Plant Soil

DOI 10.1007/s11104-016-2965-3

REGULAR ARTICLE

Improving magnesium uptake, photosynthesis

and antioxidant enzyme activities of watermelon
by grafting onto pumpkin rootstock under low magnesium
Yuan Huang & Yanyan Jiao & Muhammad Azher Nawaz & Chen Chen &
Li Liu & Zhen Lu & Qiusheng Kong & Fei Cheng & Zhilong Bie

Received: 17 April 2016 / Accepted: 14 June 2016

# Springer International Publishing Switzerland 2016

Abstract
Background and aims Magnesium (Mg) is an essential
macronutrient that plays an important role in numerous
physiological and biochemical processes of plant. However,
Mg deficiency commonly occurs worldwide. Watermelon
is an important crop that often suffers from Mg deficiency.
This study aims to test whether watermelon performance
can be improved by grafting onto rootstocks under low Mg
and to clarify the underlying physiological mechanism.
Methods Self-grafted, bottle gourd (Jingxinzhen No.1)
and pumpkin (Jingxinzhen No.4) rootstock-grafted
plants were treated with three Mg concentrations:
2.0 mM (normal condition), 0.4 mM (moderate stress),
and 0.04 mM (severe stress) for 16 days under hydroponic conditions. Ungrafted watermelon and pumpkin
were treated with 2.0 mM and 0.04 mM for 12 days.
Results The growth of the plants was not affected by
0.4 mM Mg; however, plant growth decreased under
0.04 mM Mg in all graft combinations compared with
Yuan Huang and Yanyan Jiao contributed equally to this paper

control (2.0 mM Mg). Pumpkin rootstock grafting significantly increased watermelon growth under low Mg stress
(0.04 mM Mg), compared with self-grafted and bottle
gourd-grafted plants. The Mg2+ uptake of watermelon
plants was increased by grafting onto pumpkin rootstocks,
however, root-to-shoot transport capacity of Mg2+ was
similar compared with self-grafted plants under
0.04 mM Mg. Gene expression analysis showed that
magnesium transporter genes MGT1, MGT3, MGT4,
and MGT5 may play an important role in higher Mg2+
uptake of pumpkin root. The photosynthetic parameters
and activities of superoxide dismutase, peroxidase and
catalase were significantly higher, but malonaldehyde
(MDA) content were lower in the pumpkin rootstock
grafted plants compared with other graft combinations
under 0.04 mM Mg.
Conclusion Our results provide strong evidence that
pumpkin rootstock Jinxinzhen No. 4 grafting can improve watermelon performance under low Mg stress.
The enhanced plant performance is attributed to higher
root Mg2+ uptake and the improvement of photosynthesis and antioxidant enzyme activities.

Responsible Editor: Ismail Cakmak.

Electronic supplementary material The online version of this
article (doi:10.1007/s11104-016-2965-3) contains supplementary
material, which is available to authorized users.

Keywords Watermelon . Grafting . Mineral nutrition .

Magnesium deficiency . Photosynthesis . Antioxidant
system

Y. Huang : Y. Jiao : M. A. Nawaz : C. Chen : L. Liu :

Z. Lu : Q. Kong : F. Cheng : Z. Bie (*)
College of Horticulture and Forestry Sciences, Key Laboratory of
Horticultural Plant Biology, Ministry of Education, Huazhong
Agricultural University, Wuhan 430070, Peoples Republic of
China
e-mail: biezhilong@hotmail.com

Introduction
The adsorption of magnesium (Mg) to soil particles is
relatively weak because of its small hydration shell and

Plant Soil

thus results in high leaching losses leading to Mg deficiency in agricultural lands across the world (Maathuis 2009).
Mg is an essential macronutrient that is required for important functions related to chlorophyll synthesis, enzyme
activation, and membrane stability in plants (Knoop et al.
2005). It plays an important role in numerous physiological and biochemical processes affecting plant growth and
development (Bose et al. 2011; Verbruggen and Hermans
2013). Therefore, the performance of crops under low Mg
should be improved for agricultural production.
Mg deficiency decreases the Mg2+ concentration in
plants. The performance of plants under low Mg is often
influenced by their Mg2+ concentration (Verbruggen
and Hermans 2013; Mao et al. 2014). For instance,
Mg concentrations below 12 mg g1 leaf dry weight
are frequently leads to the onset of chlorosis (Hermans
et al. 2004; Ding et al. 2006; Verbruggen and Hermans
2013). While below this threshold level, plant growth is
reduced and biomass allocation among organs is modified (Verbruggen and Hermans 2013). However, K+ and
Ca2+ uptake is also affected by Mg deficiency (Ohno
and Grunes 1985; Ding et al. 2006).
Mg deficiency inhibits photosynthesis, it is a widely
observed phenomenon in many plant species including
Pinus radiata (Laing et al. 2000), sugar beet (Hermans
et al. 2004) and citrus (Yang et al. 2012). Mg deficiency
induced decrease in CO2 assimilation can be caused by
stomatal and non-stomatal factors (Laing et al. 2000;
Yang et al. 2012). In addition, photosynthesis can also
be reduced by an accumulation of assimilates in leaves
due to impaired phloem loading under low Mg (Cakmak
et al. 1994; Sheen 1994). Photosystem II (PSII) is implicated in the response of photosynthesis to environmental stress. The maximum photochemical efficiency
of photosystem II (Fv/fm) is an important parameter
used to monitor PSII function. In sugar beet, Mg deficiency decreases Fv/fm (Hermans et al. 2004).
The impairment in photosynthetic CO2 fixation in
Mg deficient leaves causes the photosynthetic electron
transport to use less amount of the absorbed light energy
captured by the light-harvesting system. In this case, the
photosynthetic electron transport chain is greatly reduced; under these conditions, reactive oxygen species
(ROS) in Mg deficient leaves are likely generated at a
high rate; as a consequence, the photosynthetic apparatus is damaged (Yang et al. 2012). ROS are scavenged
by an integrated system composed of antioxidant enzymes, such as superoxide dismutase (SOD, EC
1.15.1.1), peroxidase (POD, 1.11.1.7), and catalase

(CAT, EC 1.11.1.16) (Apel and Hirt 2004). Enhanced

antioxidant enzyme activities have been detected in the
Mg deficient leaves of mulberry (Tewari et al. 2006),
rice (Chou et al. 2011), citrus (Yang et al. 2012), and
coffee (da Silva et al. 2014).
Grafting of vegetable crops has developed very quickly in the last 50 years, in the past, grafting was widely
used in vegetable crops to limit the effects of soil-borne
pathogens (Albacete et al. 2015). However in the recent
years, grafting can be used as an effective method to
increase plant performance under abiotic stresses, such
as low/high temperature (Li et al. 2014), drought
(Pedroso et al. 2014), copper toxicity (Rouphael et al.
2008), salinity (Edelstein et al. 2005; Huang et al. 2010)
and alkalinity (Colla et al. 2010). In addition, rootstock
grafting can improve N, P, K and Fe uptake and utilization efficiency of plants (Pulgar et al. 2000; Rivero et al.
2005; Zhang et al. 2012; Huang et al. 2013a). Watermelon is an important crop that often suffers from Mg
deficiency. One way to reduce losses in the plant performance caused by Mg deficiency in plants would be to
graft Mg deficient cultivars onto rootstocks with high
efficiency of Mg uptake. Nevertheless, no published data
is available regarding the effects of rootstock grafting on
the plant performance under low Mg (Savvas et al. 2010).
This study aimed to investigate 1) whether rootstock
grafting can increase watermelon performance under
low Mg and 2) whether the increased growth is attributed
to improved Mg uptake and enhanced photosynthesis
and antioxidant enzyme activities.

