J Plant Growth Regul (2017) 36:60–70

DOI 10.1007/s00344-016-9618-x

The Possible Roles of Priming with ZnO Nanoparticles

in Mitigation of Salinity Stress in Lupine (Lupinus termis) Plants
Arafat Abdel Hamed Abdel Latef1,2 • Mona Fawzy Abu Alhmad1 •

Khaled Ebnalwaled Abdelfattah3

Received: 6 April 2016 / Accepted: 4 May 2016 / Published online: 12 July 2016
Ó Springer Science+Business Media New York 2016

Abstract To our knowledge, little attention has been paid
to evaluating ZnO nanoparticles (ZNPs) roles in plants
grown under salinity stress. In this study, seeds of lupine
(Lupinus termis) plants were grown in plastic pots and
exposed to 0 (control) and 150 (S) mM NaCl with or
without priming with different concentrations of ZnO
[20 mg L-1 (ZNPs1), 40 mg L-1 (ZNPs2), and 60 mg
L-1 (ZNPs3)] for 20 days. Salinized plants showed a
reduction in plant growth parameters (root length, shoot
length, fresh weight, and dry weight) and in the contents
of photosynthetic pigments (chlorophyll a and b, and
carotenoids) and Zn, as well as in the activity of catalase
(CAT) against control plants. On the other side, salinity
stress boosted the contents of organic solutes (soluble
sugar, soluble protein, total free amino acids, and proline),
total phenols, malondialdehyde (MDA), ascorbic acid and
Na, as well as the activities of superoxide dismutase
(SOD), peroxidase (POD), and ascorbate peroxidase
(APX) in stressed plants over control plants. However,
seed-priming with ZNPs mostly stimulated growth of
stressed plants, which was accompanied by reinforcement
in the levels of photosynthetic pigments, organic solutes,
total phenols, ascorbic acid and Zn, as well as in the
& Arafat Abdel Hamed Abdel Latef
moawad76@gmail.com; arafat.moawad@sci.svu.edu;
a.moawd@tu.edu.sa
1 others 2007).
Botany Department, Faculty of Science, South Valley
University, Qena 83523, Egypt
2 (NPs) and nanoconjugates have been in high demand over
Biology Department, College of Applied Medical Science,
Turabah Branch, Taif University, Taif 21955, Saudi Arabia
3 agriculture, or pharmaceutical industries (Gerber and
Electronics & Nano Devices Lab., Physics Department,
Faculty of Science, South Valley University, Qena 83523,
Egypt
activities of SOD, CAT, POD, and APX enzymes over
stressed plants alone. On the contrary, priming with ZNPs
caused a decrement in the contents of MDA and Na in
stressed plants relative to salinized plants alone. It is
worthy to mention that, this improvement in salt tolerance
of plants primed with ZNPs was more obvious in plants
primed with ZNPs3 and grown both in unstressed and
stressed regimes. Thus, our findings suggest that seed-
priming with ZNPs, especially 60 mg L-1 ZnO is an
effective strategy that can be used to enhance salt toler-
ance of lupine plants.
Keywords Antioxidant enzymes  Ascorbic acid 
Nanoparticles  Organic solutes  Salinity  Zinc oxide
Introduction
Salinity is a prevalent abiotic stressor that severely affects
crop yield in some specific geographical regions, especially
in arid and semiarid regions such as Egypt. Egypt is a
country that has a history of 5000 years of experience in
irrigation (Mohamed and others 2007). However, a major
proportion of governmental funds is invested in addressing
serious salinity issues, because 33 % of the cultivated lands
are already salinized throughout the country. Effective
remediation of the salinity stress hence, is a major initiative
to be taken to secure sustainable crop yield (Mohamed and
Multimodal applications of engineered nanoparticles
the last few decades in the textile, cosmetic, electronic,
Lang 2006; Nel and others 2006; Choudhury and others
2011, 2012; Siddiqui and others 2015). In particular,

