Andrea Balažová

Wounding triggers complex secondary metabolic pathways in plants, including benzylisoquinoline alkaloid (BIA) biosynthesis in opium poppy (Papaver somniferum L.). This study explores transcriptional and metabolic responses to wounding and methyl jasmonate (MeJA) treatment, focusing on BIA biosynthesis and regulatory mechanisms. Real-time expression analysis revealed significant up-regulation of transcripts in the (S)-reticuline and papaverine biosynthetic pathway, while the noscapine pathway was suppressed. The morphinan pathway also showed transcriptional activation, except in the case of codeinone reductase (COR), which remained unresponsive to both wounding and MeJA, suggesting a partially uncoupled mechanism. Metabolite profiling using HPLC-MS demonstrated a rapid accumulation of morphine post wounding, further supporting the hypothesis of independent regulatory control over COR. The role of phospholipase C (PLC) in modulating wound-induced BIA accumulation was investigated, revealing that PLC inhibition reduced morphine production and suppressed COR expression. These findings highlight the importance of phospholipid-dependent signalling in activating morphine biosynthesis, potentially at the expense of other BIAs. This study provides insights into plant stress responses and suggests strategies for enhancing BIA production through targeted interventions, offering potential applications in improving alkaloid yield.

Plants produce a wide variety of secondary metabolites, many of which have complex structures. Wild and cultivated plants currently remain the primary sources of most pharmaceutically significant secondary metabolites. However, the production of these compounds in plants is limited by the capacity of their biosynthetic pathways. This limitation has prompted efforts to develop methods to increase the yield of desired secondary metabolites. Advances in "omic" techniques have accelerated the development of approaches that utilize plants as bio-factories to produce these valuable compounds. This review provides an overview of strategies for producing high-value plant products and using plants as heterologous hosts to produce foreign recombinant proteins for medical applications.

Jasmonates, derivatives of jasmonic acid, are lipid-derived oxylipins with cyclopentanone ring, produced as a result of dioxygenation mediated by lipoxygenases. The biosynthesis of jasmonic acid is catalysed by a series of different enzymes, starting from lipoxygenase, allene oxide synthase, allene oxide cyclase and 12-oxophytodienoic acid reductase to the final three rounds of β-oxidation. Consequently, a few metabolites known as jasmonates are formed from the resulting jasmonic acid. Some of these metabolites are active and able to work on the jasmonate receptor and thus start the jasmonate signalling pathway. Products of gene expression resulting from this pathway have a great significance in plant growth, development, reproduction, senescence and also in defence and adaptation to various adverse environmental conditions.

Plants are considered as the "masters" of epigenetic regulation. Being exposed to many environmental factors during the vegetation period, they, have developed mechanisms on the molecular level that help them rapidly and reversibly change their epigenetic status and survive environmental stress. Recent studies document the importance of epigenetic control in plant responses to environmental stress through changes in gene activity and expression, resulting in metabolic pathways modifications as well. This brief review focuses on epigenetic modifications induced by an abiotic stress and on the effects of changes in phospholipid signalling, especially concerning the formation of secondary metabolites in plants.

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4′-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl₂ (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4′-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl₂ in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Aim of the study was to evaluate activity of plant extracts of Agrimonia eupatoria L. (agrimony – AE) and Cynara cardunculus L. (artichoke – CC) in streptozotocin-induced diabetic rats. Male rats were divided into four groups: control group (C), untreated diabetic group (UD), AE and CC treated diabetic groups. During a five-week period, changes in blood glucose and body weight were monitored. Then rats were sacrificed and subjected to the assessment of changes in the reactivity of aortas and measurement of butyrylcholinesterase (BuChE) activity (Table 1) [1]. Long-term administration of extracts did not reverse the negative influence of streptozotocin administration on the body weight. To complete the panel of experiments, α-glucosidase activity was assessed in vitro. Results showed that AE water extract is a potent α-glucosidase inhibitor with IC₅₀= 46.31 ± 8.76 µg/mL. IC₅₀ of CC water extract on α-glucosidase activity was not determined. Our results demonstrate that both compared extracts exert similar activity on selected DM parameters in streptozotocin-induced diabetic rats. Acknowledgements: This work was supported by the Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA-1/0299/13, VEGA-1/0646/14, VEGA-1/0048/15). Keywords: Diabetes mellitus, agrimony, artichoke. References: [1] Marek CB, Itinose AM, Marinho Jorge TC, Parizotto RA, Tebaldi NR, Reichert AM. Influence of leaf extracts from Melia azedarach L. on butyrylcholinesterase activity in rat liver. J Med Plants Res 2012; 6: 3931 – 3938