Materials and methods

Plant materials and treatments
Experiment 1 response of grafted watermelon to low Mg
The experiment was conducted in a greenhouse at the
National Center of Vegetable Improvement in Huazhong
Agricultural University, Central China (latitude 30 27 N,
longitude 114 20 E and altitude 22 m above sea level).
Seeds of rootstocks Zaojia 8424 [Citrullus lanatus
(Thunb.) Matsum. and Nakai., Xinjiang Academy of
Agricultural Sciences, China], Jingxinzhen No.1
(Lagenaria siceraria Standl., Beijing Vegetable Research
Center, China), and Jingxinzhen No.4 (Cucurbita
moschata Duch., Beijing Vegetable Research Center, China) were sown in 50-cell plug trays filled with a 1:1:1 (v/v)

Plant Soil

mixture of peat, vermiculite, and perlite; after 5 days seeds

of scion Zaojia 8424 were sown. When the seedlings of
rootstock had developed one true leaf, scion was grafted
onto the rootstock by using the Binsertion grafting^ procedure as described by Hassell et al. (2008). In order to
maintain high humidity, seedlings were covered with a
layer of transparent plastic film, and seedlings were placed
in shade for 72 h. The plastic film was removed for a short
time during initial days to control relative humidity, and it
was completely removed after 7 d of grafting. The plants
were irrigated with tap water before grafting and
transplanting to hydroponic cultivation, and no nutrient
solution was used during this period. The self-grafted
Zaojia 8424 plants were used as control. When the third
true leaf emerged, the grafted plants were transplanted
into a 20 L plastic container (55 cm 33 cm 16 cm),
each containing nine plants. The plants were directly
exposed to different Mg treatments. The hydroponic nutrient solution formula developed by Hoagland and Arnon
(1950) was used as the base solution. The nutrient solution
was prepared with distilled water.
Three Mg concentrations [2 mM (control, normal condition), 0.4 mM (moderate stress) and 0.04 mM (severe
stress)] were used, using MgSO4 as the substance source.
Afterward, 1.6 mM and 1.96 mM Na2SO4 were added to
0.4 mM and 0.04 mM Mg treatment nutrient solutions,
respectively, to maintain the SO42 balance induced by
MgSO4 deficiency; in the base solution, MgSO4 was the
only source of SO42. In the nutrition solution, the MgSO4
concentrations under 2 mM, 0.4 mM, and 0.04 mM Mg
treatments were 240.83 mg L1, 48.17 mg L1, and
4.82 mg L1, respectively. The other macronutrients and
micronutrients were the same in the solutions with three
Mg levels except Na+.
In this experiment, nine treatments were adopted
with three graft combinations [Zaojia 8424/Zaojia
8424 (Z/Z), Zaojia 8424/Jingxinzhen No.1 (Z/JX1),
Zaojia 8424/Jingxinzhen No.4 (Z/JX4)] and three Mg
concentrations. These treatments were replicated four
times with nine plants in each replicate and were arranged in a randomized complete block design. The
nutrient solutions were renewed at an interval of 5 days
to avoid the excessive depletion of any particular ions.
The nutrient solutions were aerated using an air pump
every other hour during the day and were not aerated
during the night. During the culture, the plants were
grown under light conditions at an average photosynthetic photon-flux density of 300 mol m2 s1. The day
temperature of the greenhouse varied between 16 C

and 30 C, and its day relative humidity ranged from

55 % to 95 %. The plant samples were collected at day
16 after the low Mg treatment.
Experiment 2 response of ungrafted watermelon
and pumpkin to low Mg
In this experiment, ungrafted watermelon (Zaojia 8424)
and ungrafted pumpkin (Jingxinzhen No.4) were used as
materials. Seeds of Zaojia 8424 and Jingxinzhen No.4
were germinated in a dark chamber at 30 C, and then the
seeds were sown in 50-cell plug trays with mixed
substrate (peat:vermiculite:perlite =1:1:1, v/v). The plants
were irrigated with tap water before transplanting. Seven
days after seed sowing, the uniform seedlings
were transplanted into 10 L plastic containers
(32 cm 24 cm 14 cm), each container having six
plants. The plants were exposed to 2 mM and 0.04 mM Mg
treatments. The composition and management of nutrient
solution for 2 mM and 0.04 Mg treatment was the same as
in Experiment 1. Each treatment was replicated four times
with six plants in each replication. Plant samples were
harvested for measurement of plant growth, Mg2+ concentrations, and relative expression of magnesium transport genes at day 12 of low Mg treatment.
Determination of plant growth
Four plants per treatment were harvested and rinsed in
deionized water and were blotted carefully with tissue
paper. The grafted plants were divided into two parts,
namely, shoot (the part above graft union) and root (the
part below graft union). For ungrafted plants, the part
above the cotyledon node was regarded as the Bshoot^,
and the part below was the Broot^.
The shoot and root were placed in a forced air oven at
105 C for 15 min and at 70 C for 72 h to determine the
dry weights of the shoot, root, and whole plant (shoot +
root). The root dry weight/shoot dry weight (R/S) ratio
was subsequently calculated. Necrotic symptom was
evidently observed in the lower leaves of the selfgrafted and Jingxinzhen No.1 grafted plants at day 16
under 0.04 mM Mg. The ratio of necrotic leaf number to
the total leaf number was then calculated.
To investigate the Mg2+ concentration and size of
the rootstock before grafting, whole plant dry weight
and Mg 2+ concentration were also measured for
rootstock Z (Zaojia 8424), JX1 (Jingxinzhen No. 1)
and JX4 (Jingxinzhen No. 4).

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Analysis of mineral nutrition

Determination of leaf antioxidant enzyme activities

The dried samples of the roots and shoots were ground

using an IKA A11 analytical mill (IKA, Staufen, Germany) and were filtered through a 100-mesh sieve. The
Mg2+, K+, Ca2+, and Na+ concentrations were determined using a Varian Spectra AA 220 flame atomic
absorption spectrometer (Varian spectra AA 220,
Varian, Palo Alto, CA, USA) after digestion with
H2SO4-H2O2 (5:1) was performed. Then whole plant
Mg2+ uptake and root-to-shoot transport of Mg2+ were
calculated. Whole plant Mg2+ uptake = [(root dry weight
root Mg2+ concentration) + (shoot dry weight shoot
Mg2+ concentration)], and root-to-shoot transport of
Mg2+ = [(shoot dry weight shoot Mg2+ concentration)
/ Whole plant Mg2+ uptake] 100 %.

The fourth leaf from the top was used. The content of
malondialdehyde (MDA) was determined through the
thiobarbituric acid reaction as described by Heath and
Packer (1968). Protein content was determined according to Bradford (1976) using bovine serum albumin as
the standard. SOD activity was analyzed according to
the procedure defined by Dhindsa et al. (1981). POD
activity was assayed as described by Kochba et al.
(1977), and CAT activity was determined using the
method of Cakmak and Marschner (1992). The activities of SOD, POD, and CAT were expressed as unit/mg
protein (U/mg protein).

Determination of leaf photosynthesis

The fourth leaf from the top was used. The leaf chlorophyll relative content was measured by using a
SPAD-502 chlorophyll meter (Minolta Camera Co.
Ltd., Japan). Chlorophyll fluorescence was identified
using a portable pulse-modulated fluorometer (FMS-2,
Hansatech, Kings Lynn, Norfolk, UK) to determine
the state of PSII. The maximum efficiency of PSII
photochemistry (Fv/fm) was calculated, as described
by Maxwell and Johnson (2000). The leaves were kept
in the dark for 20 min before measurements were
conducted. During the chlorophyll fluorescence measurement, the intensities of the actinic and saturating
light settings were 400 and 3000 mol m2 s1 photosynthetically active radiation, respectively. The net
photosynthetic rate (Pn), stomatal conductance (Gs),
intercellular CO2 concentration (Ci), and transpiration
rate (Tr) were measured using a gas exchange system
(CIRAS-2, PP-systems, Hitchin, UK). The assimilatory chamber was controlled to maintain the leaf temperature, CO 2 concentration, and photosynthetic
photon-flux density at 25 C, 360 mol mol1, and
800 mol m2 s1, respectively. For the measurements
of Fv/fm and gas exchange parameters, four replicate
plants per treatment were measured between 10:00 and
11:30 AM on sunny days, the temperature and humidity were relatively uniform during the measured time
in the greenhouse. The greenhouse was equipped with
a fan and pad system, so the temperature can be
controlled to an appropriate level (around 25 C).

Total RNA extraction and gene expression analysis

Total RNA was isolated from roots of ungrafted watermelon and pumpkin with Trizol (Toyobo, Osaka, Japan)
according to the manufacturers instructions, and treated
with RNase-free DNase to remove contaminated DNA.
First-strand cDNA was synthesized using M-MuLV reverse transcriptase, and oligo-(dT)18 was used as a
primer following the manufacturers recommendation
(Fermentas, Shenzhen, China). Expression of the target
genes was measured by quantitative real-time PCR
(qRT-PCR). To investigate the putative magnesium
transporters (MGT) in watermelon and pumpkin, we
used MGT1 (AT1G80900), MGT3 (AT2G03620),
MGT4 (AT3G19640), MGT5 (AT4G28580), and MGT
7 (AT5G09690) genes sequence of Arabidopsis as
queries, and performed BLAST searches against the
Watermelon Genomics Database (http://www.icugi.
org/cgi-bin/ICuGI/tool/blast.cgi) and the transcriptome
sequences of pumpkin (NCBI/SRA database with
accession numbers SRP049398). Five putative
watermelon MGT genes (cla017218, cla022028,
cla001611, cla013012, and cla007547) and pumpkin
MGT genes (Unigene14430, CL8452.Contig3,
CL1285.Contig4, CL3760.Contig2, and CL8861.
Contig5) were found. The specific primers (Table 1)
were designed as described by Kong et al. (Kong et al.
2014) using Primer 3 software. The specificity of the
qRT-PCR reaction was checked with the melting curve
analysis (Online Resource 1). All the primers show high
specificity for each gene. In addition, the alignment of
experimental sequenced PCR product of MGT genes
and the sequence from bioinformatics analysis of watermelon and pumpkin (Online Resource 2), and the