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J Plant Growth Regul (2017) 36:60–70 61

applications of NPs as pesticides and fertilizers in crop Materials and Methods

fields are in high demand among farmers and profit-
finding industries. Nanotechnology based on new gener- Synthesis of ZnO Nanocrystals
ations NPs might offer enhanced food productivity by
lowering or minimizing pathogenic invasions (Choudhury ZnO nanocrystals (ZNCs) were synthesized by the direct
and others 2011). These nanoproducts allow small quan- chemical precipitation method. The preparation was started
tities of fertilizers/pesticides to be used effectively over a by solution A that contains 0.1 M of zinc nitrate dissolved
given period of time, while their design allows for sus- in distilled water. Simultaneously, another solution B
tained release and resistance from severe environmental (0.4 M of sodium hydroxide dissolved in distilled water)
hardness, compared to the conventional chemicals which was prepared. Next, solution B was added dropwise to
are prone to frequent leaching, evaporation, photolytic– solution A under gentle stirring and at a temperature of
hydrolytic damages, and microbial degradation (Kah 60 °C. This step takes approximately 40 min. The resultant
2015). mixture was then sealed and stirred for 2 h under contin-
Zinc oxide (ZnO) NPs (ZNPs), one of the most fre- uous heating (60 °C). The obtained precipitate was sepa-
quently used nanoproducts, are used in food packaging rated and washed several times with deionized water.
and drugs due to their superior antimicrobial efficacy Finally, the products were dried at 60 °C for about 8 h.
(Brayner and others 2006; Jones and others 2008). ZnO The shape and actual size of the ZNCs were primarily
NPs are also being frequently used in sun-protective determined with high-resolution transmission electron
lotions, wall paints, ceramic manufactures, or sporting microscopy (HRTEM). The average particle size was
goods (Fan and Lu 2005; Singh and Nanda 2014). The 21.3 nm for the prepared samples (Fig. 1).
increased popularity of using Zn in fertilizers and pesti-
cides is also commissioned due to its natural demand as a Plant Growth Conditions
micronutrient in the body (Prasad and others 2012).
Moreover, Zn is also an important cofactor in essential Lupine seeds, used for the present study were procured
biocatalytic enzymes including oxidoreductases, trans- from the local seed center, Qena, Egypt. Healthy lupine
ferases, hydrolases, ligases, and isomerases (Auld 2001). seeds were surface sterilized with 70 % ethanol for 2 min,
Previous literature has already reported both positive and then washed three times with sterilized water. Then the
negative effects of ZNPs on the physio-biochemical seeds were divided into four groups according to the
attributes of plants using different model systems (Ma- priming solution as follows:
hajan and others 2011; Prasad and others 2012; de la Rosa
1. The 1st (control) and 2nd [salinity (S)] sets were
and others 2013; Patra and others 2013; Raliya and
priming with distilled water.
Tarafdar 2013; Sedghi and others 2013; Ramesh and
2. The 3rd (ZNPs1) and 4th (ZNPs1 ? S) sets were
others 2014). However, the impact of nanoparticles on
primed with 20 mg L-1 ZnO for 12 h.
plants vary with age, species, and the features of the
3. The 5th (ZNPs2) and 6th (ZNPs2 ? S) sets were
nanoparticles (Burman and others 2013).
primed with 40 mg L-1 ZnO for 12 h.
Lupine (Lupinus termis) is grown in Egypt or in the
general Mediterranean region for its edible seeds (Khodary
2004). Lupine is one of the most vital plants from nutri-
tional and medical points of view (Khodary 2004).
Little attention has been paid to studying the impact of
ZNPs application on plants grown under saline conditions.
To our knowledge, this is the first report dealing with the
impact of priming with ZNPs on salinized lupine. There-
fore, this study was carried out to investigate whether the
phytoremediation properties of ZNPs could be used on
lupine plants for their healthy growth, by lowering the
persistent saline stress. Growth parameters, photosynthetic
pigments, organic solutes, total phenols, oxidative stress,
antioxidant enzymes, and ascorbic acid in lupine seeds
treated with sodium chloride (NaCl) or NaCl plus priming
with different concentrations of ZNPs were assessed to
evaluate the possible roles of ZNPs in mitigating the Fig. 1 Transmission electron microscopy (TEM) images of the ZnO
adverse impacts of salt stress. nanoparticles (ZNPs) samples

