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Imaging of bioluminescence responses of different plant species under L-DOPA allelochemical condition

Mohammad Masud Parvez1, Syeda Shahnaz Parvez2, Shoji Hagiwara3 and Yoshiharu Fujii1

1Chemical Ecology Unit, Department of Biological Safety, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba Science City, Ibaraki 305-8604, Japan, www.niaes.affrc.go.jp Email parvez@affrc.go.jp
2
Food Science and Technology Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba Science City, Ibaraki 305-8686, Japan, www.jircas.affrc.go.jp
3
Instrumental Engineering Laboratory, National Food Research Institute, 2-1-2 Kannondai, Tsukuba Science City, Ibaraki 305-8642, Japan, www.nfri.affrc.go.jp

Abstract

Production of O2.- of different plant species belonging several families under L-DOPA (L-3,4-dihydroxyphenylalanine) addition was investigated using bioluminiscence. Bioassay using different concentration of L-DOPA (50, 100 and 200 g/ml) caused three distinct responses (black - Gramineae and Hydrophyllaceae, no change - Leguminosae, Brassicaceae, Pedaliaceae, Solanaceae and Cucurbitaceae, and gray - Liliaceae and Compositae) in-terms of coloration changes on filter paper during early seed germination. Radicle growth of the tested species was strongly inhibited with an increase in L-DOPA concentration. Among the species tested, total photon counting at different concentration of L-DOPA showed that plant species of Gramineae family had a continued increment of total photon during the whole experimental period while others showed a decline after day 3. Imaging of bioluminescence using MCLA (2-Methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo, [1,2-α]pyrazin-3-hydrochloride) clearly showed a strong accelerated photon emission responses in all species of Gramineae family while others had either very weak or no responses. These results suggested that particularly each species of Gramineae family had special mechanism to protect the cell from L-DOPA damage.

Media summary

A unique method using MCLA-mediated imaging of bioluminescence responses showed that species of Gramineae family had special mechanism to protect the cell from L-DOPA damage.

Key Words

Allelochemical, Bioluminescence, L-DOPA, MCLA, Photon Emission.

Introduction

First isolation of L-DOPA (L-3,4-dihydroxyphenylalanine) (Figure 1) has been carried out in 1913 from a legume crop - Vicia faba L. Since 1961, L-DOPA has been proved to be the most efficacious drug in the treatment of Parkinson’s disease, and also known to be one of the strongest allelochemical compounds in nature (Dougan et al. 1975). Velvetbean (Mucuna spp.) is an example of a successful cover crop with several highly biologically active natural products. Besides using as a source of traditional legume food, small-scale farmers in the tropics have traditionally used Mucuna as a cover crop to suppress weeds. The genus Mucuna is large (>100 species) and includes 5 or more cultivated species. The most important cultivated species, Mucuna pruriens (L.) DC. var. utilis, produces the toxic L-DOPA, and the leaves and seeds contain 1% and 4 to 10% of L-DOPA, respectively (Fujii et al. 1991, Prakash et al. 2001, Siddhuraju and Becker 2001). Our recent studies revealed that L-DOPA exuded from the roots of Mucna spp. exhibited strong allelochemical activity (Nishihara et al. 2002, 2004).

More than a decade ago, Fujii and his co-workers (1992) reported that allelopathic activity of L-DOPA is variable among the plant species, and specific to the species concerned, thereby concluded that Gramineae family is more resistant than other families. However, since then no investigation was performed to elucidate the tolerance mechanism of specific families to L-DOPA, although the protective responses of some families are considered to the result from the oxidation of L-DOPA. Chemically, L-tyrosine is converted to L-DOPA and then to dopamine in a two-step process: the first, rate-limiting step is catalyzed by tyrosine hydroxylase while the second step is by the L-DOPA decarboxylase.

MCLA (2-Methyl-6-(4-methoxyphenyl) -3, 7-dihydroimidazo, [1,2-α]pyrazin-3-hydrochloride) (Figure 2) is a luciferin analog functioning as an excellent luminescence probe to detect O2·-. Luminescence of MCLA involves reaction with O2·- to form an unstable dioxetane whose decomposition emits light (Nakano et al. 1986, Tampo et al. 1998).

Figure 1. Structure of L-DOPA.

Figure 2. Structure of MCLA.

In this study, we attempted to elucidate the tolerance mechanism of different plant families to L-DOPA through imaging of MCLA-mediated bioluminescence responses using computer based analytical methods.

Methods

Plant materials and chemicals

Seeds of 25 plant species belonging 9 different families were examined in this study. L-DOPA (Nakalai Tesque, Japan), MCLA (Tokyo Kasie Kogyo, Co. Ltd., Japan), and other chemicals used in this study were purchased locally.

Experiment 1 – bioassay

All the seeds of test species were subjected to bioassay at different concentrations of L-DOPA (50, 100 and 200 g/ml). Appropriate numbers of seeds of each species were germinated on the filter paper (Advantec No. 1, Toyo Roshi Kaisha Ltd., Japan) placed on 45 mm Petri-dishes containing 1 ml of either distilled water (control) or L-DOPA solutions. The seeds in the Petri-dishes were incubated at 25C under complete darkness for 3 days in an incubator (Biotech 300L, Shimadzu, Japan). At day 3, the coloration changes on filter paper in each dish were recorded and the radicle length of each species was measured, and finally the growth is expressed as the percentage to control. Data obtained from 4 replications in the randomised complete block design.

