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Light-mediated fava bean (Vicia faba) response to phytochemical and protein elicitors and consequences on nutraceutical enhancement and seed vigour
Process Biochemistry 38 (2003) 945 /952 www.elsevier.com/locate/procbio
Light-mediated fava bean (Vicia faba) response to phytochemical and protein elicitors and consequences on nutraceutical enhancement and seed vigour Reena Randhir, Kalidas Shetty * Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, MA 01003, USA Received 13 May 2002; accepted 13 July 2002
Abstract Fava bean seedlings are a rich source of levo dihydroxy phenylalanine (L-DOPA), the precursor of the neurotransmitter dopamine. The nutraceutical value and seed vigour of light germinated fava bean seedlings were significantly improved by priming with natural elicitors like fish protein hydrolysates (FPH), lactoferrin (LF) and oregano extract (OE). These elicitors stimulate the phenylpropanoid pathway through the pentose phosphate and shikimate pathway enhancing the production of total phenolics and seed vigour. Previous research in our laboratory has shown that the best elicitor concentration for fava bean was 2 ml/l FPH, 50 ppm LF and 5 ml/l of OE. A 100% germination rate was observed in seeds primed with FPH, 97% with OE, and 92% in LF primed and control. Priming with FPH improved growth by 35 and 25.5% by OE when compared to control. The seeds primed with LF showed no significant increase compared to the control. A significant boost in total phenolics was seen in primed seedlings and this correlated with enhanced seedling height and weight. On day 16 the total phenolics was 25, 9 and 17% higher in FPH, LF and OE primed, respectively, when compared to control. On all days measured the G6PDH activity was higher in control than the elicited seedlings. Previous experiments have shown that for all elicitor treatments a high G6PDH activity was recorded during early germination (24 h) with a concurrent increase in total phenolics and was then reduced by day 20. In the case of control seedlings, phenolics and G6PDH activity was highest on day 20 corresponding to potential delayed developmental demand for metabolites. A steady increase in the GPX activity was observed as the germination progressed both in the case of control and primed seedlings reflecting the plants need for phenolics for lignification and structural development during growth. The antioxidant activity was maximal on day 20 for all treatments demonstrating that perhaps polymerization of phenolics has an antioxidant enhancing effect. The L-DOPA content in fava bean primed with FPH showed a 120% increase over that of the control, corresponding with the high phenolic production on day 16. The elicitors LH and OE showed a 97 and 30% increase respectively over that of the control. The present study demonstrates that elicited fava bean seedlings containing enhanced levels of L-DOPA has excellent potential for use as a functional food, nutraceutical and as an antioxidant. This research also holds agronomic importance suggesting the use of FPH and OE as elicitors to improve fava bean seed vigour and plant productivity. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Antioxidant activity; Fava bean (Vicia faba ); Levo-dihydroxy phenylalanine; Elicitors; Fish protein hydrolysates; Glucose-6-phosphate dehydrogenase; Guaiacol peroxidase; Lactoferrin; Oregano extract; Pentose phosphate pathway; Phenolics; Light-mediated response
1. Introduction Fava beans (Vicia faba ) have potential for treatment of Parkinson disease, hypertension, renal failure and liver cirrhosis [1,2]. Levo-amino dihydroxy phenylala-
* Corresponding author. Tel.: /1-413-545-1022; fax: /1-413-5451262 E-mail address: [email protected] (K. Shetty).
nine (L-DOPA) the precursor of the neurotransmitter dopamine is naturally found in its seedlings, green pods and beans. Wong and Geklim have shown that the LDOPA content of the fava bean seedling is much higher than that of the fava beans [3]. Studies have shown the potential that the ingestion of fava bean seedlings improved substantially the clinical symptoms in Parkinsonian patients [4]. The germination of the seedlings is easy and not season-dependent, and their ingestion does
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 1 9 - 4
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not cause flatulence. Earlier research in our laboratory showed that the total phenolic and L-DOPA content in fava bean sprouts were significantly improved by priming the seeds with phytochemical, bacterial and peptide elicitors [5,6]. The present research is to investigate if a similar stimulation by elicitors can also be achieved in light germinated fava bean seedlings. Seed priming or osmoconditioning is a common agronomic practice to improve seed vigour, synchronize and accelerate germination, stress resistance, plant growth and productivity [7,8]. It is a physiological seed treatment where the seeds are soaked in osmotic solutions for imbibition. This initiates the germinationrelated processes, but prevents the emergence of the radical. Seed vigour is defined as those seed properties that determine the activity and performance of the seed during germination and seedling emergence [9]. Among the aspects of performance are: (a) biochemical processes and reactions during germination such as enzyme reactions and respiration activity, (b) rate and uniformity of seed germination and seedling growth, (c) rate and uniformity of seedling emergence and growth in the field, and (d) emergence ability of seedlings under favourable environmental conditions. Factors that influence the level of seed vigour include the genetic constitution of the seed, environment and nutrition of the mother plant, stage of maturity during harvest, seed size, weight, specific gravity, mechanical integrity, deterioration, aging, pathogens and priming. Priming bitter gourd seeds with selenium solution enhanced germination and antioxidative traits such as free radical and peroxide scavenging activities [10]. Priming treatments advances the embryonic root and shoot tip cells into S and G2 phases of the cell cycle as measured by the increase in the DNA content [11]. In plants the phenylpropanoid pathway (PPP) produces secondary metabolites like phenolics and LDOPA. This pathway can be stimulated through the pentose phosphate and shikimate pathway by natural elicitors [12] (Fig. 1). The natural elicitors chosen for this study were fish protein hydrolysates (FPH), lactoferrin (LF) and oregano extract (OE). FPH are small partially soluble peptides rich in proline and glutamic acid [13] obtained from seafood waste processed with papain and acid treatment. They have a wide spectrum of applications from high value peptones, food ingredients and fertilizer production. Earlier research in this laboratory demonstrated that FPH being rich in proline and hydroxyproline elicits the proline linked-pentose phosphate pathway, shikimate and phenylpropanoid pathways and therefore increases phenolic synthesis in peas [14]. Similar FPH treatment have been shown to improve organogenesis in in vitro tissue culture of melon [15]. Lactoferrin (LF) is an iron-binding glyco-protein found naturally in milk, saliva mucosal surfaces and
within white blood cells. Research has shown LF to be a natural antibiotic, antioxidant, antifungal, antiviral, antitumor and immune booster. Other unique functions attributed to LF include protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, RNAse activity and as a transcriptional factor [16]. Research shows that LF enters the cell and is transported to the nucleus where it binds to specific DNA sequences and induce transcription of the reporter gene [17]. OE is high in phenolic compounds such as protocatechuic acid and its phenyl glucoside, caffeic acid, gallic acid, tocopherol and rosmarinic acid [18]. It has significant antibacterial, antiviral, antimutagenic and antioxidant activity. Elite clonal lines of oregano that produce higher amounts of phenolics and rosmarinic acid in response to Pseudomonas inoculation have been developed previously [19]. The hypothesis of this research is that the total phenolics and L-DOPA content of light germinated, fava beans seedlings can be increased by priming seeds with natural elicitors of the pentose phosphate pathway, thereby improving the nutraceutical value and seed vigour. We hope that this research will show the potential use of natural L-DOPA in fava bean seedlings as a functional food to support Parkinson’s diet with no side effects in its treatment. In parallel, plant growth may be improved as a consequence of the enhanced seed vigour due to stimulation of the phenolic content of fava bean seedlings by the elicitors. The parameters measured to characterize the effect of these elicitors were total phenolics, antioxidant activity, glucose-6-phosphate dehydrogenase (G6PDH), guaiacol peroxidase (GPX), antioxidant activity and L-DOPA. Agronomic parameters such as germination rate, average plant height and fresh weight were determined to reflect the improvement in seed vigour.
