Scientia Horticulturae 250 (2019) 329–343

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Comprehensive biochemical insights into the seed germination of walnut T

under drought stress
⁎
Naser Lotfia, Ali Soleimania, , Kourosh Vahdatib, Ramazan Çakmakçıc
a
Department of Horticulture, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
b
Department of Horticulture, Faculty of Agriculture, University of Tehran, Tehran, Iran
c
Department of Agronomy, Faculty of Agriculture, Çanakkale Onsekiz Mart Üniversitesi Çomü, Çanakkale, Turkey

A R T I C LE I N FO A B S T R A C T

Keywords: The evaluation of physiological and biochemical aspects is essential to identify clues that can assist breeders in
Juglans regia selecting Persian walnut genotypes with considerable tolerance to drought stress. This objective can be obtained
Hormone by analyzing seed biochemical compounds from different genetic resources, e.g. seeds from individual trees that
Phenolic compounds are commonly exposed to drought stress. In the current research, we evaluated the effects of drought stress, as
Polyamine
induced by PEG6000, on the germination of walnut seeds collected from six promising genotypes (i.e. 'SS2', 'TT2',
Seed germination
'TT1', 'ZM1', 'Haward' and 'Chandler'). A photoperiod of 12 h was provided during the experiments, along with an
average temperature of 25 ± 1 °C. According to the different indexes of germination, a greater tolerance was
observed among three genotypes, namely, 'ZM1', 'Haward' and ‘Chandler’. In response to drought stress, the
contents of proline and total soluble sugars increased, especially at intense levels of stress, whereas the amount
of starch decreased. The levels of antioxidant activity significantly increased by POD, APX, CAT, SOD and LOX
enzymes in the radicle and plumule tissues as the water potential reached −1.5 MPa. Three genotypes, namely,
'TT2', 'TT1' and 'SS2' were susceptible to cell wall damage (i.e. by lipid peroxidation). High levels of Spd and Spm
in tolerant genotypes were accompanied by significant increases in the zeatin, endogenous IAA, zeatin riboside
and ABA in the seeds. Our results suggest that, under drought stress, seed germination in walnuts can benefit
from the accumulation of free Spd in seeds during the phases of germination. However, the accumulation of free
Put appeared to be an inhibitory factor which suppressed the germination of walnut seeds.

1. Introduction the physiological and biochemical responses of plants to drought stress

Plants are the most important nutrient source for livings. Especially
fruits have important biochemicals for human's health. Walnuts have
been chiefly used in traditional medicine in China and other Asian
countries (Okatan, 2018; Mezeyova et al., 2018; Sabijon and Sudaria,
2018; Golak-Siwulska et al., 2018). During a plant’s life cycle, abiotic
stresses may frequently occur. Drought is potentially a primary source
of stress that can suppress the growth of fruit trees, and it accounts for a
considerable reduction in the yield of orchards (Munne-Bosch and
Muller, 2013). Due to the widespread shortage of water in certain Asian
and Middle-Eastern countries, it is important for breeders to distinguish
and to arrange novel approaches which can enhance the tolerance of
different genotypes. Natural germplasm resources are ideal materials
that can reveal the mechanisms of plant responses and their survival
under conditions of water deficiency.
Persian walnut (Juglans regia L.) is considered as an important food
source in some Middle-Eastern countries and also in various other re-
gions of the world. Seed germination, growth and the fruit yield of
mature walnut trees could be limited by the natural distribution of this
species due to its relative susceptibility to water shortages (Lotfi et al.,
2010).
The phases of germination are commonly regarded as decisive

Abbreviations: ABA, abscisic acid; AsA, ascorbate; APX, ascorbate peroxidase; CAT, catalase; CKs, cytokinins; TDNB, 5,5′-dithio-bis 2-nitrobenzoic acid; DW, dry
weight; FGP, final germination percentage; FW, fresh weight; GI, germination index; GRI, germination rate index; GSI, germination stress index; GA3, gibberellin;
GSH, glutathione; GPX, glutathione peroxidase; GR, glutathione reductase; HPLC, high performance liquid chromatography; IAA, indole-3-acetic acid; LOX, li-
poxygenase; MDA, malondialdehyde; MGT, mean germination time; NBT, nitroblue tetrazolium; PCA, perchloric acid; POD, peroxidase; PAs, polyamines; PEG,
polyethylene glycol; PVP, polyvinylpyrrolidone; Put, putrescine; ROS, reactive oxygen species; RC, relative conductivity; RWC, relative water content; SPE, solid
phase extraction; Spd, spermidine; Spm, spermine; SOD, superoxide dismutase; TPC, total phenolic compounds; TW, turgor weight
⁎
Corresponding author.
E-mail address: asoleimani@znu.ac.ir (A. Soleimani).

