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Plant Physiol. (1991) 96, 198-2070032-0889/91/96/0198/1 0/$01 .00/0

Received for publication September 24, 1990Accepted December 26, 1990

The Synergistic Effect of Drought and Light Stresses inSorghum and Pearl Millet1

JiOi Masojldek2*, Shailja Trivedi3, Lesley Halshaw, Anna Alexiou, and David 0. HallDivision of Biosphere Sciences, King's College London, Campden Hill Road, London W8 7AH, United Kingdom

ABSTRACT

The effects of drought stress and high irradiance and theircombination were studied under laboratory conditions usingyoung plants of a very drought-resistant variety, ICMH 451, ofpearl millet (Pennisetum glaucum) and three varieties of sorghum(Sorghum blcolor)-one drought-resistant from India, onedrought-tolerant from Texas, and one drought-sensitive varietyfrom France. CO2 assimilation rates and photosystem 11 fluores-cence in leaves were analyzed in parallel with photosyntheticelectron transport, photosystem 11 fluorescence, and chlorophyll-protein composition in chloroplasts isolated from these leaves.High irradiance slightly increased CO2 assimilation rates andelectron transport activities of irrigated plants but not fluores-cence. Drought stress (less than -1 megapascal) decreased CO2assimilation rates, fluorescence, and electron transport. Underthe combined effects of drought stress and high irradiance, CO2assimilation rates and fluorescence were severely inhibited inleaves, as were the photosynthetic electron transport activitiesand fluorescence in chloroplasts (but not photosystem I activity).The synergistic or distinctive effect of drought and high irradianceis discussed. The experiments with pearl millet and three varietiesof sorghum showed that different responses of plants to droughtand light stresses can be monitored by plant physiological andbiochemical techniques. Some of these techniques may have apotential for selection of stress-resistant varieties usingseedlings.

Drought is one ofthe most important environmental factorslimiting photosynthesis. The degree of the damage can beamplified if plants are exposed to PPFD and temperatureshigher than those experienced during growth. The rate ofphotosynthesis declines as water stress, photoinhibition, andother stresses (or their combination) increase (25). Limitationof photosynthesis by strong light was demonstrated over 50years ago (1 1), and it has become clear that the primary targetof photoinhibition is PSII (16). There has been sustainedinterest in trying to understand how drought stress affectsphotosynthesis. In the early 1970s it was found that low leafwater potential affects photosynthesis in at least three ways:by closure ofstomata, by inhibition ofdark fixation processes,

' This work was supported by the Science and Technology forDevelopment Programme of the European Community, contractTS2.0121.M(H).

2 Present address: Institute of Microbiology, 379 81 Tiebofi,Czechoslovakia.

3 Present address: University of Jodhpur, Jodhpur, India.

and through an inhibition of electron transport (5). Photosyn-thesis of drought-stressed plants under high irradiance wasshown to be limited by reduced photochemical activity ofleaves at water potentials below -1.1 to -1.2 MPa (15).Bjcrkman and Powles (3) assayed the 770K fluorescence andCO2 assimilation rate in leaves and electron transport inchloroplasts during high irradiance and drought of exposedand shaded leaves of Nerium oleander. They concluded thatboth kinds of stress probably cause similar inactivation of thePSII photochemistry and that drought predisposes leaves tophotoinhibition. However, from recent results, the responseto drought seems to be a more complex event. Genty et al.(12) concluded that drought mainly slowed down the rate ofplastoquinone reoxidation and that it inhibited PSI-mediatedtransport more than PSII photochemistry. Thus, it is still amatter of controversy as to which chloroplast processes aremost affected by drought. This can be attributed partly to thedifferent experimental conditions and plant material used, i.e.temperature, PPFD, degree and duration of drought, age ofplants, etc.According to Boyer (4), shortage of water limits crop pro-

duction in semi-arid regions more than any other factor. Abetter understanding of the mechanisms that enable plants toadapt to water deficit and maintain growth and productivityduring drought periods will ultimately help in the selection ofdrought-tolerant varieties. This is especially relevant to twodrought-resistant plants, namely pearl millet and sorghumwhich are predominantly grown in dry regions of developingcountries and with which relatively little physiological re-search has been done compared with major world crops likecorn, wheat, rice, etc. Sorghum and pearl millet are well-adapted to growth under unfavorable conditions such as highlight, high temperature, and drought. The varieties ofsorghumchosen for our work show different sensitivities to drought inthe field: R109 appears to have intermediate characteristicsbetween the Aralba (drought-sensitive) and ICSV 112(drought-resistant) varieties.Ludlow and Powles (22) were the first to assess the agro-

nomic significance of photoinhibition induced by drought onthe grain yield of field-grown sorghum. Field work undercontrolled drought and light conditions would be ideal butnot very easy to achieve. Our work examined plant behaviorunder laboratory conditions similar to those that can be foundon an average day in the semi-arid tropics between morningand early afternoon in order to investigate how drought andhigh irradiance interact on sorghum and pearl millet plants.We made no attempt to assess high temperature damage andtherefore maintained temperature in the range from 31° to

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DROUGHT AND LIGHT STRESSES IN SORGHUM AND PEARL MILLET

33TC. A detailed study ofphotoinhibition at different temper-atures on sorghum was carried out in our laboratory bySharma and Hall (27), who showed that only chilling temper-atures during photoinhibition strongly enhanced the inhibi-tory effect of high irradiance.

