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Biochemical and Biophysical Research Communications 276, 970–973 (2000)

doi:10.1006/bbrc.2000.3564, available online at http://www.idealibrary.com on

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ensitivity of Duckweed (Lemna major)o Ultraviolet-B Radiation

. Farooq, G. Suresh Babu, R. S. Ray, R. B. Misra, U. Shankar, and R. K. Hans1

hotobiology Laboratory, Industrial Toxicology Research Centre, Post Box 80,ahatma Gandhi Marg, Lucknow 226 001, India

eceived August 10, 2000

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The sensitivity of an important aquatic macrophyte,uckweed (Lemna major), to UV-B radiation was studiednder experimental conditions at three different dosesesignated as no, mild, and severe injury dose by observ-

ng visible injury symptoms and estimating levels ofhlorophyll, pheophytin, carotenoids, protein, starch,ree sugar, and peroxidase activity. Laboratory-grownuckweed plants were exposed to UV-B radiation at 0.4W/cm2 intensity for different time periods. Mild and

evere injury were developed at 6.48 and 8.64 J, respec-ively. Peroxidase activity increased at all the exposureevels. Dose-dependent decrease in chlorophyll andtarch with drastic depletion in protein and free sugarontent were observed. Pheophytin and carotenoidsontent increased at no injury level, but decreased atigher exposure level. The results indicate that ambientV-B radiation at the indicated level acts as a physiolog-

cal stress in Lemna major. © 2000 Academic Press

Key Words: UV-B radiation; duckweed; Lemna major;hlorophyll; pheophytin; carotenoids metabolism; per-xidase; aquatic ecosystem.

The gradual destruction of ozone in the stratosphereas resulted in an increase in UV radiation on thearth surface (Bjorn et al., 1999) affecting the ecosys-em, both directly and indirectly (Bjorn, 1996). Theffects of enhanced UV-B have been studied primarilyn agricultural species (Ficus and Booker, 1995; Dai etl., 1997) but comparatively little attention has beeniven to plants belonging to the nonagricultural eco-ystem, such as forest, grassland, and aquatic plantsTeramura et al., 1991; Caldwell and Flint, 1994).emna major, commonly known as duckweed, an im-ortant food species for aquatic herbivores, is a goodietary supplement and nutrient source (Oron et al.,985) for humans (Majid et al., 1984), livestock, andsh (Lehman et al., 1981) and is used as a good fertil-

To whom correspondence should be addressed. Fax: 0522-228227,28471. E-mail: intox@itrc.sirnetd.ernet.in.

970006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

s an indicator of water pollution (Nasu and Kugimoto,981). The floating thalloid leaves receive maximumurface UV radiation. Recent studies have indicatedhat exposure to UV radiation causes inhibition inhotosynthesis and reduction in pigments, biomass,nd protein content and alteration in metabolic processFranklin and Forster, 1997; Hader and Figueroa,997; Dohler et al., 1998; Dohlor, 1998). Suresh Babu etl. (1998) found that exposure of cyanobacteria with aV-B dose corresponding to an increase or decrease of0% with its environmental flux has drastic effects oniomass production, photosynthetic rate, protein con-ent, and nitrogen fixation. Dohlar (1997) studied theffects of low-dose UV-B irradiation and found depres-ion in the biomass of Thalassiosira rotula. Protein andigment contents were also reduced after UV-B expo-ure in Bellerochea yucatanesis. In this study, we re-ort the UV-B dose producing mild and severe visiblenjury symptoms along with effect on peroxidase, pig-

ents, chlorophyll, pheophytin, carotenoid, protein,tarch, and free sugar content in Lemna major exposedor different time periods at an intensity correspondingo a 3-year average solar UV-B output (0.4 mW/cm2),etermined near our laboratory (26° 459N latitude and0° 509E longitude at 140 m above the mean sea level.

