Proline-linked Pentose Phosphate Pathway Research Articles

Global profiling of ultraviolet-induced metabolic disruption in Melissa officinalis by using gas chromatography-mass spectrometry

Melissa officinalis contains various secondary metabolites that have health benefits. Generally, irradiating plants with ultraviolet (UV)-B induces the accumulation of secondary metabolites in plants. To understand the effect of UV-B irradiation on the metabolism of M. officinalis, metabolomics based on gas chromatography-mass spectrometry (GC-MS) was used in this study. The GC-MS analysis revealed 37 identified metabolites from various chemical classes, including alcohols, amino acids, inorganic acids, organic acids, and sugars. The metabolite profiles of the groups of M. officinalis irradiated with UV-B were separated and differentiated according to their irradiation times (i.e., 0, 1, and 2h), using principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The PCA score plots of PC1 and PC2 showed that the three groups with different irradiation times followed a certain trajectory with increasing UV-B irradiation. HCA revealed that metabolic patterns differed among the three groups, and the 1h-irradiated group was more similar to the control group (0h) than the 2h-irradiated group. In particular, UV-B irradiation of plants led to a decrease in sugars such as fructose, galactose, sucrose, and trehalose and an increase in metabolites in the tricarboxylic acid cycle, the proline-linked pentose phosphate pathway, and the phenylpropanoid pathway. This study demonstrated that metabolite profiling with GC-MS is useful for gaining a holistic understanding of UV-induced changes in plant metabolism.

Metabolite profiling of sucrose effect on the metabolism of Melissa officinalis by gas chromatography-mass spectrometry

The effect of sugar on plant metabolism, which is known to be similar to hormone-like signaling, was metabolomically studied using Melissa officinalis (lemon balm). The metabolite profiles of M. officinalis treated with sucrose were analyzed by gas chromatography-mass spectrometry (GC-MS) and principal component analysis (PCA). A total of 64 metabolites from various chemical classes including alcohols, amines, amino acids, fatty acids, inorganic acids, organic acids, phosphates, and sugars were identified by GC-MS. Three groups treated with different sucrose concentrations were clearly separated by PCA of their metabolite profiles, indicating changes in the levels of many metabolites depending on the sucrose concentration. Metabolite profiling revealed that treatment with a higher sucrose level caused an increase in the levels of metabolites such as sugars, sugar alcohols, and sugar phosphates, which are related to the glycolytic pathway of M. officinalis. Furthermore, proline and succinic acid, which are associated with the proline-linked pentose phosphate pathway, the shikimic acid pathway, and the biosynthesis of phenylpropanoids, also increased with increasing sucrose concentration. Therefore, these metabolic changes induced by sucrose ultimately led to the increased production of flavonoids such as caffeic acid via the biosynthetic pathway of phenylpropanoids. This study demonstrated that the abundance changes in some primary and secondary metabolites were somewhat interlocked with each other in response to sucrose.

OVER-EXPRESSION OF PROLINE-LINKED ANTIOXIDANT PATHWAY AND MODULATION OF PHENOLIC METABOLITES IN LONG LIFE SPAN CLONAL LINE OFORIGANUM VULGAREIN RESPONSE TO ULTRAVIOLET RADIATION