https://doi.org/10.1016/j.scienta.2019.02.060
Received 31 December 2018; Received in revised form 17 February 2019; Accepted 19 February 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
N. Lotfi, et al.
Table 1
Morphological and phenological traits of the studied genotypes.
Genotype Leafing date Tree Vigor Dichogamy
'ZM1' Early High Protandrous
'SS2' Early Medium-low Protandrous
'TT1' Early Very High Protandrous
'TT2' Early Medium Protandrous
'Chandler' Late Low Protandrous
'Haward' Late High-medium Protandrous
stages during seedling establishment, and it can be substantially wea-
kened by drought stress (Hubbard et al., 2012). In seeds, the responses
to different quantities of water in the growth medium and its shortage
can inhibit the germination and the process surrounding it (Cardoso,
2012). Previous cases of research have considered different osmotic
potentials of wet substrates by using solutions of polyethylene glycol
(PEG6000), whereby the conditions of water deficiency are simulated
for seeds (Almeida et al., 2014; Shen et al., 2015). This method is
broadly applied to numerous species of commercial seeds.
The assessment of seedling characteristics pertaining to the plumule
and radicle, such as the plumule length, radicle length and dry mass can
be applied to measure the initial vigor of plants (Vanzolini et al., 2007),
and this may also involve the analysis of seed reserves (Henning et al.,
2010). Furthermore, Pereira et al. (2015) suggested that biochemical
compounds in seeds can affect the seed germination, and it can corre-
late significantly with plant hormones such as IAA and ABA. However,
the mechanisms of these hormones and their exact effects are not well
known regarding seed germination in walnuts and their initial growth.
Radicle expansion can be delayed by ABA, thereby resulting in the in-
hibition of normal seed growth (Graeber et al., 2010). The antagonists
for the effect of ABA are gibberellins (GAs) when seeds are ready to
germinate (Miransari and Smith, 2014). GAs are stimulators for the
creation of hydrolases, especially α-amylase, which help the seeds
germinate. Cytokinins (CKs) and auxins also cause seeds to germinate.
During all stages of seed germination, CKs are known to be active
(Yamaguchi, 2008).
The most recent evidence obtained from stress physiology research
highlights that polyamines are key players in signaling responses in-
volved in central metabolisms, sugar/lipid homeostasis, maintenance,
the induction of antioxidant capacity and osmotic regulation. Plant
growth is usually regulated by a significant relationship between PAs
and hormones (Yang et al., 2008). The biosynthetic precursor of S-
adenosyl-L-methionine (SAM) occurs with a share of PAs and ethylene,
and the rate of ethylene synthesis is likely to be related to the increase
in the biosynthesis of Spd and Spm. Furthermore, it has been reported
that Zeatin and zeatin riboside in rice grains increase significantly by
applications of exogenous Spd and Spm (Yang et al., 2008). In grape
rootstocks, the responses to salinity are reportedly associated with the
presence of PAs and ABA in tissues (Upreti and Murti, 2010).
The accumulation of ROS is a common biochemical change that
occurs as a result of drought stress (Ozgur et al., 2013). Homeostatic
levels of ROS in cells are maintained by a series of non-enzyme anti-
oxidants and antioxidant enzymes. In particular, antioxidant enzymes
that scavenge ROS include SOD, catalase CAT, POD, APX, GR and GPX
(Mittler et al., 2004). Also, non-enzymatic antioxidants include GSH
and AsA. Previous cases of research have indicated that NaCl or PEG
can increase enzymatic activities of CAT, APX, SOD and POD in the
radicles and plumules of alfalfa (Wang et al., 2009). The presence of
SOD, CAT, GPX, POD and GR activities, along with AsA and GSH
contents, are reportedly increased in the radicles and leaves of Gly-
cyrrhiza uralensis under silicon applications. Silicon is intended to al-
leviate salt stress and drought stress together (Pereira et al., 2015).
These criteria are of prime value when evaluating the available genetic
variability of germplasms for the purpose of breeding programs. Ac-
cordingly, it would be valuable to select and use the seeds of genotypes
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Scientia Horticulturae 250 (2019) 329–343
Seed size (g) Kernel percent Yield
Medium (10-12) > 55 Medium
Medium (10-12) 45-50 Medium-Low
Very big (> 16) 50-55 Medium-Low
Big (12-14) 45-50 High
Medium (10-12) 40-45 Very high
Medium (10-12) 40-45 Very high
which genetically inherit tolerance to stress.
In Persia, there are many old walnut trees that have been planted on
the banks of rivers. These trees have survived for long, indicating that
they possess valuable genes for tolerance to stress, while it is likely that
their progeny could also acclimatize also acclimatize and cope with
unfavorable environmental conditions (Vahdati, 2003). In a previous
study, a significant variation was observed in the germination of seeds
among different walnut genotypes under drought stress (Lotfi et al.,
2009, 2010). However, the hormonal and biochemical aspects of
walnut seeds have not been evaluated so far in response to drought
stress, especially with a focus on different maternal resources and a
diverse genetic variation. So, the current study was conducted to pro-
vide clear evidence that the physiological and biochemical compounds
in walnut seeds can influence germination under osmotic drought
stress. It is hope that the promising results of the current work will shed
some light to advance walnut breeding programs and its improvement.
2. Materials and methods
2.1. Plant materials, seed germination and preparation of osmotic media
Half-sib seeds of six genotypes of walnuts ('SS2', 'TT2', 'TT1′, 'ZM1′,
'Haward' and 'Chandler') were collected from open-pollinated native
trees, and the seeds were screened for apparent quality. The morpho-
logical and phenological traits of the studied genotypes are presented in
Table 1. Seeds of the 'ZM1′ and 'SS2' genotypes were collected from the
Ghoshchi and the Anzal regions of Urmia located in northwest of Iran.
The Ghoshchi region is characterized by an altitude of 1421 m above
sea level, with geographic coordinates of 37.59.32 N, 45.02.18 E. The
climate of the region has an average annual precipitation of 212 mm. It
usually covers three months of severe drought in the summer, and the
winters can experience dry, cold conditions. The minimum and max-
imum temperatures are -18 and 32 °C, respectively. In certain circum-
stances, there could be up to 5 months of frost (from October to April).
The genotypes 'TT2' and 'TT1′ were collected from the suburbs of
Bonab, which is located in East Azerbaijan Province, in northwest of
Iran and the geographical coordinates of which are 37.20.0 N, 46.03.0
E. The altitude of Bonab is 1290 m. The area has an average annual
precipitation of 246.47 mm. There are 4 months of dry and hot sum-
mers, but cold and dry winters. The minimum and maximum of annual
temperatures are -14 and 35 °C, respectively. The average annual
temperature is 13 °C, and each year may include up to six months of
frost (from October to March). The walnut seeds of exotic genotypes
'Haward' and 'Chandler' were collected commercial orchards belonging
to specific research fields in from commercial orchards belonging to
specific research fields in Ankara and Konia at central and south regions
of Turkey, where the climate is dry and semi-arid in these region
Walnut seeds were soaked in water for ten days. Then, they were
treated with 5% Captan fungicide before being exposed to the chilling
treatment which involved storing the seeds in a refrigerator for four
weeks at 4 to 6 °C (to break the seeds). Then, the seeds were grown in
small polyethylene pots containing perlite with medium granularity.
The control group of this experimeφnt was distilled water. Polyethylene
glycol (PEG6000) was obtained from Duchefa Biochemie (Haarlem,
Netherlands). Different concentrations of PEG solutions that
N. Lotfi, et al. Scientia Horticulturae 250 (2019) 329–343

corresponded with the osmotic potentials (Ψs) of -0.5, -1.00, -1.50, -2, (IGV) = ΣG/t (7)
and 2.5 MPa were prepared, and 50 mL aliquots of PEG solutions were
Where, the percentage of seeds germinated at one day interval was
added to each pot as described by Michel and Kaufmann (1973):
showed by G and t is the total germination period (days).
φ = - (1.1810–2) × C - (1.1810–4) × C2 + (2.6710–4) × CT +
(8.3910-7) × C2 (1) 2.2. Relative water content, relative conductivity and Malondialdehyde
1 contents of seedlings
Where the concentration of PEG is represented by C (g L of water),
T refers to the temperature (°C), and the water potential (bar) is re-
The plumule and radicle samples (which weighed about 0.1 g of FW
presented by φ or Ψs. The verification of water potential was performed
were washed five times and then left to float in water for 24 h.
using an automatic cryoscopic osmometer (‘WP4 Dewpoint Meter’,
Subsequently, they were drained at room temperature, and the weight
Decagon Device, Inc.).
of the turgor (TW) was determined. The dry weight (DW) was recorded
Polyethylene pots were weighed, and their surface was covered with
after drying the tissues at 80 °C for 24 h (Pattanagul, 2011). The relative
a plastic coating to prevent surface evaporation. The pots were then
water content (RWC) was calculated by the following forluma:
placed in a room at 25 ± 1 °C and at a relative humidity of 57%. They
were weighed each morning, and a certain amount of distilled water RWC = (FW − DW) / (TW − DW) × 100%.
was added after calculating the evaporation rate. During the experi-
In order to measure the RC, the calculations adhered to the method
ment, no nutrient solution was added to the treated pots. When the
outlined by Chu et al. (2016). The peroxidation level of membrane li-
radicle length in the control seedlings (≈ 0 Ψs) reached 4 cm, the fol-
pids was determined by the MDA concentration, according to a slightly
lowing growth indices were measured in all seedlings: seed germination
modified method which uses thiobarbituric acid (Talaat et al., 2015).
percentage, radicle and Plumule length, fresh and dry weight, diameter
and plumule to radicle ratio. The MGT and FGP were calculated re-
spectively based on Eq. (2) (Refka et al., 2013) and Eq. (3) (Ren and 2.3. Concentrations of soluble sugars, starch and osmolytes in seeds that
Tao, 2004). germinate