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Table 1 List of primer sequences of watermelon and pumpkin used for qRT-PCR analysis
Gene

Accession Number

Forward primer(5-3)

Reverse primer(5-3)

MGT1

cla017218

TTCAGAAGGTTAGGGATG

ACAGGAGAAACAGGAGCA

MGT3

cla022028

GAAACCTGCTTCTGCTGT

ATCTAAGTCCCTGCCCTC

MGT4

cla001611

TGATAGCACCCTGAATAA

ACAAATGTTGCGGTAGTC

MGT5

cla013012

TTAGAGTGGGACAAGAATG

AAGAGGGTCAAGTAATAGGA

Watermelon

MGT7

cla007547

ACTGGAGGCTTACTTTATG

AGTTGATTTCGGTGATTG

ClACT*

Cla007792

CCATGTATGTTGCCATCCAG

GGATAGCATGGGGTAGAGCA

MGT1

Unigene14430

TGTTGCCCTGACTCGTAG

CAGACTGATCGCCATAAA

MGT3

CL8452.Contig3

GAAACCTGCTTCTGCTGT

ATCTAAGTCCCTGCCCTC

MGT4

CL1285.Contig4

GGACGAAATGGACGATGA

CGCTATGGGTAGTGCTTG

MGT5

CL3760.Contig2

GGTGCGTGACGAGATAGA

CAGATGAGGAACGGTGGC

Pumpkin

MGT7

CL8861.Contig5

GACTGCCTCCTTCTAATG

TTGCGGCTACTAATCTTG

Cm-EF1a* (ACT)

CL10326.contig1

GCCTCAAACTCCAAGGATGA

GGCTCCTTCTCGAGTTCCTT

* is reference gene (ACT) for qRT-PCR analysis

phylogenetic analysis of MGT gene in Arabidopsis,

watermelon, and pumpkin demonstrated that we are
studying real magnesium transport genes (Online
Resource 3). qRT-PCR was performed using a
LightCycler480 SYBR Green I Master kit (Roche Diagnostics, Mannheim, Germany) according to the protocols. PCR amplification included a 3 min preincubation step at 95 C, followed by 40 cycles of
95 C for 10 s, 54 C for 15 s, and 72 C for 15 s. The
PCR products were quantified by the LightCycler480
real time PCR detection system with a SYBR Green I
master kit (Roche Diagnostics, Mannheim, Germany)
and the data were analyzed by using 2-Ct method
(Livak and Schmittgen 2001). Ungrafted watermelon
and pumpkin under 2 mM Mg treatments were used as
control to calculate the relative gene expression.
Statistical analysis
Data were presented as the means of four replicates. A
two-factorial ANOVA was performed to examine the
effects of graft combination, Mg treatment, and their
interactions on the plant samples. Significance levels
were determined at * P 0.05, ** P 0.01, and
***
P 0.001; ns denoted non-significant differences.
Duncans multiple range test was used to evaluate significant differences between treatments at P 0.05. Statistical analyses were performed using SAS version 8.0
(SAS Institute Inc., Cary, NC, USA).

Results
Plant growth
For grafted plants, the shoot, root, and whole plant dry
weights and the R/S ratio were significantly affected by
graft combination, Mg treatment, and their interaction
(Table 2). The shoot, root, and whole dry weights of
Z/JX1 and Z/JX4 plants were significantly higher than
those of Z/Z (self-grafted) plants (Table 2). Among the
graft combinations, Z/JX4 yielded the highest shoot and
whole plant dry weights under 0.04 mM Mg (Table 2).
The shoot, root, and whole plant dry weights of the
plants treated with 0.4 and 0.04 mM Mg were lower
than those of the plants treated with 2 mM Mg (Table 2).
Compared with 2 mM Mg treatment, 0.04 mM Mg
treatment only slightly decreased the plant shoot dry
weight in Z/JX4 (27 %) than in Z/JX1 (51 %) and Z/Z
(31 %). Compared with the treatment with 2 mM Mg,
0.04 mM Mg treatment significantly increased the R/S
ratio of Z/Z and Z/JX1 plants. The R/S ratio of Z/JX4
was significantly lower than those of Z/JX1 and Z/Z
plants under 0.04 mM Mg (Table 2).
Under 0.4 mM Mg, no necrotic symptom induced by
Mg deficiency was observed in the leaf of plants
(Fig. 1). However, 0.04 mM Mg treatment induced
obvious leaf necrosis except Z/JX4 plants (Figs. 1, and
2). The necrotic leaf number/total leaf number per plant
was 29 % and 52 % in Z/Z and Z/JX1 plants,

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Table 2 Effect of graft combination and Mg treatment on the shoot, root, whole plant dry weight, and R/S ratio of the watermelon plants
Graft combination

Mg (mM)

Shoot dry weight (S)

Root dry weight (R)

Whole plant dry weight

R/S

g plant1

g plant1

g plant1

ratio

Zaojia 8424/Zaojia 8424

1.83 cd

100

0.23e

100

2.06 cd

0.131e

(Z/Z)

0.4

1.68de

92

0.20e

87

1.88de

0.121e
0.197ab

0.04

1.26e

69

0.19e

83

1.45e

Zaojia 8424/Jingxinzhen No.1

3.31a

100

0.61a

100

3.92a

0.184bc

(Z/JX1)

0.4

3.02ab

91

0.49b

80

3.50ab

0.166 cd
0.221a

0.04

1.63de

49

0.35d

57

1.98de

Zaojia 8424/Jingxinzhen No.4

3.10ab

100

0.42c

100

3.52ab

0.137e

(Z/JX4)

0.4

2.77b

89

0.39 cd

93

3.16b

0.142de

0.04

2.25c

73

0.33d

79

2.58c

0.147de

Analysis of variance
Graft combination (G)

***

***

***

***

Magnesium (M)

***

***

***

***

GM

***

**

**

The values represent the means of four plants

*, **, and *** denote P 0.05, 0.01, and 0.001, respectively; the values in each column followed by different letters are significantly
different (P 0.05) according to Duncans multiple range tests

respectively (Fig. 2). Bottle gourd (JX1) and pumpkin

(JX4) rootstocks were larger at grafting compared with
rootstock of Z (Zaojia 8424) (Fig. 3a). The whole plant
dry weight of JX1 and JX4 were about two times of Z
rootstock (Fig. 3b).
Ungrafted pumpkin show better plant growth than
watermelon under 2 mM and 0.04 mM Mg (Figs. 4,
and 5). Low Mg (0.04 mM) treatment significantly
reduced shoot and root dry weight of ungrafted pumpkin plants compared with 2 mM Mg, however,
ungrafted watermelon was not affected by 0.04 mM Mg
(Fig. 5). In fact, we observed low Mg2+ induced leaf
necrosis in pumpkin plants at day 10 after 0.04 mM Mg
treatment (Fig. 4), however, the leaf necrosis appeared
2 mM Mg

Z/Z

Z/JX1

much later (at day 14) in the ungrafted watermelon

plants (data not shown).
Mineral nutrition
For grafted plants, the Mg2+ and Na+ concentrations of the
shoot and root were significantly affected by graft combination, Mg treatment, and their interaction (Table 3).
Likewise, the shoot and root Ca2+ concentrations were
significantly influenced by graft combination and Mg
treatment (Table 3). The shoot and root K+ concentrations
were significantly affected by Mg treatment but were not
affected by grafting combination (Table 3). Compared
with the plants under 2 mM Mg, the plants treated with

0.4 mM Mg

Z/JX4

Z/Z

Z/JX1
2+

Fig. 1 Growth of grafted watermelon plants under different Mg

conditions; self-grafted (Zaojia 8424 grafted onto Zaojia 8424,
Z/Z), grafted onto Jingxinzhen No.1 (Z/JX1), and Jingxinzhen

Z/JX4

0.04 mM Mg

Z/Z

Z/JX1

Z/JX4

No.4 (Z/JX4) at day 16 after the exposure to 2 mM, 0.4 mM,

and 0.04 mM Mg conditions

Necrotic leaf number/total leaf

number per plant (%)

Plant Soil
60

50
40

30
20
10

0
Z/Z

Z/JX1

Z/JX4

Fig. 2 Ratio of necrotic leaf number to total leaf number per plant
under 0.04 mM Mg; the watermelon plants were self-grafted
(Zaojia 8424 grafted onto Zaojia 8424, Z/Z), grafted onto
Jingxinzhen No. 1 (Z/JX1), and Jingxinzhen No. 4 (Z/JX4); the

values are represented as means of four replicates; different letters

denote significantly different (P 0.05) according to Duncans
multiple range tests

