©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at
Phyton (Austria)
Special issue:
"Free Radicals"
Vol. 37
Fasc. 3
(71)-(80)
1.7. 1997
Non-Optimal Growth Temperatures and
Antioxidants in the Leaves of Sorghum bicolor (L.)
Moench. I. Long Term Acclimation
By
A. FUSARI1}, A.R. PAOLACCI1}, M. BADIANI 0 , R. D'OVIDIO 0 , J.G. SCANDALIOS2),
E. PORCEDDU 0 & G. GlOVANNOZZI SERMANNI 0
Key w o r d s : Sorghum bicolor (L.) Moench. cvs. Aralba and ICSV 112, non-optimal
temperature, antioxidants, photosynthesis.
Summary
FUSARI A., PAOLACCI A.R.,
BADIANI M., D'OVIDIO R.,
SCANDALIOS J.G.,
PORCEDDU
E. & GlOVANNOZZI SERMANNI G. 1997. Non-optimal growth temperatures and antioxidants in the
leaves of Sorghum bicolor (L.) Moench. I. Long term acclimation. - Phyton (Horn, Austria) 37 (3):
(71)-(80).
The foliar antioxidant status and the photosynthetic capacity were compared in two
sorghum [Sorghum bicolor (L.) Moench] cultivars of different agroclimatic provenance, namely
Aralba and ICSV 112, which were grown at near-optimal, 27±0.3 °C, suboptimal, 17±0.4 °C, or
supraoptimal, 37±0.1 °C, temperatures. Both non-optimal growth temperatures, although unable to
cause visible symptoms of stress, affected gas echange parameters and antioxidant levels both in cv.
Aralba and in cv. ICSV 112. Compared to controls, plants grown at 17±0.4 °C or at 37±0.1 °C had
higher contents of photosynthetic pigment, an increased size of the ascorbate pool and an enhanced
monodehydroascorbate reductase activity. On the other hand, suboptimal and supraoptimal growth
temperatures, respectively, decreased and increased the glutathione pool and on the capacities of
ascorbate- and guaiacol peroxidases, and of catalase. In cv. Aralba, but not in cv. ICSV 112, the
expression of Superoxide dismutase, in terms of both enzymic activity and mRNA transcripts
abundance, was downregulated by the growth at non-optimal temperature. Adaptation to nonoptimal growth temperature might involve antioxidant responses which could be different in part
from those evoked by genuine temperature stress.
1)
Dipartimento di Agrobiologia e Agrochimica, Universitä di Viterbo, Via S.C. De Lellis,
1-01100 Viterbo, Italy.
2)
Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614,
USA.
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Introduction
In order to evaluate the resulting functional impairment and/or to evoke the
maximal expression of adaptative responses, the studies on temperature stress have
been frequently conducted by exposing plant material to thermic regimes which are
rather distant, both in physical and physiological terms, from those considered
optimal for growth (FEIERABEND & al. 1992, KRAUS & FLETCHER 1994). Also,
many experimental protocols purposely adopted ex abrupto exposure of plant
material to disthermia, thus departing to a considerable extent from the real-world
circumstances. The massive elicitation of stress responses obtained through the
above treatments, however, could hinder the recognition of those early and
subliminal adjustment processes allowing plant metabolism to gradually prepare to
face adverse thermic regimes. The ubiquitous and perceptive nature of the plant
antioxidant systems renders them good candidates to play a role in the abovecited
preparatory responses. Therefore, it is of interest to study the antioxidant status of
photosynthetic tissues during the transition from optimal to adverse temperatures,
i.e. in the presence of non-optimal temperatures which, though not yet stressing,
could act in nature as signals inducing specific paths of metabolic changes. In the
present work, the foliar antioxidant status and the photosynthetic capacity were
compared in two sorghum [Sorghum bicolor (L.) Moench] cultivars of different
agroclimatic provenances, namely Aralba and ICSV 112, which were grown at
near-optimal, 27±0.3 °C, suboptimal, 17±0.4 °C, or supraoptimal, 37±0.1 °C,
temperatures, under moderate light intensity and ad libitum water and mineral
nutrition.
