SHORT COMMUNICATION

Acclimation of clusterbean cotyledon to UV-B radiationin the presence of UV-A: partial restoration of photosyntheticenergy balance and redox homeostasis

Padmanava Joshi • Sasmita Gartia •

Manoj Kumar Pradhan • Srikanta Panigrahi •

Lalitendu Nayak • Basanti Biswal

Received: 20 May 2012 / Revised: 17 February 2013 / Accepted: 18 February 2013 / Published online: 7 March 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract The photosynthetic responses of clusterbean

(Cyamopsis tetraganoloba) cotyledons exposed to UV-A,

UV-B or UV-A ? UV-B radiation for 1 h daily until day

10 have been compared. The loss in the rate of O2 evolu-

tion and CO2 assimilation (Pn) are incommensurate with

each other in both UV-A and UV-B exposed samples

indicating the occurrence of loss in photostasis of photo-

synthesis by these two radiation bands. The alteration in

redox status of QA further suggests about a loss in redox

homeostasis in the photosynthetic electron transport chain.

However, both photochemical efficiency of PS II and Pn

are well maintained in UV-A ? UV-B exposed cotyledons

in spite of reduction in water-use efficiency. The accli-

matization of clusterbean cotyledon to UV-B radiation in

the presence of UV-A has been attributed to accumulation

of flavonoids, increase in stomatal conductance (gs) and

reduction in functional size of PS II.

Keywords Chlorophyll fluorescence induction �Photosystem II � Photostasis � Redox homeostasis �Thermoluminescence � Thylakoid

Introduction

The photosystem II (PS II) of the photosynthetic apparatus

has been identified to be the primary target of ultraviolet-B

(UV-B) radiation and the electron carriers on both reduc-

ing and oxidizing sides of PS II are damaged to different

levels (Melis et al. 1992; Vass et al. 2005; Joshi et al.

2011). Consequently, the rate at which PS II reaction

centers are closed through photochemistry is different from

the rate at which they are opened through redox bio-

chemistry. This difference induces a loss in balance

between the energy absorbed by PS II through photo-

physical process and the energy utilized for carbon

assimilation resulting in a loss in photostasis of photo-

synthesis leading to the metabolism of reactive oxygen

species (ROS) (Joshi et al. 2011).

Some topical findings, however, suggest that the pres-

ence of UV-A radiation mitigates the negative impact of

UV-B radiation on photosynthesis (Lud et al. 2002; Joshi

et al. 2007). The alleviation of UV-B-induced damage

could be brought forth through acclimation, defense and/or

repair mechanisms and these mechanisms are modulated

by UV-A radiation (Mohr and Schopfer 1995). The plant

sensitivity and photomorphogenic responses to UV-B

radiation are also influenced by UV-A radiation (Krizek

2004).

However, no attempt has been made to examine if

UV-A radiation, a natural concomitant of UV-B, does play

any role in the acclimation of photosynthetic apparatus to

UV-B-radiation. Therefore, our objectives have been to

differentiate the effects of UV-A on the redox homeostasis

and the energy source and sink relationship of the photo-

synthetic apparatus from those of UV-B discretely and

to examine if UV-A exposure modulates the process of

acclimation of clusterbean cotyledons to UV-B radiation.

Communicated by K. Trebacz.

P. Joshi (&) � S. Gartia � M. K. Pradhan � S. Panigrahi �L. Nayak

Anchal College, Padampur, Rajborasambar,

Bargarh 768036, Odisha, India

e-mail: padmanava.joshi@gmail.com

S. Gartia � B. Biswal

School of Life Sciences, Sambalpur University,

Jyotivihar, Sambalpur 768019, Odisha, India

123

Acta Physiol Plant (2013) 35:2323–2328

DOI 10.1007/s11738-013-1245-6

Materials and methods

Plant materials and UV exposures

Clusterbean (Cyamopsis tetragonoloba L.) seedlings were

grown in different Petri plates on cotton soaked with dis-

tilled water under white light and were exposed to either

UV-A (Philips TL 20, Type 09, 320-400 nm with peak at

365 nm, 75 lmol m-2 s-1), UV-B (Philips TL-20, Type

05, 280-315 nm with peak at 315 nm, 60 lmol m-2 s-1)

or UV-A ? UV-B (UV-A and UV-B radiation simulta-

neously) for 1 h daily from day 1 to 10 as described by

Joshi et al. (2007). The intensity of UV-B used in this work

is much above the level of UV-B in nature so that its

damaging effects are not marginalized subsequently during

growth of seedlings in white light. In a previous work

(Joshi et al. 2007), it has been shown that the cotyledons

follow the developmental pattern of juvenile phase up to

day 6 and steady phase from day 7 to 10 followed by

declining senescence phase. Thus, different biochemical

and biophysical measurements were conducted on day 10

within an hour of the exposures.

