J.

Agronomy & Crop Science (2011) ISSN 0931-2250

DROUGHT STRESS

Fulvic Acid Application Improves the Maize Performance

under Well-watered and Drought Conditions
S. A. Anjum1, L. Wang1, M. Farooq2, L. Xue1 & S. Ali3
1 College of Agronomy and Biotechnology, Southwest University, Chongqing, China
2 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan
3 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

Keywords Abstract
antioxidant enzymes; drought; fulvic acid; gas
exchange; maize Water deficit is perhaps the most severe threat to sustainable crop production
in the conditions of changing climate. Researchers are striving hard to develop
Correspondence resistance against water deficit in crop plants to ensure food security for the
M. Farooq coming generations. This study was conducted to establish the role of fulvic
Department of Agronomy,
acid (FA) application in improving the performance of hybrid maize (Zea mays
University of Agriculture,
Faisalabad, Pakistan
L.) under drought. Maize plants were grown under normal conditions till tas-
Tel.: +92 41 9200161-9/2931 selling and were then subjected to drought by cessation of water followed by
Fax: +92 41 9200605 foliar application of FA (1.5 mg l)1). Drought stress disrupted the photosyn-
E-mail: wanglc2003@163.com thetic pigments and reduced the gas exchange leading to reduction in plant
growth and productivity. Nonetheless, exogenous FA application substantially
L. C. Wang ameliorated the adversities of drought by sustaining the chlorophyll contents
College of Agronomy and Biotechnology,
and gas exchange possibly by enhanced levels of antioxidant enzyme (superox-
Southwest University,
Chongqing 400716, China
ide dismutase (SOD), peroxidase (POD), and catalase (CAT)) activities and
Tel.: +86 23 68250285 proline. These beneficial effects yielded in terms of plant growth and allometry,
Fax: +86 23 68250764 and grain yield. It is interesting to note that FA application also improved the
E-mail: wanglc2003@163.com crop performance under well-watered conditions. Hence, FA may be applied to
improve the crop performance under drought and well-watered conditions.
Accepted June 20, 2011

doi:10.1111/j.1439-037X.2011.00483.x

processes of accumulation, transport and distribution of

Introduction
To feed the ever-increasing population is the biggest chal-
lenge for the people engaged in research in plant sciences;
nonetheless, this confront is becoming more severe in
days to come owing to climate change. It has been esti-
mated that agricultural production has been restricted in
about one-third of the worlds arable land owing to cycli-
cal or unpredictable droughts (Chaves and Oliveira 2004).
The effects of drought on plants have many facets; the
roots are the first to respond water stress and send chem-
ical signals through the stem to leaves, where stomatal
conductance is reduced (Schachtman and Goodger 2008).
Stomatal closure deprives the leaves of carbon dioxide,
and photosynthetic carbon assimilation is decreased in
favour of photorespiration. The non-stomatal mecha-
nisms include changes in chlorophyll synthesis, functional
and structural changes in chloroplasts and disturbances in
assimilates (Farooq et al. 2009a,b). Drought can also lead
to pigment degradation (Hendry et al. 1987), thus causing
irreversible damage to the photosynthetic apparatus.
Water stress distresses the water relations of plants at cel-
lular, tissue and organ levels (Farooq et al. 2009a,b). A
decrease in the relative leaf water content (RLWC) in
response to drought stress has been noted in a wide vari-
ety of crop plants (Farooq et al. 2009a). Osmotic adjust-
ment is an important physiological mechanism by which
plants synthesize and accumulate solutes that function as
osmolytes in cells in response to water deficit (Seki et al.
2007), maintaining the structure of cellular components
and protecting cellular functions (Bartels and Sunkar
2005).
Drought induces oxidative stress in plants by genera-
tion of reactive oxygen species (ROS) (Farooq et al.
2009a). The ROS such as O2), H2O2 and OH radicals