2. Materials and methods 2.1. Elicitors and treatments The three elicitors used in this study were FPH, LF and OE. Earlier research in this laboratory has shown that the best elicitor concentration in fava bean was 2 ml/l FPH, 50 ppm LF and 5 ml/l of OE [5]. These elicitor concentrations were used in the present study. FPH emulsion, a byproduct of mackerel processing was obtained from Conolly Seafood, Gloucester, MA. LF was obtained from Sigma Chemical Co., St. Louis, MO. The Oregano extract was prepared by soaking 1 g of crushed dried oregano leaves in 50 ml of 95% ethanol for 3 days. The mixture was pulverized in a blender and centrifuged at 13 000 rpm for 10 min. The supernatant was transferred to a beaker and the ethanol was allowed
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Fig. 1. Pentose phosphate pathway for synthesis of phenolic compounds and L-DOPA.
to evaporate. The residue was then dissolved in 50 ml of distilled water.
off from above the soil. About 20 samples were measured for each treatment.
2.2. Seed treatment and light germination
2.3. Total phenolics assay
Dry seeds of fava bean (V. faba ) were purchased from Stop and Shop Supermarkets, Hadley, MA. The seeds were soaked in distilled water for the control and in distilled water plus FPH (2 ml/l)/LF (50 ppm)/OE (5 ml/ l). Approximately 20 seeds were placed in 500 ml of the soak solution in 1000-ml conical flasks. The flasks were then placed in a rotary shaker at 150 rpm for 24 h. The pre-soaked seeds were washed in distilled water and germinated in sterilized potting soil at 25 8C. Germination was carried out for 20 days and the seed vigour properties such as germination rate, plant height and fresh weight was measured on day 12, 16 and 20. The total germination rate was calculated based on the total number of seedling emergence on day 7. The shoot height above the soil was directly measured. The fresh weight of seedlings was measured after the plant was cut
Total phenolics assay developed by Chandler and Dodds [20] and modified by Shetty [21] was followed. Samples were taken on the 12th day, 16th day and 20th day of growth. Approximately 50 mg of the leaf sample was immersed in 2.5 ml of 95% ethanol and kept in the freezer for 48/72 h. Samples were homogenized using a tissue tearor (Biospec Products, Bartleville, OK) and centrifuged at 13 000 rpm for 10 min. One ml of the supernatant was transferred to a test tube and 1 ml of 95% ethanol, 5 ml of distilled water and 0.5 ml of FolinCiocalteu phenol reagent (Sigma Chemical Co, St. Louis, MO) were added. After an incubation period of 5 min 1 ml of 5% Na2CO3 was added mixed well and kept in the dark for an hour. The samples were then vortexed and absorbance was measured at 725 nm using a UV spectrophotometer (Spectonic Genesys 5; Milton
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Roy Company, Rochester, NY). Phenolics were expressed as mg/g fresh weight. 2.4. Antioxidant assay The antioxidant activity of fava bean leaf extract was determined by the b-carotene oxidation model system as described by Miller with some modifications [22]. The bcarotene solution was prepared by dissolving 10 mg of b-carotene in 50 ml chloroform in amber coloured flask to prevent light oxidation. One ml of this solution was pipetted to a flask covered with aluminum foil. Chloroform was then evaporated under vacuum at 40 8C for 5 min. The b-carotene was dissolved in 20 ml of linolenic acid and 184 ml of Tween 40 emulsifier. Fifty milliliters of H2O2 solution (176 ml H2O2 in 100 ml distilled water) was added and mixed thoroughly until the b-carotene was completely dissolved. To 100 ml of the phenolic extracts 5 ml of this prepared b-carotene solution was added. Control tubes had 100 ml of 95% ethanol. As soon as the emulsion was added the zero time absorbance was read at 470 nm. Subsequent absorbance readings were recorded after a 30 min incubation period in a 50 8C water bath. The protection factor was used to express antioxidant activity as a ratio of sample absorbance at 30 min to that of the control.
50 ml of the enzyme extract added, and the change in absorbance monitored over a period of 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction co-efficient of NADPH2 (6.22 mM 1 cm 1). The enzyme was quantified in nanomoles per minute per milligram of protein. 2.7. Guaiacol peroxidase (GPX) assay The assay followed was a modified version developed by Laloue and George [25]. The enzyme reaction mixture containing 0.1 M potassium phosphate buffer (pH 6.8), 50 mM guaiacol solution and 0.2 mM hydrogen peroxidase was prepared and used to to blank the spectrophotometer at 470 nm. One milliliter of this reaction mixture was taken in a 1.5 ml plastic cuvette and 50 ml of the diluted enzyme extract (1:10) was added. The change in absorbance was monitored for a period of 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction co-efficient of the oxidized product tetraguaiacol (26.6 mM 1 cm 1). The GPX enzyme was quantified in nanomoles per minute per milligram of protein. 2.8. HPLC analysis of L-DOPA
2.5. Total protein assay A cold pestle and motor was used to grind 100 mg of the fava bean leaf sample in cold enzyme extraction buffer (0.5% polyvinylpyrrolidone (PVP), 3mM EDTA. 0.1 M potassium phosphate buffer of pH 7.5). The sample was centrifuged at 13 000 rpm for 15 min at 2/ 5 8C and stored on ice. The supernatant was used in the estimation of total protein, G6PDH and GPX enzyme assays. The protein content was measured by the Bradford method [23]. The Bradford dye reagent was prepared by diluting the commercial dye concentrate in a 1:4 ratio with distilled water. To 100 ml of the sample and blank (extraction buffer only) in test tubes 5 ml of the dye was added and incubated at room temperature for 5 min. The samples were mixed and the absorbance was read at 595 nm using a UV spectrophotometer. 2.6. Glucose-6-phosphate dehydrogenase (G6PDH) assay A modified version of the assay described by Deutsch [24] was followed. The enzyme reaction mixture containing 5.88 mmol b-NADP, 88.5 mmol MgCl2 and 53.7 mmol glucose-6-phosphate, 0.77 mmol malemide was prepared. This mixture was used to obtain the basal blank of the spectrophotometer at 339 nm. One milliliter of this mixture was taken in 1.5 ml plastic cuvettes and
Fava bean leaf samples (200 mg) were immersed in 95% ethanol and kept in the freezer for 48/72 h. They were then homogenized in a tissue tearer (Biospec Products, Bartleville, OK) and centrifuged at 13 000 rpm for 10 min. Supernatants were transferred to a 25 ml beaker and the ethanol evaporated. The residue was dissolved in 10 ml of buffer solution (32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Na2EDTA, 0.215 mM octyl sulphate pH 4). This solution was filtered through 0.45 mm disposable syringe filters (Schleicher & Schuell, Keene, NH). High performance liquid chromatography (HPLC) was performed using a Agilent 1100 liquid chromatograph equipped with a variable wavelength detector. The analytical column was a reverse phase Supleco Discovery C18, 250 mm /4.6 mm with a packing material of 5 mm particle size. The total composition of the mobile phase was 18% methanol and 82% buffer consisting of 0.01 M ammonium acetate at pH 5.4 at the flow rate of 1 ml min 1. Tyrosine and catecholamines standards [L-DOPA, dopamine, norepinephrine and epinephrine; Sigma Chemicals, St. Louis, MO] were chromatographed separately and in mixture. The sample was chromatographed under the same conditions. Retention time and spectrum was compared with that of the standard L-DOPA. The amount of L-DOPA in the fava bean hypocotyls was measured from the peak height obtained at 280 nm, computed automatically
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Fig. 2. Effect of priming fava beans with FPH, LF and OE on seedling vigor.