Measurement of photosynthetic pigment and isolation

of chloroplasts

Pigments extracted from cotyledons of clusterbean seed-

lings with 100 % chilled acetone were used for determi-

nation of chlorophyll (Chl) and carotenoids (Car) as

described by Wellburn and Lichtenthaler (1984).

Chloroplasts were isolated from cotyledons of cluster-

bean seedlings following the method of Izawa and Good

(1968)

Estimation of malondialdehyde (MDA) and flavonoids

Thylakoid lipid peroxidation was quantified in terms of

accumulation of MDA as described by Panda et al. (1987).

Accumulation of flavonoids in cotyledons was estimated

following the methods of Flint et al. (1985).

Measurement of O2 evolution from cotyledons

and from isolated chloroplasts

Photosynthetic oxygen evolution was measured directly

from the cotyledons with a leaf disk electrode unit (LD 2/3,

Leaf lab 2, Hansatech, UK).

O2 evolution from isolated chloroplasts in 30 mM Na/K

phosphate basic assay buffer (pH 7.2) containing 30 mM

NaCl and 200 mM sucrose with 0.3 mM of 2,6-dichloro-

benzoquinone (DCBQ), as an electron acceptor, was

measured with a Clark-type oxygen electrode at 21 �C in

rate-saturating red light. Chloroplasts containing 40 lg Chl

were placed in 2 mL of reaction mixture for the measure-

ment. Gramicidin (2.5 lM) was used as an uncoupler.

Measurement of PAM fluorescence

Fluorescence transient and different fluorescence parame-

ters were measured with a PAM fluorometer (FMS-1,

Hansatech, UK) from cotyledons of clusterbean seedlings

according to Schreiber et al. (1986).

Gas exchange measurement

The stomatal conductance (gs), internal CO2 concentration

(Ci), rate of evaporation (E) and the rate of CO2 assimi-

lation (Pn) were measured from the cotyledons of cluster-

bean seedlings with the help of an infrared gas analyzer

(CIRCAS 2 pp system, Hitchin, UK) fitted with an ergo-

nomic Parkinson’s universal leaf cuvette PLC6 (U). Water-

use efficiency (WUE) and intrinsic water-use efficiency

(IWUE) were calculated as Pn/E and Pn/gs respectively.

Measurement of thermoluminescence (TL) glow curves

Isolated chloroplasts containing 100 lg Chl mL-1 in

the presence of 3-(3,4-dichlorophenyl)-l,1-dimethyl urea

(DCMU) at a concentration of 10-5 M were frozen to liquid

nitrogen temperature under continuous illumination of

4.5 lmol m-2 s-1 and then TL curves were obtained by

heating the sample at 0.22 �C s-1 following the method of

Desai et al. (1971). Activation energy (EA) of different peaks

was calculated using Chen formula:

EA ¼ 2:29 kT2m=DT

where DT is the temperature difference at half intensity of

glow peak (Tatake and Desai 1981).

Statistical analyses were conducted following the

method described by Simpson et al. (1960).

Results and discussion

The changes in gs, Ci and Pn in 10-day-old cotyledons of

clusterbean seedlings with UV-A, UV-B or UV-A ? UV-

B exposure are described in Table 2. The level of Pn

decreased by 14.5 % (p \ 0.01) in UV-B exposed sample

but it increased marginally in UV-A exposed one (2.4 %;

p \ 0.05). No significant effect on Pn was observed in UV-

A ? UV-B exposed cotyledons. Analyses of gas exchange

data reveal that UV-B-exposure diminishes Pn in spite of a

significant rise in Ci (35.6; p \ 0.005) and gs (38.31;

p \ 0.005). The finding is in conformity with the earlier

report of Sullivan and Teramura (1990) in soybean. On the

contrary, Albert et al. (2005) have observed no change in

2324 Acta Physiol Plant (2013) 35:2323–2328

123

Pn despite a significant rise in Ci and gs in response to UV-

B radiation. Thus, it is perceptible that the inhibition of PS

II and structural damage of chloroplasts have major con-

tribution to UV-B-induced decline in photosynthesis of

clusterbean cotyledon.