2011 Blackwell Verlag GmbH, 197 (2011) 409417 409

Anjum et al.
can directly attack membrane lipids and increase lipid
peroxidation, inactivate metabolic enzymes and damage
the nucleic acids and ultimately leads to cell death (Mit-
tler 2002). The content of malondialdehyde (MDA) has
been considered an indicator of oxidative damage (Mller
et al. 2007). In fact, MDA is one of the ultimate products
of lipid peroxidation caused by free radicals. Plants have
developed efficient enzymatic antioxidant systems to cope
with drought stress and to avoid oxidative damage (Hor-
vath et al. 2007). The enzymatic components may directly
scavenge ROS or may act by producing a non-enzymatic
antioxidant. Maintaining a higher level of antioxidative
enzyme activities may contribute to drought tolerance by
increasing the capacity against oxidative damage (Sharma
and Dubey 2005, Farooq et al. 2009d,e).
Maize (Zea mays L.), as a main food and forage crop, is
grown all over the world under a wide range of climates.
To improve the agricultural productivity within the water-
limited areas, it is imperative to induce tolerance against
drought stress. Various agronomic and physiological prac-
tices are being applied for this purpose. One such
approach is the exogenous application of growth-regulat-
ing substances to increase the crop ability to withstand
stressful environments (Farooq et al. 2008, 2009b,c,d,e).
Humic substances (fulvic acid and humic acid) have
been identified to regulate the plant growth under well-
watered and drought conditions (Nardi et al. 2002). In
many systems, humic substances behave similar to auxins,
but until recently, it has not been confirmed either they
contain auxin-like substances or not (Nardi et al. 1996,
2002).
Although some researchers have suggested its role in
improving plant water relations (Xudan 1986), and resis-
tance against abiotic stresses (Cimrin and Yilmaz 2005,
Yldrm 2007), the physiological mechanism of fulvic
acid (FA)induced growth promotion and resistance
against abiotic stresses is rarely known. It is hypothesized
that FA application improves the maize performance
under drought by modulating antioxidant system, gas
exchange and maintenance of tissue water status.
Material and methods
This experiment was carried out during summer 2009 in
net house at Department of Agronomy and Biotechnol-
ogy, Southwest University, Chongqing, China. The experi-
mental area lies between latitudes 29 49 32 N,
longitudes 106 26 02 E and altitude 220 m.
Plant material and growth conditions
The seeds of maize (Zea mays L. cv. dong-dan No. 60, a
local hybrid variety) were obtained from Liaoning Dongya
410
Seed Company Ltd, Liaoning, China. Seeds were first sown
in nursery trays on 16 April 2009. Two-week (14 days)-
old seedlings were transplanted into plastic pots (34 cm in
diameter, 24 cm in depth) filled with sandy loam soil con-
taining organic matter 16.83 g kg)1, total nitrogen
2.04 g kg)1, total phosphorus 3.83 g kg)1, total potassium
10.33 g kg)1, alkali-hydronitrogen content 90.77 mg kg)1,
available phosphorus 32.69 mg kg)1, readily available
potassium 56.47 mg kg)1 and pH 6.47. Total weight of
each pot was 14 kg after filling with soil and organic mat-
ter. Two maize seedlings were transplanted per pot; the
pots were arranged in a completely randomized design
(CRD) and received equal amount of water until imposi-
tion of stress treatments. All the pots were applied with
fertilizer at the rate of 15 g pot)1 (5 g at sowing, 5 g
24 days after transplanting (DAP), 5 g after 45 DAP)
using NPK compound fertilizer with 15 %, 5 % and 5 %
N, P2O5 and K2O, respectively. Data on temperature and
relative humidity were recorded using thermocouples and
hygrometers (Vaissala HMP35, Helsinky, Finland). The
average night and day net house temperature was
24.77 0.29 C and 33.11 0.40 C, respectively, whereas
average night and day net house relative humidity was
70.09 1.43 % and 47.83 1.77 %, stress.
Moisture stress and fulvic acid (FA) application
The plants were grown with normal water supply till
tasselling and then were divided into four sets (i) set at
75 % soil field capacity (well-watered) receiving distilled
water (W), (ii) set at 75 % soil field capacity (well-
watered) receiving FA application (WFA), (iii) set at
35 % soil field capacity (drought) receiving distilled
water (D) and (iv) set at 35 % soil field capacity
(drought) receiving FA application (DFA). Total volume
of spray, water (for control treatment) and FA was
25 ml per plant. There were 20 pots per treatment and
three replications of each experimental unit. Moisture
treatments were monitored everyday by TRIME-EZ/-IT
(IMKO Micromodultechnik GmbH, Germany). The pots
were weighed daily to maintain the desired soil water
levels by adding appropriate volumes of water. Fulvic
acid (FA) (Xinjiang Huitong Handilong Humic Acid
Liability Co., Ltd, Xinjiang 839001, China) was applied
1.5 mg l)1 as foliar spray 6 days after the imposition of
moisture stress.
Observations
Gas exchange parameters were measured 12 days after FA
application. The maize plants were sampled (3rd leaf
from top) at 7, 12 and 17 days after FA application
to assess the photosynthetic pigments, protein, RLWC,
2011 Blackwell Verlag GmbH, 197 (2011) 409417

