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South African Journal of Botany 75 (2009) 34 – 42

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Physiological responses of three maize cultivars to drought stress

and recovery
B. Efeoğlu a , Y. Ekmekçi b,⁎, N. Çiçek b
a
.
Abant I zzet Baysal University, Faculty of Arts and Sciences, Department of Biology, Gölköy 14280 Bolu, Turkey
b
Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe-Ankara, Turkey
Received 20 April 2007; received in revised form 3 April 2008; accepted 26 June 2008

Abstract

Water shortages and soil water losses due to environmental change and land use change are challenges to maize production. An experiment was
conducted to investigate the physiological responses of the maize (Zea mays L.) cultivars Doge, Vero and Luce to drought conditions. Drought stress was
imposed on the plants 12 days after sowing by withholding irrigation for 12 days and then rewatering for 6 days. Growth of all cultivars was retarded
under drought stress conditions and regained speed during the recovery stage. RWC decreased in all cultivars by drought and reached the control values
during the recovery period. Fresh and dry biomass of the cultivars significantly decreased in all cultivars. Drought affected the minimum fluorescence
(Fo) of all cultivars, but a significant effect was only found in Doge. Drought also caused decreases in FM, FV/FM, FV′/FM′, ϕPSII and qL; and an increase
of non-photochemical quenching (NPQ), but those returned to control values during the recovery stage in all three cultivars. Chlorophyll (chl) a, chl b,
total chl (a + b) and carotenoid contents of all maize cultivars were significantly reduced under drought, but a recovery was observed following
rewatering. Anthocyanin and proline contents increased in all cultivars. Although Doge was affected from drought more than the other cultivars, it could
probably withstand drought with better upregulating its protective mechanisms. As a result of that Doge was classified as less drought tolerant, but others
as tolerant.
© 2008 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Biomass; Drought; Maize; Proline; PSII activity; Recovery

1. Introduction Nayyar and Gupta, 2006; Yang et al., 2006). Nutrient uptake by
plants is decreased under drought stress conditions due to reduced
Water is a major limiting factor affecting plant growth, transpiration, impaired active transport and membrane permeability
development and yield mainly in arid and semiarid regions where resulting in reduced root absorbing power (Tanguilig et al., 1987).
plants are often exposed to periods of water deficit stress also Most of the damaging effect of drought is associated with the
known as drought stress. Drought is one of the major causes for photosynthetic process of the plant. Many studies have shown that
crop loss worldwide, reducing average yields with 50% and over the decrease of the photosynthetic activity under drought stress
(Wang et al., 2003). Responses to drought are multiple and can be attributed to both stomatal and non-stomatal limitations
interconnected. It is well established that drought stress impairs (Shangguan et al., 1999; Yordanov et al., 2003; Zlatev and
numerous metabolic and physiological processes in plants (Levitt, Yordanov, 2004). One of the earliest responses to drought is
1980). It leads to growth reduction, reduction in the content of stomatal closure. Stomatal closure allows plants to limit trans-
chlorophyll pigments and water, and changes in fluorescence piration, but it also limits CO2 absorption, which leads to a
parameters (Lu and Zhang, 1999; Lima et al., 2002; Colom and decreased photosynthetic activity (Nayyar and Gupta, 2006; Yang
Vazzana, 2003; Souza et al., 2004; Zlatev and Yordanov, 2004; et al., 2006). Nonetheless, limitations to CO2 absorption imposed
Ekmekçi et al., 2005; Mohsenzadeh et al., 2006; Li et al., 2006; by stomatal closure may promote an imbalance between
photochemical activity of photosystem II (PSII) and the electron
⁎ Corresponding author. Tel.: +90 312 297 6166; fax: +90 312 299 20 28. requirement of the Calvin–Benson cycle, leading to an excess of
E-mail address: [email protected] (Y. Ekmekçi). absorbed excitation energy and subsequent photoinhibitory
0254-6299/$ - see front matter © 2008 SAAB. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.sajb.2008.06.005
 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42 35