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62
4. The 7th (ZNPs3) and 8th (ZNPs3 ? S) sets were
primed with 60 mg L-1 ZnO for 12 h.
After priming, the thoroughly washed seeds were sown
(5 five seeds/pot) in plastic pots filled with 2 kg of dried
soil. The pots were kept in the wire house of the experi-
mental farm of South Valley University, Qena, Egypt
located at latitude 26°110 2500 N and longitude 32°440 4200 E
under natural conditions of temperature, light, and
humidity during the growing season of 2015. The pots were
arranged in a completely randomized design in a factorial
arrangement with three replications. At the time of sowing,
the seeds were irrigated at field capacity with 0 (control)
and 150 (S) mM NaCl. The salt solution was added once
and leaching was avoided by maintaining soil water below
field capacity at all times. The pots were then irrigated at
field capacity with normal water through the whole
experimental period (20 days). Twenty days after sowing,
the lupine plants were harvested for further analyses.
Growth Traits
The lengths of roots and shoots were measured using a
measuring scale. Additionally, fresh weights of full-length
plants were recorded, followed by drying of the samples at
80 °C in an oven, and dry weights of the plants were then
measured.
Photosynthetic Pigments
According to Lichtenthaler and Wellburn (1983), the
contents of photosynthetic pigments (chlorophyll a and b,
and carotenoids) in fresh leaves were assessed spec-
trophotometrically. The pigment extract was determined
versus a blank of pure 80 % acetone at 663, 644, and
452.5 nm for chlorophyll a, chlorophyll b, and carotenoid
contents, respectively.
Organic Solutes (Soluble Sugar, Soluble Protein,
Total Free Amino Acids, and Proline)
The anthrone sulfuric acid method described by Irigoyen
and others (1992) was used to estimate soluble sugar
content. The absorbance was measured spectrophotomet-
rically at 620 nm against a blank (distilled water ? an-
throne reagent). The Bradford (1976) method using bovine
serum albumin as a standard was used to assess soluble
protein content. Total free amino acids content was mea-
sured using the method of Lee and Takanashi (1966). The
absorbance was read at 570 nm against a blank (only dis-
tilled water and the same reagent). The proline content was
estimated according to Bates and others (1973) and the
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J Plant Growth Regul (2017) 36:60–70
absorbance was measured at 520 nm using toluene as a
blank.
Total Phenols
Total phenol content was assayed by the Folin–Ciocalteu
reagent (Skerget and others 2005). The absorbance was
measured spectrophotometrically at 760 nm.
Malondialdehyde (MDA)
The thiobarbituric acid (TBA) reaction cited in Abdel Latef
and Tran (2016) was used to determine MDA content in
fresh leaf samples. The absorbances were read at 532, 600,
and 450 nm.
Assays of Antioxidant Enzyme Activities
Samples were extracted from fresh leaves based on the
method, as cited by Ahmad and others (2016). The activity
of superoxide dismutase (SOD; EC 1.15.1.1) was deter-
mined using the nitro blue tetrazolium (NBT) method
described by Giannopolitis and Ries (1977). Catalase
(CAT; EC 1.11.1.6) activity was assessed according to the
method previously described by Aebi (1984). Peroxidase
(POD; EC 1.11.1.7) activity was estimated according to the
method described by Maehly and Chance (1954) and
Klapheck and others (1990). Ascorbate peroxidase (APX;
EC 1.11.1.11) activity was measured by the method of
Chen and Asada (1992).
Ascorbic Acid
Ascorbic acid content of fresh leaves was estimated
according to Mukherjee and Choudhuri (1983). The
absorbance was estimated spectrophotometrically at
525 nm.
Na and Zn
The determination of Na and Zn contents was carried out in
ground-dried samples and analyzed by atomic absorption
spectrometry according to (Allen 1989).
Statistical Analysis
All data shown are the mean values. Data were statistically
analyzed by the analysis of variance (ANOVA) with SAS
software (Version 9.1; SAS Institute, Cary, NC, USA) and
Duncan’s multiple range test was calculated at the 0.05
level of significance (P \ 0.05). Data represented in the
J Plant Growth Regul (2017) 36:60–70 63

tables and figures are mean ± standard deviation (SD) of under salt stress (Fig. 2a, b). Accordingly, the three concen-
three independent replicates of each treatment. trations of ZNPs can be arranged in the following order from
most to least effective: ZNPs3 [ ZNPs2 [ ZNPs1.