proline, MDA and antioxidant enzyme activities. After
washing, the leaves were frozen in liquid nitrogen and
stored at )80 C until biochemical analysis. The remain-
ing plants were harvested on 14 August 2009, i.e.
118 days after planting for plant biomass components
and yield-related traits.
Leaf gas exchange
Leaf gas exchange attributes were measured with a portable
infrared gas exchange analyzerbased photosynthesis sys-
tem (Li-6400; Li-Cor, Lincoln, NE, USA) during 8:30
10:15 am. Fifteen leaves were selected for each treatment
with the following adjustments: molar flow of air per unit
leaf area was 407.62 mmol m)2 s)1, water vapour pressure
into leaf chamber was 3.4 mbar, photosynthetically active
radiation at leaf surface was up to 1201 lmol m)2 s)1,
temperature of leaf ranged from 37.03 to 41.67 C, ambi-
ent temperature was 36.3940.98 C, ambient CO2 con-
centration was 399.3 mol mol)1 and relative humidity
(RH) was 52.32 %. Water-use efficiency (WUE) was calcu-
lated as ratio between net photosynthesis (A) and transpi-
ration rate (E). Intrinsic water-use efficiency (WUEi) was
calculated as ratio between net photosynthesis (A) and sto-
matal conductance (gs).
Pigments
Chlorophyll a (Chl a), chlorophyll b (Chl b) and chloro-
phyll a + b (Chl a + b) were determined following the
method of Arnon (1949). Leaf sample (0.1 g) was grin-
ded and putted into 15-ml centrifuge tube, along with
10 ml of miscible liquids by 95.5 % acetone and abso-
lute ethyl alcohol in 1 : 1 ratio and then covered with
black plastic bag and kept at dark place until the sample
changed into white. The Chl a, Chl b and chl a + b
concentrations were measured using a UV-visible spec-
trophotometer, and absorbance was measured at 645,
652 and 663 nm.
Proline
Proline content of leaves was measured following the
method of Bates et al. (1973). Leaf samples (0.5 g) were
homogenized with 5 ml sulphosalicylic acid (3 % w/v)
and centrifuged at 4000 g for 10 min, 2 ml of filtrate was
mixed with 2 ml acid ninhydrin and 2 ml of glacial acetic
acid in a test tube. The mixture was placed in a water
bath for 30 min at 98 C and then allowed to cool at
room temperature. The reaction mixture was extracted
with 5 ml toluene, and the chromophore containing tolu-
ene was aspirated, cooled to room temperature and
absorbance was measured at 520 nm.
2011 Blackwell Verlag GmbH, 197 (2011) 409417
Fulvic Acid Improves the Maize Performance
Malondialdehyde
The level of membrane damage was estimated by deter-
mining the activity of Malondialdehyde (MDA) following
De Vos et al. (1991). Leaf samples (0.5 g) were homoge-
nized in 5 ml 5 % trichloroacetic acid. The homogenate
was centrifuged at 4000 g for 10 min at 25 C, and 3 ml
2-thiobarbituric acid in 20 % trichloroacetic acid was
added to 2-ml aliquot of the supernatant. The mixture
was heated at 98 C for 10 min and cooled rapidly in an
ice bath. After centrifugation at 4000 g for 10 min, the
absorbance was recorded at 532 nm.
Relative leaf water contents
To determine Relative leaf water contents (RLWC), fresh
leaves (1.5 g) were weighed to get fresh weight (Wf).
Later, these leaves were floated on water for 18 h, and
saturated weight (WS) was determined. These leaves were
oven dried for 72 h at 70 C to have dry weight (Wd).
RLWC were calculated as follows:
RLWC = (Wf Wd =Ws Wd  100:
Soluble protein and antioxidant enzymes assay
The protein contents were determined using bovine serum
albumin (BSA) as a standard, according to the method of
Bradford (1976). Frozen leaf samples (0.5 g) were ground
in liquid nitrogen with a mortar and pestle and homoge-
nized in 10 ml 50 mm sodium phosphate buffer (pH 7.0)
containing 1 mm EDTA-Na2 and 2 % (w/v) polyvinylpyrr-
olidine-40 (PVP-40). The homogenate was centrifuged at
11 000 g for 15 min at 4 C. The supernatant was col-
lected and used for antioxidant enzyme activity analysis.
One millilitre of Bradford solution was added to 100 ll
crude extract and absorbance recorded at 595 nm for the
estimation of total protein content. The protein concen-
tration was calculated from a BSA standard curve.
The superoxide dismutase (SOD) activity was assayed by
ready kits provided by Nanjing Jiancheng Bioengineering
Institute, China. Assay for SOD was carried out following
the protocol mentioned with the detection kit. One unit of
SOD activity was defined as the amount of enzyme required
for 1 mg tissue proteins in 1 ml of a reaction mixture SOD
inhibition rates to 50 % as monitored at 550 nm.
The peroxidase (POD) activity was determined by fol-
lowing the method of Upadhyaya et al. (1985) in a 3.9-ml
reaction mixture containing 50 mm phosphate buffer (pH
7.0), 28 ll guaiacol, 100 ll enzyme extract and 19 ll
H2O2. At 420 nm, at least 2 min per 30 s read the
absorbance and used the absorbance change 0.01 as a
POD activity.
411
Anjum et al.

