Variability of Chalcone Synthase in Chamomile Accessions (Matricaria chamomilla).

Chamomile (Matricaria chamomilla) is an important medicinal plant whose beneficial activities partly rely on certain flavonoids. The first dedicated step in flavonoid biosynthesis is chalcone synthase (CHS, EC 2.3.1.74). The genomic DNA of CHS was studied in six chamomile specimens from different genotypes to describe interspecimen, as well as interspecific, variability. One specimen of M. discoidea was included as an outgroup. The two exons of CHS of M. chamomilla (McCHS) and M. discoidea (MdCHS) were 188 bp and 1,011 bp long, separated by an intron of variable length between 192 and 199 bp in McCHS and 201 bp in MdCHS, respectively. The two exons with 5.3 and 6.2 mutations per 100 bp, respectively, were more conserved than the intron with 11.5 mutations per 100 bp. In total, 96 SNPs were detected in both species, of which 12 SNPs were only present in MdCHS and 80 SNPs only in McCHS. Overall, 70 haplotypes (multilocus genotypes, MLGs) were detected. The samples could be classified into two groups, a 'compact' group of a low number and diversity of haplotypes and a 'variable' group of a high number and diversity of haplotypes. Of the 74 SNPs in McCHS, only six SNPs were non-synonymous. However, the amino acid changes did not affect critical areas of the enzyme. The combination of the six SNPs resulted in nine translated amino acid MLGs. The CHS network located MdCHS, due to the crossing barrier, quite distant from chamomile. MdCHS docked to McCHS at a position from where McCHS divergently evolved into two directions.

In vitro characterization of a (E)-ß-farnesene synthase from Matricaria recutita L. and its up-regulation by methyl jasmonate.

(E)-ß-farnesene is a sesquiterpene semiochemical that is used extensively by both plants and animals for communication. This acyclic olefin is found in the essential oil of chamomile (Matricaria recutita) and was demonstrated that it could attract natural enemies to reduce cabbage aphids in the Chinese cabbage fields. However, little is known regarding the sequence and function of (E)-ß-farnesene synthase in M. recutita. In this study, we reported a new full-length cDNA encoding (E)-ß-farnesene synthase from M. recutita (Mr-ßFS). The cDNA of Mr-ßFS consisted of 2010bp including 1725bp of coding sequence encoding a protein of 574 amino acids with a molecular weight of 67kDa. The deduced amino acid sequence exhibits a considerably higher homology to ßFS from Artemisia annua (about 92% identity) than to ßFSs from other plants (about 20-40% identity). The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, (E)-ß-farnesene, from farnesyl diphosphate. Real-time quantitative PCR (qRT-PCR) analysis showed that Mr-ßFS expression was highest in leaves and lowest in disk florets. The treatment of M. recutita with methyl jasmonate (MeJA) significantly enhanced the transcriptional level of ßFS gene and the content of (E)-ß-farnesene in M. recutita. The transcriptional level of ßFS gene was approximately 11.5-fold higher than the control sample and the (E)-ß-farnesene emission level ranged from approximately from 0.082 to 0.695µg/g after 24h induction. Our results laid a solid foundation for later improving crop aphid resistance by transgenic technology and provided an important basic data for the regulation of valuable products from M. recutita.

Cloning and characterization of a farnesyl pyrophosphate synthase from Matricaria recutita L. and its upregulation by methyl jasmonate.

Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.

Enantioselective microbial synthesis of the indigenous natural product (-)-α-bisabolol by a sesquiterpene synthase from chamomile (Matricaria recutita).

(-)-α-Bisabolol, a sesquiterpene alcohol, is a major ingredient in the essential oil of chamomile (Matricaria recutita) and is used in many health products. The current supply of (-)-α-bisabolol is mainly dependent on the Brazilian candeia tree (Eremanthus erythropappus) by distillation or by chemical synthesis. However, the distillation method using the candeia tree is not sustainable, and chemical synthesis suffers from impurities arising from undesirable α-bisabolol isomers. Therefore enzymatic synthesis of (-)-α-bisabolol is a viable alternative. In the present study, a cDNA encoding (-)-α-bisabolol synthase (MrBBS) was identified from chamomile and used for enantioselective (-)-α-bisabolol synthesis in yeast. Chamomile MrBBS was identified by Illumina and 454 sequencing, followed by activity screening in yeast. When MrBBS was expressed in yeast, 8 mg of α-bisabolol was synthesized de novo per litre of culture. The structure of purified α-bisabolol was elucidated as (S,S)-α-bisabolol [or (-)-α-bisabolol]. Although MrBBS possesses a putative chloroplast-targeting peptide, it was localized in the cytosol, and a deletion of its N-terminal 23 amino acids significantly reduced its stability and activity. Recombinant MrBBS showed kinetic properties comparable with those of other sesquiterpene synthases. These data provide compelling evidence that chamomile MrBBS synthesizes enantiopure (-)-α-bisabolol as a single sesquiterpene product, opening a biotechnological opportunity to produce (-)-α-bisabolol.