Results Effect of Priming with ZNPs on Photosynthetic

Effect of Priming with ZNPs on Growth Traits
of Lupine Plants under Normal and Salt Stress
Conditions
The lengths of roots and shoots and weights (fresh and dry)
of lupine-treated plants were assessed to evaluate the
impact of priming with different concentration of ZNPs on
plant growth under salinity stress (Fig. 2a, b). Salinity
pressure provoked a significant suppression in root length
(51.00 %), shoot length (33.33 %), fresh weight (32.45 %),
and dry weight (47.92 %) versus control. Seed-priming
with ZNPs mostly induced an elevation in the formerly
mentioned growth characteristics (Fig. 2a, b). This pro-
motion influence of ZNPs was not only observed in the
growth of salinized plants, but also elevated the growth in
plants subjected to unstressed regimes. Interestingly, the
maximum increment in growth parameters was recorded in
stressed plants primed with ZNPs3 and reached in
ZNPs3 ? S plants to 80.07, 43.30, 36.71, and 52.00 % in
root length, shoot length, fresh weight, and dry weight,
respectively over S plants alone. This indicates that ZNPs3 is
probably the best effective nanoparticle to boost plant growth
Pigments of Lupine Plants under Normal and Salt
Stress Conditions
Chlorophyll a, chlorophyll b and carotenoids decreased by
41.13, 44.85, and 39.13 %, respectively in salinized plants
relative to control plants (Table 1). However, seed-priming
with ZNPs alone as well as in combination with NaCl
mostly enhanced photosynthetic pigments in unstressed
and stressed plants. An increase by 68.67, 43.28, and
85.71 % in chlorophyll a, chlorophyll b, and carotenoids,
respectively was observed in the ZNPs3 ? S treatment
compared to the S treatment alone (Table 1).
Effect of Priming with ZNPs on Organic Solutes
and Total Phenols of Lupine Plants under Normal
and Salt Stress Conditions
NaCl stress resulted in a marked accumulation in soluble
sugar (21.63 %), soluble protein (39.18 %), total free amino
acids (44.29 %), proline (60.78 %), and total phenols
(58.33 %) over control plants (Fig. 3a, b). Seed-priming
with ZNPs especially ZNPs3 alone as well as in combination
with NaCl induced a further accumulation in organic solutes

Fig. 2 Effects of salinity stress

and seed-priming with ZnO
nanoparticles (ZNPs) on a root
length and shoot length and
b fresh weight and dry weight in
20-day-old lupine plants. Bars
represent standard deviation
(±SD) of the means (n = 3).
Different letters indicate
significant differences among
the treatments at P \ 0.05,
according to Duncan’s multiple
range test. S, 150 mM NaCl;
ZNPs1, 20 mg L-1 ZnO;
ZNPs2, 40 mg L-1 ZnO;
ZNPs3, 60 mg L-1 ZnO

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64 J Plant Growth Regul (2017) 36:60–70

Table 1 Effects of salinity stress and seed-priming with ZnO nanoparticles (ZNPs) on photosynthetic pigments (mg g-1 FW) in 20-day-old
lupine leaves
Traits Control Salinity (S) ZNPs1 ZNPs1 ? S ZNPs2 ZNPs2 ? S ZNPs3 ZNPs3 ? S

Chlorophyll 2.82 ± 0.16b 1.66 ± 0.20d 2.86 ± 0.25b 2.22 ± 0.42c 2.90 ± 0.23b 2.57 ± 0.38bc 3.64 ± 0.31a 2.80 ± 0.11b
a
Chlorophyll 2.43 ± 0.21d 1.34 ± 0.25h 2.72 ± 0.26c 1.50 ± 0.18g 2.92 ± 0.21b 1.67 ± 0.16f 3.24 ± 0.24a 1.92 ± 0.24e
b
Carotenoids 0.23 ± 0.25c 0.14 ± 0.19d 0.27 ± 0.27bc 0.17 ± 020d 0.30 ± 0.29b 0.25 ± 0.21c 0.35 ± 0.32a 0.26 ± 0.23bc
Values are means standard deviation (± SD) of three independent replications (n = 3)
Different letters within the row indicate statistically significant differences between treatments, according to Duncan’s multiple range test
(P \ 0.05)
FW, fresh weight; S, 150 mM NaCl; ZNPs1, 20 mg L-1 ZnO; ZNPs2, 40 mg L-1 ZnO; ZNPs3, 60 mg L-1 ZnO

