Ozone Treatment as an Approach to Induce Specialized Compounds in Melissa officinalis Plants.

Plants are constantly subjected to environmental changes that deeply affect their metabolism, leading to the inhibition or synthesis of "specialized" compounds, small organic molecules that play a fundamental role in adaptative responses. In this work, Melissa officinalis L. (an aromatic plant broadly cultivated due to the large amounts of secondary metabolites) plants were exposed to realistic ozone (O3) dosages (80 ppb, 5 h day-1) for 35 consecutive days with the aim to evaluate its potential use as elicitor of specialized metabolite production. Ozone induced stomatal dysfunction throughout the whole experiment, associated with a low photosynthetic performance, a decrease in the potential energy conversion activity of PSII, and an alteration in the total chlorophyll content (-35, -36, -10, and -17% as average compared to the controls, respectively). The production of hydrogen peroxide at 7 days from the beginning of exposure (+47%) resulted in lipid peroxidation and visible injuries. This result suggests metabolic disturbance within the cell and a concomitant alteration in cell homeostasis, probably due to a limited activation of antioxidative mechanisms. Moderate accumulated doses of O3 triggered the accumulation of hydroxycinnamic acids and the up-regulation of the genes encoding enzymes involved in rosmarinic acid, phenylpropanoid, and flavonoid biosynthesis. While high accumulated doses of O3 significantly enhanced the content of hydroxybenzoic acid and flavanone glycosides. Our study shows that the application of O3 at the investigated concentration for a limited period (such as two/three weeks) may become a useful tool to stimulate bioactive compounds production in M. officinalis.

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.

MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Development of a project for interprofessional collaboration between medical and pharmacy students to improve medication safety in polypharmacy (PILLE).

Aim: Interprofessional collaboration is particularly relevant to patient safety in outpatient care with polypharmacy. The educational project "PILLE" is meant to give medical and pharmacy students an understanding of the roles and competencies needed for cooperation in the provision of healthcare and to enable interprofessional learning. Method: The curriculum is aimed at pharmacy and medical students and was developed in six steps according to the Kern cycle. It is comprised of an interprofessional seminar, a joint practical training in a simulated pharmacy, and a tandem job shadowing at a primary care practice. The project was implemented in three stages due to the pandemic: The interprofessional online seminar based on the ICAP model and the digital inverted classroom was held in the 2020 winter semester; the interprofessional practical training was added in the 2021 summer semester; and the interprofessional tandem job shadowing at a primary care practice in the 2021 winter semester. Attitudes toward interprofessional learning, among other things, was measured in the evaluation using the SPICE-2D questionnaire (Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education). Results: In the first three semesters, a total of 105 students (46 pharmacy, 59 medicine) participated in the project, of which 78 participated in the evaluation (74% response rate). The students stated, in particular, that they had learned about the competencies and roles of the other profession and desired additional and more specific preparatory materials for the course sessions. The SPICE-2D questionnaire showed high values for both groups of students already in the pre-survey and these increased further as a result of the project. Conclusion: Joint case-based learning could be implemented under the conditions imposed by the pandemic. Online teaching is a low-threshold means to enable interprofessional exchange.

BAHD Company: The Ever-Expanding Roles of the BAHD Acyltransferase Gene Family in Plants.

Plants' ability to chemically modify core structures of specialized metabolites is the main reason why the plant kingdom contains such a wide and rich array of diverse compounds. One of the most important types of chemical modifications of small molecules is the addition of an acyl moiety to produce esters and amides. Large-scale phylogenomics analyses have shown that the enzymes that perform acyl transfer reactions on the myriad small molecules synthesized by plants belong to only a few gene families. This review is focused on describing the biochemistry, evolutionary origins, and chemical ecology implications of one of these families-the BAHD acyltransferases. The growth of advanced metabolomic studies coupled with next-generation sequencing of diverse plant species has confirmed that the BAHD family plays critical roles in modifying nearly all known classes of specialized metabolites. The current and future outlook for research on BAHDs includes expanding their roles in synthetic biology and metabolic engineering.

The first step into phenolic metabolism in the hornwort Anthoceros agrestis: molecular and biochemical characterization of two phenylalanine ammonia-lyase isoforms.

