Effects of Melatonin on Growth，Photosynthetic Characteristics and Antioxidant System in Seedling of Wheat under Drought Stress

Abstract

Abstract：The objective of the study is to reveal the effects of melatonin(MEL)on wheat seedlings
growth under drought stress．A winter wheat(Triticum aestivum L．)cultivar，Xinong 9871(drought
sensitive)，was selected as the experimental material and was planted in pot under soil conditions．Melatonin(100μmol·L-1)was applied to the soil. The effects of MEL on wheat seedling growth，the photosynthetic characteristics and active oxygen metabolism were investigated． Results showed that drought stress inhibited the seedling growth，decreased the chlorophyll content，stomatal conductance，net photosynthetic rate and maximum PSⅡ quantum yield；resulted in H2O2 and MDA accumulation；and enhaneed the activity of antioxidant enzymes and antioxidant content．Under drought stress condition，MEL application increased the biomass and root—shoot ratio；maintained a higher level in RWC，ch1orophy11 content，net photosynthetic rate and the photochemical efficiency；increased the activity of CAT，APX，POD and AsA ，GSH content；and reduced the H2 02 and M DA accumulation．After rewatering，the wheat seedlings with MEL application recovered growth quickly．This study indicated that MEL application increased the root/shoot ratio under drought stress；promoted the water absorption；improved leaf water status；enhanced antioxidant capacity；and alleviated the active oxygen damage．Meiatonin—treated plants showed a well water status and slight oxidative damage，which was contributed to maintain higher photosynthetic rate，SO as to improve the wheat drought tolerance．

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Melatoninincreasedmaize(ZeamaysL.)
seedlingdroughttolerancebyalleviating
drought-inducedphotosyntheticinhibitionand
oxidativedamage
ArticleinActaPhysiologiaePlantarum·February2016
ImpactFactor:1.58·DOI:10.1007/s11738-015-2045-y
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NorthwestA&FUniversity
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ORIGINAL ARTICLE
Melatonin increased maize (Zea mays L.) seedling drought
tolerance by alleviating drought-induced photosynthetic inhibition
and oxidative damage
Jun Ye
1
•Shiwen Wang
1,2
•Xiping Deng
1,2
•Lina Yin
1,2
•Binglin Xiong
1
•
Xinyue Wang
1
Received: 8 June 2015 / Revised: 6 December 2015 / Accepted: 9 December 2015
ÓFranciszek Go
´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako
´w 2016
Abstract The effect of melatonin application on
enhancing plant stress tolerance is already known, but the
specifics of its performance and its underlying mechanism
are still poorly understood. The influences of foliar-
sprayed melatonin (100 lmol/L) on maize (Zea mays L.)
seedlings growth during drought stress were investigated
in this study. The growth of seedlings was not affected by
melatonin application under normal conditions. After 8
days of drought stress, growth was significantly inhibited,
but this inhibition was alleviated by foliar-spraying with
melatonin. After rehydration, melatonin-treated plants
recovered more quickly than untreated plants. Further
investigation showed that, under drought condition,
melatonin-treated plants showed higher photosynthetic
rates, stomatal conductances and transpiration rates than
those untreated plants. Compared with untreated plants,
the melatonin-treated plants exhibited low osmotic
potential under drought stress, which contributed to the
maintenance of high turgor potential and relative water
content. Drought stress induced the accumulation of
hydrogen peroxide and malondialdehyde, but the
accumulation was decreased by melatonin application.
Also, both enzymatic and nonenzymatic antioxidant
activity were enhanced by melatonin application under
drought stress. These results imply that the effects of
melatonin on enhancing drought tolerance can be ascribed
to the alleviation of drought-induced photosynthetic
inhibition, improvement in plant water status, and miti-
gation of drought-induced oxidative damage. The results
suggest that melatonin could be considered as a potential
plant growth regulator for the improvement of crop
drought tolerance in crop production.