In the present experiments, we examined the effect ofdrought stress, high irradiance, and their combination on theleaves of three different varieties of sorghum (Sorghum bico-lor) and one of pearl millet (Pennisetum glaucum). The CO2assimilation rate and modulated fluorescence in leaves andthe electron transport activities, fluorescence induction, elec-tron spin resonance signal II, and CP4 composition in chlo-roplasts from the stressed leaves were monitored. We studiedthe correlation between inhibition at the leaf level and inchloroplasts from these leaves, i.e. the relationship betweenleaf photochemical activities and chloroplast biochemical andstructural changes seen during photoinhibition of drought-stressed plants. Considerable changes were found in leavesafter high irradiance ofdrought-stressed plants. In chloroplastsfrom these leaves, not only may the site of action of the stressoccur in PSII, but several processes can be affected. Differ-ences in varieties were clearly seen in 2-week-old seedlings.

MATERIALS AND METHODS

Cultivation of Plants and Drought Treatment

Plants of sorghum (Sorghum bicolor [L.] Moench), adrought-sensitive variety Aralba (France), and two drought-resistant varieties from the United States and India-R109A(Cargill Seeds, Minneapolis, MN) and ICSV 112 (Interna-tional Crop Research Institute for Semi-arid Tropics, Hyder-abad, India)-were used as well as a drought-resistant hybridof pearl millet (Pennisetum glaucum [L.] R.Br.), var ICMH451 (International Crop Research Institute for Semi-aridTropics). The plants were grown in vermiculite (Micafil) inplastic pots (10 cm diameter) and watered with half-strengthHoagland solution. The day/night regime in the growth cham-ber was 16 h light at 230C and 8 h dark at 16'C. A combinationof fluorescent tubes and tungsten bulbs was used for cultiva-tion providing a PPFD of 120 to 150 ,umol m2 -s-'. Plantswere used at day 15 or day 16 after sowing when they were13 to 15 cm high and had usually three leaves. In droughtexperiments, watering was stopped at day 5 or day 6 aftersowing when plants were about 1.5 cm high. The height ofdrought-stressed plants was about 10 cm at day 15 or day 16.They had two or three leaves, smaller and darker than thoseof irrigated plants.

Leaf Water Potential

Leaves were cut close to the stem and the water potentialmeasured immediately using a pressure chamber (PMS In-strument Co., Corvallis, OR).

4Abbreviations: CP, Chl-protein; F,, F., Fm, variable, constant,and maximum fluorescence; CP47, CP43, PSII core Chl proteins;EPR, electron paramagnetic resonance; LHCII, light-harvesting an-tenna of PS11; D, the tyrosine 160 residue in the D2 protein of thePS11 reaction center.

Photoinhibition

Photoinhibition treatment of plants was performed for 6 hunder three 2-kW tungsten-halogen tubular lamps (Thorn,UK) mounted in an aluminum reflector providing a PPFDof 2500 gmol m-2s-' at the leaf surface. An open perplextank with running tap water was placed between the lampsand plants. The equipment was installed in a temperature-controlled room with intensive cooling and ventilation. Thetemperature during the experiments was maintained at 31 to33°C: no high-temperature damage was seen.

Recovery from Photoinhibition

After photoinhibition, the plants were placed in the labo-ratory (PPFD 20-30 4mol.m-2 .s' at 20-25°C) and rewa-tered, and fluorescence characteristics (described later) of theleaves were measured during the subsequent 2 to 3 h. Theplants were then transferred to the growth chamber (PPFD120-150 umol * m-2 * s-') and the fluorescence characteristicsrecorded again 24 h after the start ofthe initial photoinhibitiontreatment.

Gas Exchange Measurement in Leaves

Gas exchange was studied with a portable, open type IRCO2 analyzer (LCA-2; Analytical Development Co. Ltd.,Hoddesdon, UK). Two recently matured leaves (second orthird) of two different plants were placed in a leaf chamberand illuminated at a PPFD of 1200 ,mol *m2 *- s- with a slideprojector. The leaves were attached to the plants. The differ-ential of CO2 concentration was recorded at the steady state(after about 5 min). The CO2 assimilation rate was calculatedaccording to Long and Hallgren (21). Inlet humidity wasadjusted so that the value in the chamber was close to theambient level in order to avoid any stomatal closure inresponse to dry air. The air temperature inside the chamber(-30°C) did not change markedly since an IR filter and a fanwere used to avoid heating up.

Chl Fluorescence of Leaves (Modulated Fluorescence)The F. and F, components of fluorescence were measured

from the upper surface of leaves attached to plants at labora-tory temperature after 15 to 25 min of dark adaptation in aleaf clip using a modulated fluorimeter (Dual ChannelMFMS; Hansatech, King's Lynn, UK). During measure-ments, the bottom stopper of the leaf clip was open to ensurefree gas exchange around the leaves. The modulated lightsource provided a PPFD 3 to 4 ,umol.m-2.s-' at 4.8 kHz withan interference filter with a peak transmission wavelength of585 nm. The intensity was sufficiently low so as not to induceany significant variable fluorescence. Fluorescence was de-tected with a 695-nm interference filter. No detectable signalwas recorded in an empty clip when actinic light was switchedon. To achieve Fm (i.e. saturated irradiance), the leaves, afterdark adaptation, were illuminated with white light of PPFD3000 to 3500 4mol.m-2.s-' (Dual Light Source PLS 2; Han-satech) using a fiberoptic cable. This light intensity was satu-rated to reach maximum fluorescence. The ratio ofFv/Fm (Fm= F. + F,) was calculated from the traces and plotted againsttime.