MAIN CONCLUSION: Two isoforms of phenylalanine ammonia-lyase (PAL) have been isolated as cDNA sequences from the hornwort Anthoceros agrestis. The encoded enzymes convert L-phenylalanine and to lower extents L-tyrosine and L-histidine. Thus, the functional presence of the general phenylpropanoid pathway in one of the earliest land plant groups is established. The hornwort Anthoceros agrestis has an elaborated phenolic metabolism resulting in phenolic compounds, such as rosmarinic acid or megacerotonic acid. The general phenylpropanoid pathway is involved in the biosynthesis of these compounds. Two phenylalanine ammonia-lyase (PAL) genes, AaPAL1 and AaPAL2, have been identified in Anthoceros agrestis and the protein with an N-terminal 6xHis-tag heterologously synthesized in Escherichia coli for a full biochemical characterization. Both PAL proteins accept L-phenylalanine, L-tyrosine as well as L-histidine as substrates, although the activity is explicitly the highest with L-phenylalanine. Km values as well as catalytic efficiencies were determined for phenylalanine (Km AaPAL1 39 µM, AaPAL2 18 µM) and tyrosine (Km AaPAL1 3.3 mM, AaPAL2 3.5 mM). In suspension cultures of Anthoceros agrestis, PAL genes were transcribed in parallel to rosmarinic acid (RA) accumulation and both showed highest abundance in the early growth phase. In a phylogenetic tree, both AaPAL amino acid sequences grouped within a clade with PAL amino acid sequences of diverse origin ranging from non-vascular to vascular plants, while most PALs from eudicots and monocots were mainly found in two other clades. The similarity of the hornwort PAL amino acid sequences to PAL sequences from vascular plants is more than 80% showing a strong conservation within the land plants. With this characterization of PALs from Anthoceros agrestis together with former investigations concerning cinnamic acid 4-hydroxylase and 4-coumaric acid CoA-ligase, the functional presence of the general phenylpropanoid pathway in this hornwort is proven.

Using an unmanned aerial system to analyse environmental impacts of charcoal production on tropical savanna ecosystems in northwestern Kenya.

In many regions of Sub-Saharan Africa, charcoal plays an important role as energy source but is widely perceived as a major driver of deforestation and forest degradation. This narrative, however, is mostly based on research within primary production regions. Though space-borne remote sensing applications can be useful in monitoring such large-scale production modes, environmental effects of household-level production are less easy to assess. Therefore, the present study employs an unmanned aerial system (UAS) to assess the impact of small-scale charcoal production on the vegetation density in the immediate vicinity of production sites. The UAS data was complemented by field measurements and very high-resolution WordView-2 satellite imagery. This approach revealed only small differences between charcoal production sites and reference plots which were usually evened out after 20-25-m distance to the plot centre using a concentric ring analysis. Results further show that a distinction between different land-use practices is difficult, even with the high spatial resolution provided by a UAS. Thus, more research and new approaches are needed to evaluate the role of small-scale charcoal production in deforestation and forest degradation processes against the background of other human activities. However, to exploit the full potential of UAS for monitoring environmental effects in charcoal producing areas, official regulations need to be clearer and more reliable.

Hydroxycinnamoyltransferase and CYP98 in phenolic metabolism in the rosmarinic acid-producing hornwort Anthoceros agrestis.

MAIN CONCLUSION: Anthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered. Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.

The evolution of the phenylpropanoid pathway entailed pronounced radiations and divergences of enzyme families.

Land plants constantly respond to fluctuations in their environment. Part of their response is the production of a diverse repertoire of specialized metabolites. One of the foremost sources for metabolites relevant to environmental responses is the phenylpropanoid pathway, which was long thought to be a land-plant-specific adaptation shaped by selective forces in the terrestrial habitat. Recent data have, however, revealed that streptophyte algae, the algal relatives of land plants, have candidates for the genetic toolkit for phenylpropanoid biosynthesis and produce phenylpropanoid-derived metabolites. Using phylogenetic and sequence analyses, we here show that the enzyme families that orchestrate pivotal steps in phenylpropanoid biosynthesis have independently undergone pronounced radiations and divergence in multiple lineages of major groups of land plants; sister to many of these radiated gene families are streptophyte algal candidates for these enzymes. These radiations suggest a high evolutionary versatility in the enzyme families involved in the phenylpropanoid-derived metabolism across embryophytes. We suggest that this versatility likely translates into functional divergence, and may explain the key to one of the defining traits of embryophytes: a rich specialized metabolism.

Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts.

