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Environmental and Experimental Botany 66 (2009) 160–171

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Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

hotosynthesis, fluorescence, shoot biomass and seed weight responses of threeowpea (Vigna unguiculata (L.) Walp.) cultivars with contrasting sensitivity toV-B radiation

iridara-Kumar Surabhi1, K. Raja Reddy ∗, Shardendu Kumar Singhepartment of Plant and Soil Sciences, Mississippi State University, Box 9555, Mississippi State, MS 39762, USA

r t i c l e i n f o

rticle history:eceived 22 April 2008eceived in revised form 12 January 2009ccepted 5 February 2009

eywords:hlorophyll fluorescenceowpeahotosynthesisV-B radiationV-B absorbing compounds

a b s t r a c t

Current and projected increases in ultraviolet-B (UV-B) radiation (280–320 nm) may alter crop produc-tion. An experiment was conducted to study the influence of UV-B radiation on photosynthetic CO2

assimilation, photosystem II (PSII) photochemistry, pigments, UV-B absorbing compounds, vegetativeand reproductive growth responses of three cowpea cultivars, California Blackeye (CB)-5, CB-27, andMississippi Pinkeye (MPE). Cowpea cultivars were subjected to four levels of biologically effective UV-Bradiation of 0 (control), 5, 10 and 15 kJ m−2 d−1 from 8 days after emergence (DAE) to maturity of the cropin sunlit, controlled environment chambers. Increase in UV-B radiation caused a decrease in maximumlight-saturated photosynthesis (Amax) in all the three cultivars, but the magnitude of decrease varied, 12%in MPE, 37% in CB-27, and 43% in CB-5 at 15 kJ of UV-B. The lower damaging effect of UV-B on Amax in MPEwas supported by a smaller decrease in maximum rate of Rubisco activity (Vcmax) coupled with higherlevels of light-saturated rate of electron transport (Jmax) compared to other sensitive cultivars, CB-5 andCB-27. The tolerance nature of MPE was further evidenced by less or no effect of UV-B on photosyntheticelectron transport rate (ETR), photochemical quenching (qP), and quantum yield of PSII photochemistry(˚PSII) compared to CB-5 and CB-27 across the UV-B treatments. Further, maximal electron transportrates (ETRmax) estimated at saturated light substantially decreased with increase in UV-B in CB-5 and CB-27, while there was no or less influence on MPE. All cultivars showed linear relationship between ˚PSII

and quantum yield of CO2 fixation (˚CO2 ) derived from light–response curves and internal-CO2-response

curves of chlorophyll a (Chla) fluorescence. MPE maintained significantly higher concentration of UV-Babsorbing compounds compared to the CB-5 and CB-27 across UV-B levels. Plants grown at elevated UV-Bradiation produced significantly lower plant and seed dry weights in both the sensitive cultivars (CB-5and CB-27) while the effect on MPE was minimal. This study revealed that the current and projected UV-Bradiation could significantly decrease the growth and yield of sensitive cowpea cultivars, possibly causedby reduction in the photosynthetic efficiency of plants. In general, MPE showed more tolerance to UV-B

of the

radiation based on most

. Introduction

Over the last 50 years, stratospheric ozone has decreased

y about 5%, mainly due to the release of ozone-destroyingnthropogenic pollutants such as chlorofluorocarbons (Pyle, 1997)esulting higher levels of UV-B (280–320 nm) radiation at thearth’s surface. Increases in solar UV-B have raised concernsegarding the damaging impact of UV-B radiation on crop plants

∗ Corresponding author. Tel.: +1 662 325 9463; fax: +1 662 325 9461.E-mail address: krreddy@pss.msstate.edu (K.R. Reddy).

1 Current address: Division of Biology and Ecological Genomics Institute, Kansastate University, Manhattan, KS 66506, USA.

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.02.004

parameters studied as compared to the CB-5 and CB-27.© 2009 Elsevier B.V. All rights reserved.

(Caldwell et al., 1998; Kakani et al., 2003). Current globalterrestrial UV-B radiation range between 2 and 12 kJ m−2 d−1

on a given day with near equator and mid-latitudes receiv-ing higher doses (total ozone mapping spectrometer 2002,http://toms.gsfc.nasa.gov/ery uv/euv v8.html), which includes anincrease of 6–14% since 1980s (UNEP, 2002). The three dimensionalChemistry-Climate models estimates indicate that ground-levelUV-B radiation is currently near its maximum levels and is expectedto revert to the pre-1980s level at the mid-latitudes by 2040–2070,if all member countries implement the Montreal Protocol (WMO,

2007). An examination of more than 200 plant species reveals thatapproximately 20% are sensitive, 50% are mildly sensitive or toler-ant and 30% are completely insensitive to UV-B radiation (Teramura,1983). Therefore, further studies are needed to understand themechanisms of UV-B radiation on major crop species.

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Photosynthetic organisms need sunlight and are inevitablyxposed to UV-B radiation. Even a small increase in incident UV-radiation can have significant biological effects because UV-B is

eadily absorbed by a number of important macromolecules suchs nucleic acids, proteins, lipids and phytohormones (Rozema etl., 1997). Failure to protect from UV-B may result in a wide rangef morphological, physiological and metabolic responses includ-ng altered plant growth, reduced yield, damage to photosystemI (PSII), and decrease in chlorophyll content (Caldwell and Flint,994; Saile-Mark and Tevini, 1997; Germ et al., 2005). In general, theV-B protective mechanisms in plants are elucidated by increase

n biosynthesis of UV-B absorbing compounds, quenching of freeadicals and DNA-repair system (Rozema et al., 1997; Kakani etl., 2004). Of the various parameters studied from the 62 pub-ished papers, Searles et al. (2001) reported an average increasef about 10% UV-B absorbing compounds was the most apparenthenomenon in plants.

UV-B radiation can impair all major processes of photosynthesisncluding photochemical reactions in thylakoid membranes, enzy-

atic processes in the Calvin cycle, stomatal limitations to CO2iffusion (Bornman, 1989; Allen et al., 1998), destruction of aminocid residues, and oxygen mediated damage to unsaturated fattycids in plant cell membranes. Higher levels of UV-B radiation haseen shown to inhibit photosynthesis in pea (Nogues and Baker,995), cotton (Reddy et al., 2003; Zhao et al., 2004) and oilseedape (Allen et al., 1997). However, other studies simulating 15–25%zone depletion or 30% increase in UV-B radiation have reportedo significant changes in plant growth and photosynthetic perfor-ance in legumes and other crops (Allen et al., 1998; Chimphango

t al., 2003; Sullivan et al., 2003). Reports of UV-B radiation influ-nce on photosynthesis are inconsistent probably due to differencesn species or cultivars of the same species, different growth environ-

ents, the UV-B dosages and the exposure duration of UV-B dosesRozema et al., 1997; Kakani et al., 2003).