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Plant Physiol. Vol. 96, 1991

Isolation of Chloroplasts

Leaves (25 g; second and third) were cut into 0.5-cm piecesand ground in 200 mL of grinding medium (medium A: 330mM sorbitol, 50 mm Tricine, 4 mM MgCl2, 2 mM MnCl2, 10

mM NaCl, 0.25% [w/v] BSA, and 5 mM DTT, pH 7.8) for 7to 10 s using a modified kitchen blender (model MX 32;Braun, FRG) with replaceable razor blades (Schick single-edged). Grinding of millet leaves was easier than that ofsorghum. The homogenate was filtered through two layers ofmuslin and then through a nylon cloth (72-,um mesh). Aftercentrifugation (1200 g for 8-9 min), the surface of the chlo-roplast pellet was washed twice with grinding medium withoutDTT (medium B) and resuspended to give about 4 to 6 mg.Chl mL-'. All operations were performed at 0 to 4°C. Thedegree of chloroplast intactness was about 50 to 60% (meas-ured using potassium ferricyanide as electron acceptor beforeand after hypotonic shock). We used the same procedure forpreparation of chloroplasts from drought-stressed plants, butthe yield was lower since leaves were wilted and not easy togrind. The intactness of chloroplasts was similar as in the caseof irrigated plants. The concentration of Chl and carotenoidswas determined in 80% (v/v) acetone as described by Lich-tenthaler and Wellburn (20).

Assay of Electron Transport Activities

Electron transport activities of chloroplasts, after breakageby hypotonic shock, were followed polarographically as 02

evolution or uptake at 20°C using a water-jacketed Clark type02 electrode chamber (Rank Bros., Cambridge, UK). TwomL of the measuring medium (medium C: 330 mM sorbitol,50 mM Tricine, 4 mm MgCl2, 10 mm NaCl, 0.25% [w/vJ BSA,pH 7.8) containing chloroplasts (50 ,ug of Chl) were illumi-nated by two projectors with an orange filter with a cut offwavelength of 550 to 750 nm (No 105; Lee Co., Andover,UK) at PPFD 800 to 900 Mmol m-2-s-. Artificial donorsand acceptors were added immediately before or during illu-mination. The following electron transport activities were

assayed in Amol 02 * mg-'Chl *h-' (14).

Water to Potassium Ferricyanide: The Whole Chain or PSIIActivity

Potassium ferricyanide (5 mm final concentration) acceptselectrons mainly from PSI, but to some extent it can also bereduced by PSII depending on the degree of damage to thechloroplasts. The reaction was uncoupled from photophos-phorylation by adding 5 mM NH4Cl.

Water to Methyl Viologen: The Whole Chain ElectronTransport

Methyl viologen (0.05 mm final concentration) is reducedby PSI and immediately auto-oxidized. This can be followedexperimentally as oxygen uptake since 1 mM NaN3 was addedto inhibit the endogenous catalase activity. NH4Cl (5 mM)was present to uncouple photophosphorylation.

Water to Phenylenediamine: PSI! Activity

Phenylenediamine (0.75 mm final concentration) was in-jected in the light to a mixture already containing 5 mmNH4C1.

Reduced TMPD to Methyl Viologen: PSI Activity

N,N,N',N'-Tetramethyl-p-phenylenediamine (0.3 mm finalconcentration) and sodium ascorbate (2 mm final concentra-tion) were added simultaneously in the dark to a reactionmixture already containing 0.05 mm methyl viologen, 1 mMNaN3, 5 mm NH4Cl, and 0.005 mm DCMU (in order to blockelectron transport from PSII). The slow dark oxygen uptakewas subtracted from the reaction after the light was turnedon. Since electron transport depends on the quality of chlo-roplast preparation, it varies somewhat from experiment toexperiment. The values shown in the tables are usually amean of several experiments.

Fluorescence Induction in Chloroplasts

The course of fluorescence induction of intact chloroplastswas measured in a DW 2/2 cuvette using a TR1 transientrecorder (Hansatech) since a modulated system is not suitablefor measurement ofF. and F, in suspensions. The experimen-tal recording time after switching on the actinic light (50-100umol . m-2 * s-') was 60 ms. This was provided by a LED lightsource, LS 1 (Hansatech), with a narrow band width (maxi-mum at 660 nm). The increase in light intensity from thelight-emitting diodes is very short (several nanoseconds), andfor fluorescence induction measurement can be used withouta shutter. A fast detector probe was situated perpendicular tothe light source and protected against stray actinic light by acut-off filter with a wavelength above 710 nm. The signal wasrecorded by a transient recorder (TRl; Hansatech) with abuilt-in trigger and replayed (1 min) to a chart recorder (1 Vrange, chart speed, 12 cm/min). Before measurement, thechloroplasts were resuspended in the reaction medium andstirred for 5 min in the dark at 20'C and then 1 min withDCMU (0.005 mM).

EPR Signal 11 in Chloroplasts

Samples of chloroplasts (4-6 gg Chl * mL-') were placed inquartz tubes (2.3-2.5 mm i.d.) immediately after preparationand kept in the dark at 0C for 1.5 h. They were then frozenand stored in liquid N2. EPR spectra were measured at 30'Kin the dark using a Varian E-4 EPR or a Bruker ESP 300spectrophotometer equipped with an Oxford Instrumentscryostat and temperature controller. EPR conditions were asfollows: microwave frequency, 9.35 GHz; microwave power,20 mW (20 dB); modulation amplitude, 2.018 G; center field,3335 G. The amplitude of signal II was measured betweenthe minimum and maximum points and corrected for con-centration of Chl and tube diameter.