Fig. 3 Effects of salinity stress and seed-priming with ZnO nanopar-
ticles (ZNPs) on a organic solutes (soluble sugar, soluble protein,
total free amino acids, and proline) and b total phenols in 20-day-old
lupine plants. Bars represent standard deviation (±SD) of the means
(n = 3). Different letters indicate significant differences among the
treatments at P \ 0.05, according to Duncan’s multiple range test.
DW, dry weight; GAE, gallic acid equivalent; S, 150 mM NaCl;
ZNPs1, 20 mg L-1 ZnO; ZNPs2, 40 mg L-1 ZnO; ZNPs3, 60 mg
L-1 ZnO

and total phenols compared to control and NaCl-stressed free amino acids, proline, and total phenols, respectively was
plants (Fig. 3a, b). An increase by 36.84, 68.40, 71.56, observed in stressed plants primed with ZNPs3 compared to
32.31, and 45.65 % in soluble sugar, soluble protein, total the plants treated with NaCl alone (Fig. 3a, b).

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J Plant Growth Regul (2017) 36:60–70
Effect of Priming with ZNPs on MDA of Lupine
Plants under Normal and Salt Stress Conditions
Exposure of lupine plants to 150 mM NaCl dramatically
accumulated MDA content by 83.76 % over control plants
(Fig. 4). Priming with ZNPs1, ZNPs2, and ZNPs3 reduced
MDA content by 11.96, 39.24, and 74.23 %, respectively
compared to control (Fig. 4). In salinized plants, priming
with ZNPs1, ZNPs2, and ZNPs3 hampered the accumula-
tion of MDA by 11.56, 21.12, and 41.91 %, respectively
against salinized plants alone (Fig. 4).
Effect of Priming with ZNPs on Antioxidant
Enzymes of Lupine Plants under Normal and Salt
Stress Conditions
The data in Table 2 illustrated that salinity stress resulted
in an increment in the activity of SOD, POD, and APX by
44.50, 79.30, and 81.25 % over control. On the other side,
CAT activity decreased by 35.96 % under salinity stress in
comparison to control. Seed-priming with ZNPs alone or
plus 150 mM NaCl mostly stimulated the activity of all
tested antioxidant enzymes and this stimulation was more
obvious in ZNPs3 plants than ZNPs1 and ZNPs2 plants,
respectively (Table 2). In ZNPs3 ? S treatment, the stim-
ulation in SOD, CAT, POD, and APX reached to 46.95,
100.73, 47.52, and 61.66 %, respectively over S treatment
alone (Table 2).
Effect of Priming with ZNPs on Ascorbic Acid
of Lupine Plants under Normal and Salt Stress
Conditions
Figure 5 showed that, treatment with NaCl caused a sig-
nificant increase (44.54 %) in the content of ascorbic acid
over control. Ascorbic acid content of control plants
Fig. 4 Effects of salinity stress and seed-priming with ZnO nanopar-
ticles (ZNPs) on malondialdehyde content (MDA) in 20-day-old
lupine leaves. Bars represent standard deviation (±SD) of the means
(n = 3). Different letters indicate significant differences among the
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
65
primed with ZNPs1, ZNPs2, and ZNPs3 increased by
23.12, 44.54, and 71.21 % over control plants alone,
respectively. Ascorbic acid content of salinized plants
primed with ZNPs1, ZNPs2, and ZNPs3 was enhanced by
25.02, 52.60, and 100.03 % over salinized plants alone,
respectively (Fig. 5).
Effect of Priming with ZNPs on Na and Zn
of Lupine Plants under Normal and Salt Stress
Conditions
Na content accumulated by 2.22-fold over control
(Fig. 6a). However, seed-priming with ZNPs decreased the
accumulation of Na content. The maximum decrement in
Na content was recorded in seeds primed with ZNP3 and
this reduction in Na content of ZNPs3 ? S plants was
45.29 % lower than S plants alone (Fig. 6a). In contrast to
the Na pattern, Zn content markedly decreased under
salinity stress by 58.06 % compared to control (Fig. 6b).
Seed-priming with ZNPs increased Zn content and the
maximum increase was recorded in ZNPs3 plants. In
ZNPs3 ? S plants, the increase in Zn content reached to
2.24-fold over S plants alone (Fig. 6b).
Discussion
Zn is an essential element necessary for growth and
development of plants (Pathak and others 2012). The most
common impact of salt pressure on plant physiology is a
depression in the growth which is necessary for the sur-
vival of a plant exposed to this pressure. In this study,
salinity stress caused depression in plant growth by less-
ening growth attributes due to water deficit which leads
to abnormal changes in plant morphology (Jiang and
others 2014), osmotic stress, nutritional disorders, and
treatments at P \ 0.05, according to Duncan’s multiple range test.
FW, fresh weight; S, 150 mM NaCl; ZNPs1, 20 mg L-1 ZnO;
ZNPs2, 40 mg L-1 ZnO; ZNPs3, 60 mg L-1 ZnO
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Table 2 Effects of salinity stress and seed-priming with ZnO nanoparticles (ZNPs) on the activities of superoxide dismutase (SOD) (U g-1 FW),
catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) (U min-1 g-1 FW) in leaves of 20-day-old lupine plants
Traits Control Salinity (S) ZNPs1 ZNPs1 ? S ZNPs2 ZNPs2 ? S ZNPs3 ZNPs3 ? S