damage to PSII reaction centers (Foyer and Noctor, 2000; Baker some physiological parameters associated with better drought
and Rosenqvist, 2004). Several in vivo studies reported that water tolerance among maize cultivars.
deficit stress resulted in damage to the PSII oxygen evolving
complex (Lu and Zhang, 1999; Skotnica et al., 2000) and to 2. Materials and methods
inactivation of PSII reaction centers associated with degradation
of D1 protein (He et al., 1995; Cornic, 2000). Thus, over reduction 2.1. Plant materials and stress treatments
of photosynthetic electron chain or inactivation of oxygen
evolving manganese complex, which predisposes PSII to damage Three maize (Zea mays L.) cultivars: Doge, Luce and Vero were
caused by long-lived P680+ or stress on the concurrent synthesis used in this study. Seeds were surface sterilized with a 2%
of new D1 protein may result in the formation of reactive oxygen sodiumhypochloride (NaOCl) solution for 10 min. Then they were
species (ROS), which can lead to photoinhibition and oxidative washed and imbibed in distilled water for 24 h. After incubation,
damage (Asada, 1999; Hakala et al., 2005; Nishiyama et al., the seeds were planted in PVC pots holding 1000 g air-dried soil.
2006). Plants have several protection mechanisms in order to The soil had the following properties: field capacity 30%, texture
prevent the damaging effect of ROS by an increase in dissipation clay–sand, CaCO3 1.41%, pH 7.45, EC 3020 µS/cm, organic
of excess excitation energy or by the production of sun-screen matter 7.4% and total N 0.37%. Plants were grown under well-
pigments (carotenoids and anthocyanin) (Young, 1991; Sherwin watered conditions. Seedlings of the cultivars were grown under a
and Farrant, 1998; Gould et al., 2002; Pietrini et al., 2002). In constant temperature regime of 23± 1 °C for 14 h photoperiod at
addition to that, many plants cope with drought stresses by 40 ± 5% relative air humidity and at a photosynthetic photon flux
synthesizing and accumulating some substances. They most density (PPFD) of 250 µmol m− 2 s− 1 light intensity in a controlled
relate osmotic adjustment (accumulation of ions and of growth room. Drought stress was imposed on plants 12 days after
compatible solutes), water circulation (aquaporins), reaction to sowing by withholding irrigation for 12 days followed by
oxidative stress, detoxification, protection or degradation of rewatering for 6 days. Measurements were made at day 12 of
proteins or different cellular structures (chaperons, dehydrins, the stress treatment and at day 6 following the start of rewatering.
proteases and antiproteases), abscisic acid (ABA) synthesis, For each treatment irrigated plants were used as controls.
carbon and nitrogen metabolism and signal transduction (Hong et
al., 2000; Hien et al., 2003; Pinheiro et al., 2004; Riccardi et al., 2.2. Growth parameters
2004; Mahajan and Tuteja, 2005).
The reaction of the plants to drought differs significantly at At the end of each treatment, the seedling length (the
various organizational levels depending upon intensity and dura- distance from soil surface to upper end of the longest leaves) of
tion of stress as well as plant species and the stage of development the maize cultivars was measured (cm/plant). The fresh (g/FW)
(Chaves et al., 2003). Maize is one of the major summer crops and dry (g/DW) biomass of the seedlings was also determined.
grown in the Mediterranean region and also it is the third most
important crop following wheat and barley in Turkey and raised in 2.3. Chlorophyll a fluorescence measurements
an approximate area of 800.000 ha (FAOSTAT, 2006). The scarce
and highly variable precipitation in this region make efficient Chlorophyll a fluorescence measurements were performed in a
planning of water use for irrigation necessary for most summer growth cabinet at 24 °C with a portable, modulated fluorescence
crops. Maize has been reported to be very sensitive to drought monitoring system, (FMS-II-Hansatech, UK), on randomly
(Farre et al., 2000). Early studies showed that genotypic selected leaves (third leaf) of the cultivars. Following 30 min
differences occur in growth response of maize to drought stress. dark adaptation, the minimum chlorophyll a fluorescence (Fo)
Characteristic differences in response to drought stress have also was determined using a measuring beam of 0.2 µmol m− 2 s− 1
been identified for a range of morphological and physiological intensity. A saturating pulse (1 s white light with a PPFD of
characteristics, including root development, stomatal activity, 7500 µmol m− 2 s− 1) was used to obtain the maximum fluores-
osmotic adjustment, abscisic acid and proline levels in the whole cence (FM) in the dark-adapted state. The quantum efficiency of
plant (Li and Van Staden, 1998a,b; Selmani and Wasson, 2003). open PSII reaction centers in dark-adapted plants (FV/FM) was
In order to improve the agricultural productivity within the water calculated from (FM − Fo)/FM. Light-induced changes in chlor-
limited areas, it is imperative to ensure higher crop yields against ophyll a fluorescence following actinic illumination (300 µmol
drought stress. There has only been a limited number of pub- m− 2 s− 1) were recorded prior to measurement of Fo′ (minimum
lications related to simultaneous comparison of photochemical chlorophyll a fluorescence in light saturated state) and FM′
efficiencies of PSII and physiological response which could be (maximum fluorescence in light saturated stage). The quantum
useful for identifying differences in maize cultivars under drought efficiency of open PSII reaction centers in the light-adapted state,
stress and recovery. Therefore, it may be possible to determine if referred to as ФPSII (FM′ −FS/FM′), was determined from FM′ and
there is damage to light reaction systems in photosynthetic FS values and also the quantum efficiency of excitation energy
machinery during drought and recovery by analysis of chlor- trapping of PSII, (FV′/FM′), was calculated according to Genty et al.
ophyll fluorescence which can serve as easy and rapid indicators (1989). After turning off the actinic light the leaves were illu-
of stress conditions in plants. minated with far-red light (7 µmol m− 2 s− 1) to oxidize the PQ-
The present study was planned to identify the effect of drought pool in order to be able to determine the minimum fluorescence
stress on PSII photochemistry by analyses of fluorescence and by level of the light-adapted state (Fo′). The fraction of open PSII
36 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42

