ORIGINAL ARTICLE Physiological characterization of maize tolerance to low dose of aluminum, highlighted by promoted leaf growth Liang Wang 1 • Xian-Wei Fan 1 • Jian-Long Pan 1 • Zhang-Bao Huang 1 • You-Zhi Li 1 Received: 9 March 2015 / Accepted: 17 July 2015 / Published online: 8 August 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Main conclusion Effects of a low aluminum (Al) dose were characterized. The Al supplement inhibited root growth but enhanced leaf growth in maize lines with different Al sensitivities. High levels of Al are phytotoxic especially in acidic soils. The beneficial effects of low Al levels have been reported in some plant species, but not in maize. Maize is relatively more sensitive to Al toxicity than other cereals. Seedlings, at the three leaf stage, of four Chinese maize foundation parent inbred lines with different Al tolerances, were exposed to complete Hoagland’s nutrient solution at pH 4.5 supplemented with 48 lM Al 3? under controlled growth conditions, and then the Al stress (AS) was removed. The leaf and root growth, root cell viability, superoxide dis- mutase (SOD), peroxidase (POD), catalase (CAT), ions (K ? , Ca ?? and Mg ?? ), photosynthetic rate and chloro- phyll, protein and malondialdehyde contents in tissues were assayed. In conclusion, a low Al dose inhibits root growth but enhances leaf growth in maize. The Al-pro- moted leaf growth is likely a result of increased protein synthesis, a lowered Ca ?? level, and the discharge of the growth-inhibitory factors. The Al-promoted leaf growth may be a ‘memory’ effect caused by the earlier AS in maize. Al causes cell wall rupture, and a loss of K ? , Ca ?? and Mg ?? from root cells. CAT is an auxiliary antioxidant enzyme that works selectively with either SOD or POD against AS-related peroxidation, depending on the maize tissue. CAT is a major antioxidant enzyme responsible for root growth, but SOD is important for leaf growth during AS and after its removal. Our results contribute to under- standing how low levels of Al affect maize and Al-resistant mechanisms in maize. Keywords Acidic soils Á Aluminum toxicity Á Aluminum benefit Á Maize Á Root Abbreviations AS Al stress CAT Catalase MDA Malondialdehyde POD Peroxidase RAS Removal of AS ROS Reactive oxygen species SOD Superoxide dismutase SOR Superoxide radicals Introduction Aluminum (Al) is the third most abundant chemical ele- ment in the earth’s crust (Pilon-Smits et al. 2009). It is phytotoxic, especially in acidic soils with a pH in the 4.5–5.0 range (Matsumoto 2000). The acidic soils account for *50 % of the world’s cultivable area (Panda et al. 2009). The phytotoxicity of Al has been studied exten- sively in organs, tissues, and cells (Kochian 1995; Ma L. Wang and X.-W. Fan contributed equally to this work. & You-Zhi Li [email protected]1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning 530004, Guangxi, People’s Republic of China 123 Planta (2015) 242:1391–1403 DOI 10.1007/s00425-015-2376-3
13
Embed
Physiological characterization of maize tolerance to low ... · Physiological characterization of maize tolerance to low dose of aluminum, highlighted by promoted leaf growth Liang
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
ORIGINAL ARTICLE
Physiological characterization of maize tolerance to low doseof aluminum, highlighted by promoted leaf growth
The decreased activity levels of SOD and POD in the
roots and leaves of all of the AS-treated maize lines
(Fig. 2a–d) were not in agreement with the results previ-
ously reported in maize under AS (Boscolo et al. 2003).
Change in CAT activity
The CAT activity levels in the roots of the AS-treated
maize lines started to significantly decrease 24 h after AS,
and then remained almost unchanged during further AS
treatment (Fig. 2e). After RAS, the activity levels signifi-
cantly increased but did not reach the activity levels in the
respective control roots (Fig. 2e). During AS and RAS, the
changes in the CAT activity levels in the leaves of the AS-
treated maize lines (Fig. 2f) were similar to the changes in
SOD activity levels in the leaves of the AS-treated maize
lines (Fig. 2b).