The catalase (CAT) activity was determined by assay
kits provided by Nanjing Jiancheng Bioengineering
Institute, China. One unit of CAT activity was defined as
1 mg tissue proteins consumed 1lmol H2O2 at
405 nm s)1. CAT activity was expressed as enzyme units
per mg of protein.
Growth and yield components
Leaf area of maize plants was measured with leaf area
meter (Li-Cor 3100, Li-Cor, Lincoln, NE, USA) 15 days
after FA application. At harvest, 30 plants (10 plants from
each replicate) representing each treatment were sampled
randomly and quantified to determine the growth, yield
and yield components.
Statistical analysis
Data set was statistically analysed by analysis of variance
(anova) technique using computer software spss 16.0,
and NewmanKeuls test was applied for mean separation.
Graphical presentation of the data was made by Microsoft
Excel.
Results
Growth and yield
Drought stress substantially reduced the maize growth,
yield and yield-related components (Table 1). Nonethe-
less, FA application substantially improved the leaf area,
number of leaves/plant, cob length and fresh and dry
weight of maize plants under both drought and well-
watered conditions (Table 1). However, FA did not
improve the plant height under both drought and well-
watered conditions (Table 1). Likewise, FA treatment sub-
stantially improved the yield and yield-related traits
including kernel rows/cob, kernel number/row, 100-kernel
weight, grain yield, biological yield and harvest index
under stress and normal conditions (Table 2). However,
FA did not improve the cob length (Table 1) and kernel
numbers per row (Table 2) under well-watered conditions
and 100-kernel weight under both drought and well-
watered conditions (Table 2).
Leaf gas exchange
Drought stress led to considerable decline in gas exchange
attributes in maize plants under drought (Table 3). Water
stress led to partial stomatal closure and resulted in low-
ered gas exchange attributes. However, exogenous appli-
cation of FA significantly increased the CO2 assimilation
rate in both the stressed and non-stressed maize plants.
Furthermore, exogenous application of FA was effective
in improving the photosynthesis, transpiration rate,
water-use efficiency and intrinsic water-use efficiency
under both stressed and well-watered conditions
(Table 3). However, FA did not improve the stomatal
conductance under both drought and well-watered condi-
tions (Table 3).