MGT=Σni-ti /N, (2) Primarily, lyophilization and the reconstitution of flow were ana-
lyzed by the method described by Janeczko et al. (2010). Agilent
Where the number of seeds that germinated within consecutive in-
Technologies 1200 HPLC was used for analyzing the samples, and the
tervals of time is represented by ni, the time between the beginning of
HPLC device was equipped with an amperometric detector ESA (Cou-
the test and the end of a particular interval is represented by ti and the
lochem II Analytical Cell 5040) and a golden electrode. The column in
total number of germinated seeds is shown as N.
this experiment was the Hamilton RCX-10 250 × 4.1 mm which be-
(FGP) % = (n/nt) ×100 (3) longs to the HPLC model Hamilton, Reno, NV, USA. Furthermore,
100 mM sodium hydroxide was solubilized in water. Then, it formed a
Where ‘n’ shows the number of germinated seeds and the total
mobile phase which was characterized by a flow rate of 1.5 ml min−1.
number of seeds represented by nt.
The analytical potential of 200 mV was selected for the detector,
The indices of germination are namely: GRI, GI and GSI as calcu-
thereby adjusting the oxidizing and reducing potentials to 800 m and
lated respectively by Eqs. (4), (5) (Salehzade et al., 2009) and (6)
−900 mV respectively. Controlling the apparatus was regulated by
(Ahmad et al., 2009).
Agilent Technologies ChemStation B04 software, and the method of
Coomassie brilliant blue G250 was used for assessing a collection of
nd nd nd nd
(GI ) = ⎛ 1 ⎞ + ⎛ 2 ⎞ + ⎛ 3 ⎞ + ...⎛ i ⎞ data before processing them. The concentration of soluble proteins is
⎝ 1 ⎠ ⎝ 2 ⎠ ⎝ 3 ⎠ ⎝ i ⎠ (4)
already known in the scientific literature (Wang et al., 2013), and their
Where the germination speed is indicated by (GI), and the nd1, nd2, levels of absorbance were assessed at a colorimetric wavelength of
…., ndi marks indicate the number of seeds that germinated on day 1, 2, 595 nm. The amount of proline was measured using the methods of
3, … and i Chołuj et al. (2008), and a wavelength of 520 nm was applied to
measure its absorbance. The final calculations included the standard
G1 − GI − 1 curve with proline.
GRI (%) = ∑⎛ i
⎞ × 100
(5)
⎝ ⎠
2.4. Antioxidants, reactive oxygen species, and phenolic compounds
Where the percentage of germination on each day of the germina-
tion period is indicated by Eq. (5) (a higher GRI shows a faster ger- First, the homogenizations of Plumule and radicle samples were
mination), the percentage of seeds that germinated on the day (i) is performed using a Brinkmann homogenizer (Polytron-Kinematica
represented by Gi and the percentage of seeds that germinated on the GmbH) with a methanol/water ratio of 70:30. The homogenizations
previous day is shown by Gi-1. were carried out in an ice bath, and by reduced pressure of the filter
paper model (Whatman No 1). Both superoxide radicals (O·–2) and the
(GSI) = [PI (stressed seeds) / PI (control seeds)] × 100 = [(nd2 × 1)
hydrogen peroxide (H2O2) content were analyzed using the Bu et al.
+ (nd4 × 0.75) + (nd6 × 0.5) + (nd8 × 0.25) (stressed seeds)] /
(2016) methods. Each 0.5 g of the radicle and plumule tissues were
[(nd2 × 1) + (nd4 × 0.75) + (nd6 × 0.5) + (nd8 × 0.25) (control
obtained from a germinated seed and were used for assaying the ac-
seeds)], (6)
tivity of antioxidant enzymes. Accordingly, the homogenization of
This equation is an expression of drought tolerance, and where PI samples occurred in an extraction buffer (10 mL) supplemented with
indicates the promptness index, the nd2, nd4, nd6, and nd8 express the 1.0 mM EDTA, 50 mM of phosphate buffer (pH 7.8), 1.0 g PVP and 0.5%
number of seeds that germinated on day 2, 4, 6, and 8 respectively. The (v/v) Triton X-100. The extracted homogenate was centrifuged at
relationship between the germination rate and water potential was 12,000 rpm for 20 min. This supernatant was used for analyzing the
described by regression analysis using SIGMAPLOT®9.0 (software activities of superoxide dismutase (SOD), catalase (CAT) and perox-
Systat Software Inc., San Jose, CA, USA). In the end, the speed of ger- idase (POD). The LOX activity was measured by using the method de-
mination was calculated by Eq. (7), as indicated by the index of ger- scribed by Kosakivska et al. (2014). All stages in this measurement were
mination velocity (IGV) (i.e. where greater values show faster germi- carried out at 0–4 °C. Furthermore, the ability of SOD to inhibit NBT, as
nation rates) (Luan et al., 2014): a photochemical reduction, was measured and detected at 560 nm. It is

