Ftemeh Etemdi,Msoud Hshemi,*,Reen Rndhir,Omid ZndVkili,Ali Edi

aStockbridge School of Agriculture,University of Massachusetts,Amherst,MA 01003,USA

bSpringfield Technical Community College,Springfield,MA 01102,USA

cUniversity of Mohaghegh Ardabili,Ardabil,Iran

Keywords:

A B S T R A C T Faba bean(Vicia faba L.)has been identified as a rich source of L-DOPA,which is used in treating Parkinson's disease.Biosynthesis and accumulation of active substances such as LDOPA in plant tissues may interact with growing conditions and processing methods.Accumulation trends of L-DOPA in various faba bean organs and the effect of drought stress and N fertilization on L-DOPA content were studied in a field and two greenhouse

1.Introduction

Faba bean is a cool-season legume crop with high nutritional value[1]and has also been identified as a medicinal plant,given that it accumulates a large amount of L-DOPA(3,4-dihydroxyphenyl-L-alanine), a precursor of dopamine currently used in treating Parkinson's disease(PD). L-DOPA is synthesized from the amino acid L-tyrosine in the mammalian body and brain[2—4].Synthesized L-DOPA is expensive and often causes a variety of side effects including nausea,vomiting,low blood pressure,drowsiness,and restlessness[5—7].For this reason,use of natural sources of L-DOPA to avert potential side effects has been recommended[5,8].The world demand for L-DOPA is estimated to be as high as 250 t year?1[9]with an annual market value of about$100 billion[5,10,11].Different parts of faba bean accumulate L-DOPA with different rates and trends[12].

Environmental conditions influence the accumulation of secondary metabolites in plants[13—15].Environmental stresses may influence metabolic pathways involved in the synthesis of secondary metabolites,thus changing the synthesis and accumulation of natural compounds in plants[16].Adaptation of plants to biotic and abiotic stresses is due primarily to the stimulation of protective biochemical systems and synthesis of secondary metabolites such as phenolics derived from the phenylpropanoid pathway[2,17].Plants grown under high temperature[18,19]and nutritional disorders[2,20]produced phenylpropanoid metabolites such as flavonoids,isoflavonoids,anthocyanins,and polyphenols.Both environmental and genetic factors play roles in L-DOPA production[21,22].An earlier report indicated that the nature of the N source and the presence/absence of illumination affected the production of L-DOPA by cell suspension cultures of velvet bean[23].Randhir and Shetty[2]reported that under microwave-induced stimulation, L-DOPA content of germinated sprouts increased by 59%.

Plants exposed to some degree of drought stress accumulate higher concentrations of secondary metabolites[24,25].Such enhancement is reported to occur in nearly all classes of natural products including simple and complex phenols,numerous terpenes,and also N-containing substances such as alkaloids,cyanogenic glucosides,and glucosinolates.Owing to the beneficial effects of drought stress on accumulation of secondary metabolites,especially phenolic compounds,exposing plants to some degree of water stress has been suggested as a potential approach to increasing the production of these active substances[26].There is limited information available on the response of L-DOPA content of faba bean to drought stress.

Although faba bean is an N2-fixing plant,there is a lack of information about the effect of supplemental N fertilization on its L-DOPA concentration.Deficiencies in N and P directly influenced the accumulation of phenylpropanoids[27]and lignification[28].

Because fresh faba beans are not available at all times in all locations,Parkinson patients may store relatively large numbers of plants for consumption.Plants may be processed by various methods,including chopping frozen tissues and powdering dried plants.However,it is not known to what extent processing influences the L-DOPA content of faba bean plants.Earlier reports[29,30]indicated that L-DOPA in legumes can be destroyed by cooking and soaking in alkaline solutions.Dahouda et al.[31]concluded that the L-DOPA in velvet bean was degraded by cooking but not by roasting.More information about the potential negative impact of processing methods on L-DOPA concentration is needed.

The main objectives of this study were to investigate:

1)the accumulation trend and concentration of L-DOPA in various organs of faba bean plant

2)the influence of drought stress on L-DOPA concentration

3)whether supplemental N fertilization can improve L-DOPA content

4)the influence of processing methods on L-DOPA content of faba bean

2.Materials and methods

2.1.Measuring L-DOPA concentration in plant parts

A two-year field study was conducted at the University of Massachusetts Amherst Agricultural Experiment Station,Crops and Animal Research and Education Farm in South Deerfield(42°28′37″N,72°36′2″W)in 2013 and 2014.The soil type at the experimental site was a Hadley fine sandy loam(nonacid,mesic Typic Udifluvent).Seeds were inoculated with peat-based Rhizobium leguminosarum(VERDESIAN,N.DURE,Carry,NC)prior to sowing in both experiments.Seeds were planted on March 5 in 2013 and March 10 in 2014 in three replications.Faba bean plants were harvested at two stages of growth.The first harvest was at the six-leaf stage(15 days after germination)and the second harvest was at 95 days after germination when pods were fully grown.In the first harvest,whole seedlings were used for L-DOPA determination.However,in the second harvest,plants were divided into various organs including roots,stems,leaves,terminal buds,flowers,young pods,and mature seeds.All organs were digested separately and analyzed for L-DOPA using high performance liquid chromatography(HPLC),a reliable method to measure L-DOPA[32].