Keywords Antioxidant activity Drought tolerance 
Melatonin Photosynthesis Oxidative damage Water
status
Abbreviations
Fv/Fm Maximal quantum yield of PSII photochemistry
UPSII Effective PSII quantum yield
NPQ Non-photochemical quenching coefficient
ETR Electron transport rate
RWC Relative water content
FW Fresh weight
TW Turgid weight
DW Dry weight
H
2
O
2
Hydrogen peroxide
MDA Malondialdehyde
SOD Superoxide dismutase
CAT Catalase
APX Ascorbate peroxidase
POD Peroxidase
AsA Ascorbic acid
DPPH 1,1-Diphenyl-2-picryl-hydrazyl
ROS Reactive oxygen species
Communicated by A. Gniazdowska-Piekarska.
&Shiwen Wang
shiwenwang@nwsuaf.edu.cn
1
State Key Laboratory of Soil Erosion and Dryland Farming
on the Loess Plateau, Institute of Soil and Water
Conservation, Chinese Academy of Science/Northwest A&F
University, Xinong Road No. 26, Yangling 712100, Shaanxi,
China
2
University of Chinese Academy of Sciences, Beijing 100049,
People’s Republic of China
123
Acta Physiol Plant (2016) 38:48
DOI 10.1007/s11738-015-2045-y

Introduction
Drought stress adversely influences crop growth and pro-
ductivity worldwide (Lobell et al. 2014). To improve
agricultural productivity, it is imperative that we enhance
crop drought tolerance by various approaches. Exogenous
application of plant growth regulators (such as osmopro-
tectants, antioxidant compounds and growth promoters)
has been considered as an efficient way to enhance plant
drought tolerance in crop production (Singh and Usha
2003). Therefore, finding new plant growth regulators that
improve crop drought tolerance is an effective approach to
improving crop production. Melatonin (N-acetyl-5-meth-
oxytryptamine) has widely existed in living organisms
(Tan et al. 2012). It is also widely found in a wide range of
concentrations in plants (Paredes et al. 2009; Posmyk and
Janas 2009; Arnao 2014). Melatonin has been reported to
play critical functions in regulating plant growth and
development, including such processes as vegetative
growth promotion, seed germination, rooting and flowering
(Arnao and Herna
´ndez-Ruiz 2014; Hardeland 2015).
Melatonin has also been observed to improve tolerance of
multiple stresses, including drought, heavy metals, salinity,
ultraviolet radiation, chilling, heat, pathogens, and herbicides
(Park 2011; Janas and Posmyk 2013; Arnao and Herna
´ndez-
Ruiz 2014,2015; Wei et al. 2014; Chan and Shi 2015; Reiter
et al. 2015). Melatonin is a well-documented antioxidant in
both animals and plants (Zhang and Zhang 2014). A com-
monly proposed explanation for melatonin’s beneficial effect
on plant stress tolerance is that it enhances plant antioxidant
ability (Arnao and Herna
´ndez-Ruiz 2015; Zhang et al. 2015).
Exogenous application of melatonin has also been found to
improve plant drought tolerance. Zhang et al. (2013)showed
that melatonin-treated cucumber (Cucumis sativus L.) plants
had higher rates of seed-germination and root growth when
exposed to drought stress. Melatonin also ameliorated drought
stress in grape (Vitis vinifera) cuttings (Meng et al. 2014).
Meanwhile, melatonin application delayed drought-induced
leaf senescence in apple (Malusdomesticus Bokh.) trees under
long-term drought stress (Wang et al. 2013).
Although multiple studies have shown that melatonin
application can improve drought tolerance, its specific
performance and the underlying mechanism of melatonin’s
effect on crop drought tolerance are poorly understood.