MAIN CONCLUSION: Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for L-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of L-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying L-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC-MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

The Biosynthesis of Phenolic Compounds is an Integrated Defence Mechanism to Prevent Ozone Injury in Salvia officinalis.

Specialized metabolites constitute a major antioxidant system involved in plant defence against environmental constraints, such as tropospheric ozone (O3). The objective of this experiment was to give a thorough description of the effects of an O3 pulse (120 ppb, 5 h) on the phenylpropanoid metabolism of sage, at both biochemical and molecular levels. Variable O3-induced changes were observed over time among the detected phenylpropanoid compounds (mostly identified as phenolic acids and flavonoids), likely because of their extraordinary functional diversity. Furthermore, decreases in the phenylalanine ammonia-lyase (PAL), phenol oxidase (PPO), and rosmarinic acid synthase (RAS) activities were reported during the first hours of treatment, probably due to an O3-induced oxidative damage to proteins. Both PAL and PPO activities were also suppressed at 24 h from the beginning of exposure, whereas enhanced RAS activity occurred at the end of treatment and at the recovery time, suggesting that specific branches of the phenolic pathways were activated. The increased RAS activity was accompanied by the up-regulation of the transcript levels of genes like RAS, tyrosine aminotransferase, and cinnamic acid 4-hydroxylase. In conclusion, sage faced the O3 pulse by regulating the activation of the phenolic biosynthetic route as an integrated defence mechanism.

Phenolic metabolism in the hornwort Anthoceros agrestis: 4-coumarate CoA ligase and 4-hydroxybenzoate CoA ligase.

KEY MESSAGE: 4-Coumarate coenzyme A ligase and 4-hydroxybenzoate coenzyme A ligase from the hornwort Anthoceros agrestis expressed in E. coli were characterized on biochemical and molecular levels and showed interesting substrate specificities. Acyl-activating enzymes are associated with the biosynthesis or degradation of various metabolic products such as lipids, amino acids, sugars, and natural compounds. In this work, cDNA sequences encoding 4-coumarate coenzyme A ligase (4CL) and 4-hydroxybenzoate coenzyme A ligase (4HBCL) were amplified from the hornwort Anthoceros agrestis. The coding sequences were expressed in E. coli and purified by Ni-chelate chromatography. The CoA ligases exhibited different substrate specificities. 4CL catalyzed the activation of 4-coumaric acid, 3-coumaric acid, 2-coumaric acid, caffeic acid, isoferulic acid, ferulic acid, and cinnamic acid but lacked activities towards sinapic acid and benzoic acids. In contrast, 4HBCL preferred 4-hydroxybenzoic acid and benzoic acid, but also accepted other benzoic acid derivatives except salicylic acid and 3-aminosalicylic acid. Furthermore, 4HBCL also activated isoferulic acid, cinnamic acid, 2-coumaric acid, 3-coumaric acid, 4-coumaric acid and caffeic acid, but lacked affinity for ferulic acid and sinapic acid. These substrate specificities could be related to the phenolic compounds identified in Anthoceros agrestis.

Functional expression and characterization of cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis in Physcomitrella patens.

KEY MESSAGE: Cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis (AaC4H) was functionally expressed in the moss Physcomitrella patens and characterized at biochemical and molecular levels. Cinnamic acid 4-hydroxylase (C4H), a cytochrome P450-dependent hydroxylase, catalyzes the formation of 4-coumaric acid (=4-hydroxycinnamic acid) from trans-cinnamic acid. In the hornwort Anthoceros agrestis (Aa), this enzyme is supposed to be involved in the biosynthesis of rosmarinic acid (a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid) and other related compounds. The coding sequence of AaC4H (CYP73A260) was expressed in the moss Physcomitrella patens (Pp_AaC4H). Protein extracts from the transformed moss showed considerably increased C4H activity driven by NADPH:cytochrome P450 reductase of the moss. Since Physcomitrella has own putative cinnamic acid 4-hydroxylases, enzyme characterization was carried out in parallel with the untransformed Physcomitrella wild type (Pp_WT). Apparent Km-values for cinnamic acid and NADPH were determined to be at 17.3 µM and 88.0 µM for Pp_AaC4H and 25.1 µM and 92.3 µM for Pp_WT, respectively. Expression levels of AaC4H as well as two Physcomitrella patens C4H isoforms were analyzed by quantitative real-time PCR. While PpC4H_1 displayed constantly low levels of expression during the whole 21-day culture period, AaC4H and PpC4H_2 increased their expression during the first 6-8 days of the culture period and then decreased again. This work describes the biochemical in vitro characterization of a cytochrome P450-dependent enzyme, namely C4H, heterologously expressed in the haploid model plant Physcomitrella patens.