Materials
and
Methods
The sorghum cultivar Aralba is a low-tannin, intermediate class, commercial Fl hybrid
normally grown in Mediterranean environments; the cv. ICSV 112 is a "tan" cultivar developed in
India by the International Crop Research Institute for the Semi-Arid Tropics (ICRISAT). The
sorghum seedlings were grown from seeds sown on a mixture of agriperlite and coarse vermiculite
(1:1, vokvol). Water and nutrients were supplied in the form of aerated Hoagland solution
(HOAGLAND & ARNON 1939), given twice a week during seedlings growth. The seeds were
germinated under controlled environmental conditions at a constant temperature regime of 27±0.3
°C, a relative humidity of 60±5%, a 16 h photoperiod, and a photosynthetic photon flux density at
plant height of 500-550 umol nr 2 s"1, obtained through a set of Sylvania Grolux wide spectrum
incandescent lamps. Established seedlings (5-7 d-old) were either left at the optimal temperature of
27±0.3 °C or gradually exposed, at a 2 °C per day change rate, to the suboptimal temperature of
17±0.4 °C or to the supraoptimal temperature of 37±0.1 °C until the fifth leaf was fully expanded.
Each of the above treatments was replicated in three separate experiments. Maize (Zea mays L. cv.
Samantha) plants to be used for the extraction of genomic DNA (see below) were grown at 27±0.3
°C under the aforementioned controlled conditions. Non-destructive measurements (see below) were
conducted on the third, fourth and fifth fully expanded leaf belonging to 6 representative plants,
uniform in size, for each sorghum cultivar. Then, the same leaves were excised at their collar from
each individual plant, pooled together, wrapped in aluminum foil, dipped in liquid N 2 within 30 s
from excision and finally stored at -80 °C until needed for destructive measurements (see below).
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Equipment, protocol, and microenvironmental conditions adopted for measuring gas exchange rates
and chlorophyll a fluorescence have been given in a previous paper (BADIANI & al. 1993b). The
determinations of: chlorophyll (Chi) and total carotenoids (Car); ascorbic (As A) and
dehydroascorbic (DHAsA) acids; reduced glutathione (GSH) and glutathione disulfide (GSSG);
total ascorbate (APX , EC 1.11.1.11) and guaiacol (GPX, EC 1.11.1.7) peroxidases, catalase (CAT,
EC 1.11.1.6), DHAsA reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR,
EC 1.6.5.4), GSSG reductase (GR, EC 1.6.4.2), and total Superoxide dismutase (SOD, EC
1.15.1.1) activities; water soluble protein content (TSP); thiobarbituric acid-reactive substances
(TBARS), and leaf dry weight (DW) were performed following the procedures reported in previous
papers (BADIANI & al. 1993a, 1993b). Each analyte was measured at least in triplicate. For each of
the above measurements, the same number of sample replicates was adopted at 27±0.3, 17±0.4 and
37±0.1 °C. Total RNA from sorghum leaves was extracted by using the RNA Fast kit (Molecular
System, San Diego, CA, USA). Poly(A)+ RNA was purified by using the PolyATtract mRNA
Isolation System (Promega, Madison, WI, USA). Both procedures were carried out according to the
manifacturers instructions. Genomic DNA from sorghum and maize leaves was extracted as
reported by D'OVIDIO & al. 1992. DNA and mRNA (8 |ug) electrophoretic fractionation, transfer to
nylon membrane, prehybridization, hybridization and washing of the nylon filters were performed
following standard procedures (SAMBROOK & al. 1989). Southern and Northern blot hybridizations
were performed by using as a probe the 71 lbp EcoR I Sod2.7 insert in pUC12. This full-length
cDNA encodes one of the four cytosolic Cu,Zn-isoforms of the enzyme (SOD-2) in maize leaves
(SCANDALIOS 1990). pUC12 DNA (50-100 ng) was labelled with digoxigenin by the Polymerase
Chain Reaction following the procedure reported by D'OVIDIO & ANDERSON 1994.
Results
and
Discussion
Constitutive differences among cv. Aralba and cv. ICSV 112 plants grown at of
27±0.3 °C (optimum) are shown in Table 1. Under non-optimal thermic regimes,
the plants of both cvs. lacked any visible symptom of stress or damage and were
visually indistinguishable from their control counterparts grown at 27±0.3 °C.
Thus, the adopted gradual and limited deviations from the optimal growth
temperature apparently failed to cause manifest symptoms of temperature stress.
Figure 1 coherently shows that neither reductions of leaf DW and TSP nor
pigments bleaching and changes in Chi a fluorescence characteristics (as reported
by SCHÖNER & KRAUSE 1990, PAULSEN 1994) occurred in sorghum plants grown
at 17±0.4 °C or at 37=1=0.1 °C. In spite of this, the growth at non-optimal
temperatures affected both the gas exchange parameters and the antioxidant status
of the sorghum plants (Fig. 1) Unadvertant exposure of the plant material to excess
radiative energy, interfering with the S. bicolor responses to non-optimal growth
temperature, appeared unlikely. In fact, on a DW basis, the growth at both nonoptimal temperatures led to a general increase in the levels of photosynthetic
pigments, most conspicuous for Chi b (Fig. 1). Moreover, no temperaturedependent effect was observed on the main Chi a fluorescence characteristics.