Firstly, the performance of melatonin on plant drought
tolerance has been investigated in only a few plant species,
and only a quite small number of these studies have
focused on highly important crops. Secondly, these studies
have typically administered melatonin by either putting it
into the soil or adding it into a nutrient solution, both of
which are inconvenient in field crop production. Third,
most of the studies have been conducted under environ-
mentally controlled conditions, such as in growth chambers
or greenhouses, so that their results cannot accurately
reflect the performance of melatonin with regard to stress
tolerance in the field environment. Therefore, the perfor-
mance and mechanism of melatonin’s effect on drought
tolerance needs further study, especially in highly impor-
tant crops under field environmental conditions.
Maize (Zea mays L.) is sensitive to drought stress, and
its average annual yield loss due to drought is around 15 %
of its potential yield (Ziyomo and Bernardo 2013). The
present study was carried out to investigate the perfor-
mance and mechanism of the effect of melatonin applica-
tion on drought tolerance in maize seedlings under field
environmental conditions. Five-week old seedlings in pots
were sprayed with melatonin and then subjected to drought
stress. Their growth, photosynthetic parameters, antioxi-
dant ability and water status were investigated.
Materials and methods
Plant cultivar and drought and melatonin
treatments
The experiments were conducted during June and July
2014 at the Institute of Soil and Water Conservation,
Chinese Academy of Sciences. Seedlings of the maize
cultivar ‘‘Cheng Yu 888’’, a relatively drought-sensitive
cultivar, were sown in pots (diameter 20 cm, depth 30 cm)
each containing 15 kg air-dried brown soil. As base fer-
tilizers, N, P
2
O
5
and K
2
O were present at concentrations of
0.22, 0.15 and 0.05 g kg
-1
dried soil, respectively. Soil
water content was expressed as a percent maximum pot
capacity (Ogbaga et al. 2014). All pots were watered to
85 % before sowing and were placed under a rain shed in
the field. Five weeks after sowing, four uniform plants
were maintained in each pot. Half of the pots were then
exposed to drought treatment and sprayed with either
melatonin (100 lM) or water. The sprayed melatonin
solution was prepared as follows: 2.3 g melatonin was
dissolved in 50 mL ethyl alcohol as a stored solution. 1 mL
of this stored solution was diluted to 2 L with deionized
water and 0.05 % (V/V) Tween-20 as a surfactant. Every
pot was sprayed with 100 mL prepared solution. The
experiment included four treatments: (1) well-watered, (2)
well-watered?melatonin, (3) drought, and (4)
drought?melatonin. The control of well-watered and
drought treatments was according to Chen et al. (2015).
The soil water contents are shown in Fig. 1. After 8 days of
drought treatment, all plants in the drought treatment group
were rehydrated and permitted to grow for another 1 week.
The fifth and tenth leaves from the bottom were marked at
the beginning of the drought treatment. On days 0, 4 and 8
of drought treatment and days 1 and 7 of subsequent
48 Page 2 of 13 Acta Physiol Plant (2016) 38:48
123

rehydration, the physiological parameters of the marked
leaves were measured. Simultaneously, the same leaves
were gathered, and put into liquid nitrogen for 30 min, then
stored at -80 °C for the following measurements. The
osmotic potential, hydrogen peroxide, lipid peroxidation,
antioxidant enzyme activity and DPPH-radical scavenging
activity were measured.
Determination of shoot dry weight and individual
leaf area
Plants from each group were sampled after 0, 4 and 8 days
of drought stress and 1 and 7 days of subsequent rehy-
dration. Plant tissues were dried in and oven (80 °C) for 3
days and then the shoot dry weight was determined. Each
treatment included twelve replicates. The leaf area was
estimated according to Wang et al. (2012), as follows: leaf
area =leaf length 9maximum leaf width 90.75.
Analysis of leaf gas exchange
Gas exchange parameters (photosynthetic rate, stomatal
conductance and transpiration rate) were determined with a
portable photosynthesis system (LI-6400XT; LI-COR Bio-
sciences, Lincoln, NE, USA) between 9:00 and 11:00 AM.