using an Agilent Chemstation 4.0 and was expressed in terms of milligrams per gram fresh weight. 2.9. Statistical analysis Analysis of variance was conducted using ANOVA single factor test using Microsoft Excel 2000. Both F statistics and P values were calculated to quantify levels of significance for each treatment type.
3. Results and discussion Seed priming is a widely accepted agronomic practice to enhance seed vigour and plant growth. Upon imbibition, the embryonic cells switch from quiescence to a highly active metabolic state [26]. Proteome analysis of Arabidopsis seed priming and germination showed a total of 1300 seed proteins, and 74 proteins were observed during imbibition and radical protrusion [27]. This shows that there was active gene expression and that the genes involved could certainly be elicited by priming. Germination rate is an important index of seedling vigour, and it is always targeted for enhancing overall seed performance and production of plants by physical, chemical and biological approaches. Poor emergence has been attributed to low seed vigour, high susceptibility to seed and soil borne diseases and imbibitional damage [28]. In fava beans, enhanced germination rate was observed in seeds primed with FPH and OE. After 7 days a 100% germination rate was observed in seeds primed with FPH, 97% with OE, and 92% in LF primed and control (Fig. 2). After 12 days of growth a 35% improvement in plant height and weight was observed in seeds primed with FPH and 25.5% with OE when compared to control. The same improved trend was noticed with measurements on day 16 and day 20. The seeds primed with LF showed no significant increase compared to the control. FPH is also commonly used as a plant fertilizer and this study supports this practice based on enhanced seed vigour response. Phenolics are secondary metabolites produced as a result of the plants interaction with the environment. They play a vital role in plant growth, regulation of plant metabolism and lignin synthesis [29]. They have been reported to exhibit medicinal properties such as antitumor, antiviral, antimicrobial, anti-inflammatory,
Fig. 3. Effect of priming fava beans with FPH, LF and OE on the phenolic content. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) or P B/0.10 (#) by ANOVA single factor F test.
hypotensive and antioxidant [12,30]. Phenolics are primarily produced through the pentose phosphate pathway (PPP), shikimate and phenylpropanoid pathways (Fig. 1). In the case of peas, an increase in total phenolics was observed in correlation with enhanced seedling vigour in seeds subjected to low pH treatments [31]. In the present study also a significant boost in total phenolics was seen and this correlated with enhanced seedling height and weight. On day 12 and 16 the phenolic content of fava bean leaves were significantly higher in the elicited seedlings than the control with the maximal amount observed on day 16 (Fig. 3). On day 16 the total phenolics was 25, 9 and 17% higher in FPH, LF and OE primed respectively, when compared to control. However, on day 20 the phenolic content in the control was 30% higher than that of the primed sample and this may correspond to the demand from delayed plant growth. These results demonstrate the initial beneficial effect of the elicitors during the early stages of germination in enhancing seed vigour or perhaps their role is limited for only as long as the elicitor concentration in the seeds remain at a threshold level. The reduction in total phenolics on day 20 may be due to the partitioning of phenolics towards lignin or antioxidants production in the elicited seedlings, while the control just begins its requirement of phenolics. G6PDH is the first committed and rate limiting enzyme in driving the PPP towards the production of phenolics and L-DOPA. Its activity is controlled by the ratio of NADP to NADPH2. It regulates the flux of
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carbon through the PPP providing precursors and cofactors for other biosynthetic routes. This step drives the conversion of glucose-6-phosphate to 6-phosphoglucono lactone and the concurrent reduction of NADP to NADPH2. This enzyme exists in two isoforms in the plant cell namely the cytosolic form, which is active in the dark and the plastidic form in the chloroplast, which is active in the light [32]. In the present study its activity increased with the growth of the fava bean seedlings. On all days measured the activity was higher in the case of the control than the elicited seedlings (Fig. 4). Previous experiments have shown that for all elicitor treatments a high G6PDH activity was recorded during early germination (24 h), which may be due to the general mobilization of carbohydrates to the growing hypocotyls [5] and then reduced by day 12 as in this study. This indicated that G6PDH likely mobilized carbohydrates in the cotyledons during the priming period directing them towards the phenylpropanoid pathway in response to the elicitor as reflected by the increase in phenolics on day 12 and 16. By day 16 an allosteric feedback inhibition by sugar-phosphates and related downstream end products is possible which is relieved by day 20 as G6PDH returns to higher activity. In the case of control seedlings phenolics and G6PDH activity was highest on day 20 corresponding to potential delayed developmental demand for metabolites. It can be hypothesized that in elicited priming the elicitors act as a driving force pushing the PPP pathway forward towards product formation as reflected by increased phenolic content during early growth. These metabolites are regulated by G6PDH based on feedback control by intermediary and secondary metabolites linked to the Pentose Phosphate Pathway and Phenylpropanoid pathways. GPX is an isoenzyme of peroxidase which catalyses the conversion of phenolics from the phenylpropanoid
Fig. 4. The effect of priming fava beans with FPH, LF and OE on G6PDH activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) or P B/0.10 (#) by ANOVA single factor F test.
pathway to lignin and lignans. Peroxidase is known to be responsible for the cross linking of phenolic moieties during the biosynthesis of lignins in the plant cell wall [33]. It is also involved in numerous processes such as ethylene production, wound healing, aromatic compound degradation, pathogen defense, stiffening and degradation of indole-3-acetic acid. Increase in peroxidase activities in plants following treatments with pathogens, elicitors or biotic and abiotic stress is welldocumented [34]. In fava bean a steady increase in the GPX activity was observed as the germination progressed in the case of control and the primed seedlings reflecting the plants requirement for phenolics for lignification and structural development during growth (Fig. 5). The low peroxidase activity during early stages of growth shows a minimal need for lignification with the phenolics being channeled to other growth related activities such as antioxidant and defence responses. The elicited priming did not have a significant effect on the GPX activity compared to control. However, on day 16 the FPH and LF elicited seedlings showed slightly higher activity than the control. Antioxidants are free radical scavengers that intercept and neutralize the effect of destructive oxygen free radicals. Singlet oxygen, superoxides and other activated oxygen species have been implicated in biological oxidation processesed as aging. Recent interest in phenolics has increased due to its antimicrobial, antimutagenic and antioxidant properties. They are effective in the inhibition of all phases of the peroxidative process: first neutralizing free radicals, then blocking the peroxidation catalysis by iron and finally through interruption of lipid-radical chain reactions [35]. The antioxidant activity of fava seedlings was high on day 12 and day 20 for all treatments (Fig. 6). This shows that initially phenolics are antioxidant in nature and lower proportions of phenolics are being partitioned to
Fig. 5. Effect of priming fava beans with FPH, LF and OE on GPX activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) by ANOVA single factor F test.
