Journal of Photoscience (2001), Vol. 8(1), pp. 1−7
Photosynthetic Response and Protective Regulation To Ultraviolet-B Radiation In Green Pepper (Capsicum annuum L.) Leaves
Dae Whan Kim1, Sung-Soo Jun and Young-Nam Hong
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea
Kumho Life and Environmental Science Laboratory, Kwangju 500-712, Korea
The deteriorative effect of ultraviolet-B (UV-B) radiation on photosynthesis was assessed by the simultaneousmeasurement of O evolution and chlorophyll (Chl) fluorescence in green pepper. UV-B was given at the intensity
of 1 W·m-2, a dosage often encountered in urban area of Seoul in Korea, to the detached leaves. Both Pmax andquantum yield of O evolution was rapidly decreased, in a parallel phase, with increasing time of UV-B treatment.
Chl fluorescence parameters were also significantly affected. Fo was increased while both Fm and Fv weredecreased. Photochemical efficiency of PS II (Fv / Fm) was also declined, although to a lesser extent than Pmax. Both qP and NPQ were decreased similarly with increasing time of UV-B treatment. However, PS I remainedstable. The addition of lincomycin prior to UV-B treatment accelerated the decline in Fv / Fm to some extent,suggesting that D1 protein turnover may play a role in overcoming the harmful effect of UV-B. The amount ofphotosynthetic pigments was less affected than photosynthetic response in showing decline in Chl a and carotenoidsafter 24 h-treatment. Presumptive flavonoid contents, measured by changes in absorbance at 270 nm, 300 nm and330 nm, were all increased by roughly 50% after 8 h-treatment. Among antioxidant enzymes, activities of catalaseand peroxidase were steadily increased until 12 h of UV-B treatment whereas ascorbate peroxidase, dehydroascorbatereductase and glutathione reductase did not show any significant change. The results indicate that deteriorativeeffect of UV-B on photosynthesis precedes the protection exerted by pigment synthesis and antioxidant enzymes. key words: UV-B radiation, pepper, photosynthesis, chlorophyll fluorescence, flavonoid, antioxidant enzyme INTRODUCTION
absorbing biomolecules, namely proteins and nucleic acidswere photomodified [9,10]. In addition, UV-B radiation resulted
Plants in nature are continuously affected by various natural
in changes in pigment composition by reducing Chl content
environmental factors such as light, water and temperature
and increasing flavonoid content, which was especially
and respond to changes in those factors to their advantage.
After rapid industrialization, anthropogenic environmental
Decrease in photosynthesis due to UV-B radiation was
pollutants such as heavy metals and airborne pollutants presented
mainly attributed to the inactivation of PS II [12-15]. UV-B
a new set of adverse factors directly or indirectly affecting
treated leaves showed reduction in O evolution and Chl
plants growth. Among these factors, UV-B (between 280 and
fluorescence originating from PS II . The same results
320 nm) has drawn attention in recent years since the heavy
were observed in the isolated chloroplasts from UV-B treated
usage of Freon (chloroflurocarbon) gas for aerosol propellants
plants . UV-B mostly affected PS II reaction center itself
and refrigeration resulted in the rapid erosion of stratospheric
and water oxidizing complex [16,17]. The outcome was due
ozone layer on a global scale [1,2]. In consequence, UV-B
to the denaturation of proteins by UV-B in PS II complex,
level reaching on the ground has greatly increased . For
especially in D1 protein and LHCP II and subsequent damage
example, UV-B radiation was in the range of 0.9 W· m-2 during
in Q was followed [13,17]. D2 protein, generally thought to
the midday in Seoul according to a recent report of Korea
be unaffected, was also shown harmed . Destruction of
Trp residue in the ATPase protein resulted in the inactivation
Increased UV-B radiation was shown to affect harmfully
of ATPase . UV-B reduced RNA transcription of cab and
most physiological and biochemical processes in plants including
psb A . On the other hand, PS I and cytochrome b-f
photosynthesis [4,5], dark respiration [4,5], transpiration ,
complex were hardly affected by UV-B .
biomass allocation , and leaf expansion [7,8]. UV-B
UV-B affected the dark reaction as well by reducing
irradiation could also be damaging when essential UV-
Rubisco activities [15,20]. Both large (55 kDa) and small (15kDa) subunits of Rubisco were degraded and synthesis of
*To whom correspondence should be addressed.
their mRNA was declined after long exposure to UV-B .