Electrophoresis

Chloroplasts were washed twice with 10 mm sodium pyro-phosphate (pH 7.4) and twice with 65 mM Tris-HCl buffer

200 MASOADEK ET AL.




Table I. Values of Leaf Water Potentials of Control and Drought-stressed Plants of Three Different Varieties of Sorghum and PearlMillet at the Beginning of Experiments

Species Control DS

MPa + SESorghum var Aralba -0.42 ± 0.04 -2.65 ± 0.05Var R109A -0.43 ± 0.06 -1.39 ± 0.07Var ICSV 112 -0.31 ± 0.05 -0.75 ± 0.04

Pearl millet -0.27 ± 0.03 -0.91 ± 0.05

(pH 7.8). For CP analysis, the samples of chloroplast mem-branes were solubilized in 65 mm Tris-HCl buffer (pH 7.8)containing n-octyl-f3-D-glucopyranoside and SDS for 1 to 3min at 0°C. The ratio ofn-octyl-/3-D-glucopyranoside:SDS:Chlwas 20:1:1 (w/w/w). CPs were separated under nondenaturingconditions at 2 to 3C using a 10 to 15% (w/v) polyacrylamidegel (acrylamide:methylenebisacrylamide ratio = 40:1) in com-bination with the Laemmli buffer system (19). The contentof SDS in the upper reservoir buffer, stacking, and separatinggels was 0.05% (w/v). To each slot 30 to 35 ,ug of Chl were

applied. Immediately after electrophoresis the CP pattern wasscanned (a Chromoscan 3 Scanner; Joyce-Loebl, UK).For polypeptide analysis, the samples of thylakoid mem-

branes were dissolved at laboratory temperature for 20 minin 300 mM Tris-HCl buffer (pH 9), which contained 2.5% (w/v) SDS. After addition of solid DTT to a final concentrationof 4% (w/v), the extract was heated for 5 min at 65C. Thepolypeptide pattern was obtained using a 10 to 20% (w/v)linear gradient polyacrylamide gel (acrylamide:methylene-bisacrylamide ratio = 33:1), which contained 6 M urea and0.1% (w/v) SDS (19). About 100 gg of protein material was

added to each well. The gels were stained with 0.01% (w/v)Coomassie brilliant blue.

RESULTS

Our results are in two parts, those taken with leaves (invivo) and those measured in isolated chloroplasts from theseleaves (in vitro). We attempt to correlate leaf and chloroplastresponses and to discover the site(s) of action of drought andhigh-irradiance stresses on chloroplast membranes. The var-

ious methods were examined using pearl millet and three

sorghum varieties having different response to drought stressin order to select appropriate monitoring techniques.

Leaves

As a measure of water status, leaf water potential was

measured at the beginning of all experiments. The waterpotential of drought-stressed sorghum ICSV and R109A va-

rieties and pearl millet was higher than that of Aralba variety(Table I). Water potentials between -1 and -2 MPa in 15-d-old plants cause severe drought stress.The CO2 assimilation rates of leaves of irrigated plants of

all sorghum varieties were between 5.5 and 8.5 Amol CO2.m2.s '. In our experiments, the highest CO2 assimilation

rate was found in ICSV 112 and the lowest in Aralba; theassimilation rate for millet was lower-about 4.9 Amol CO2.m-2 s' (Table II). After 6 h of high irradiance, the assimila-tion rate of irrigated plants of sorghum increased by 2 to 22%and in millet plants by 5%, which indicates that the Calvincycle is fully functional. The results suggest that the young,

fully photosynthesizing leaves can acclimatize when exposedto high irradiance, probably by mobilizing their protectiveand repair mechanisms. The assimilation rates of all drought-stressed plants were lower than in control plants: in Aralbathey were as low as 17% ofthe control plants. The assimilationrate of the drought-stressed ICSV 1 12 variety and pearl milletdeclined to only 65 and 61%, respectively. However, in Aralbaand R109A plants, a combination of high irradiance anddrought stress resulted in CO2 assimilation rates of only 6%of control plants but in ICSV 112 and millet it dropped to 34and 15%, respectively.

In parallel with the CO2 assimilation rates, modulatedfluorescence was also measured in leaves. In nonstressedleaves of higher plants, the Fv/Fm ratio is close to 0.83, whichis typical for healthy plants (2). The ratio expresses the poten-tial yield of the photochemical reaction (18). The measure-

ments were taken every half hour from control and drought-stressed plants in rotation. Each experiment was repeatedseveral times to confirm a trend, and then one typical curve

is shown for each variety. Modulated fluorescence curves were

recorded during the high light treatment and for several hoursafter light stress (recovery), and the final value was taken 24h after the start of the high light treatment (Fig. 1). The timecourse of Fv/Fm in non-drought-stressed plants showed a

Table II. Changes in C02 Assimilation Rates during Drought Stress and/or High Irradiance of ThreeSorghum Varities and Pearl Millet

Conditions of high irradiance (6 h): temperature, 31-33IC; irradiance, 2500 Amol. m-2. s-. The lastmature leaf was used for measurements. Numbers in parentheses are percent of control.

CO2 Assimilation RatesSpecies Control + Drought stress +

Control 6 h high irradiance Drought stress 6 h high irradiance

pmol C02-m 2s 1 + SE

Sorghum var Aralba 5.67 ± 0.65 6.94 ± 1.62 (122) 0.94 ± 0.56 (17) 0.39 ± 0.21 (7)Var R109A 7.61 ± 1.30 7.73 ± 1.30 (102) 2.30 ± 0.90 (30) 0.52 ± 0.17 (7)Var ICSV 1 12 8.23 ± 0.75 9.76 ± 0.42 (119) 5.31 ± 0.31 (65) 2.79 ± 0.94 (34)

Pearl millet 4.39 ± 0.52 4.63 ± 1.04 (105) 2.67 ± 0.78 (61) 0.68 ± 0.09 (15)

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E

SAM

II.