SOD 8.27 ± 0.50g 11.95 ± 0.26d 8.30 ± 0.40g 12.73 ± 0.50c 9.27 ± 0.46f 14.13 ± 0.18b 10.77 ± 0.28e 17.56 ± 0.38a
de f d ef b d a
CAT 4.06 ± 0.38 2.60 ± 0.45 4.52 ± 0.40 3.31 ± 0.68 8.48 ± 0.44 5.05 ± 1.22 12.13 ± 0.61 7.11 ± 0.23c
f d f c e b d
POD 10.00 ± 0.20 17.93 ± 0.44 11.00 ± 1.51 20.74 ± 1.51 13.61 ± 0.72 24.50 ± 0.37 16.96 ± 0.34 26.45 ± 0.40a
g cd f c e b de
APX 2.72 ± 0.46 4.93 ± 0.29 3.52 ± 0.34 5.32 ± 0.28 4.10 ± 0.18 6.22 ± 0.11 4.55 ± 0.29 7.97 ± 0.13a
Values are means standard deviation (± SD) of three independent replications (n = 3)
Different letters within the row indicate statistically significant differences between treatments, according to Duncan’s multiple range test
(P \ 0.05)
FW, fresh weight; S, 150 mM NaCl; ZNPs1, 20 mg L-1 ZnO; ZNPs2, 40 mg L-1 ZnO; ZNPs3, 60 mg L-1 ZnO

Fig. 5 Effects of salinity stress and seed-priming with ZnO nanopar- at P \ 0.05, according to Duncan’s multiple range test. FW, fresh
ticles (ZNPs) on ascorbic acid content in 20-day-old lupine leaves. weight; S, 150 mM NaCl; ZNPs1, 20 mg L-1 ZnO; ZNPs2, 40 mg
Bars represent standard deviation (± SD) of the means (n = 3). L-1 ZnO; ZNPs3, 60 mg L-1 ZnO
Different letters indicate significant differences among the treatments

physiological and biochemical imbalance (Soliman and
others 2015; Ahmad and others 2016). Seed-priming with
ZNPs positively affects the growth traits in NaCl-stressed
lupine as Zn plays a vital role in (1) building natural auxin
(IAA) and consequently activating cell division and
enlargement (Ali and Mahmoud 2013), (2) maintenance
of the structural integrity of biomembranes (Weisany and
others 2012), (3) phospholipids accumulation (Jiang and
others 2014), (4) improvement in protein synthesis
(Ebrahimian and Bybordi 2011), (5) scavenging free
oxygen radicals (Jiang and others 2014), (6) translocation
of nutrients from the aged cells to newborn cells (Rock-
enfeller and Madeo 2008; Jiang and others 2014), and (7)
decreasing the uptake of excess of Na? and Cl- (Weisany
and others 2012; Ibrahim and Faryal 2014; Jiang and
others 2014). Our finding is compatible with the results
obtained by Laware and Raskar (2014) on onion,
Mukherjee and others (2014) on green pea (Pisum sati-
vum), Rezaei and Abbasi (2014) on cotton (Gossypium
hirsutum L.) and Soliman and others (2015) on moringa
(Moringa peregrina). It is worth mentioning that the
maximum increment in growth parameters under normal
and saline regimes was noticed in plants priming with
ZNPs3, which means that ZNPs3 has a superior promot-
ing impact either in unstressed or stressed regimes and
could be the subject of future studies. Prasad and others
(2012) reported that treating groundnut seeds with
nanoscale ZnO particles with a concentration of
1000 ppm has induced a marked increase in germination,
root and shoot length, and vigor index over other con-
centrations of the same material. They reported that, the
main reason for these influences is not known but it is
likely to be due to the higher concentrations of zinc in the
seed when treated with nanoscale ZnO particles.
The decrement in photosynthetic pigments of L. termis
leaves under the saline condition is in full agreement with
the finding of Weisany and others (2011). They stated that,
the reduction of pigment content was ascribed to enhanced
chloroplast structure damage, pigment-protein complex
instability, and chlorophyllase activity (Singh and Dubey

