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j.envexpbot.2018.08.016

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Accepted Manuscript
Title: Long-term exogenous application of melatonin
improves nutrient uptake fluxes in apple plants under
moderate drought stress
Authors: Bowen Liang, Changqing Ma, Zhijun Zhang, Zhiwei
Wei, Tengteng Gao, Qi Zhao, Fengwang Ma, Chao Li
PII:
DOI:
Reference:
S0098-8472(18)31102-X
https://doi.org/10.1016/j.envexpbot.2018.08.016
EEB 3542
To appear in:
Environmental and Experimental Botany
Received date:
Revised date:
Accepted date:
23-7-2018
15-8-2018
15-8-2018
Please cite this article as: Liang B, Ma C, Zhang Z, Wei Z, Gao T, Zhao Q, Ma F, Li
C, Long-term exogenous application of melatonin improves nutrient uptake fluxes in
apple plants under moderate drought stress, Environmental and Experimental Botany
(2018), https://doi.org/10.1016/j.envexpbot.2018.08.016
This is a PDF file of an unedited manuscript that has been accepted for publication.
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Long-term exogenous application of melatonin improves nutrient uptake fluxes in apple plants under
moderate drought stress
Bowen Liang, Changqing Ma, Zhijun Zhang, Zhiwei Wei, Tengteng Gao, Qi Zhao, Fengwang Ma*, Chao Li*
Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China
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Bowen Liang: lbwnwsuaf@126.com;
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State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of
Changqing Ma: macqing@126.com;
Zhijun Zhang: 15030127393@163.com
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Zhiwei Wei: weizhiwei89@126.com;
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Tengteng Gao: gaotengteng@nwsuaf.edu.cn;
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Qi Zhao: zhaoqi93@nwsuaf.edu.cn
*
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To whom correspondence should be addressed. E-mail: lc453@163.com, fwm64@nwsuaf.edu.cn &
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fwm64@sina.com; Tel: 86-29-87082648; Fax: 86-29-87082648
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Highlights
The effect of drought-induced stress can be alleviated by melatonin.
Melatonin positively influenced the growth and physiological parameter of Malus.
Melatonin increased 15N uptake, utilization and accumulation.
Activity of enzymes involved in N-metabolism can be increased by melatonin.
Melatonin increased nutrient uptake by increasing related genes transcription.
Abstract
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To examine the potential roles of melatonin in nutrient uptake, we investigated the influence of its long-term
exogenous application on ?Naganofuji No.2? apple (Malus domestica Borkh.) under moderate drought conditions.
Both growth and the uptake of macro- and micronutrients were generally decreased in stressed plants. However,
the application of exogenous melatonin significantly mitigated this growth reduction and enabled plants to
maintain uptake fluxes. This addition of melatonin also markedly alleviated the inhibitory effects of drought on
photosynthesis, stomatal apertures, chlorophyll levels, and relative water content, and it controlled the burst of
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relative electrolyte leakage and hydrogen peroxide. Our investigation with stable isotopes further verified that
exogenous melatonin was associated with significant increases in the uptake, utilization, and accumulation of
?15N under drought conditions. Stress sharply reduced the activity of enzymes involved in nitrogen metabolism
(NR, NiR, GS, and GOGAT), but the application of melatonin substantially reversed that response. We also
examined whether melatonin might control the expression of genes for N-metabolism and transport. Here, the
transcript levels of metabolic genes (NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT) and uptake genes (AMT1;2,
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AMT1;5, AMT1;6, AMT2;1, NRT1;1, NRT2;4, NRT2;5, and NRT2;7) were greatly up-regulated in the leaves.
Exogenous melatonin treatment also significantly increased the concentration of endogenous melatonin. Thus,
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understanding the role of melatonin in nutrient uptake introduces new possibilities to use this compound for
agriculture purposes and provides a valuable foundation for enhancing plant tolerance and adaptability to future
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drought stress.
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Keywords: Melatonin, Drought stress, Ionome, Nutrient uptake, 15N-labeling, Apple
Abbreviations: AMT, ammonium transporter; Ci, intercellular CO2 concentration; GOGAT, glutamate synthase;
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Gs, stomatal conductance; GS, glutamine synthetase; HPLC-MS/MS, high performance liquid
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chromatography-tandem mass spectrometry; NiR, nitrite reductase; NR, nitrate reductase; NRT, nitrate
transporter; Pn, net photosynthesis rate; qRT-PCR, quantitative real-time PCR; REL, relative electrolyte leakage;
rate
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1. Introduction
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RGR, relative growth rate; RWC, relative water content; SEM, scanning electron microscope; Tr, transpiration
Water stress is becoming a crucial global challenge because of warming climates and increasingly limited
water resources (Jury and Vaux, 2005). Consequently, farmers will face longer and more intense periods of
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drought, especially in arid and semi-arid regions (Shao et al., 2008). Water is one of the most limiting factors that
influence many morphological, physiological, biochemical, metabolic, transcriptomic, and proteomic processes,
thereby affecting plant survival, development, and crop yields (Alam et al., 2017). Adaptive agricultural
strategies are urgently needed in these changing environments. Because different organs show various levels of
sensitivity to water stress, a whole-plant approach is required in research rather than focusing only on single
components (Chaves et al., 2003).
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Mineral elements are indispensable for plant growth, productivity, and water relations, and they are
involved in many metabolic activities. They have structural, electrochemical, and catalytic roles in all biological
organisms (Goldstein et al., 2013), and are fundamental to hydraulic systems. For example, long-distance water
transport in plants can be modulated by altering the concentration of cations in the xylem sap (Nardini et al.,
2011). This phenomenon, the ?ionic effect?, is likely due to ion-mediated changes in the volume of pectins found
in pit membranes and/or the electroviscous properties of pit apertures (Santiago et al., 2013). It also has a major
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role in optimizing the delivery of water and nutrients to different plant sectors and in regulating tolerance to
drought stress (Oddo et al., 2011).
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Nutrient uptake is highly affected by soil water potential and is restricted under drought conditions (Salehi
et al., 2016). The process by which nitrogen (N) is absorbed greatly depends upon the mobility of water in the
soil because NH4+ or NO3- is initially dissolved in water and subsequently taken up by the roots before being
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transported to the aboveground portions of the plant (Garwood and Williams, 1967). Drought stress reduces the
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absorption of ammonium and nitrate in plants (Rennenberg et al., 2006). Uptake of N is largely determined by
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specific genes, e.g., ammonium transporters (AMTs) and nitrate transporters (NRTs) (Wang et al., 2012b). Some
of those genes have been functionally elucidated in Arabidopsis (Xu et al., 2012) and Populus (Couturier et al.,
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2007). We have reported previously that members of the AMT family (AMT1;2, AMT1;5, AMT1;6, and AMT2;1)
and NRT family (NRT1;1, NRT2;4, NRT2;5, NRT2;7) are involved in the response by apple plants (Malus spp.)
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to PEG-induced drought stress (Huang et al., 2018a; Huang et al., 2018b). After NH4+ and/or NO3- is taken up
into the roots, a large amount of NH4+ is assimilated locally while the remainder is translocated to leaves or other
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parts of the plant. In contrast, only a limited amount of NO3- is assimilated in the roots and most is transported to
the leaves (Xu et al., 2012). During this assimilation process, NO3- is converted to NH4+ by nitrate reductase (NR)
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and nitrite reductase (NiR) (Xu et al., 2012). Afterward, NH4+ is assimilated to glutamine and glutamate via
glutamine synthetase (GS) and glutamate synthase (GOGAT) (Huang et al., 2018b). Water deficits also influence
the activities of enzymes and the transcriptional abundance of genes involved in N-metabolism (Huang et al.,
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2018a; Huang et al., 2018b; Meng et al., 2016). However, the effects of melatonin
(N-acetyl-5-methoxytryptamine) on the transcriptional regulation of genes related to N-uptake, -reduction, and
-metabolism under drought stress have, to our knowledge, not been thoroughly studied. Stable isotope techniques,
such as 15N-labeling, can provide important insight into the uptake of N by roots within the soil profile
(Bakhshandeh et al., 2016). Transformation and absorption of N has also been investigated by using 15N tracers
(Dijkstra et al., 2015). Therefore, the research described here also applied such tracers to examine the impact
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melatonin has on nutrient uptake under drought conditions.
As a highly evolutionarily conserved molecule, melatonin is present in almost all organisms, both plant and
animal, and shows exceptional multiplicity of properties (Tan et al., 2012). It primarily functions in widespread
antioxidant actions (Tan et al., 2007, 2015). In mammals, it has several important physiological roles in
modulating circadian rhythms, seasonal reproduction, immunomodulation, and anti-inflammatory activity, as
well as detoxifying free radicals (Tan et al., 2010). Since it was first identified in plants (Dubbels et al., 1995;
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Hattori et al., 1995), its physiological functions have been further explored with regard to the regulation of root
development (Park and Back, 2012), seed germination (Tiryaki and Keles, 2012), leaf senescence (Wang et al.,
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2012a), and fruit formation/maturation (Lei et al., 2013). Evidence has been found that exogenously applied
melatonin improves plant growth and crop yields, and can also advance post-harvest fruit ripening and perhaps
enhance fruit quality (Reiter et al., 2015). Finally, melatonin has been shown to alleviate the adverse effects of
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abiotic stresses in plants, primarily serving as the first line of defense against environmental challenges such as
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temperature extremes (Shi and Chan, 2014), salinity (Li et al., 2012), UV radiation (Afreen et al., 2006), heavy
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metals (Posmyk et al., 2008), alkaline stress (Gong et al., 2017), and nutrient deficiencies (Li et al., 2016).
Melatonin positively influences plant responses to water stress, promoting seed germination and seedling
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growth in PEG-stressed cucumber (Cucumis sativus L.) (Zhang et al., 2013) and retarding drought-induced leaf
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senescence (Ma et al., 2018; Wang et al., 2013). Treatment with this molecule can also significantly increase
whole-plant drought tolerance (Li et al., 2015; Wang et al., 2017). However, most research with melatonin has
focused on electrolyte leakage, chlorophyll levels, photosynthetic performance, and the activity of antioxidant
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enzymes, with less attention being paid to its effects on the relationship between stress tolerance and ionome
concentrations, nutrient uptake, and utilization under drought stress. In addition to taking a whole-plant approach
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rather than examining individual organs in isolation, it is of paramount importance to understand the complex
interrelations among the physiological processes involved in within-tree nutrient uptake and organ growth when
exposed to a water deficit (Rahmati et al., 2018). Therefore, the objective of our study was to investigate whether
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melatonin supplementation could increase drought tolerance by regulating nutrient uptake and utilization.
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2. Materials and methods
2.1 Plant materials and growing conditions
These trials were conducted at the Northwest A&F University, Yangling (34�?N, 108�?E), Shaanxi,
China, where the climate is semi-arid. In mid-March 2017, buds of cv. ?Naganofuji No.2? were grafted onto
one-year-old rootstock of Malus hupehensis and grown in plastic containers (38� cm) filled with cultivation
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soil and sand (1:1, v:v). The plants were located in a greenhouse under ambient light, at 20 to 35癈, and with a
relative humidity of 50 to 75%. To eliminate position effects, we rotated the containers weekly. Standard
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horticultural practices were followed for disease and pest control.