MDA content and SOR production
The MAD contents in the roots of the AS-treated maize
lines started to significantly increase 24 h after AS
(Fig. 3a). After the RAS treatment, the contents in the
roots of the AS-treated maize lines significantly declined
when compared with those of the respective roots at the
72-h time point of AS. However, only the MDA content
in the roots of the AS-treated Z58 line was similar to that
in the corresponding control after RAS treatment
(Fig. 3a).
The MDA contents in the leaves of the AS-treated maize
lines started to significantly increase 48 h after AS
(Fig. 3b), lagging behind the changes in the MDA contents
in the roots of the AS-treated maize lines (Fig. 3a). How-
ever, the MDA contents in the leaves of the 72-h-stressed
maize lines almost recovered after RAS treatment to the
respective control level (Fig. 3b).
The increased MDA contents in the roots and leaves of
the AS-treated maize lines did not support the previous
conclusion that the presence of Al did not cause lipid
peroxidation (Boscolo et al. 2003).
With the increase in MDA contents, SOR production in
the roots and leaves of all the AS-treated maize lines
increased at 24 h, decreased at 48 h, and then increased
again at 72 h after AS (Fig. 3c, d).
0200400600800
10001200140016001800200022002400
0 24 48 72 24 48
Al i
on in
the
root
s (μ
g g-
1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0102030405060708090
100
0 24 48 72 24 48
Al i
on in
the
leav
es (μ
g g-
1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0 24 48 72 24 48
RAS (h)AS (h)
HZ4
C7-2
Y478
Z58
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 24 48 72 24 48
Rel
ativ
e gr
owth
of t
he ro
ots
(%)
H4; Stress C7-2; Stress
Y478; Stress Z58; Stress
1
1.4
1.8
2.2
2.6
3
3.4
0 24 48 72 24 48Rel
ativ
e gr
owth
of t
he le
aves
(%)
H4; Stress C7-2; Stress
Y478; Stress Z58; Stress
AS (h) RAS (h) AS (h) RAS (h)
AS (h) RAS (h) AS (h) RAS (h)
(a)
(e)
(g)
(f)
(h)
(b)
(c)
(d)
Fig. 1 The growth of maize seedlings under AS and after RAS. The
phenotypes of the different maize lines (a–d), the relative growth of
roots (e) and leaves (f), and the Al ion contents in roots (g) and leaves
(h). The values are mean ± standard error (SE) from at least five
individual seedlings
Planta (2015) 242:1391–1403 1395
123
Viability of the root cells
The Evans blue staining indicated that decreased root cell
viability under AS occurred in cells near the root tips at
24 h after AS, and then was found in the cells in the
upper tissues with AS, being more obvious in the roots of
the AS-treated H4 and C7-2 lines (Fig. 4). Notably, a
decreased root cell viability in Z58 line during AS
seemed to be limited to the cells near the root tip zone
(Fig. 4). The decrease in root cell viability in the AS-
treated maize lines could be alleviated by RAS treatment,
especially in the roots of AS-treated Y478 and Z58 lines
(Fig. 4).
The cells in the root tip zones of the AS-treated maize
lines showed plasmolysis and cell wall rupture, and had
concentrated and enlarged nuclei, while the cellular con-
tents leaked (Fig. 4). These symptoms started 48 h after
AS, and were more serious in H4, C7-2 and Y478 lines
than in Z58 line (Fig. 4). Interestingly, the symptoms were
greatly alleviated by 48 h of RAS treatment (Fig. 4).
K, Ca, and Mg ions in the tissues
The K, Ca, and Mg ion contents in the roots and leaves of
all the AS-treated maize lines declined when compared
with the levels in the respective controls (Fig. 5a–f). The
contents of these ions in tissues of the AS-treated maize
lines were significantly enhanced by RAS treatment
(Fig. 5a–f). These results echoed the changes in cell
structure in the root tip zone (Fig. 4).