The photomodification of Trp residue by UV-B in the large
Received 10 December 2000; accepted 25 January 2001
subunit produced 66 kDa subunit instead of 55 kDa subunit
. All of them led to the decline in Rubisco activities and
subsequent reduction in CO assimilation. Measurement of O evolution and Chl fluorescence. Chl
UV-B was absorbed directly by DNA to form UV-induced
fluorescence and O evolution were measured simultaneously
photoproducts such as cyclobutane pyrimidine dimer and
using leaf discs of 3.5 cm-diameter, taken from leaves of randomly
pyrimidine (6,4) pyrimidone dimer, which caused serious
selected plants, in Hansatech (Kings Lynn, UK) LD2 leaf disc
errors in replication and transcription [9,22]. To ameliorate
chamber. After UV-B treatment, leaves were dark-adapted for 30
these deteriorative effects, plants developed two types of
min in a floating state on DW. Each leaf disc was chosen to have
protective mechanisms: DNA repair and shielding to reduce
approximately equal Chl content, when measured by Minolta Chl
DNA damage. Photolyase was shown to be involved in repairing
meter (SPAD-502, Minolta, Japan) and normalized to have equal
UV-B damaged DNA in plants [23, 24]. Thickening of leaves
Chl content. The measurement of Chl content by SPAD-502 was
and formation of cuticular waxes were manifested to prevent
reliably matched with that of conventional extraction method
the penetration of UV-B . Accumulation of proline, ferulic
using acetone. Chl fluorescence was measured using Walz PAM
acid, and flavonoids was observed in the leaves [26,27]. Among
Chl fluorometer (Effeltrich, Germany) while O evolution was
them UV-absorbing flavonoids and transcripts involved in
polarographically measured with a Clark type electrode at 25oC
flavonoid biosynthesis were increased after long exposure to
using Hansatech O electrode control box. To measure Pmax and
UV-B [27-29]. Increase in flavonoid content decreased the
quantum yield of O evolution, actinic light was given at three
UV-induced dimer formation . Furthermore, UV-B radiation
different intensities of 30, 50, and 270 µmol · m-2· s-1 using Schott
led to the formation of biologically active oxygenic species
illuminator (Schott Glass, Stafford, UK). Fv/Fm, NPQ (defined
that could damage the photosynthetic apparatus and adversely
as Fm/Fm’-1), and qP were calculated as described before .
affect the enzyme activities. Antioxidant enzymes such as
PS I oxidation and reduction kinetics was measured by monitoring
catalase, ascorbate peroxidase, and glutathione peroxidase
changes in absorbance at 820 nm (∆A ) using PAM Chl
were activated against oxidative stresses . Accordingly,
fluorometer connected to 830 nm LED (type ED 800T) as
activities of various antioxidant genes and enzymes were
increased after UV-B treatment [32,33]. Lincomycin treatment. Lincomycin was treated by immersing
In the present study, the multiple effects of UV-B and plants’
the petiole of each detached leaf in a Eppendorf tube containing
response against it were studied in green pepper, a major
1.2 mM lincomycin solution for 4 h in the growth chamber. UV-B
produce in Korea. We first attempted to evaluate the deteriorative
treatment was subsequently done to the lincomycin-imbibed leaves.
effect of realistic UV-B dosage encountered often in Korea,
Determination of photosynthetic pigments. The photosynthetic
on photosynthesis. Next the lesion sites were identified and
pigments were extracted following the method of Hiscox and
assessed by indices of Chl fluorescence parameters. Finally,
Israelstham . In 15 mL tube, leaf segments were put with 5
the physiological protective responses in terms of pigment
mL of DMSO and incubated for 1 h at 65°C. The extracted
composition and regulation of antioxidant enzymes were
solution was diluted twice with the same volume of DMSO and
absorbance was read at 470, 645, and 663 nm, respectively. Theamount of Chl a, Chl b, and total carotenoids was calculatedaccording to the formula of Lichtenthaler  as given below. MATERIALS AND METHODS Plant material. Green pepper plants (Capsicum annuum L. cv.