2.2 Experimental design
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The experimental layout was completely randomized and consisted of combined watering and melatonin
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treatments. After three months of growth under well-watered conditions, 200 uniform and healthy ?Naganofuji
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No.2? trees were divided into four groups (50 plants per treatment) to render the following regimes: (1) normal
control, irrigated daily to maintain 75 to 85% field capacity (CK); (2) moderate drought, irrigated daily to
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maintain 45 to 55% field capacity (DT); (3) melatonin control, irrigated daily to maintain 75 to 85% field
capacity plus 100 ?M melatonin (MCK); and (4) melatonin combined with moderate drought, irrigated daily to
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maintain 45 to 55% field capacity plus 100 ?M melatonin (MDT). Irrigation was withheld from the
drought-stressed plants beginning on 15 June 2017 while normal irrigation continued for the well-watered plants.
N-enriched urea (CO(15NH2)2, produced by the Shanghai Research Institute of Chemical Industry (abundance
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Tests with 15N-labeling were performed at the same time. Ten trees per group were labeled with 1.5 g of
of 10.14%), while the other 40 trees in each group were fertilized with 1.5 g of normal urea (CO(NH 2)2).
Transpiration water losses were evaluated gravimetrically by weighing all pots and calculating the changes in
weight that occurred between watering events. Afterward, the amount of water lost was added back to each pot
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every other day at 18:00 h. For half of the plants in either the well-watered or drought treatments, exogenous
melatonin was applied with a 100 ?M solution replacing the same amount of water added back to the soil every
10 d. To minimize soil evaporation, we covered the soil surface of each pot with a 3-cm-thick layer of sieved
sand. The experiment was terminated after 60 d, on 15 August. Plant growth measurements were made on Days
0 and 60, while gas exchange, chlorophyll concentrations, and gene expression were determined on Days 0, 15,
30, 45, and 60. Leaf stomata were observed with a scanning electron microscope (SEM) on Day 60, and mineral
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elements were analyzed on Days 0 and 60.
2.3 Growth measurements
Plant lengths (PLs) were measured from the base of the stem, at soil level, to the terminal bud of the main
stem. Trunk diameter (TD) was measured with a digital micrometer (0.001 mm) 10 cm above the graft union.
Whole plants from each treatment were harvested and divided into root, stem, and leaf portions. The roots were
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first rinsed with tap water, and then all samples were washed in tap water, 0.1 mol L -1 of HCL, and distilled
water. After the total fresh weight (TFW) was recorded, each sample was fixed at 105癈 for 15 min, then dried
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in a forced-air oven at 75癈 for 48 h to a constant weight. Total dry weight (TDW) of the biomass was computed
as the sum of the values for root, stem, and leaf dry masses. The relative growth rate (RGR) was calculated by
the equation of Radford (Radford, 1967): RGR= (ln DW2- ln DW1) / (t2- t1), where DW1 is plant dry weight at
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Day 0 (t1), and DW2 is plant dry weight at Day 60 (t2).
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2.4 Quantification of gas exchange
Gas exchange parameters, including net photosynthesis rate (Pn), transpiration rate (Tr), stomatal
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conductance (Gs), and intercellular CO2 concentration (Ci), were monitored with a Li-Cor portable
photosynthesis system (Li6400; LICOR, Huntington Beach, CA, USA) on sunny days between 09:00 and 11:00
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h. All photosynthetic readings were taken at 1000 ?mol photons m-2 s-1 and a constant airflow rate of 500 ?mol
s-1. The concentration of cuvette CO2 was set at 400 ?mol CO2 mol-1 air. For all treatments, data were recorded
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from 10 mature, fully exposed leaves from the same position of each selected plant.
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2.5 Observations of leaf stomata by SEM
Ten leaves were collected from the same position per treatment group. The samples were immediately fixed
with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS; pH 6.8) to avoid any damage or
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alterations during sample preparation. They were first rinsed five times with PBS (for 5, 10, 15, 20, and 30 min),
and then dehydrated in a graded ethanol series, vacuum-dried, and gold-coated. The SEM observations were
made with an S-4800 microscope (Hitachi Led., Tokyo, Japan). Stomata were counted at random in 30 visual
sections on the abaxial epidermis, and final tallies were used to calculate stomatal density. We used Image J
software for measuring stomatal lengths, widths, and apertures.
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2.6 Measurements of relative electrolyte leakage, relative water content, H 2O2, and chlorophyll concentrations
Relative electrolyte leakage (REL) was determined from the leaves according to the method described by
Dionisio-Sese and Tobita (Dionisio-Sese and Tobita, 1998). Relative water content (RWC) was determined
gravimetrically pre-dawn and calculated as: RWC= [(FM-DM) / (TM-DM)] � 100, where FW is leaf fresh mass
and DM is leaf dry mass. The turgid mass (TM) was recorded after leaves were floated for 24 h in distilled water
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in a closed container at 4癈 under darkness. Hydrogen peroxide was extracted with 5% trichloroacetic acid and
measured as described previously (Patterson et al., 1984). On each sampling date, chlorophyll (Chl) was
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extracted from harvested leaves with 80% acetone, and concentrations were determined spectrophotometrically
according to the method of Arnon (Arnon, 1949), using a UV-1750 spectrophotometer (Shimadzu, Kyoto,
Japan).
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2.7 Melatonin extraction and analysis
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Leaf samples were collected 60 d after the exogenous melatonin or water tests began. Melatonin was
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extracted from leaves in three biological replicates by a method modified from that of Pothinuch and
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Tongchitpakdee (Pothinuch and Tongchitpakdee, 2011). Briefly, approximately 0.5 g of frozen tissue were
ground to a fine powder in a mortar with liquid nitrogen, then suspended in 5 mL of methanol and
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ultra-sonicated (80Hz) for 35 min at 4癈. After centrifugation at 10,000g at 4癈 for 15 min, the supernatants
were collected and dried by nitrogen gas. The melatonin in each supernatant was further extracted and detected
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by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), as described by Zhao
et al. (Zhao et al., 2013). The MS/MS detection was performed using an API 5500 Q-TRAP tandem MS
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instrument (AB SCIEX, Framingham, MA, USA).
2.8 Determination of mineral elements
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After being individually ground and sieved, 0.2-g samples of roots, stems, and leaves were digested with
concentrated sulfuric acid (H2SO4, AR, 98%) and H2O2 (GR, ?30%). From the resulting digestion and after the
addition of 100 mL of deionized H2O, N and P concentrations were obtained with an Auto Analyzer 3 (AA3)
continuous flow analyzer (SEAL Analytical, Norderstedt, Germany), while the K concentration was analyzed by
a flame photometer (M410; Sherwood Scientific Ltd., Cambridge, UK). Other 0.1-g samples were digested with
nitric acid (HNO3, AR, 65%) using the microwave reaction system (Multiwave PRO; Anton Paar GmbH, Graz,
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Austria). Elemental analyses of Ca, Mg, Fe, Mn, Cu, Zn, and B were performed by inductively coupled
plasma-atomic emission spectroscopy (iCAP Q ICP-MS; Thermo Fisher Scientific Co., Waltham, MA, USA).
2.9 Determination of nutrient uptake fluxes
Over a 60-d period, nutrient uptake fluxes were calculated based on values for RGR, DW, and the total
concentrations of nutrients in samples from the roots (r), stems (s), and leaves (l), as follows (Kruse et al., 2007):
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(Total Nutrient)r = RGR譊Wr�(Nutrient)r
(Total Nutrient)s = RGR譊Ws�(Nutrient)s
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(Total Nutrient)l = RGR譊Wl�(Nutrient)l
Jupt Nutrient = (Total Nutrient)r + (Total Nutrient)s + (Total Nutrient)l. The uptake flux was expressed as either
units of milligrams per plant per day or micrograms per plant per day.
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2.10 Stable isotope analyses
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Nitrogen stable isotope ratios for the samples were examined using an elemental analyzer (Flash EA
1112HT, Thermo Fisher Scientific, Inc., USA) coupled with an isotope ratio mass spectrometer (Finnigan Delta
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V Advantage, Thermo Fisher Scientific, Inc.) in the joint Laboratory of Stable Isotope Ratio Mass Spectrometry
between Shenzhen HuaKe Precision Testing Technology, Inc., and the Graduate School at Shenzhen, Tinghua
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University. The ratios were expressed in ?-unit notation, which is defined as follows (Feng et al., 2018): ?X (?)
= [(Rsample/Rstandard)-1] � 1000, where X= 15N, and R is the 15N/14N ratio for nitrogen. The Rstandard for the 15N tests
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was Atm-N2. To correct for any instrument drift (rarely necessary), laboratory working standards (protein,
glycine, and/or urea) were run regularly during the tests. Analytical precision was �2? for ? 15N. The total
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accumulation of 15N in a particular organ type (leaf, stem, or root) was calculated as the product of DW and the
N concentration in that organ. Uptake activity was recorded as the amount of 15N taken up per unit weight of
roots per unit time (Hu et al., 2015). The utilization rate was determined as the ratio of total 15N content in the
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tree to that occurring in the fertilizer (Zheng et al., 2018).
2.11 Determination of activities of enzymes involved in N-assimilation
Activity of NR in the leaves was analyzed based on the method previously described (H鰃berg et al., 1986).
The activity of NiR was measured as the reduction in the amount of NO 2- in the reaction mixture, based on a
method previously described (Seith et al., 2010). Activity of GS was assayed spectrophotometrically (Wang et al.,
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2008), while that of GOGAT was measured based on the method described by Lin and Kao (Lin and Kao, 1996).
2.12 qRT-PCR analysis
Total RNA was extracted from leaf samples using a Wolact� plant RNA isolation kit (Vicband, Hong Kong,
China) according to the manufacturer?s instructions. Quantitative real-time PCR (qRT-PCR) was performed on
an ABI StepOnePlus real-time PCR system (Applied Biosystems, Singapore), using SYBR Premix Ex Taq II
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(Takara, Kyoto, Japan). Transcripts of the Malus elongation factor 1 alpha gene (EF-1a; DQ341381) were used
to standardize the cDNA samples for different genes (Li et al., 2015). All primers used for qRT-PCR are shown
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in Table S1. Three biological replicates with three technical replicates were assayed for each sample.
2.13 Statistical analysis
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All data were analyzed with SPSS 20.0 software. One-way analysis of variance (ANOVA) was used to
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compare the means from each treatment group. We then applied two-way ANOVA [model: ?drought?,
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?melatonin?, and ?drought � melatonin], using a general linear model to confirm whether the effects of drought
and melatonin, individually and combined, had any significant influence on the results. Tukey?s multiple range
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tests were used at a significance level of P0.05, and data were presented as the means � standard deviation (SD) of
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three to 10 replicate samples.