2.2.Processing method influence on L-DOPA concentration

Five plants were harvested randomly from each experimental plot replication when the lower pods were physiologically mature.Pods and leaves were separated and processed by various methods including air drying,oven drying,boiling,and freezing.

Air drying:samples were placed on paper bags and left to dry at room temperature for two,four,and seven days.

Oven drying:samples were placed on metal trays and dried in a forced-air oven at 50°C for 24 h.

Freezing:samples were chopped and stored in a freezer immediately after harvest at?23 °C for 48 h prior to analysis.

Boiling:samples of leaves and seeds were cooked in boiling water(five times the sample weight)for 40 min.

After each processing method,200-mg samples from each treatment were taken.For the drying method,samples were ground to pass a 0.42 mm screen and prepared for extraction.For the other processing methods,samples were ground with mortar and pestle prior to analysis.

2.3.Drought-stress influence on L-DOPA concentration

A greenhouse experiment was conducted at the College of Natural Sciences,University of Massachusetts Amherst.Seeds of faba bean(cv.Windsor)were inoculated and planted two times,on October 14 and December 15,2014,in 25 cm×50 cm trays with 3.2-cm cells filled with a pro-mix soil.Seedlings were transferred to individual pots filled with sterilized play sand after nine days.Greenhouse temperature was set at 15°C with 16 h light during daytime and 12 °C at night with a relative humidity of 50%±5%.The experiment was laid out in a randomized complete block design with three soil moisture treatments and replicated three times. Soil moisture treatments were planned to expose plants to moderate and severe drought stress,based on soil moisture depletion.Soil moisture holding characteristics,including the volumetric soil moisture content at?0.03 MPa(field capacity,FC)is the amount of soil moisture or water content held in the soil after excess water has drained away,and?1.5 MPa(permanent wilting point,PWP)is defined as the minimal value of soil moisture the plant requires not to wilt and was determined with a pressure-plate apparatus.The difference between FC and PWP was taken as the available soil water content.Soil moisture content in all pots was monitored twice a day using time-domain reflectometry(TDR)(MiniTrase System,Soil Moisture Equipment Corp.,Santa Barbara,CA)fitted with 20-cm probe rods.Watering of control pots(nonstress)was based on maintaining soil water content at 80% FC to avoid drought stress.Pots under moderate and severe levels of water deficit received 60%(moderate stress)and 40%(severe stress)of the amount of irrigation water used in the control pots.

Plants were harvested at four vegetative growth stages:two,four,six,and eight leaves.At each stage,three plants were cut at the soil surface and oven-dried at 50°C.Dried plant samples were ground to pass a 0.42 mm screen and prepared for extraction.

2.4.Nitrogen stress influence on L-DOPA concentration

Seeds of faba bean(cv.Windsor)were inoculated and planted twice:October 14 and December 15,2014.Growing conditions and layout of this experiment were similar to those of the drought stress experiment.After seedlings were transplanted to the main pots,N fertilizer(calcium ammonium nitrate with 27%N)was applied at five rates:0,0.250,0.050,0.075,and 0.100 g N pot?1(equivalent to 0,25,50,75,and 100 kg ha?1).Faba bean seedlings were harvested at four vegetative growth stages:2,4,6,and 8 leaves.At each growth stage,three plants were harvested at the soil level and oven-dried at 50°C.Dried plant samples were ground to pass a 0.42 mm screen and prepared for extraction.

2.5.Plant preparation procedure

The procedure described by Shetty et al.[32]for the extraction of L-DOPA was used with some modifications.Dry samples of 200 mg were immersed in 95%ethanol and kept in the freezer for72 h.Samples were then homogenized using a Bio Homogenizer(BIOSPEC PRODUCTS,Bartlesville,OK)and centrifuged at 13,000 r min?1for 10 min.The liquid portion was left under a fume hood until the ethanol evaporated.The residue was dissolved in a buffer solution(32 mmol L?1citric acid,54.3 mmol L?1sodium acetate,0.074 mmol L?1EDTA,0.215 mmol L?1octyl sulfate pH 4)and refrigerated until particles settled.The supernatant was passed through a 0.45 μm syringe filter for analysis by HPLC(LC 2010A,Shimadzu Prominence HPLC with DAD detector(SPD-M20A Photodiode Array Detector,Shimadzu,Japan,Tokyo),Phenomenex Gemini C18 column).