The 6-cm
2
leaf chamber was used and the photo flux density
was 1000 lmol m
-2
s
-1
. The fifth or tenth leaf was used for
measurement. Each treatment included six replicates.
Chlorophyll concentration
The chlorophyll concentration was determined through
measuring the SPAD value by a SPAD meter (SPAD-502,
Konica-Minolta, Tokyo, Japan). Each leaf was measured at
ten locations and each treatment included six replicates.
Analysis of chlorophyll fluorescence
Chlorophyll fluorescence was measured with a pulse
amplitude modulated chlorophyll fluorescence system
(Imaging PAM, Walz, Effeltrich, Germany) at room tem-
perature. The following parameters were obtained using
Imaging Win software (Version 2.40, Walz): maximal
quantum yield of PSII photochemistry (Fv/Fm), effective
PSII quantum yield (UPSII), non-photochemical quenching
coefficient (NPQ) and electron transport rate (ETR). Each
treatment included four replicates.
Determination of relative water content (RWC), leaf
water potential, osmotic potential and turgor
pressure
Leaf RWC was measured on days 4 and 8 of drought
treatment according to the method of Turner (1981). Leaf
water potential and osmotic potential were measured
Fig. 1 Changes in soil water content. Values are mean ±SE from
thirteen replicates
Fig. 2 Shoot dry weight and leaf area of maize plants growing in
well watered conditions, under drought stress or of seedlings treated
with melatonin and growing in well watered or drought stress
conditions. Values are mean ±SE from twelve replicates. Significant
differences between different treatments on the same day of the
experimental period are indicated by different letters (P\0.05)
Acta Physiol Plant (2016) 38:48 Page 3 of 13 48
123

according to the methods of Chen et al. (2014). The turgor
pressure was calculated as the difference between leaf
water potential and osmotic potential. Each treatment
included six replicates.
Determination of hydrogen peroxide (H
2
O
2
)
and lipid peroxidation
The H
2
O
2
concentration was measured following the
method described by Loreto and Velikova (2001). Leaf
lipid peroxidation was determined according to the method
of Heath and Packer (1968) by measuring the amount of
malondialdehyde (MDA). Each treatment included three
replicates.
Determination of antioxidant enzyme activity
Superoxide dismutase (SOD) activity was determined
according to the methods described by Beauchamp and
Fridovich (1973). Catalase (CAT) activity was assayed
Fig. 3 Photosynthetic rate (a,b), stomatal conductance (c,d) and
transpiration rate (e,f) in the fifth or tenth leaves of maize plants
growing in well watered conditions, under drought stress or in leaves
of seedlings treated with melatonin and growing in well watered or
drought stress conditions. Values are mean ±SE from six replicates.
Significant differences between different treatments on the same day
of the experimental period are indicated by different letters
(P\0.05)
48 Page 4 of 13 Acta Physiol Plant (2016) 38:48
123

according to the method of Hamurcu et al. (2013).
Ascorbate peroxidase (APX) activity was determined
according to the methods described by Nakano and Asada
(1981). Peroxidase (POD) activity was assayed based on
the methods described by Kochba et al. (1977). Soluble
protein content was measured according to the method of
Bradford (1976). Each treatment included three replicates.
Determination of DPPH-radical scavenging activity
DPPH-radical scavenging activity was measured and cal-
culated according to Matsuura et al. (2003). Each treatment
included three replicates.
Statistical analysis
Data were analyzed by analysis of variance (ANOVA) and
the least significant differences (LSD) test using the SPSS
Data Processing System. Statistical significance was set at
P\0.05.