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3. Results
3.1 Plant growth and evaluation of drought tolerance
Drought stress had a strong inhibitory effect on overall plant growth, leading to significant decreases in
values for PL (by 25.8%), TD (22.9%), TDW (40.2%), and RGR (36.6%) (Figure 1, Table 1). However,
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exogenous melatonin significantly alleviated those declines when compared with drought-stressed plants that
had received no such supplementation. Those decreases were then only 19.8% for PL, 17.1% for TD, 35.9% for
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TDW, and 31.6% for RGR when compared with control values (Table 1). Three parameters typically used for
assessing drought tolerance were evaluated. Under well-watered conditions, values for RWC, REL, and H2O2 did
not differ significantly among no-melatonin and melatonin-applied plants. However, after 60 d of drought stress,
the RWC of melatonin-applied plants was 1.79% higher than the level calculated for the no-melatonin plants.
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The calculation of REL also revealed how drought stress can affect leaf membranes, with values being
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significantly increased, by 11.62%, in DT plants, but by only 5.94% in the MDT plants when compared with the
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CK. Finally, our comparison of H2O2 data showed that values were significantly increased, by 110.9% in DT
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plants, but only by 58.2% in the MDT plants when compared with the CK (Table 1).
Figure 1 Plants after 60 d of exposure to different watering and melatonin treatments: CK, irrigated daily to
maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
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Table 1 Plant length (PL), trunk diameter (TD), total dry weight (TDW), relative growth rate (RGR),
relative
electrolyte leakage (REL), relative water content (RWC), and H2O2 concentration for plants grown 60 d under
different watering and melatonin treatments. Data are means � SD (n=10). Within a column, values not followed
by the same letter indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK,
irrigated daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK,
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irrigated daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain
45-55% field capacity plus 100 ?M melatonin. Significance of effects due to main factors drought (DT),
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melatonin (MT), and their interaction (DT譓T) are represented by ns, not significant; *, P <0.05; **, P <0.01;
and ***, P <0.001.
PL
TD
TDW
RGR
REL
RWC
H2O2
(mm)
(g plant )
(g kg d )
(%)
(%)
(?mol g-1 FW)
CK
104.6�11a
8.3�45a
60.0�88a
23.4�52a
11.6�31c
91.1�45a
1.7�06c
DT
77.7�04c
6.4�45c
35.9�87c
14.9�41c
13.0�48a
86.7�83c
3.5�25a
MCK
106.7�03a
8.4�39a
60.4�98a
23.5�54a
11.6�25c
91.2�51a
1.5�13c
MDT
83.9�941b
6.9�43b
38.5�56b
16.0�24b
12.3�06b
88.2�84b
2.6�10b
***
***
***
***
*
***
***
***
*
**
**
Significance of effects
***
***
MT
***
*
**
DT譓T
*
ns
*
***
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***
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DT
-1
N
-1
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(cm)
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Treatment
3.2 Gas exchange and total Chl concentrations
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In response to drought stress, Pn, which represents the assimilation efficiency of CO2, was decreased in all
treatments throughout the experimental period, with rates being significantly lower for no-melatonin than for
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melatonin-applied plants. On Day 60, Pn from melatonin-applied plants was 1.05 and 2.34 times higher than the
rate for well-watered and drought-stressed control plants, respectively (Figure 2A). Values for Gs followed a
similar trend, indicating that the process of photosynthesis was somewhat dependent on the action of the stomata
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(Figure 2D). Values for two other gas exchange parameters, Tr and Ci, were also decreased during the treatment
period, but these declines were not as severe under the influence of melatonin (Figure 2B and C). These data
suggested that melatonin enabled plants to maintain a more normal photosynthetic system under drought
conditions. After 60 d of stress, total Chl concentrations were significantly lower than the levels measured in
well-watered controls. In particular, total Chl was reduced by 31.7% and 24.4% in no-melatonin and
melatonin-applied plants, respectively (Figure 2E).
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Figure 2 Effects of drought stress and melatonin on net photosynthesis rate (Pn; A), transpiration rate (Tr; B),
intercellular CO2 concentration (Ci; C), stomatal conductance (Gs; D), and total chlorophyll concentration (TCC;
E). Data are means � SD of 5 replicate samples. Time points not labeled with same letter indicate significant
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differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85%
field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85%
field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M
melatonin.
3.3 Stomatal behavior
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Drought stress altered stomatal parameters, and clear structural differences were observed between
treatments with or without melatonin. Stomatal density was higher in drought-stressed leaves than in the control,
but the stomatal lengths, widths, and apertures of the former type were significantly decreased (Figure 3).
Although exogenous melatonin inhibited stomatal density (13.9%), it did increase stomatal lengths (12.6%),
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widths (9.9%), and apertures (58.0%) significantly under drought conditions.
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Figure 3 Properties of stomata from plants after 60 d of exposure to different watering and melatonin treatments.
(A) density, (B) width, (C) length, and (D) aperture. Data are means � SD of 30 images. For each panel, bars not
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labeled with same letter indicate significant differences at P0.05, based on Tukey?s multiple range tests.
Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field
capacity; MCK, irrigated daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated
A
daily to maintain 45-55% field capacity plus 100 ?M melatonin. Significant effects of main factors drought (DT),
melatonin (MT), and interactions (DT譓T) are shown: ns, not significant; *, P <0.05; **, P <0.01; and ***, P
<0.001.
3.4 Concentrations and uptake fluxes of ionome
The concentrations of various minerals in the leaf ionome are shown in Table S2. Drought conditions were
13
associated with significant reductions in the levels of N, P, K, Ca, and B but increases in Fe, Mn, and Zn. Stress
had no critical influence on Mg and Cu concentrations. Under drought conditions, exogenous melatonin
significantly increased concentrations of N, P, K, and Ca by a range of 3.3% (for N) to 10.4% (for P) but led to
marked declines for Fe, Mn, Cu, Zn, and B (by 6.4% for Mn to 19.8% for Cu) in melatonin-applied plants when
compared with no-melatonin plants. When plants were exposed to stress, exogenous melatonin increased stem
concentrations of P, Cu, and Zn by 4.2% (Zn) to 15.5% (Cu); reduced the levels of N, Mg, Fe, and B by 9.4% (N)
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to 16.3% (B); but had no significant effect on K, Ca, and Mn (Table S3). Under such stress conditions, a
comparison between the roots of no-melatonin and melatonin plants showed that the addition of this molecule
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increased the concentration of P by 13.8%; reduced the levels of N, Ca, Fe, and Mn by 3.1% (Fe) to 21.7% (Mn);
but had no significant effect on K, Mg, Cu, Zn, and B (Table S4).
Data for mineral nutrient uptake are presented in Table 2. The effect of drought was highly significant.
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When comparing between well-watered and stressed plants, reductions were noted for all of the elements
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analyzed here. The uptake of N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, and B was diminished by 59.5%, 74.0%, 66.8%,
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62.8%, 67.1%, 64.0%, 70.0%, 70.5%, 72.5%, and 68.4%, respectively. However, exogenous melatonin was
associated with increases in the uptake of these nutrients under drought conditions. When the comparison was
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made between melatonin and no-melatonin plants under stress, uptake of N, P, K, Ca, Mg, Cu, Zn, and B was
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improved by 9.7%, 29.2%, 24.0%, 5.3%, 9.3%, 9.5%, 10.7%, and 8.1%, respectively.
Table 2 Uptake fluxes of nutritional elements in plants after 60 d of growth under different watering and
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melatonin treatments. Data are means � SD (n=5). Unit of measure: mg plant-1 day-1 for N, P, K, Ca, and Mg; ?g
plant-1 day-1 for Fe, Mn, Cu, Zn, and B. Within a row, values not followed by the same letter indicate significant
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differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85%
field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85%
field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M
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melatonin. Significance of effects due to main factors drought (DT), melatonin (MT), and their interaction
(DT譓T) are represented by ns, not significant; **, P <0.01; and ***, P <0.001.
Element
CK
DT
MCK
MDT
Significance of effects
DT
MT
DT譓T
N
21.5�09a
8.7�01d
19.8�24b
9.5�01c
***
***
***
P
2.5�03a
0.7�01c
2.6�03a
0.8�01b
***
***
***
K
14.1�24b
4.7�13d
14.5�19a
5.8�06c
***
***
**
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Ca
17.2�16a
6.4�02d
16.8�13b
6.8�03c
***
ns
***
Mg
3.3�04a
1.1�04b
3.2�05a
1.2�02b
***
ns
**
Fe
855.1�.44a
307.5�47c
750.2�17b
234.0�37d
***
***
ns
Mn
56.4�42a
16.9�18c
40.9�38b
16.4�05c
***
***
***
Cu
17.8�08a
5.3�11d
16.7�22b
5.8�11c
***
**
***
Zn
23.5�67a
6.5�19c
21.7�63b
7.2�18c
***
ns
**
B
63.4�88a
20.0�27c
58.5�14b
21.6�46c
***
**
***
3.5
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Effects of exogenous melatonin application on endogenous melatonin concentrations
To quantify the endogenous level of melatonin under normal and drought conditions we measured the
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levels of melatonin at 60 d after the stress treatment began (Figure 4). The level of melatonin produced in the
well-watered trees, without exogenous melatonin application, was 0.28 ng g -1 FW. Although endogenous
melatonin concentrations were lower in stressed plants, we found no statistically significant difference between
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the DT and CK groups. However, such supplementation led to significant elevations in endogenous melatonin
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concentrations under both well-watered and drought conditions, with increases of 25.0 and 165.3 ng g -1 FW in
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MCK and MDT plants, respectively (Figure 4F).
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Figure 4 HPLC-MS/MS spectra (A-E) and endogenous level of melatonin in leaves (F) of apple trees under
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different treatment conditions. HPLC-MS/MS spectra of melatonin standard sample (A), CK sample (B), DT
sample (C), MCK sample (D), and MDT sample (E), both retention times are 6.53. Treatments: CK, irrigated
daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated
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daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55%
field capacity plus 100 ?M melatonin. Significant effects of main factors drought (DT), melatonin (MT), and
interactions (DT譓T) are shown by ***, P <0.001.
3.6 15N-urea concentration, accumulation, uptake activity, and utilization rate
Stress treatment noticeably reduced the concentration of 15N-urea in the leaves (40.6%) but increased that
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component in the roots (35.3%) when compared with samples collected under normal conditions (Figure 5A-F).
Under induced drought, the accumulation of 15N-urea was decreased in the leaf (67.3%), stem (47.8%), and root
(7.3%). Uptake and utilization of 15N-urea were also reduced in response to stress, which explained the smaller
accumulation of 15N-urea in DT plants. However, exogenous melatonin was associated with significant increases
in the uptake, utilization, and accumulation of 15N-urea under drought conditions. When melatonin and
no-melatonin plants were compared under stress treatment, uptake activity and the utilization rate of 15N-urea
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were improved by 12.6% (Figure 5G) and 14.2% (Figure 5H). Furthermore, the overall accumulation of 15N-urea
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rose by 14.2%.
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Figure 5 Effects of drought stress and melatonin on ?15N concentrations and accumulation in leaves (A, D),
stems (B, E), and roots (C, F), and on uptake activity (G), and utilization rate (H). Data are means � SD
(n=3~10). For each panel, bars not labeled with same letter indicate significant differences at P0.05, based on
Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT, irrigated
daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85% field capacity plus 100 ?M
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melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M melatonin. Significant
effects of main factors drought (DT), melatonin (MT), and interactions (DT譓T) are shown: ns, not significant;
*, P <0.05; **, P <0.01; and ***, P <0.001.