ABSTRACT Tissue culture-generated shoot-based clonal lines of oregano that have a short and long life span during in vitro culture were investigated to determine the role of proline-linked pentose phosphate pathway in modulating phenolic response following ultraviolet (UV) exposure. This novel reductant cycling pathway for energy and oxidative pentose phosphate pathway (PPP) is more efficient in long life span O-17 clonal line in response to UV and this supported antioxidant response. UV treatment stimulated rosmarinic acid and associated antioxidant enzyme response in O-17 clonal line compared with short life span O-3 clonal line. Additionally, O-17 clonal line had higher glucose-6-phosphate dehydrogenase and guaicol peroxidase activity in response to UV exposure compared with O-3 clonal line, indicating stimulation of anabolic pathway supporting PPP during UV treatment. The stimulation of proline dehydrogenase (PDH) activity in O-17 clonal line in response to UV indicated a switch to PDH-linked energy pathway via proline oxidation. PRACTICAL APPLICATIONS This oregano clonal model of ultraviolet (UV)-induced oxidative stress management has implications for designing dietary phytochemicals to manage oxidation-linked disease in humans. Further, the correlation between UV-linked inducibility of phenolic-linked antioxidant enzyme response through proline-linked pentose phosphate pathway and life span of in vitro shoot cultures of specific oregano clonal lines can be a good model for aging studies. The metabolic response link to aging and protective phytochemicals can be investigated in oregano tissue cultures, and phenolic extracts from various responding clonal lines could be explored in other eukaryotic aging models.

ELICITATION OF THE PROLINE-LINKED PENTOSE PHOSPHATE PATHWAY METABOLITES AND ANTIOXIDANT ENZYME RESPONSE BY ASCORBIC ACID IN DARK GERMINATED FAVA BEAN SPROUTS

ABSTRACT Elicitation by ascorbic acid stimulated higher phenolic content in late stages of germination and higher antioxidant activity during early and late germination in fava bean sprouts. The high free radical scavenging-linked antioxidant activity on day 1 coincided with high levo-dihydroxy phenylalanine and total phenolics. A positive correlation between the stimulation of phenolic synthesis and glucose-6-phosphate dehydrogenase (G6PDH) activity was also observed, indicating its likely role in carbohydrate mobilization. Improved guaiacol peroxidase (GPX) activity in response to elicitation indicated the likely polymerization of soluble phenolics. Higher GPX, superoxide dismutase and catalase linked antioxidant enzyme response correlated to increase in proline and phenolics. A proline-linked stimulation likely provided nicotinamide adenine dinucleotidephosphate from pentose phosphate pathway (PPP) as indicated by an increase in G6PDH, the first committed step of PPP, which then supported phenolic synthesis and antioxidant enzyme response. PRACTICAL APPLICATIONS This research indicates that ascorbic acid is an effective elicitor of the pentose phosphate pathway (PPP) in dark germinated fava bean sprouts. It increases synthesis of health-relevant phenolics and levo-dihydroxy phenylalanine relevant to Parkinson's disease management. The stimulation of PPPwas linked to accumulation of free proline thereby supporting the proline-linked pentose phosphate pathway model. It provides an understanding of the role of phenolic antioxidants on redox modulation in the cell by being effective free radical scavengers and also by inducing the antioxidant enzyme responses through providing the reducing equivalents required for their activity. This knowledge can be used as a biochemical strategy to design seed sprouts for functional food applications.

Rhodiola‐induced inhibition of adipogenesis involves dysfunctional antioxidant response associated with proline‐linked pentose phosphate pathway

Recent research suggests that increased reactive oxygen species production from accumulated fat in obesity leads to elevated systemic oxidative stress, contributing to the development of obesity-linked diseases. The aim of this study was to investigate dietary phenolic-linked changes of key enzymes associated with endogenous antioxidant enzyme response (AER) and energy-linked pathways during the differentiation of 3T3-L1 adipocytes based on the proline-linked pentose phosphate pathway (PLPPP) model. We hypothesized that, in differentiating preadipocytes, proline dehydrogenase (PDH) activity may increase due to requirement of energy, which also can be coupled to enhanced glucose-6-phosphate dehydrogenase (G6PDH) activity required for NADPH generation necessary for stimulation of AER. Treatment of Rhodiola extracts high in phenolics inhibited the activity of PDH and G6PDH as well as the differentiation of 3T3-L1 adipocytes. The inhibition of PDH and G6PDH by Rhodiola likely prevents proline oxidation required for critical ATP generation that is coupled to AER via PPP. This indicates that Rhodiola can disrupt proline-linked energy generation and AER via PPP, leading to suppression of adipogenesis. This provides a biochemical rationale for studies to identify the roles of phenolic phytochemicals as anti-adipogenic agents that can modulate cellular redox environment in adipocytes.