0.4 and 0.04 mM Mg significantly decreased the Mg2+

concentrations in the shoot and root of the graft

combinations (Table 3). However, the Mg2+ concentrations in the shoot and root of Z/JX4 were significantly

JX1

JX4

27 cm

B
0.12

Whole plant dry weight (g/plant)

a
0.10
0.08
0.06
b
0.04
0.02
0.00

JX1

JX4

C
Whole plant Mg2+ concentration (mg/g DW)

Fig. 3 Plant growth (a, b) and

Mg2+ concentration (c) of
rootstock Z (Zaojia 8424), JX1
(Jingxinzhen No. 1) and JX4
(Jingxinzhen No. 4) before
grafting; the values are
represented as means of four
replicates; different letters denote
significantly different (P 0.05)
according to Duncans multiple
range tests

9
8

ab

JX1

JX4

b
7
6
5
4
3
2
1
0

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2 mM Mg

Fig. 4 Growth of ungrafted

watermelon (Zaojia 8424, Z) and
pumpkin (Jingxinzhen No. 4,
JX4) plants at day 10 after the
exposure to 2 mM and
0.04 mM Mg conditions

0.04 mM Mg

2 mM
2 mM
Mg
Mg

0.04
0.04
mM
mM
Mg
Mg

15cm
Zaojia 8424
(Z)

higher than those of Z/Z and Z/JX1 plants under 0.4 and
0.04 mM Mg. Under 0.04 mM Mg, the Mg2+ concentration in the shoot of Z/JX4 increased by 39.29 % and

34.48 % compared with those of Z/Z and Z/JX1, respectively, while the increased value of Mg2+ concentrations
in the root were 40.30 % and 23.68 % (Table 3). Whole

A
1.80
a

Shoot dry weight (g/plant)

1.60

2 mM

1.40

0.04 mM

1.20

1.00
0.80

0.60
0.40
0.20
0.00
Z

JX4

B
0.40
a

0.35

Root dry weight (g/plant)

Fig. 5 Shoot (a) and root (b) dry

weight of ungrafted watermelon
(Zaojia 8424, Z) and pumpkin
(Jingxinzhen No. 4, JX4) plants
under 2 mM and 0.04 mM Mg
conditions; the values are
represented as means of four
replicates; different letters denote
significantly different (P 0.05)
according to Duncans multiple
range tests

Jingxinzhen
No.4 (JX4)

2 mM

0.30

0.04 mM
b

0.25
0.20

c
c

0.15
0.10
0.05
0.00
Z

JX4

Plant Soil
Table 3 Effect of graft combination and Mg treatment on the shoot and root Mg2+, K+, Ca2+ and Na+ concentrations of the watermelon
plants
Graft combination

Mg (mM)

Shoot (mg g1 DW)

Mg2+

Zaojia 8424/Zaojia 8424 (Z/Z)

Root (mg g1 DW)

K+

Ca2+

Na+

Mg2+

K+

Ca2+

Na+

3.71b

48.97a

8.14bc

0.49de

2.62d

33.19a

2.30d

2.77d

0.4

1.49d

43.16b

9.72abc

0.85ab

1.75fe

16.45b

3.57bcd

5.14c

0.04

0.84 g

36.93 cd

5.33d

1.00a

1.34 g

16.03b

4.84ab

9.40b

Zaojia 8424/Jingxinzhen No.1

4.32a

43.56ab

9.81ab

0.36e

4.44b

30.28a

2.52 cd

2.08d

(Z/JX1)

0.4

0.99f

43.85ab

9.28abc

0.88ab

1.79e

18.86b

4.27abc

4.44c

0.04

0.87gf

34.09d

8.38abc

0.73bc

1.52 fg

13.37b

4.52ab

8.96b

Zaojia 8424/Jingxinzhen No.4

4.36a

40.75bc

9.66abc

0.36e

5.00a

30.17a

3.28bcd

2.22d

(Z/JX4)

0.4

1.77c

41.82bc

10.37a

0.46de

3.32c

15.82b

5.00ab

4.58c

0.04

1.17e

36.86 cd

7.62c

0.57 cd

1.88e

14.02b

6.03a

12.41a
*

Analysis of variance
Graft combination (G)

***

ns

***

***

ns

***

Magnesium (M)

***

***

***

***

***

***

***

***

GM

***

ns

ns

**

***

ns

ns

**

The values represent the means of four replicates

*, **, and *** denote P 0.05, 0.01, and 0.001, respectively, and ns as Bnot significant^; the values in each column followed by different
letters are significantly different (P 0.05) according to Duncans multiple range tests

plant Mg2+ uptake was enhanced when watermelon plans

were grafted onto pumpkin rootstock; however, root-toshoot Mg2+ transport capacity was similar compared with

Whole plant Mg2+ uptake (mg/plant)

A
20.00

15.00
10.00

2 mM
0.4 mM

5.00

0.04 mM

cde

cd

de

e
0.00
Z/Z

Z/JX1

Z/JX4

B
Root - to - shoot transport of Mg2+ (%)

Fig. 6 Whole plant Mg2+ uptake

(a) and root-to-shoot transport of
Mg2+ under 2 mM, 0.4 mM, and
0.04 mM Mg (b); the watermelon
plants were self-grafted (Zaojia
8424 grafted onto Zaojia 8424,
Z/Z), grafted onto Jingxinzhen
No. 1 (Z/JX1), and Jingxinzhen
No. 4 (Z/JX4); the values are
represented as means of four
replicates; different letters denote
significantly different (P 0.05)
according to Duncans multiple
range tests

self-grafted plants (Fig. 6). Compared with the plants with

2 mM Mg, the K+ concentration in the shoot and root of
the graft combinations was evidently decreased under 0.4

100.00

ab

80.00

cd

bc

ab
de

cd
2 mM
0.4 mM
0.04 mM

60.00
40.00
20.00
0.00
Z/Z

Z/JX1

Z/JX4

Plant Soil

and 0.04 mM Mg treatment except the shoot K+ concentration of Z/JX1 and Z/JX4 plants (Table 3). Compared
with 2 mM Mg, 0.04 mM Mg treatment obviously decreased the Ca2+ concentration in the shoot, and 0.4 mM
and 0.04 mM Mg treatments evidently increased the Ca2+
concentrations in the root of the graft combinations
(Table 3). The Ca2+ concentrations in the shoot of Z/JX1
and Z/JX4 plants were significantly higher than those of
Z/Z plants under 0.04 mM Mg (Table 3). The Na+ concentrations in the shoot of Z/JX4 were significantly lower
than those of Z/JX1 and Z/Z plants, but the Na+ concentration in the root of Z/JX4 was significantly higher than
those of Z/JX1 and Z/Z plants (Table 3).
Bottle gourd (JX1) show high Mg2+ concentration at
grafting compared with Z; however, no significant difference was observed for the pumpkin (JX4) rootstock
(Fig. 3c). Shoot Mg2+ concentration and root-to-shoot
transport capacity of Mg2+ in ungrafted watermelon and
pumpkin was similar under 2 mM and 0.04 mM Mg
(Fig. 7a, d); however, ungrafted pumpkin had significantly higher root Mg2+ concentration and whole plant
Mg2+ uptake compared with ungrafted watermelon
plants under 0.04 mM Mg (Fig. 7b, c).

Relative expression of root magnesium transport (MGT)

genes was conducted in ungrafted watermelon and
pumpkin plants. According to the BLAST searches
against the Watermelon Genomics Database and the
transcriptome sequences of pumpkin, we identified five
magnesium transport genes in watermelon and pumpkin
(Table 1). The mRNA levels of MGT1, MGT3, MGT4,
MGT5 in the roots of ungrafted pumpkin were up regulated significantly under 0.04 mM Mg treatment compared with the plants under 2 mM Mg (Fig. 8). On the
opposite, relative gene expression of MGT3 and MGT4
were significantly down regulated, while MGT1, MGT5
and MGT7 were not affected in ungrafted watermelon
roots under 0.04 mM Mg (Fig. 8).
Leaf photosynthesis
The leaf photosynthetic parameters were affected by
graft combination, Mg treatment, and their interaction
(Table 4). Compared with 2 mM Mg treatment,
0.4 mM Mg treatment did not influence the measured

C
5.00

2 mM
0.04 mM

2+

4.00
3.00
2.00
b

1.00

0.04 mM

5.00
4.00

3.00
2.00

1.00

D
2 mM
0.04 mM
b

2.50
2.00

1.50
d

1.00
0.50
0.00

2+

Root- to- shoot transport of Mg (%)

2+

Root Mg concentration (mg/g DW)

2 mM

6.00

JX4

3.50
3.00

7.00

0.00

0.00

8.00

6.00

Whole plant Mg uptake (mg/plant)

2+

Shoot Mg concentration (mg/g DW)

