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Introduction
Drought stress is the main cause of reduced plant growth
and productivity in semi-arid regions and causes a complex
of responses at molecular, cellular, physiological and
developmental levels (Ingram and Bartels 1996).
Very low water contents, resulting from severe
dehydration, are often associated with increased levels of
activated oxygen species (AOS), such as superoxide anion
(O2 ), hydrogen peroxide (H2 O2 ), hydroxyl radical
(HO ) and singlet oxygen (1 O2 ), which, in turn, damage
cellular structures and macromolecules (Smirnoff 1993),
or act as signal molecules that activate multiple defence
responses (Van Breusegem et al. 2001; Vranova et al. 2002).
Chloroplasts, mitochondria and peroxisomes are the
major sources of AOS in plant cells (Asada 1999). Plants
use enzymatic and non-enzymatic antioxidant defence
Abbreviations used: AOS, activated oxygen species; APX, ascorbate peroxidase; CAT, catalase; CP, control plants; FR, ne roots; IAAox, indoleacetate
oxidase; LWP, leaf water potentials (predawn); MR, medium roots; POD, guaiacol peroxidase; PPO, polyphenol oxidase; SOD, superoxide dismutase;
SP, drought-stressed plants; VPD, vapour pressure decit.
CSIRO 2005
10.1071/FP04003
1445-4408/05/010045
46
A. Sofo et al.
47
The supernatant was recovered and used for the enzyme activity
assay. CAT activity was assayed according to Aebi (1984). The
decomposition of H2 O2 was followed spectrophotometrically by the
decrease in A240 . One unit of CAT activity corresponded to the amount
of enzyme that decomposes 1 mol of H2 O2 per minute, according to
Havir and McHale (1987).
POD, IAAox, PPO extraction and activity
An aliquot (1.0 g) of frozen powder was added to 10.0 mL of cold
200 mM NaPi , pH 7.0, 5 mM Na2 EDTA, 0.1% (w / v) PVPP, 3 mM
dithiothreitol, 15 mM -mercaptoethanol, 10 mM sodium metabisulte,
prepared and stored at 4 C the day before and, after 30 min, centrifuged
at 15 000 g for 30 min. The supernatant was recovered and used for the
enzyme activity assay.
POD activity was measured according to Chance and Maehly (1955).
The activity of the mixture was determined spectrophotometrically
at 470 nm after 10 min at 20 C. IAAox activity, due to POD, was
spectrophotometrically measured at 247 and 254 nm according to Ricard
and Job (1974). PPO activity was assayed according to Canal et al.
(1988) by reading absorbance at 420 nm.
Total activities for POD, IAAox and PPO were expressed as increases
in absorbance per minute.
Statistical analysis
The values of physiological parameters were represented as means
of nine measurements ( SE) from three selected plants (three
measurements per plant), while the values of enzyme activities were
expressed as means of three measurements ( SE) from three plants
having a similar level of drought stress (one measurement per plant
and three replications of each measurement). Statistical analysis was
performed using ANOVA. Signicant differences between values of
enzyme activity in CP and SP were determined at P0.05, according to
Duncans multiple range test.
Results
Environmental conditions and physiological parameters
The highest value of maximum air temperature was 33.8 C
on 12 July and the mean of all the daily maximum values
was 31.2 C. Minimum relative humidity pattern showed
the highest value (40.8%) on 5 July, with a mean of
30.1%. Vapour pressure decit range was between 2.2
(on 5 July) and 4.1 kPa (on July 22), with a mean value of
3.2 kPa. PPFD levels uctuated within a range from 22.21
to 28.34 mol m2 d1 (on 7 July and 4 July, respectively)
(Fig. 1). The mean value of predawn LWP in CP was
0.36 MPa. LWP values in SP declined during the whole
period of water decit, reaching a minimum of 5.73 MPa
after 20 d of stress (Fig. 2).
Stressed plants showed a gradual and continuous decrease
of net photosynthesis rate at the mild stress level, followed
by a rapid decline at the moderate stress level to almost
zero value reached at the severe stress level (Fig. 3A).