3.7 Activity of enzymes involved in N-metabolism
After the absorption of NH4+ and NO3-, enzymes such as NR, NiR, GS, and GOGAT play key roles in
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nitrate reduction and N-assimilation. Our monitoring of those enzymes indicated that drought stress sharply
reduced activities for all of them (Figure 6). When comparing between well-watered and stressed plants,
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activities of NR, NiR, GS, and GOGAT declined by 68.1%, 23.6%, 79.0%, and 76.3%, respectively. However,
when melatonin was applied to either well-watered or droughty soils, enzyme activity was obviously increased,
especially in leaves from stressed plants. By Day 60, the activity of NR, NiR, GS, and GOGAT in MDT leaves
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rose by 168.1%, 23.5%, 74.2%, and 104.6%, respectively, when compared with the corresponding DT plants. All
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of these results demonstrated that drought stress slowed the process of nitrate reduction and N-assimilation in the
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leaves. However, supplementation with melatonin substantially alleviated this response.
Figure 6 Effects of melatonin on activities of nitrate reductase (NR; A), nitrite reductase (NiR; B)
glutamine
synthetase (GS; C), and glutamate synthase (GOGAT; D) in leaves under different treatment conditions. Data are
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means � SD (n=3~5). For each panel, bars not labeled with same letter indicate significant differences at P0.05,
based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT,
irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85% field capacity plus
100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M melatonin.
Significant effects of main factors drought (DT), melatonin (MT), and interactions (DT譓T) are shown by ***,
3.8 Transcriptional regulation of genes involved in N-metabolism and -uptake
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P <0.001.
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In addition to examining the physiological responses to drought stress and the application of melatonin, we
studied internal molecular responses, as manifested by alterations in the transcriptional regulation patterns of key
genes implicated in N-uptake and -metabolism. In leaves, the induced water deficit affected the transcript levels
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of N-metabolic genes NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT and genes for N-uptake, i.e., AMT1;2,
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AMT1;5, AMT1;6, AMT2;1, NRT1;1, NRT2;4, NRT2;5, and NRT2;7. During the 60-d drought period, we found
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that transcript levels for all investigated genes related to N-reduction and -assimilation in the leaves of the DT
plants were also significantly decreased compared with those of the CK plants (Figure 7). Furthermore, the
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transcript abundance of all AMTs and NRTs was suppressed in the leaves of DT plants when compared with CK
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plants (Figure 8). However, their relative expression abundance was higher in the MDT plants than in the DT
plants, indicating that melatonin promoted the expression of genes associated with N-uptake and -metabolism.
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conditions.
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This led us to conclude that melatonin could, to a certain degree, increase nutrient uptake under drought
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Figure 7 Effects of melatonin on expression of key genes involved in N-assimilation in leaves under different
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treatment conditions. Total RNA was isolated from samples at specified time points (0-60 d), converted to cDNA,
and subjected to qRT-PCR. Expression levels were calculated relative to expression of Malus elongation factor 1
alpha gene (EF-1a; DQ341381) mRNA. Data are means � SD (n=4). Time points not labeled with same letter
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indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to
maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
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Figure 8 Effects of melatonin on expression of key genes involved in N-uptake in leaves under different
treatment conditions. Total RNA was isolated from samples at specified time points (0-60 d), converted to cDNA,
and subjected to qRT-PCR. Expression levels were calculated relative to expression of Malus elongation factor 1
alpha gene (EF-1a; DQ341381) mRNA. Data are means � SD (n=4). Time points not labeled with same letter
indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to
22
maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
4. Discussion
Drought is the most common environmental stress factor that limits plant development, and productivity by
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interrupting a series of physiological, biochemical, and molecular mechanisms (Gupta et al., 2014). Growth is
affected through depressed leaf water potential, higher electrolyte leakage, reduced biomass production, and
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altered plant water and nutrient relationships (Boomsma and Vyn, 2008). Our results showed that the induction
of drought was associated with significant decreases in PL, TD, DW, RWC, and RGR. In accordance with
previous findings (Zhang et al., 2015), our data also indicated that pretreatment with melatonin significantly
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alleviated this growth inhibition. Solid evidence has implicated melatonin as a growth promoter that also
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enhances plant tolerance to both abiotic and biotic stresses (Antoniou et al., 2017; Shi et al., 2015a). Synthesized
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by plants, this molecule acts as a potent antioxidant and/or regulator of growth and development (Arnao and
Hernandez-Ruiz, 2018). Plants can also absorb exogenously applied melatonin and accumulate it in their organs
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(Zhang et al., 2014), resulting in positive outcomes under some abiotic stress conditions (Zhang et al., 2013). We
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demonstrated here that exogenous melatonin treatment led to a significant boost in endogenous melatonin
concentrations, especially under the water deficit. This exogenous supplementation can improve plant tolerance
to extreme temperatures, salinity, ultraviolet radiation, heavy metals, and chemical stresses. It also markedly
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enhances drought tolerance by Triticum aestivum (wheat) (Cui et al., 2017), Zea mays (maize) (Fleta-Soriano et
al., 2017), Glycine max (Wei et al., 2015), Cucumis sativus (cucumber) (Zhang et al., 2013), and Malus plants
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(Li et al., 2015), as demonstrated by less electrolyte leakage, greater growth, and higher biomass production.
Although many stress factors suppress plant growth by targeting more than one physiological process,
photosynthesis is the most severely affected, as reflected by changes in biomass production that may be due to a
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lower demand for assimilates (Kalaji et al., 2014). We noted that drought treatment was associated with
significantly diminished photosynthetic performance, similar to that previously described (Sapeta et al., 2013;
Sun et al., 2018). Under such conditions, photosynthesis is influenced by several mechanisms, including
stomatal limitations, the primary drought-stress response that leads to reduced stomatal conductance (Warren et
al., 2011). When plants close their stomata to minimize water losses, that action also inhibits atmospheric CO 2
diffusion into the leaf and chloroplasts, thereby decreasing the rate of photosynthesis and limiting carbon
23
assimilation, which then reduces plant growth (Karimi et al., 2015). We investigated a possible correlation
between stomatal limitations and decreased photosynthesis and observed that, under drought conditions, the
stomatal apertures were smaller. Therefore, because of that vital physiological mechanism, plants that
demonstrate better capacity to control their stomatal behavior survive and grow well during periods of stress
(Saliendra et al., 1995). Another mechanism is a non-stomatal, or mesophyll, limitation that involves a decrease
in the leaf chlorophyll concentration (Pagter et al., 2005). A decline in the rate of photosynthesis is associated not
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only with stomatal closure but also with disturbances in the levels of leaf photosynthetic pigments (Li et al.,
2013). Pigments such as Chl a and Chl b have central roles in photosynthetic light reactions and are used as an
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index of drought tolerance. However, water deficits can induce the generation of reactive oxygen species in the
leaves and promote chlorophyll degradation. Similar to the results we have previously reported (Li et al., 2015),
the TCC was decreased in our current experiments. We noted a concomitant drop in both the rate of
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photosynthesis and behavior of the stomatal apertures, as well as lower chlorophyll concentrations, all of which
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suggest that the stomata and chlorophyll have critical roles in inhibiting photosynthetic activity under drought
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stress.
When plants are exposed to drought conditions, net photosynthesis is reduced due to regulatory
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mechanisms that include a decrease in stomatal conductance and chlorophyll concentrations (Bohnert and Jensen,
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1996). However, we found that, in the presence of melatonin, the rate of photosynthesis was maintained at a
higher level throughout the experimental period when compared with plants that did not receive exogenous
supplementation. Moreover, after 60 d of drought treatment, the stomatal apertures and Chl levels were larger in
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leaves from melatonin-treated plants. This might explain why plants in that treatment group were better able to
maintain higher photosynthetic values under stress. Therefore, even though photosynthesis might be reduced
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under drought conditions, exogenous supplementation with melatonin significantly eases those negative effects
by maintaining stable stomatal apertures and Chl levels. Levels of Chl in melatonin-treated plants and the
efficiency of Photosystem II are also maintained at levels similar to those measured in non-stressed plants (Wang
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et al., 2013). When stomatal conductance remains stable, exogenous melatonin can cause the stomata to reopen
in cucumber and Malus (Li et al., 2015; Zhang et al., 2013). Melatonin contributes to a more efficient
Photosystem II in apple trees, alleviating the inhibition in photosynthesis caused by drought stress and allowing
the leaves to maintain better water conservation, less electrolyte leakage, stable levels of Chl, greater stomatal
conductance, and higher photosynthetic performance (Li et al., 2015; Wang et al., 2013). Some protective effects
of melatonin on Chl concentrations have been reported for wheat (Turk et al., 2014), Cynodon dactylon (Shi et
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al., 2015b), Lycopersicon esculentum (tomato) (Liu et al., 2015), and Citrus aurantium (Kostopoulou et al.,
2015). Similar results have been obtained with drought-stressed cucumber seedlings, in which the application of
melatonin reduced Chl degradation and increased the photosynthetic rate, thereby reversing the adverse effects
of a water deficit (Zhang et al., 2013). All of these reports, along with our current results, suggest that melatonin
benefits the photosynthetic process by influencing stomatal activity and photosynthetic pigments.
Drought stress reduces plant productivity by inhibiting the uptake of common minerals (Huang et al., 2007).
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Although water uptake and nutrient absorption are independent processes in the roots, the need for water to
support nutrient transport and plant growth makes those processes closely related. Most nutrients are absorbed
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by plant roots as ions, and water acts as a medium for their movement. Water deficits inhibit this flow of
nutrients within the soil, the absorption of those elements, and their uptake by roots (Fageria et al.,
2002).Therefore, drought-stressed plants will have lower nutrient absorption because less water is available and
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the diminished power of the roots hinders the uptake process (Sardans and Penuelas, 2012). Adequate absorption
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of minerals is important for the maintenance of plant structural integrity and key physiological processes, and
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any changes in mineral uptake may negatively affect plant metabolism (Dumlupinar et al., 2011). This might
explain why a lower RGR ultimately reduces biomass accumulations under a drought scenario (Ludlow and
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Muchow, 1990). He and Dijkstra (He and Dijkstra, 2014) have indicated that drought conditions negatively
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affect plant nutrition because the reduction in nutrient uptake is larger than the decrease in growth. This stress
also prevents the absorption of macro- and micronutrients by cherry tomato plants (Sanchez-Rodriguez et al.,
2010). A decline in soil moisture is linked with decreased uptake of N and P (Sardans and Penuelas, 2012) and it
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also prevents the uptake of Fe, Mn, Zn, etc. (Yasar et al., 2014). In this research, our data revealed that the
uptake of macro- and microelements are significantly decreased by drought-induced stress, but those inhibitions
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are not as severe when exogenous melatonin is part of the treatment. This molecule has an important modulating
influence on the mineral element composition of plants and mitigates stress by helping to up-regulate those
elements. For example, melatonin supplementation significantly ameliorates reductions in the concentrations of
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K, P, S, Mg, Fe, Cu, Mn, and Zn, and further increases Ca and B concentrations in maize seedlings under
low-temperature stress (Turk and Erdal, 2015). Likewise, we showed here that supplemental melatonin promoted
the uptake of nutrients under drought conditions.