331
N. Lotfi, et al.
a common approach to define one unit of SOD as the amount of enzyme
that causes a 50% reduction in the inhibition of NBT (Zhang et al.,
2015). Further assessments were aimed at measuring the activity of
CAT, according to a modified version of the method used by Sohn et al.
(2005). Changes in the absorbance within the reaction solution were
recorded at 240 nm for 5 min. One unit of CAT activity was defined as
0.01 units per minute, as required for the change in absorbance. The
available method described by Pan et al. (2006) was applied for the
measurement of POD activity. Accordingly, 2.7 mL of the 50 mM
phosphate buffer (pH 7.8) was mixed with 0.2 mL of the enzyme ex-
tract. This resultant solution was mixed with one mL H2O2 (2%) and
0.1 mL of 50 mM guaiacol. Then, the changes in absorbance were
measured at 470 nm within 5 min.
In 1 mL of the extraction buffer [50 mM phosphate buffer (pH 7.0),
fresh seedlings (0.5 g) were homogenized by pestle and mortar at 4 °C.
The buffer contained 1 mM dithiothreitol, 1 mM GSH, 1 mM AsA and
1 mM MgCl2. Then, the homogenate was centrifuged at 12,000 rpm for
20 min and the supernatant was used as a crude extract for the mea-
surement of GR and APX. The activity of APX was measured according
to the method a method described by Kang and Saltveit (2002). As
ascorbate is oxidized, the absorbance decreases at 240 nm. The GR
activity was analyzed by using the decrease in absorbance at 340 nm by
the oxidation of NADPH (Kang and Saltveit, 2002). The method de-
scribed by Li et al. (2015) was applied was applied to measure the AsA
content by using 0.2 g fresh seedling tissues. The extracted 0.2 g of fresh
seedling tissue was homogenized with 4 mL of 5 mM EDTA-TCA so as to
determine the GSH. The values were specifically determined by using
TDNB according to a common method described by Khan et al. (2014).
The reaction mixture was assayed for changes in its absorbance at
420 nm. The total GSH concentration was calculated by using the
standard curve in association with GSH.
The HPLC analysis operated by using materials of standard grade for
the measurement of different phenolic compounds such as juglone
(481-39-0), gallic acid (149-91- 7), chlorogenic acid (327-97-9), pyr-
ocatechin (120-80-9), catechin (154-23-4), syringic acid (530-57-4),
rutin (153- 18-4), cinnamic acid (140-10-3), epicatechin (154-234-),
vanillic acid (121-34-6), and two solvents, namely, phosphoric acid and
methanol (MeOH). These materials were purchased from Sigma Aldrich
Chemical Co. The materials provided for this (0.5 g/ml) were dissolved
by using methanol. A concentration of 100×dilution was used as the
standard for the HPLC to operate. Starter materials for the HPLC op-
eration comprised 1 g of each sample, extracted in 10 ml methanol,
which was placed in the dark at 4 °C for 12 h. The HPLC included an
Edmund Buhler SM 30 as the control shaker (200 rpm/min). The su-
pernatant was filtered on a 0.45 μm MILLEX-HV unit filter of syringe
(SLHV 013 N L, PVDF Durapore, Bedford, M.A.) before being inserted
into the HPLC apparatus. The quantities of phenolic compounds and
their components are given in units of μg/g.
2.5. Quantification of plant hormones
After the radicles and Plumules had been sampled from the treated
plants, they were immediately frozen in liquid nitrogen. The plant tis-
sues (amounting to four samples per each treatment; 50 mg of FW) were
homogenized and lyophilized in methanol, and then cooled with a cold
mixture of water and formic acid (15:4:1) according to the methods
described by Dobrev and Kaminek (2002). Also, during the homo-
genization, each sample received labeled kinetin with nitrogen 15 N,
along with deuterated IAA and ABA. This combination resulted from an
internal isotopic standard mixture. The SPE that had been created by
the columns Oasis MCX (Waters) was applied to a fraction of pre-pre-
pared extract, and then three fractions were collected: the ”acidic”
fraction was diluted with methanol and was used according to the HPLC
method of Stefancic et al. (2007) for analyses of IAA and ABA. Fur-
thermore, we used the methods of Yang et al. (2008) and Liu et al.
(2013) for the detection and measurement of Put, Spm and Spd.
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Scientia Horticulturae 250 (2019) 329–343
Accordingly, the samples which comprised 0.5 g of FW underwent the
process of homogenization in PCA with a volume of 5 mL at 5% (v/v).
This was followed by incubating the homogenates at 5 °C for an hour.
The homogenates were centrifuged at 25,000×g for 20 min. The su-
pernatant was employed in order to extract the free phenolic acids
(PAs) and the soluble-conjugated Put, as well as the extraction of PA as
an insoluble-conjugated form via the pellet (dissolved by 1 mol L-1
NaOH). Soluble-conjugated and insoluble-conjugated PAs were assayed
by 2 mL of 5% PCA supernatant which was mixed with a supernatant of
NaOH. The latter was acid-hydrolyzed since it contained 2 mL of 12 mol
L-1 HCl. It was then resuspended in 0.5 mL of 5% (v/v) PCA. Based on
the application of benzoyl chloride, specific methods were used for the
extraction of PAs in the non-hydrolyzed supernatant, the hydrolyzed
supernatant, and the hydrolyzed pellet. These were quantified by the
HPLC (Hamilton, Reno, NV, HPLC Pump/2489 UV Detector, and the
USA). The C18 column featured dimensions of (7.5 cm × 4.6 mm,
3.5 μm), along with waters symmetry which was used for the injection
of exactly 20 μL of each sample when loaded onto a column. The
column remained at a stable temperature of 30 °C, with a flow rate of
0.7 mL min–1. The absorbance operated at 230 nm for the detection of
polyamine. The extracts were prepared with a solvent mixture of iso-
propanol: H2O: concentrated HCl (2:1:0.002, v/v/v). They were cen-
trifuged and purified with a series of steps for the extraction of JA and
SA. A re-dissolved operation was carried out with the final concentra-
tion in 100 mL methanol. A half of this volume was exposed to the ESI-
triple quadrupole mass spectrometer (HPLC–ESI–MS/MS, Applied Bio-
systems, USA) equipped with a reverse-phase C18 Gemini column (150
9 2.00 mm, 5-lm particle size, Phenomenex, USA) (Ubaidillah et al.,
2015).
3. Results
3.1. Effects of osmotic stress on germination and growth indices
The cumulative count of seed germination was considered through
time at different levels of osmotic stress. This parameter determined the
values of water potential in the six genotypes within each osmotic
treatment (Table 2). In the control treatment, seeds that had begun to
germinate exhibited lower values of water potential, compared to the
ones already cultured. Seed germination among the studied genotypes
exceeded 65%. Seed germination was inhibited and delayed in seeds
that were imbibed in PEG solutions, followed by a decrease in water
potential. The 'Chandler', 'Haward' and 'ZM1′ experienced a better re-
covery at lower values of ѱs than at −1 MPa. Indeed, the seeds of these
genotypes were able to germinate at - 2.5 MPa (Table 2). Among the
studied genotypes, the 'ZM1′ showed the lowest level of susceptibility to
PEG-induced water stress, as compared to the other two tolerant gen-
otypes. This result is based on the number of seeds which germinated at
all levels of ѱs. Furthermore, as the ѱs increased, the different re-
sponses of the genotypes were revealed as significant by the ANOVA.
Significant occasions of decrease were observed in the FGP at the water
potential of -1.5 MPa. However, susceptible genotypes were not capable
of seed germination at -2.5 MPa. The highest value of FGP was observed
in the genotype of 'ZM1′ which proved to be more tolerant at all levels
of osmotic stress (Table 3). Changes in the MGT ranged from 10.81 days
(in the control) to 36.79 days at -2.5 MPa. It generally decreased par-
allel to higher water potentials. The MGT increased significantly when
the water potential was lower than - 0.5 MPa. Furthermore, it took
30–36 days for seeds to germinate at a water potential of -2.5 MPa. The
'ZM1′ and 'Chandler' showed the highest rate of germination among
genotypes, at all levels of ѱs. In this study, the speed of germination and
the number of germinated seeds were shown by the indices of GI and
GRI respectively. At a high level of PEG concentration, the two indices
exhibited a downward trend. Also, the GI index showed a downward
trend, parallel to the decrease in ѱs, from 4.54 (in the control) to 0.34
at -2.5 MPa. The GI variation was not significant at ѱs values below
N. Lotfi, et al. Scientia Horticulturae 250 (2019) 329–343