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Fig. 6. Effect of priming fava beans with FPH, LF and OE on the antioxidant activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) ANOVA single factor F test.
Fig. 7. Effect of priming fava beans with FPH, LF and OE on LDOPA content. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) ANOVA single factor F test.
lignification. This may be because during early germination there is a high demand for oxygen and therefore phenolics might be protecting the cells from potential oxidation-induced deterioration. On day 16 the antioxidant activity reduced with a concurrent increase in LDOPA. This suggests that most phenolics were being channeled towards production of L-DOPA. The antioxidant activity was maximal on day 20 for all treatments demonstrating that perhaps polymerization of phenolics has an antioxidant enhancing effect. Within treatments, priming with FPH showed the highest antioxidant activity on all days followed by OE. Biosynthesis of L-DOPA starts with the amino acid tyrosine, a product of the pentose phosphate pathway (PPP) (Fig. 1). An additional hydroxyl group is added to the aromatic ring of tyrosine by the enzyme tyrosine hydroxylase. This forms levo-dihydroxyphenylalanine (L-DOPA), which is decarboxylated by aromatic-Lamino acid decarboxylase to form dopamine in the human brain [36]. Parkinson’s disease is a progressive neurodegenerative disease in part caused by imbalance of dopamine and acetylcholine in the brain. This is usually due to degeneration of the cells that produce dopamine thus altering the brain neurotransmitters that control fine motor movement, gait, memory, intelligence, cognition, mood, sleep, appetite, and gastrointestinal function [1]. Fava bean seedling consumption increased levels of L-DOPA in the blood with a marked improvement in the motor performance of the patients without any side effects [4]. In the present study the highest L-DOPA content was observed at day 16 of germination in all treatments (Fig. 7). The L-DOPA content in fava bean primed with FPH showed a 120% increase over that of the control on day 16. The elicitors LH and OE showed a 97 and 30% increase, respectively, over that of the control. The high L-DOPA content corresponds with the high phenolic production on day 16. This is followed by an increase in GPX activity on
day 20 with low phenolics and L-DOPA, indicating a potential polymerization of total phenolics to lignin or lignan. There are two major implications from this study namely the functional food relevance and agronomic benefit through improved seed vigour. Phytochemicals and functional food components are the emerging interests in Food Science and Nutrition. Interest is being fueled by their potential role in the prevention and/or treatment of disease. Clinical studies show that management of Parkinson’s disease with the consumption of fava bean seedlings in order to get the desired amount of L-DOPA has much potential [4]. The present study demonstrates that elicited fava bean seedlings containing enhanced levels of L-DOPA has improved the potential for use as a functional food, nutraceutical and as an antioxidant. Another advantage is that the seedling would not cause flatulence in patients as the whole bean does and its production is not season dependent. Commercial seed treatments are commonly developed to increase the value of the seed with improved growth and productivity. This research also holds agronomic importance suggesting the use of FPH and OE as elicitors to improve fava bean seed vigour and production eventually. It is possible to extend this seed priming approach to other plant species to improve seed vigour and phenolics.
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[3] Wong KP, Geklim B. L-DOPA in the seedlings of Vicia faba : its identification, quantification and metabolism. Biogen Amines 1992;8:167 /73. [4] Vered Y, Rabey JM, Palevitch D, Grosskopf I, Harsat A, Yanowski A, Shabtai H, Graff E. Bioavailability of Levodopa after consumption of Vicia faba seedlings by Parkinsonian patients and control subjects. Clin Neuropharmacol 1994;17:138 /46. [5] Randhir R, Shetty P, Shetty K. L-DOPA and total phenolic stimulation in dark germinated fava bean in response to peptide and phytochemical elicitors. Process Biochem 2002;37:1247 /56. [6] Shetty P, Athalla M, Shetty K. Enhancement of phenolic content, L-DOPA and antioxidant activity in germinating fava beans in response to bacterial elicitors. Food Biotechnol 2001;15:47 /67. [7] Taylor AG, Harman GE. Concepts and technologies of selected seed treatments. Annu Rev Phytopathol 1990;28:321 /39. [8] Pattan HD, Gothkar AK, Joshi P, Chivasa A, Nyamudeza P. Onfarm seed priming; using participatory methods to revive and refine a key technology. Agric Syst 2001;69:151 /64. [9] Argerich CA, Bradford KJ. The effects of priming and aging in seed vigour in tomato. J Exp Bot 1989;40:599 /607. [10] Chen CC, Sung JM. Priming bitter gourd seeds with selenium solution enhances germinability and antioxidative responses under sub-optimal temperature. Physiol Plant 2001;111:9 /16. [11] Gurusinghe SH, Cheng ZY, Bradford KJ. Cell cycle activity during seed priming is not essential for germination advancement in tomato. J Exp Bot 1999;50:101 /6. [12] Shetty K. Biotechnology to harness the benefits of dietary phenolics; focus on Lamiaceae. Asia-Pac J Clin Nutr 1997;21:79 /102. [13] Mackie IM. Fish protein hydrolysates. Process Biochem 1982;17:26 /9. [14] Andarwulan N, Shetty K. Improvement of pea (Pisum sativum ) seed vigour response by fish protein hydrolysates in combination with acetyl salicylic acid. Process Biochem 1999;35:159 /65. [15] Milazzo MC, Zheng Z, Kellett G, Haynesworth K, Shetty K. Stimulation of benzyladenine-induced in vitro shoot orangogenesis and endogenous proline in melon by fish protein hydrolysates in combination with proline analogues. J Agric Food Chem 1999;47:1771 /5. [16] He J, Furmanski P. Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature 1995;373:721 /4. [17] Kanyshkova TG, Semenov DV, Buneva VN, Nevinsky GA. Lactoferrin and its biological function. Biochemistry 2001;66:1 / 5. [18] Cervato G, Carabelli M, Gervasio S, Cittera A, Cazzola R, Cestaro B. Antioxidant properties of oregano (Origanum vulgare ) leaf extracts. J Food Biochem 2000;24:453 /65. [19] Perry PL, Shetty K. A model for involvement of proline during Pseudomonas -mediated stimulation of rosmarinic acid levels in oregano shoot clones. Food Biotechnol 1999;13:137 /54.