Does the degree of osteoarthritis influence the clinical outcome after anatomic total shoulder arthroplasty?

BACKGROUND: The degree of preoperative osteoarthritis has been shown to influence the postoperative outcome and the patients' satisfaction rate in hip and knee joint replacement surgery. However, no corresponding information is available for total shoulder arthroplasty (TSA). We therefore set out to evaluate the influence of preoperatively measured end-stage osteoarthritis on the postoperative clinical outcome of TSA. METHODS: A retrospective analysis of 103 anatomic total shoulder replacements (96 patients) was performed. Patients were evaluated radiologically with X-rays in two planes and clinically using the Constant and Murley score (CS) and the self-reported satisfaction with the result. The degree of osteoarthritis was radiographically analyzed with the aid of the classifications according to Kellgren/Lawrence, Gerber, Guyette, and Allain and according to whether complete narrowing of the glenohumeral joint was present or not [bone-on-bone contact (BOB) or no bone-on-bone contact (No BOB)]. RESULTS: The clinical results of TSA did not differ significantly among the various stages of osteoarthritis in any of the classifications (p > 0.05). The CS was significantly higher postoperatively for both the BOB and the No BOB group (p < 0.0001). Patients with BOB had a significantly lower CS preoperatively than patients with No BOB (p = 0.0172). In addition, the preoperative pain level was significantly higher in patients with BOB (p = 0.014). Postoperatively, no significant difference in CS (p = 0.6738) was found between the BOB group and the No BOB group. The mean improvement in CS was not statistically significant (p = 0.2218). CONCLUSION: In contrast to hip and knee joint replacement procedures, a milder grade of osteoarthritis does not adversely influence the functional result or subjective satisfaction rate after TSA. The degree of osteoarthritis on conventional X-rays has no bearing on the postoperative clinical outcome. Therefore, the decision on when to carry out anatomic total shoulder arthroplasty should depend on the patient's pain level and loss of quality of life.

A BAHD hydroxycinnamoyltransferase from Actaea racemosa catalyses the formation of fukinolic and cimicifugic acids.

MAIN CONCLUSION: The nucleotide sequence of a BAHD hydroxycinnamoyltransferase was amplified from Actaea racemosa (Ranunculaceae) and expressed in E. coli. The protein catalysed the formation of cimicifugic acids and thus is named hydroxycinnamoyl-CoA:piscidic acid hydroxycinnamoyltransferase (ArHPT1; cimicifugic acid synthase). Actaea racemosa (syn. Cimicifuga racemosa) is known to contain triterpene lactone glycosides and cimicifugic acids. The latter are esters of various hydroxycinnamic or benzoic acids with piscidic or fukiic acid. Amplification of a nucleotide sequence from A. racemosa, that was already known as HCT1 from an EST approach, and its expression in E. coli resulted in a protein that was able to catalyse the formation of several cimicifugic acids. For the characterisation of this hydroxycinnamoyltransferase (hydroxy)cinnamoyl-coenzyme A thioesters were synthesised as donor substrates and piscidic acid isolated as acceptor substrate. The lowest Km-value with 6.8 µM was determined for p-coumaroyl-CoA. More than 30 possible acceptor substrates were tested, but only piscidic acid and putatively fukiic acid were accepted. The apparent Km-value for piscidic acid was 32.3 µM. High expression of the hydroxycinnamoyltransferase gene was found in roots, but the content of cimicifugic acids was higher in leaves and flowers than in roots. This work describes for the first time a biosynthetic step in the formation of cimicifugic acids catalysed by a so far uncharacterised hydroxycinnamoyltransferase accepting piscidic acid as acceptor substrate thus being a hydroxycinnamoyl-CoA:piscidic acid hydroxycinnamoyltransferase (ArHPT1; cimicifugic acid synthase).

The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells.

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

Variation in the flavonolignan composition of fruits from different Silybum marianum chemotypes and suspension cultures derived therefrom.