2.6.HPLC

L-DOPA was analyzed by HPLC equipped with an autosampler and diode array detector.Data were acquired and processed by an LC Solution Data System(Shimadzu,Japan).Chromatographic separation was performed on a PhenomenexC18column (250.0 mm × 4.6 mm,5 μm)(Phenomenex,CA,USA)with an isocratic flow of 1 mL min?1.The mobile phase was 0.1%acetic acid(98%)and methanol(2%).The mobile phase was filtered through 0.2 μm filters and degassed by sonication for 30 min.Injection volume was 20 μL and L-DOPA detection was at 283 nm.

2.7.Calibration curve

Four concentrations of stock solution(50,100,200,and 400 mg L?1)were prepared and injected with mobile phase.The calibration curve was obtained by plotting the absorbance peak area against the concentrations of the standard solution.A linearity graph of control solution of L-DOPA is shown in Fig. 1

2.8.Statistical analysis

Collected data were subjected to ANOVA using PROC GLM of SAS[33,34].Effects were considered significant at P≤.05 by the F-test,and when the F-test was significant,Fisher's least significant difference(LSD)test was used for mean separation.The variability of the data in all of the figures is indicated by corresponding error bars.

Regression equations for faba bean biomass and L-DOPA concentration under drought stress and N application rates were determined by fitting the data to linear or quadratic trend using PROC REG.

Fig.1–Control chromatogram with Retention time(min)3.18 for L-DOPA.

Fig.2– L-DOPA concentration(a)and yield(b)of various organs of faba bean.Values are means of two experiments and three replications.The bars are representing standard error of the means.

3.Results and discussion

3.1. L-DOPA concentration in various plant parts

L-DOPA concentration differed significantly among organs of faba bean(Fig.2-a).The highest L-DOPA concentration(13.3 mg g?1)was extracted from young seedlings harvested 15 days after germination. L-DOPA content decreased as plants matured.When plants were harvested at physiological maturity,the highest L-DOPA content was detected in leaves(10.5 mg g?1)followed by flowers,young pods,mature seeds,and roots.Earlier reports focused mainly on L-DOPA content of seeds[2,35].Results from the present study indicated that mature seeds were not the richest part of faba bean and contained on average 7.2 mg g?1,a concentration lower than reported previously[2].The L-DOPA yield of each plant part is a product of its concentration and the dry weight of the organ.Accordingly,the highest L-DOPA yield was obtained from leaves(Fig.2-b).Recently[36]we reported that L-DOPA yields of seeds differed markedly among cultivars,with Aquadulce producing the highest L-DOPA yield with 38 kg ha?1.

3.2.Influence of processing method on L-DOPA concentration

Processing methods influenced the L-DOPA content of seeds and leaves(Fig.3).As expected,the highest L-DOPA was extracted from freshly harvested plants.The highest L-DOPA concentrations extracted from fresh leaves and seeds were 22.4 mg g?1and 16.1 mg g?1,respectively.When leaves and seeds were chopped and kept frozen for 48 h,their L-DOPA content decreased by 24.1%and 21.1%,respectively.The reduction in L-DOPA due to processing was much higher when plant parts were processed by methods other than freezing.When leaves or seeds were oven-or air-dried for two,four,or seven days,the L-DOPA content decreased similarly and the extracted concentrations were as low as 50%of those of fresh-harvested leaves and seeds.Among various processing methods,boiling caused the greatest reduction in L-DOPA concentration in leaves and seeds.

L-DOPA is a simple phenolic secondary metabolite that is synthesized in the shikimic acid pathway.It is derived from L-tyrosine by the action of tyrosinase(Fig.4).In general,activity of enzymes is negatively impaired by extreme changes in their normal conditions.Storing plant materials for relatively short period of time in room temperatures may result in significant decrease in L-DOPA concentration.We found a decrease of up to 50% in L-DOPA concentration when leaves and seeds of faba bean were kept at room temperature for 48 h or oven-dried.However,we found no further reductions when plant samples were kept for four and seven days.Drying and heating of plant parts may result in accelerating the degradation and/or reduction of enzyme activities,owing mainly to protein denaturation[37].

Fig.3–The influence of various processing methods on L-DOPA content of faba bean leaves and seeds.Values are means of two experiments and three replications.The bars are representing standard error of the means.

Fig.4–The two initial steps of L-DOPA biosynthesis.

Nyirenda et al.[38]reported that boiling,which is a combination of soaking and high temperature,severely reduced the L-DOPA content of velvet bean.In the present study we extracted less L-DOPA by boiling plant materials than by applying other processing methods(Fig.3).The result was in accord with earlier reports indicating that L-DOPA in legumes is destroyed by cooking or roasting[29,30,39].