Chlorophyll a (Chla) fluorescence has proved to be a useful,uantitative, rapid and non-invasive technique to study differentspects of photosynthesis. Chla fluorescence has been used forcreening cowpea cultivars for various abiotic stresses such asalinity and water deficits (Souza et al., 2004; Murillo-Amador etl., 2005) and UV-B radiation (Hideg et al., 2006). Linear correla-ion between ˚PSII and ˚CO2 observed by Fracheboud et al. (1999)mplies that ˚PSII can be used as a tool to select plants with differenthotosynthetic capacity under stressful environments.

Several recent reviews have summarized the effects andonsequences of UV-B radiation on major agricultural and non-gricultural species (Allen et al., 1998; Kakani et al., 2003; Caldwellt al., 2007). The conclusions from these studies and reviews arehat the sensitivity of crop plants to UV-B radiation varies depend-ng on species and cultivars, growth conditions, as well as theimultaneous occurrence of the other stress conditions.

Cowpea [Vigna unguiculata (L.) Walp.] is an important cropargely grown in warm and hot regions of Africa, Asia and theorth and South Americas, and is often regarded as being well-dapted to high temperatures and drought compared with otherrop species (Ehlers and Hall, 1997). Due to its wide range ofeographical distribution, cowpea plants are currently experienc-ng various levels of ambient UV-B radiation. In a recent study,

usil et al. (2002) reported that cowpea as one of the most UV-sensitive legume among the 17 species largely grown in South

frica. Few studies exist on cowpea subjected to different abiotictresses such as drought (Souza et al., 2004), salinity (Cavalcanti

t al., 2007) and high temperature (Ehlers and Hall, 1998). Toate, very few studies have addressed the effects of UV-B radia-ion on cowpea (Premkumar and Kulandaivelu, 2001; Chimphangot al., 2003). An increased UV-B (simulating 20% O3 depletion)adiation markedly alleviated the adverse effect of magnesium defi-

erimental Botany 66 (2009) 160–171 161

ciency in cowpea (Premkumar and Kulandaivelu, 2001), and nosignificant change in plant height, leaf area and dry matter pro-duction were observed in cowpea (Chimphango et al., 2003). Thesestudies have represented a smaller set of plant characteristics usu-ally measured from part of plant organs and growth stages. Wehypothesized that, being tropical in origin, UV-B tolerant char-acteristics are present in cowpea with genotypic variability andwhen exposed to UV-B. Therefore, the objectives of this studywere to evaluate cowpea cultivar responses to a range of UV-B radiation, and to gain deeper understanding of UV-B responsemechanisms on photosynthesis by studying the functionality of thephotosynthetic apparatus as assessed by chlorophyll fluorescencemeasurements. Additionally, shoot biomass, seed weight, pigmentsand UV-B absorbing compounds will simultaneously be investi-gated to determine the possible relationship with photosyntheticparameters and to detect possible cultivar differences in stress pro-tection.

2. Materials and methods

2.1. Experimental conditions, plant culture and treatments

The experiment was conducted at the R.R. Foil Plant ScienceResearch Center, Mississippi State University, Mississippi State(38◦28′N, 88◦47′W), MS, USA in 2005, using controlled environ-ment chambers known as Soil–Plant–Atmosphere-Research (SPAR)units. Each SPAR chamber consists of a steel soil bin (1 m deepby 2 m long by 0.5 m wide) to accommodate the plant root sys-tem, and a Plexiglas chamber (2.5 m tall by 2.0 m long by 1.5 mwide) to accommodate aerial plant system. Details of operationand control of SPAR chambers have been described by Reddy etal. (2001).

In the present study, we have selected three different cowpeacultivars with varying levels of biotic and abiotic stress tolerance.Mississippi Pinkeye is a new variety with the multiple disease resis-tance capacity. CB-27 is a high yielding variety and has a number ofother desirable features, such as heat and disease resistance char-acteristics than variety CB-5 (Ehlers et al., 2002). Seeds of threecowpea cultivars (California Blackeye-5, CB-5; California Blackeye-27, CB-27, and Mississippi Pinkeye, MPE) were sown in pots (15 cmdiameter and 15 cm in height) filled with fine sand on 26 July 2005.Five pots of each cultivar were arranged randomly in each SPARchamber. Temperatures in all units were controlled and maintainedat 30/22 ◦C (day/night), and the average day/night temperatureacross the UV-B treatments were 29.3 ± 0.12/21.6 ± 0.15 ◦C. Thechamber CO2 concentration [CO2] was controlled and maintained at360 �L L−1, and the average [CO2] across the UV-B treatments was364 ± 0.84 �L L−1. Emergence was observed 5 days after sowing inall units. Pots were thinned to two plants per pot at 5 days afteremergence (DAE). Plants were watered three times a day with full-strength Hoagland’s nutrient solution delivered at 08:00, 12:00 and17:00 h to ensure favorable water and nutrient conditions for plantgrowth through an automated- and computer-controlled drip irri-gation system. Variable-density black shade cloths (Hummert SeedCo., St. Louis, MO, USA) around the edges of plants in each SPAR unitwere adjusted regularly to match plant height in order to simulatenatural shading in the presence of other plants.

Four treatments of biologically effective UV-B (280–320 nm)radiation intensities of 0 (control), 5, 10 and 15 kJ m−2 d−1 wereimposed from 10 to 62 DAE. Square-wave UV-B supplementation

system was used to provide respective UV-B treatments using thegeneralized plant response action spectrum normalized at 300 nm(Caldwell, 1971) at near ambient solar radiation regimes. The Plex-iglas chambers are completely opaque to solar UVB radiation,but transmits 12% UV-A and >95% incoming daily solar radia-

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ion (285–2800 nm) outside of the SPAR units measured with ayranometer (Model 4–8; The Eppley Laboratory Inc., Newport,I, USA), ranged from 1.5 to 24 MJ m−2 d−1 with an average of8 ± 4 MJ m−2 d−1. The UV-B radiation was imposed for 8 h per day,rom 08:00 to 16:00 h by eight fluorescent UV-313 lamps (Q-Panelompany, Cleveland, OH, USA) driven by 40 W dimming ballasts.he lamps were wrapped with presolarized 0.07-mm celluloseiacetate film to filter UV-C (<280 nm) radiation. The cellulosecetate film was changed 3–4-d intervals. The UV-B radiation deliv-red at the top of the plant canopy was checked daily at 09:00 hith a UVX digital radiometer (UVP Inc., San Gabriel, CA) calibratedsing an Optronic Laboratory (Orlando, FL, USA) model 754 Spec-roradiometer, which was used initially to quantify lamp output.he lamp output was adjusted as needed to maintain the respec-ive UV-B radiation levels. A distance of 0.5 m from the lamps to theop of plant canopy was maintained throughout the experiment.he actual biologically effective UV-B radiation was measured dailyuring the crop growth period at 10 different locations in eachPAR unit corresponding to the pots arranged in rows. The aver-ge UV-B radiation dosages for the whole experimental periodor the four levels of the UV-B radiation treatments were 0.0,.79 ± 0.15, 9.82 ± 0.12 and 14.62 ± 0.19 for the planned 0, 5, 10 and5 kJ m−2 d−1 set points, respectively. The simulated O3 depletionf the four UV-B doses was 0, 6, 12 and 24 %, respectively, at thisocation.