Proline was extracted from leaf samples (20 mg DW) according to

Weimberg (1987) with minor modifications. The absorbance of the
sample extract was spectrophotometrically determined at 520 nm.
The proline concentration was determined as (µmol g/DW) using a
standard curve.

2.7. Data analysis

The experiments were performed in a randomized block

design with three replicates. Differences among the treatments
as well as between the cultivars were tested using the SPSS
statistical program. Statistical variance analysis was performed
using ANOVA and compared with least significant differences
(LSD) at 5% level.

3. Results

3.1. Growth
Fig. 1. Effect of drought stress and recovery on seedling length of maize
cultivars. The error bars represent standard error (±SE) for 12 plants (n = 12).
Arrow indicates rewatering. D-c, Doge-control, D-t, Doge treatment (drought Growth of the seedlings was retarded under drought stress
stress for 12 d and then recovery for 6 d), L-c, Luce-control, L-t, Luce-treatment conditions in Doge and Luce. During the recovery stage the
(drought stress for 12 d and then recovery for 6 d), V-c, Vero-control, V-t, Vero- seedlings increased their growth rate again but the length of the
treatment (drought stress for 12 d and then recovery for 6 d).

reaction centres as qL = (FM′ −FS/FM′ − Fo′). (Fo′/FS) were calculated

using the ‘lake’ model according to Kramer et al. (2004) and non-
photochemical quenching NPQ = (FM′ − FM)/(FM) were also
calculated according to Bilger and Björkman (1990).

2.4. Pigment analysis

Photosynthetic pigments were extracted from leaf samples in

100% acetone. The absorbance of the extracts was measured at
470, 644.8 and 661.6 nm using a Shimadzu Mini-1240 UV–Vis
spectrophotometer. The content of chlorophyll (a and b) and total
carotenoids (xanthophylls and carotenes, x + c) was calculated
using adjusted extinction coefficients (Lichtenthaler, 1987).
Anthocyanins were extracted from leaf material in 1 ml of
acidified methanol for 48 h at 4 °C. The absorbance was measured
at 530 and 657 nm and the anthocyanin content was calculated
according to Mancinelli et al. (1975). The pigment content was
expressed as mg g/DW.

