melatonin increased maize (zea mays l.) seedling drought...

ORIGINAL ARTICLE

Melatonin increased maize (Zea mays L.) seedling droughttolerance by alleviating drought-induced photosynthetic inhibitionand oxidative damage

Jun Ye1 • Shiwen Wang1,2 • Xiping Deng1,2 • Lina Yin1,2 • Binglin Xiong1 •

Xinyue Wang1

Received: 8 June 2015 / Revised: 6 December 2015 / Accepted: 9 December 2015

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 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

H2O2 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

[email protected]

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

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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 Hernandez-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 Hernandez-

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 Hernandez-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 (Malus domesticusBokh.) 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, P2O5 and K2O 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 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 9 maximum leaf width 9 0.75.

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-cm2 leaf chamber was used and the photo flux density

was 1000 lmol m-2s-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 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 (H2O2)

and lipid peroxidation

The H2O2 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)


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

values could be obtained for the fifth leaves at the end of

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)


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



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).

H2O2 and MDA contents

Under drought treatment, H2O2 largely accumulated in the

fifth and the tenth leaf, but this accumulation of H2O2 was

markedly reduced by melatonin application. After 4 and 8

days of drought treatment, the H2O2 content of the tenth

leaf was 15.14 and 25.56 % lower in melatonin-treated

seedlings. Similarly, the H2O2 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)


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




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 CO2

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 CO2 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 H2O2 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)


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)


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 H2O2 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 Hernandez-

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




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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