ATERIALS AND METHODS

Lemna major was collected from local ponds washed axenically,ultured and maintained in Hoagland medium (EPA, 1975) in arowth chamber at 28 6 2°C under fluorescent light (2500 Lux) andark period for 16/8 h/day and controlled humidity (60%). The younglants of third generation were transferred to sterilized petri dishesnd irradiated at 0.4 mW/cm2 UV-B radiation for 3.0, 4.5, and 6.0 horresponding to a dose of 4.32, 6.48, and 8.64 J, respectively, usingV-B emitting fluoroscent tube (T-40M) manufactured by Vilberourmat, Marne Lavalle, France. The spectral emission of UV-Bource ranged from 280 to 320 nm with a peak at 312 nm. Thentensity of UV-B was measured with a RMX-3W radiometer (Vilberourmat) equipped with a UV-B detecting probe. The plants werebserved for 48 h for any visible injury symptoms. The dose causingisible mild injury symptoms (MI) was determined. One-third above

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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

severe injury, SI) and below (no injury, NI) of this dose was selectedor comparative study of metabolic alterations.

The plants (20) of each petri dish were pooled and analysed im-ediately after exposure. The plants were homogenized (10% w/v) in

hilled Milli Q water using pestle and mortar, muslin filtered and theomogenate was centrifuged in cold at 500g for 20 min. The super-atant was used for the assay of peroxidase activity (Srivastava etl., 1972) and protein (Lowry et al., 1951) content.Fresh tissue was extracted in 80.0% acetone for the spectroscopic

stimation of chlorophyll (Strain et al., 1971), pheophytin (Vernon,960) and carotenoids (Duxbury and Yentech, 1956) content. Totalree sugar was estimated colorimetrically in alcoholic extract (Mont-omery, 1957). Residue left after alcoholic extraction was hydrolyzedith perchloric acid (Agarwal et al., 1977) and starch was estimateds free sugar. For recovery experiments, other groups of the exposedlants at 4.32 J (no injury dose) were returned to the culture mediumor 24 h and extracted with 80.0% acetone. The two extracts ofxposed plants (immediately and after 24 h) were scanned separatelyetween 400 and 700 nm in spectrometer (Unicam UV2-100 UV/Vis)nd compared with control.

ESULTS AND DISCUSSION

Table 1 lists the visible injury symptoms and theeroxidase activity in Lemna major immediately afterxposure to UV-B radiations. No visible injury (NI)ymptom was observed at 4.32 J. Mild injury (MI)ymptoms in the form of chlorosis with marginal ne-rosis was observed at 6.48 J. Severe injury (SI) symp-oms of chlorosis followed by necrosis was observed at.64 J. Young leaves were more damaged than intensereen colored matured, comparatively old leaves. Theeroxidase activity increased at all the exposure level.he increase was 1.64-, 1.56-, and 1.91-fold at no, mild,nd severe injury doses, respectively.The metabolic alterations in Lemna major on expo-

ure to UV-B are reported in Fig. 1. The chlorophyllontent decreased dose dependently by 19.03, 31.41,nd 34.34%, respectively, at NI, MI, and SI dose. Pheo-hytin content first increased by 22.52% at NI dose andhen decreased by 19.49 and 36.93% at MI and SIoses, respectively. The pheophytin/chlorophyll ratioas higher in the exposed plants at NI and MI dosehile it was little lower in SI dose exposed plants.arotenoid content increased at NI dose by 17.36% andecreased by 12.63 and 21.05% at MI and SI dose level.rastic depletion in protein content was found in UV-Bxposed plants. The depletion was dose dependent. The

Injury Symptoms and Peroxidase Activity in Lemna major

Sl No. Dose (J)

Control1 No injury exposed 4.322 Mild injury exposed 6.483 Severe injury exposed 8.64

Note. The data are the average of 3 values. f, fold.a (OD change/min/g fresh wt).

971

ecrease in protein content was 35.88, 50.85, and5.29%, respectively, at NI, MI, and SI dose level.tarch content was also decreased dose dependently,ut not as severe as the protein and free sugar content.

9.00, 14.02, and 24.01% decrease in starch contentas observed. Total free sugar content was also de-

reased dose dependently by 49.15% at NI and MI doseevel and its content was further reduced.

The absorption spectra of control and UV-B-exposedlants both immediately and after 24 h of exposure arehown in Fig. 2. The pattern of absorption spectra in

er UV-B (280–320 nm) Exposure at 0.4 mW/cm2 Intensity

Peroxidasea Visible injury symptoms

59.2597.50 (1.64 f) No visible symptoms92.5 (1.56 f) Chlorosis and marginal necrosis113.5 (1.91 f) Chlorosis followed by necrosis

FIG. 1. Metabolic alteration in Lemna major on exposure toV-B radiation.