Effect of vitamin C and folic acid on seed vigour response and phenolic-linked antioxidant activity

Folic acid and vitamin C were used in the concentration range of 0–500 μM as exogenous growth enhancers to stimulate pea ( Pisum sativum) seedling vigour. The results suggest that a concentration of 50 μM folic acid and 500 μM vitamin C were optimum in maximally enhancing seed vigour and potentially seedling performance according to both agronomic and biochemical seed vigour parameters. Results indicated that germination percentage, shoot weight, shoot height, and root length were enhanced in folic acid and vitamin C treated plants compared to control plants. The levels of enhanced phenolic content in response to folic acid and vitamin C treatments were highest on days 8 and 10. Evaluation of critical biochemical parameters indicated that the average glucose-6-phosphate dehydrogenase (G6PDH) activity and proline content in response to treatments were higher than control and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Key enzymes, guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) were also higher in response to treatments and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Taken together, these studies support the hypothesis that the proline-linked pentose phosphate pathway stimulates phenolic synthesis and related free-radical scavenging antioxidant activity. Further, this proline-linked pentose phosphate pathway stimulation in response to folic acid and vitamin C was also correlated to antioxidant enzyme response indicated by the stimulation of GPX, SOD, and CAT activities. Therefore, this study indicates the enhancement of seed vigour response by folic acid and vitamin C as reflected in both agronomic and biochemical responses, and this occurred through the stimulation of phenolic-linked antioxidant response that is likely positively modulated through the proline-linked pentose phosphate pathway.

Cranberry phenolics-mediated antioxidant enzyme response in oxidatively stressed porcine muscle

The antioxidant response mechanism by which phenolic phytochemicals show their positive benefits in animal systems is not very well understood. The ability of cranberry juice powder (CP), ellagic acid (EA), rosmarinic acid (RA) and their synergies to mediate a cellular antioxidant response in oxidatively stressed porcine muscle tissue was investigated. Results indicated that treatment with CP, EA, RA and their synergies reduced or helped counter oxidative stress as indicated by the formation of malondialdehyde (MDA). It was also observed that CP, EA, RA and their synergies stimulated the pentose phosphate pathway (PPP) linked to the accumulation of free proline suggesting a possible coupling of proline biosynthesis with PPP. This coupling of proline-linked pentose phosphate pathway could be involved in the stimulation of cellular antioxidant enzymic response by replenishing the cellular needs for NADPH 2. As a consequence these exogenous phenolic treatments resulted in the stimulation of cellular antioxidant enzyme systems involving superoxide dismutase (SOD), catalase (CAT) and peroxidase, which correlated well with the decreased MDA formation. This suggested that exogenously treated phenolic phytochemicals could be reducing the oxidative stress in porcine muscle by stimulating the PPP linked to proline biosynthesis and by the activation of the cellular antioxidant enzyme system. The results also suggest that pure exogenous phenolics, EA and RA appeared to be effective when they were present in a cranberry phenolic background, suggesting a possible synergistic mode of action between EA, RA and cranberry phenolics in mediating a cellular antioxidant enzyme response.

A Hypothetical Model for Action of Soybean Isoflavonoids Against Cancer Involving a Shift to Proline-Linked Energy Metabolism Through Activation of the Pentose-Phosphate Pathway