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J Plant Growth Regul (2017) 36:60–70
Fig. 6 Effects of salinity stress
and seed-priming with ZnO
nanoparticles (ZNPs) on a Na
content and b Zn content in
20-day-old lupine. Bars
represent standard deviation
(± SD) of the means (n = 3).
Different letters indicate
significant differences among
the treatments at P \ 0.05,
according to Duncan’s multiple
range test. DW, dry weight; S,
150 mM NaCl; ZNPs1, 20 mg
L-1 ZnO; ZNPs2, 40 mg L-1
ZnO; ZNPs3, 60 mg L-1 ZnO
1995). In this investigation, a similar increased trend of
soluble sugar, soluble protein, total free amino acids, pro-
line, and total phenols was pronounced in lupine plants
exposed to NaCl stress. Increased accumulation of total
soluble sugar and soluble protein in response to salt stress
was reported by Ahmad and others (2016) in chickpea. Total
free amino acids and proline were also reportedly boosted
under salt stress in wheat (Abdel Latef 2010). Total phenol
content was increased in wheat (Yasmeen and others 2013)
under salinity stress. Soluble sugar acts as an important
organic solute to keep the cell homeostasis (Ahmad and
others 2016). Soluble protein plays a pivotal role in
osmoregulation under saline conditions and can supply a
storage form of nitrogen (Ahmad and others 2016). Accu-
mulation of soluble protein content under stress may be the
result of improved synthesis of specific stress-related proteins
(Ahmad and others 2016). Total free amino acids and proline
are important organic solutes that assist in cell osmoregula-
tion under salinity stress (Azooz and others 2004). Phenolic
compounds played a vital role in safeguarding the plants
against harmful impacts induced by various pressures. Total
phenols have the antioxidant feature because they are elec-
tron-donating agents (Ahmad and others 2016), thus elimi-
nating reactive oxygen species (ROS).
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
67
Priming with ZNPs at different concentrations resulted
in a significant increment in photosynthetic pigments,
organic solutes and total phenols in unstressed and stressed
lupine plants. This increment reached its maximum value
in plants primed with ZNPs3. Zn probably keeps chloro-
phyll synthesis through the protection of the sulfydryl
group, a function primarily associated with Zn (Cakmak
2000; Weisany and others 2011). Moreover, it shares in
chlorophyll synthesis (Li and others 2006; Weisany and
others 2011). Zn also plays a main role in sugar formation
and enzyme structure involved in the biosynthesis of amino
acids (Soliman and others 2015). These findings are in
agreement with Soliman and others (2015). The manifest
accumulation of organic solutes and total phenols due to
priming with ZNPs3 might boost salt tolerance of cells
through osmotic adjustment, consequently improve plant
growth.
Membrane lipid peroxidation is a sign of membrane
destruction under saline stress regimes (Katsuhara and
others 2005; Abdel Latef and Chaoxing 2011; Ahmad and
others 2016). Lipid peroxidation is generally estimated in
terms of MDA content (Abdel Latef 2011; Ranjit and
others 2016). MDA is a secondary end product of
polyunsaturated fatty acid oxidation and is widely used to
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68
assess the extent of lipid peroxidation as an indicator of
oxidative stress (Lin and Kao 2000; Abdel Latef and
Chaoxing 2014; Zheng and others 2016). The present study
showed that there was high accumulation in MDA content
under saline conditions, suggesting that salt stress could
destroy the integrity of the cellular membrane, as well as
cellular compounds, like proteins and lipids. ZNPs appli-
cation, especially ZNPs3, reduced MDA content, thus
ameliorating the injury normally induced by salinity stress.
This is consistent with the results of Burman and others
(2013) who reported that ZNPs induced defensive impacts
on biomembranes versus alternations of membrane per-
meability and oxidative stress in chickpea seedlings.
It has been reported in many studies that exposure to salt