Indeed, the mean values obtained for the ratios FM/F0 and FV/FM were similar to
those obtained from plants grown at 27±0.3 °C (Fig. 1). These results tend to
exclude the occurrence of both photoinhibitory and photooxidative processes and
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ICSV 1 12
Aralba
i
i
i
WUE
-
i mi
-
* Bb
i
i
i
200
400
600
i
1
1
200
1
1
—Fv/Fm—
—Fm/FQ —
— Fm
—Fv
—F0
+00
^^^^
h"
F
a»—
H
250
0
500
*
1
1
1
-y-i
250
750
500
750
DW
+^
J=—
=
1
-TChl/Car-Chl a/b —
—Car
Chi b —
—Chi a - —TChl
% S?
t EÜ
600
'
—TSP
•
-
—TBARS— -
* m^
-
300
600
t mt
0
900
300
GSH/GSSG
-~ *u
t
M
0
*
*
*
-
—G5SG—
-
—GSH
-
GSH+GSSG
>
*
*
H
i
i
i
600
900
<
L
H
H—'
400
150
*
m
—DHAR—
*
m
—MDHAR—
t
GR
-
•—•
Ü• •
i
-
900
1—i-
300
m
600
•
300
<
i
i
600
900
-
800
300
450
0
15D
% change respect to value at 27 °C
|
| 17 vs 27 ° C | H 37 vs 27 DC
300
450
©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at
(75)
thus suggest that the observed effects on Pn-associated parameters and antioxidants
levels were specifically due to the thermic treatments adopted. In previous work on
non-hardened seedlings of a C3 Gramineae, Triticum durum Desf. cv. Duilio
(BADIANI & al. 1993b, PAOLACCI & al. in press), it was found that, compared to
the optimum of 25 °C, both a suboptimal (10 °C) and a supraoptimal (30 °C)
growth temperature, in the absence of stress symptoms, evoked a common set of
antioxidant responses consisting of: a) accumulation of photo synthetic pigments,
especially Chi b; b) enhanced consumption of non-enzymic antioxidants, such as
AsA and GSH, and c) decrease or steadiness in the extractable capacities of ROSscavenging enzymes, such as GPX, CAT, and SOD. It was suggested that nonoptimal, non-stressing growth temperatures might induce overproduction of ROS,
especially H2O2, dealt with by non-enzymic antioxidants, acting as a first defense
line, and without the intervention of ROS-scavenging enzymes. It was also
speculated that the above responses might be the first step of a rather efficient
biphasic adaptative strategy, aimed both at saving resources in the presence of
moderate metabolic perturbations and at producing the maximal compensative
effort only if and when it is effectively needed. According to this hypothesis,
should a further deviation from the normative growth temperature enhance the risk
of oxidative stress, then more robust but more energetically-expensive responses
would be required, such as the induction of scavenging- and antioxidantregenerating enzymes. Indeed, the induction of antioxidant enzymes has been
repeatedly reported to be caused by, and to take part in, the acclimation to both
low (SCHÖNER & KRAUSE 1990) and high (KRAUS & FLETCHER 1994) temperature
stress.
Unlike in T. durum, in S. bicolor the patterns of antioxidants responses to
suboptimal and supraoptimal growth temperatures were mostly divergent (Fig. 1).
However, an analysis of these responses reveals that the above biphasic adaptation
hypothesis might be still tenable. In fact, moderate hyperthermia, unable to affect
the P n and to stimulate lipid peroxidation (Fig. 1), caused in both sorghum
cultivars notable increases in pigments levels, especially Chi b, and in the pool
sizes of AsA and GSH, more accentuated for the oxidized forms, DHAsA and
GSSG, respectively (Fig. 1). In further agreement with the T. durum results (see
above), H2O2- scavenging enzymes, as well as DHAR , were either unaffected of
Fig. 1. Comparison between the mean relative changes induced in the leaves of two
sorghum cultivars by the growth at 17±0.4 °C or at 37±0.1 °C with the values obtained from control
plants grown at 27±0.3 °C made = 100. Each result is the mean ± standard deviation (vertical bars)
of the values obtained from third, fourth and fifth fully expanded leaves and measurement
replication is the same as reported in Table 1 for each plant parameter. The asterisks denote
statistically significant differences from control values (P<0.05). Acronyms for plant parameters are
the same as in Table 1.