Soybean isoflavonoids and related phenolic antioxidants from other dietary plant species have long been associated with numerous biological activities linked to the chemoprevention of cancers and oxidation-linked diseases. But until now, no mechanisms or models have been put forth to explain how all of these activities of dietary phenolics could work together to promote health and protect from disease. Here, we present a hypothetical model based on the soybean isoflavonoid genistein for the beneficial action of dietary phenolic antioxidants that incorporates not only the known molecular activities, but also the known metabolic effects that dietary phenolics have on cellular systems such as energy metabolism and the antioxidant enzyme response to reactive oxygen species. Our model presents in a clear manner how a cancerous cell, although mutated and dysfunctional on both biochemical and genetic levels, cannot escape its beginnings as a normal cell and the underlying response mechanisms that are retained from that heritage. By our model, we present how dietary phenolics such as genistein may trigger a switch in energy metabolism from an NADH-supported system to one supported by proline (via the proline-linked pentose-phosphate pathway) through the activation of a stress-response mechanism known to occur in normal cells during stress conditions and which may still remain in cancerous cells. Further, we postulate how an inability of cancer cells to disengage this stress response-induced change in energy metabolism due to biochemical and/or genetic mutations could result in the death of the diseased cell. This metabolic model has implications for the further understanding of how dietary phenolics may confer their beneficial effects and also for the design of chemopreventive agents and functional foods.

Phenolic Antioxidant Biosynthesis in Plants for Functional Food Application: Integration of Systems Biology and Biotechnological Approaches

We are applying a dynamic systems biology approach to the development of several phenolic phytochemicals in food-grade plants as ingredients for functional food applications. Phenolic antioxidant phytochemicals from food-grade plants will be an important part of a healthy diet in a global population that is projected to reach 9 billion in the next 50 years. Such phytochemicals are being targeted for designing conventional foods with added health benefits (functional foods). Such value-added foods are needed for dietary support to manage major oxidation-linked diseases, such as diabetes, cardiovascular disease, arthritis, cognition diseases and cancer. Plants produce phenolic metabolites as a part of growth, developmental and stress-adaptation response. These stress and developmental-modulated phenolic phytochemicals can be targeted for the design of functional foods. In order to design consistent food-grade phytochemical profiles for safety and clinical relevancy, novel tissue culture and bioprocessing technologies have been developed. The strategy for designing these phenolic phytochemicals is based on the model that phenolic metabolites in plants are efficiently produced through an alternative mode of metabolism that links proline synthesis to activity of the pentose–phosphate pathway. Using the proline-linked pentose–phosphate pathway model, techniques have been developed to isolate high phenolic clonal lines of food-grade plants from single heterozygous seeds. Further, using the same model, elicitation concepts and techniques have been applied to over-produce phenolic metabolites in seeds and sprouts. In both clonal and seed sprout systems, exogenous treatment of phenolic phytochemicals from a non-target species elicited endogenous stimulation of phenolic synthesis and, potentially, an antioxidant response. From these investigations, a hypothetical model has been proposed in which the proline-linked pentose–phosphate pathway is critical for modulating protective antioxidant response pathways in diverse biological systems, including humans. This model, when confirmed precisely, may provide a mechanism for understanding the mode of action of phenolic phytochemicals in modulating antioxidant pathways in relation to human health. An understanding of the interconnection of the proline-linked pentose–phosphate pathway and antioxidant response pathway can provide dietary and nutritional mechanisms as well as new strategies to manage oxidation-linked diseases through improvement of host physiological response. In this review we have focused on clonal herbs, fava bean sprouts and cranberry bioprocessing as 3 model systems for understanding biosynthesis of phenolic metabolites for functional food application.

Stimulation of total phenolics, L-DOPA and antioxidant activity through proline-linked pentose phosphate pathway in response to proline and its analogue in germinating fava beans ( Vicia faba)