stress could induce ROS formation causing increased
activity of antioxidative enzymes as a defense system
(Weisany and others 2012; Soliman and others 2015;
Ahmad and others 2016). This is harmonious with our
results that showed that growing lupine in saline conditions
led to a marked increase in the antioxidant enzymes (ex-
cept CAT), which can be considered as good evidence of
ROS production. The high activity of SOD, POD and APX
under salt stress is a good signal of lupine ability to adapt
with ROS. Therefore, it suggested that the increase in the
activities of antioxidant enzymes may be attributed to the
adaptive defense system of L. termis against the harmful
impact imposed by NaCl. On the other hand, the decrease
in CAT might be due to the increasing rate of ROS scav-
enging by the other antioxidant enzymes.
Beside these antioxidant enzymes, there are metabolites
that act as ROS scavengers either in conjunction with the
antioxidative enzymes or independently. Nonenzymatic
components of the antioxidative defense system include the
major cellular redox buffers ascorbic acid and glutathione.
They are involved in many cellular processes and not only
have critical roles in plant tolerance and act as enzyme
cofactors, but also affect plant growth and development
from earlier growth stages to senescence (Hajiboland
2013). Ascorbic acid is a water soluble antioxidant that acts
to prevent injury induced by ROS in plants (Gill and Tuteja
2010). It plays a main role in the detoxification of ROS due
to its ability to donate electrons in a wide range of enzy-
matic and nonenzymatic reactions. Ascorbic acid is able to
reduce H2O2 to H2O via the APX reaction (Hajiboland
2013). In this work, exposure to salt stress increased the
level of ascorbic acid in lupine leaves as compared to the
control. Our results are in agreement with findings of
Soliman and others (2015). The results of the present study
showed that seeds primed with ZNPs, especially ZNPs3,
had high antioxidant enzymes and nonenzymatic activity
when compared with salinized plants indicating that the
further increase in enzyme activity in response to ZNPs
treatment was due to extreme oxidative stress caused by
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
J Plant Growth Regul (2017) 36:60–70
NaCl and the protection against salt stress by high levels of
antioxidant enzymes induced by ZNPs especially ZNPs3.
Probably, Zn is able to assist the enzymes and nonenzy-
matic antioxidant biosynthesis (Weisany and others 2012;
Rezaie and Abbasi 2014; Soliman and others 2015).
Salt-stressed L. termis accumulated lower content of Na
and higher content of Zn upon priming application of
ZNPs. The accumulation of less Na is a great sign of salt
resistance in plants treated with ZNPs. Our results are
consistent with the findings of Soliman and others (2015)
who reported that foliar application of ZNPs could mitigate
Na injury in M. peregrina.
Conclusions
It is inferred from the results of the current research that
priming with ZNPs, especially ZNPs3 (60 mg L-1 ZnO),
lessened the negative impacts of NaCl on lupine plants
through enhancing photosynthetic pigments, adjusting
osmoregulation, and lowering the contents of MDA and
Na. Further, protection under NaCl stress was achieved via
ameliorated total phenols and activities of antioxidant
enzymes. Thus, application of ZNPs could be a strategy to
energize the growth and economic yield in plants growing
in salinized soils. Further efforts are required to gain a full
understanding of how zinc oxide nanoparticles alleviate the
adverse effects of salinity stress in plants.
Author contributions Arafat Abdel Hamed Abdel Latef conceived
and designed the experiments. Khaled Ebnalwaled Abdelfattah pre-
pared ZnO nanoparticles. Arafat Abdel Hamed Abdel Latef and Mona
Fawzy Abu Alhmad conducted the experiments. Mona Fawzy Abu
Alhmad collected the data. Arafat Abdel Hamed Abdel Latef ana-
lyzed the data and wrote the manuscript.
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