Both UV-A and UV-B radiations have damaging effects

on the photosynthetic apparatus of green plants (Melis et al.

1992; Vass et al. 2002, 2005; Ivanova et al. 2008; Joshi et al.

2011). In the present work also, the damaging effects could

be discernible from the results of Car and Car/chlorophyll a

(Chla) as described in Fig. 1. The level of Car per pair of

cotyledons declined by 4.74 (p \ 0.25), 28 (p \ 0.005) and

7.9 (p \ 0.05) % in UV-A, UV-B and UV-A ? UV-B

exposed seedlings, respectively. On the other hand, Car/

Chla ratio increased insignificantly (0.27 %, p \ 0.1) in the

cotyledons of UV-A exposed seedlings, while it declined by

8.09 % (p \ 0.001) and 1.03 % (p \ 0.01) in UV-B and

UV-A ? UV-B exposed seedlings, respectively. UV-B-

mediated loss in Car could be correlated to the disorgani-

zation of thylakoid membrane as evident from the decline in

Car and Car/Chla and enhancement in MDA accumulation

(36 %, p \ 0.005) (Fig. 1).

The alterations in Chla fluorescence transient parameters

such as photochemical potential of dark adapted (FV/FM)

and light adapted (F0V=F0M) cotyledons, PS II quantum yield

(APS II) and the excitation pressure (1 - qp) (where qp is

the photochemical quenching coefficient) in UV-A, UV-B

and UV-A ? UV-B exposed seedlings are described in

Table 1, while the changes in O2 evolution in response to

these radiation are depicted in Fig. 2. The mode of

alteration in different parameters associated with Chla

fluorescence (Table 1) and O2 evolution (Fig. 2) as induced

by UV-A is observed to be different from those induced by

UV-B radiation. Marginal but parallel loss in FV/FM

(4.6 %; p \ 0.01) and F0V=F0M (6.09 %; p \ 0.01), APS II

(8.63 %; p \ 0.005) (Table 1) and O2 evolution (9 %;

p \ 0.01) (Fig. 2) in UV-A exposed sample indicate that

both OEC and PS II reaction center are inactivated uni-

formly. On the other hand, a sweeping reduction in O2

evolution (46 %; p \ 0.005) and a moderate decrease in

FV/FM (14.11 %; p \ 0.005) or F0V=F0M (16.5 %;

p \ 0.005) in UV-B exposed sample signify an uneven

damage on acceptor and reducing sides of PS II resulting in

a UV-B-induced enhancement in the excitation pressure

(37 %; p \ 0.005) on PS II (Table 1).

0

25

50

75

100

125

150

Car Car/Chla MDA Flavonoid

Rel

ativ

e am

ount

(%

)

Fig. 1 Relative changes in the level of total Car, Car/Chla, accumu-

lation of MDA and accumulation of flavonoids per pair of cotyledons

of clusterbean seedlings on day 10 with UV-A (bar with vertical

lines), UV-B (bar with crossed lines) or UV-A ? UV-B (bar with

diamond points) exposure of 1 h duration from day 1. Control: black.

Each value is a mean of three or four independent measurements.

(n = 3 for Car and Car/Chla; n = 4 for MDA and flavonoids) ± SD.

100 % = 0.038 mg/pair of cotyledons for Car, 0.2608 for Car/Chla,

50 nmol g-1 fresh weight for MDA and 14 unit g-1 fresh weight for

flavonoids. For details, see ‘‘Materials and methods’’

Table 1 Changes in different parameters associated with fast Chla

fluorescence kinetics as measured from cotyledons of clusterbean

seedlings with UV-A, UV-B or UV-A ? UV-B exposure on day 10

Parameters Control UV-A UV-B UV-A ? UV-B

FV/FM 0.850 ± 0.01 0.811 ± 0.013 0.730 ± 0.016 0.814 ± 0.017

F0V=F0M 0.821 ± 0.011 0.771 ± 0.017 0.686 ± 0.007 0.763 ± 0.008

APS II 0.8 ± 0.003 0.731 ± 0.019 0.6 ± 0.015 0.76 ± 0.009

1 - qp (%) 100 ± 3.65 95.12 ± 6.09 137 ± 4.51 112 ± 1.22

Each value is a mean of 3 independent measurements (n = 3) ± SD. For details, see