0 5 10 15 20 25

TIME (h)0 5 10 15 20 25

TIME (h)Figure 1. The curves of the Fv/Fm ratio during photoinhibition of control (A) and drought-stressed (0) plants. Modulated fluorescence wasmeasured on leaves of three sorghum varieties (Aralba, A; R109A, B; ICSV 112, C) and pearl millet (D), as described in "Materials and Methods."Arrows show the end of photoinhibition where recovery started.

biphasic decrease (with a slight reverse 1-2 h after the start)to a ratio of about 0.6 to 0.7 after 6 h of light stress. In somecurves, a slow increase of FV/Fm appeared at the end of highlight treatment (Fig. 1, A and C). The recovery in moderatelight after photoinhibition was very fast and was completedwithin 24 h. In drought-stressed plants, the decrease of Fv/Fmduring high irradiance did not have a pronounced biphasiccharacter, and the Fv/Fm ratio dropped to 0.50 to 0.53 at theend of 6 h high light. In the ICSV 112 variety (less stressedthan Aralba and R109A), a rise in Fv/Fm was also observedtowards the end of high irradiance period. The recovery ofFv/Fm after high irradiance showed a slower rise to a lowervalue in drought-stressed plants (except ICSV 112) than inwell-irrigated plants where the Fv/Fm ratio of about 0.8 was

seen after several hours. ICSV 112 and R109A plants re-

covered most rapidly, whereas in Aralba plants, which were

the most damaged by drought stress, the recovery of the F,/Fm was very slow and only to about 0.73 after 24 h from thestart of the light stress (Fig. 1A).

Chloroplasts

In all experiments, chloroplasts were isolated from controland drought-stressed plants just prior to and after the end ofthe high irradiance treatment. After 6 h high irradiance, theelectron transport activities ofchloroplasts from non-drought-stressed plants of the three sorghum varieties and millet wereup to 36% higher than the beginning of the treatment (Table

III). This indicates that, as in the case of the CO2 assimilationrates in young, healthy plants, high irradiance at moderatetemperatures can stimulate the electron transport activities,especially of PSII. The activity of PSI appeared, however, tobe decreased by about 5 to 10% by high irradiance. It can becaused by a lower ability of PSI to adapt to light changes.

Electron transport activities of chloroplasts from drought-stressed plants were lowered from 8 to 70% (Table III), withthe R109A and ICSV 112 sorghum varieties showing the leastdamage to electron transport. After combined drought andlight stresses, all plants showed greatly decreased electrontransport activities. The greater PSII inhibition was seen inAralba-only 11 to 31% of control activities remained. PSIactivity was affected much less (53% of control for Aralba,whereas with ICSV 1 2 and millet plants, PSI activity slightlyincreased).

Chloroplast fast fluorescence induction curves were re-corded immediately after preparation of the chloroplasts si-multaneously with measurement of electron transport activi-ties. The curves of fluorescence induction have two phases: afast phase (within 1 ms) so-called constant or initial F., and aslow phase reached in several tens of milliseconds to give theso-called Fm. The fast rise is the minimum fluorescence ofPSII (all PSII primary electron acceptor molecules in theoxidized state) and the slow rise reflects the accumulation ofprimary electron acceptor of PSII in reduced state as theplastoquinone pool is reduced (7). Fluorescence characteris-tics are very sensitive to chloroplast quality, as is electron

202 MASOJiDEK ET AL.




Table III. Changes in Electron Transport Activities of Chloroplasts Prepared from Three SorghumVarieties and Pearl Millet Plants after Drought Stress and/or High Irradiance

Conditions of high irradiance as in Table II. Numbers in parentheses are percent of control. Electrontransport activities: H20 -* FeCy,a whole chain; H20 -- PD, PSII; H20 -* MV, whole chain; TMPDMV, PSI.

Electron Transport Activity

Activity Control + Drought stress +Control 6 h high irradiance Drought stress 6 h high irradiance

JUlMOI 02-mg1Chl h -'+ SE

Sorghum var AralbaH20-. FeCyt 111 ± 7.6 120 ± 6.2 (109) 65 ± 5.5 (58) 32 ± 7.5 (28.5)H20--PD 146 ± 10.6 161 ± 9.5 (110) 101 ± 9.1 (69) 46 ± 6.2 (31)H20 - MV 101 ± 11.5 104 ± 12.1 (103) 30 ± 5.2 (30) 12 ± 2.8 (12)TMPD - MV 276 ± 23 269 ± 26 (98) 165 ± 21 (60) 148 ± 20 (54)

Var R109AH2O- ,FeCy 131 ± 7.1 178 ± 8.5 (136) 120 ± 6.1 (92) 70 ± 5.3 (53)H20 -PD 166 ± 11.0 190 ± 18.4 (115) 144 ± 9.0 (87) 92 ± 5.8 (55)H20 -, MV 190 ± 20.2 127 ± 7.9 (120) 87 ± 5.9 (46) 63 ± 4.9 (34)TMPD -, MV 310 ± 25 295 ± 21 (95) 278 ± 22 (90) 234 ± 21 (76)

Var ICSV 1 12H20- FeCy 75± 6.2 88± 5.1 (116) 66± 3.5 (87) 39± 2.4 (51)H20 PD 78 ± 7.0 77 ± 5.4 (99) 61 ± 4.1 (78) 47 ± 3.8 (60)H2O - MV 102 ± 9.3 122 ± 7.4 (120) 92 ± 8.9 (90) 70 ± 8.1 (69)TMPD , MV 321 ± 25 288 ± 20 (90) 203 ± 22 (63) 351 ± 28 (109)

Pearl milletH20 -, FeCy 71 ± 3.4 74 ± 5.4 (105) 40 ± 4.3 (56) 34 ± 5.1 (48)H20 -, PD 72 ± 10.1 68 ± 8.2 (94) 40 ± 6.0 (56) 34 ± 13.0 (46)H20 - MV 96 ± 13.2 118 ± 12.4 (124) 40 ± 6.2 (41) 19 ± 3.1 (20)TMPD -* MV 229 ± 25 215 ± 27 (94) 164 ± 20 (72) 246 ± 30 (107)

a FeCy, potassium ferricyanide; PD, phenylenediamine; MV, methyl viologen; TMPD, N,N,N,N-tetra-methyl-p-phenylenediamine.