2.5. Relative water content

The RWC content was calculated using the standard formula

[(FW−DW)/(HydW−DW)*100] previously determined by Farrant
(2000), where FW, HydW and DW are the leaf fresh weight,
hydrated (full turgor) and dry weights, respectively. The hydrated
weight was determined by weighing the leaf after 24 h of immersion
in distilled water in a sealed flask at room temperature. Dry weight
was determined gravimetrically after 48 h at 70 °C in an oven.

2.6. Proline content

Fig. 2. Fresh (A) and dry (B) biomass of maize seedlings subjected to drought
The proline content of drought stressed and irrigated (control) stress and recovery. The error bars represent standard error (±SE) for 6 plants
plants was determined using the method of Bates et al. (1973). (n = 6). ⁎ See Fig. 1. for explanation of legends.
 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42 37

seedlings was significantly shorter than the length of the rewatering analysing several fluorescence parameters deter-
controls. The growth of the seedlings continued under irrigated mined under dark-adapted and steady state conditions (Fig. 4).
and drought stress conditions in all genotypes but growth was There was no significant change in Fo under irrigated and
significantly less in drought stressed plants during recovery than drought stress conditions in Luce and Vero, but drought caused
in control plants for the cultivar Vero. While the seedling length a significant increase in Fo in Doge. There was no significant
was not significantly different among cultivars with respect to difference in Fo between recovery and control in all cultivars
the control seedlings, Vero had the longest seedlings both (Fig. 4A). Further a significant reduction in FM in Doge and
during the stress treatment and during recovery (Fig. 1). Luce exposed to drought was observed (Fig. 4B). In control
Maize cultivars exposed to drought had a lower fresh and dry leaves, the quantum efficiency of open PSII reaction centers in
biomass than their controls due to a significant drought-induced the dark-adapted state (FV/FM) was approximately 0.81–0.82
reduction in growth (Fig. 2). Fresh biomass of cultivars was (Fig. 4C). This parameter decreased significantly in response to
significantly reduced under drought stress conditions but it was drought stress in all cultivars. Following rewatering a full
significantly increased during recovery. There was no significant recovery of the FV /FM-value was observed (Fig. 4C).
difference among cultivars neither during the stress treatment Additionally, drought stress induced reduction in the quantum
nor during recovery (Fig. 2A). In addition, dry biomass was yield of PSII electron transport, ϕPSII, in all cultivars. The
significantly decreased under drought stress and recovery reduced ϕPSII was a result of the decrease in the efficiency of
conditions in all genotypes compared to their controls. There excitation energy trapping of PSII reaction centers, FV′/FM′
was no significant difference among cultivars at the beginning of (Fig. 4D, E). A significant decrease in qL was also observed in
stress, but Doge had the highest dry biomass after 12 and 18 days all drought stressed cultivars (Fig. 4F) indicating that the
of growth under control conditions (Fig. 2B). balance between excitation rate and electron transfer rate has
changed leading to a more reduced state of the PSII reaction
3.2. Relative water content centers. The effect on the qL was larger than the effect on the FV
′/FM′ in Vero and Luce. On the other hand, increase of NPQ in
RWC was significantly reduced under drought stress conditions, drought conditions reflecting the non-photochemical energy
but it significantly increased during recovery. Whereas Luce had dissipation in all cultivars was determined significant compared
the highest RWC following Doge and Vero at control, there was no to their controls (Fig. 4G). This value was highest in Doge
significant difference among cultivars at stress and recovery stage followed by Vero and Luce. At end of 6 days rewatering, all
(Fig. 3). These low RWC (approx. 45%) values of leaves of all fluorescence parameters in leaves of all cultivars returned to
cultivars rapidly recovered following rewatering and reached their control values.
control levels after 6 d of rewatering. Moreover, leaves of the maize
cultivars showed wilt symptoms and leaf rolling throughout the 3.4. Pigment analyses
drought stress treatment.
The chlorophyll content (a, b, a + b) of all maize cultivars
3.3. Chlorophyll a fluorescence measurements was significantly reduced under stress, but it increased and
reached the control values during recovery (Table 1). The
Drought tolerance of three maize cultivars was evaluated on strong drought-induced decrease of the chl a content indicates
the basis of their response to 12 days of stress and 6 days of that the drought stress induced a strong loss of photosynthetic
reaction centers (PSI and PSII). At the same time the chl a/b
ratio increased during the treatment both in the control and
treated plants indicating a decrease in the antenna size as the
seedlings develop. However, the calculated values were too
variable to observe any clear drought stress-related trends. The
carotenoid content decreased in parallel with the chlorophyll
content. However, calculating the ratio between the two
parameters there are considerably more carotenoids per
chlorophyll molecule in drought stressed Doge-plants. In
Vero, on the other hand, the drought stress treatment did not
increase the carotenoid to chlorophyll ratio relative to the
control plants.
The anthocyanin content of all cultivars significantly
increased under drought stress conditions compared to the
control plants; it significantly decreased in Doge and Vero
during recovery. Table 1 shows that the plants with the lowest
control level of anthocyanins (Vero) showed the strongest
Fig. 3. Effect of drought stress and recovery on relative water content of leaves. drought-induced increase. On the other hand, the plants with the
The error bars represent standard error (± SE) for 6 plants (n = 6). ⁎ See Fig. 1. for highest control level of anthocyanin (Luce) had the lowest
explanation of legends. drought-induced increase.
38 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42