The rationale behind the study was to enhance azetedine-2-carboxylate (A2C)-linked stress in the germinating seeds to which it responds by increased proline synthesis to overcome inhibition of proline dehydrogenase (PDH). A2C is a competitive inhibitor of proline that inhibits its transport from cytosol to the mitochondria for further metabolic recycling by binding on to the active sites of PDH. The enhanced synthesis of proline would increase the ratio of the cofactors NADP/NADPH 2. The increase in the cofactors would result in the over-expression of the phenypropanoid pathway required for the phenolic acids and L-DOPA synthesis via pentose phosphate pathway through the activity of glucose-6-phosphate dehydrogenase (G6PDH). Fava bean were chosen since it has high phenolic and L-DOPA levels and could be an important part of the diet especially for patients suffering from Parkinson's Disease. The objectives were investigated by assaying for total phenolic content, the corresponding antioxidant activity by β-carotene oxidation method, proline levels and enzymes such as G6PDH and guaiacol peroxidase (GPX) using spectrophotometric methods. L-DOPA was quantified using HPLC. The fava bean seeds were primed with water, 200 μM A2C, 0.25 mM proline and a combination of A2C and proline treatments. After the priming stage, seeds were dark germinated and grown for a period of 8 days, for biochemical analysis. L-DOPA levels did not change in comparison to the control treatments while total phenolic content, proline and G6PDH were all enhanced by the treatments. During the early stages of germination the phenolic acids were antioxidant in nature, which later was reduced as they become polymerized to lignins and lignans via the GPX activity. Total phenolic synthesis was both coupled and uncoupled to PLPPP depending on the treatments. The three treatments over-expressed PLPPP since large difference between control and the treatments were observed for all parameters, except L-DOPA content.

Effects of UV treatment on the proline-linked pentose phosphate pathway for phenolics and L-DOPA synthesis in dark germinated Vicia faba

Fava beans ( Vicia faba) have a high phenolic content. Antioxidant properties of phenolics are extensively studied for their ability to inhibit lipid oxidation in food and for improved health. This legume has the potential to be an effective dietary source of polyphenolics for health. The rationale behind this study was to observe the stimulation of nutraceutically relevant phenolic metabolites when germinating fava beans are exposed to ultra violet (UV) and the potential role of the proline-linked pentose phosphate pathway (PPP). Studies have shown phenolic phytochemicals can be stimulated with UV light. The objective was to over-express the PPP through the regulation of the key enzyme, glucose-6-phosphate dehydrogenase (G6PDH), for enhanced phenolics and L-DOPA production and investigate the link to proline metabolism. Guaiacol peroxidase (GPX) activity was monitored to understand the extent to which the UV-stimulated phenolics could be converted to polymeric phenols. L-DOPA is the precursor of dopamine (DA), the neuromodulator and used for treatment of Parkinson's disease. Seeds of fava beans were pretreated in water, exposed to 5, 10 and 15 h UV light and germinated for 8 days in dark. Total phenolics, proline levels and enzyme activities were measured spectrophotometrically. L-DOPA was measured by High-performance liquid chromatography (HPLC). A total of 15 h UV treatment stimulated L-DOPA on day 1, which reduced over next 8 days of germination. Antioxidant activity of the total phenolic acids was determined by inhibition of the β-carotene oxidation and there was stimulation of antioxidant activity in response to 10 h UV treatment on day 2. Total phenolic content increased 10-fold after 15 h UV light exposure on day 6 along with the highest activity of G6PDH. All treatments stimulated GPX activity indicating polymerization of free phenolics in the late stage of germination. Proline levels were also stimulated in response to all treatments. The stimulation of proline concurrent with stimulation of G6PDH and total phenolics in response to 15 h UV indicates a role of proline-linked PPP. Results indicate an increase in nutraceutically-relevant phenolic metabolites along with enhanced antioxidant activity in response to UV stress. This novel approach provides a mechanism to understand and enhance biosynthesis of important phenolic compounds in plant and legume systems for use in nutraceutical applications.