Tissue and method specificities of phenylalanine ammonia-lyase assay.

A large number of studies have estimated phenylalanine ammonia-lyase (PAL) activity because it strongly reacts to various stimuli. Activity of this enzyme has been assayed mainly by means of spectrophotometry, but the precision of this method is poorly known. We compared assays of PAL activity using spectrophotometry and high performance liquid chromatography (HPLC) in two species (Matricaria chamomilla and Arabidopsis thaliana). Additionally, copper-exposed M. chamomilla plants and buffer with additive were also tested. Our data indicate that spectrophotometry both overestimates (leaves of M. chamomilla) and underestimates (leaves and roots of A. thaliana) PAL activity in comparison with HPLC, suggesting interference of UV-absorbing metabolites. HPLC also showed more accurate detection of cinnamic acid in Cu-exposed chamomile roots. Addition of dithiothreitol to the extraction buffer enhanced PAL activity but reduced proteins, indicating an artificial negative effect. A comparison of PAL activity in selected species is also provided.

Significance of phenols in cadmium and nickel uptake.

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.

Nitric oxide signals ROS scavenger-mediated enhancement of PAL activity in nitrogen-deficient Matricaria chamomilla roots: side effects of scavengers.

Owing to the abundance of phenolic metabolites in plant tissue, their accumulation represents an important tool for stress protection. However, the regulation of phenolic metabolism is still poorly known. The regulatory role of reactive oxygen species (ROS) in the activity of phenylalanine ammonia-lyase (PAL) in nitrogen (N)-deficient chamomile roots treated for 24 h was studied using three ROS scavengers [dithiothreitol (DTT), salicylhydroxamic acid, and sodium benzoate]. Scavengers decreased the level of hydrogen peroxide and/or superoxide (and up-regulated ascorbate/guaiacol peroxidase and glutathione reductase), but, surprisingly, stimulated PAL activity. This up-regulation was correlated with increases in nitric oxide (NO) content, total soluble phenols, selected phenolic acids, and, partially, lignin (being expressed the most in DTT-exposed roots). We therefore tested the hypothesis that NO may be involved in these changes. Application of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) decreased PAL activity and the accumulation of soluble phenols in all treatments. Exogenous H(2)O(2) and NO also stimulated PAL activity and the accumulation of phenols. We conclude that NO, in addition to hydrogen peroxide, may regulate PAL activity during N deficiency. The anomalous effect of PTIO on NO content and possible mechanism of ROS scavenger-evoked NO increases in light of the current knowledge are also discussed.

Phenolic metabolism of Matricaria chamomilla plants exposed to nickel.

We examined accumulation of phenolic acids, total soluble phenolics and flavonoids, and activities of phenolic metabolism-related enzymes (shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO)) in Matricaria chamomilla plants exposed to 3, 60 and 120 microM of nickel (Ni) for 10 days. Ni showed low toxicity as indicated by unaltered content of total soluble phenolics in the leaf rosettes. In the roots, the effects of Ni were more visible, including increased total phenolics and PAL activity, but a decrease in PPO activity was observed. CAD activity was not affected by any of the Ni concentrations. Cinnamic acid derivatives were affected more than benzoic acid derivatives. Accumulation of chlorogenic acid, an important antioxidant compound, was enhanced by Ni treatment (ca. 4-fold in 120 microM Ni). Accumulation of protocatechuic acid, a phenol with high chelating strength, even decreased in the leaf rosettes. These observations are discussed in connection to antioxidative properties of phenolic metabolites and previously tested metals (cadmium and copper).

Oxidative status of Matricaria chamomilla plants related to cadmium and copper uptake.

Cadmium (Cd) and copper (Cu) uptake by the plants of Matricaria chamomilla and relation to activities of guaiacol peroxidase (GPX, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) up to 7 days of exposure to 3, 60 and 120 microM Cd or Cu was studied. Cd content in rosettes was ca. 10-fold higher in comparison to Cu while Cu was preferentially accumulated in the roots. In line with this observation, increase of CAT and GPX activity was similar in rosettes of Cd and Cu-treated plants, indicating non-redox active properties of Cd and low Cu accumulation. In the roots, Cu showed strong pro-oxidant effect, as judged from extreme stimulation of CAT and GPX, followed by increase of hydrogen peroxide and malondialdehyde. However, GPX seemed to be more important for alleviation of oxidative stress (ca. 93-250-fold higher activity in 120 microM Cu-treated roots). Cd had substantially lower influences and stimulated GR activity more than Cu. Activities of hydrogen peroxide-scavenging enzymes in relation to its accumulation are also discussed.