Fig. 4. Chlorophyll fluorescence responses of maize cultivars to imposed drought stress and recovery. (A) Fo, minimum fluorescence (B) FM maximum fluorescence (C)
FV/FM, the quantum efficiency of PSII in dark-adapted state (D) FV′/FM′, the quantum efficiency of excitation energy trapping of PSII in light-adapted state (E)ФPSII, the
quantum efficiency of PSII in light adapted state (F) qL, the fraction of open PSII reaction centres (G) NPQ, non-photochemical quenching. The error bars represent standard
error (±SE) for 3 plants (n = 3). ⁎ See Fig. 1. for explanation of legends.
 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42 39

Table 1
Pigment contents of maize cultivars exposed to drought stress and recovery, where “0 day” treatment corresponds to 12 days-old maize seedlings before drought
Cultivars Treatments Chl a content Chl b content Chl a + b content Chl a/b Carotenoid content Anthocyanin content
(mg g DW- 1) (mg g DW- 1) (mg g DW- 1) (mg g DW- 1) (mg g DW- 1)
Doge 0 day 17.34 ± 1.55 4.82 ± 0.23 22.17 ± 1.72 3.59 ± 0.25 3.40 ± 0.23 0.042 ± 0.003
12 days control 31.45 ± 4.41 7.36 ± 0.80 38.81 ± 5.13 4.26 ± 0.24 7.23 ± 1.08 0.055 ± 0.003
12 days drought 10.94 ± 1.21 2.32 ± 0.28 13.26 ± 1.46 4.80 ± 0.29 3.03 ± 0.42 0.092 ± 0.018
18 days control 31.00 ± 3.32 6.66 ± 0.87 37.66 ± 4.18 4.74 ± 0.16 6.25 ± 0.29 0.054 ± 0.005
12 days drought + 6days recovery 22.19 ± 1.43 5.19 ± 0.30 27.39 ± 1.79 4.28 ± 0.10 5.51 ± 0.33 0.059 ± 0.005
Luce 0 day 17.27 ± 2.03 7.47 ± 1.03 36.90 ± 2.96 3.94 ± 0.30 5.70 ± 0.63 0.051 ± 0.006
12 days control 32.72 ± 1.54 7.37 ± 0.56 39.91 ± 2.02 4.39 ± 0.19 7.75 ± 0.33 0.048 ± 0.007
12 days drought 13.07 ± 0.86 2.69 ± 0.22 14.34 ± 0.99 4.43 ± 0.26 3.06 ± 0.23 0.079 ± 0.011
18 days control 24.98 ± 1.39 6.28 ± 0.66 37.75 ± 2.00 5.05 ± 0.26 7.23 ± 0.34 0.060 ± 0.005
12 days drought + 6days recovery 24.36 ± 2.67 6.35 ± 0.68 34.29 ± 3.33 4.40 ± 0.15 6.68 ± 0.63 0.089 ± 0.017
Vero 0 day 29.43 ± 4.99 5.89 ± 1.16 23.17 ± 6.12 3.16 ± 0.17 3.47 ± 0.90 0.036 ± 0.003
12 days control 32.54 ± 3.58 7.20 ± 0.60 39.92 ± 4.12 4.61 ± 0.23 7.96 ± 0.89 0.049 ± 0.004
12 days drought 11.65 ± 0.79 3.29 ± 0.29 16.36 ± 1.05 4.02 ± 0.21 3.31 ± 0.23 0.100 ± 0.018
18 days control 31.47 ± 3.50 6.00 ± 0.79 30.97 ± 4.29 4.30 ± 0.08 5.85 ± 0.82 0.044 ± 0.005
12 days drought + 6days recovery 27.95 ± 1.68 5.22 ± 0.33 29.58 ± 2.00 4.73 ± 0.08 6.17 ± 0.39 0.028 ± 0.003
LSD 5% 6.26 1.68 8.31 0.54 1.50 0.021
Each value represents the mean of six replicates (n = 6) and its standard errors (±SE).