Diabetes mellitus (DM) is frequently diagnosed at a time when patients already suffer from several cardiovascular complications. Our previously published data (Molecules 201520 (11): 20538-50) on the anti-oxidative properties of Agrimonia eupatoria L. (AE) and Cynara cardunculus L. (CC) prompted us to extend the available evidence on their possible protective activities on selected DM-related parameters in a streptozotocin-induced DM rat model and in a series of in vitro experiments. Male rats were divided into four groups: control group, untreated diabetic group, AE and CC treated diabetic groups. During a five-week period, changes in blood glucose and body weight were monitored. Then, rats were sacrificed and subjected to the assessment of changes in the reactivity of aortas and measurement of butyrylcholinesterase activity. To complete the panel of experiments, α-glucosidase activity was assessed in vitro. Our results demonstrate that both tested extracts exert similar anti-diabetic activities. However, better anti-oxidant activity of the A. eupatoria extract indicates its higher clinical potential in the prevention and/or adjuvant therapy of developing cardiovascular complications related to DM and diseases associated with oxidative stress.

Our recent study is focussed on the investigation of the influence of biotic (Botrytis cinerea, methyl jasmonate) and abiotic (cadmium and cobalt chloride) elicitors on Melissa officinalis L. (lemon balm) suspension cultures production properties. The plant material was treated with different concentrations of methyl jasmonate (10 and 100 μmol.l⁻¹), with a phytopathogenic fungus hydrolyzate (Botrytis cinerea, 1 and 2 ml with glucose equivalent 24 μg.ml⁻¹), cadmium and cobalt (II) chloride (both at concentrations 100 μmol.l⁻¹ and 1 μmol.l⁻¹). Elicited suspension cultures were harvested after 24, 48 and 72 h of elicitor treatment. The elicitation effect was evaluated based on hydroxycinnamic acid derivatives content expressed as rosmarinic acid in dose- and time-dependent manners. The Botrytis cinerea hydrolyzate exhibited the best effect on Melissa officinalis L. suspension cultures production properties among biotic elicitors (glucose equivalent 24 mg.ml⁻¹, 1 ml, after 72 h of treatment). The hydroxycinnamic acid derivatives content increased in triplicate compared to non-elicited samples. Methyl jasmonate affected the suspension cultures production properties moderately at concentration 10 μmol.l⁻¹. Chlorides salts of cobalt and cadmium stimulated phenolic compounds production effectively at concentrations 100 μmol.l⁻¹. The presence of cobalt and cadmium ions in suspension cultures growth media increased the hydroxycinnamic acid derivatives content twofold and quadruple, respectively.

Greater celandine, similarly as other plants of the family Papaveraceae, produces benzylisoquinoline alkaloids, primarily benzophenanthridines. Polyphenoloxidase (PPO) is most probably involved in the formation of dopamine, which is one of the precursors of norcoclaurine, the first intermediate with the benzylisoquinoline structure. This study has revealed that PPO present in the latex of greater celandine is localized in the organelles, which serve to store alkaloids (the so-called 1000 g organelles). The enzyme was purified by means of affinity chromatography into electrophoretic homogeneity. It possesses a relative molecular mass of approximately 65 kDa and exerts two activities, the monophenolase and diphenolase ones. With the use of a polymerase chain reaction, it was possible to amplify a part of the PPO gene from the region of the active site.