©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at
Parameter O
n
cv. Aralba
±6.1 1
± 3.52
±0.97
± 2.04
±0.36
cv. 1CSV 112
n.s
n.s.
n.s.
n.s.
n.s.
P
O.524± 0.061
1.770± 0.230
2.296±0.224
4.438± 0.610
0.769± 0.036
n.s.
n.s.
n.s.
n.s.
n.s.
19.69
24.66
1.86
5.78
2.12
0.550i 0.084
1.654± 0.245
2.204±0.229
4.089± 0.681
0.746± 0.048
n.s.
n.s.
n.s.
n.s.
n.s.
17.61 ± 3.63
25.0 1 ± 1.48
1.79 ±0.79
5.92 ± 1.76
2.37 ±0.68
4.41 ± 0.91
3.71 ± 1.16
0.70 ±0.19
5.1 1 ±1.09
4.25 x 1.71
0.86 ±0.31
1.20 ±0.41
4.94 ± 0.53
4.57 ± 0.82
...
...
n.s..
n.s.
•
5.30 ± 0.89
4.72 ± I ,14
1.12±0.31
1.02 ±0.58
0.10 ± 0.06
9.15 ± 1.33
1.49 ± 0 . 5 1
1.25 ±0.22
0.94 ± 0.23
0.31 ± 0.01
3.00 ± 0.58
GSH+GSSG
Parameter (• >
±0.56
± 1.40
± 0.79
±10.09
0.27 ±0.06
1.39 ±0.27
0.52 ±0.01
0.34 ±0.08
0.21 ±0.08
0.06 ±0.01
3.36±0.65
cv. Aralba
12.66 ± 4 . 1 5
0.69 ± 0.23
2.07 ± 0.84
1.47 ± 0.23
0.22 ±0.09
0.47 ± 0.17
0.1 1 ±0.06
0.10 ±0.04
0.07 ± 0.01
0.0I± 0.004
5.14± 1.41
cv. 1CSV 112
...
**
n.s
••*
...
»•«
P
GSSG
GSH/GSSG
GR
MDI1AR
DHAR
APX
GPX
CAT
SOD
TBARS
TSP
DW
n.s
n.s.
n.s
#• •
»•»
• »*
1.43
3.39
2.84
34.92
17.75 ±2.17
14.81 ±0.86
15.42 ±2.02
GSH
18.81 ± 3.61
16.66 ±3.99
15.02 ± 1.54
Table 1. Mean values (± standard deviation of the mean) measured in the leaves of cv. Aralba and cv. [CSV 1 12 sorghum seedlings grown at
the optimal temperature of 27± 0.3 °C. P denotes the number of replicate measurements for each cultivar and the nominal significance level of the
difference; n = 18. * = 0.05> P >0.01; ** ~- 0.01> P >0.001; *** = P <0.001; n.s. = not significant at P = 0.05. For parameters acronyms, see the text.
P
PY
r
l
WUE
E
v
Fn
!
>M
•M^O
F
V
V M
Chi a+b
Cilia
Chi b
Car
Chi alb
Chi a+b! Car
AsA+DHAsA
AsA
DI-IAsA
AxA/DIIAsA
Units: P n . nmol CO m"2 s"'; PY. (imol CO (mol photons)"1; r. s m"1; F.|. umol H^O m" 2 s"1; WUE. mol CO9(mol H-^O)'1: F o , Fy, Ff^. arbiträr)' units;
Chi a. Chi b. Car. AsA. DHAsA. and TSP. g (kg dry weigTit)"1; GSH and GSSG. mmol (kg dry weight)"1; MDHAR. DHAR^GR, APX, and GPX, units (mg protein)"1;
CAT, units* 10"3 (nig protein)"'; SOD. Superoxide dismutase. g (kg protein)"1; TBARS. mg (kg dry weight)"1; DW, %of fresh weight.