Saemaeul Kumjang #3) were grown in pots filled with soil(Bioplug #2, Hungnong Seeds Co, Ltd., Korea) for 4 to 5 weeks
Total carotenoid = (1000 A − 1.82 Chl a − 85.02 Chl b) / 198
in a growth chamber maintained at 25 ± 1oC under the photoperiodof 16 h-light and 8 h-dark. Light was provided by True-lite II
Measurement of UV-B absorbing pigments. The amount of
fluorescent lamps (Durotest, USA) at the intensity of 200
UV-B absorbing flavonoids was estimated according to Krizek
µmol ·m-2·s-1. Randomly selected leaves of similar size were
et al. . Leaf slices were extracted in 15 mL of solution
sampled for subsequent UV-B treatment.
containing 40% (V/V) methanol and 1% (V/V) HCl and boiled
UV-B treatment. UV-B, at the intensity of 1 W · m-2 was
for 3 min in a water bath. The extracted solution was left at RT
directly cast on the detached leaves floating on DW at RT. The
for 24 h and the absorbance values at 270 nm, 300 nm, and 330
intensity of UV-B was adjusted to be slightly higher than the
nm were monitored. The absorbance values were calculated
daily maximal dosage (0.9 W ·m-2) in urban area of Seoul in May.
UV-B radiation was provided by the additional attachment of a
Activities of antioxidant enzymes. To measure the activities of
UV-B lamp (XX-15B, Spectronics Corporation, USA), covered
antioxidant enzymes, crude extract was obtained by grinding 1 g
with cellulose acetate membrane to eliminate UV below 280 nm,
of leaf slices with 2 mL of solution containing 85 mM K-PO4
to the fluorescent lamps in the growth chamber. UV-B intensity
buffer (pH 7.8) and 1% (W/V) polyvinylpyrrolidone in a mortar
was determined by Digital Radiometer (DRC-100X, Spectronics
and centrifuging at 18000 g for 25 min. CAT, GR, and POD were
Photosynthetic Response and Protective Regulation to UV-B Radiation
assayed according to Rao et al. . CAT activity was determinedspectrophotometrically by the decrease of absorbance at 240 nmin a cuvette with 1 mL of reaction mixture containing 100 mMK-PO (pH 7.0), 40 µL of crude extract, and 10 mM H O , which
was added just prior to the initiation of reaction. GR was assayedin 1 mL of reaction mixture containing 100 mM K-PO (pH 7.8),
0.2 mM NADPH, 0.5 mM GSSG (oxidized glutathione), 2 mMEDTA, and 100 µL of crude extract by monitoring decrease ofabsorbance at 340 nm. The assay was initiated by the addition ofNADPH. POD activity was quantified by measuring the rate ofguajacol tetramerization shown as the increase in absorbance at470 nm in a cuvette containing 1 mL of reaction mixture of 100mM K-PO (pH 6.5), 16 mM guajacol, 10 mM H O , and 100 µL
of crude extract. The reaction was initiated by adding crudeextract and followed for 10 min.
APX and DHAR were measured by following the method of
Asada . APX was monitored by the decrease in absorbance at
Figure 1. Changes in the Pmax and quantum yield of O evolution
in the UV-B treated leaves. Data presented are mean values ± S.E.
290 nm in 1 mL of reaction mixture composed of 50 mM K-PO4
(pH 7.0), 0.5 mM ascorbate, 0.1 mM H O , and 5 µL of crude
extract. DHAR was assayed in 1 mL of reaction mixture containing50 mM K-PO (pH 6.5), 0.5 mM dehydroascorbate, 5 mM GSH
yield implies that UV-B treatment may not make green pepper
(reduced glutathione), 0.1 mM EDTA, and 15 µL of crude extract
more susceptible to photoinhibition. Pmax was decreased more
by monitoring increase of absorbance at 256 nm.
rapidly than quantum yield after UV-B treatment in coleus .