Table 1 Influence of fulvic acid (FA) application on agronomic traits of maize under drought and well-watered conditions

Shoot fresh Shoot dry

Treatments Plant height (cm) Leaf area (cm2) weight/plant (g) weight/plant (g) No. of leaves/plant Cob length (cm)

W 206.62 2.33 ab 238.01 0.99 b 251.86 1.67 b 54.16 2.53 b 13.02 0.18 b 19.71 0.34 a
WFA 214.27 1.49 a 256.04 2.23 a 279.43 2.22 a 63.72 2.60 a 13.26 0.14 a 20.64 0.22 a
D 191.92 2.82 c 199.03 2.50 d 198.20 1.86 c 37.71 2.19 c 10.27 0.20 d 17.63 0.32 c
DFA 202.29 2.64 bc 221.49 3.74 c 221.80 2.31 b 48.86 1.73 b 11.60 0.06 c 18.66 0.35 b

Values in the table are mean SE. Values followed by the same letter within columns do not differ significantly at P < 0.05 according to Newman
Keuls test. W, well-watered; WFA, FA application under well-watered conditions; D, drought stress; DFA, FA application under drought conditions.

Table 2 Influence of fulvic acid (FA) application on yield and yield-related traits of maize under drought and well-watered conditions

Kernel Kernel 100-kernel Biological Grain Harvest

Treatments rows/cob number/row weight (g) Kernels/cob yield/plant (g) yield/plant (g) index (%)

W 14.06 0.07 bc 37.63 0.46 a 30.67 0.62 a 553.96 1.10 b 310.33 4.35 b 176.66 1.27 b 54.49 0.29 b
WFA 14.73 0.06 a 38.50 0.43 a 31.77 1.17 a 607.10 1.87 a 336.31 2.90 a 192.32 1.30 a 55.98 0.07 a
D 13.73 0.07 c 32.30 0.32 b 25.45 0.33 b 386.90 3.06 d 233.67 2.90 d 121.52 1.55 d 49.34 0.70 d
DFA 14.33 0.18 b 33.66 0.26 c 27.47 0.31 b 448.06 2.43 c 269.69 4.01 c 144.33 1.76 c 51.58 0.47 c

Values in the table are mean SE. Values followed by the same letter within columns do not differ significantly at P < 0.05 according to Newman
Keuls test. W, well-watered; WFA, FA application under well-watered conditions; D, drought stress; DFA, FA application under drought conditions.