Table 2
The effect of PEG-induced osmotic stress on the seed germination traits of walnut genotypes. Radicle Length (RL), Plumule Length (PL), Radicle Fresh Weight (RFW),
Plumule Fresh Weight (PFW), Radicle Thickness (RT), Plumule Thickness (PT), Radicle Dry Weight (RDW), Plumule Dry Weight (PDW), Plumule / Radicle Length
ratio (P/RL ratio), Plumule / Radicle Fresh Weight ratio (P/RFW ratio), Plumule / Radicle Thickness ratio (P/RT ratio), Plumule / Radicle Dry Weight ratio (P/RDW
ratio).
Genotype Stress level (MPa) Seed germination traits

RL (mm) PL (mm) RFW (g) PFW (g) RT (mm) PT (mm) RDW (g) PDW (g) P/RL ratio P/RFW ratio P/RT ratio P/RDW ratio

*
'ZM1' 0 61.48 29.28 0.96 0.22 9.79 7.75 0.36 0.08 0.49 0.23 0.81 0.24
0.5 51.49 13.28 0.67 0.12 8.20 3.51 0.25 0.04 0.25 0.18 0.41 0.18
1 49.94 10.04 0.47 0.05 7.95 2.65 0.18 0.02 0.20 0.11 0.33 0.11
1.5 47.15 8.28 0.46 0.04 7.51 2.19 0.17 0.01 0.17 0.10 0.29 0.11
2 48.20 5.95 0.37 0.02 6.63 1.57 0.14 0.01 0.12 0.06 0.25 0.07
2.5 45.11 4.62 0.25 0.01 3.10 1.26 0.09 0 0.10 0.03 0.40 0.03
'SS2' 0 57.08 19.63 1.05 0.19 13.32 8.61 0.39 0.07 0.35 0.21 0.67 0.22
0.5 46.34 9.84 0.72 0.09 10.81 4.32 0.27 0.03 0.22 0.13 0.42 0.14
1 35.20 9.82 0.46 0.08 8.21 4.31 0.17 0.03 0.28 0.20 0.54 0.20
1.5 29.31 6.44 0.29 0.03 6.83 2.83 0.11 0.01 0.23 0.12 0.44 0.13
2 24.59 4.89 0.21 0.02 5.73 2.14 0.08 0.01 0.20 0.09 0.38 0.09
2.5 16.30 3.60 0.15 0.01 3.80 1.58 0.05 0.00 0.22 0.10 0.42 0.10
'TT2' 0 96.97 93.10 4.37 1.41 13.92 6.95 1.66 0.55 0.99 0.36 0.51 0.37
0.5 72.73 55.26 3.97 0.78 13.06 6.98 1.51 0.30 0.83 0.20 0.54 0.20
1 62.02 35.94 2.33 0.64 11.20 5.78 0.89 0.19 0.56 0.27 0.52 0.22
1.5 48.72 10.36 0.74 0.04 2.91 1.47 0.28 0.02 0.21 0.08 0.51 0.09
2 22.77 11.27 0.29 0.03 2.30 1.07 0.11 0.01 0.51 0.12 0.47 0.13
2.5 13.65 3.73 0.18 0.01 1.77 0.54 0.04 0.01 0.29 0.06 0.33 0.25
'Haward' 0 58.89 39.67 1.45 0.56 8.31 12.76 0.55 0.22 0.70 0.40 1.59 0.42
0.5 53.32 29.46 1.19 0.54 7.53 9.48 0.45 0.21 0.57 0.47 1.30 0.49
1 40.37 14.53 0.64 0.28 5.70 4.67 0.24 0.11 0.38 0.42 0.87 0.44
1.5 39.55 10.57 0.43 0.07 5.58 3.40 0.16 0.02 0.27 0.16 0.61 0.17
2 34.46 9.26 0.37 0.04 4.86 2.98 0.14 0.02 0.27 0.13 0.63 0.14
2.5 31.16 9.78 0.37 0.04 4.40 2.88 0.14 0.01 0.31 0.12 0.66 0.12
'TT1' 0 103.10 106.72 3.17 2.06 13.58 10.23 1.20 0.81 1.03 0.64 0.75 0.67
0.5 61.97 55.62 1.82 0.57 8.16 5.33 0.69 0.22 0.90 0.31 0.65 0.33
1 51.52 40.93 0.75 0.36 6.78 3.92 0.28 0.14 0.79 0.48 0.58 0.50
1.5 42.18 32.81 0.59 0.25 5.55 3.14 0.22 0.10 0.78 0.43 0.57 0.44
2 34.36 10.99 0.33 0.11 4.52 1.05 0.12 0.04 0.32 0.35 0.23 0.36
2.5 18.12 8.81 0.20 0.04 2.38 0.84 0.07 0.01 0.49 0.21 0.36 0.22
'Chandler' 0 61.48 29.28 0.96 0.22 9.79 7.75 0.36 0.08 0.49 0.23 0.81 0.24
0.5 51.49 13.28 0.67 0.12 8.20 3.51 0.25 0.04 0.25 0.18 0.41 0.18
1 49.94 10.04 0.47 0.05 7.95 2.65 0.18 0.02 0.20 0.11 0.33 0.11
1.5 47.15 8.28 0.46 0.04 7.51 2.19 0.17 0.01 0.17 0.10 0.29 0.11
2 48.20 5.95 0.37 0.02 6.63 1.57 0.14 0.01 0.12 0.06 0.25 0.07
2.5 47.61 4.62 0.25 0.01 3.10 1.26 0.09 0.00 0.10 0.03 0.40 0.03
LSD 11.94 7.23 0.41 0.15 1.88 1.16 0.16 0.05 0.05 0.16 0.08 0.19

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1%
level.

-0.5 MPa, indicating that germination was not significantly suppressed
at these levels of drought stress. Significantly lower values of GI were
observed at a water potential of -1.5 MPa (Table 3). The 'ZM1′ genotype
exhibited the highest value of GI (Table 2). The rise in ѱs was accom-
panied by greater variations in GRI which varied from 35.85 (in the
control) to 2.74 at -2.5 MPa (Table 3). The 'ZM1′ showed the highest
values of GRI among the studied genotypes. Tolerance to drought which
was indicated by the GSI index showed a downward trend with the
decrease of water potential and reached a minimum amount at
-2.5 MPa, whereas the highest values were observed in the control and
at -0.5 MPa. For all genotypes, we observed high values (> 90%) when
ѱs was lower than −1 MPa. Furthermore, GSI values lower than 50%
were observed at −2 MPa. Accordingly, the 'ZM1′ showed values
of > 50% at these levels of water stress. For the six genotypes, the re-
lationship between the rate of germination and the water potential was
identifiable (Table 3). Significant interactions were observed among
these traits by the ANOVA (P ≤ 0.01).
Higher levels of osmotic stress significantly reduced the growth
indices, including the radicle and plumule length, their fresh and dry
masses (data not shown), and their thicknesses, especially in the tissues
of the plumule (Table 2). Also, similar trends were observed for the
ratio of these traits (Plumule / radicle ratio) (Table 2). In the genotypes
'ZM1′, 'Haward' and 'Chandler', there were variations among these traits
at different levels of osmotic stress, but the extents of variation were
less than those observed in the other genotypes (Table 2). Plumules that
were produced from germinated seeds at different osmotic stress levels
were significantly smaller than those of the control (P ≤ 0.01). In the
'ZM1′, 'Haward' and 'Chandler', the plumule length decreased slightly
when the seeds germinated at -1.5 MPa, but the suppressive effect of
water stress was observed noticeably (by 70% reduction in plumule
length) at -2.5 MPa (Table 2). Similar results were achieved with regard
to radicle growth. In particular, the radicle length in 'Haward' was re-
duced significantly (by up to 50% or more) only in seedlings that ger-
minated at -1.5 MPa. However, the radicle elongation in 'TT1′, 'TT2' and
'SS2' declined at all levels of the osmotic stress, except at 0 and
-0.5 MPa. Among the genotypes, water stress levels caused significant
interactions which were detected by ANOVA. These effects were seen in
the thickness of the radicles and plumules, their fresh and dry weights,
and other evaluated traits (Table 2).
3.2. RWC, RC, and MDA of seedlings
The reduction in water potential significantly reduced the RWC
values by all of the ѱs levels (p ≤0.01) (Table 2). Substantial levels of
decrease in the RWC of radicles were obtained because there was an
increase in the osmotic stress level (Table 2). Seedlings of the genotypes