3.5. Proline content relatively more than root growth in both the plant types (Nayyar
and Gupta, 2006). In this study, growth of seedlings was
Proline content was significantly increased under drought retarded under drought stress conditions and regained during
stress conditions and significantly decreased during recovery in recovery stage in all cultivars (Fig. 1). All maize cultivars
all genotypes examined. Doge and Luce had the highest drought exposed to drought stress, had lower fresh and dry biomass
stress-induced proline contents (Fig. 5). During recovery the compared to control plants, as happens in many species exposed
proline content fell back again to the levels observed before the to drought stress (Fig. 2A, B). The relative water content in
treatment in all three cultivars. leaves of drought stressed cultivars decreased significantly
(Fig. 3). Many investigations have shown that when leaves are
4. Discussion subjected to drought leaves exhibit large reductions in relative
water content and water potential (Kyparissis et al., 1995;
Drought, like other environmental stresses, affects many Scarascia-Mugnozza et al., 1996; Li and Van Staden, 1998a,b;
physiological and metabolic processes within plants. Growth Decov et al., 2000; Nayyar and Gupta, 2006). It is known that
was unaffected at mild stress levels (− 0.4 MPa) but largely dehydration is often reversible. This is compatible with this
inhibited in plants root and leaves of wheat and maize at study that RWC was significantly reduced under drought, but it
moderate and high stress levels. Leaf growth was inhibited significantly increased at recovery stage comparing to drought
conditions in all cultivars (Fig. 3).
Stomatal closure is one of the first responses to drought
stress. It causes primarily a decline in the rate of photosynthesis
(Mahajan and Tuteja, 2005). The measurement of chlorophyll
fluorescence emitted by intact, attached leaves is thought to be a
reliable, non-invasive method for monitoring photosynthetic
events and for judging the physiological status of the plant.
Fluorescence induction patterns and derived indices have been
used as empirical diagnostic tools in plant physiological studies
(Strasser et al., 2000; Baker and Rosenqvist, 2004; Kocheva
et al., 2004). In the C4 species maize the fluorescence quenching
parameters in light are directly reflecting the photosynthetic
activity during the drought stress, without being obscured by the
buffering effect of photorespiration during the initial stomatal
closure, which is the case in C3 species as Baker and Rosenqvist
(2004 and references therein) emphasized in their paper. Of the
cultivars tested only Doge showed an increase of Fo (Fig. 4A).
An Fo-increase may have a variety of causes. It has been
Fig. 5. Leaf proline content in maize cultivars exposed to drought stress and associated with a dissociation of light harvesting complex
recovery. The error bars represent standard error (± SE) for 3 plants (n = 3), LSD (LHC) II from the reaction centers, it may due to the presence of
5% = 0.13. ⁎ See Fig. 1. for explanation of legends. photoinhibited reaction centers, however also a more reduced
40 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42