Transpiration rate and stomatal conductance patterns in SP
showed a similar trend, as both displayed a marked decrease at
the mild stress level and subsequently a gradual drop during
moderate and severe drought stress (Fig. 3BC). Substomatal
CO2 concentration gradually increased at mild stress level,
subsequently reached a plateau during moderate stress and
48
A. Sofo et al.
Max air
temperature (C)
35
30
Min RH (%)
45
35
25
VPD (kPa)
15
5
4
3
PPFD
(mol m2 d1)
2
30
25
20
3 July
8 July
13 July
18 July
23 July
7
6
5
4
3
2
1
0
0
12
16
20
18
15
12
9
6
3
Transpiration rate
(mmol m2 s1)
8
6
4
2
Stomatal conductance
(mmol m2 s1)
0
250
200
150
100
50
C
0
Substomatal CO2
(L L1)
300
250
200
150
100
50
0
0
12
16
20
49
Day
SP
CP
SP
CP
29.21 1.24
30.73 1.35
39.06 1.97*
45.80 1.77*
48.17 2.01*
40.66 0.73*
28.72 1.17
27.43 0.95
28.84 0.91
27.56 1.28
28.52 1.05
28.67 0.85
CP
13.23 0.13
11.27 0.45
12.70 0.56
13.76 0.46
10.61 0.28
11.49 0.24
IAAox
14.30 0.49
16.15 0.75*
19.00 0.47*
22.56 0.60*
16.28 0.34*
16.50 0.41*
SP
CP
34.66 1.70
33.42 1.29
35.21 0.96
35.73 0.85
34.13 1.20
33.26 0.66
PPO
33.49 0.87
31.87 1.06
26.55 0.85*
25.47 1.04*
24.13 1.34*
20.36 0.91*
SP
0
8
16
20
None
Mild
Moderate
Severe
CP
6.96 0.26
7.56 0.10
6.31 0.18
6.42 0.17
SP
7.23 0.15
17.32 0.38*
19.37 0.66*
25.00 1.12*
SOD
0.23 0.01
0.24 0.01
0.32 0.02*
0.34 0.01*
SP
CP
0.19 0.01
0.20 0.01
0.21 0.01
0.22 0.01
APX
2.86 0.09
4.57 0.20*
8.16 0.13*
5.98 0.13*
2.98 0.05
3.26 0.09
3.74 0.13
3.14 0.12
18.77 0.31
26.51 0.34*
39.37 1.37*
39.52 0.90*
SP
CP
SP
PPO
CP
7.54 0.12 30.31 0.42 32.12 0.12
7.28 0.11 28.47 0.47 31.79 0.48
8.73 0.38 10.90 0.34* 31.72 0.34
6.77 0.11 7.16 0.12* 33.63 0.79
IAAox
Day
CP
8.12 0.38
7.61 0.35
8.27 0.20
6.85 0.09
SP
7.99 0.20
11.20 0.28*
15.94 0.59*
17.19 0.49*
SOD
0.36 0.01
0.42 0.01*
0.56 0.02*
0.51 0.02*
SP
CP
0.37 0.02
0.33 0.01
0.40 0.02
0.35 0.02
APX
2.08 0.05
3.10 0.07*
2.74 0.10*
2.58 0.07*
2.35 0.04
2.36 0.07
2.57 0.22
2.25 0.13
23.88 0.34
39.05 1.56*
47.77 2.11*
44.56 1.27*
25.62 0.85
23.52 1.35
25.09 0.67
22.97 0.85
IAAox
CP
SP
CP
19.76 1.22
40.46 1.28
39.61 1.60
41.88 1.84
PPO
11.22 0.13 10.67 0.35 38.19 0.99
15.74 0.51* 9.36 0.48 35.63 0.74
24.00 1.04* 10.04 0.13 21.43 0.43*
23.17 0.56* 11.81 0.41 19.05 0.33*
SP
None
0
Mild
8
Moderate 16
Severe
20
Degree
of
drought
Table 3. Antioxidant enzyme activities of medium roots from drought stressed (SP) and irrigated (CP) plants
Samples were collected, at 06000700 h, at 0, 8, 16 and 20 d from the beginning of the drought stress period. Each value represents the mean of three measurements ( SE) from three plants
having a similar level of drought stress. *Signicant differences at the 5% level between values of control and drought stressed plants (P0.05, according to Duncans multiple range test)
Day
Degree
of
drought
Table 2. Antioxidant enzyme activities of ne roots from drought stressed (SP) and irrigated (CP) plants
Samples were collected, at 06000700 h, at 0, 8, 16 and 20 d from the beginning of the drought stress period. Each value represents the mean of three measurements ( SE) from three plants
having a similar level of drought stress. *Signicant differences at the 5% level between values of control and drought stressed plants (P0.05, according to Duncans multiple range test)
4.45 0.15
4.50 0.12
4.87 0.12
5.33 0.10
6.53 0.15
7.36 0.14
APX
SOD
None
0 15.21 0.82
Mild
4 18.24 0.50
Mild
8 30.73 0.89*
Moderate 12 34.34 1.27*
Moderate 16 31.69 1.90*
Severe
20 25.17 1.06*
Degree
of
drought