Protein and chlorophyll contents,
and the photosynthetic rate
The total protein contents were significantly higher in the
roots and leaves of the AS-treated maize lines than in the
roots and leaves of respective control lines (Fig. 6a, b). The
protein content started to significantly increase 24 h after
AS in the roots (Fig. 6a) and 48 h after AS in the leaves
(Fig. 6b). After RAS treatment, the protein contents in the
tissues of the AS-treated maize lines significantly declined
AS (h) RAS (h) AS (h) RAS (h) AS (h) RAS (h)
AS (h) RAS (h) AS (h) RAS (h) AS (h) RAS (h)
0
40
80
120
160
200
240
280
0 24 48 72 24 48
SOD
act
ivity
in th
e ro
ots
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0
50
100
150
200
250
300
350
400
450
0 24 48 72 24 48
POD
act
ivity
in th
e ro
ots
(A 4
70m
in-1
mg-
1pr
otei
n)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0
2
4
6
8
10
12
14
0 24 48 72 24 48
CA
T ac
tivity
in th
e ro
ots
(O
D24
0m
in-1
mg-
1pr
otei
n) H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
08
162432404856647280
0 24 48 72 24 48
SOD
act
ivity
in th
e le
aves
-1
(U m
gpr
otei
n)-1
(U m
gpr
otei
n)
H4 Control H4 StressC7-2 Control C7-2 StressY478 Control Y478 StressZ58 Control Z58 Stress
0
4
8
12
16
20
24
28
0 24 48 72 24 48
CA
Tac
tivity
in th
e le
aves
(O
D24
0m
in-1
mg-
1pr
otei
n) H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
048
12162024283236404448
0 24 48 72 24 48
POD
act
ivity
in th
e le
aves
(
A 470
min
-1m
g-1 pr
otei
n)H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2 Activities of antioxidant enzymes in the maize tissues under
AS and after RAS. The SOD activities in roots (a) and leaves (b), thePOD activities in roots (c) and leaves (d), and the CAT activities in
roots (e) and leaves (f). The values are mean ± SE from at least five
individual seedlings
1396 Planta (2015) 242:1391–1403
123
when compared with protein content levels in corre-
sponding AS-treated maize lines before RAS (Fig. 6a, b).
The chlorophyll contents in the leaves of the AS-treated
Z58 line decreased slightly within 48 h of AS and signif-
icantly 72 h after AS when compared with the chlorophyll
levels in the control line (Fig. 6c). A significant decrease in
the chlorophyll content was found in three AS-treated
maize lines (H4, C7-2 and Y478), starting 24 h or 48 h
after AS depending on the lines. After RAS treatment, the
chlorophyll contents in the leaves of the AS-treated maize
lines reached the levels in the respective controls (Fig. 6c).
The photosynthetic rates in the leaves of all the AS-
treated maize lines started to significantly decrease 24 h
after AS (Fig. 6d). After RAS treatment, the photosynthetic
rates in the leaves of the AS-treated maize lines obviously
increased when compared with the photosynthetic rate
levels in respective maize lines treated by AS for 72 h
(Fig. 6d).
Discussion
Al toxicity in plants occurs in acidic soils (Matsumoto
2000). However, the beneficial effects of low Al doses on
plants in acidic soils may occur in both Al-tolerant plants
and many Al-stimulated plants (Osaki et al. 1997), and is
characterized by growth promotion. The seedlings of Al-
tolerant triticale and alfalfa showed large root regrowth
during AS (Zhang et al. 1999, 2007). Additionally, lower
Al concentrations significantly stimulated the root growth
of Al-tolerant soybean PI 416937 (Du et al. 2010).