Biosynthesis and Medical Applications of Rosmarinic Acid

ABSTRACT Plant phenolics are a significant group of secondary metabolites which have diverse medicinal applications. Among various phenolics, rosmarinic acid is an important caffeoyl ester with proven medicinal properties and well-characterized physiological functions. Rosmarinic acid is found in substantial quantities in the family Lamiaceae with medicinal uses in several cultures. The major problem in the use of phytopharmaceuticals from the family Lamiaceae is the plant-to-plant variability of specific metabolites like rosmarinic acid. This is due to genetic heterogeneity, which results from cross-pollination common to all species in this family. Genetically uniform shoot-based clonal lines of several species in the family Lamiaceae have been isolated using tissue culture approaches. These genetically uniform clones can be used to breed elite varieties which target enhancement of specific metabolites. Such clonal lines are also useful for biochemical pathway characterization to provide genetic access to specific metabolites of value in medicine. Currently, genetically uniform clonal fines of oregano and thyme are being used to analyze the potential role of proline-linked pentose phosphate pathway in regulating rosmarinic acid biosynthesis. Characterization of this pathway will help to identify key regulatory genes involved in rosmarinic acid biosynthesis.

A model for the role of the proline-linked pentose phosphate pathway in polymeric dye tolerance in oregano

Polymeric dyes are aromatic hydrocarbon compounds that are similar to aromatic soil pollutants and inhibit plant growth. Polymeric dye-tolerant plants may be useful in phytoremediation because they could provide a rhizosphere suitable for colonization by microbes that are efficient degraders of aromatic compounds. Plants that produce high levels of phenolic compounds may be more tolerant of aromatic contaminants because of their inherent mechanisms for degrading or sequestering endogenous aromatic compounds. A clonal line of oregano ( Origanum vulgare) O-1 that is highly tolerant of the polymeric dye R-478 has been isolated. Over a time course of exposure to 0.1% Poly R-478, O-1 plants showed a progressive decrease in total phenolic compounds, whereas peroxidase activity and free proline levels increased. The inverse relationship between phenolics and peroxidase activity suggests a role for peroxidase-mediated lignification in polymeric dye tolerance. The increased level of free proline in response to polymeric dye exposure suggests that phenolic synthesis requires proline synthesis and/or oxidation. These results support our working model that aromatic pollutant-tolerance may be regulated through the proline-linked pentose phosphate pathway, a primary metabolic pathway.

INFLUENCE OF ACETYL SALICYLIC ACID IN COMBINATION WITH FISH PROTEIN HYDROLYSATES ON HYPERHYDRICITY REDUCTION AND PHENOLIC SYNTHESIS IN OREGANO (ORIGANUM VULGARE) TISSUE CULTURES

Tissue culture-generated shoot-based clonal lines of oregano are being used to investigate the role of proline-linked pentose phosphate pathway in stimulating the phenolic antioxidant, rosmarinic acid (RA) and lignification, which was reported to be linked to guaiacol peroxidase (GPX) activity. One of the problems in oregano tissue culture is hyperhydricity and modifications of media are being used to control this physiological malformation. This study reports the reduction of hyperhydricity, stimulation of RA biosynthesis and lignification in oregano clonal line O-1 and O-5 in response to acetyl salicylic acid (ASA), fish protein hydrolysate (FPH, standardized mackerel hydrolysates) and combination of FPH and ASA (FPH/ASA). All treatments reduced hyperhydricity of both clonal lines compared to control. Following exogenous treatment with ASA and FPH/ASA, enhanced total phenolic content, RA content and concurrently higher levels of GPX activity were observed compared to control and FPH treatments. The concentration of total phenolics and RA as well as GPX activity in O-5 clonal line was higher than in O-1 clonal line either on day 15 or day 30. Antioxidant activity of the phenolic extracts of all cultures was high on day 30 compared to day 15 and FPH/ASA treatment had the highest activity on both days. The concentration of chlorophyll in O-1 was higher than in 0–5 and the concentration in both lines were similar in response to respective treatments. The stimulation of RA synthesis in response to ASA and FPH/ASA provided strong clues that ASA can be used as an abiotic elicitor for RA stimulation and mobilization of proline and/or glutamic acid in FPH and this may be linked to stimulation of the pentose phosphate pathway, driving key precursor metabolites towards shikimate and phenylpropanoid pathways. RA-stimulating compounds also enhanced total phenolics and hardened stem tissues, which correlated with higher GPX activity, indicating possible lignification due to polymerization of phenolic metabolites. This research also provides strategies to prevent hyperhydricity in tissue culture by ASA and combination of FPH/ASA and these were linked to lignification, high levels of total phenolics and RA. This improvement is important for efficiency and quality of in vitro plant tissue propagation and outdoor transplanting of elite phenolic antioxidant-producing oregano cultures. This research also provides insight into regulation of rosmarinic acid, a phenolic antioxidant relevant for food preservative applications.