Gene expression of magnesium transport genes

JX4

100.00
90.00

2 mM

ab
bc

80.00

0.04 mM

70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00

JX4

Fig. 7 Shoot (a) and root (b) Mg2+ concentration, whole plant
Mg2+ uptake (c), and root-to-shoot transport of Mg2+ (d) of
ungrafted watermelon (Zaojia 8424, Z) and pumpkin (Jingxinzhen
No. 4, JX4) plants under 2 mM and 0.04 mM Mg conditions; the

JX4

values are represented as means of four replicates; different letters

denote significantly different (P 0.05) according to Duncans
multiple range tests

Plant Soil

2 mM
2.50
2.00
1.50

0.04 mM
a

ab
b

1.00
0.50
0.00
ZJ

JX4

Relative expression level of M GT3

Relative expression level of M GT5

D
2 mM
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00

0.04 mM
a

ZJ

JX4

E
2 mM
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00

0.04 mM
a

b
c

ZJ

JX4

Relative expression level of M GT7

Relativ e ex pres s ion lev el of M GT1

2 mM
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

ab

ZJ

0.04 mM
a

ab

JX4

Relative expression level of M GT4

C
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00

2 mM

0.04 mM
a

b
c

ZJ

JX4

Fig. 8 Relative expression level of magnesium transport genes

MGT1 (a), MGT3 (b), MGT4 (c), MGT5 (d), and MGT 7 (e) in the
root of ungrafted watermelon (Zaojia 8424, Z) and pumpkin
(Jingxinzhen No. 4, JX4) plants under 2 mM and 0.04 mM Mg

conditions; the values are represented as means of four replicates;

different letters denote significantly different (P 0.05) according
to Duncans multiple range tests

photosynthetic parameters in the graft combinations.

However, 0.04 mM Mg treatment significantly decreased SPAD, Fv/fm, Pn, Gs, and Tr in Z/Z and
Z/JX1 plants (Table 4). By contrast, SPAD, Fv/fm, Pn,
Gs, and Tr in Z/JX4 plants were not affected by
0.04 mM Mg treatment. The values of these parameters
under 0.04 mM Mg treatment were significantly higher
in Z/JX4 than in Z/Z and Z/JX1 (Table 4).

0.04 mM Mg significantly increased the MDA and

protein content of all graft combinations. The increased extent in MDA content was smaller in
Z/JX4 than in Z/Z and Z/JX4 plants (Table 5). The
SOD and POD activities were affected by graft combination, Mg treatment, and their interaction (Table 5).
The CAT activity was affected by graft combination
and its interaction with Mg treatment (Table 5). Compared with 2 mM Mg treatment, 0.4 mM Mg treatment significantly increased the SOD and CAT activities in Z/Z and the POD activity in Z/JX4. Under
0.04 mM Mg treatment, the SOD, POD and CAT
activities in Z/JX4 plants significantly increased
(Table 5). In particular, the SOD, POD, and CAT
activities in Z/JX4 plants were significantly higher
than those in Z/Z and Z/JX1 plants (Table 5).

Leaf antioxidant system

The MDA and protein contents were significantly
affected by Mg treatment and its interaction with graft
combination (Table 5). Compared with 2 mM Mg
treatment, 0.4 mM Mg treatment did not influence
the MDA and protein content. Nonetheless,

Plant Soil
Table 4 Effect of graft combination and Mg treatment on the leaf photosynthesis of the watermelon plants
Graft combination

Mg (mM)

Chlorophyll relative
content (SPAD)

Fv/fm

Pn (mol
CO2 m2 s1)

Gs (mol
H2O m2 s1)

Ci (mol
CO2 mol1)

Tr (mmol
H2O m2 s1)

Zaojia 8424/Zaojia 8424

44.73a

0.72a

17.97a

0.79a

300.77a

8.29ab

(Z/Z)

0.4

44.50a

0.72a

16.76a

0.74a

289.89ab

7.63ab

0.04

39.22b

0.69b

7.79b

0.13d

255.96c

3.65c

Zaojia 8424/Jingxinzhen No.1

44.33a

0.73a

18.56a

0.67abc

268.02bc

7.31ab

(Z/JX1)

0.4

43.83a

0.72a

17.47a

0.44c

258.47c

6.95b

0.04

39.67b

0.68b

8.52b

0.15d

248.01c

3.86c

Zaojia 8424/Jingxinzhen No.4

47.05a

0.73a

17.26a

0.71ab

286.58ab

7.57ab

(Z/JX4)

0.4

44.83a

0.72a

16.49a

0.49bc

268.9bc

6.92b

0.04

46.65a

0.73a

18.39a

0.68ab

284.59ab

8.42a
**

Analysis of variance
Graft combination (G)

***

**

**

**

**

Magnesium (M)

**

***

***

***

**

***

GM

***

***

**

***

The values represent the means of four replicates. Maximum photochemical efficiency of photosystem II (Fv/fm), net photosynthetic rate
(Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr)
*, **, and *** denote P 0.05, 0.01, and 0.001, respectively; the values in each column followed by different letters are significantly
different (P 0.05) according to Duncans multiple range tests

Discussion
Pumpkin rootstock grafting increases watermelon
performance under low Mg
Mg deficiency can inhibit plant growth and development (Maathuis 2009; Yang et al. 2012). Considering
that Mg is mobile within plants, its deficiency symptoms
first appear on lower (older) leaves before they appear
on upper (younger) leaves. One of the typical symptoms
of Mg deficiency is leaf necrosis (Cakmak and Kirkby
2008). The results of this study demonstrated that rootstock grafting could affect the growth of watermelon
under low Mg, but its efficiency is dependent on rootstock. Pumpkin rootstock (Jingxinzhen No.4) grafting
could increase the growth of watermelon under low Mg
(0.04 mM Mg). However, the bottle gourd (Jingxinzhen
No.1) rootstock grafting could not increase the watermelon performance under low Mg (Table 2, Figs. 1, and
2). Previous studies suggested that grafting onto pumpkin and wild watermelon rootstocks could improve the
watermelon performance under low potassium stress.
Nevertheless, no obvious positive effect was observed
on the bottle gourd grafted plants (Huang et al. 2013a).
Grafting onto pumpkin and bottle gourd could increase
performance of cucumber under salt stress (Colla et al.
2012; Huang et al. 2013a). Therefore, rootstock grafting

is an effective approach to increase watermelon performance under low Mg. However, the rootstock should be
carefully selected.

Mg uptake of watermelon was enhanced by grafting

onto pumpkin rootstock under low mg
Rootstock grafting could increase the N, P, and K uptake
of watermelon under low N, P, and K conditions (Pulgar
et al. 2000; Zhang et al. 2012; Huang et al. 2013a).
Several researchers have demonstrated that rootstock
grafting could also increase plant performance under
salt stress by limiting the Na+ uptake (Esta et al.
2005; Edelstein et al. 2011; Huang et al. 2013b). In this
study, pumpkin grafting increases watermelon plant
performance under low Mg through mainly through
higher Mg2+ uptake, since the root-to-shoot transport
capacity of Mg2+ between self-grafted watermelon and
pumpkin grafted plants were similar. The Mg2+ concentrations in the shoot of self-grafted (Z/Z) and bottle
gourd rootstock grafted (Z/JX1) plants were less than
1 mg/g dry weight, a value that can cause leaf necrosis
(Verbruggen and Hermans 2013). In the present study,
we can see the leaf necrosis in the self-grafted and bottle
gourd rootstock grafted plants at day 16 after
0.04 mM Mg treatment.

Plant Soil
Table 5 Effect of graft combination and Mg treatment on the leaf antioxidant system of the watermelon plants
Graft combination

Mg (mM)

MDA
(mol/g FW)

Protein
(mg/g FW)

SOD
(U/mg protein)

POD
(U/mg protein)

CAT
(U/mg protein)

Zaojia 8424/Zaojia 8424

6.36c

7.23d

31.57f

60.19 cd

56.86e

(Z/Z)

0.4

6.96c

6.73d

57.05ab

66.93bc

65.60 cd

0.04

11.66a

9.08a

36.85ef

57.86d

59.20de

Zaojia 8424/Jingxinzhen No.1

6.19c

7.36 cd

46.46cde

63.18 cd

69.85bc

(Z/JX1)

0.4

6.72c

7.18d

55.75abc

63.75 cd

69.08bc

0.04

10.42a

8.37b

48.68bcd

64.82 cd

64.82 cd

Zaojia 8424/Jingxinzhen No.4

6.83c

7.33d

40.26def

62.10 cd

71.24bc

(Z/JX4)

0.4

7.23bc

7.35 cd

49.46bcd

72.87ab

73.87ab

0.04

8.60b

8.04bc

59.69a

76.19a

80.62a

Analysis of variance
Graft combination (G)

ns

ns

**

***

Magnesium (M)

***

***

***

ns

GM

**

**

The values represent the means of four replicates

*, **, and *** denote P 0.05, 0.01, and 0.001, respectively, and ns as Bnot significant^; the values in each column followed by different
letters are significantly different (P 0.05) according to Duncans multiple range tests