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Table 3
The effect of PEG-induced osmotic stress and genotype interaction on the germination indexes of walnut genotypes. Final Germination Percentage (FGP), Mean
Germination Time (MGT), Germination Index (GI), Germination Rate Index (GRI), Germination Stress Index (GSI), Radicle Water Content (RWC), Plumule Water
Content (PWC), Relative Water Content (RWC), Radicle Relative Conductivity (RRC), Plumule Relative Conductivity (PRC).
Genotype Stress level (MPa) Germination indexes

FGP (%) MGT (days) GI (%) GRI (% day−1) GSI (%) RWC (%) PWC (%) RWC (%) RRC (%) PRC (%)

*
'ZM1' 0 85.25 16.53 25.79 10.04 95.22 104.75 2.71 3.13 16.23 93.79
0.5 77.73 16.66 26.70 9.92 96.05 105.66 2.65 2.92 15.01 90.82
1 69.43 20.55 25.27 8.17 89.97 98.97 2.67 2.77 14.59 87.55
1.5 61.47 21.02 22.53 7.41 85.36 93.90 2.27 2.63 12.85 85.89
2 50.44 21.77 17.50 6.48 82.64 90.91 2.22 2.45 11.81 83.13
2.5 44.51 24.35 15.27 6.23 80.41 88.46 2.10 2.13 10.47 76.57
'SS2' 0 89.76 16.14 23.19 8.79 89.03 97.93 2.85 2.95 16.23 88.28
0.5 82.83 16.37 20.95 8.69 84.39 92.83 2.36 2.74 15.01 84.57
1 61.52 20.88 19.53 6.57 78.38 86.22 2.04 2.45 12.61 80.13
1.5 47.10 23.69 17.19 4.20 70.22 77.24 1.66 2.13 11.44 69.11
2 37.81 26.20 12.44 2.65 59.88 65.87 1.31 1.48 9.49 61.38
2.5 23.27 30.15 9.87 3.04 49.93 54.92 1.07 1.13 8.11 52.24
'TT2' 0 78.09 17.50 21.86 8.08 87.84 96.63 2.83 3.70 17.32 87.13
0.5 72.07 17.76 19.76 7.26 83.27 91.59 2.34 3.45 16.02 83.47
1 53.52 22.65 18.42 5.49 77.34 85.07 2.03 3.27 13.45 79.08
1.5 40.98 25.69 16.21 3.29 69.28 76.21 1.65 3.10 12.21 68.22
2 32.89 28.41 11.72 2.07 59.09 65.00 1.30 2.90 10.13 60.58
2.5 20.24 32.70 9.31 1.48 49.26 54.19 1.06 2.52 8.66 51.57
'Haward' 0 92.07 14.83 25.43 8.67 88.81 97.70 2.69 3.40 17.32 92.57
0.5 84.43 14.94 26.32 10.80 105.17 115.69 2.63 3.18 16.02 89.63
1 74.98 18.43 24.91 8.89 100.34 110.37 2.65 3.01 15.57 86.41
1.5 66.38 18.85 22.21 8.07 93.66 103.03 2.25 2.85 13.72 84.77
2 54.47 19.53 17.26 7.06 90.66 99.72 2.20 2.66 12.61 82.05
2.5 48.07 21.85 15.06 6.78 88.12 96.93 2.09 2.32 11.17 75.58
'TT1' 0 70.91 15.92 19.61 9.28 95.08 104.59 2.24 3.19 16.77 86.75
0.5 65.44 16.16 17.72 9.18 92.89 102.18 1.92 2.96 15.51 83.11
1 48.60 20.60 16.52 6.94 87.33 96.06 1.59 2.65 13.02 7.74
1.5 37.21 23.37 14.54 4.44 78.49 86.34 1.24 2.30 11.82 67.92
2 29.87 25.85 10.52 2.80 68.52 75.37 1.00 1.60 9.80 60.31
2.5 18.38 29.75 8.35 3.21 65.16 71.67 1.36 1.22 8.38 51.34
'Chandler' 0 84.35 18.10 22.81 11.27 84.47 92.92 2.34 3.39 16.77 92.16
0.5 77.83 18.37 23.61 11.14 80.61 88.67 2.39 3.16 15.51 89.24
1 69.43 23.43 28.60 9.17 73.88 81.27 2.21 2.99 15.07 86.04
1.5 61.47 26.58 29.61 8.33 65.94 72.53 1.99 2.84 13.28 84.40
2 50.44 29.39 28.02 7.28 54.84 60.32 1.92 2.65 12.20 81.69
2.5 44.51 33.82 24.98 6.99 47.33 52.07 1.84 2.31 10.82 75.25
LSD 0.08 1.61 1.45 2.24 10.76 11.83 0.31 0.08 0.61 2.19

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1%
level.