‘‘Materials and methods’’

0

25

50

75

100

125

Cotyledon Chloroplasts

Oxy

gen

Evo

luti

on (

%)

Fig. 2 Relative changes in the level of O2 evolution as measured

from cotyledons and chloroplasts isolated from cotyledons of

clusterbean seedlings on day 10 with UV-A (bar with vertical lines),

UV-B (bar with crossed lines) or UV-A ? UV-B (bar with diamond

points) exposure of 1 h duration from day 1. Control: black. Each

value is a mean of three independent measurements. (n = 3) ± SD

100 % = 15.2 lmol O2 evolved m-2 s-1 for cotyledons and

250 lmol O2 evolved/mg Chl/h for isolated chloroplasts. For details,

see ‘‘Materials and methods’’

Acta Physiol Plant (2013) 35:2323–2328 2325

123

Alterations in fluorescence transient parameters and O2

evolution in response to UV exposure, as discussed above,

and decline in the ratio in vivo O2 evolution to net pho-

tosynthesis (O2/Pn) (Table 2) may indicate a loss in the

coordinated flow of electron extracted from H2O to elec-

tron consuming sink. The ratio O2/Pn, used here to assess

the photosynthetic source–sink equilibrium (Falkowski and

Chen 2003; Joshi et al. 2011), declined by 20.9

(p \ 0.005), 36.87 (p \ 0.005) and 27.9 % in response to

UV-A, UV-B and UV-A ? UV-B exposure, respectively.

A close analysis of the pattern of decline in the ratio in

response to UV-A and UV-B exposure reveals a difference

in the mode of loss in the balance between rate of electron

released by oxygen evolving complex of PS II and the rate

of electron consumed for assimilation of CO2 and the UV-

B-induced loss is partially restored by UV-A radiation in

UV-A ? UV-B exposed sample. The difference in the

pattern of alteration in 1 - qp (Table 1), that reflects the

redox status of QA (Dietz et al. 1985; Wilson et al. 2006),

could be a manifestation of the difference in the mode of

loss in photostasis of photosynthesis. Further, the mode

of change in 1 - qp indicatesQ�A

QAþQA[ QA

QAþQAin UV-B and

QA

Q�AþQA[ Q�A

Q�AþQAin UV-A exposed samples. Plausibly, at a

low excitation pressure, the decrease in FV/FM (Table 1) in

UV-A or UV-A ? UV-B exposed samples could hardly

affect Pn as observed in this work (Table 2).

The redox state of QA in PS II is further examined by TL

technique. Table 3 describes the relative changes in the

parameters associated with TL curve (not shown) obtained

from DCMU-treated isolated chloroplasts. Bands ‘Q’ and

‘C’ are known to arise from recombination of charge pairs

[S2, QA-] and [D?, QA

-] (Demeter et al. 1993) at 13 and

40 �C, respectively. UV-B exposure, besides diminishing

the bands height, upshifted the peak temperature of band Q

by 7.5 �C. UV-A ? UV-B exposure also diminished the

height of these bands but fairly less than that of UV-B

exposed one and the peak temperature of Q band was up-

shifted by only 2 �C. The energy of activation (EA) of band

Q increased by 3.21 (p \ 0.01), 16.3 (p \ 0.005) and 10.2

(p \ 0.05) % in UV-A, UV-B and UV-A ? UV-B exposed

samples, respectively. Further the ratio of the area under

the TL curve containing both Q and C bands to O2 evo-

lution measured from isolated chloroplasts (A/O2:) has

been calculated and normalized to 100 % for control

sample. The ratio increased by 18 % (p \ 0.005) in UV-B

exposed sample, while it decreased by 15 % (p \ 0.001)

and 8 % in UV-A and UV-A ? UV-B exposed sample,

respectively. A significant up-shift in the peak temperature

of Q band observed here (Table 3) is contrary to the

findings of Hideg et al. (1993) and the difference may be

attributed to higher level of UV-B used in this work. The

number of active reaction center, that is responsible for QA

reduction, is different in different samples as chloroplasts

containing equal amount of Chl has been used for TL

measurement. Therefore, the parameter ‘‘A/O2:’’ has been

used as a measure of QA reduction. The enhancement of the

ratio in UV-B exposed sample indicating an accumulation

of reduced QA could be due to blockage of electron transfer

between QA and QB (Melis et al. 1992; Joshi et al. 2011)

leading to the loss in redox homeostasis.