.2. Photosynthetic CO2 assimilation and fluorescenceeasurements

.2.1. Light–response curves of photosynthesis (A/PAR)Gas exchange measurements were made on intact leaves using

n open gas exchange system, LI-6400 photosynthesis (LICOR Inc.,incoln, NE, USA) fitted with a leaf chamber fluorometer (LCF)hat provided LED-based fluorescence and light. The youngest, fullyxpanded third or fourth leaf from the main axis terminal, weresed to measure A/PAR curves at 20–24 days after treatment (DAT).hen measuring A/PAR curves, the temperature inside the leaf

uvette was set to 30 ◦C, and [CO2] was set to 360 �L L−1. Artifi-ial illumination was applied to leaves from a red-blue LED lightource attached to the sensor head. The PAR was gradually increasedrom 0 to 2000 �mol m−2 s−1 by 10 steps (0, 50, 100, 200, 400,00, 1000, 1500, 1750, 2000 �mol m−2 s−1). The A/PAR curves weretted using nonrectangular hyperbola least square curve fitting pro-edure (Lambers et al., 1998) as described in Eq. (1) where ˚ ishe apparent quantum efficiency, Q is the PAR, Amax is the light-aturated rate of CO2 assimilation, � is the convexity or curvatureactor, and Rd is the dark respiration and A is the photosyntheticate.

= ˚Q + Amax −√

(˚Q + Amax)2 − 4˚Q�Amax

2�− Rd (1)

.2.2. Internal-CO2-response curves of photosynthesis (A/Ci)Measurements were taken at constant PAR of 1500 �mol

−1 s−1, ambient relative humidity (50%), and leaf cuvette tem-erature of 30 ◦C (24–28 DAT). The [CO2] in leaf cuvette washanged by 11 steps (400, 300, 200, 100, 50, 0, 400, 400, 600, 800,000 �L L−1). Lower [CO2] were measured first to prevent stomatallosure effects. The maximum rate of Rubisco activity (Vcmax), light-

aturated rate of electron transport (Jmax), and the triose-phosphatetilization rate (TPU) were derived from A/Ci curves with the help ofHOTOSYN software version 1.1.2 (Dundee Scientific Ltd., Dundee,K) that employs the Farquhar biochemical model (von Caemmerernd Farquhar, 1981) as modified by Harley et al. (1992).

erimental Botany 66 (2009) 160–171

2.2.3. Light– and internal-CO2-response curves of chlorophyll a(Chla) fluorescence

The measurement of light–response curves of Chla fluorescence(F-PAR) as well as internal-CO2-response curves of Chla fluores-cence (F–Ci) was performed by using LI-6400-40 leaf chamberfluorometer (LCF) attached to the LI-6400 photosynthesis system,which provided LED-based fluorescence and light source. The fol-lowing parameters were calculated from these measurements asdescribed by Rosenqvist and van Kooten (2003). The F ′

v/F ′m or

˚PSIIopen, which is the efficiency of energy harvested by oxidized(open) PSII reaction centers in light, was calculated by the Eq. (2),where F ′

o, minimum fluorescence of a light-adapted leaf that hasmomentarily been darkened and F ′

m, maximum florescence duringa saturating light flash estimated by providing a saturating flashintensity of >6000 �mol m−2 s−1 and flash duration of 0.8 s. Thefraction of absorbed photons that are used for photochemistry forlight-adapted leaf (˚PSII) was calculated by using Eq. (3), whereFs is “steady-state” fluorescence. Two competing processes thatquench the level of chlorophyll fluorescence in the light referredas photochemical (qP) and non-photochemical (qN) quenching werecomputed from the Eq. (4). Photochemical quenching includes pho-tosynthesis and photorespiration, and tends to be largest under lowlight conditions, since that is where leaves use light most efficiently.The actual flux of photons (�mol m−2 s−1) driving PSII is called anelectron transport rate (ETR), and is calculated by the Eq. (5), wheref is the fraction of absorbed quanta that is used by PSII, and is typi-cally assumed to be 0.5 for C3 and 0.4 for C4 plants, and ˛leaf is leafabsorptance. Quantum yield can also be inferred from gas exchangemeasurements, and is given the symbol ˚CO2 , was calculated byusing Eq. (6). The maximum value of ETR (ETRmax) and saturatedvalues of ETR (ETRsat) were calculated by fitting exponential rise tomaximum function to the corresponding ETR with F-PAR and F–Cicurves, respectively, by using the Eq. (7) where, Y is the electrontransport rate and x is PAR. The ETRmax and ETRsat were calculatedas (a + c).

F ′v

F ′m

= (F ′m − F ′

o)F ′

m(2)

˚PSII = F ′m − Fs

F ′m

= �F

F ′m

(3)

qP = (F ′m − Fs)

(F ′m − F ′

o)(4)

ETR =(

F ′m − Fs

F ′m

)fI˛leaf (5)

˚CO2 = (A − Adark)I˛leaf

(6)

Y = a(1 − e−bx) + c (7)

2.3. Quantification of pigments and UV-B absorbing compounds

Pigments (chlorophyll a, chlorophyll b and carotenoids) andUV-B absorbing compounds (phenolics) were extracted from theleaves used for photosynthetic measurements (20 DAT). Pigmentanalysis was performed according to the method as described byChappelle et al. (1992). Briefly, five leaf discs (38.5 mm2 each)were punched from the leaf blades used in photosynthetic mea-surements and placed in a vial with 5 mL of dimethyl sulphoxidesolution for pigments and a solution containing methanol:distilledwater:hydrochloric acid (v/v/v) in 79:20:1 ratio for phenolic extrac-

tion. Three samples were collected from each treatment andincubated at room temperature for 24 h in the dark to allowcomplete extraction of chlorophyll, carotenoids and UV-B absorb-ing compounds into the respective solutions. The absorbance ofthe extracts was measured at 470, 648 and 664 nm for pigments

d Experimental Botany 66 (2009) 160–171 163

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nd 300 nm for phenolics using SmartSpec-3000 UV–vis spec-rophotometer (Bio-Rad Laboratories, Hercules, CA, USA) and thebsorbance was used to calculate total chlorophyll (Chla + Chlb),arotenoid and phenolic concentrations and expressed on leaf areaasis (�g cm−2). The concentration of phenolics was calculatedsing the equation, C = 16.05 × A, where A is absorbance at 300 nmnd C is concentration of UV-B absorbing compound (�g mL−1 ofxtract) and expressed as equivalents of p-coumaric acid.