Because plants are sessile organisms that cannot actively choose between alternative environments, they
may require a larger range of magnesium transport
functionality (e.g., in adapting to Mg2+ availability in
the soil) (Gebert et al. 2009). Mg uptake has shown at
least two types of uptake system, considered to be low
and high affinity transport systems. Low affinity transporters play an important role in the Mg uptake under
normal Mg (several mM) conditions (Tanoi et al. 2014);
while under low Mg (1 mM or less) uptake by plant
roots is mainly mediated by a high affinity transport
system that is dependent on metabolic energy (Rengel
and Robinson 1989; Tanoi et al. 2014).
A CorA-like gene family in Arabidopsis thaliana,
namely, MGT/MRS2, encodes several putative Mg2+
transport proteins that regulate Mg2+ uptake (Schock
et al. 2000; Li et al. 2001). The Mg2+ transport activities
of several MGTs have been characterized in heterologous
systems, including bacteria or yeast mutants lacking
Mg2+ transport capacity (Gebert et al. 2009). In these
systems, members of the MGT family vary in their
affinities for Mg2+ transport. Some of the MGT genes
have been functionally characterized. MGT1 is localized
in the plasma membrane and is expressed in the root hair
and elongation zones, in the vascular tissues, suggesting a
role in Mg2+ uptake in the root or translocation in the
above tissues (Li et al. 2001; Gebert et al. 2009). MGT3

are tonoplast localized and possibly involved in Mg2+

homeostasis (Gebert et al. 2009; Conn et al. 2011).
MGT5 is localized in the mitochondria and plays an
essential role in pollen development (Li et al. 2008),
and recent publication demonstrated that it was also
involved in Mg2+ uptake and transport (Xu et al. 2015).
MGT7 is involved in the regulation of root Mg2+ uptake,
compartmentalization in the cell and thereby the regulation of Mg2+ homeostasis (Gebert et al. 2009). However,
most of the studies related to Mg transporters were conducted on Arabidopsis thaliana; the expression pattern
and function of Mg transporters in other plants are quite
unknown. In the present study, response of MGT gene to
low Mg was quite different between ungrafted watermelon and pumpkin, higher expression of MGT1, MGT3,
MGT4, and MGT5 was found in the roots of pumpkin
under low Mg stress (Fig. 8). The pumpkin rootstock
grafted (Z/JX4) plant had a higher ability to uptake Mg2+
from the nutrient solution than the self-grafted (Z/Z) and
bottle gourd rootstock grafted (Z/JX1) plants. Therefore,
different expression level of Mg transporter genes may be
partially responsible for the differences between watermelon and pumpkin, the MGT homologous genes
(MGT1, MGT3, MGT4, and MGT5) may involved in
the higher Mg2+ uptake in the pumpkin root. However,
other Mg2+ transporter genes may also be involved in the
high uptake of Mg2+ in pumpkin roots, which needs

Plant Soil

further investigations. Such as recently it was found that

MGT6 functions as a plasma membrane Mg2+ transporter
in Arabidopsis thaliana, mediating Mg2+ uptake in the
roots especially when plants encounter a low Mg2+ environment (Mao et al. 2014).
In the present study, the increased Mg uptake is not
due to enhanced growth but that Mg uptake is the
primary cause for improved growth of pumpkin rootstock grafted plants. There are two lines of evidence for
this: (1) increased Mg concentrations in roots and shoots
and (2) increased transcription of Mg transporter genes.
The Mg deficiency in this study decreased the Mg2+
and K+ concentrations. This event was probably caused
by the synergistic effect between Mg2+ and K+ (Ding
et al. 2006). The synergistic behavior between Mg and
K could be explained partially by the role of Mg in
plants in Mg-ATP complexes, which are required for
active absorption of K across the root cells (Narwal et al.
1985). Na2SO4 was added to the Mg deficient treatment
to supply SO42 and the osmoticum of the nutrient
solution, which inevitably produced Na+ in the nutrient
solution. However, the damage induced by Mg deficiency could not be attributed to the high Na+ uptake in selfgrafted (Z/Z) and bottle gourd rootstock grafted (Z/JX1)
plants, at least to the short term experiment (16 days
after low Mg), as the shoot Na+ concentration was very
low (1 mg/g dry weight) under 0.04 mM Mg condition.
This situation cannot induce Na+ toxicity in plants.
However, it is likely that the plants that accumulated
higher root Na+ under low Mg conditions (0.04 mM)
were undergoing adjustments because of salinity stress.
In the present study, we used full strength Hoagland and
Arnon nutrient solution, so the high concentration of
nutrients probably has osmotic stress on small seedlings.
This might be the reason why we got high sodium
concentrations in the roots under low Mg conditions.
The shift from the osmotic phase of salinity to ion
toxicity is not time dependent. It can take place in a
short time, depending upon the plant species and the
environmental conditions (Munns and Tester 2008;
Tavakkoli et al. 2010). In the root, Mg deficient
(0.04 mM Mg) pumpkin rootstock grafted plants accumulated around 10 mg of Na+ per g of dry weight,
because pumpkin plants are relatively tolerant to salt
stress, the highly vacuolated root cortical cells of pumpkin roots could sequester more Na+, and thus limiting
the transport of Na+ to the shoot and increase scion
performance under salt stress. That is why pumpkin is
often used as salt tolerant rootstock (Huang et al. 2013b;

Lei et al. 2014). Under 0.04 mM Mg treatment, the Ca2+

concentration in the shoot was decreased. Similar results
have been reported in wheat (Ohno and Grunes 1985).
Pumpkin rootstock grafted plants have higher
photosynthesis under low Mg
Photosynthesis is related to the dry matter accumulation
of plants. Plants need Mg to harvest solar energy and
drive photochemistry. This case is probably one of the
most important physiological functions of Mg as the
central atom of chlorophyll (Hrtensteiner 2009). This
study suggests that chlorophyll bleaches by ROS was
accelerated by the Mg deficiency, as indicated by the
relative chlorophyll contents, except for the pumpkin
rootstock grafted plants (Z/JX4) (Table 4). PSII plays a
key role in the response of photosynthesis to environmental perturbations (Maxwell and Johnson 2000). In
their study, Hermans et al. (2004) realized that the Mg
deficient leaves in sugar beet had decreased maximum
Fm and Fv/fm. Our study showed that 0.04 mM Mg
treatment decreased the Fv/fm of the self-grafted (Z/Z)
and bottle gourd rootstock grafted (Z/JX1) plants, but
not of the pumpkin rootstock grafted (Z/JX4) plants.
This observation indicates that pumpkin rootstock
grafted plants have better performance under low Mg.
Mg is involved in the modulation of the activity of
key photosynthetic enzymes with the best illustrated
example of ribulose-1, 5-bisphosphate carboxylase/oxygenase, which catalyzes the first major step of carbon
fixation (Cakmak and Kirkby 2008). Low Mg levels
affect the net rate of photosynthesis across a wide variety of plant species (Laing et al. 2000; Hermans et al.
2004; Yang et al. 2012). However, in this study, the
decrease in CO2 assimilation of self-grafted (Z/Z) and
bottle gourd rootstock grafted (Z/JX1) plants might be
caused by stomatal limitation, as it was not accompanied
by high intracellular CO2 concentration upon Mg depletion. However, further direct evidence on stomatal aperture such as SEM (scanning electron microscope)
observation should be provided to support this statement. Some studies suggested that Mg deficiency increases the sugar concentration and changes sucrose
export from source leaves before any loss in photosynthetic activity (Hermans and Verbruggen 2005). Therefore, in the present study, the decline in photosynthetic
activity might also be due to some other reasons, such as
the increased sugar concentration in leaves, although the
sugar concentration was not measured in the study.

Plant Soil

Compared with 2 mM Mg treatment, 0.04 mM Mg

treatment did not influence the net photosynthesis rate
of the pumpkin rootstock grafted plants (Z/JX4). This
finding suggests that pumpkin grafted watermelon could
adapt to low Mg stress better than the self-grafted (Z/Z)
and bottle gourd rootstock grafted (Z/JX1) plants.