Changes in Chl fluorescence parametersRESULTS AND DISCUSSION
The initial fluorescence (Fo) and maximal photochemical
efficiency (Fv/Fm) are general indicators for PS II functionality. General effect of UV-B on leaf morphology
Damages to PS II are often accompanied with rise in Fo level
The detached leaves did not show any significant change in
and drop in Fv/Fm. Changes in Fo reflect structural alterations
appearance after 24 h of UV-B treatment. However, the leaves
at the antenna of PS II while those in Fv/Fm indicate varied
became more darkened and glossy after a few h of UV-B
energy capturing efficiency in PS II . As shown in Fig.
treatment. Morphological alteration such as thickening of
2A, after UV-B treatment Fo was increased whereas both Fm
leaves and formation of cuticular waxes were shown to be the
and Fv were decreased rapidly in the initial 2 h. Photochemical
primary response to UV-B in terrestrial plants . In green
efficiency of PS II, represented by Fv/Fm, was linearly declined,
pepper, the leaves also became glossy and dark, possibly due
although to a lesser extent than Pmax, showing 12%, 23%, 29%
to the accumulation of waxes and some UV-B absorbing
and 39% reduction after 1, 2, 3 and 4 h of UV-B treatment
(Fig. 2B). The linear decrease in Fv/Fm was also observed inDunaliella after UV-B treatment . Effect of UV-B on Pmax and quantum yield of O evolution
After onset of illumination, the maximal Chl fluorescence
To evaluate the extent of deteriorative effect of UV-B on the
declines as photosynthetic reaction proceeds due to the
photosynthetic capacity, Pmax and quantum yield of O
photosynthetic electron flow represented as qP, and nonradiative
evolution were measured after direct treatment of UV-B on
energy dissipation and ∆pH formation manifested as NPQ
the detached leaves. Pmax was determined under saturating
. Both qP and NPQ were decreased linearly in a parallel
PFR (270 µmol· m-2·s-1) while quantum yield was determined
phase by the increased time of UV-B treatment (Fig. 3).
under low PFR (30 and 50 µmol· m-2· s-1). As shown in Fig. 1,
Decreased qP indicates that the acceptor side of PS II (Q ) is
Pmax was decreased about 28% after 1 h, 47% after 2h, 49%
overly reduced by hindered electron flow .NPQ usually
after 3 h, and 64% after 4 h while quantum yield was decreased
increases when qP is decreased . Decreased NPQ
about 18%, 42%, 46%, and 59%, respectively. Although
accompanied with decreased qP may reflect the damage in
decrease in Pmax preceded decrease in quantum yield, both
ATPase by UV-B, which would block DpH formation by
Pmax and quantum yield were linearly decreased in a parallel
increased back pressure. APTase was shown harmed in earlier
phase in relation to the increased time of UV-B treatment,
demonstrating both maximal photosynthetic activity and
Changes in absorbance at 820 nm reflects the oxidation and
efficiency in the photosynthetic apparatus were similarly
reduction state of P-700 that reflects electron flow from PS II
affected by UV-B. Parallel decrease in Pmax and quantum
to PS I and from PS I to NADP+ . After 4 h of UV-B
Figure 2. Changes in various Chl fluorescence parameters in theUV-B treated leaves: (A) the maximal (Fm) and variable (Fv)fluorescence, (B) the initial (Fo) and the maximal photochemicalefficiency of PSII (Fv/Fm). Fo, Fm, and Fv are given in arbitraryunits. Data presented are mean values ± S.E. for 5 measurements.
Figure 4. Changes in the Pmax (A), Fv/Fm (B), and Fo (C) in thecontrol and lincomycin-treated leaves after UV-B treatment. Datapresented are mean values ± S.E. for 5 measurements.
effect in photoinhibition. Similarly, UV-B treatment damagedD1 proteins . In view of these, UV-B was treated to thelincomycin-infiltrated leaves to test the role of D1 proteinturnover in alleviating the adverse effect of UV-B. As shownin Fig. 4A, decrease in Pmax was identical in control andlincomycin-treated leaves. However, decrease in Fv/Fm andincrease in Fo was accelerated in lincomycin-treated leaves(Fig. 4B and 4C). The decrease in qP and NPQ was alsoaccelerated in lincomycin-treated leaves (Fig. 3). The results
Figure 3. Changes in the photochemical quenching (qP) and
implicate that D1 protein turnover would play a role in
nonphotochemical quenching (NPQ) in the control and lincomycin-
treated leaves after UV-B treatment. Data presented are mean values
Using antisensor lines of Arabidopsis plants with reduced
content in xanthophyll, the effect of UV-B treatment was alsotested. Antisensor plants showed more rapid decline in Fv/
treatment absorbance at 820 nm was not changed in P-700
Fm. In addition, decrease in qQ and NPQ occurred faster
oxidation-reduction kinetics (data not shown). Therefore, PS I
(data not shown). These results indicate that xanthophyll
appeared to be resistant to UV-B damage as previously
cycle is also involved in relieving the deteriorative effect of
UV-B. Therefore, photoprotective mechanisms appear to bebeneficiary for reducing the adverse effect of UV-B. Effect of lincomycinTurnover of D1 protein plays a significant role in overcoming
Effect on the contents of photosynthetic pigments
photoinhibition as lincomycin-infiltrated leaves show exacerbating
The adverse effect of UV-B on photosynthesis may result in
Photosynthetic Response and Protective Regulation to UV-B Radiation
Table 1. Changes in the amount of photosynthetic pigments afterUV-B treatment.