©Verlag Ferdinand Berger & Söhne Ges.m.b.H., Horn, Austria, download unter www.biologiezentrum.at
(77)
drastically depressed in sorghum leaves developed at 37±0.1 °C (Fig. 1). On the
other hand, and again sticking to the biphasic adaptation hypothesis, Fig. 1
suggests that the growth temperature of 17±0.4 °C must be, for the species S.
bicolor, at the boundary between moderate hypothermia and genuine cold stress. In
fact, albeit the levels of light intercepting- and protective pigments were enhanced,
the P n was severely decreased and oxidative stress was stimulated, at least by
judging from AsA and GSH depletion and TBARS oveiproduction (Fig. 1). Being
unable to cope with the temperature-dependent oxidative stress with the sole "first
move", i.e. at the expense of non-enzymic antioxidants (see above), to sustain or
even to increase the activities of H2O2-scavenging enzymes and of DHAR (Fig. 1)
could have become mandatory for sorghum plants grown at 17±0.4 °C. The above
interpretive framework does not apply to the results obtained for GR, MDHAR and
SOD (Fig. 1). The first enzyme tended to reflect the effects of non-optimal growth
temperatures on its substrate, namely GSSG, thus being unaffected or increased at
37±0.1 °C and clearly depressed at 17±0.4 °C. The temperature-dependent
stimulation of MDHAR activity was dramatic, especially at 37±0.1 °C and in cv.
ICSV 112, and deserves further investigation, particularly in the light of the
accumulating evidence pointing to the relevance of the monodehydroascorbate
radical as the main oxidation product arising from the complex AsA redox
chemistry within the plant cell (SANO & ASADA 1994). The effects of non-optimal
temperatures on the total SOD activity were mirrored by those on Sod transcripts
abundance, albeit in cv. Aralba Sod expression was much more reduced at 37±0.1
°Cthanatl7±0.4°C(Fig. 2).
_I 100
Aralba
- "IFBT" " -1
_
Hi
80
1
60 H
Q.
u> 4 0
X>
20
0
ICSV 1 12
- 30
-_
_
-
i
40
T
I
3
(£!
•f
20 13
s
o
CD
10 £
I
27 37
17 27
Growth temperature, °C
0
37
Fig. 2. The effects of different growth temperature on the expression of Superoxide
dismutase in two sorghum cultivars. Inner panel: typical Northern hybridization signals obtained by
using the heterologous cDNA probe Sod2.1 from maize (see text for details); each lane contained 8
ug of total mRNA. Outer panel: total enzyme activity.
Although no attempt was made in the present work to discriminate among
the different Sod genes, the results in Fig. 2 confirm the high cellular turnover of
the enzyme (SCANDALIOS 1990). In the cv. Aralba the differences among the
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profiles of enzyme activity and of mRNA population suggest that both
transcriptional and post-translational regulation intervene in the response of the
Sod gene(s) to non-optimal growth temperatures. Southern analysis of sorghum
and maize genomic DNA (Fig. 3) suggests a certain degree of similarity in the
organization of the Sod genes in the two C4 Gramineae. However, the
hybridization signal with sorghum genomic DNA was more intense than that with
maize, suggesting the presence of a higher number of SOD-coding sequences in the
SORGHUMMAIZE
El Hill PI EV Bl El HID PI EV Bl
Fig. 3. Typical southern blotting patterns of genomic DNA from sorghum [Sorghum
bicolor (L.) Moensch cv. Aralba] and maize (Zea mays L. cv. Samantha) leaves probed with the
Sod2.7 cDNA probe (see figure 2). Acronyms for restriction endonucleases are: Bl, Bam HI; El,
Eco RI; EV, Eco RV; Hill, Hind III; PI, Pst I.
former species. Differences among cultivars in their response patterns to nonoptimal temperatures were scanty, SOD expression being the exception (Fig. 1 and
2). This could imply that, because of the mildness of the temperatre treatments, the
compensatory ability of both cv. Aralba and cv. ICSV 112 was never exceeded.
However, it should be noted that at suboptimal temperature, and despite of its
origin and of its intrinsically lower antioxidant potential (see Table 1), cv. ICSV
112 performed even slightly better than cv. Aralba in terms of P n , lipid
peroxidation, GSH level and total SOD activity. Also, the depressive effects of
supraoptimal temperature on ROS-scavenging enzymes were less intense in cv.
ICSV 112 than in cv. Aralba. Moreover, unless in cv. Aralba, no temperaturedriven downregulation of the Sod gene(s) was observed in the cv. ICSV 112
leaves. It could be therefore suggested that, at least in S. bicolor and in response to
disthermia, the ability to control oxidative processes and to sustain endogenous
defense systems is more important than the intrinsic, constitutive antioxidant level.
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Acknowledgements
We gratefully acknowledge Dr. F. BlDINGER (ICRISAT) for kindly providing the seeds of
cv. ICSV 112 and Dr. C. PERANI for photographs. Work supported by the National Research
Council of Italy, Special Project RAISA.
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