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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ontrol and exposed plants was similar. Nine absorp-ion peaks at 381.0, 422.0, 435.0, 530.0, 545.0, 566.0,85.0, 612.0, and 664.0 nm were observed in controllants which were reduced to seven in UV-B-exposedlants (378.0, 434.0, 532.0, 546.0, 562.0, 576.0, and64.0 nm). It has been observed that there was slighthift in peaks of exposed plants; however, the absor-ance was different at different wavelengths. In thistudy, although each wavelength has not been workedut in detail, the observation made at 664.0 nm (chlo-ophyll peak) was selected for chlorophyll recoverytudy after a 24-h waiting period. The chlorophyll ab-orbance at 664.0 nm was 0.968 in control plants whichas reduced to 0.803 at NI dose. After a 24-h waitingeriod the absorbance increased to 0.913 showing aartial recovery of chlorophyll.UV-B injury to plants mainly depends on intensity

nd exposure periods which may damage cellularembrane architecture, resulting in permeability

hanges (Farooq and Hans, unpublished data). As theellular contents begin to leak into the intracellularpaces, enzymes combating oxidation stress react withhenols converting them to quinones. O-Quinones po-ymerize with amino acids, amines and sulfhydrylroups of protein to form low-molecular-weight reddishrown pigments in exposed leaves (Howell, 1974). Thisnvolvement of phenol appears to be the cause of visibleecrotic lesions in injured leaves (Howell, 1974). In theresent study severe injury produced by the UV-B wasndicated by the necrosis of large areas of leaf tissue.xposure to UV-B causes reactive oxygen species gen-ration, like singlet oxygen, superoxide and hydroxyladicals which on interconversion form hydrogen per-xide (Joshi and Pathak, 1984); however, the plantsan overcome its phytotoxic effects by readily detoxify-ng it with increased peroxidase activity. Peroxidase

FIG. 2. The absorption spectra of control and UV-B-exposedemna major.

972

nd acts as a determining factor for plants tolerance toV-B radiation.It is also clear that metabolic alterations in plants

re initiated at the concentration much below thateeded for visible injury. A decrease in chlorophyllontent by 19.03% and a corresponding 22.52% in-rease in pheophytin showed conversion of chlorophyllnto pheophytin as also is evident by increase inheophytin/chlorophyll ratio (1.38 in control to 2.08 inxposed plants), indicating that UV-B induced senes-ence in the plant. At this dose carotenoid content alsoncreased by 17.36%, showing enhanced carotenoidynthesis under UV-B stress conditions. Enhancementf carotenoid content was attributed to the protectionf photosynthetic apparatus. This observation is ingreement with that of Dohler (1998). However, whenlants exposed to NI dose were returned back to theulture medium for 24 h, this resulted in the partialecovery of chlorophyll, demonstrating that UV-B-nduced damage was reversible at low dose.

At the MI dose, chlorophyll pheophytin and carot-noid contents were decreased and the pheophytin/hlorophyll ratio was increased from 1.38 in control to.61 in exposed plants. These results showed pigmentestruction at visible injury dose and a part of chloro-hyll conversion into pheophytin. A decreased carot-noid content showed reduced ability of the plant toope with oxidative stress.A high dose of UV-B irradiance disturbs the normal

hysiological and metabolic process as evident byarked decrease in different metabolites at SI dose

evel. The energy requirement for various physiologicalnd biochemical processes is met through the in-reased hydrolysis of macromolecules such as proteinnd starch. Proteins have their peak absorbance in theV region (280 nm) making them very sensitive tolteration or damage by UV radiation (Caldwell, 1979)rotein decreased in UV-B exposed plants is a generalbservation (Vu et al., 1982; Yuichi et al., 1989; Dohler,998; Dohler et al., 1998; Hader and Figuerea, 1997;uresh Babu et al., 1998), and the present study alsoorroborates that the UV-B radiation causes decreasen protein in Lemna major also.

The results demonstrated that UV-B radiation in-uced oxidative stress in Lemna major at all the expo-ure levels. The pigment damage at the metabolic levelas partially reversible at a dose level below threshold.owever, higher doses and longer periods of sunshines in tropical and subtropical regions could producerreversible damage to this important aquatic macro-hyte.

CKNOWLEDGMENTS

The authors thank Dr. P. K. Seth, Director, ITRC, Lucknow, for hiseen interest and suggestions in this study. We also thank Mr. A. K.igam for secretarial assistance.

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