Results
Plant growth
Under normal (well-watered) condition, application of
melatonin showed no effect on the shoot dry weight and
leaf area. Drought stress significantly reduced shoot dry
weight, diminishing it by 10.5 and 20.8 % after 4 and 8
days of drought treatment, respectively. In contrast, in
plants that treated with melatonin, shoot dry weight was
reduced by only 5.5 and 9.9 % after 4 and 8 days of
drought treatment. After rehydration for 7 days, shoot dry
weight of melatonin-treated seedlings was 7.5 % greater
than that of untreated seedlings (Fig. 2a). In untreated
plants, leaf area was reduced by 13.8 and 24.2 % after 4
and 8 days of drought treatment, respectively; in mela-
tonin-treated plants, leaf area was reduced by only 6.1 and
12.0 %. After rehydration, leaf area increased faster in
melatonin-treated plants than that untreated ones (Fig. 2b).
Gas exchange parameters
Under normal (well-watered) conditions, application of
melatonin had no obvious effect on photosynthetic rate,
stomatal conductance or transpiration rate (Fig. 3).
Drought stress significantly decreased all of those
parameters. The photosynthetic rate decreased by 69.3
and 72.7 % in the tenth and fifth leaves, respectively, at
the end of the drought treatment. This decrease was partly
reversed by melatonin application (Fig. 3a, b). Similarly,
leaf stomatal conductance and transpiration rates were
higher in melatonin-treated maize seedlings than that
untreated ones under drought conditions. After rehydra-
tion for 7 days, these two parameters were measured
again: in the tenth leaf, they had recovered to normal
levels in both melatonin-treated plants and untreated
plants, but melatonin-treated plants exhibited faster
recovery. In the fifth leaf, on the other hand, these
parameters continued to decrease, but this decrease
occurred more slowly in melatonin-treated plants. No
valuescouldbeobtainedforthefifthleavesattheendof
the experiment due to leaf death.
Fig. 4 Chlorophyll content (SPAD units) in the fifth or tenth leaves
of maize plants growing in well watered conditions, under drought
stress or in leaves of seedlings treated with melatonin and growing in
well watered or drought stress conditions. Values are mean ±SE
from six replicates. Significant differences between different treat-
ments on the same day of the experimental period are indicated by
different letters (P\0.05)
Acta Physiol Plant (2016) 38:48 Page 5 of 13 48
123

Fig. 5 Chlorophyll fluorescence parameters: maximum PSII quan-
tum yield (Fv/Fm), effective quantum yield of PSII (UpsII), non-
photochemical quenching (NPQ) and electron transport rate (ETR) in
the tenth or fifth leaves of maize plants growing in well watered
condition, under drought stress or in leaves of maize seedlings treated
with melatonin and growing in well watered condition or under
drought stress. Values are mean ±SE from four replicates. Signif-
icant differences between different treatments on the same day of the
experimental period are indicated by different letters (P\0.05)
48 Page 6 of 13 Acta Physiol Plant (2016) 38:48
123

Chlorophyll concentration
Leaf chlorophyll concentration (assessed in the form of
SPAD values) was not affected by melatonin under normal
conditions. After 8 days of drought stress, chlorophyll
concentration was significantly decreased, but it was higher
in melatonin-treated plants than that untreated ones in both
the fifth and the tenth leaf (Fig. 4). After rehydration for 7
days, chlorophyll concentration had recovered to normal
levels in the tenth leaf while continuing to decrease in the
fifth leaf. However, chlorophyll concentration was higher
in melatonin-treated plants than that untreated ones in both
the fifth and the tenth leaf.
Chlorophyll fluorescence parameters
Melatonin application did not influence Fv/Fm, UspII,
NPQ or ETR under well-watered conditions (Fig. 5).
Drought stress significantly decreased Fv/Fm, UspII and
ETR and increased NPQ in both the fifth and the tenth leaf.
After 4 days of drought treatment, the chlorophyll fluo-
rescence parameters was not affected by melatonin in
maize seedlings. By the end of the drought stress period,
however, melatonin application significantly moderated the
decreases in Fv/Fm, UspII and ETR and the increase in
NPQ in both the fifth and the tenth leaf.