The stable isotope investigation further verified that the concentration, uptake activity, and utilization rate
of ?15N were obviously increased by the addition of exogenous melatonin under drought conditions. In general,
the absorption of nitrogen declines in response to a water deficit, and activity of enzymes and transcript levels of
25
genes related to N-assimilation are reduced at the same time (Meng et al., 2016). This includes activity by NR,
GS, and GOGAT (Huang et al., 2018a; Rubio-Wilhelmi et al., 2012). Although we also found all of these
outcomes to be true in our current study, supplementing the irrigation solution with melatonin enabled plants to
maintain those parameters at a higher, more normal status. Furthermore, the transcript levels of genes related to
N-metabolism, i.e., NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT, were up-regulated by the addition of
melatonin. In contrast, the transcript levels of AMT1;2, AMT1;5, AMT1;6, and AMT2;1 as well as NRT1;1,
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NRT2;4, NRT2;5, and NRT2;7 were suppressed by drought stress. Those results are consistent with those
reported by Huang et al. (2018a, b), who showed that most representative genes related to N-uptake were
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down-regulated in Malus in response to drought stress. In that research, the addition of melatonin again induced
higher expression of these genes, with both physiological and molecular data indicating that this exogenous
supplementation could promote the uptake of nitrogen under drought conditions. Nutrient uptake is a vital factor
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in conferring plant tolerance to a water deficit (Samarah et al., 2004). Thus, it is likely that melatonin helped
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improve drought tolerance by increasing the uptake of mineral elements in our plants.
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5. Conclusions
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In conclusion, we believe that our research group is the first to study the long-term effects that exogenous
melatonin has on the ionome and on uptake fluxes in apple plants under moderate drought stress. Application of
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100 ?M melatonin was linked to a significant boost in nutrient uptake. The presence of this molecule also
effectively slowed the drought-related declines in RGR and photosynthesis. Moreover, melatonin increased the
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concentration, uptake activity, and utilization rate of ?15N. Our data also provided evidence that melatonin
elevated the activity of enzymes related to N-assimilation. Results from qRT-PCR analysis showed that
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melatonin has an important role in regulating the metabolism and uptake of that nutrient. We suggest that this
positive influence of melatonin offers new opportunities in the field of agriculture, so that plants can be
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developed with greater drought tolerance and enhanced capacity to adapt to future environmental challenges.
Author Statement
Bowen Liang: Conceptualization, Methodology, Software, Formal Analysis, Investigation, Resources, Data
Curation, Writing ? Original Draft, Writing ? Review & Editing, Visualization. Changqing Ma: Methodology,
Software, Investigation. Zhijun Zhang: Investigation. Zhiwei Wei: Investigation. Tengteng Gao: Investigation.
Qi Zhao: Investigation. Fengwang Ma: Conceptualization, Validation, Resources, Writing ? Review & Editing,
Supervision, Project Administration, Funding Acquisition. Chao Li: Conceptualization, Validation, Resources,
26
Writing ? Review & Editing, Supervision, Project Administration, Funding Acquisition.
Declaration of interest
The authors declare that they have no conflict of interest.
Acknowledgements
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This work was supported by the State Key Program of the National Natural Science Foundation of China
(31330068) and by the earmarked fund for the China Agriculture Research System (CARS-27). The authors are
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grateful to Priscilla Licht for help in revising our English composition.
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potential biological functions in the fruits of sweet cherry. J. Pineal Res. 55, 79-88.
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Zhao, Y., Tan, D.X., Lei, Q., Chen, H., Wang, L., Li, Q.T., Gao, Y.A., Kong, J., 2013. Melatonin and its
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N
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317, 15-22.
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er deficits also influence
the activities of enzymes and the transcriptional abundance of genes involved in N-metabolism (Huang et al.,
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2018a; Huang et al., 2018b; Meng et al., 2016). However, the effects of melatonin
(N-acetyl-5-methoxytryptamine) on the transcriptional regulation of genes related to N-uptake, -reduction, and
-metabolism under drought stress have, to our knowledge, not been thoroughly studied. Stable isotope techniques,
such as 15N-labeling, can provide important insight into the uptake of N by roots within the soil profile
(Bakhshandeh et al., 2016). Transformation and absorption of N has also been investigated by using 15N tracers
(Dijkstra et al., 2015). Therefore, the research described here also applied such tracers to examine the impact
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melatonin has on nutrient uptake under drought conditions.
As a highly evolutionarily conserved molecule, melatonin is present in almost all organisms, both plant and
animal, and shows exceptional multiplicity of properties (Tan et al., 2012). It primarily functions in widespread
antioxidant actions (Tan et al., 2007, 2015). In mammals, it has several important physiological roles in
modulating circadian rhythms, seasonal reproduction, immunomodulation, and anti-inflammatory activity, as
well as detoxifying free radicals (Tan et al., 2010). Since it was first identified in plants (Dubbels et al., 1995;
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Hattori et al., 1995), its physiological functions have been further explored with regard to the regulation of root
development (Park and Back, 2012), seed germination (Tiryaki and Keles, 2012), leaf senescence (Wang et al.,
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2012a), and fruit formation/maturation (Lei et al., 2013). Evidence has been found that exogenously applied
melatonin improves plant growth and crop yields, and can also advance post-harvest fruit ripening and perhaps
enhance fruit quality (Reiter et al., 2015). Finally, melatonin has been shown to alleviate the adverse effects of
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abiotic stresses in plants, primarily serving as the first line of defense against environmental challenges such as
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temperature extremes (Shi and Chan, 2014), salinity (Li et al., 2012), UV radiation (Afreen et al., 2006), heavy
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metals (Posmyk et al., 2008), alkaline stress (Gong et al., 2017), and nutrient deficiencies (Li et al., 2016).
Melatonin positively influences plant responses to water stress, promoting seed germination and seedling
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growth in PEG-stressed cucumber (Cucumis sativus L.) (Zhang et al., 2013) and retarding drought-induced leaf
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senescence (Ma et al., 2018; Wang et al., 2013). Treatment with this molecule can also significantly increase
whole-plant drought tolerance (Li et al., 2015; Wang et al., 2017). However, most research with melatonin has
focused on electrolyte leakage, chlorophyll levels, photosynthetic performance, and the activity of antioxidant
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enzymes, with less attention being paid to its effects on the relationship between stress tolerance and ionome
concentrations, nutrient uptake, and utilization under drought stress. In addition to taking a whole-plant approach
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rather than examining individual organs in isolation, it is of paramount importance to understand the complex
interrelations among the physiological processes involved in within-tree nutrient uptake and organ growth when
exposed to a water deficit (Rahmati et al., 2018). Therefore, the objective of our study was to investigate whether
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melatonin supplementation could increase drought tolerance by regulating nutrient uptake and utilization.
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2. Materials and methods
2.1 Plant materials and growing conditions
These trials were conducted at the Northwest A&F University, Yangling (34�?N, 108�?E), Shaanxi,
China, where the climate is semi-arid. In mid-March 2017, buds of cv. ?Naganofuji No.2? were grafted onto
one-year-old rootstock of Malus hupehensis and grown in plastic containers (38� cm) filled with cultivation
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soil and sand (1:1, v:v). The plants were located in a greenhouse under ambient light, at 20 to 35癈, and with a
relative humidity of 50 to 75%. To eliminate position effects, we rotated the containers weekly. Standard
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horticultural practices were followed for disease and pest control.
2.2 Experimental design
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The experimental layout was completely randomized and consisted of combined watering and melatonin
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treatments. After three months of growth under well-watered conditions, 200 uniform and healthy ?Naganofuji
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No.2? trees were divided into four groups (50 plants per treatment) to render the following regimes: (1) normal
control, irrigated daily to maintain 75 to 85% field capacity (CK); (2) moderate drought, irrigated daily to
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maintain 45 to 55% field capacity (DT); (3) melatonin control, irrigated daily to maintain 75 to 85% field
capacity plus 100 ?M melatonin (MCK); and (4) melatonin combined with moderate drought, irrigated daily to
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maintain 45 to 55% field capacity plus 100 ?M melatonin (MDT). Irrigation was withheld from the
drought-stressed plants beginning on 15 June 2017 while normal irrigation continued for the well-watered plants.
N-enriched urea (CO(15NH2)2, produced by the Shanghai Research Institute of Chemical Industry (abundance
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15
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Tests with 15N-labeling were performed at the same time. Ten trees per group were labeled with 1.5 g of
of 10.14%), while the other 40 trees in each group were fertilized with 1.5 g of normal urea (CO(NH 2)2).
Transpiration water losses were evaluated gravimetrically by weighing all pots and calculating the changes in
weight that occurred between watering events. Afterward, the amount of water lost was added back to each pot
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every other day at 18:00 h. For half of the plants in either the well-watered or drought treatments, exogenous
melatonin was applied with a 100 ?M solution replacing the same amount of water added back to the soil every
10 d. To minimize soil evaporation, we covered the soil surface of each pot with a 3-cm-thick layer of sieved
sand. The experiment was terminated after 60 d, on 15 August. Plant growth measurements were made on Days
0 and 60, while gas exchange, chlorophyll concentrations, and gene expression were determined on Days 0, 15,
30, 45, and 60. Leaf stomata were observed with a scanning electron microscope (SEM) on Day 60, and mineral
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elements were analyzed on Days 0 and 60.
2.3 Growth measurements
Plant lengths (PLs) were measured from the base of the stem, at soil level, to the terminal bud of the main
stem. Trunk diameter (TD) was measured with a digital micrometer (0.001 mm) 10 cm above the graft union.
Whole plants from each treatment were harvested and divided into root, stem, and leaf portions. The roots were
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first rinsed with tap water, and then all samples were washed in tap water, 0.1 mol L -1 of HCL, and distilled
water. After the total fresh weight (TFW) was recorded, each sample was fixed at 105癈 for 15 min, then dried
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in a forced-air oven at 75癈 for 48 h to a constant weight. Total dry weight (TDW) of the biomass was computed
as the sum of the values for root, stem, and leaf dry masses. The relative growth rate (RGR) was calculated by
the equation of Radford (Radford, 1967): RGR= (ln DW2- ln DW1) / (t2- t1), where DW1 is plant dry weight at
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Day 0 (t1), and DW2 is plant dry weight at Day 60 (t2).
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2.4 Quantification of gas exchange
Gas exchange parameters, including net photosynthesis rate (Pn), transpiration rate (Tr), stomatal
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conductance (Gs), and intercellular CO2 concentration (Ci), were monitored with a Li-Cor portable
photosynthesis system (Li6400; LICOR, Huntington Beach, CA, USA) on sunny days between 09:00 and 11:00
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h. All photosynthetic readings were taken at 1000 ?mol photons m-2 s-1 and a constant airflow rate of 500 ?mol
s-1. The concentration of cuvette CO2 was set at 400 ?mol CO2 mol-1 air. For all treatments, data were recorded
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from 10 mature, fully exposed leaves from the same position of each selected plant.