transport, and therefore we always assayed freshly isolatedchloroplasts since rethawing after storage in liquid nitrogendecreases the Fv/Fm ratio by about 10%.The Fv/Fm ratio of control (non-drought stressed) chloro-

plasts of two sorghum varieties was between 0.64 and 0.69and for millet 0.75; after high irradiance it decreased by 7 to13%, i.e. 0.56 to 0.64 (Table IV). We do not include R109Ain Table IV since the values were similar to those of Aralba.In chloroplasts from drought-stressed plants, the Fv/Fm ratiodeclined similarly as after high irradiance treatment, by 3 to16% compared with control plants; the lowest Fv/Fm valuewas that of the Aralba (Fv/Fm = 0.56) and the highest of pearlmillet (Fv/Fm = 0.69), suggesting again that the most detri-mental influence of drought stress occurs in Aralba. After 6 hof high irradiance with drought-stressed plants, the Fv/Fmratio dropped to 67 to 86% of the control for pearl millet andthe ICSV 112 sorghum variety, respectively. However thelowest Fv/Fm ratio was found in the Aralba variety, 56% ofcontrol. In all measurements, the F0 value slightly increasedin drought-stressed plants (by 3-19%) but decreased againafter light stress in the case of Aralba. The highest rise of Foappeared in drought-stressed pearl millet, a 23% increase afterlight stress.Measurement of EPR signal II (dark-stable) is one of the

methods for monitoring the donor side ofPSII since the signalis generated by D (or YD) (9). D is probably not directlyinvolved in electron transfer to P680 but it is located in the

alternative way of electron transport from the water-splittingsystem to P680. It may become functional when electrontransport through the primary electron donor of PSII fails.Therefore, it has been suggested that D plays a role in thestability and protection of the oxygen-evolving complex (23).The amplitude of signal II (in relative units) from chloroplastsfrom all plants did not alter much after 6 h of high irradiance.It either slightly decreased by 3 to 6% in Aralba, R109A, andmillet or remained unaltered in the case of ICSV 112 (TableV). The values of signal II for drought-stressed plants werelower by 3 to 9%, similar to non-drought-stressed plants afterlight stress. However, after a combination oflight and droughtstress, signal II was decreased by 11 to 32% (the greatestinhibition again found in Aralba). It suggests that the donorside of PSII is also affected by drought stress and highirradiance.

Analysis of the CP components of the thylakoid membranecan provide information about the structural organization ofthe core of both photosystems and their light harvestingantennae. CP47 and CP43, the two CPs of the PSII core, serveas the connecting antennae between the main light harvestingcomplex LHCII and reaction center of PSIT. CPI is a mon-omer of the PSI core CP and CPI* contains CPI and a partof light-harvesting antenna of PSI, LHCI. The ratio betweenLHCII (i.e. LHCPII + LHCPII*) and PSII (i.e. CP47 + CP43)can be taken as a measure ofthe PSII unit size, which indicatesthe efficiency of energy transfer. In the present experiments,

203




Table IV. Changes of Fluorescence Parameters Resolved from Fast Induction Curves of ChloroplastsPrepared from Leaves of Two Varieties of Sorghum and Pearl Millet after Drought Stress and/or HighIrradiance

Conditions of high irradiance as in Table II. Fluorescence components: F0, constant; Fv, variable; Fm,maximum (Fm = F, + F,). Numbers in parentheses are percent of irrigated plants (control). Data are ±SE.

Component Control Control + Drough Stress Drought Stress +6 h High Irradiance 6 h High Irradiance

Sorghum var AralbaFo 41.5 ± 1.1 38.5 ± 2.1 (93) 44.5 ± 4.5 (107) 41.4 ± 3.6 (99)Fv 74.5 ± 5.5 49.7 ± 3.1 (67) 53.0 ± 4.3 (71) 23.5 ± 3.2 (32)Fv/Fm 0.64 0.56 (87) 0.56 (84) 0.36 (56)

Var ICSV 1 12Fo 36.0 ± 3.2 36.0 ± 2.8 (100) 37.0 ± 4.4 (103) 37.5 ± 3.9 (104)F, 76.0 ± 6.2 63.5 ± 4.3 (84) 76.0 ± 5.4 (100) 54.3 ± 3.1 (71)Fv/Fm 0.69 0.64 (93) 0.67 (97) 0.59 (86)

Pearl milletFo 26.0 ± 1.1 31.0 ± 3.5 (119) 31.0 ± 0.8 (119) 32.0 ± 5.3 (123)Fv 75.0 ± 5.1 56.0 ± 6.9 (75) 66.0 ± 4.8 (88) 34.0 ± 5.5 (45)Fv/Fm 0.75 0.65 (88) 0.69 (93) 0.51 (69)

the PSII core CPs (CP47 and CP43) of control thylakoidcontained 7.8 to 10.8% of the total Chl present in the gel; the

lowest amount was found in pearl millet (Table VI). Theresults obtained with R109A being similar to those of ICSV112 were not included in Table VI. The PSI CP complexes(CPI and CPI*) had 28.5 to 31.1% (the highest in pearl millet).The PSII light-harvesting antennae contained 35.8 to 42.9%(the highest in ICSV 112). After exposure to high irradiance,the amount of PSII antenna increased up to 11%. In non-water-stressed plants, the ratio of LHCII/PSII decreased afterhigh irradiance, suggesting a reduction in size ofthe PSII unit,which is consistent with the demonstration that sun-exposedplants have smaller antenna than shade-adapted plants (1).After high irradiance, the amount of PSI CPs increased in allplants by about 3 to 14% compared with control thylakoids.