412 2011 Blackwell Verlag GmbH, 197 (2011) 409417

 Fulvic Acid Improves the Maize Performance

Table 3 Impact of fulvic acid (FA) application on gas exchange traits and water-use efficiency of maize under drought and well-watered conditions

Transpiration Stomatal Water-use Intrinsic water-use

Net photosynthesis rate conductance efficiency efficiency Intercellular
Treatments (lmol m)2 s)1) (mmol m)2 s)1) (lmol m)2 s)1) (lmol mmol)1) (lmol mmol)1) CO2 (lmol mol)1)

W 20.58 0.71 b 3.49 0.10 b 0.234 0.01 ab 7.80 0.13 b 108.25 2.27 b 274 2.03 b
WFA 23.01 0.32 a 3.92 0.07 a 0.268 0.02 a 9.18 0.38 a 113.26 1.17 a 283 1.76 a
D 15.48 0.80 d 2.58 0.02 d 0.192 0.01 b 5.12 0.19 d 93.87 1.05 d 259 0.88 d
DFA 17.91 0.17 c 2.84 0.07 c 0.217 0.01 ab 7.02 0.16 c 102.14 1.23 c 267 2.18 c

Values in the table are mean SE. Values followed by the same letter within columns do not differ significantly at P < 0.05 according to
NewmanKeuls test. W, well-watered; WFA, FA application under well-watered conditions; D, drought stress; DFA, FA application under drought
conditions.

Chlorophyll contents
W WFA
The biosynthesis of photosynthetic pigments (Chl a, Chl 2.95
(a)
D DFA
b and Chl a + b) was significantly affected by drought 2.85
stress. However, foliar application of FA was found to be

Chl a (mg g1 FW)

2.75
effective in alleviating, partially or completely, the inhibi-
2.65
tory effects of moisture stress on pigment biosynthesis
2.55
(Fig. 1). There was considerable improvement in photo-
synthetic pigments of maize plants owing to foliar FA 2.45

treatment as compared to the untreated plants when 2.35

grown under water stress conditions, and the values were 2.25
only slightly lower than those of plants grown in well- 7 12 17
watered conditions. Under normal conditions, the treat-
2.3 W WFA
ment of plants with FA also enhanced the chlorophyll (b)
D DFA
contents in the maize plants. 2.2
Chl b (mg g1 FW)

2.1

Protein, RLWC, proline and MDA 2

Protein contents were significantly increased from day 7 1.9

onwards, reached its maximum at day 12, then started to 1.8

declined, but the protein content kept at higher levels 1.7
during the period of drought stress compared with well-
1.6
watered control. Nonetheless, application of FA led to
7 12 17
pronounced production of proteins under drought.
RLWC was reduced subsequently under prolonged W WFA
(c)
drought stress but was improved by foliar spray of FA 4.25 D DFA
under water-deficit and well-watered conditions (Fig. 2).
Chl a +b (mg g1 FW)

4.05
Water stress stimulated the proline accumulation in
stressed maize plants. Nonetheless, the FA application fur- 3.85
ther improved the proline contents and reached peak at
3.65
day 12 after FA application and then decreased as the
drought stress progressed. FA application also improved 3.45
the proline accumulation under well-watered conditions
3.25
(Fig. 3). 7 12 17
Membrane stability showed a highly significant
Sampling date (days after stress)
decrease with increasing drought level in maize plants.
The inhibitory effects of water stress on the membrane Fig. 1 Chl a (a), Chl b (b) and Chl a + b (c) contents in maize under
stability of maize plants were partially alleviated by well-watered and drought conditions in response to FA applica-
exogenous application of FA (Fig. 3). The MDA contents tion SE. W, well-watered; WFA, fulvic acid (FA) in well-watered con-
of maize plants increased in response to water stress. ditions; D, drought stress; DFA, fulvic acid (FA) in drought conditions.

2011 Blackwell Verlag GmbH, 197 (2011) 409417 413

Anjum et al.