[20] Chandler SF, Dodds JH. The effect of phosphate nitrogen and sucrose on the production of phenolics and solosidine in callus cultures of Solanum tuberosum . Plant Cell Rep 1983;2:105 /8. [21] Shetty K, Curtis OF, Levin RE, Withowsky RW. Prevention of vitrification associated with in vitro shoot cultures of oregano (Origanum vulgare ) by Pseudomonas spp. J Plant Physiol 1995;147:447 /51. [22] Andurwulan N, Fardiaz D, Wattimenia GA, Shetty K. Antioxidant activity associated with lipid and phenolic mobilization during seed germination of Pangium edule Reinw. J Agric Food Chem 1999;47:3158 /63. [23] Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein /dye binding. Anal Biochem 1976;72:248 /54. [24] Deutsch J. Glucose-6-phosphate dehydrogenase. In: Methods of enzymatic analysis, 3rd ed.. Berlin: Verlag Chemie, 1983:191 /7. [25] Laloue H, Weber-Lofti F, Lucau- Danila A, Gullemat P. Identification of ascorbate and guaiacol peroxidase in needle chloroplasts of spruce trees. Plant Physiol Biochem 1997;35:341 / 6. [26] Bradford KJ. Manipulation of seed water relations via osmotic priming to improve germination under stress conditions. Hort Sci 1986;21:1105 /12. [27] Gallardo K, Job C, Groot PC, Puype M, Demol H, Vandekerckhove J, Job D. Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol 2001;126:835 /48. [28] Styer RC, Cantliffe DJ. Relationship between environment during seed development and seed vigour of two endosperm mutants of corn. J Am Soc Hort Sci 1983;108:717 /20. [29] Lewis NG, Yamamoto E. Lignins: occurrence, biosynthesis and biodegradation. Annu Rev Plant Physiol 1990;41:455 /96. [30] Robbins R. Medical and nutritional aspects of citrus bioflavonoids. In: Nagy S, Attaway J, editors. Citrus nutrition and quality. Washington, DC: American Chemistry Society, 1980:43 / 59. [31] McCue P, Zheng Z, Pinkhan J, Shetty K. A model for enhanced pea seedling vigour following low pH and salicylic acid treatments. Process Biochem 2000;35:603 /13. [32] Scheibe R, Geissler A, Fickenscher K. Chloroplast glucose-6phosphate dehydrogenase: Km shift upon light modulation and reduction. Arch Biochem Biophys 1989;274(1):290 /7. [33] Moorales M, Barcelo AR. A basic peroxidase isoenzyme from vacuoles and cell walls of Vitis vinifera . Phytochemistry 1997;45:229 /32. [34] Kwak SS, Kim SK, Park IH, Liu JR. Enhancement of peroxidase activity by stress related chemicals in sweet potato. Phytochemistry 1996;43:565 /8. [35] Cervato G, Carabelli M, Gervasio S, Cittera A, Cazzola R, Cestaro B. Antioxidant properties of oregano (Origanum vulgare ) leaf extracts. J Food Biochem 2000;24:453 /65. [36] Elsworth JD, Roth RH. Dopamine synthesis, uptake metabolism, and receptors: relevance to gene therapy of Parkinson’s disease. Exp Neurol 1997;144:4 /9.
Light-mediated fava bean (Vicia faba) response to phytochemical and protein elicitors and consequences on nutraceutical enhancement and seed vigour Reena Randhir, Kalidas Shetty * Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, MA 01003, USA Received 13 May 2002; accepted 13 July 2002
Abstract Fava bean seedlings are a rich source of levo dihydroxy phenylalanine (L-DOPA), the precursor of the neurotransmitter dopamine. The nutraceutical value and seed vigour of light germinated fava bean seedlings were significantly improved by priming with natural elicitors like fish protein hydrolysates (FPH), lactoferrin (LF) and oregano extract (OE). These elicitors stimulate the phenylpropanoid pathway through the pentose phosphate and shikimate pathway enhancing the production of total phenolics and seed vigour. Previous research in our laboratory has shown that the best elicitor concentration for fava bean was 2 ml/l FPH, 50 ppm LF and 5 ml/l of OE. A 100% germination rate was observed in seeds primed with FPH, 97% with OE, and 92% in LF primed and control. Priming with FPH improved growth by 35 and 25.5% by OE when compared to control. The seeds primed with LF showed no significant increase compared to the control. A significant boost in total phenolics was seen in primed seedlings and this correlated with enhanced seedling height and weight. On day 16 the total phenolics was 25, 9 and 17% higher in FPH, LF and OE primed, respectively, when compared to control. On all days measured the G6PDH activity was higher in control than the elicited seedlings. Previous experiments have shown that for all elicitor treatments a high G6PDH activity was recorded during early germination (24 h) with a concurrent increase in total phenolics and was then reduced by day 20. In the case of control seedlings, phenolics and G6PDH activity was highest on day 20 corresponding to potential delayed developmental demand for metabolites. A steady increase in the GPX activity was observed as the germination progressed both in the case of control and primed seedlings reflecting the plants need for phenolics for lignification and structural development during growth. The antioxidant activity was maximal on day 20 for all treatments demonstrating that perhaps polymerization of phenolics has an antioxidant enhancing effect. The L-DOPA content in fava bean primed with FPH showed a 120% increase over that of the control, corresponding with the high phenolic production on day 16. The elicitors LH and OE showed a 97 and 30% increase respectively over that of the control. The present study demonstrates that elicited fava bean seedlings containing enhanced levels of L-DOPA has excellent potential for use as a functional food, nutraceutical and as an antioxidant. This research also holds agronomic importance suggesting the use of FPH and OE as elicitors to improve fava bean seed vigour and plant productivity. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Antioxidant activity; Fava bean (Vicia faba ); Levo-dihydroxy phenylalanine; Elicitors; Fish protein hydrolysates; Glucose-6-phosphate dehydrogenase; Guaiacol peroxidase; Lactoferrin; Oregano extract; Pentose phosphate pathway; Phenolics; Light-mediated response
1. Introduction Fava beans (Vicia faba ) have potential for treatment of Parkinson disease, hypertension, renal failure and liver cirrhosis [1,2]. Levo-amino dihydroxy phenylala-
* Corresponding author. Tel.: /1-413-545-1022; fax: /1-413-5451262 E-mail address: [email protected] (K. Shetty).