In drought-stressed plants that were not exposed to highirradiance, the amount of PSII CPs slightly decreased or didnot change and the amount ofPSI CPs (the PSI core) generallyincreased (by 3-10%). However, after 6 h of high irradianceof drought-stressed plants the amount of PSII core CPs di-minished by 8 to 17%. After high irradiance of drought-stressed plants, the content of PSI complexes (mainly CPI)increased, the ratio of LHCII/PSII increased and PSII de-

creased, whereas the amount of LHCII did not change. Inboth nonstressed and drought-stressed plants, the ratio ofcarotenoids/Chl a + b increased, which probably correlateswith their protective role against high irradiance (e.g. 10, 27).

Analyses of the thylakoid polypeptides ofthe three varietiesof sorghum after drought and light stress were inconsistentand an interpretation was therefore not possible. There were

only very small changes in the polypeptide spectrum of pearlmillet (not shown).

DISCUSSION

Several mechanisms of protection against high irradianceoccur at different levels (18). They can be classified as: (a)short-term events (in seconds or minutes), which include statetransition mechanism (reversible phosphorylation of the cer-tain population of LHCII) and thermal dissipation of excess

energy related to reversible energy-dependent fluorescencequenching; (b) intermediate-term events (in minutes or hours)represented by various reaction systems scavenging radicalsor preventing their formation, particularly those derived fromoxygen; (c) long-term events, e.g. energy-consuming carbonmetabolism including photorespiration, dissipation of excess

Table V. Changes in the Amplitude of EPR Signal 11 Measured in Chloroplasts Isolated from Leaves ofThree Different Varieties of Sorghum and Pearl Millet after Drought Stress and/or High Irradiance

Conditions of high irradiance as described in Table II. Numbers in parentheses are percent of control.Samples were kept in the dark for 1.5 h at 0C.

Amplitude of EPR Signal 11

Material Control + Drought stress +Control 6 h high irradiance Drought stress 6 h high irradiance

relative units

Sorghum var Aralba 13.8 + 1.3 13.3 ± 0.4 (96) 12.6 ± 1.0 (91) 9.4 ± 1.2 (68)var R109A 12.7 0.6 12.0 ± 1.4 (95) 12.3 ± 1.2 (97) 11.3 ± 0.9 (89)var ICSV 112 12.9 0.9 13.1 ± 1.5 (101) 12.5 ± 0.5 (97) 11.0 ± 0.6 (86)

Pearl millet 14.2 ± 1.2 13.8 ± 0.9 (97) 13.5 ± 1.5 (95) 12.8 ± 1.9 (90)

204 MASOJIDEK ET AL.




Table VI. Changes in Distribution of Chl in CPs of Thylakoid Membranes Isolated from Two Varietiesof Sorghum and Pearl Millet after Drought Stress and/or High Irradiance

Conditions of high irradiance as in Table II. CP denomination: CPl* + CPI = PSI complex; CPI = PSIcore; CP47 + CP43 = PSII core; LHCII* + LHCII = PSII antenna. Numbers in parentheses are percentof control (irrigated plants). LHC/PSII is the ratio of LHCII* + LHCII to CP47 + CP43.

Distribution of Chi

CP Control + Drought stress +Control 6 h high irradiance Drought stress 6 h high irradiance

Sorghum var AralbaCPI*+ CPI 28.6 ± 0.9 25.3 ± 1.1 (89) 31.7 ± 1.0 (110) 31.2 ± 1.2 (109)CPI 6.8 ± 0.5 4.4 ± 0.4 (44) 8.6 ± 0.4 (127) 8.8 ± 0.5 (129)CP47 + CP43 10.5 ± 0.5 11.8 ± 0.4 (109) 10.9 ± 0.4 (101) 9.9 ± 0.2 (92)LHCII* + LHCII 41.1 ± 0.8 41.0 ± 1.0 (100) 39.1 ± 0.9 (95) 39.0 ± 0.9 (95)Ratio LHC/PSII 3.81 3.47 3.59 3.94Ratio carotenoids/ 0.229 0.243 0.241 0.245

Chi a + bVar ICSV 1 12

CPI* + CPI 28.5 ± 1.2 27.8 ± 1.0 (97) 30.4 ± 1.3 (107) 30.1 ± 0.9 (106)CPI 8.3 ± 0.6 8.8 ± 0.5 (106) 9.8 ± 0.8 (118) 10.8 ± 0.7 (131)CP47 + CP43 10.3 ± 0.3 10.4 ± 0.4 (101) 9.7 ± 0.4 (94) 8.6 ± 0.5 (83)LHCII* + LHCII 42.9 ± 1.2 41.8 ± 0.9 (97) 41.8 ± 0.9 (97) 42.4 ± 1.1 (99)Ratio LHC/PSII 4.15 4.03 4.29 4.93Ratio carotenoids/ 0.257 0.292 0.259 0.269

Chi a + bPearl millet

CPI* + CPI 31.1 ± 1.0 26.7 ± 0.3 (86) 32.0 ± 0.7 (103) 30.9 ± 0.9 (99)CPI 5.6 ± 0.4 6.2 ± 0.2 (111) 6.0 ± 0.7 (108) 7.5 ± 1.0 (135)CP47 + CP43 7.8 ± 0.1 8.7 ± 0.5 (111) 8.1 ± 0.6 (103) 6.7 ± 0.1 (86)LHCII* + LHCII 35.8 ± 0.7 38.2 ± 0.5 (107) 36.4 ± 0.5 (102) 38.5 ± 0.9 (107)Ratio LHC/PSII 4.6 4.4 4.5 5.7Ratio carotenoids/ 0.237 0.243 0.245 0.258

ChI a + b

energy via photoinhibitory quenching, or changes of charac-teristics of the light harvesting system. In older plants ofsorghum, physical protection by extensive leaf rolling is alsoobserved, which causes shading of the exposed upper leafsurface and also decreases water vapor loss (8, 22), but thisphenomenon rarely occurs in seedlings.