Experiment 2 – photon emission

Seeds of 12 plant species belonging 6 families were selected from the observation of coloration changes (no change, black, gray) on the filter paper in the Experiment 1, and further analyzed for the photon emission experimentation. Appropriate numbers of seeds of each species were germinated on the filter paper (Advantec No. 1, Toyo Roshi Kaisha Ltd., Japan) placed on 50 mm stainless steel dishes containing 1 ml of either distilled water (control) or L-DOPA solutions at 25C under complete darkness for 5 days in a computer connected rotary growth chamber equipped with a two-dimensional photon counting unit and a photo-multiplier (Hamamatsu Phonotics Corp., Japan). The experiment was set in randomised complete block design with 4 replications.

Experiment 3 – luminescence response

MCLA-mediated luminescence responses due to the free radical scavenging in 4 representative samples of plant species treated with L-DOPA during early seed germination stage were detected through the emitted photon using an ICCD camera (C2400-35H, Hamamatsu Phonotics Corp., Japan) equipped with a software (ARGUS 20, Hamamatsu Phonotics Corp., Japan) linked image processor in a computer controlled system (Hamamatsu Phonotics Corp., Japan). Luminescence responses were detected keeping the samples at 25C in a dark chamber with integrated time 30 min after MCLA application to the samples. The experiment was set in randomised complete block design with 4 replications.

Results

Table 1 shows the radicle growth inhibition (% to control) of different plant species and coloration change on filter paper due to L-DOPA treatment (50, 100 and 200 g/ml).

Table 1. Growth inhibition (% to control) of different plant spe cies and coloration change of filter paper due to L-DOPA treatment. indicates standard

Radicle growth of the tested species was strongly inhibited with an increase in L-DOPA concentration. Bioassay using different concentration of L-DOPA caused three distinct responses (black - Gramineae and Hydrophyllaceae, no change - Leguminosae, Brassicaceae, Pedaliaceae, Solanaceae and Cucurbitaceae, and gray - Liliaceae and Compositae) in-terms of coloration changes on filter paper during early seed germination.

At different concentration of L-DOPA, plant species of Gramineae family had a continued increment of total photon counting during the whole experimental period while others showed a decline after day 3 (Figure 3). Imaging of bioluminescence using MCLA clearly showed a strong accelerated luminescence emission responses in all species of Gramineae family (Figure 4) while others had either very weak or no responses (data not shown). Enhanced luminescence emission responses in the species of Gramineae family were strongly correlated with the increased total photon counting (for compare, please see Figures 3 and 4). Imaging of bioluminescence responses of different plant species clearly showed that the members of Gramineae family liberates free radicals during their early seed germination under L-DOPA allelochemical condition, thus displaying the enhanced luminescence emission through the scavenging of liberated free radicals by the antioxidant property of MCLA.

Figure 3. Total photon counting of different plant species at different L-DOPA concentration.

Figure 4. Bioluminescence response of different plant species with L-DOPA treatment (200 g/ml). Upper panel – control, lower panel – corresponding treatment.

Conclusion

This study provides a new perspective to explore the tolerance mechanism of allelopathy. The results presented here suggested that particularly each species of Gramineae family possess high capability of liberating free radicals for scavenging during their early seed germination under L-DOPA allelochemical condition, thus, displaying special tolerance mechanism to protect the cell from L-DOPA damage.

References

Dougan D, Wade D and Mearric, KP (1975) Effects of L-DOPA metabolites at a dopamine receptor suggest a basis for “on-off” in Parkinson’s disease. Nature 254, 71-72.

Fujii Y, Shibuya T and Yasuda, T (1991) L-3,4-dihydroxyphenylalanine as an allelochemical candidate from Mucuna pruriens (L.) DC. var. utilis. Agriculture, Biology, Chemistry 55, 617-618.

Fujii Y, Shibuya T and Yasuda, T (1992) Allelopathy of velvetbean: Its discrimination and identification of L-DOPA as a candidate of allelopathic substances. JARQ 25, 238-247.

Nakano M, Sugioka K, Ushijima Y and Goto, T (1986) Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-α]pyrazin-3-hydrochloride, for estimating the ability of human granulocytes to generate O2·-. Analytical Biochemistry 159, 363-369.

Nishihara E, Parvez MM, Araya H and Fujii, Y (2004) Germination growth response of different plants species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA). Plant Growth Regulation, 42: 181-189.

Nishihara E, Araya H, Hiradate S and Fujii, Y (2002) The inhibition of lettuce growth by diffused L-3,4-dihydroxyphenylalanine (L-DOPA) in Mucuna accessions. Proceedings of the 3rd World Congress on Allelopathy, Tsukuba, Japan.

Prakash D, Niranjan A and Tewari, SK (2001) Some nutritional properties of the seeds of three Mucuna species. International Journal of food Science and Nutrition 52, 79-82.

Siddhuraju P and Becker K (2001) Rapid reversed-phase high performance of L-DOPA (L-3,4-dihydroxyphenylalanine), non-methylated and methylated tetrahydroisoquinoline compounds from Mucuna beans. Food Chemistry 72, 389-394.

Tampo Y, Tsukamoto M and Yonaha, M (1998) The antioxidant action of (2-Methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo, [1,2-α]pyrazin-3-hydrochloride), a chemiluminescence probe to detect superoxide anions. FEBS Letters 430, 348-352.

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