.4. Shoot biomass and seed weight

Total dry weight of five plants per genotype in each treatmentas determined 18 DAT by harvesting one plant per pot from the

urface of the soil, and dry weight was determined by oven dryingor 72 h at 70 ◦C. The final remaining plants were harvested at the

aturity (53 DAT). Seed of cowpea pods were separated by handnd dried at the room temperature for several weeks. Total seedeight plant−1 (seed wt.) was determined from five plants for each

f the cultivar.

.5. Statistical analysis

Four UV-B treatments were randomly arranged in four identicalPAR units. Except for the treatment factors of UV-B radiation, thether growth conditions were identical in all four units. ANOVAas performed using the general linear model “PROC GLIMMIX”rocedure of SAS software 9.1 (Statistical Analysis System Institute

nc., 2002). The least square means (LSMEANS) were obtained forach class of effect and combination of effects, and was comparedsing t-test at P = 0.05 level of significance. Levels of significance

n tables are given by NS, *, **, *** for not significant, significant at< 0.05, 0.01 and 0.001, respectively. The standard errors of meansere also calculated and presented in the graphs and tables as errorars.

. Results

.1. Light response of photosynthetic (A/PAR) and fluorescenceF-PAR)

Leaf photosynthetic rates (A) increased with increase in PAR inll three cowpea cultivars across all UV-B levels (Fig. 1). The shapesf the A/PAR curves, however, differed significantly among cultivarsnd UV-B treatments within the cultivar. Even though higher A wereecorded in CB-5 and CB-27 compared to MPE under 0 UV-B, theesponses to increasing UV-B levels were greater in CB-5 and CB-27han in MPE (Fig. 1).

Amax declined significantly (P < 0.001) with increase in UV-Bosage in all cowpea cultivars (Table 1). Maximum decrease in Amax

as observed at 15 kJ of UV-B; 43, 37 and 12% for CB-5, CB-27 andPE, respectively. Cowpea cultivars also showed significant cultivar

P < 0.001) and UV-B × cultivar (P < 0.05) interactions for Amax.Light compensation point (LCP) values were significantly

ncreased (P < 0.01) in all the three cowpea cultivars. Further,owpea showed significant cultivar (P < 0.001) and UV-B × cultivarP < 0.001) interactions for LCP (Table 1). There was significant UV-× cultivar (P < 0.001) interaction for the light saturation estimate

LSE). Compared to control, LSE decreased at elevated UV-B in CB-5nd CB-27. However, LSE exhibited an increase in MPE at all ele-ated levels of UV-B radiation (Table 1). Leaf dark respiration (Rd)owever, increased at all levels of UV-B treatments in all the three

ultivars relative to 0 UV-B levels. A significant UV-B (P < 0.05) andultivar (P < 0.01) differences were observed for Rd (Table 1).

Compared with controls, the 5 and 10 kJ UV-B caused significantP < 0.05) increase in internal-CO2 concentration (Ci) values in allowpea cultivars (Table 1). Cultivars differed significantly (P < 0.01)

Fig. 1. Photosynthesis light response curves (A/PAR) of three cowpea cultivars sub-jected to different levels of UV-B radiation. Error bars are ±S.E. of three replications.

for Ci. Transpiration rate (E) decreased significantly (P < 0.05) in CB-5 and CB-27 across all UV-B levels. In MPE, on the other hand, E wasslightly increased at 5 and 10 kJ and decreased at the 15 kJ of UV-B.Cultivars, however, varied significantly (P < 0.01) for E (Table 1).

The parameters derived from the F-PAR curves clearly indicatedthe functionality of photosystems. UV-B radiation significantly(P < 0.05) affected photosynthetic ETR (Fig. 2). The averaged ETR val-ues decreased with increase UV-B levels in CB-5 and CB-27 whileETR was unaffected in MPE. Typically, qP decreased with increase inPAR irrespective of UV-B levels (Fig. 2). At low PAR, there was littledifference in qP values between 0 and 15 kJ UV-B-treated leaves,but when PAR increased, the decrease in qP was much greater.Averaged over PAR levels, the qP of the 15 kJ UV-B-treated leaveswas 0.617, 0.676 and 0.713 in CB-5, CB-27 and MPE, respectively.

The ˚PSII is a product of two parameters: PSII maximum efficiencyin light (F ′

v/F ′m), which is primarily related to non-photochemical

processes, and the photochemical quenching. Similar to qP, ˚PSIIdecreased significantly (P < 0.05) at higher UV-B levels both in CB-

164 G.-K. Surabhi et al. / Environmental and Experimental Botany 66 (2009) 160–171

F ochemm AR) in

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(c

ig. 2. Influence of UV-B radiation and PAR on electron transport rate (ETR), photaximal fluorescence (F ′

v/F ′m) from light–response curves of Chla fluorescence (F-P

and CB-27. Averaged over PAR levels, plants grown at the 15 kJ ofV-B recorded 21 and 15% less ˚PSII in CB-5 and CB-27, respectively.

F ′v/F ′

m) did not differ significantly among the cultivars and levels ofV-B radiation (Fig. 2). Similarly, apparent quantum efficiency (˚)

nd convexity (�) were also not affected by UV-B radiation amonghe cultivars (Table 1).

ETRmax, derived from the F-PAR curves, showed significantP < 0.001) UV-B × cultivar interaction. Increasing UV-B radiationaused linear decrease in ETRmax in CB-5 (Fig. 3). In contrast, CB-27

ical quenching (qP), quantum yield of PSII photochemistry (˚PSII) and variable tothree cowpea cultivars. Error bars are ±S.E. of three replications.

and MPE showed slightly higher levels of ETRmax at 5 and 10 kJ ofUV-B. Exposure to higher UV-B (15 kJ) caused decreased ETRmax inCB-27, but not in MPE as compared to control.

The allocation of electrons produced by the oxygen evolving

complex can be studied by simultaneous measurement of the quan-tum yield of electron transfer at PSII (˚PSII) measured by chlorophyllfluorescence and quantum yield of CO2 fixation (˚CO2 ) measuredby gas exchange. The relationships between ˚PSII and ˚CO2 derivedfrom F-PAR curves were linear (Fig. 4). Increase in UV-B radiation

G.-K

.Surabhietal./Environm

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entalBotany66

(2009)160–171

165

Table 1UV-B radiation effects on photosynthetic light–response curves (A/PAR) parameters in three cowpea cultivars, leaf respiration (Rd), apparent quantum efficiency (˚), light saturated maximum photosynthesis (Amax), convexity (�),light compensation point (LCP), light saturation estimate (LSE), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (E). ± Represents S.E. of three measurements.