Stimulation of benzyladenine-induced in vitro shoot organogenesis and endogenous proline in melon (Cucumis melo L.) by fish protein hydrolysates in combination with proline analogues.

A previous study demonstrated that proline is beneficial for improving melon in vitro shoot organogenesis. A natural source of proline and proline precursors can be obtained from fish protein hydrolysates (FPH), a byproduct of the fishery industry. Proline analogues azetidine-2-carboxylate and hydroxyproline in combination with standardized FPH were used to stimulate proline synthesis and benzyladenine-induced shoot organogenesis by exploiting the proposed proline-linked pentose phosphate pathway (PPP). In the presence of elevated levels of endogenous proline, potential stimulation of cytokinins and auxin may occur via the PPP and shikimate pathways, respectively. Treatments with FPH singly and in combination with the above proline analogues significantly increased the endogenous proline content and the extent of differentiation, suggesting that in vitro organogenesis is closely linked to proline synthesis, strengthening the hypothesis that purine metabolism via the proline-linked PPP may be important for organogenesis. Thioproline addition resulted in increased proline levels but without corresponding stimulation of organogenesis. This study also provides potential use of fishery waste for value-added application in plant micropropagation industry.

SOMATIC EMBRYOGENESIS IN ANISE (PIMPINELLA ANISUM L.): THE EFFECT OF PROLINE ON EMBRYOGENIC CALLUS FORMATION AND ABA ON ADVANCED EMBRYO DEVELOPMENT

Somatic embryos of anise (Pimpinella anisum) are an excellent system to investigate the biosynthesis of food-related phenolic metabolites like anethol which is linked to differentiation. Somatic embryogenesis in anise is a two part system. The first is a 2,4-D (2,4-dichlorophenoxy acetic acid)-induced embryogenic callus formation which requires optimal levels of endogenous auxin and cytokinin levels. The second is the advanced embryo development stage in the absence of 2,4-D which requires abscisic acid to stimulate embryo growth. Our work has shown that proline and the precursors of proline (ornithine and arginine), in combination with the proline analog, azetidine-2-carboxylate (A2C) significantly stimulated 2,4-D-induced embryogenic callus formation and subsequent somatic embryo development in hormone-free medium. Further, abscisic acid stimulated somatic embryo development in anise in the second stage. Total phenolic assays done on embryogenic tissue during 2,4-D-induced embryogenic callus, formation, and during abscisic acid-mediated embryo development showed that levels were highest in embryogenic tissue induced in the presence of proline plus A2C. Total phenolic levels of developing embryos in the absence of 2,4-D and in the presence of abscisic acid were higher than that of the control. A hypothesis for further investigation is proposed based on the potential role of the proline-linked pentose phosphate pathway in stimulating the biosynthesis of purines and phenolic metabolites during anise somatic embryogenesis.

Stimulation of Rosmarinic Acid in Shoot Cultures of Oregano (Origanumvulgare) Clonal Line in Response to Proline, Proline Analogue, and Proline Precursors