All of the maize lines tested in this study showed similar
changes in leaf and root growth rates, root cell viability,
SOD, POD, and CAT activities, of K, Ca and Mg ion
contents, protein contents, chlorophyll and MDA contents,
and photosynthetic rates under AS and after RAS, but the
magnitudes and response time of the changes differed
depending on the maize line, suggesting differences in AS-
1
2
3
4
5
6
7
0 24 48 72 24 48Prod
uctio
n ra
te o
f SO
Rs
in th
e le
aves
(nM
min
-1m
g-1
prot
ein)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
00.5
11.5
22.5
33.5
44.5
5
0 24 48 72 24 48
MD
A in
the
leav
es (μ
M g
-1 F
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
1
2
3
4
5
6
7
8
9
0 24 48 72 24 48Prod
uctio
n ra
te o
f SO
Rs
in th
e ro
ots
( nM
min
-1m
g-1
prot
ein)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 24 48 72 24 48
MD
A in
the
root
s ( μ
M g
- 1FW
) H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
AS (h) RAS (h) AS (h) RAS (h)
AS (h) RAS (h) AS (h) RAS (h)
(a) (c)
(b) (d)
Fig. 3 The MAD contents, and SOR production rate in the maize tissues under AS and after RAS. The MDA contents in roots (a) and leaves (b).The SOR production rates in roots (c) and leaves (d). The values are mean ± SE from at least five individual seedlings
Planta (2015) 242:1391–1403 1397
123
tolerant mechanisms. The increased MAD contents in the
tissues of the AS-treated maize lines (Fig. 3a, b) indirectly
supported the previous conclusion that the Al treatment
could trigger lipid peroxidation in the sensitive maize lines
(Giannakoula et al. 2008), but they did not corroborate the
view that Al treatments did not induce lipid peroxidation in
both sensitive and tolerant maize lines (Boscolo et al.
2003). Our results showed that in maize, AS can cause
decreases in the Ca and Mg ion contents in Al-tolerant
Y478 and Z58 lines and Al-sensitive H4 and C7-2 lines
(Fig. 5c–f), which supported previous conclusions (Gian-
nakoula et al. 2008; Mihailovic et al. 2008). However, AS
caused a significant decrease in K ion contents (Fig. 5a, b),
which was in contrast to the previous conclusion (Yu et al.
2011). The discrepancies in the above-mentioned results
likely resulted from the differences among the maize lines
and/or partly from the experimental conditions, such as
applied Al3? concentrations and/or stress duration. During
C7-2
Z58
Y478
C7-2
H4
Z58
Y478
C7-2
Z58
Y478
C7-2
Z58
Y478
C7-2
Z58
Y478
Not examined
Not examined
Not examined
Not examined
0 24 48 72 24 48
AS (h) RAS (h)
H4H4 H4 H4
H4 H4H4 H4 H4 H4
C7-2C7-2 C7-2 C7-2 C7-2C7-2
Y478Y478 Y478 Y478 Y478Y478
Z58Z58 Z58 Z58 Z58Z58
Fig. 4 Viability and structure of the fresh root cells under AS and
after RAS. The root cell viability was assayed by using Evans blue
staining method. For observation of cell structure, the tissues of the
root tip zone were sectioned lengthwise by using a paraffin slicing
machine and then photographed by light microscopy. The detailed
procedures were indicated in ‘‘Materials and methods’’
1398 Planta (2015) 242:1391–1403
123
AS or after RAS, changes in the contents of Ca, Mg and K
ions (Fig. 5) were closely related to changes in the cell
structure in the root tip zone (Fig. 4), suggesting that low
external Al concentrations can also lead to the loss of Ca,
Mg, and K ions by disrupting the cell’s integrity. Addi-
tionally, the decreased ion contents in the roots of the AS-
treated maize lines may be partially ascribed to impaired
root uptake capacity during AS.
Considering the promotion of leaf growth (Fig. 1a–c)
during AS as well as the recovery of AS-damaged cell
walls in the root tip zone after RAS (Fig. 4) and other
parameters of AS-treated maize lines after RAS treatment,
we conclude that low doses of Al only decrease root
growth rate and that the AS-caused inhibition of root
growth of maize can be alleviated by appropriate RAS
treatments.
For AS-treated maize lines, changes in the chlorophyll
contents (Fig. 6c) did not correspond with changes in the
photosynthetic rates (Fig. 6d), suggesting that the differ-
ences among photosynthetic rates in maize lines under AS
result from differences in photosystems rather than
chlorophyll contents. This reasoning partly confirms a
previous finding that AS led to a severe decrease in activity
of photosystem 2 activity (Mihailovic et al. 2008).
For an in-depth analysis of the correlation among the
parameters, we conducted a multiple factor correlation
analysis of the data resulting from AS and RAS treatments
(Tables 1, 2).