PQ-pool in dark-adapted leaves will lead to an increase of the Çakırlar (2008) reported for soya bean that salt stress affected
measured Fo. It may even represent an artefact, if the PSI the chl a/b ratio in several cultivars. Some of the cultivars
content would increase relative to the PSII content leading a seemed to adapt to the salt stress by reducing their chl a/b ratio
relatively larger contribution of PSI-fluorescence to Fo. It is (indicating a larger antenna size). In the maize cultivars studied
interesting that despite the measured strong decrease of the here this effect was not so strong. During the experiment, the chl
chlorophyll content no decrease of the Fo was observed. It a/b ratio increased both in the control and the treated plants. As
suggests that the modulated measuring equipment simply shown in Table 1, the changes in the chl a/b ratio in the control
probes somewhat deeper in the stressed plants monitoring the (in both directions) were quite substantial making it e.g. difficult
same number of reaction centers as in the control plants. to judge the significance of the relatively high chl a/b ratio
Changes in light scattering related to changes in the RWC seem found in the cultivar Doge at the end of the drought treatment.
to have had little impact on the fluorescence measurements. Mullineaux and Emlyn-Jones (2005) have suggested that the
Zlatev and Yordanov (2004) reported that drought stress rate of PSII photo damage will be reduced by minimizing PSII
induced in their bean plants always an increase in Fo antenna size and thereby reducing the absorptive cross section
accompanied by a decrease in FM. In Doge the drought stress of the reaction centers.
treatment led to a drastic decrease of FM (Fig. 4B) associated Plants contain substantial amounts of carotenoids that serve
with a Fo increase (Fig. 4A) leading to a strong decrease of the as non-enzymatic scavengers of active oxygen species (Jung et
FV/FM value. A sustained decrease of the FV/FM may indicate al., 2000). Carotenoids are responsible for scavenging of singlet
the occurrence of photoinhibitory damage (Maxwell and oxygen hence comparatively high carotenoid levels in geno-
Johnson, 2000; Colom and Vazzana, 2003). Normally, photo- types have been suggested to be a measure of their tolerance
inhibited reaction centers are quickly de-assembled and (Chandrasekar et al., 2000). Drought stress caused a strong loss
replaced by new ones (Aro et al., 1993). The data presented of photosynthetic reaction centers (loss of chl a, see Table 1).
for Doge only make sense if it is assumed that the drought stress Since carotenoids are mainly found in association with
treatment led to an inhibition of the repair cycle accompanied by photosynthetic reaction centers, the observed carotenoid-loss
an accumulation of damaged reaction centers (Nishiyama et al., was to be expected. However, if the carotenoid content is
2006). An inactivation mechanism for PSII favoured recently is determined on a chlorophyll basis, drought-induced an increase
an inactivation of the oxygen evolving complex, which predis- in the cultivar Doge. This could give this cultivar a means to
poses PSII to damage caused by long-lived P680+ or stress on the dissipate more excitation energy. In this respect we note that
concurrent synthesis of new D1 protein (Hakala et al., 2005; Doge was also the cultivar with the highest NPQ-value
Ohnishi et al., 2005). The decrease of the quantum efficiency of following drought stress. Anthocyanins are located in the
open reaction centers in the light (FV′/FM′) observed in Fig. 4D can vacuoles. They also have the potential to scavenge active
be interpreted to represent an NPQ-related increase of the oxygen species like for example long-lived hydrogen peroxide
probability of heat emission lowering the trapping efficiency of (Gould et al., 2002; Pietrini et al., 2002). The anthocyanin
open reaction centers (Lu and Zhang, 2000). Increases in NPQ in responses differed quite strongly between Doge and Vero on the
all cultivars under drought conditions were indeed detected one hand and Luce on the other hand. In Luce the anthocyanin
(Fig. 4G). The quantum yield of electron transfer at PSII (ϕPSII) is levels were quite high independent of the presence of a stress. In
the product of the efficiency of the open reaction centers (FV′/FM′) Doge and Vero there was a quite strong drought stress induced
and the photochemical quenching (qP) (Genty et al., 1989; Lu and increase but before and after the stress the measured levels were
Zhang, 1999; Sinsawat et al., 2004). In all cultivars a decrease of low. The inducibility of the response by drought stress in Doge
the qL was observed in response to the drought stress treatment, and Vero suggests that anthocyanin levels play a role in the
indicating that a larger percentage of the PSII reaction centers was defence of plants to drought stress.
closed at any time, which in turn indicates that the balance The synthesis of osmolytes including proline is widely used
between excitation rate and electron transfer rate had changed. by plants to stabilize membranes and maintain the conformation
Drought stress leads to a strong reduction of the uptake of of proteins at low leaf water potentials. The synthesis and
CO2 and therefore decreases also the demand for the products of accumulation of osmolytes varies among plant species as well
photochemistry: ATP and NADPH. In that respect a reduction as among different cultivars of the same species. Proline is also
of the reaction center content would make sense for the plant known to be involved in reducing the photo damage in the
from a regulatory point of view. The rapid recovery of the plants thylakoid membranes by scavenging and/or reducing the
following rewatering also suggests that the reaction center loss production of 1O2 (Reddy et al., 2004). Increase in proline
may have played a regulatory role and did not just represent accumulation observed under drought stress in Doge, Luce and
damage (Table 1). Leaf rolling of cultivars during drought has Vero (Fig. 5) was in accordance with earlier observations made
been proposed to limit the absorption of light by the on maize (Chandrasekar et al., 2000).
photosynthetic antenna systems. In addition, leaf rolling may In conclusion, the three maize cultivars investigated showed
have limited the loss of water via transpiration. Previous studies similar responses against drought stress. Although Doge was
indicated that drought-tolerant genotypes were able to maintain the most affected cultivar in terms of some chlorophyll
a higher chlorophyll content than susceptible genotypes. fluorescence parameters such as Fo, FM, FV/FM; all cultivars
Chandrasekar et al. (2000) reported this phenomenon for recovered after 6 days of rewatering. Doge may have coped
drought resistant and susceptible wheat cultivars. Çiçek and with stress by upregulating protective mechanisms such as
 B. Efeoğlu et al. / South African Journal of Botany 75 (2009) 34–42 41