Enhancement of leaf antioxidant enzyme activities

of pumpkin rootstock grafted plants under low Mg
It is well known that decreased demand for photosynthates (because of inhibited assimilate transport and/or
decreased growth) downregulates carbon assimilation
and thus triggers oxidative stress, leading to the overproduction of ROS in plants, which are highly reactive, and
toxic, thereby damaging proteins, lipids, carbohydrates,
and DNA and ultimately resulting in oxidative stress (Gill
and Tuteja 2010). However, plants possess a welldeveloped antioxidant defense system comprising antioxidant enzymes, including SOD, POD, and CAT. The
combined action of these enzymes converts the potentially dangerous superoxide radical (O2) and hydrogen
peroxide (H2O2) into water (H2O) and molecular oxygen
(O2), thereby averting the cellular damage (Apel and Hirt
2004). The increase in antioxidant enzyme activities under Mg deficient conditions has been reported in many
species (Tewari et al. 2006; Chou et al. 2011; Yang et al.
2012; da Silva et al. 2014). Higher tolerant plants often
display a high antioxidant system under Mg stress (da
Silva et al. 2014). Nonetheless, the Mg deficiencyinduced up regulation in leaf antioxidant system does
not always sufficiently protect the Mg deficient leaves
from oxidative damage (Yang et al. 2012). Mg deficient
plants could accumulate a significant high amount of
MDA, a general indicator of lipid peroxidation (Tewari
et al. 2004). In this study, we determined that all plants
could protect leaves from oxidative damage under
0.4 mM Mg. Nevertheless, a higher increase in SOD,
POD, and CAT activities, but lower increase in MDA
content occurred in the plants grafted onto Jingxinzhen
No.4, but not in the self-grafted (Z/Z) and Jingxinzhen
No.1 Z/JX1 plants treated with 0.04 mM Mg. This
observation indicates the induction of antioxidant systems to reduce oxidative stress in pumpkin grafted plants.
Similar results have been reported in grafted cucumber
and tomato plants under salt stress (He et al. 2009; Huang
et al. 2010). Therefore, the enhancement of the activities
of leaf antioxidant enzymes is an important factor that

affects the performance of pumpkin rootstock grafted

(Z/JX4) plants to low Mg.
In the present study, the enzymes activities were measured in a less optimal way, which is by the enzyme assay
over the total protein concentration. This procedure has the
potential to introduce systemic bias into the results, because the change of total protein concentration can affect
the normalized enzyme activity. In an optimal case activities need to be normalized by the specific protein abundance measured by Western blotting. However, our results
strongly demonstrated that the performance of watermelon
plants can be obtained by grafting onto pumpkin rootstock
grafted (Z/JX4) plants under low Mg conditions.

Root Mg2+ concentration of pumpkin rootstock is

an important parameter to predict grafted watermelon
performance under low Mg
Generally, rootstocks are vigorous compared with cultivated varieties, and this feature promotes the widespread
use of rootstocks (Savvas et al. 2010; Albacete et al.
2015). Pumpkin is used as a rootstock for watermelon,
because of its higher tolerance to soil-borne diseases and
abiotic stress (Davis et al. 2008). In the present study, we
can observe that the ungrafted pumpkin is vigorous than
ungrafted watermelon. However, higher plant growth
reduction was also observed in the ungrafted pumpkin
plants under low Mg, suggesting that pumpkin itself
seems sensitive to low Mg stress than watermelon. In
fact, it is difficult to evaluate the tolerance of different
species to stress, since the vigor was quite different. If
watermelon and pumpkin with similar vigor were exposed to low Mg at the same time, an earlier damage
symptoms induced by low Mg may be observed in
watermelon plants. Grafting is the practice of joining
two parts (rootstock and scion) together permanently,
the use of root of rootstock during grafting is the main
source of the advantage. In the present study, the root
Mg2+ concentration was significantly higher (37 %) in
the ungrafted pumpkin than ungrafted watermelon under low Mg, which is consistent with the result of
watermelon plants grafted onto pumpkin rootstock.
Thus, root Mg2+ concentration of rootstock itself can
be used as an indicator to predict the performance of
grafted watermelon plants to low Mg. Although we have
investigated the expression of Mg2+ transport genes,
however molecular mechanism for efficient Mg2+ uptake of pumpkin roots needs further investigations.

Plant Soil

The plant performance under low Mg may be affected

by the preconditions when the Mg deficiency treatment
was started. In the present study, the size of the rootstock
JX1 and JX4 were larger at the time of grafting compared
to rootstock of Z (Fig. 3a, b). In addition, the rootstock JX1
had higher Mg2+ concentration than Z, while no significant
difference was found in the rootstock JX4 (Fig. 3c). Meanwhile, root Mg2+ concentration was significantly lower in
the ungrafted pumpkin plants than that in the ungrafted
watermelon plants under normal Mg condition (Fig. 7 b).
However, grafting onto JX1 cannot enhance watermelon
performance under low Mg condition, but the performance
of watermelon can be improved by grafting onto JX4
(pumpkin rootstock). These results suggested that relationship between plant size and Mg2+ concentration of rootstock before low Mg treatment and plant performance after
low Mg treatment was complex. However, further investigations are required to clarify the mechanism.

Conclusion
In conclusion, Jingxinzhen No.4 pumpkin rootstock
grafting can increase watermelon plant performance under
low Mg (0.04 mM). To the best of our knowledge, this
study is the first to examine the performance and physiological mechanism of grafted plants under low Mg. The
physiological mechanism of the improved performance of
grafted watermelon under low Mg is related to higher Mg
uptake, enhanced photosynthesis and antioxidant enzyme
activities. Higher expression level of Mg transport genes
(MGT1, MGT3, MGT4, and MGT5) may be partially
responsible for the higher uptake capacity of Mg2+ in
pumpkin roots compared with watermelon roots. Therefore, Jingxinzhen No.4 pumpkin rootstock grafting can
decrease the risk of yield loss of watermelon induced by
magnesium deficiency, and could be considered as a
promising rootstock for farmers under low magnesium
conditions. In addition, Jingxinzhen No.4 pumpkin rootstock grafting, which exhibits a highly efficient Mg uptake, may decrease environmental pollution.

Acknowledgments This work was supported by National Natural Science Foundation of China (31201660, 31471919), China
Agriculture Research System (CARS-26-16), the International
Science and Technology Cooperation Program of China
(2015DFG32310), and the Fundamental Research Funds for the
Central Universities (2013PY086).

References
Albacete A, Martnez-Andjar C, Martnez-Prez A, Thompson
AJ, Dodd IC, Prez-Alfocea F (2015) Unravelling rootstock
scion interactions to improve food security. J Exp Bot 66:
22112226
Apel K, Hirt H (2004) Reactive oxygen species: metabolism,
oxidative stress, and signal transduction. Annu Rev Plant
Biol 55:373379
Bose J, Babourina O, Rengel Z (2011) Role of magnesium in
alleviation of aluminium toxicity in plants. J Exp Bot 62:
22512264
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254
Cakmak I, Kirkby EA (2008) Role of magnesium in carbon
partitioning and alleviating photooxidative damage. Physiol
Plant 133:692704
Cakmak I, Marschner H (1992) Magnesium deficiency and high
light intensity enhance activities of superoxide dismutase,
ascorbate peroxidase and glutathione reductase in bean
leaves. Plant Physiol 98:12221227
Cakmak I, Hengeler C, Marschner H (1994) Changes in phloem
export of sucrose in leaves in response to phosphorus, potassium and magnesium deficiency in bean plants. J Exp Bot 45:
12511257
Chou TS, Chao YY, Huang WD, Hong CY, Kao CH (2011) Effect
of magnesium deficiency on antioxidant status and cadmium
toxicity in rice seedlings. J Plant Physiol 168:10211030
Colla G, Rouphael Y, Cardarelli M, Salerno A, Rea E (2010) The
effectiveness of grafting to improve alkalinity tolerance in
watermelon. Environ Exp Bot 68:283291
Colla G, Rouphael Y, Rea E, Cardarelli M (2012) Grafting cucumber plants enhance tolerance to sodium chloride and
sulfate salinization. Sci Hort 135:177185
Conn SJ, Conn V, Tyerman SD, Kaiser BN, Leigh RA, Gilliham
M (2011) Magnesium transporters, MGT2/MRS2-1 and
MGT3/MRS2-5, are important for magnesium partitioning
within Arabidopsis thaliana mesophyll vacuoles. New
Phytol 190:583594
da Silva DM, Brando IR, Alves JD, de Santos MO, de Souza
KRD, de Silveira HRO (2014) Physiological and biochemical impacts of magnesium-deficiency in two cultivars of
coffee. Plant Soil 382:133150
Davis AR, Perkins-Veazie P, Sakata Y, Lopez-Galarza S, Maroto JV,
Lee SG, Huh YC, Sun ZY, Miguel A, King SR, Cohen R, Lee
JM (2008) Cucurbit grafting. Crit. Rev. Plant Sci 27:5074
Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot 32:93101
Ding Y, Luo W, Xu G (2006) Characterisation of magnesium
nutrition and interaction of magnesium and potassium in rice.
Ann Appl Biol 149:111123
Edelstein M, Ben-Hur M, Cohen R, Burger Y, Ravina I (2005)
Boron and salinity effects on grafted and non-grafted melon
plants. Plant Soil 269:273284
Edelstein M, Plaut Z, Ben-Hur M (2011) Sodium and chloride
exclusion and retention by non-grafted and grafted melon and
Cucurbita plants. J Exp Bot 62:177184