2.7 W WFA 100 W WFA

(a) (a)
D DFA D DFA
Total protein (mg g1 FW)

2.5
80

Total proline (g g1)

2.3
60
2.1
40
1.9

1.7 20

1.5
0
7 12 17 7 12 17

100 (b) W WFA 26 (b) W WFA

Malondialdehyde contents (gmol g1 FW)

D DFA
Relative leaf water contents (%)

90 D DFA

22
80

70 18

60

14
50

40
10
7 12 17
7 12 17
Sampling date (days after stress)
Sampling date (days after stress)

Fig. 2 Total protein (a) and relative leaf water contents (b) in maize
Fig. 3 Proline (a) and malondialdehyde contents (b) in maize under
under well-watered and drought conditions in response to FA applica-
well-watered and drought conditions in response to FA applica-
tion SE. W, well-watered; WFA, fulvic acid (FA) in well-watered con-
tion SE. W, well-watered; WFA, fulvic acid (FA) in well-watered con-
ditions; D, drought stress; DFA, FA in drought conditions.
ditions; D, drought stress; DFA, FA in drought conditions.

However, treatment with FA exerted a retarding effect on
the MDA contents in water-stressed as well as well-
watered maize plants.
Antioxidant enzymes
Under drought, activities of antioxidant enzymes (SOD
and POD and CAT) were significantly increased.
Exogenously applied, FA caused a further increase in the
activities of antioxidant enzymes under drought and
well-watered conditions. The patterns of SOD and POD
activities were parallel, showing a linear increase in
activity with the progression of drought stress, while CAT
activity decline as the water stress progressed (Fig. 4).
Discussion
Drought is one of the major constrains limiting the crop
performance worldwide. Maize crop was used in this
experiment because of its importance as one of the best
model plants to combine physiological and agronomic
studies. Drought-related reduction in growth, yield and
yield components of maize plants could be ascribed to
stomatal closure in response to low soil water content,
which decreased the intake of CO2 and, as a result, pho-
tosynthesis decreased, whereas foliar application of FA
alleviated the water deficit-induced inhibition in growth
and yield. FA has been found to increase the permeability
of root membranes (Delfine et al. 2005) and hydraulic
conductance (Xudan 1986), thereby increasing the nutri-
ent and water uptake and enabling the plants to perform
well even under the conditions of water deficit.
Drought stress reduced the gas exchange traits possibly
by decrease in leaf expansion (Table 1) and oxidation of
chloroplast lipids (Fig. 1) (Menconi et al. 1995). The
decrease in chlorophyll content under drought stress has
been considered a typical symptom of oxidative stress and
may be the result of pigment photo-oxidation and chlo-
rophyll degradation. The linear decline in chlorophyll
contents with progression of drought stress in our study
is in accordance with Gadallah (1995) who reported that
Chl a, Chl b and Chl a + b content decreased with