3.3.Influence of drought stress on faba bean biomass and LDOPA concentration

Under all irrigation treatments,change in biomass of faba bean showed a quadratic response with a typical sigmoid trend(Table 1).The amount of biomass production decreased significantly as drought stress intensified.At eight-leaf stage,faba bean plants grown under moderate and severe drought stress produced 27%and 42%less biomass than well-watered plants.Reduction in biomass of plants under drought stress condition is not surprising,given that growth of plant organs requires maximum turgor pressure.Such pressure is not available when irrigation water is limited,resulting in reduced plant height,leaf size,and stem diameter[40,41].Other than cell expansion,photosynthesis is also considered to be one of the earliest physiological functions impaired when plants experience drought stress,owing mainly to stomatal closure[15,24].

L-DOPA contents of well-watered faba bean seedlings and those grown under stress conditions increased until the fourleaf stage followed by a declining trend(Table 2).Imposing drought stress elevated the L-DOPA concentration to its peak value of 23.3 mg g?1under the severe-stress condition,almost twofold that of well-watered seedlings.A similar result reported by Khang et al.[42]was an increased accumulation of phenolic compounds in six different legumes subjected to drought stress.Production of secondary metabolites in plants interacts strongly with growing condition and environmental stress[13,43].It appears that enhancement of L-DOPA accumulation in faba bean seedlings is part of the defense mechanism in plants experiencing drought stress.The defense mechanism helps plants to tolerate the stress condition for a longer period of time[27,37].

Reactive oxygen species(ROS),also called active oxygen species(AOS)or reactive oxygen intermediates(ROI)are theresult of the partial reduction of atmospheric O2,which is increased by lipid per oxidation,chlorophyll destruction,protein oxidation,and nucleic acid damage under stress situations during plant growth[24,44].Plants have an innate ability to biosynthesize a wide range of non-enzymatic antioxidants capable of attenuating ROS-induced oxidative damage[44,45].

Table 1–Influence of drought stress on faba bean biomass harvested in early growth stages.

Table 2–Influence of drought stress on faba bean L-DOPA concentration in early growth stages.

L-DOPA yield is a product of its concentration and plant biomass.Although drought stress elevated L-DOPA concentrations in faba bean seedlings, L-DOPA yield was compromised by a large adverse effect of drought stress on biomass production(Table 1),so that well-watered plants produced the highest L-DOPA yield(Fig.5).Thus,imposing drought stress should not be considered a viable option for increasing LDOPA yield in faba bean.

3.4.Influence of N application rate on faba bean biomass and L-DOPA concentration

Faba bean biomass increased with N application rate up to the equivalent of 75 kg ha?1(Table 3).As a legume,faba bean meets its N need by biological fixation.However the results of the present study revealed that due to the conditions of the experiment,the amount of biological fixation was not enough to satisfy faba bean N requirements.Even under field conditions,many legumes often respond positively to supplemental N[46—49].

Given that L-DOPA is an N-containing compound,we assumed that higher availability of N in the soil stimulates L-DOPA synthesis,so that more L-DOPA can be extracted.The results of our study,however,did not fully confirm this hypothesis.At all growth stages,the lowest L-DOPA concentrations were found in plants grown with no supplementary N.At the four-leaf stage,maximum L-DOPA content was extracted from plants receiving the equivalent of 100 kg N ha?1,but in later stages of growth,no significant differences in L-DOPA concentration were detected among the N application rates(Table 4).

4.Conclusions

Fig.5–Influence of drought stress on L-DOPA yield of faba bean in early stages of growth.Values are means of three replications and two experiments.

Table 3–Influence of N application rate on faba bean biomass in early growth stages.

Table 4 – Influence of N application rate on faba bean L-DOPA concentration in early growth stages. Values are means of three replications and two experiments.

The highest L-DOPA concentration was accumulated in seedlings. However, given that total extractable L-DOPA is a function of both concentration and biomass,the highest L-DOPA yield was extracted from mature plants. The highest L-DOPA content was found in fresh leaves. Immediate freezing of plant tissues preserved more L-DOPA than other processing methods.Although drought stress stimulated L-DOPA synthesis,especially at the early stages of growth,plants grown under well-watered conditions yielded higher L-DOPA than stressed plants.We conclude that application of exogenous N cannot be used as a strategy to stimulate L-DOPA synthesis,but that the use of higher N application rates may improve L-DOPA yield by promoting biomass production.

Acknowledgments

This research project was partially funded through a graduate student grant received from Northeast Sustainable Agriculture Research and Education (GNE14-078).The authors gratefully acknowledge assistance with HPLC analysis in Dr.Baoshan Xing's Laboratory.

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