Plant Soil
Esta MT, Martinez-Rodriguez MM, Perez-Alfocea F, Flowers TJ,
Bolarin MC (2005) Grafting raises the salt tolerance of
tomato through limiting the transport of sodium and chloride
to the shoot. J Exp Bot 56:703712
Gebert M, Meschenmoser K, Svidov S, Weghuber J, Schweyen
R, Eifler K, Lenz H, Weyand K, Knoop V (2009) A rootexpressed magnesium transporter of the MRS2/MGT gene
family in Arabidopsis thaliana allows for growth in lowMg2+ environments. Plant Cell 21:40184030
Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant
machinery in abiotic stress tolerance in crop plants. Plant
Physiol Biochem 48:909930
Hassell RL, Memmott F, Liere DG (2008) Grafting methods for
watermelon production. Hortscience 43:16771679
He Y, Zhu Z, Yang J, Ni X, Zhu B (2009) Grafting increases the
salt tolerance of tomato by improvement of photosynthesis
and enhancement of antioxidant enzymes activity. Environ
Exp Bot 66:270278
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189198
Hermans C, Verbruggen N (2005) Physiological characterization
of Mg deficiency in Arabidopsis thaliana. J Exp Bot 418:
21532161
Hermans C, Johnson GN, Strasser RJ, Verbruggen N (2004)
Physiological characterisation of magnesium deficiency in
sugar beet: acclimation to low magnesium differentially affects photosystems I and II. Planta 220:344355
Hoagland DR, Arnon DS (1950) The water culture method for
growing plants without soil. Calif Agric Exp Stat Circ 347:132
Hrtensteiner S (2009) Stay-green regulates chlorophyll and
chlorophyll-binding protein degradation during senescence.
Trends Plant Sci 14:155162
Huang Y, Bie ZL, He SP, Hua B, Zhen A, Liu ZX (2010)
Improving cucumber tolerance to major nutrients induced
salinity by grafting onto Cucurbita ficifolia. Environ Exp
Bot 69:3238
Huang Y, Li J, Hua B, Liu ZX, Fan ML, Bie ZL (2013a) Grafting
onto different rootstocks as a means to improve watermelon
tolerance to low potassium stress. Sci Hort 149:8085
Huang Y, Bie ZL, Liu PY, Niu ML, Zhen A, Liu ZX, Lei B, Gu DJ,
Lu C, Wang BT (2013b) Reciprocal grafting between cucumber and pumpkin demonstrates the roles of the rootstock
in the determination of cucumber salt tolerance and sodium
accumulation. Sci Hort 149:4754
Knoop V, Groth-Malonek M, Gebert M, Eifler K, Weyand K
(2005) Transport of magnesium and other divalent cations:
evolution of the 2-TM-GxN proteins in the MIT superfamily.
Mol Gen Genomics 274:205216
Kochba J, Lavee S, Spiegel-Rey P (1977) Differences in peroxidase activity and isoenzymes in embryogenic and nonembryogenic Shamouti orange ovular callus lines. Plant
Cell Physiol 18:463467
Kong QS, Yuan JX, Gao LY, Zhao S, Jiang W, Huang Y, Bie ZL
(2014) Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.
PLoS One 9(2):e90612. doi:10.1371/journal.pone.0090612
Laing W, Greer D, Sun O, Beets P, Lowe A, Payn T (2000)
Physiological impacts of Mg deficiency in Pinus radiata:
growth and photosynthesis. New Phytol 146:4757

Lei B, Huang Y, Sun JY, Xie JJ, Niu ML, Liu ZX, Fan ML, Bie ZL
(2014) Scanning ion-selective electrode technique and X-ray
microanalysis provides direct evidence of contrasting Na+
transport ability from root to shoot in salt-sensitive cucumber
and salt-tolerant pumpkin under NaCl stress. Physiol Plant
152:738748
Li L, Tutone AF, Drummond RS, Gardner RC, Luan S (2001) A
novel family of magnesium transport genes in Arabidopsis.
Plant Cell 13:27612775
Li LG, Sokolov LN, Yang YH, Li DP, Ting J, Pandy GK, Luan S
(2008) A mitochondrial magnesium transporter functions in
Arabidopsis pollen development. Mol Plant 1:675685
Li H, Wang F, Chen XJ, Shi K, Xia XJ, Considine MJ, Yu JQ,
Zhou YH (2014) The sub/supra-optimal temperature-induced
inhibition of photosynthesis and oxidative damage in cucumber leaves are alleviated by grafting onto figleaf gourd/luffa
rootstocks. Physiol Plant 152:571584
Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2
C
T method. methods 25:402408
Maathuis FJM (2009) Physiological functions of mineral macronutrients. Curr Opin Plant Biol 12:250258
Mao D, Chen J, Tian L, Liu Z, Yang L, Tang R, Li J, Lu C, Yang Y,
Shi J, Chen L, Li D, Luan S (2014) Arabidopsis transporter
MGT6 mediates magnesium uptake and is required for growth
under magnesium limitation. Plant Cell 26:22342248
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence a
practical guide. J Exp Bot 51:659668
Munns R, Tester M (2008) Mechanisms of salinity tolerance.
Annu. Rev. Plant Biol 59:651681
Narwal RP, Kumar V, Singh JP (1985) Potassium and magnesium
relationship in cowpea (Vigna unguiculata (L.) Walp.). Plant
Soil 86:129134
Ohno T, Grunes DL (1985) Potassium-magnesium interactions
affecting nutrient uptake by wheat forage. Soil Sci Soc Am
J 49:685690
Pedroso FK, Prudente DA, Bueno ACR, Machado EC, Ribeiro
RV (2014) Drought tolerance in citrus trees is enhanced by
rootstock-dependent changes in root growth and carbohydrate availability. Environ Exp Bot 101:2635
Pulgar G, Villora G, Moreno DA, Romero L (2000) Improving the
mineral nutrition in grafted watermelon plants: nitrogen metabolism. Biol Plant 43:607609
Rengel Z, Robinson DL (1989) Competitive Al3+ inhibition of net
Mg2+ uptake by intact Lolium multiflorum roots. Plant
Physiol 91:14071413
Rivero RM, Ruiz JM, Romero L (2005) Iron metabolism in tomato
and watermelon plants: influence of grafting. J Plant Nutr 27:
22212234
Rouphael Y, Cardarelli M, Rea E, Colla G (2008) Grafting of
cucumber as a means to minimize copper toxicity. Environ
Exp Bot 63:4958
Savvas D, Colla G, Rouphael Y, Schwarz D (2010) Amelioration
of heavy metal and nutrient stress in fruit vegetables by
grafting. Sci Hort 127:156161
Schock I, Gregan J, Steinhauser S, Schweyen R, Brennicke A,
Knoop V (2000) A member of a novel Arabidopsis thaliana
gene family of candidate Mg2+ ion transporters complements
a yeast mitochondrial group II intron-splicing mutant. Plant J
24:489501

Plant Soil
Sheen J (1994) Feedback control of gene expression. Photosynth
Res 39:427438
Tanoi K, Kobayashi NI, Saito T, Iwata N, Kamada R, Iwata R,
Suzuki H, Hirose A, Ohmae Y, Sugita R, Nakanishi TM
(2014) Effects of magnesium deficiency on magnesium uptake activity of rice root, evaluated using 28Mg as a tracer.
Plant Soil 384:6977
Tavakkoli E, Rengasamy P, McDonald GK (2010) High concentrations of Na+ and Cl ions in soil solution have simultaneous detrimental effects on growth of faba bean under
salinity stress. J Exp Bot 61:44494459
Tewari RK, Kumar P, Tewari N, Srivastava S, Sharma PN (2004)
Macronutrient deficiencies and differential antioxidant
responses-influence on the activity and expression of superoxide dismutase in maize. Plant Sci 166:687694
Tewari RK, Kumar P, Sharma PN (2006) Magnesium deficiency
induced oxidative stress and antioxidant responses in mulberry plants. Sci Hort 108:714

Verbruggen N, Hermans C (2013) Physiological and molecular

responses to magnesium nutritional imbalance in plants.
Plant Soil 368:8799
Xu XF, Wang B, Lou Y, Han WJ, Lu JY, Li DD, Zhu J, Yang ZN
(2015) Magnesium transporter 5 plays an important role in
Mg transport for male gametophyte development in
Arabidopsis. Plant J 84:925936
Yang GH, Yang LT, Jiang HX, Li Y, Wang P, Chen LS (2012)
Physiological impacts of magnesium-deficiency in citrus
seedlings: photosynthesis, antioxidant system and carbohydrates. Trees 26:12371250
Zhang L, Meng XX, Liu N, Yang JH, Zhang MF (2012) Effects of
grafting on phosphorus uptake and utilization of watermelon
at early stage under low phosphorus stress. J Fruit Sci 29:
120124