynthesis were downregulated in the transgenic lines, which might be correlated with the phenotype changes in roots. Moreover, the upregulation of plant growth regulation genes, such as UGT73C4 (zeatin), ARR15, GH3.5, ETR2, ERS2, APH4, and SAG113 might be responsible for massive leaf growth. In addition, transgenic lines shown high anthocyanin accumulation due to the upregulation of the (1) 3AT1 and (3) GSTF, particularly, GSTF12 genes in the flavonoid biosynthesis pathway. However, the TFs such as, CCoAMT, bHLH, WRKY, CYP, and other MYBs were also significantly upregulated in transgenic lines, which increased the content of anthocyanins in A. thaliana seedlings. In conclusion, a BAHD acyltransferase (TEA031065) was identified, which might play a vital role in tea growth and secondary metabolites regulation. This study increases our knowledge concerning the combined functionality of the tea BAHD acyltransferase gene (TEA031065).

Project description:Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5' and 3' rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thioesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species' pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.

Dataset Information

Identification and Characterization of Five BAHD Acyltransferases Involved in Hydroxycinnamoyl Ester Metabolism in Chicory.

Publications

Identification and Characterization of Five BAHD Acyltransferases Involved in Hydroxycinnamoyl Ester Metabolism in Chicory.

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