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2.5 Observations of leaf stomata by SEM
Ten leaves were collected from the same position per treatment group. The samples were immediately fixed
with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS; pH 6.8) to avoid any damage or
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alterations during sample preparation. They were first rinsed five times with PBS (for 5, 10, 15, 20, and 30 min),
and then dehydrated in a graded ethanol series, vacuum-dried, and gold-coated. The SEM observations were
made with an S-4800 microscope (Hitachi Led., Tokyo, Japan). Stomata were counted at random in 30 visual
sections on the abaxial epidermis, and final tallies were used to calculate stomatal density. We used Image J
software for measuring stomatal lengths, widths, and apertures.
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2.6 Measurements of relative electrolyte leakage, relative water content, H 2O2, and chlorophyll concentrations
Relative electrolyte leakage (REL) was determined from the leaves according to the method described by
Dionisio-Sese and Tobita (Dionisio-Sese and Tobita, 1998). Relative water content (RWC) was determined
gravimetrically pre-dawn and calculated as: RWC= [(FM-DM) / (TM-DM)] � 100, where FW is leaf fresh mass
and DM is leaf dry mass. The turgid mass (TM) was recorded after leaves were floated for 24 h in distilled water
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in a closed container at 4癈 under darkness. Hydrogen peroxide was extracted with 5% trichloroacetic acid and
measured as described previously (Patterson et al., 1984). On each sampling date, chlorophyll (Chl) was
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extracted from harvested leaves with 80% acetone, and concentrations were determined spectrophotometrically
according to the method of Arnon (Arnon, 1949), using a UV-1750 spectrophotometer (Shimadzu, Kyoto,
Japan).
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2.7 Melatonin extraction and analysis
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Leaf samples were collected 60 d after the exogenous melatonin or water tests began. Melatonin was
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extracted from leaves in three biological replicates by a method modified from that of Pothinuch and
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Tongchitpakdee (Pothinuch and Tongchitpakdee, 2011). Briefly, approximately 0.5 g of frozen tissue were
ground to a fine powder in a mortar with liquid nitrogen, then suspended in 5 mL of methanol and
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ultra-sonicated (80Hz) for 35 min at 4癈. After centrifugation at 10,000g at 4癈 for 15 min, the supernatants
were collected and dried by nitrogen gas. The melatonin in each supernatant was further extracted and detected
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by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), as described by Zhao
et al. (Zhao et al., 2013). The MS/MS detection was performed using an API 5500 Q-TRAP tandem MS
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instrument (AB SCIEX, Framingham, MA, USA).
2.8 Determination of mineral elements
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After being individually ground and sieved, 0.2-g samples of roots, stems, and leaves were digested with
concentrated sulfuric acid (H2SO4, AR, 98%) and H2O2 (GR, ?30%). From the resulting digestion and after the
addition of 100 mL of deionized H2O, N and P concentrations were obtained with an Auto Analyzer 3 (AA3)
continuous flow analyzer (SEAL Analytical, Norderstedt, Germany), while the K concentration was analyzed by
a flame photometer (M410; Sherwood Scientific Ltd., Cambridge, UK). Other 0.1-g samples were digested with
nitric acid (HNO3, AR, 65%) using the microwave reaction system (Multiwave PRO; Anton Paar GmbH, Graz,
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Austria). Elemental analyses of Ca, Mg, Fe, Mn, Cu, Zn, and B were performed by inductively coupled
plasma-atomic emission spectroscopy (iCAP Q ICP-MS; Thermo Fisher Scientific Co., Waltham, MA, USA).
2.9 Determination of nutrient uptake fluxes
Over a 60-d period, nutrient uptake fluxes were calculated based on values for RGR, DW, and the total
concentrations of nutrients in samples from the roots (r), stems (s), and leaves (l), as follows (Kruse et al., 2007):
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(Total Nutrient)r = RGR譊Wr�(Nutrient)r
(Total Nutrient)s = RGR譊Ws�(Nutrient)s
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(Total Nutrient)l = RGR譊Wl�(Nutrient)l
Jupt Nutrient = (Total Nutrient)r + (Total Nutrient)s + (Total Nutrient)l. The uptake flux was expressed as either
units of milligrams per plant per day or micrograms per plant per day.
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2.10 Stable isotope analyses
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Nitrogen stable isotope ratios for the samples were examined using an elemental analyzer (Flash EA
1112HT, Thermo Fisher Scientific, Inc., USA) coupled with an isotope ratio mass spectrometer (Finnigan Delta
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V Advantage, Thermo Fisher Scientific, Inc.) in the joint Laboratory of Stable Isotope Ratio Mass Spectrometry
between Shenzhen HuaKe Precision Testing Technology, Inc., and the Graduate School at Shenzhen, Tinghua
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University. The ratios were expressed in ?-unit notation, which is defined as follows (Feng et al., 2018): ?X (?)
= [(Rsample/Rstandard)-1] � 1000, where X= 15N, and R is the 15N/14N ratio for nitrogen. The Rstandard for the 15N tests
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was Atm-N2. To correct for any instrument drift (rarely necessary), laboratory working standards (protein,
glycine, and/or urea) were run regularly during the tests. Analytical precision was �2? for ? 15N. The total
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accumulation of 15N in a particular organ type (leaf, stem, or root) was calculated as the product of DW and the
N concentration in that organ. Uptake activity was recorded as the amount of 15N taken up per unit weight of
roots per unit time (Hu et al., 2015). The utilization rate was determined as the ratio of total 15N content in the
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tree to that occurring in the fertilizer (Zheng et al., 2018).
2.11 Determination of activities of enzymes involved in N-assimilation
Activity of NR in the leaves was analyzed based on the method previously described (H鰃berg et al., 1986).
The activity of NiR was measured as the reduction in the amount of NO 2- in the reaction mixture, based on a
method previously described (Seith et al., 2010). Activity of GS was assayed spectrophotometrically (Wang et al.,
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2008), while that of GOGAT was measured based on the method described by Lin and Kao (Lin and Kao, 1996).
2.12 qRT-PCR analysis
Total RNA was extracted from leaf samples using a Wolact� plant RNA isolation kit (Vicband, Hong Kong,
China) according to the manufacturer?s instructions. Quantitative real-time PCR (qRT-PCR) was performed on
an ABI StepOnePlus real-time PCR system (Applied Biosystems, Singapore), using SYBR Premix Ex Taq II
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(Takara, Kyoto, Japan). Transcripts of the Malus elongation factor 1 alpha gene (EF-1a; DQ341381) were used
to standardize the cDNA samples for different genes (Li et al., 2015). All primers used for qRT-PCR are shown
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in Table S1. Three biological replicates with three technical replicates were assayed for each sample.
2.13 Statistical analysis
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All data were analyzed with SPSS 20.0 software. One-way analysis of variance (ANOVA) was used to
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compare the means from each treatment group. We then applied two-way ANOVA [model: ?drought?,
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?melatonin?, and ?drought � melatonin], using a general linear model to confirm whether the effects of drought
and melatonin, individually and combined, had any significant influence on the results. Tukey?s multiple range
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tests were used at a significance level of P0.05, and data were presented as the means � standard deviation (SD) of
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three to 10 replicate samples.
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3. Results
3.1 Plant growth and evaluation of drought tolerance
Drought stress had a strong inhibitory effect on overall plant growth, leading to significant decreases in
values for PL (by 25.8%), TD (22.9%), TDW (40.2%), and RGR (36.6%) (Figure 1, Table 1). However,
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exogenous melatonin significantly alleviated those declines when compared with drought-stressed plants that
had received no such supplementation. Those decreases were then only 19.8% for PL, 17.1% for TD, 35.9% for
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TDW, and 31.6% for RGR when compared with control values (Table 1). Three parameters typically used for
assessing drought tolerance were evaluated. Under well-watered conditions, values for RWC, REL, and H2O2 did
not differ significantly among no-melatonin and melatonin-applied plants. However, after 60 d of drought stress,
the RWC of melatonin-applied plants was 1.79% higher than the level calculated for the no-melatonin plants.
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The calculation of REL also revealed how drought stress can affect leaf membranes, with values being
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significantly increased, by 11.62%, in DT plants, but by only 5.94% in the MDT plants when compared with the
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CK. Finally, our comparison of H2O2 data showed that values were significantly increased, by 110.9% in DT
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plants, but only by 58.2% in the MDT plants when compared with the CK (Table 1).
Figure 1 Plants after 60 d of exposure to different watering and melatonin treatments: CK, irrigated daily to
maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
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Table 1 Plant length (PL), trunk diameter (TD), total dry weight (TDW), relative growth rate (RGR),
relative
electrolyte leakage (REL), relative water content (RWC), and H2O2 concentration for plants grown 60 d under
different watering and melatonin treatments. Data are means � SD (n=10). Within a column, values not followed
by the same letter indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK,
irrigated daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK,
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irrigated daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain
45-55% field capacity plus 100 ?M melatonin. Significance of effects due to main factors drought (DT),
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melatonin (MT), and their interaction (DT譓T) are represented by ns, not significant; *, P <0.05; **, P <0.01;
and ***, P <0.001.
PL
TD
TDW
RGR
REL
RWC
H2O2
(mm)
(g plant )
(g kg d )
(%)
(%)
(?mol g-1 FW)
CK
104.6�11a
8.3�45a
60.0�88a
23.4�52a
11.6�31c
91.1�45a
1.7�06c
DT
77.7�04c
6.4�45c
35.9�87c
14.9�41c
13.0�48a
86.7�83c
3.5�25a
MCK
106.7�03a
8.4�39a
60.4�98a
23.5�54a
11.6�25c
91.2�51a
1.5�13c
MDT
83.9�941b
6.9�43b
38.5�56b
16.0�24b
12.3�06b
88.2�84b
2.6�10b
***
***
***
***
*
***
***
***
*
**
**
Significance of effects
***
***
MT
***
*
**
DT譓T
*
ns
*
***
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***
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DT
-1
N
-1
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(cm)
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Treatment
3.2 Gas exchange and total Chl concentrations
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In response to drought stress, Pn, which represents the assimilation efficiency of CO2, was decreased in all
treatments throughout the experimental period, with rates being significantly lower for no-melatonin than for
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melatonin-applied plants. On Day 60, Pn from melatonin-applied plants was 1.05 and 2.34 times higher than the
rate for well-watered and drought-stressed control plants, respectively (Figure 2A). Values for Gs followed a
similar trend, indicating that the process of photosynthesis was somewhat dependent on the action of the stomata
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(Figure 2D). Values for two other gas exchange parameters, Tr and Ci, were also decreased during the treatment
period, but these declines were not as severe under the influence of melatonin (Figure 2B and C). These data
suggested that melatonin enabled plants to maintain a more normal photosynthetic system under drought
conditions. After 60 d of stress, total Chl concentrations were significantly lower than the levels measured in
well-watered controls. In particular, total Chl was reduced by 31.7% and 24.4% in no-melatonin and
melatonin-applied plants, respectively (Figure 2E).
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Figure 2 Effects of drought stress and melatonin on net photosynthesis rate (Pn; A), transpiration rate (Tr; B),
intercellular CO2 concentration (Ci; C), stomatal conductance (Gs; D), and total chlorophyll concentration (TCC;
E). Data are means � SD of 5 replicate samples. Time points not labeled with same letter indicate significant
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differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85%
field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85%
field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M
melatonin.