Cultivar UV-B(kJ m−2 d−1)

Rd

(�mol m−2 s−1)˚ (�mol CO2 �mol−1

photon)Amax

(�mol m−2 s−1)� LCP

(�mol m−2 s−1)LSE(�mol m−2 s−1)

gs(�mol m−2 s−1)

Ci

(�mol m−2 s−1)E(mol m−2 s−1)

CB-5 0 2.73 ± 0.24 0.08 ± 0.003 41.07 ± 3.40 0.02 ± 0.01 48.3 ± 3.90 537.7 ± 21.83 0.273 ± 0.013 261 ± 6.10 5.61 ± 0.055 4.39 ± 0.55 0.08 ± 0.010 29.80 ± 1.65 0.18 ± 0.10 55.5 ± 1.81 438.3 ± 25.44 0.238 ± 0.006 291 ± 9.10 4.46 ± 0.26

10 3.86 ± 0.76 0.07 ± 0.010 34.40 ± 1.81 0.01 ± 0.00 62.7 ± 1.59 474.0 ± 2.89 0.227 ± 0.038 280 ± 5.46 4.06 ± 0.6015 3.83 ± 0.15 0.05 ± 0.010 23.47 ± 2.11 0.25 ± 0.14 77.4 ± 5.52 546.3 ± 15.38 0.263 ± 0.079 294 ± 5.34 4.40 ± 0.07

CB-27 0 3.95 ± 0.58 0.06 ± 0.003 47.43 ± 3.24 0.37 ± 0.02 60.5 ± 6.21 792.0 ± 21.00 0.326 ± 0.053 269 ± 11.46 6.78 ± 0.825 4.56 ± 1.19 0.07 ± 0.000 44.77 ± 1.07 0.13 ± 0.05 65.8 ± 3.00 711.7 ± 22.26 0.235 ± 0.016 286 ± 6.09 4.70 ± 0.11

10 5.09 ± 0.16 0.07 ± 0.000 40.30 ± 0.65 0.00 ± 0.00 70.9 ± 2.73 633.7 ± 20.73 0.324 ± 0.016 296 ± 2.83 5.57 ± 0.0915 4.24 ± 0.23 0.07 ± 0.000 29.83 ± 2.40 0.13 ± 0.13 57.7 ± 5.30 461.8 ± 36.37 0.226 ± 0.043 275 ± 12.16 5.35 ± 0.70

MPE 0 1.85 ± 0.51 0.07 ± 0.010 36.37 ± 1.88 0.11 ± 0.05 30.2 ± 0.92 471.0 ± 6.35 0.242 ± 0.023 258 ± 8.44 4.45 ± 0.265 3.49 ± 0.69 0.07 ± 0.010 33.70 ± 1.10 0.20 ± 0.04 56.4 ± 2.25 647.5 ± 5.48 0.217 ± 0.025 270 ± 10.95 4.62 ± 0.38

10 3.80 ± 0.82 0.07 ± 0.010 36.50 ± 3.26 0.27 ± 0.18 61.0 ± 4.10 594.3 ± 4.26 0.272 ± 0.040 280 ± 6.35 4.91 ± 0.3715 2.73 ± 0.25 0.06 ± 0.000 32.13 ± 1.53 0.44 ± 0.04 46.5 ± 1.07 597. 7 ± 11.14 0.164 ± 0.004 244 ± 2.48 3.99 ± 0.13

UV-B * NS *** NS ** NS NS * *

Cultivar ** NS *** NS *** *** NS ** **

UV-B × cultivar NS NS * * *** *** NS NS NS

NS, non-significant.* P = 0.05 level of significance.

** P = 0.01 level of significance.*** P = 0.001 level of significance.

Fig.3.In

flu

ence

ofU

V-B

radiation

onm

aximu

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ortrate

(ETRm

ax )d

erivedfrom

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signifi

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levelsofA

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tedin

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re.Errorbars

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reerep

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Fig.4.Th

erelation

ship

between

quan

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PSIIph

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1 d Experimental Botany 66 (2009) 160–171

looi

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66 G.-K. Surabhi et al. / Environmental an

evels caused elevation in ˚PSII/˚CO2 ratios in CB-5. In contrast, thether two cultivars, CB-27 and MPE, showed slight increase at 5 kJf UV-B and maintained almost same values with further increasen UV-B levels (10 and 15 kJ).

.2. Internal-CO2-response of photosynthetic (A/Ci) anduorescence (F–Ci)

A/Ci response curves showed that three cowpea cultivarsesponded differently to UV-B radiation (Fig. 5). In CB-5, 5 kJ ofV-B treatment caused significant decrease in A compared to theontrol and the decrease was smaller at 10 and 15 kJ compared tohe decrease observed at 5 kJ. In contrast, compared to controls,he A for CB-27 and MPE did not differ for the 5, and 10 kJ of UV-, but the 15 kJ of UV-B caused marked decrease in A in both theultivars.

Cowpea cultivars differed significantly (P < 0.01) for maximumarboxylation velocity of Rubisco (Vcmax), and either UV-B or UV-× cultivar interactions were insignificant for Vcmax (Table 2).

urther, the decrease in Vcmax at 15 kJ UV-B in CB-5, CB-27 andPE was 36%, 38% and 17%, respectively. In consistent with the

cmax, cultivars also varied significantly (P < 0.05) for Jmax. No signif-cant differences were observed for TPU between cultivars or UV-Breatments (Table 2).

Increase in Ci (F–Ci curves) caused gradual increase in ETR, qP,PSII and (F ′

v/F ′m) in all cultivars irrespective of UV-B levels (Fig. 6).

he reduction in ETR was greater in CB-5 whereas, CB-27 and MPEhowed none or minimal effect of UV-B radiation on ETR. Thereas a significant (P < 0.05) UV-B × cultivar interaction for ETR. qP

lso exhibited a significant (P < 0.001) UV-B × cultivar interaction.n CB-5, elevated levels of UV-B radiation decreased qP values com-ared to control. This decrease, however, was limited to the highest15 kJ) UV-B in the cultivars CB-27 and MPE. UV-B, cultivar and UV-× cultivar interactions were not significant for ˚PSII (Fig. 6). UV-B

adiation significantly affected the F ′v/F ′

m values in all the cowpeaultivars

Similar to the ETRmax derived from the F-PAR curves, ETRsat,erived from the F–Ci response curves, decreased linearly in CB-

as UV-B radiation increased (Fig. 7). However, a quadratic trendas observed for CB-27 and MPE. ETRsat exhibited a significant

P < 0.001) UV-B × cultivar interaction. The relationships between

able 2V-B radiation effects on internal-CO2 (Ci)-response curves of photosynthesis (A/Ci) curves parameters in three cowpea cultivars, maximum rate of carboxylation by Rubisco

Vcmax), PAR saturated rate of electron transport (Jmax) and triose phosphate utilization capacity (TPU). ± Represents S.E. of three measurements.

ultivar UV-B (kJ m−2 d−1) Vcmax (�mol m−2 s−1) Jmax (�mol m−2 s−1) TPU (�mol m−2 s−1)

B-5 0 125.4 ± 6.68 227.5 ± 12.41 12.4 ± 1.665 85.0 ± 19.01 217.0 ± 65.36 8.4 ± 0.77

10 86.7 ± 26.20 187.5 ± 44.70 9.9 ± 1.8015 80.6 ± 4.81 168.0 ± 2.31 8.7 ± 1.14

B-27 0 114.7 ± 3.70 262.0 ± 17.90 13.8 ± 2.105 96.0 ± 14.57 261.0 ± 53.51 14.3 ± 2.98

10 115.3 ± 18.30 242.5 ± 62.10 12.0 ± 5.0015 70.7 ± 7.80 158.0 ± 29.10 9.5 ± 1.00

PE 0 126.5 ± 11.08 168.5 ± 12.96 10.7 ± 1.765 151.0 ± 18.40 331.0 ± 17.20 13.8 ± 0.20

10 145.5 ± 14.70 341.5 ± 30.30 12.3 ± 0.6015 105.3 ± 23.01 254.3 ± 30.44 13.1 ± 1.24

V-B NS NS NSultivar * ** NSV-B × Cultivar NS NS NS

S, non-significant (P > 0.05).* P <0.01 level of significance.