within the control group did not show significant differences in their
RWC values. Under severe drought conditions, the seedlings showed
significantly lower values of RWC than those of the control. After ex-
posure to osmotic stress, the seedlings exhibited a greater membrane
permeability which correlated significantly with the MDA content.
MDA levels in the plumule samples were higher, compared to the ra-
dicle samples in all genotypes and among all osmotic stress levels. At
the maximum level of osmotic stress, the MDA levels increased sig-
nificantly by about four times greater than those observed in the control
seedlings (Table 4).
However, an opposite trend was observed with regard to the RC and
MDA under each osmotic stress level and among the different genotypes
(Table 4). In both tissues of the radicles and plumules, the RC decreased
in all genotypes. However, the gradients of decline were slower in the
genotypes of 'ZM1′, 'Haward' and 'Chandler', compared to other geno-
types.
3.3. Starch, soluble sugars and osmolytes
The concentrations of starch, proline and soluble sugars in the ra-
dicles and plumules varied significantly under osmotic stress, as com-
pared with the control (Table 4). The proline concentration in radicles
and plumules multiplied by 2.68 and 3.56, respectively, under PEG
treatment (-2.5 MPa) compared to the control. Also, proline levels in the
plumule were higher than in the radicles among all genotypes. The
proline content significantly increased, especially in the three geno-
types 'ZM1′, 'Haward' and 'Chandler'. It reached a maximum amount at
-2.5 MPa, but the differences between the mean values of proline con-
centrations were not significant at different levels of osmotic stress
among other genotypes. Similar trends were observed with regard to
soluble sugars in the radicles and plumules of samples in all of the
studied genotypes. The starch content decreased in the genotypes
'ZM1′, ' Haward ' and 'Chandler' (0.41 times) but remained almost
constant in other genotypes (Table 4).
3.4. Antioxidant status and phenolic compounds
Superoxide radicals and hydrogen peroxide contents in walnut
seedlings increased under osmotic stress levels, as compared to the
control (Table 4). Parallel to increases in the PEG concentration, the
O−2 and H2O2 contents increased significantly. The peroxidase (POD),
catalase (CAT), superoxide dismutase (SOD), lipoxygenase (LOX), glu-
tathione reductase (GR) and ascorbate peroxidase (APX) activity in-
creased under osmotic stress, but the trend of increase was different
among the six genotypes and depended on the concentration of PEG
(Table 3). A significant increase in the activity of these enzymes was
observed under a low PEG concentration (- 0.5 MPa) and reached the
maximum at −2 MPa of ѱs before declining at -2.5 MPa (Table 3). The

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Table 4
The effect of PEG-induced osmotic stress and genotype interaction on the biochemical compounds and antioxidant enzyme activity of radicles and plumule of walnut
genotypes. Radicle Malondialdehyde content (R MDA), Plumule MDA content (P MDA), Radicle Lipoxygenase activity (R LOX), Plumule LOX activity (P LOX),
Radicle Peroxidase activity (R POD), Plumule POD activity (P POD), Radicle Ascorbate peroxidase activity (R APX), Plumule APX activity (P APX), Radicle
Glutathione content (R GSH), Plumule GSH content (P GSH), Radicle Proline content (R PRO), Plumule PRO content (P PRO), Radicle Starch content (R STA),
Plumule STA content (P STA).
Genotype Stress Biochemical compounds and antioxidant enzyme activity
level
(MPa)
R MDA P MDA R LOX P LOX R POD P POD R APX P APX R GSH P GSH R PRO P PRO R STA P STA(mg/
(μmol/g (μmol/g (U/g (U/g (U/g (U/g (U/g (U/g (μg/g (μg/g (μg/g) (μg/g) (mg/g g FW)
FW) FW) min) min) min) min) min) min) FW) FW) FW)

*
'ZM1' 0 43.71 77.28 22.02 58.56 125.62 168.96 113.20 135.50 0.88 1.08 25.06 33.38 3.61 5.74
0.5 56.74 100.32 36.60 97.36 150.23 202.05 127.79 152.96 1.08 1.33 28.04 38.82 2.84 4.53
1 78.90 139.49 45.83 121.89 192.87 259.41 169.11 202.43 1.48 1.83 38.31 49.91 2.60 4.14
1.5 101.36 179.21 81.03 215.52 238.16 320.32 201.77 241.52 1.90 2.34 51.69 64.42 2.21 3.51
2 122.65 216.85 113.46 301.75 291.94 392.67 274.61 328.71 2.54 3.14 73.35 74.90 1.88 2.99
2.5 146.36 258.76 167.47 445.40 343.34 461.79 259.82 311.01 2.97 3.66 89.24 89.55 1.50 2.39
'SS2' 0 44.96 79.46 24.31 64.67 131.15 176.40 109.89 131.53 0.80 0.99 20.58 49.04 3.40 5.41
0.5 48.11 85.06 24.81 66.00 144.12 193.84 115.35 138.08 0.84 1.04 21.64 34.47 3.46 5.51
1 46.51 82.30 24.02 63.89 135.99 182.91 117.19 140.28 0.80 0.99 21.61 37.30 3.50 5.57
1.5 45.21 79.94 25.81 68.65 143.64 193.19 116.54 139.50 0.83 1.02 23.29 36.24 3.26 5.19
2 48.76 86.22 25.67 68.27 138.75 186.62 116.71 139.70 0.86 1.06 23.60 37.57 3.33 5.31
2.5 50.20 88.75 22.72 60.44 142.53 191.70 116.04 138.90 0.81 1.00 23.14 35.78 3.54 5.63
'TT2' 0 46.96 83.03 26.49 70.47 120.27 125.71 114.65 137.24 0.81 1.01 20.82 34.02 3.52 5.40
0.5 50.27 88.88 27.04 71.92 132.16 127.00 120.88 144.70 0.85 1.05 21.90 36.73 3.58 5.50
1 48.64 86.01 26.17 69.62 124.71 124.76 121.52 145.46 0.81 1.00 21.87 37.22 3.62 5.56
1.5 47.25 83.53 28.13 74.81 131.71 130.54 119.01 142.46 0.84 1.03 23.57 38.75 3.37 5.18
2 50.96 90.10 27.97 74.40 127.23 125.09 120.03 143.68 0.87 1.08 23.88 37.95 3.45 5.30
2.5 52.46 92.74 24.76 65.86 130.70 121.58 115.15 137.83 0.82 1.01 23.41 36.30 3.66 5.62
'Haward' 0 45.68 80.76 21.55 57.31 115.19 126.24 117.11 140.18 0.89 1.10 25.35 34.15 3.73 5.73
0.5 59.29 104.83 35.82 95.28 137.76 156.71 132.20 158.24 1.09 1.35 28.37 39.71 2.94 4.52
1 82.45 145.77 44.85 119.28 176.86 195.58 174.95 209.42 1.50 1.85 38.77 51.05 2.69 4.13
1.5 105.93 187.28 79.30 210.90 218.39 237.27 208.73 249.85 1.92 2.37 52.31 65.90 2.28 3.51
2 128.17 226.61 111.03 295.30 267.71 287.50 284.08 340.05 2.57 3.17 74.23 76.62 1.94 2.98
2.5 152.94 270.41 163.88 435.87 314.84 313.69 268.79 321.74 3.01 3.71 90.31 91.61 1.55 2.38
'TT1' 0 40.26 71.19 23.79 63.28 137.09 188.36 185.91 222.53 0.79 0.98 20.31 50.24 3.35 5.53
0.5 43.10 76.21 24.28 64.59 138.49 206.98 204.29 244.54 0.83 1.02 21.36 35.32 3.42 5.64
1 41.71 73.74 23.51 62.52 136.06 195.32 113.20 135.50 0.79 0.97 21.33 38.22 3.45 5.70
1.5 40.51 71.62 25.26 67.18 142.36 206.29 127.79 152.96 0.82 1.01 22.98 37.13 3.22 5.31
2 43.69 77.25 25.12 66.81 136.41 199.27 109.89 131.53 0.85 1.05 23.29 38.49 3.29 5.43
2.5 44.98 79.52 22.24 59.15 137.02 203.61 115.35 138.08 0.80 0.99 22.83 36.66 3.49 5.76
'Chandler' 0 39.16 69.24 23.99 63.81 137.66 180.42 178.07 213.15 0.87 1.07 24.73 34.20 3.55 5.88
0.5 50.83 89.88 39.89 106.09 170.90 215.76 212.95 254.90 1.07 1.31 27.67 39.77 2.81 4.63
1 70.69 124.98 49.94 132.82 213.28 277.00 273.40 327.26 1.46 1.81 37.82 51.13 2.57 4.24
1.5 90.82 160.57 88.30 234.85 258.75 342.04 337.59 404.10 1.87 2.31 51.02 65.99 2.18 3.60
2 109.89 194.30 123.64 328.82 313.52 419.29 413.84 495.36 2.51 3.10 72.40 76.73 1.86 3.06
2.5 131.13 231.85 182.49 485.35 342.19 493.10 486.69 582.57 2.93 3.62 88.08 91.74 1.48 2.44
LSD 2.96 7 2.78 7.41 20.19 20.27 10.60 12.69 0.10 0.13 3.60 13.67 0.37 0.58