3.3 Stomatal behavior
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Drought stress altered stomatal parameters, and clear structural differences were observed between
treatments with or without melatonin. Stomatal density was higher in drought-stressed leaves than in the control,
but the stomatal lengths, widths, and apertures of the former type were significantly decreased (Figure 3).
Although exogenous melatonin inhibited stomatal density (13.9%), it did increase stomatal lengths (12.6%),
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widths (9.9%), and apertures (58.0%) significantly under drought conditions.
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Figure 3 Properties of stomata from plants after 60 d of exposure to different watering and melatonin treatments.
(A) density, (B) width, (C) length, and (D) aperture. Data are means � SD of 30 images. For each panel, bars not
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labeled with same letter indicate significant differences at P0.05, based on Tukey?s multiple range tests.
Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field
capacity; MCK, irrigated daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated
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daily to maintain 45-55% field capacity plus 100 ?M melatonin. Significant effects of main factors drought (DT),
melatonin (MT), and interactions (DT譓T) are shown: ns, not significant; *, P <0.05; **, P <0.01; and ***, P
<0.001.
3.4 Concentrations and uptake fluxes of ionome
The concentrations of various minerals in the leaf ionome are shown in Table S2. Drought conditions were
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associated with significant reductions in the levels of N, P, K, Ca, and B but increases in Fe, Mn, and Zn. Stress
had no critical influence on Mg and Cu concentrations. Under drought conditions, exogenous melatonin
significantly increased concentrations of N, P, K, and Ca by a range of 3.3% (for N) to 10.4% (for P) but led to
marked declines for Fe, Mn, Cu, Zn, and B (by 6.4% for Mn to 19.8% for Cu) in melatonin-applied plants when
compared with no-melatonin plants. When plants were exposed to stress, exogenous melatonin increased stem
concentrations of P, Cu, and Zn by 4.2% (Zn) to 15.5% (Cu); reduced the levels of N, Mg, Fe, and B by 9.4% (N)
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to 16.3% (B); but had no significant effect on K, Ca, and Mn (Table S3). Under such stress conditions, a
comparison between the roots of no-melatonin and melatonin plants showed that the addition of this molecule
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increased the concentration of P by 13.8%; reduced the levels of N, Ca, Fe, and Mn by 3.1% (Fe) to 21.7% (Mn);
but had no significant effect on K, Mg, Cu, Zn, and B (Table S4).
Data for mineral nutrient uptake are presented in Table 2. The effect of drought was highly significant.
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When comparing between well-watered and stressed plants, reductions were noted for all of the elements
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analyzed here. The uptake of N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, and B was diminished by 59.5%, 74.0%, 66.8%,
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62.8%, 67.1%, 64.0%, 70.0%, 70.5%, 72.5%, and 68.4%, respectively. However, exogenous melatonin was
associated with increases in the uptake of these nutrients under drought conditions. When the comparison was
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made between melatonin and no-melatonin plants under stress, uptake of N, P, K, Ca, Mg, Cu, Zn, and B was
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improved by 9.7%, 29.2%, 24.0%, 5.3%, 9.3%, 9.5%, 10.7%, and 8.1%, respectively.
Table 2 Uptake fluxes of nutritional elements in plants after 60 d of growth under different watering and
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melatonin treatments. Data are means � SD (n=5). Unit of measure: mg plant-1 day-1 for N, P, K, Ca, and Mg; ?g
plant-1 day-1 for Fe, Mn, Cu, Zn, and B. Within a row, values not followed by the same letter indicate significant
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differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85%
field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85%
field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M
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melatonin. Significance of effects due to main factors drought (DT), melatonin (MT), and their interaction
(DT譓T) are represented by ns, not significant; **, P <0.01; and ***, P <0.001.
Element
CK
DT
MCK
MDT
Significance of effects
DT
MT
DT譓T
N
21.5�09a
8.7�01d
19.8�24b
9.5�01c
***
***
***
P
2.5�03a
0.7�01c
2.6�03a
0.8�01b
***
***
***
K
14.1�24b
4.7�13d
14.5�19a
5.8�06c
***
***
**
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Ca
17.2�16a
6.4�02d
16.8�13b
6.8�03c
***
ns
***
Mg
3.3�04a
1.1�04b
3.2�05a
1.2�02b
***
ns
**
Fe
855.1�.44a
307.5�47c
750.2�17b
234.0�37d
***
***
ns
Mn
56.4�42a
16.9�18c
40.9�38b
16.4�05c
***
***
***
Cu
17.8�08a
5.3�11d
16.7�22b
5.8�11c
***
**
***
Zn
23.5�67a
6.5�19c
21.7�63b
7.2�18c
***
ns
**
B
63.4�88a
20.0�27c
58.5�14b
21.6�46c
***
**
***
3.5
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Effects of exogenous melatonin application on endogenous melatonin concentrations
To quantify the endogenous level of melatonin under normal and drought conditions we measured the
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levels of melatonin at 60 d after the stress treatment began (Figure 4). The level of melatonin produced in the
well-watered trees, without exogenous melatonin application, was 0.28 ng g -1 FW. Although endogenous
melatonin concentrations were lower in stressed plants, we found no statistically significant difference between
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the DT and CK groups. However, such supplementation led to significant elevations in endogenous melatonin
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concentrations under both well-watered and drought conditions, with increases of 25.0 and 165.3 ng g -1 FW in
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MCK and MDT plants, respectively (Figure 4F).
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Figure 4 HPLC-MS/MS spectra (A-E) and endogenous level of melatonin in leaves (F) of apple trees under
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different treatment conditions. HPLC-MS/MS spectra of melatonin standard sample (A), CK sample (B), DT
sample (C), MCK sample (D), and MDT sample (E), both retention times are 6.53. Treatments: CK, irrigated
daily to maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated
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daily to maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55%
field capacity plus 100 ?M melatonin. Significant effects of main factors drought (DT), melatonin (MT), and
interactions (DT譓T) are shown by ***, P <0.001.
3.6 15N-urea concentration, accumulation, uptake activity, and utilization rate
Stress treatment noticeably reduced the concentration of 15N-urea in the leaves (40.6%) but increased that
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component in the roots (35.3%) when compared with samples collected under normal conditions (Figure 5A-F).
Under induced drought, the accumulation of 15N-urea was decreased in the leaf (67.3%), stem (47.8%), and root
(7.3%). Uptake and utilization of 15N-urea were also reduced in response to stress, which explained the smaller
accumulation of 15N-urea in DT plants. However, exogenous melatonin was associated with significant increases
in the uptake, utilization, and accumulation of 15N-urea under drought conditions. When melatonin and
no-melatonin plants were compared under stress treatment, uptake activity and the utilization rate of 15N-urea
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were improved by 12.6% (Figure 5G) and 14.2% (Figure 5H). Furthermore, the overall accumulation of 15N-urea
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N
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rose by 14.2%.
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Figure 5 Effects of drought stress and melatonin on ?15N concentrations and accumulation in leaves (A, D),
stems (B, E), and roots (C, F), and on uptake activity (G), and utilization rate (H). Data are means � SD
(n=3~10). For each panel, bars not labeled with same letter indicate significant differences at P0.05, based on
Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT, irrigated
daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85% field capacity plus 100 ?M
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melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M melatonin. Significant
effects of main factors drought (DT), melatonin (MT), and interactions (DT譓T) are shown: ns, not significant;
*, P <0.05; **, P <0.01; and ***, P <0.001.
3.7 Activity of enzymes involved in N-metabolism
After the absorption of NH4+ and NO3-, enzymes such as NR, NiR, GS, and GOGAT play key roles in
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nitrate reduction and N-assimilation. Our monitoring of those enzymes indicated that drought stress sharply
reduced activities for all of them (Figure 6). When comparing between well-watered and stressed plants,
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activities of NR, NiR, GS, and GOGAT declined by 68.1%, 23.6%, 79.0%, and 76.3%, respectively. However,
when melatonin was applied to either well-watered or droughty soils, enzyme activity was obviously increased,
especially in leaves from stressed plants. By Day 60, the activity of NR, NiR, GS, and GOGAT in MDT leaves
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rose by 168.1%, 23.5%, 74.2%, and 104.6%, respectively, when compared with the corresponding DT plants. All
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of these results demonstrated that drought stress slowed the process of nitrate reduction and N-assimilation in the
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leaves. However, supplementation with melatonin substantially alleviated this response.
Figure 6 Effects of melatonin on activities of nitrate reductase (NR; A), nitrite reductase (NiR; B)
glutamine
synthetase (GS; C), and glutamate synthase (GOGAT; D) in leaves under different treatment conditions. Data are
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means � SD (n=3~5). For each panel, bars not labeled with same letter indicate significant differences at P0.05,
based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to maintain 75-85% field capacity; DT,
irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to maintain 75-85% field capacity plus
100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field capacity plus 100 ?M melatonin.
Significant effects of main factors drought (DT), melatonin (MT), and interactions (DT譓T) are shown by ***,
3.8 Transcriptional regulation of genes involved in N-metabolism and -uptake
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P <0.001.
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In addition to examining the physiological responses to drought stress and the application of melatonin, we
studied internal molecular responses, as manifested by alterations in the transcriptional regulation patterns of key
genes implicated in N-uptake and -metabolism. In leaves, the induced water deficit affected the transcript levels
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of N-metabolic genes NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT and genes for N-uptake, i.e., AMT1;2,
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AMT1;5, AMT1;6, AMT2;1, NRT1;1, NRT2;4, NRT2;5, and NRT2;7. During the 60-d drought period, we found
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that transcript levels for all investigated genes related to N-reduction and -assimilation in the leaves of the DT
plants were also significantly decreased compared with those of the CK plants (Figure 7). Furthermore, the
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transcript abundance of all AMTs and NRTs was suppressed in the leaves of DT plants when compared with CK
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plants (Figure 8). However, their relative expression abundance was higher in the MDT plants than in the DT
plants, indicating that melatonin promoted the expression of genes associated with N-uptake and -metabolism.
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conditions.
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This led us to conclude that melatonin could, to a certain degree, increase nutrient uptake under drought
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Figure 7 Effects of melatonin on expression of key genes involved in N-assimilation in leaves under different
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treatment conditions. Total RNA was isolated from samples at specified time points (0-60 d), converted to cDNA,
and subjected to qRT-PCR. Expression levels were calculated relative to expression of Malus elongation factor 1
alpha gene (EF-1a; DQ341381) mRNA. Data are means � SD (n=4). Time points not labeled with same letter
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indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to
maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
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Figure 8 Effects of melatonin on expression of key genes involved in N-uptake in leaves under different
treatment conditions. Total RNA was isolated from samples at specified time points (0-60 d), converted to cDNA,
and subjected to qRT-PCR. Expression levels were calculated relative to expression of Malus elongation factor 1
alpha gene (EF-1a; DQ341381) mRNA. Data are means � SD (n=4). Time points not labeled with same letter
indicate significant differences at P0.05, based on Tukey?s multiple range tests. Treatments: CK, irrigated daily to
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maintain 75-85% field capacity; DT, irrigated daily to maintain 45-55% field capacity; MCK, irrigated daily to
maintain 75-85% field capacity plus 100 ?M melatonin; and MDT, irrigated daily to maintain 45-55% field
capacity plus 100 ?M melatonin.