**P < 0.001 level of significance.

Fig. 5. Internal-CO2 (Ci)-response curves of photosynthesis (A/Ci) of three cowpeacultivars subjected to different levels of UV-B radiation. Error bars are ±S.E. of threereplications.

G.-K. Surabhi et al. / Environmental and Experimental Botany 66 (2009) 160–171 167

F ranspo( s of Ca ±S.E.

˚l˚h

3

(Uiiwttw

ig. 6. Influence of UV-B radiation and internal-CO2 concentration (Ci) on electron t˚PSII) and variable to maximal fluorescence (F ′

v/F ′m) derived from Ci response curve

nalysis of UV-B, cultivar and UV-B × cultivar are presented in figure. Error bars are

PSII and ˚CO2 derived from the A/Ci response curves were alsoinear for all the cultivars under each treatment conditions. The

PSII/˚CO2 ratio increased at elevated UV-B in all the genotypes,owever this increment was greater in CB-5 (Fig. 8).

.3. Pigments and UV-B absorbing compounds

Total chlorophyll concentration decreased significantlyP < 0.001) in all the three cowpea cultivars with increase inV-B radiation (Table 3). CB-5 showed the highest decrease (24%)

n chlorophyll concentration at 15 kJ UV-B. In contrast, Chla/b ratio

ncreased significantly (P < 0.05) in all the three cowpea cultivars

ith increase in UV-B radiation. Carotenoid concentration, onhe other hand, did not show any significant change across UV-Breatments. A significant (P < 0.001) UV-B × cultivar interactionas observed for UV-B absorbing compounds. Compared to the

rt rate (ETR), photochemical quenching (qP), quantum yield of PSII photochemistryhla fluorescence (F–Ci) in three cowpea cultivars. The significance levels of ANOVAof three replications.

control, UV-B absorbing compounds were significantly increasedat all levels of UV-B radiation in CB-27 and MPE; however, CB-5did not show a distinct pattern. Averaged over UV-B levels, theincrease in UV-B absorbing compounds was greater in MPE (43%)compared to CB-27 (16%) and CB-5 (6%).

3.4. Shoot biomass and seed weight

Cowpea cultivars showed significant (P < 0.001) UV-B and UV-B × cultivar interactions for shoot biomass (Fig. 9). Elevated UV-Bradiation caused a decrease in shoot biomass in CB-5 and CB-27.

Shoot biomass was significantly higher under UV-B treatments inMPE compared 0 UV-B-treated plants. UV-B radiation showed sig-nificant (P < 0.05) reduction on seed weights. The reduction in seedweight was higher in CB-27 (56%) and CB-5 (41%) compared to MPE(1.5%) at 15 kJ of UV-B (Fig. 10).

168 G.-K. Surabhi et al. / Environmental and Experimental Botany 66 (2009) 160–171

Table 3UV-B radiation induced effects on cowpea leaf total chlorophylls, chlorophyll a/b ratio, carotinoids and UV-B absorbing compounds. ± Represents S.E. of three measurements.

Cultivar UV-B (kJ m−2 d−1) Total chlorophyll (�g cm−2) Chlorophyll a/b Carotenoids (�g cm−2) UV-B absorbing compounds (�g cm−2)

CB-5 0 37.1 ± 1.33 1.9 ± 0.04 6.4 ± 0.37 57.4 ± 4.915 36.3 ± 0.90 2.0 ± 0.05 7.3 ± 0.21 76.9 ± 1.4310 32.5 ± 1.41 2.0 ± 0.05 6.1 ± 0.20 60.7 ± 5.6215 28.1 ± 0.72 2.1 ± 0.07 5.9 ± 0.30 44.3 ± 2.62

CB-27 0 38.8 ± 1.26 1.9 ± 0.00 6.8 ± 0.83 49.7 ± 3.965 36.1 ± 2.39 2.0 ± 0.05 6.5 ± 0.39 53.9 ± 3.9010 34.8 ± 1.74 2.0 ± 0.01 6.3 ± 0.44 64.0 ± 3.2215 31.2 ± 1.03 2.0 ± 0.04 5.4 ± 0.13 52.9 ± 2.26

MPE 0 34.9 ± 1.43 1.9 ± 0.02 5.9 ± 0.16 37.5 ± 4.295 32.2 ± 2.49 2.1 ± 0.06 5.6 ± 0.52 51.0 ± 0.2610 34.3 ± 1.32 2.0 ± 0.05 6.1 ± 0.31 55.9 ± 3.7115 28.7 ± 1.41 2.0 ± 0.05 5.5 ± 0.08 53.8 ± 3.47

UV-B *** * NS *

Cultivar NS NS NS ***

UV-B x Cultivar NS NS NS ***

N

4

rTtttr(tupUdrdtrdcre

FdcU

activities (Nogues et al., 1998). In the same study, however, theyfound 43% increase in UV-B absorbing compounds, which mightexplain the stability of photosynthetic system of pea plants to theelevated UV-B, similar to our finding in MPE.

S, non-significance (P > 0.05).* P < 0.05 level of significance.

*** P < 0.001 level of significance.

. Discussion

In the present study, cowpea cultivars exhibited differentialesponses in CO2 assimilation to the intensities of UV-B radiation.he magnitude, however, varied significantly among the cultivarshat can be exploited in cowpea breeding programs. Net photosyn-hetic rate saturated at lower PAR in all cultivars when exposedo UV-B radiation. Similar photosynthetic responses have beeneported in cotton and other legumes with increased UV-B radiationReddy et al., 2003, 2004; Amudha et al., 2005). Amax is an impor-ant photosynthetic parameter that represents the maximal photontilization capacity of plants and thus, reflects the net primaryroductivity. In the current study, Amax decreased at all elevatedV-B levels, but the decrease was comparatively lower in MPE. Theecrease in Amax under UV-B radiation is in agreement with othereports (Reddy et al., 2004; Zhao et al., 2004; Koti et al., 2007). Aifference in Amax among the cowpea cultivars explains the exis-ence of differential tolerance mechanisms in response to UV-B

adiation. Cultivar MPE responded slightly less to UV-B possiblyue to observed rapid increase in the synthesis of UV-B protectiveompounds in the leaf epidermal cells that absorb the damagingadiation (Sullivan et al., 2003). Contrary to these results, pea plantsxposed to even higher doses of UV-B radiation (32 kJ m−2 d−1)

ig. 7. Influence of UV-B radiation on maximum electron transport rate (ETRsat))erived from internal-CO2 (Ci)-response curves of Chla fluorescence (F–Ci) in threeowpea cultivars. The significance levels of ANOVA analysis of UV-B, cultivar andV-B × cultivar are presented in figure. Error bars are ±S.E. of three replications.

did not show marked differences in light-saturated photosynthetic

Fig. 8. The relationship between quantum yield of PSII photochemistry (˚PSII) andthe quantum yield of CO2 assimilation (˚CO2 ) internal-CO2 (Ci)-response curves ofChla fluorescence (F–Ci) in three cowpea cultivars. Lines represent the linear fitbetween ˚PSII and ˚CO2 . Each value is a mean of three replications, and for clarity,±S.E. are not included.