nine (L-DOPA) the precursor of the neurotransmitter dopamine is naturally found in its seedlings, green pods and beans. Wong and Geklim have shown that the LDOPA content of the fava bean seedling is much higher than that of the fava beans [3]. Studies have shown the potential that the ingestion of fava bean seedlings improved substantially the clinical symptoms in Parkinsonian patients [4]. The germination of the seedlings is easy and not season-dependent, and their ingestion does
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 1 9 - 4
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not cause flatulence. Earlier research in our laboratory showed that the total phenolic and L-DOPA content in fava bean sprouts were significantly improved by priming the seeds with phytochemical, bacterial and peptide elicitors [5,6]. The present research is to investigate if a similar stimulation by elicitors can also be achieved in light germinated fava bean seedlings. Seed priming or osmoconditioning is a common agronomic practice to improve seed vigour, synchronize and accelerate germination, stress resistance, plant growth and productivity [7,8]. It is a physiological seed treatment where the seeds are soaked in osmotic solutions for imbibition. This initiates the germinationrelated processes, but prevents the emergence of the radical. Seed vigour is defined as those seed properties that determine the activity and performance of the seed during germination and seedling emergence [9]. Among the aspects of performance are: (a) biochemical processes and reactions during germination such as enzyme reactions and respiration activity, (b) rate and uniformity of seed germination and seedling growth, (c) rate and uniformity of seedling emergence and growth in the field, and (d) emergence ability of seedlings under favourable environmental conditions. Factors that influence the level of seed vigour include the genetic constitution of the seed, environment and nutrition of the mother plant, stage of maturity during harvest, seed size, weight, specific gravity, mechanical integrity, deterioration, aging, pathogens and priming. Priming bitter gourd seeds with selenium solution enhanced germination and antioxidative traits such as free radical and peroxide scavenging activities [10]. Priming treatments advances the embryonic root and shoot tip cells into S and G2 phases of the cell cycle as measured by the increase in the DNA content [11]. In plants the phenylpropanoid pathway (PPP) produces secondary metabolites like phenolics and LDOPA. This pathway can be stimulated through the pentose phosphate and shikimate pathway by natural elicitors [12] (Fig. 1). The natural elicitors chosen for this study were fish protein hydrolysates (FPH), lactoferrin (LF) and oregano extract (OE). FPH are small partially soluble peptides rich in proline and glutamic acid [13] obtained from seafood waste processed with papain and acid treatment. They have a wide spectrum of applications from high value peptones, food ingredients and fertilizer production. Earlier research in this laboratory demonstrated that FPH being rich in proline and hydroxyproline elicits the proline linked-pentose phosphate pathway, shikimate and phenylpropanoid pathways and therefore increases phenolic synthesis in peas [14]. Similar FPH treatment have been shown to improve organogenesis in in vitro tissue culture of melon [15]. Lactoferrin (LF) is an iron-binding glyco-protein found naturally in milk, saliva mucosal surfaces and
within white blood cells. Research has shown LF to be a natural antibiotic, antioxidant, antifungal, antiviral, antitumor and immune booster. Other unique functions attributed to LF include protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, RNAse activity and as a transcriptional factor [16]. Research shows that LF enters the cell and is transported to the nucleus where it binds to specific DNA sequences and induce transcription of the reporter gene [17]. OE is high in phenolic compounds such as protocatechuic acid and its phenyl glucoside, caffeic acid, gallic acid, tocopherol and rosmarinic acid [18]. It has significant antibacterial, antiviral, antimutagenic and antioxidant activity. Elite clonal lines of oregano that produce higher amounts of phenolics and rosmarinic acid in response to Pseudomonas inoculation have been developed previously [19]. The hypothesis of this research is that the total phenolics and L-DOPA content of light germinated, fava beans seedlings can be increased by priming seeds with natural elicitors of the pentose phosphate pathway, thereby improving the nutraceutical value and seed vigour. We hope that this research will show the potential use of natural L-DOPA in fava bean seedlings as a functional food to support Parkinson’s diet with no side effects in its treatment. In parallel, plant growth may be improved as a consequence of the enhanced seed vigour due to stimulation of the phenolic content of fava bean seedlings by the elicitors. The parameters measured to characterize the effect of these elicitors were total phenolics, antioxidant activity, glucose-6-phosphate dehydrogenase (G6PDH), guaiacol peroxidase (GPX), antioxidant activity and L-DOPA. Agronomic parameters such as germination rate, average plant height and fresh weight were determined to reflect the improvement in seed vigour.
2. Materials and methods 2.1. Elicitors and treatments The three elicitors used in this study were FPH, LF and OE. Earlier research in this laboratory has shown that the best elicitor concentration in fava bean was 2 ml/l FPH, 50 ppm LF and 5 ml/l of OE [5]. These elicitor concentrations were used in the present study. FPH emulsion, a byproduct of mackerel processing was obtained from Conolly Seafood, Gloucester, MA. LF was obtained from Sigma Chemical Co., St. Louis, MO. The Oregano extract was prepared by soaking 1 g of crushed dried oregano leaves in 50 ml of 95% ethanol for 3 days. The mixture was pulverized in a blender and centrifuged at 13 000 rpm for 10 min. The supernatant was transferred to a beaker and the ethanol was allowed
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Fig. 1. Pentose phosphate pathway for synthesis of phenolic compounds and L-DOPA.
to evaporate. The residue was then dissolved in 50 ml of distilled water.
off from above the soil. About 20 samples were measured for each treatment.
2.2. Seed treatment and light germination
2.3. Total phenolics assay
Dry seeds of fava bean (V. faba ) were purchased from Stop and Shop Supermarkets, Hadley, MA. The seeds were soaked in distilled water for the control and in distilled water plus FPH (2 ml/l)/LF (50 ppm)/OE (5 ml/ l). Approximately 20 seeds were placed in 500 ml of the soak solution in 1000-ml conical flasks. The flasks were then placed in a rotary shaker at 150 rpm for 24 h. The pre-soaked seeds were washed in distilled water and germinated in sterilized potting soil at 25 8C. Germination was carried out for 20 days and the seed vigour properties such as germination rate, plant height and fresh weight was measured on day 12, 16 and 20. The total germination rate was calculated based on the total number of seedling emergence on day 7. The shoot height above the soil was directly measured. The fresh weight of seedlings was measured after the plant was cut
Total phenolics assay developed by Chandler and Dodds [20] and modified by Shetty [21] was followed. Samples were taken on the 12th day, 16th day and 20th day of growth. Approximately 50 mg of the leaf sample was immersed in 2.5 ml of 95% ethanol and kept in the freezer for 48/72 h. Samples were homogenized using a tissue tearor (Biospec Products, Bartleville, OK) and centrifuged at 13 000 rpm for 10 min. One ml of the supernatant was transferred to a test tube and 1 ml of 95% ethanol, 5 ml of distilled water and 0.5 ml of FolinCiocalteu phenol reagent (Sigma Chemical Co, St. Louis, MO) were added. After an incubation period of 5 min 1 ml of 5% Na2CO3 was added mixed well and kept in the dark for an hour. The samples were then vortexed and absorbance was measured at 725 nm using a UV spectrophotometer (Spectonic Genesys 5; Milton
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Roy Company, Rochester, NY). Phenolics were expressed as mg/g fresh weight. 2.4. Antioxidant assay The antioxidant activity of fava bean leaf extract was determined by the b-carotene oxidation model system as described by Miller with some modifications [22]. The bcarotene solution was prepared by dissolving 10 mg of b-carotene in 50 ml chloroform in amber coloured flask to prevent light oxidation. One ml of this solution was pipetted to a flask covered with aluminum foil. Chloroform was then evaporated under vacuum at 40 8C for 5 min. The b-carotene was dissolved in 20 ml of linolenic acid and 184 ml of Tween 40 emulsifier. Fifty milliliters of H2O2 solution (176 ml H2O2 in 100 ml distilled water) was added and mixed thoroughly until the b-carotene was completely dissolved. To 100 ml of the phenolic extracts 5 ml of this prepared b-carotene solution was added. Control tubes had 100 ml of 95% ethanol. As soon as the emulsion was added the zero time absorbance was read at 470 nm. Subsequent absorbance readings were recorded after a 30 min incubation period in a 50 8C water bath. The protection factor was used to express antioxidant activity as a ratio of sample absorbance at 30 min to that of the control.