Leaf water status
Melatonin application had no effect on the RWC of maize
seedlings under well-watered conditions (Fig. 6). In the
tenth leaf, the RWC of untreated plants was 82.5 and
68.9 % after 4 and 8 days of drought stress, respectively,
while that of melatonin-treated plants was 86.9 and 75.2 %.
In the fifth leaf, similarly, RWC was higher in melatonin-
treated plants than that untreated ones under drought stress.
Application of melatonin had no significant effect on
leaf water potential under either well watered or drought
conditions, but drought stress significantly decreased the
leaf water potential after 4 days of drought treatment in the
tenth and the fifth leaf (Fig. 7a, b). The osmotic potential
was not affected by melatonin in well-watered plants.
Drought stress decreased leaf osmotic potential in plants
with and without melatonin application, but this decrease
was larger in melatonin-treated maize seedlings. The
osmotic potential in the tenth leaf of untreated plants was
decreased by 19.6 and 30.8 % after 4 and 8 days of drought
treatment, respectively. In melatonin-treated plants, how-
ever, it was decreased by 33.3 and 36.7 %. The leaf turgor
pressure increased markedly in the tenth leaf by melatonin
application under drought stress (Fig. 7e).
H
2
O
2
and MDA contents
Under drought treatment, H
2
O
2
largely accumulated in the
fifth and the tenth leaf, but this accumulation of H
2
O
2
was
markedly reduced by melatonin application. After 4 and 8
days of drought treatment, the H
2
O
2
content of the tenth
leaf was 15.14 and 25.56 % lower in melatonin-treated
seedlings. Similarly, the H
2
O
2
content of the fifth leaf was
also 19.40 and 10.35 % lower in melatonin-treated plants
(Fig. 8a, b).
Fig. 6 Relative water content (RWC) in the fifth or tenth leaves of
maize plants growing in well watered conditions, under drought stress
or in leaves of seedlings treated with melatonin and growing in well
watered or drought stress conditions. Values are mean ±SE from six
replicates. Significant differences between different treatments on the
same day of the experimental period are indicated by different letters
(P\0.05)
Acta Physiol Plant (2016) 38:48 Page 7 of 13 48
123

MDA content was not affected by melatonin application
under well-watered conditions. An obvious trend toward
increasing MDA content was observed in drought-stressed
plants, but melatonin application significantly decreased
MDA accumulation under drought stress conditions
(Fig. 8c, d).
Activity of antioxidant enzymes and DPPH-radical
scavenging activity
Antioxidant enzyme activities (SOD, CAT, APX and POD)
were increased by drought treatment. However, melatonin
application resulted in much higher activities of SOD,
CAT, APX and POD under drought stress conditions
(Fig. 9). The DPPH-radical scavenging activity decreased
remarkably under drought stress, but this reduction was
significantly alleviated by melatonin application (Fig. 10).
Discussion
Drought stress critically inhibits plant growth. In this study,
however, the severity of drought-induced growth inhibition
in maize seedlings was reduced by foliar spraying of
melatonin. In addition, melatonin-treated plants recovered
more quickly after rehydration than untreated plants did
Fig. 7 Water potential (a,b), osmotic potential (c,d) and turgor
pressure (e,f) in the fifth or tenth leaves of maize plants growing in
well watered conditions, under drought stress or in leaves of seedlings
treated with melatonin and growing in well watered or drought stress
conditions. Values are mean ±SE from six replicates. Significant
differences between different treatments on the same day of the
experimental period are indicated by different letters (P\0.05)
48 Page 8 of 13 Acta Physiol Plant (2016) 38:48
123

(Fig. 2). The results showed that melatonin application
enhanced maize drought tolerance in field conditions. The
results of this study are in agreement with the previous
reports showing that melatonin application can enhance
drought tolerance in tomato (Solanum lycopersicum)
seedlings (Liu et al. 2015).