G.-K. Surabhi et al. / Environmental and Exp

Fig. 9. . Influence of UV-B radiation on shoot dry weights of three cowpea cultivars(18 DAT). Error bars are ±S.E. of five replications.

Fb

tclatppetpdp(

stcitCutfcciHaainMrims

irradiation (Hader et al., 1996; Hideg et al., 2006). In the present

ig. 10. Influence of UV-B radiation on seed weights of three cowpea cultivars. Errorars are ±S.E. of five replications.

In general, increased UV-B radiation also increased LCP in allhe three cowpea cultivars which are similar to the observations inotton (Zhao et al., 2004). From the results, it is clear that higherevels of UV-B radiation lower the LSE in sensitive cultivars (CB-5nd CB-27) whereas it was not affected or even slightly increased inhe tolerant cultivar (MPE). Increased LCP in the present study mayossibly due to the reductions in quantum yield or increase in res-iratory losses. The UV-B-induced reduction in apparent quantumfficiency (˚) in the sensitive cultivar (Table 1) provides an addi-ional evidence of the role of non-stomatal mechanisms in limitinghotosynthesis, which is consistent with the earlier reports underifferent environmental stresses such as flooding conditions in oaklants (Gardiner and Krauss, 2001), and UV-B radiation in cottonZhao et al., 2004).

In the present study, neither UV-B radiation nor cultivars didhow significant changes in stomatal conductance (gs) similar tohe observations of Allen et al. (1999) in pea grown under fieldonditions. However, Nogues et al. (1998) found a 65% decreasen stomatal conductance in pea plants raised under supplemen-al UV-B in greenhouse-grown plants. In the present study, higheri values were recorded in CB-5 than in the other two cultivarsnder all levels of UV-B radiation. Reddy et al. (2003) have reportedhat decreased A and gs were not associated with the Ci, and there-ore elevated Ci in the sensitive cowpea cultivars (CB-5 and CB-27)ould be an indirect evidence of reduced mesophyll photosyntheticapacity. This was supported by the low light utilization efficiencyn CB-5 and CB-27 compared to MPE under higher UV-B radiation.owever, it was widely reported that any UV-B effects on stom-ta do not affect CO2 assimilation (Teramura et al., 1991; Ziskand Teramura, 1992). UV-B radiation caused significant increasen transpiration (E) in all the three cultivars; however, the mag-itude of increase in E was much smaller in the tolerant cultivar,PE. In contrast to the present study, Hideg et al. (2006) have

eported that 10.3 kJ m−2 d−1 UV-B caused an increase in E bothn tolerant and sensitive barley cultivars. They explained that it

ay be due to the possible water stress caused by UV-B radiationince frequent side effects under UV-B radiation is water stress as

erimental Botany 66 (2009) 160–171 169

a result of modified stomatal opening (Jensen and van den Noort,2000).

The analysis and interpretation of A/Ci curves has become acommon practice to understand environmental stress effects onbiochemical limitation of photosynthesis (Farquhar et al., 1980;Zhao et al., 2004). According to the model of von Caemmerer andFarquhar (1981), analysis of relationship between net CO2 assim-ilation (A) and (Ci) allows separation of the relative limitationsimposed by stomata, Rubisco carboxylation velocity (Vcmax) and thecapacity for regeneration of ribulose 1,5-bisphotate (RuBP) or PAR-saturated rate of electron transport (Jmax) on leaf photosynthesis.In the current study, MPE showed marked increase in Vcmax at 5and 10 kJ of UV-B, and the 15 kJ caused slightly decrease in Vcmax

which is in agreement with the earlier reports on several other cropspecies (rape-Allen et al., 1998; cotton-Zhao et al., 2004). However,Vcmax was highly reduced in CB-5 and CB-27 at elevated UV-B. Thisdecrease in Vcmax might be attributed to the deactivation or loss ofthe Rubisco enzyme (Quick et al., 1991). Allen et al. (1997) whileperforming in vitro biochemical assays of Rubisco activity in Bras-sica demonstrated that decreased Rubisco activity at higher UV-Bwas due to reduction in the amount of Rubisco and not simply adeactivation as the specific activity of Rubisco carboxylation.

Allen et al. (1997) reported that analysis of mature oilseed rapeleaves subjected to UV-B radiation also revealed a decline in max-imum rate of electron transport contributing to RuBP regeneration(Jmax). Further, RuBP regeneration could be limited either by aninability to supply reductants and ATP from electron transport oran inactivation or loss of Calvin cycle enzymes other than Rubisco(Allen et al., 1998). Similarly, decreased RuBP regeneration (Jmax) indifferent plant species subjected to UV-B radiation were also evi-dent in other studies (Allen et al., 1997; Zhao et al., 2004). In thepresent study, neither reductions in TPU was significantly affectedby UV-B nor cultivars varied for TPU.

In the present study, increasing PAR levels caused linear decreasein the F ′

v/F ′m irrespective of UV-B radiation. However, F ′

v/F ′m was not

significantly affected either in the tolerant (MPE) or in the sensitive(CB-5 and CB-27) cowpea cultivars by UV-B treatments, which isin agreement with the studies with barley cultivars (Hideg et al.,2006). One may address the actual utilization of energy absorbedby PSII antennae in photochemistry by assessing the magnitude ofphotochemical quenching (qP) as affected by UV-B radiation. Photo-chemical quenching can be approximately expressed as an indicatorof the proportion of open PSII centers (Genty et al., 1989). In thepresent study, an increase in qP induced by UV-B radiation in tol-erant cultivar (MPE) suggests that tolerant cultivar kept more PSIIcenters in an open state so that more excitation energy can be usedfor electron transport. In contrast, sensitive cultivars (CB-5 and CB-27) failed to maintain more PSII centers in open state, and thusshowed decreased qP.