In our experiments, plants were grown under conditions(lower temperature and irradiance) that could be natural atthe beginning of growing season. In any case, irrigated seed-lings could quickly acclimate to much higher irradiance atelevated temperature and increase their leaf and chloroplastactivities (Tables II and III). Our experiments accomplishedtheir aim: the more detailed study of drought and irradiancestress mechanisms with respect to their monitoring. Althoughwe used young plants, it is unlikely that something principallydifferent could happen in mature plants grown in the fieldwhen conditions become unfavorable.The mechanism by which drought stress inhibits the pho-

tosynthetic electron transport system is still unclear, althougha number of authors have attempted an explanation (3, 12,15, 17, 26). Ogren and Oquist (24) concluded from theirresults that the drought-induced decline in CO2 uptake wasinitially equally attributable to stomatal and nonstomatalfactors, but the further decline as drought continued wassolely due to nonstomatal factors. There is no doubt now thatdrought stress predisposes the photosynthetic apparatus to

photoinhibitory damage (3, 22), but it is difficult to assess anapproximate share between drought stress and photoinhibi-tion and a possible mechanism.

Concerning the site of drought stress action, several authorsreported that PSII photochemistry is predisposed by droughtstress to photoinhibitory damage (e.g. Refs. 3, 24). In contra-diction, Genty et al. (12) concluded that PSI-mediated elec-tron transport was inhibited by drought, whereas PSII electrontransport remained the same. They also reported that droughtdoes not induce sensitization to photoinhibition.

It is obvious from our results that even moderate droughtstress enhances sensitivity of plants to high irradiance, and itbecomes even more pronounced when drought stress in-creases. In drought-stressed plants, stomata close due to waterdeficit as lately reported for example by Steinberg et al. (28).In our experiments, the values of stomatal conductance werecalculated from water vapor relations using an IR gas ana-lyzer, which we do not consider very reliable for obtainingabsolute results. However, we found values of about 0.3 cms-' for irrigated plants and values of about 0.1 cm *s-' or lessfor drought-stressed plants before high irradiance, suggestingpartial or complete stomatal closure. Drought-stressed plantsof Aralba and R109A showed great changes since electrontransport activities vary between 30 to 69% and 46 to 92% ofcontrols, respectively, and the CO2 assimilation rates are 17and 30%, respectively. In pearl millet, we could see a smaller

205



MASOJiDEK ET AL.

percentage decrease of CO2 assimilation rate than electrontransport activities. It does not exclude the possibility thatlower photosynthetic assimilation causes a decline of photo-chemical activities (or even fluorescence), but there may existan independent nonstomatal mechanism affecting photo-chemical reactions under drought stress. Lower stomatal con-ductance and thus lower CO2 concentration can enhance theeffect of high irradiance. When the Calvin cycle does notwork, a surplus of reduced ferredoxin causes the generationof reactive radicals via the Mehler reaction, and they cancause modification or degradation of proteins. The differencebetween the decrease of CO2 assimilation rates and electrontransport activities among sorghum varieties and pearl milletand a signal II decrease may suggest another mechanism ofdrought affecting photochemical activities. A decrease of theamplitude of signal II in drought-stressed plants after highirradiance treatment indicates that the donor side of PSII isaffected and the donation of electrons from the oxygen evolv-ing complex to the reaction center of PSII as reported by Theget al. (29) or Callahan et al. (6). This can probably cause adecrease of photochemical activities as electron transport isinterrupted. Some damage to the PSII polypeptides mayhappen due to a generation of reactive species (radicals), whichresults in the subsequent disassembly of the PSIH complex aswe could see from CP analysis. This hypothesis is supportedby recent results of Hundal et al. (13), who proposed themodel for photoinhibitory (and other) stress in which thecollapse of PSII complex was preceded by the Dl-proteindisassembly (and/or degradation) due to the 02 evolvingcomplex damage. Methods for a more precise assay of the Dl-)rotein that plays a crucial role in PSIH photoinactivationwere not available in the laboratory, but some indication ofits degradation can be deduced from a disappearance of thePSII CPs. There still exists a possibility that both mechanisms(i.e. the effect of low CO2 concentration and a direct decreaseof PSII activity) work together. Some other structural changesprobably appear in thylakoid membranes during photoinhi-bition ofdrought-stressed plants. We usually detected a highercontent of PSI CPs than in irrigated plants and much less ofthe PSII core (CP43 + CP47). This trend continued asdrought-stressed plants were exposed tohigh irradiance, sup-porting the observation of changes in PSII and PSI activityduring drought. We found only small changes in thylakoidpolypeptides of sorghum and no changes in millet.

Interesting results that can also be pertinent to answeringthe question about the extent of photoinhibition on the back-ground of drought stress can be deduced from the Fv/Fmcurves (Fig. 1). They suggest that, in both irrigated anddrought-stressed plants, the Fv/Fm ratio decreases with highirradiance but in drought-stressed plants the inhibition isgreater. The recovery of the FV/Fm ratio was fast even indrought-stressed plants without watering after photoinhibitorytreatment, showing that the first phase of the rise is attribut-able to recovery from photoinhibition and the second mightbe due to recovery from drought stress.Our experiments with pearl millet and three varieties of

sorghum show that the extent of photoinhibition depends onthe degree ofdrought stress. It can be detected by physiologicaland biochemical techniques in 2-week-old seedlings. Themethods employed here might be used for monitoring envi-

ronmental stresses in field-grown plants and help in selectingstress-resistant varieties for growth under unfavorableconditions.

ACKNOWLEDGMENTSWe thank Professor Richard Cammack and Andrew White for

help with EPR measurements and Drs. Ivan Setlik, Janet Corlett, andHamlyn Jones for their valuable discussion and reading of themanuscript.

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the synergistic of and stresses in pearl millet1 · anopen perplex tank with running tap water was...

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