Mature fruits collected from different milk thistle (Silybum marianum (L.) Gaertn.) plants, grown in various habitats in Europe, were analysed for silymarin content and variation in component composition. Two different German and Polish cultivars each, as well as fruits from Hungary and Bulgaria have been compared with respect to their ratio of flavonolignan regioisomers. Besides differences in total silymarin content (0.8%-4.9%), three distinct chemotypical variations in fruit flavonolignan regioisomer composition in the cultivars have been observed. Although the differences in the diastereomer ratios of silybin A/B and isosilybin A/B were not significant, they never appeared in a 1:1 ratio. In vitro cultures have been established from seedlings of three typical chemotypes for further insights into flavonolignan content and composition in suspension cultures and the release of these specialized compounds to the extracellular space. The differences in the three Silybum marianum chemotypes were also observed in the composition of the intracellular silymarin of suspension-cultured cells. Silymarin components released to the cell culture medium, however, showed a highly differing composition with only low amounts of silychristin and silydianin. Assays with crude protein extracts prepared from suspension cells or habituated medium of these three chemotypes did not result in differences in silymarin content or composition. In in vitro assays the formation of the regioisomers silydianin and silychristin were strongly influenced by the taxifolin:coniferyl alcohol concentration ratio.

How do background ozone concentrations affect the biosynthesis of rosmarinic acid in Melissa officinalis?

Lemon balm (Melissa officinalis; Lamiaceae) plants were exposed to background ozone (O3) dosages (80ppb for 5h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Immediately at the end of fumigation the plants appeared visually symptomless, but necrotic lesions were observed later. The biosynthesis of rosmarinic acid (RA) comprises eight enzymes, among them phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and rosmarinic acid synthase (RAS). The transcript levels of these genes have been investigated by quantitative RT-PCR. There was a quick up-regulation of all genes at 3h of O3 exposure, but at 24h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease of RA concentration in lemon balm leaves. The specific activity of PAL increased at 12h FBE to 163% in comparison to control levels. This work provides insight into the effect of O3 stress on the formation of the main phenolic ingredient of the pharmaceutically important plant M. officinalis.

How sensitive is Melissa officinalis to realistic ozone concentrations?

Lemon balm (Melissa officinalis, L.; Lamiaceae) was exposed to realistic ozone (O3) dosages (80 ppb for 5 h), because high background levels of O3 are considered to be as harmful as episodic O3 regimes. Temporal alterations of different ecophysiological, biochemical and structural parameters were investigated in order to test if this species can be considered as an O3-bioindicator regarding changes in background concentrations. At the end of ozone exposure, the plants did not exhibit any visible foliar symptoms, as only at microscopic level a small number of dead cells were found. Photosynthetic processes, however, were significantly affected. During and after the treatment, ozone induced a reduction in CO2 fixation capacity (up to 52% after 12 h from the beginning of the treatment) due to mesophyllic limitations. Intercellular CO2 concentration significantly increased in comparison to controls (+90% at the end of the post-fumigation period). Furthermore impairment of carboxylation efficiency (-71% at the end of the post-fumigation period compared to controls in filtered air) and membrane damage in terms of integrity (as demonstrated by a significant rise in solute leakage) were observed. A regulatory adjustment of photosynthetic processes was highlighted during the post-fumigation period by the higher values of qNP and (1-q(P)) and therefore suggests a tendency to reduce the light energy used in photochemistry at the expense of the capacity to dissipate the excess as excitation energy. In addition, the chlorophyll a/b ratio and the de-epoxidation index increased, showing a rearrangement of the pigment composition of the photosynthetic apparatus and a marked activation of photoprotective mechanisms.

Structural analysis of coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum reveals a novel active-site environment.

Coniferyl alcohol 9-O-methyltransferase from Linum nodiflorum (Linaceae) catalyzes the unusual methylation of the side-chain hydroxyl group of coniferyl alcohol. The protein was heterologously expressed in Escherichia coli as a hexahistidine derivative and purified for crystallization. Diffracting crystals were obtained of the pure protein and of its selenomethionine derivative, as well as of complexes with coniferyl alcohol and with S-adenosyl-L-homocysteine together with coniferyl alcohol 9-O-methyl ether (PDB entries 4ems, 4e70 and 4evi, respectively). The X-ray structures show that the phenylpropanoid binding mode differs from other phenylpropanoid O-methyltransferases such as caffeic acid O-methyltransferase. Moreover, the active site lacks the usually conserved and catalytic histidine residue and thus implies a different reaction mode for methylation. Site-directed mutagenesis was carried out to identify critical amino acids. The binding order of coniferyl alcohol and S-adenosyl-L-methionine was investigated by isothermal titration calorimetry experiments.

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.