Plants acclimatize to the environmental stresses for

optimum photosynthesis through different mechanisms.

The accumulation of flavonoid, a UV-B screening pigment

(Day 1993; Mohr and Schopfer 1995) and scavenger of free

radicals (Bor et al. 1990) is mediated through photom-

orphogenic photoreceptors as inferred from the increase in

the level of flavonoids in UV-A, UV-B and UV-A ? UV-B

exposed cotyledons by 3 % (p \ 0.1), 8.7 % (p \ 0.05)

and 22 % (p \ 0.005), respectively (Fig. 1). A significant

increase in the pigment level in UV-A ? UV-B but mar-

ginally in either UV-A or UV-B exposed samples dem-

onstrate the co-action of the light absorbed by blue/UV-A

and UV-B photoreceptors in eliciting the response (Mohr

and Schopfer 1995). Further, the results of Car/Chla

(Fig. 1), a measure of the functional size of PS II (Maxwell

et al. 1994), in UV-A and UV-A ? UV-B exposed coty-

ledons suggest about the occurrence of UV-A-specific

chloroplast movement mediated through blue/UV-A pho-

toreceptor (Jarillo et al. 2001). The response is known to

protect the photosynthetic apparatus by changing the

excitation distribution between the photosystems (Davis

Table 2 Changes in the levels of stomatal conductance (gs), CO2 assimilation (Pn), internal CO2 concentration (Ci) and O2/Pn in the cotyledons

of clusterbean seedlings with UV-A, UV-B or UV-A ? UV-B exposures on day 10

Treatment gs

(mmol m-2 s-1)

Pn

(lmol CO2 m-2 s-1)

Ci

(lmol m-2 s-1)

O2/Pn (%)

(a.u.)

WUE (%)

(Pn/E) (lmol mmol-1)

IWUE (%) (Pn/gs)

(lmol mmol-1)

Control 26 ± 0.72 8.3 ± 0.029 45 ± 2.49 100 100 100

UV-A 44.2 ± 0.84 8.5 ± 0.011 65 ± 8.72 76.6 64.8 59.4

UV-B 36.1 ± 0.71 7.1 ± 0.016 61 ± 4.2 59.39 69.2 64.4

UVA ? UVB 67.4 ± 1.9 8.22 ± 0.009 72 ± 12.3 75.12 37.11 37.8

Each value is a mean of 4 independent measurements (n = 4) ± SD. The value ‘O2/Pn’, ‘WUE’ and ‘IWUE’ of the control sample are taken as

100 %. For details, see ‘‘Materials and methods’’

2326 Acta Physiol Plant (2013) 35:2323–2328

123

and Hangarter 2012). Higher stomatal conductance in

response to UV-A radiation in UV-A and UVA ? UV-B

exposed sample, a UV-A-specific response (Boccalandro

et al. 2011) could also be discerned from the results of gas

exchange measurement (Table 2). The alterations in gs and

Ci as a result of stomata opening lead to a decline in WUE

by 35.22 % (p \ 0.005), 30.8 % (p \ 0.005) and 62.88 %

(p \ 0.005) and in IWUE by 48.65 % (p \ 0.005), 35.6 %

(p \ 0.005) and 63.2 % (p \ 0.005) in UV-A, UV-B and

UV-A ? UV-B exposed samples, respectively.

Author contribution M.K. Pradhan, S. Panigrahi and

L. Nayak conducted biochemical analysis for determina-

tion pigment content, MDA and flavonoids accumulation

while S. Gartia, P.N. Joshi and B. Biswal measured

fluorescence transient, thermoluminescence and net photo-

synthesis.

Acknowledgments We thank Prof. U.C. Biswal for going through

the manuscript and constructive suggestions. We also thank Dr. N.K.

Ramaswamy (NABD, BARC, Mumbai) and Dr. R.K. Iyer (MBAD,

BARC, Mumbai) for providing facilities to carry out TL studies. The

work is supported by UGC, New Delhi, and DST, New Delhi.

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