Photosynthesis is the physico-chemical process by
which plants use light energy to drive the synthesis of
organic compounds, and it is the basis of plant production
(Xu et al. 2014). Drought is a serious environmental stress
inhibiting photosynthesis. The limitation of ambient CO
2
diffusion to the site of carboxylation which induced by
stomatal closure, is usually considered the main reason for
the decline in photosynthetic rate under water stress
(Chaves et al. 2009; Liu et al. 2013). Previous studies
showed that apple seedlings treated with melatonin main-
tained significantly higher CO
2
assimilation rates and
stomatal conductance under drought conditions (Wang
et al. 2013; Li et al. 2015). In this study, compared with
untreated plants, the melatonin-treated plants maintained
large leaf areas and high photosynthetic rates under
drought stress, enabling a much greater supply of assimi-
lates to growing tissue (Fig. 3). Also, the enhanced
stomatal conductance associated with foliar-sprayed
melatonin may contribute to high photosynthetic rate dur-
ing drought stress.
After rehydration, melatonin application accelerated the
recovery of the photosynthetic rate in young leaves (e.g.
the tenth leaf) and retarded leaf senescence in old leaves
(e.g. the fifth leaf), suggesting that melatonin application
can alleviate drought-induced injury to the photosynthetic
system (Fig. 3). Meanwhile, melatonin-treated plants
maintained higher chlorophyll contents than untreated
plants in both old leaves and young leaves (Fig. 4). The
protective effect of melatonin on chlorophyll was also
found in cucumbers (Wang et al. 2015) and macroalga
Ulva sp. (Tal et al. 2011). Stress often induces damage of
PSII in a leaf (Maxwell and Johnson 2000). In this study,
PSII photosynthetic efficiency, represented by the expres-
sion of Fv/Fm, was kept at higher values in melatonin-
treated plants than that untreated ones (Fig. 5). Melatonin-
treated plants also maintained higher UPSII and ETR and
lower NPQ. These results suggested that melatonin could
protect the drought induced damage in photosynthetic
system. Similar result was also observed in cucumber and
apple (Wang et al. 2013; Zhang et al. 2013). The rapid
Fig. 8 H
2
O
2
and malondialdehyde (MDA) content in the fifth or
tenth leaves of maize plants growing in well watered conditions,
under drought stress or in leaves of seedlings treated with melatonin
and growing in well watered or drought stress conditions. Values are
mean ±SE from three replicates. Significant differences between
different treatments on the same day of the experimental period are
indicated by different letters (P\0.05)
Acta Physiol Plant (2016) 38:48 Page 9 of 13 48
123

recovery of growth after rehydration in melatonin-treated
plants may be due to the protective effect of melatonin on
the photosynthetic system.
Osmotic adjustment is one of strategies for a plant to
tolerant osmotic and water deficit stresses (Yin et al. 2013).
Melatonin-treated plants exhibited greater decreases in
osmotic potential than those untreated ones under drought
treatment (Fig. 7). The lower osmotic potential values
could maintain water in leaves under drought stress. This
result satisfactorily explains the mitigating effect of
Fig. 9 The activities of superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APX) and peroxidase (POD) in the
fifth or tenth leaves of maize plants growing in well watered
conditions, under drought stress or in leaves of seedlings treated with
melatonin and growing in well watered or drought stress conditions.
Values are mean ±SE from three replicates. Significant differences
between different treatments on the same day of the experimental
period are indicated by different letters (P\0.05)
48 Page 10 of 13 Acta Physiol Plant (2016) 38:48
123

melatonin on RWC in melatonin-treated plants under
drought stress (Fig. 6). Meanwhile, melatonin application
helped the plants to maintain higher turgor pressure
(Fig. 7), which contributes to keeping the stomata open and
the photosynthetic rate relatively high (Meng et al. 2014).