SODs together with PODs form the first line of
antioxidant defense against ROS (Ito-kuwa et al. 1999;
Veljovic-Jovanovic et al. 2006). In the SOD-POD system,
SODs first degrade O2-1 into O2 and H2O2, and the latter is
then degraded by POD into H2O and O2 (Boscolo et al.
2003; Wang et al. 2013). CAT scavenges photorespiratory
H2O2 by a catalytic reaction of 2H2O2 ? O2 ? 2H2O
(Willekens et al. 1997). As expected, there was a positive
correlation between SOD and POD activities in the roots
(Table 1) and leaves (Table 2) of AS-treated maize lines.
Interestingly, the CAT activity showed a positive correla-
tion with the SOD activity in the roots of the AS-treated
maize lines (Table 1) but showed a negative correlation
with the POD activity in the leaves of the AS-treated maize
lines (Table 2). This suggests that the roots of the AS-
treated maize lines require more antioxidant enzymes to
0
1
2
3
4
5
6
7
8
0 24 48 72 24 48
K io
n in
the
root
s (m
g g-1
-1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0
2
4
6
8
10
12
14
16
18
0 24 48 72 24 48
K io
n in
the
leav
es (m
g g
DW
)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0
1
2
3
4
5
6
7
8
0 24 48 72 24 48
Ca
ion
in th
e ro
ots
(mg
g-1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
0123456789
1011121314
0 24 48 72 24 48Ca
ion
in t
he le
aves
(mg
g-1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
00.10.20.30.40.50.60.70.80.9
1
0 24 48 72 24 48
Mg
ion
in th
e ro
ots
(mg
g-1D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
00.20.40.60.8
11.21.41.61.8
2
0 24 48 72 24 48Mg
ion
in th
e le
aves
( mg
g- 1 D
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
AS (h) RAS (h) AS (h) RAS (h) AS (h) RAS (h)
AS (h) RAS (h) AS (h) RAS (h) AS (h) RAS (h)
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 5 The contents of K (a, b), Ca (c, d) and Mg ions (e, f) in maize roots (a, c, e) and leaves (b, d, f) under AS and after RAS. The values are
mean ± SE from at least five individual seedlings
Planta (2015) 242:1391–1403 1399
123
cope with AS-triggered peroxidation relative to the AS-
treated leaves. This appears reasonable because SOR pro-
duction was greater in the AS-treated roots than in the AS-
treated leaves (Fig. 3c, d). Thus, CAT is likely an auxiliary
antioxidant enzyme that selectively cooperates with either
SOD or POD to play a role in antioxidation under AS and
after RAS, depending on maize tissues.
The CAT activity positively correlated with root growth
rate (Table 1), while the SOD activity showed a positive
correlation with the leaf growth rate (Table 2). This
456789
10111213141516
0 24 48 72 24 48
Prot
ein
in th
e le
aves
(mg
g- 1 F
W)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
600650700750800850900950
10001050110011501200
0 24 48 72 24 48Chl
orop
hyll
in th
e le
aves
(mg
g-1
FW)
H4 Control H4 StressC7-2 Control C7-2 StressY478 Control Y478 StressZ58 Control Z58 Stress
0
4
8
12
16
20
24
28
0 24 48 72 24 48Ph
otos
ynth
etic
rate
in th
e le
aves
(μM
CO
2m
-2s-
1 )
H4 Control H4 StressC7-2 Control C7-2 StressY478 Control Y478 StressZ58 Control Z58 Stress
AS (h) RAS (h) AS (h) RAS (h)
AS (h) RAS (h) AS (h) RAS (h)
0
0.5
1
1.5
2
2.5
3
3.5
4
0 24 48 72 24 48
Prot
ein
in th
e ro
ots
(mg
g-1
FW)
H4; Control H4; StressC7-2; Control C7-2; StressY478; Control Y478; StressZ58; Control Z58; Stress
(a) (b)
(c) (d)
Fig. 6 The protein content in
maize roots (a) and leaves (b),and chlorophyll contents (c) andphotosynthetic rate (d) in maize
leaves under AS and after RAS.
The values are mean ± SE from
at least five individual seedlings
Table 1 Correlation among affected parameters in the roots under AS and after RAS
Parameters Al Growth SOD POD CAT SOR MDA Protein K Ca Mg