414 2011 Blackwell Verlag GmbH, 197 (2011) 409417


Superoxide dismutase (U mg1 protein)
140 (a) W WFA
D DFA
130
120
110
100
90
80
7 12
45 (b) W WFA
D DFA
Peroxidase (g g1 FW min1)
40
35
30
25
20
7 12
(c)
2.6
Catalase (U mg1 protein)
2.4
2.2
2 cate that FA improved the free radical quenching system
1.8
1.6
7 12
Sampling date (days after stress)
Fig. 4 Superoxide dismutase (a), peroxide (b),
ties in maize under well-watered and drought
to FA application SE. W, well-watered; WFA,
watered conditions; D, drought stress; DFA, FA
increasing severity of water stress. The decrease in chloro-
phyll contents under water stress can be attributed to the
sensitivity of pigments to increasing stress, which has
been reported by Younis et al. (2000). FA treatment led
to enhancement in chlorophyll contents, which might
have improved the drought induction against oxidative
damage as reported by Kraus et al. (1995) that higher Chl
content has been associated with the stress tolerance of
plants.
In our study, improved water-use efficiency (WUE)
and intrinsic water-use efficiency (WUEi) ratios with FA
application substantiated that improved water economy
during photosynthesis is important to maize under
drought stress. RLWC related to water uptake by the
roots as well as water loss by transpiration. Many investi-
gations have shown that when leaves are subjected to
2011 Blackwell Verlag GmbH, 197 (2011) 409417
Fulvic Acid Improves the Maize Performance
drought, leaves exhibit large reductions in RLWC and
water potential (Nayyar and Gupta 2006). The improve-
ment in FA-induced RLWC may be attributed to the
decrease in water loss and improved water uptake by
improved root growth and root hydraulic conductance
(Li et al. 2005).
Water deficit increased the leaf proline content, which
might have contributed to osmotic adjustment and
allowed the plant to maintain turgor pressure and adapt
17
to limited water availability. This is conducive with previ-
ous reports on drought-induced proline accumulation
(Tan et al. 2006). FA treatment enhanced the proline
accumulation which in turn protected the maize plants
from adverse impacts of drought. This FA-induced
drought tolerance could be attributed to proline, rapidly
accumulated in the leaves of water-stressed plants.
When the plants suffered from water stress, the defen-
sive system needs to be activated to resist the active oxy-
gen injury. Drought conditions exhibited greater
17 accumulation in MDA contents, a product of lipid perox-
idation, in plants under stress condition (Diego et al.
W WFA
2003). In our study, FA-treated plants considerably low-
D DFA
ered MDA contents which in line with Blum and Ebercon
(1981) who reported that lower MDA contents could be
correlated with stress tolerance. In the present investiga-
tion, the lower values of MDA by application of FA indi-
that offers protection against oxidative stress.
ROS scavenging is important in imparting tolerance
against oxidative injury. Under water stress, maize plants
17
deployed an efficient antioxidative enzyme system simul-
taneously to withstand catastrophic effect of induced oxi-
and catalase (c) activi-
dative stress. The increases in antioxidative enzyme
conditions in response activities may have resulted from excess energy inter-
fulvic acid (FA) in well- cepted by photosynthetic pigments. It has been proved
in drought conditions. that efficient antioxidative characteristics can provide bet-
ter protection against oxidative stress in leaves under
drought stress (Reddy et al. 2004). Efficient destruction of
O2) and H2O2 in plant cells requires the concerted action
of antioxidants. Among antioxidant enzymes, SOD con-
verted the toxic O2) to H2O2 that is further scavenged to
the O2 and H2O by the antioxidant enzymes such as CAT
and POD. The increased SOD activity induced the higher
tolerance to oxidative stress. Maize crop was able to
maintain POD activity for detoxifying ROS in response to
water stress. The transient increase in CAT activity during
initial periods of water stress might protect the maize
plants from oxidative injury. However, the decline in
CAT activity in prolonged stress indicated that the scav-
enging function of CAT was impaired with prolonged
drought stress. These results indicated that the ability of
CAT to quench ROS was maintained during initial stress
but was limited during a prolonged period of water stress,
415
Anjum et al.
as observed in rice root exposed to salt stress (Khan et al.
2002), indicated that POD had a higher capacity than
CAT for the decomposition of H2O2 generated by SOD.
Interestingly, maize performance (in terms of most of
the traits studied) was also improved by FA application
under well-watered conditions. These observations sup-
port the assumption that FA may have auxin-like proper-
ties and roles (Nardi et al. 1996, 2002). Hence, our
hypothesis has been partially proved that FA application
improves the drought resistance in maize under drought.
To sum up, FA application protected the photosyn-
thetic apparatus, through maintenance of a balance
between ROS synthesis and destruction, and improved
the gas exchange rates under drought and well-watered
conditions, thereby leading to growth and yield improve-
ment. Therefore, FA may be applied exogenously to
improve the maize performance under both drought and
well-watered conditions.
Acknowledgements
The present research work was supported by National
Eleventh Five-Year Research and Development Project of
China (2006BAD29B08). The authors are grateful to the
Key Laboratory of Biotechnology and Crop Quality
Improvement of Ministry of Agriculture, China. The
authors say thanks to Dr. San-gen Wang, who provided
some very useful guidelines, instructions and comments
on an earlier draft of this paper.
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