In addition, a mitigating effect of melatonin on RWC has
also been found under cold stress conditions (Turk et al.
2014). In the present study, it is worth noting that mela-
tonin-treated plants maintained larger leaf areas and higher
transpiration rates, suggesting that melatonin may enhance
plant root water uptake ability.
Drought stress triggers ROS accumulation and breaks
down the balance between ROS generation and detoxifi-
cation (Gong et al. 2005). The accumulation of ROS can
induce lipid peroxidation, chlorophyll degradation, and
loss of cell membrane integrity and photosynthetic activity.
Plants have developed an enzymatic antioxidant system
and a nonenzymatic antioxidant system to protect against
ROS damage (de Souza et al. 2014). Enhancing plant
antioxidant ability has been considered the primary func-
tion of melatonin in plant stress tolerance (Zhang et al.
2015). In this study, melatonin application enhanced the
activities of antioxidant enzymes, including SOD, CAT,
APX, and POD, as well as DPPH-radical scavenging
ability, and decreased H
2
O
2
and MDA accumulation
(Figs. 8,9,10). Therefore, melatonin application enhanced
plant antioxidant ability in the present study.
Recently, a model proposed by Arnao and Herna
´ndez-
Ruiz (2014,2015) suggested that melatonin acts as an
antioxidant, a biostimulator and a plant growth regulator in
plant responses to abiotic stress. Based on this model, there
are three possible ways in which the results of the present
study may show how melatonin is involved in improving
plant drought tolerance. Firstly, melatonin enhanced
DPPH-radical scavenging activity, suggesting that it may
directly enhance antioxidant ability as an antioxidant
molecule. Secondly, melatonin application enhanced
antioxidant enzyme activities, suggesting that melatonin
may work as a biostimulator to regulate antioxidant gene
expression. These two effects could decrease drought-in-
duced ROS and help plants maintaining high chlorophyll
content and PSII photosynthetic efficiency, thus improve
plant drought tolerance and recovery ability. Thirdly,
melatonin application decreased leaf osmotic potential,
suggesting that melatonin could be involved in regulating
plant water status under drought conditions.
In summary, melatonin application enhanced the activ-
ities of antioxidative enzyme and non-enzyme antioxidants
in drought-stressed plants, which decreased ROS accumu-
lation. This reduction in ROS accumulation in turn reduced
drought-induced damage to the photosynthetic system. In
addition, melatonin moderated water stress by enhancing
osmotic adjustment ability. Reduced oxidative damage and
improved water status enabled plants to maintain higher
chlorophyll contents and photosynthetic rates and thereby
improved plant drought tolerance. The results of this study
imply that melatonin can improve plant drought tolerance
and could be considered as a potential growth regulator in
crop production.
Author contribution statement Jun Ye carried out the
whole experiment, gathered the data, analyzed the results,
and drafted the manuscript. Shiwen Wang designed the
whole experiment and was in charge of manuscript revi-
sion. Binglin Xiong and Xinyue Wang gave assistance in
the measurements of water potential and antioxidant
enzyme activities. Lina Yin and Xiping Deng helped in
interpretation of the results and preparing the manuscript.
Fig. 10 1,1-Diphenyl-2-picryl-hydrazyl (DPPH)-radical scavenging
activity in the fifth or tenth leaves of maize plants growing in well
watered conditions, under drought stress or in leaves of seedlings
treated with melatonin and growing in well watered or drought stress
conditions. Values are mean ±SE from three replicates. Significant
differences between different treatments on the same day of the
experimental period are indicated by different letters (P\0.05)
Acta Physiol Plant (2016) 38:48 Page 11 of 13 48
123

Acknowledgments This study was supported by Youth Innovation
Promotion Association of the Chinese Academy of Sciences
(2013307), National Key Technology Support Program of China
(2015BAD22B01), National Basic Research Program of China
(2015CB150402) and the 111 Project of Chinese Education Ministry
(B12007).
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