50 ml of the enzyme extract added, and the change in absorbance monitored over a period of 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction co-efficient of NADPH2 (6.22 mM 1 cm 1). The enzyme was quantified in nanomoles per minute per milligram of protein. 2.7. Guaiacol peroxidase (GPX) assay The assay followed was a modified version developed by Laloue and George [25]. The enzyme reaction mixture containing 0.1 M potassium phosphate buffer (pH 6.8), 50 mM guaiacol solution and 0.2 mM hydrogen peroxidase was prepared and used to to blank the spectrophotometer at 470 nm. One milliliter of this reaction mixture was taken in a 1.5 ml plastic cuvette and 50 ml of the diluted enzyme extract (1:10) was added. The change in absorbance was monitored for a period of 5 min. The rate of change in absorbance per minute was used to quantify the enzyme in the mixture using the extinction co-efficient of the oxidized product tetraguaiacol (26.6 mM 1 cm 1). The GPX enzyme was quantified in nanomoles per minute per milligram of protein. 2.8. HPLC analysis of L-DOPA
2.5. Total protein assay A cold pestle and motor was used to grind 100 mg of the fava bean leaf sample in cold enzyme extraction buffer (0.5% polyvinylpyrrolidone (PVP), 3mM EDTA. 0.1 M potassium phosphate buffer of pH 7.5). The sample was centrifuged at 13 000 rpm for 15 min at 2/ 5 8C and stored on ice. The supernatant was used in the estimation of total protein, G6PDH and GPX enzyme assays. The protein content was measured by the Bradford method [23]. The Bradford dye reagent was prepared by diluting the commercial dye concentrate in a 1:4 ratio with distilled water. To 100 ml of the sample and blank (extraction buffer only) in test tubes 5 ml of the dye was added and incubated at room temperature for 5 min. The samples were mixed and the absorbance was read at 595 nm using a UV spectrophotometer. 2.6. Glucose-6-phosphate dehydrogenase (G6PDH) assay A modified version of the assay described by Deutsch [24] was followed. The enzyme reaction mixture containing 5.88 mmol b-NADP, 88.5 mmol MgCl2 and 53.7 mmol glucose-6-phosphate, 0.77 mmol malemide was prepared. This mixture was used to obtain the basal blank of the spectrophotometer at 339 nm. One milliliter of this mixture was taken in 1.5 ml plastic cuvettes and
Fava bean leaf samples (200 mg) were immersed in 95% ethanol and kept in the freezer for 48/72 h. They were then homogenized in a tissue tearer (Biospec Products, Bartleville, OK) and centrifuged at 13 000 rpm for 10 min. Supernatants were transferred to a 25 ml beaker and the ethanol evaporated. The residue was dissolved in 10 ml of buffer solution (32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Na2EDTA, 0.215 mM octyl sulphate pH 4). This solution was filtered through 0.45 mm disposable syringe filters (Schleicher & Schuell, Keene, NH). High performance liquid chromatography (HPLC) was performed using a Agilent 1100 liquid chromatograph equipped with a variable wavelength detector. The analytical column was a reverse phase Supleco Discovery C18, 250 mm /4.6 mm with a packing material of 5 mm particle size. The total composition of the mobile phase was 18% methanol and 82% buffer consisting of 0.01 M ammonium acetate at pH 5.4 at the flow rate of 1 ml min 1. Tyrosine and catecholamines standards [L-DOPA, dopamine, norepinephrine and epinephrine; Sigma Chemicals, St. Louis, MO] were chromatographed separately and in mixture. The sample was chromatographed under the same conditions. Retention time and spectrum was compared with that of the standard L-DOPA. The amount of L-DOPA in the fava bean hypocotyls was measured from the peak height obtained at 280 nm, computed automatically
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Fig. 2. Effect of priming fava beans with FPH, LF and OE on seedling vigor.
using an Agilent Chemstation 4.0 and was expressed in terms of milligrams per gram fresh weight. 2.9. Statistical analysis Analysis of variance was conducted using ANOVA single factor test using Microsoft Excel 2000. Both F statistics and P values were calculated to quantify levels of significance for each treatment type.
3. Results and discussion Seed priming is a widely accepted agronomic practice to enhance seed vigour and plant growth. Upon imbibition, the embryonic cells switch from quiescence to a highly active metabolic state [26]. Proteome analysis of Arabidopsis seed priming and germination showed a total of 1300 seed proteins, and 74 proteins were observed during imbibition and radical protrusion [27]. This shows that there was active gene expression and that the genes involved could certainly be elicited by priming. Germination rate is an important index of seedling vigour, and it is always targeted for enhancing overall seed performance and production of plants by physical, chemical and biological approaches. Poor emergence has been attributed to low seed vigour, high susceptibility to seed and soil borne diseases and imbibitional damage [28]. In fava beans, enhanced germination rate was observed in seeds primed with FPH and OE. After 7 days a 100% germination rate was observed in seeds primed with FPH, 97% with OE, and 92% in LF primed and control (Fig. 2). After 12 days of growth a 35% improvement in plant height and weight was observed in seeds primed with FPH and 25.5% with OE when compared to control. The same improved trend was noticed with measurements on day 16 and day 20. The seeds primed with LF showed no significant increase compared to the control. FPH is also commonly used as a plant fertilizer and this study supports this practice based on enhanced seed vigour response. Phenolics are secondary metabolites produced as a result of the plants interaction with the environment. They play a vital role in plant growth, regulation of plant metabolism and lignin synthesis [29]. They have been reported to exhibit medicinal properties such as antitumor, antiviral, antimicrobial, anti-inflammatory,
Fig. 3. Effect of priming fava beans with FPH, LF and OE on the phenolic content. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) or P B/0.10 (#) by ANOVA single factor F test.