*Data presented are mean values ± S.E. for 5 measurements.
part from the declined photosynthetic pigments as UV-B wasshown to degrade Chl. As shown in Table 1, UV-B irradiancehad negligible effect on the content of photosynthetic pigments
Figure 5. Changes in the contents of UV-B absorbing flavonoids
up to 12 h of treatment. Total amounts in Chl a, Chl b, and
after UV-B treatment. Changes in flavonoid contents were estimated
carotenoids all remained unchanged. At this time point the
by measuring the absorbance values at 270, 300, and 330 nm of
photosynthesis presumably stops completely. Thus it appears
pigment solution extracted in ethanol. Data presented are mean
that initial decline in photosynthesis after UV-B treatment is
independent of contents in photosynthetic pigments. However,after 24 h of treatment Chl a was declined about 25% and
Effect on the activities of antioxidant enzymes
carotenoids were decreased about 40% (Table 1). In contrast,
Under UV-B radiation, high amounts of free radicals are
Chl b stayed little changed. In Pisum sativum, decrease in Chl
produced, generating, in turn, active oxygenic species such as
a and b accompanied with formation of chlorophyllides a and
H O although the exact mechanism by which they are
b was observed . Decrease in carotenoids was contradictory
generated is unknown. Under such oxidative stresses, plants
to the result in Pisum sativum in which carotenoid content
invoke the antioxidant defense systems [29,31]. One consists
was increased relative to the Chl content . The increased
of low molecular weight antioxidants such as ascorbate,
carotenoids provide photoprotection under high light from
glutathione, and carotenoids. The other is composed of various
photoinhibitory damage. In our experiment, UV-B was sup
antioxidant enzymes that are activated to scavenge the potentially
plemented with relatively low light (200 µmol· m-2· s-1)where
dangerous active species [32,33]. We tested 5 representative
photoinhibition was presumably lacking. Our results implicate
antioxidant enzymes, namely APX, CAT, DHAR, GR, and
that UV-B per se may photooxidize carotenoids to some
POD under UV-B treatment. Activities of CAT and POD
extent, resulting in their degradation. Effect on the contents of UV-B absorbing pigmentsPlants protect themselves against UV light by accumulating
screening pigments. Colorless flavonoids were accumulatedin the epidermal tissues of UV-treated plants and reduceddamage from UV radiation [27,28]. In view of this, changesin presumptive flavonoid contents after UV-B treatment wereinvestigated by monitoring changes in absorbance values at270, 300, and 330 nm. As shown in Fig. 5, absorbance valuesat three wavelengths did not change until 4 h after UV-Btreatment. However, on the 8th h, the absorbance values at allwavelengths were roughly 50% increased and stayed on the samelevel on the 12th h. The induction of flavonoid accumulationseemed to occur far after significant decline of photosynthesis,which implied that flavonoid accumulation would not helpprotecting the photosynthetic apparatus. Time course ofinduction was correlated with transcription of enzymes involved
Figure 6. Changes in the activities of various antioxidant enzymes
in flavonoid synthesis . Transcription of chalcone synthase
after UV-B treatment: CAT, catalase; DHAR, dehydroascorbate
and 4-coumaroyl-CoA ligase reached to maximal level after 6
reductase; POD, peroxidase. Data presented are mean values ± S.E.
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The Efficacy of Paxil (Paroxetine) for Panic Disorder Journal : The Current Practice of Medicine Authors : Dr Sean D. Hood, Dr Spilios Argyropoulos, Prof David J. Nutt Corresponding Author : Sean Hood ([email protected]) Introduction Substantial advances into the pharmacological treatment of Panic Disorder (PD) have been made in the lasttwo decades. Although tricyclic antidepres
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