Table 1. Antioxidant enzyme activities of leaves from drought-stressed (SP) and control (CP) plants
Samples were collected, at 06000700 h, at 0, 4, 8, 12, 16 and 20 d from the beginning of the drought stress period. Each value represents the mean of three measurements ( SE) from three
plants having a similar level of drought stress. *Signicant differences at the 5% level between control and drought-stressed plants (P0.05, according to Duncans multiple range test)
50
A. Sofo et al.
51
in the leaves of olive trees (Ryan et al. 2002) and in olive oil
(Owen et al. 2000), being found in cytoplasm, vacuoles
and cell walls. Phenolic compounds are also involved
in auxin protection or catabolism (Hrubcova et al. 2000)
and in the modulation of the cell wall plasticity (Fry
1986). Furthermore, abscisic acid-induced stomatal closure is
reversed by some phenolics (Purohit et al. 1991), suggesting
an important role of these compounds in gas exchange
dynamics. For all these reasons, the regulative action of
PPO plays an important role in the physiological aspects of
plants subjected to water decit conditions. Drought stress
can improve the antioxidant action of phenols by inhibiting
PPO activity (Tables 13) and consequently by maintaining
the phenol compounds pool in the reduced state. Additionally,
the proteolytic activity of PPO (Kuwabara and Katoh 1999)
suggests that the enzyme could be involved in removing the
proteins damaged by AOS.
Our results highlight the olive trees capacity to withstand
arid environments and to maintain high photosynthetic
rates at moderate drought stress when the transpiration
rate declines signicantly. The different values of enzyme
activities found in leaves, ne roots and medium roots
conrm their different functions: leaf tissues showed
more pronounced changes, owing to the synergical effect
of high irradiance levels and loss of cellular water; FR
were more sensitive to drought stress and its consequent
effects, while MR maintained a prolonged functionality and
presented less reactivity, even at severe drought stress. In
this investigation into antioxidant defence enzyme activities
of olive tree, we have found evidence for an up-regulation
of AOS-scavenging enzymes as plants enter water decit
conditions. The results obtained underline the important
role of some antioxidant enzymes in protecting cellular
apparatus during water decit conditions. Research into
variations in the activities of other antioxidant enzymes and
the action of other molecules with a non-enzymatic action
may give a more complete picture of the response of olive
tree to drought and better explain its high resistance to this
specic abiotic stress.
Acknowledgments
We are grateful to Dr Mike Clearwater for his important
suggestions about the manuscript. We thank Professor
Elvira Di Nardo for her contribution to the statistical analysis
and Dr Giuseppe Montanaro for his help with use of LCA-4.
References
Aebi H (1984) Catalase in vitro. Methods in Enzymology 105,
121126.
Allen RD, Webb RP, Schake SA (1997) Use of transgenic plants to
study antioxidant defenses. Free Radical Biology and Medicine 23,
473479. doi: 10.1016/S0891-5849(97)00107-X
Angelopoulos K, Dichio B, Xiloyannis C (1996) Inhibition of
photosynthesis in olive trees (Olea europaea L.) during water stress
and rewatering. Journal of Experimental Botany 47, 10931100.
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A. Sofo et al.
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