hypotensive and antioxidant [12,30]. Phenolics are primarily produced through the pentose phosphate pathway (PPP), shikimate and phenylpropanoid pathways (Fig. 1). In the case of peas, an increase in total phenolics was observed in correlation with enhanced seedling vigour in seeds subjected to low pH treatments [31]. In the present study also a significant boost in total phenolics was seen and this correlated with enhanced seedling height and weight. On day 12 and 16 the phenolic content of fava bean leaves were significantly higher in the elicited seedlings than the control with the maximal amount observed on day 16 (Fig. 3). On day 16 the total phenolics was 25, 9 and 17% higher in FPH, LF and OE primed respectively, when compared to control. However, on day 20 the phenolic content in the control was 30% higher than that of the primed sample and this may correspond to the demand from delayed plant growth. These results demonstrate the initial beneficial effect of the elicitors during the early stages of germination in enhancing seed vigour or perhaps their role is limited for only as long as the elicitor concentration in the seeds remain at a threshold level. The reduction in total phenolics on day 20 may be due to the partitioning of phenolics towards lignin or antioxidants production in the elicited seedlings, while the control just begins its requirement of phenolics. G6PDH is the first committed and rate limiting enzyme in driving the PPP towards the production of phenolics and L-DOPA. Its activity is controlled by the ratio of NADP to NADPH2. It regulates the flux of
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carbon through the PPP providing precursors and cofactors for other biosynthetic routes. This step drives the conversion of glucose-6-phosphate to 6-phosphoglucono lactone and the concurrent reduction of NADP to NADPH2. This enzyme exists in two isoforms in the plant cell namely the cytosolic form, which is active in the dark and the plastidic form in the chloroplast, which is active in the light [32]. In the present study its activity increased with the growth of the fava bean seedlings. On all days measured the activity was higher in the case of the control than the elicited seedlings (Fig. 4). Previous experiments have shown that for all elicitor treatments a high G6PDH activity was recorded during early germination (24 h), which may be due to the general mobilization of carbohydrates to the growing hypocotyls [5] and then reduced by day 12 as in this study. This indicated that G6PDH likely mobilized carbohydrates in the cotyledons during the priming period directing them towards the phenylpropanoid pathway in response to the elicitor as reflected by the increase in phenolics on day 12 and 16. By day 16 an allosteric feedback inhibition by sugar-phosphates and related downstream end products is possible which is relieved by day 20 as G6PDH returns to higher activity. In the case of control seedlings phenolics and G6PDH activity was highest on day 20 corresponding to potential delayed developmental demand for metabolites. It can be hypothesized that in elicited priming the elicitors act as a driving force pushing the PPP pathway forward towards product formation as reflected by increased phenolic content during early growth. These metabolites are regulated by G6PDH based on feedback control by intermediary and secondary metabolites linked to the Pentose Phosphate Pathway and Phenylpropanoid pathways. GPX is an isoenzyme of peroxidase which catalyses the conversion of phenolics from the phenylpropanoid
Fig. 4. The effect of priming fava beans with FPH, LF and OE on G6PDH activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) or P B/0.10 (#) by ANOVA single factor F test.
pathway to lignin and lignans. Peroxidase is known to be responsible for the cross linking of phenolic moieties during the biosynthesis of lignins in the plant cell wall [33]. It is also involved in numerous processes such as ethylene production, wound healing, aromatic compound degradation, pathogen defense, stiffening and degradation of indole-3-acetic acid. Increase in peroxidase activities in plants following treatments with pathogens, elicitors or biotic and abiotic stress is welldocumented [34]. In fava bean a steady increase in the GPX activity was observed as the germination progressed in the case of control and the primed seedlings reflecting the plants requirement for phenolics for lignification and structural development during growth (Fig. 5). The low peroxidase activity during early stages of growth shows a minimal need for lignification with the phenolics being channeled to other growth related activities such as antioxidant and defence responses. The elicited priming did not have a significant effect on the GPX activity compared to control. However, on day 16 the FPH and LF elicited seedlings showed slightly higher activity than the control. Antioxidants are free radical scavengers that intercept and neutralize the effect of destructive oxygen free radicals. Singlet oxygen, superoxides and other activated oxygen species have been implicated in biological oxidation processesed as aging. Recent interest in phenolics has increased due to its antimicrobial, antimutagenic and antioxidant properties. They are effective in the inhibition of all phases of the peroxidative process: first neutralizing free radicals, then blocking the peroxidation catalysis by iron and finally through interruption of lipid-radical chain reactions [35]. The antioxidant activity of fava seedlings was high on day 12 and day 20 for all treatments (Fig. 6). This shows that initially phenolics are antioxidant in nature and lower proportions of phenolics are being partitioned to
Fig. 5. Effect of priming fava beans with FPH, LF and OE on GPX activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) by ANOVA single factor F test.
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Fig. 6. Effect of priming fava beans with FPH, LF and OE on the antioxidant activity. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) ANOVA single factor F test.
Fig. 7. Effect of priming fava beans with FPH, LF and OE on LDOPA content. Marked data are significantly different than the control at P B/0.01 (*), P B/0.05 (/) ANOVA single factor F test.
lignification. This may be because during early germination there is a high demand for oxygen and therefore phenolics might be protecting the cells from potential oxidation-induced deterioration. On day 16 the antioxidant activity reduced with a concurrent increase in LDOPA. This suggests that most phenolics were being channeled towards production of L-DOPA. The antioxidant activity was maximal on day 20 for all treatments demonstrating that perhaps polymerization of phenolics has an antioxidant enhancing effect. Within treatments, priming with FPH showed the highest antioxidant activity on all days followed by OE. Biosynthesis of L-DOPA starts with the amino acid tyrosine, a product of the pentose phosphate pathway (PPP) (Fig. 1). An additional hydroxyl group is added to the aromatic ring of tyrosine by the enzyme tyrosine hydroxylase. This forms levo-dihydroxyphenylalanine (L-DOPA), which is decarboxylated by aromatic-Lamino acid decarboxylase to form dopamine in the human brain [36]. Parkinson’s disease is a progressive neurodegenerative disease in part caused by imbalance of dopamine and acetylcholine in the brain. This is usually due to degeneration of the cells that produce dopamine thus altering the brain neurotransmitters that control fine motor movement, gait, memory, intelligence, cognition, mood, sleep, appetite, and gastrointestinal function [1]. Fava bean seedling consumption increased levels of L-DOPA in the blood with a marked improvement in the motor performance of the patients without any side effects [4]. In the present study the highest L-DOPA content was observed at day 16 of germination in all treatments (Fig. 7). The L-DOPA content in fava bean primed with FPH showed a 120% increase over that of the control on day 16. The elicitors LH and OE showed a 97 and 30% increase, respectively, over that of the control. The high L-DOPA content corresponds with the high phenolic production on day 16. This is followed by an increase in GPX activity on
day 20 with low phenolics and L-DOPA, indicating a potential polymerization of total phenolics to lignin or lignan. There are two major implications from this study namely the functional food relevance and agronomic benefit through improved seed vigour. Phytochemicals and functional food components are the emerging interests in Food Science and Nutrition. Interest is being fueled by their potential role in the prevention and/or treatment of disease. Clinical studies show that management of Parkinson’s disease with the consumption of fava bean seedlings in order to get the desired amount of L-DOPA has much potential [4]. The present study demonstrates that elicited fava bean seedlings containing enhanced levels of L-DOPA has improved the potential for use as a functional food, nutraceutical and as an antioxidant. Another advantage is that the seedling would not cause flatulence in patients as the whole bean does and its production is not season dependent. Commercial seed treatments are commonly developed to increase the value of the seed with improved growth and productivity. This research also holds agronomic importance suggesting the use of FPH and OE as elicitors to improve fava bean seed vigour and production eventually. It is possible to extend this seed priming approach to other plant species to improve seed vigour and phenolics.
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