Tissue culture-generated shoot-based clonal lines are being used to investigate the role of proline-linked pentose phosphate pathway in stimulating rosmarinic acid (RA). In this study is reported the stimulation of RA biosynthesis in oregano clonal line O-1 in response to proline, proline precursors (ornithine and arginine), and proline analogue (azetidine-2-carboxylate, A2C). Following exogenous treatment with proline and proline precursors in the presence or absence of proline analogue A2C, significantly enhanced RA content and concurrently higher levels of endogenous proline were observed compared to control. Analogue (A2C) treatment alone stimulated highest levels of RA without any increase in endogenous proline. Endogenous proline levels on day 30, however, were significantly higher in all proline and ornithine treatments with or without A2C but not in treatments with A2C or arginine alone. The stimulation of RA synthesis in response to proline or proline precursors with or without A2C suggested that deregulation and enhancement of proline synthesis or proline oxidation may be important for RA biosynthesis in oregano. This stimulation of RA biosynthesis provided strong clues that proline synthesis may be linked to stimulation of the pentose phosphate pathway, driving key precursor metabolites toward shikimate and phenylpropanoid pathways. RA-stimulating compounds also enhanced total phenolics and hardened stem tissues, indicating possible lignification due to polymerization of phenolic metabolites. Keywords: Arginine; azetidine-2-carboxylate; Origanum vulgare; ornithine; phenolics; proline; rosmarinic acid; shoot culture

Regulation of Benzyladenine-Induced in Vitro Shoot Organogenesis and Endogenous Proline in Melon (Cucumis melo L.) by Exogenous Proline, Ornithine, and Proline Analogues

Melon (Cucumis melo L.) is an excellent system to study the role of proline in regulating benzyladenine-induced shoot organogenesis, which is also an important system in our laboratory for investigating flavor and antioxidant metabolite synthesis in differentiated tissues. We have hypothesized that proline stimulated in vitro shoot organogenesis through the activation of the proposed proline-linked pentose phosphate pathway, which may regulate endogenous cytokinin and auxin biosynthesis. To further investigate this hypothesis, it is essential to first confirm whether benzyladenine-induced shoot organogenesis can be stimulated by enhancing proline synthesis through the use of proline analogues and ornithine (proline precursor). In this study, benzyladenine-induced shoot organogenesis was substantially stimulated by exogenous proline and/or ornithine in combination with proline analogues such as azetidine-2-carboxylate (A2C) and hydroxyproline (HP). Among these treatments, proline in combination with A2C or HP showed the highest stimulation. The stimulation of organogenesis correlated closely with increased levels of endogenous proline content, thereby strengthening the hypothesis that proline-linked activation of purine and aromatic metabolism via the stimulation of pentose phosphate pathway may be important for in vitro organogenesis in melon.

EFFECTS OF PROLINE AND PROLINE ANALOGS ON TOTAL PHENOLIC AND ROSMARINIC ACID LEVELS IN SHOOT CLONES OF THYME (Thymus vulgaris L.)

Plant phenolic compounds such as rosmarinic acid exhibit antioxidant and antimicrobial activities and have potential as functional food ingredients. Our model has proposed that a cellular redox cycle linking pyroline-5-carboxylate and proline can stimulate the pentose phosphate, shikimate and phenylpropanoid pathways, and ultimately lead to an increase in phenolic synthesis, including rosmarinic acid (RA). In this study, the response of thyme shoot clones to proline and/or proline analogs was investigated through measurement of total phenolics, rosmarinic acid and proline levels, as well as glucose-6-phosphate dehydrogenase (G6PDH) and phenylalanine ammonia-lyase (PAL) activities. A genetically uniform, high phenolic-containing thyme shoot-based clonal line T-12 was used. Results indicate that supplementation of exogenous proline did not enhance total phenolic and rosmarinic acid content. Therefore, exogenous proline may not have stimulated the proline-linked pentose phosphate pathway. However, increases in proline, total phenolics and rosmarinic acid synthesis were observed following supplementation with the proline analogs, hydroxyproline and azetidine-2-carboxylate. These analogs through proline synthesis may have activated the pentose phosphate pathway and therefore the phenylpropanoid pathway by linking steps involved in NADP/NADPH interconversions. As a result, increases in total phenolic and rosmarinic acid levels were observed. This mode of regulation via proline synthesis-linked to pentose phosphate pathway may be important for synthesis of RA and its overexpression in elite thyme clonal lines for food and medicinal applications.