Several studies have shown that PSII is often sensitive to UV-Band regarded as the most sensitive photosynthetic target (Bornman,1989; Melis et al., 1992). PSII operating efficiency of leaves has beenshown to decrease with increasing PAR in a non-linear mannerwhich is accompanied by the changes in the PSII maximum effi-ciency (F ′

v/F ′m) (Genty et al., 1989). The linear correlation between

˚PSII and ˚CO2 observed in the present study suggest that ˚PSII canbe used as a tool to select for cultivars with different photosyntheticcapacities under UV-B stress conditions.

UV-B usually results in decreased electron transport andincreased non-radioactive energy dissipation to prevent the over-excitation of PSII, depending on the intensity and duration of

study, the reductions in ETR varied highly among the cowpea cul-tivars which are in agreement with the observed responses inother leguminous and non-leguminous crops (Allen et al., 1999;Hideg et al., 2006). Similar to this study, earlier studies demon-

1 d Exp

sPiMca

EoaitochIuerilh(

pUvipiccw2(cpswel

UhUclreapo

2iAwtts(Kcsya

70 G.-K. Surabhi et al. / Environmental an

trated that the ˚PSII and the oxidation state of PSII (qP) declined asAR increased, while ETR and non-photochemical quenching (NPQ)ncreased (Greer and Jeffares, 1998; Laing et al., 2000; Burritt and

ackenzie, 2003). This decrease in ˚PSII, ETR and qP might haveaused an over-excitation of the photochemical system of the CB-5s compared to other two cultivars (Greer and Jeffares, 1998).

Previous reports have shown that UV-B radiation decreased theTRmax, and that this decrease was correlated with changes in theverall activity of Rubisco (Bischof et al., 2000). In the present study,linear decrease in ETRmax in the sensitive cultivar (CB-5) with

ncrease in UV-B could be due to the decreased ˚PSII and concomi-ant decrease in CO2 assimilation. Though linear relationships werebserved between ˚PSII and ˚CO2 for all the cultivars, the sensitiveultivar (CB-5) showed strong increase in the ˚PSII/˚CO2 ratio atigher UV-B levels (slope = 24.4) compared to control (slope = 15.3).

ncreased ratio of ˚PSII/˚CO2 could be due to either more electronssed for photorespiration or increase in the strength of alternativelectron sinks such as the superoxide radical generating Mehlereaction (Fryer et al., 1998). On the other hand, a small increasen the ˚PSII/˚CO2 ratio in the tolerant cultivar (MPE) at higherevels of UV-B radiation indicates that only minor changes mightave occurred in the photosynthetic apparatus under UV-B stressLeipner et al., 1999).

In the present study, significant decreased level of total chloro-hyll concentration in all the cowpea cultivars at higher levels ofV-B radiation are in agreement with studies in cotton under ele-ated UV-B radiation (Zhao et al., 2003; Reddy et al., 2004). Decreasen total chlorophyll concentration induced by UV-B radiation wasrobably due to the destruction of structure of chloroplasts, inhib-

ted synthesis of new chlorophyll, and increased degradation ofhlorophylls (Sakaki et al., 1983). Chlorophyll a/b ratio was signifi-antly increased by UV-B radiation in all the three cowpea cultivarshich is consistent with the previous reports in cotton (Zhao et al.,

003). However, decreased chlorophyll a/b ratio observed in wheatLi et al., 2000) was due to a stronger reduction in Chla than Chlboncentration under UV-B. Higher levels of carotenoids in the leafrovide a protective function for the plants (Bornman, 1999). In thistudy, however, no significant cultivar differences and UV-B effectsere observed. Similarly, Zhao et al. (2003) reported that lower lev-

ls of UV-B did not change the carotenoid concentration, but higherevels of UV-B radiation (15 kJ) caused a 16% reduction in cotton.

UV-B absorbing compounds are the ‘first line of defense’ againstV-B radiation-induced damage in plants. Several earlier studiesave demonstrated that there was relatively little transmission ofV-B through epidermal cells containing phenolic and flavonoidompounds (Jordan, 1996). UV-B induced greater levels of pheno-ic compounds in the current study are in agreement with similaresults in other species (Bornman, 1999; Kakani et al., 2004; Reddyt al., 2004; Hideg et al., 2006). In a recent meta-analysis, Searles etl. (2001) reported that 10% increase in the UV-B absorbing com-ounds in response to UV-B radiation as the most consistent featurebserved across species.

Lower shoot biomass in the sensitive cultivars (CB-5 and CB-7) observed in the current study in response to UV-B radiations similar to the other reports on leguminous crops (Singh, 1996;mudha et al., 2005). In the present study, reduction in seed yieldas more pronounced at the higher UV-B (15 kJ) in both the sensi-

ive cultivars (CB-5 and CB-27); these results are in agreement withhe earlier reports showing reduction in reproductive traits such aseeds per pod, pod numbers, seed weight and shelling percentageRajendiran and Ramanujam, 2004; Amudha et al., 2005). Similarly,

akani et al. (2004) reported decreased reproductive potential inotton under UV-B radiation. Germ et al. (2005) found that exclu-ion of UV-B from the solar radiation led to more than double theield of pumpkin fruits. The reason for yield reduction may be due tolterations in plant vegetative and reproductive growth, e.g., plant

erimental Botany 66 (2009) 160–171

stunting, flower suppression and/or delay of flowering and lowerpod set (Saile-Mark and Tevini, 1997; Amudha et al., 2005).

The relationship between plant photosynthetic characteristicsand yield could easily be explained by the consistent UV-B tol-erance response of cultivar MPE among these attributes. Currentstudy revealed that the UV-B tolerance mechanisms operating atthe photosynthetic process level could be useful to sustain the plantgrowth and yield under UV-B condition.

In summary, enhanced UV-B radiation caused significant dam-aging effects in the sensitive cowpea cultivars (CB-5 and CB-27)in terms of photosynthetic CO2 assimilation, photochemistry, pig-ments, UV-B radiation absorbing ability, seed weight and shootbiomass. On the other hand, no UV-B damaging effects wereobserved or UV-B influence was minimal on the tolerant cowpeacultivar (MPE). Increased tolerance in MPE was supported by higherlevels of photosynthetic CO2 assimilation, reduced damaging effecton photochemistry coupled with higher levels of pigments, and bet-ter UV-B screening ability. Therefore, significant variation in UV-Bsensitivity exists among cowpea cultivars, which is apparently dueto inherent genotypic variation. Cultivar selection for a niche envi-ronment should take consideration of UV-B tolerance mechanisms,and breeding cultivars tolerant to higher levels of UV-B will be bene-ficial in the regions where current UV-B levels are higher and wouldbe more desirable in the projected future climatic conditions.

Acknowledgements

Appreciation is expressed for the excellent technical assistanceprovided by David Brand. We thank Drs. M. Gu and Y. Yang for crit-ical comments on the manuscript and Dr. Jeff Ehlers, University ofCalifornia-Riverside, CA for the seed material. Part of this researchwas supported by the USDA-UV-B monitoring Program, Fort Collins,CO, USA. This is a contribution from Department of Plant and SoilSciences, Mississippi State University, Mississippi Agricultural andForestry Experiment Station, paper no. J-11026.

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