4. Discussion
Drought is the most common environmental stress factor that limits plant development, and productivity by
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interrupting a series of physiological, biochemical, and molecular mechanisms (Gupta et al., 2014). Growth is
affected through depressed leaf water potential, higher electrolyte leakage, reduced biomass production, and
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altered plant water and nutrient relationships (Boomsma and Vyn, 2008). Our results showed that the induction
of drought was associated with significant decreases in PL, TD, DW, RWC, and RGR. In accordance with
previous findings (Zhang et al., 2015), our data also indicated that pretreatment with melatonin significantly
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alleviated this growth inhibition. Solid evidence has implicated melatonin as a growth promoter that also
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enhances plant tolerance to both abiotic and biotic stresses (Antoniou et al., 2017; Shi et al., 2015a). Synthesized
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by plants, this molecule acts as a potent antioxidant and/or regulator of growth and development (Arnao and
Hernandez-Ruiz, 2018). Plants can also absorb exogenously applied melatonin and accumulate it in their organs
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(Zhang et al., 2014), resulting in positive outcomes under some abiotic stress conditions (Zhang et al., 2013). We
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demonstrated here that exogenous melatonin treatment led to a significant boost in endogenous melatonin
concentrations, especially under the water deficit. This exogenous supplementation can improve plant tolerance
to extreme temperatures, salinity, ultraviolet radiation, heavy metals, and chemical stresses. It also markedly
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enhances drought tolerance by Triticum aestivum (wheat) (Cui et al., 2017), Zea mays (maize) (Fleta-Soriano et
al., 2017), Glycine max (Wei et al., 2015), Cucumis sativus (cucumber) (Zhang et al., 2013), and Malus plants
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(Li et al., 2015), as demonstrated by less electrolyte leakage, greater growth, and higher biomass production.
Although many stress factors suppress plant growth by targeting more than one physiological process,
photosynthesis is the most severely affected, as reflected by changes in biomass production that may be due to a
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lower demand for assimilates (Kalaji et al., 2014). We noted that drought treatment was associated with
significantly diminished photosynthetic performance, similar to that previously described (Sapeta et al., 2013;
Sun et al., 2018). Under such conditions, photosynthesis is influenced by several mechanisms, including
stomatal limitations, the primary drought-stress response that leads to reduced stomatal conductance (Warren et
al., 2011). When plants close their stomata to minimize water losses, that action also inhibits atmospheric CO 2
diffusion into the leaf and chloroplasts, thereby decreasing the rate of photosynthesis and limiting carbon
23
assimilation, which then reduces plant growth (Karimi et al., 2015). We investigated a possible correlation
between stomatal limitations and decreased photosynthesis and observed that, under drought conditions, the
stomatal apertures were smaller. Therefore, because of that vital physiological mechanism, plants that
demonstrate better capacity to control their stomatal behavior survive and grow well during periods of stress
(Saliendra et al., 1995). Another mechanism is a non-stomatal, or mesophyll, limitation that involves a decrease
in the leaf chlorophyll concentration (Pagter et al., 2005). A decline in the rate of photosynthesis is associated not
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only with stomatal closure but also with disturbances in the levels of leaf photosynthetic pigments (Li et al.,
2013). Pigments such as Chl a and Chl b have central roles in photosynthetic light reactions and are used as an
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index of drought tolerance. However, water deficits can induce the generation of reactive oxygen species in the
leaves and promote chlorophyll degradation. Similar to the results we have previously reported (Li et al., 2015),
the TCC was decreased in our current experiments. We noted a concomitant drop in both the rate of
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photosynthesis and behavior of the stomatal apertures, as well as lower chlorophyll concentrations, all of which
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suggest that the stomata and chlorophyll have critical roles in inhibiting photosynthetic activity under drought
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stress.
When plants are exposed to drought conditions, net photosynthesis is reduced due to regulatory
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mechanisms that include a decrease in stomatal conductance and chlorophyll concentrations (Bohnert and Jensen,
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1996). However, we found that, in the presence of melatonin, the rate of photosynthesis was maintained at a
higher level throughout the experimental period when compared with plants that did not receive exogenous
supplementation. Moreover, after 60 d of drought treatment, the stomatal apertures and Chl levels were larger in
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leaves from melatonin-treated plants. This might explain why plants in that treatment group were better able to
maintain higher photosynthetic values under stress. Therefore, even though photosynthesis might be reduced
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under drought conditions, exogenous supplementation with melatonin significantly eases those negative effects
by maintaining stable stomatal apertures and Chl levels. Levels of Chl in melatonin-treated plants and the
efficiency of Photosystem II are also maintained at levels similar to those measured in non-stressed plants (Wang
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et al., 2013). When stomatal conductance remains stable, exogenous melatonin can cause the stomata to reopen
in cucumber and Malus (Li et al., 2015; Zhang et al., 2013). Melatonin contributes to a more efficient
Photosystem II in apple trees, alleviating the inhibition in photosynthesis caused by drought stress and allowing
the leaves to maintain better water conservation, less electrolyte leakage, stable levels of Chl, greater stomatal
conductance, and higher photosynthetic performance (Li et al., 2015; Wang et al., 2013). Some protective effects
of melatonin on Chl concentrations have been reported for wheat (Turk et al., 2014), Cynodon dactylon (Shi et
24
al., 2015b), Lycopersicon esculentum (tomato) (Liu et al., 2015), and Citrus aurantium (Kostopoulou et al.,
2015). Similar results have been obtained with drought-stressed cucumber seedlings, in which the application of
melatonin reduced Chl degradation and increased the photosynthetic rate, thereby reversing the adverse effects
of a water deficit (Zhang et al., 2013). All of these reports, along with our current results, suggest that melatonin
benefits the photosynthetic process by influencing stomatal activity and photosynthetic pigments.
Drought stress reduces plant productivity by inhibiting the uptake of common minerals (Huang et al., 2007).
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Although water uptake and nutrient absorption are independent processes in the roots, the need for water to
support nutrient transport and plant growth makes those processes closely related. Most nutrients are absorbed
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by plant roots as ions, and water acts as a medium for their movement. Water deficits inhibit this flow of
nutrients within the soil, the absorption of those elements, and their uptake by roots (Fageria et al.,
2002).Therefore, drought-stressed plants will have lower nutrient absorption because less water is available and
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the diminished power of the roots hinders the uptake process (Sardans and Penuelas, 2012). Adequate absorption
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of minerals is important for the maintenance of plant structural integrity and key physiological processes, and
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any changes in mineral uptake may negatively affect plant metabolism (Dumlupinar et al., 2011). This might
explain why a lower RGR ultimately reduces biomass accumulations under a drought scenario (Ludlow and
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Muchow, 1990). He and Dijkstra (He and Dijkstra, 2014) have indicated that drought conditions negatively
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affect plant nutrition because the reduction in nutrient uptake is larger than the decrease in growth. This stress
also prevents the absorption of macro- and micronutrients by cherry tomato plants (Sanchez-Rodriguez et al.,
2010). A decline in soil moisture is linked with decreased uptake of N and P (Sardans and Penuelas, 2012) and it
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also prevents the uptake of Fe, Mn, Zn, etc. (Yasar et al., 2014). In this research, our data revealed that the
uptake of macro- and microelements are significantly decreased by drought-induced stress, but those inhibitions
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are not as severe when exogenous melatonin is part of the treatment. This molecule has an important modulating
influence on the mineral element composition of plants and mitigates stress by helping to up-regulate those
elements. For example, melatonin supplementation significantly ameliorates reductions in the concentrations of
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K, P, S, Mg, Fe, Cu, Mn, and Zn, and further increases Ca and B concentrations in maize seedlings under
low-temperature stress (Turk and Erdal, 2015). Likewise, we showed here that supplemental melatonin promoted
the uptake of nutrients under drought conditions.
The stable isotope investigation further verified that the concentration, uptake activity, and utilization rate
of ?15N were obviously increased by the addition of exogenous melatonin under drought conditions. In general,
the absorption of nitrogen declines in response to a water deficit, and activity of enzymes and transcript levels of
25
genes related to N-assimilation are reduced at the same time (Meng et al., 2016). This includes activity by NR,
GS, and GOGAT (Huang et al., 2018a; Rubio-Wilhelmi et al., 2012). Although we also found all of these
outcomes to be true in our current study, supplementing the irrigation solution with melatonin enabled plants to
maintain those parameters at a higher, more normal status. Furthermore, the transcript levels of genes related to
N-metabolism, i.e., NR, NiR, GS, Fd-GOGAT, and NADH-GOGAT, were up-regulated by the addition of
melatonin. In contrast, the transcript levels of AMT1;2, AMT1;5, AMT1;6, and AMT2;1 as well as NRT1;1,
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NRT2;4, NRT2;5, and NRT2;7 were suppressed by drought stress. Those results are consistent with those
reported by Huang et al. (2018a, b), who showed that most representative genes related to N-uptake were
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down-regulated in Malus in response to drought stress. In that research, the addition of melatonin again induced
higher expression of these genes, with both physiological and molecular data indicating that this exogenous
supplementation could promote the uptake of nitrogen under drought conditions. Nutrient uptake is a vital factor
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in conferring plant tolerance to a water deficit (Samarah et al., 2004). Thus, it is likely that melatonin helped
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improve drought tolerance by increasing the uptake of mineral elements in our plants.
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5. Conclusions
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In conclusion, we believe that our research group is the first to study the long-term effects that exogenous
melatonin has on the ionome and on uptake fluxes in apple plants under moderate drought stress. Application of
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100 ?M melatonin was linked to a significant boost in nutrient uptake. The presence of this molecule also
effectively slowed the drought-related declines in RGR and photosynthesis. Moreover, melatonin increased the
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concentration, uptake activity, and utilization rate of ?15N. Our data also provided evidence that melatonin
elevated the activity of enzymes related to N-assimilation. Results from qRT-PCR analysis showed that
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melatonin has an important role in regulating the metabolism and uptake of that nutrient. We suggest that this
positive influence of melatonin offers new opportunities in the field of agriculture, so that plants can be
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developed with greater drought tolerance and enhanced capacity to adapt to future environmental challenges.
Author Statement
Bowen Liang: Conceptualization, Methodology, Software, Formal Analysis, Investigation, Resources, Data
Curation, Writing ? Original Draft, Writing ? Review & Editing, Visualization. Changqing Ma: Methodology,
Software, Investigation. Zhijun Zhang: Investigation. Zhiwei Wei: Investigation. Tengteng Gao: Investigation.
Qi Zhao: Investigation. Fengwang Ma: Conceptualization, Validation, Resources, Writing ? Review & Editing,
Supervision, Project Administration, Funding Acquisition. Chao Li: Conceptualization, Validation, Resources,
26
Writing ? Review & Editing, Supervision, Project Administration, Funding Acquisition.
Declaration of interest
The authors declare that they have no conflict of interest.
Acknowledgements
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This work was supported by the State Key Program of the National Natural Science Foundation of China
(31330068) and by the earmarked fund for the China Agriculture Research System (CARS-27). The authors are
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grateful to Priscilla Licht for help in revising our English composition.
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