increasing NPQ, chl a/b ratio (smaller antenna size), anthocya- Hakala, M., Tuominen, I., Keränen, M., Tyystjärvi, T., Tyystjärvi, E., 2005.
nin and proline content, decreasing FV′/FM′ compare to other Evidence for the role of the oxygen-evolving manganese complex in
photoinhibition of photosystem II. Biochimica et Biophysica Acta 1706,
two cultivars. Results of the study suggest that Doge is less 68–80.
drought tolerant than the other two cultivars. He, J.X., Wang, J., Liang, H.G., 1995. Effects of water stress on photochemical
function and protein metabolism of photosystem II in wheat leaves.
Acknowledgement Physiologia Plantarum 93, 771–777.
Hien, D.T., Jacobs, M., Angenon, G., Hermans, C., Thu, T.T., Son, L.V.,
Roosens, N.H., 2003. Proline accumulation and Δ1-pyrroline-5-carboxylate
The authors would like to thank to Dr. G. Schansker synthetase gene properties in three rice cultivars differing in salinity and
(University of Geneva) for the critical reading of the manuscript drought tolerance. Plant Science 165, 1059–1068.
and help with the English language. Hong, Z., Lakkineni, K., Zhang, Z., Verna, P.S., 2000. Removal of feedback
inhibition of 1–pyrroline-5-carboxylate synthetase results in increased
praline accumulation and protection of plant from osmotic stress. Plant
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Edited by PJ Houghton