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1%
level.

SOD, POD, LOX, CAT and APX activity increased by 3.56, 2.67, 7.58,
4.83 and 2.29 times, respectively, which indicates a significant increase
in PEG-treated seedlings, compared to the control. This trend was ob-
served among all antioxidant enzymes in the genotypes 'ZM1′, 'Haward'
and 'Chandler' and also in the other genotypes ('TT2', 'TT1′ and 'SS2').
Furthermore, the enzyme activities in Plumule samples were higher
than those in the radicle samples among all osmotic stress levels for all
genotypes (Table 4).
Under osmotic stress, there were significant levels of decrease in the
AsA content in the beginning, but certain PEG concentrations made the
ѱs reach −1 MPa and caused the AsA content to increase significantly.
The trends of change in the AsA content were similar to those observed
in the antioxidant enzyme activity. These trends showed an increase in
the AsA content of 'ZM1′, 'Haward' and 'Chandler' genotypes. Osmotic
stress caused significant increases in the GSH content, compared with
the control, especially in the three genotypes 'ZM1′, 'Haward' and
'Chandler'. The GSH reached its maximum amount at -2.5 MPa. In 'TT2',
'TT1′ and 'SS2', the variations in GSH amount were not significant at
different levels of osmotic stress. The GSH and AsA level increased by
3.49 and 2.25 times in PEG-treated seedlings, compared to the control
(Table 5). Similar to the enzyme activities, the GSH and AsA levels in
the plumule were higher in comparison with the radicle samples among
all osmotic stress levels and for all genotypes. Among the germinating
seeds, the TPC contents of tissues showed positive trends of change
(Table 6).
There was an increase in the content of TPC in tolerant genotypes,
reaching a maximum level when the water potential of osmotic media
was -2.5 MPa Also, the TPC content in Plumule samples was higher than
in the radicles. The results of HPLC analysis described the differences in
phenolic compounds (Table 5). Accordingly, phenolic compounds in-
creased in drought-tolerant genotypes ('ZM1′, 'Haward' and 'Chandler'),
parallel to the increase in TPC. Among these compounds, the cinnamic
acid contents of radicles and plumules were at their lowest, but in-
creased when the plants were exposed to high levels of PEG con-
centration. Catechin and pyrocatechin were at their highest levels and
significantly increased at more severe levels of osmotic stress (Table 6).
These compounds in the plumules were higher than in the radicles
among all genotypes, and the differences also depended on the severity
of the osmotic stress levels. The measurable contents of phenolic
compounds were significantly affected by the concentration of PEG, the

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Table 5
The effect of PEG-induced osmotic stress and genotype interaction on the hormones content (μg) of radicles and plumule of walnut genotypes. Radicle Jasmonic acid
content (R JA), Plumule JA content (P JA), Radicle Salicylic acid content (R SA), Plumule SA content (P SA), Radicle Abscisic acid content (R ABA), Plumule ABA
content (P ABA), Radicle Gibberellin content (R GA3), Plumule GA3 content (P GA3), Radicle Indole-3-acetic acid content (R IAA), Plumule IAA content (P IAA),
Radicle Cytokinin content (R CK), Plumule CK content (P CK).
Genotype Stress level Hormonal compounds contents
(MPa)
R JA (μg/g P JA (μg/ R SA (μg/g P SA (μg/g R ABA P ABA R GA3 P GA3 R IAA P IAA R CK P CK (μg/
FW) g FW) FW) FW) (μg/g FW) (μg/g FW) (μg/g FW) (μg/g FW) (μg/g (μg/g (μg/g g FW)
FW) FW) FW)

*
'ZM1' 0 10.67 16.72 99.38 145.00 0.23 1.12 80.38 43.65 1.47 4.63 1.75 3.60
0.5 13.58 21.29 111.74 163.03 0.46 2.21 80.54 43.73 1.35 4.19 2.27 4.66
1 17.70 27.73 127.85 186.53 0.64 3.07 84.39 45.83 1.19 3.76 2.45 5.03
1.5 23.52 36.85 147.56 215.30 0.90 4.32 76.72 41.66 0.87 2.75 2.69 5.52
2 27.87 43.68 180.19 262.89 0.79 3.80 79.50 43.17 0.68 2.14 2.34 4.81
2.5 30.32 47.51 222.99 325.35 0.67 3.22 80.86 43.90 0.51 1.61 2.09 4.29
'SS2' 0 11.32 17.74 95.63 139.53 0.22 1.06 85.21 46.27 1.42 4.48 1.65 3.38
0.5 10.57 16.57 96.41 140.66 0.23 1.12 76.47 41.52 1.29 4.06 1.59 3.27
1 11.82 18.52 96.94 141.44 0.21 1.01 78.45 42.60 1.16 3.64 1.44 2.96
1.5 10.35 16.22 97.18 141.79 0.22 1.09 79.50 43.17 0.84 2.67 1.37 2.82
2 10.41 16.31 95.08 138.72 0.23 1.11 80.86 43.90 0.66 2.07 1.28 2.63
2.5 10.86 17.02 97.19 141.80 0.23 1.13 85.21 46.27 0.49 1.56 1.11 2.27
'TT2' 0 11.46 17.96 96.95 141.46 0.22 1.08 76.47 41.52 1.30 4.11 1.82 3.37
0.5 10.71 16.78 97.74 142.60 0.23 1.14 78.45 42.60 1.13 3.56 1.67 3.43
1 11.96 18.75 98.28 143.39 0.21 1.03 80.38 43.65 0.82 2.58 1.54 3.16
1.5 10.47 16.41 98.53 143.75 0.22 1.11 81.50 44.25 0.71 2.24 1.44 2.96
2 10.53 16.51 96.39 140.63 0.23 1.13 83.71 45.46 0.64 2.00 1.35 2.78
2.5 11.00 17.23 98.53 143.76 0.23 1.15 76.45 41.51 0.48 1.50 1.19 2.45
'Haward' 0 10.80 16.93 100.76 147.00 0.23 1.14 80.38 43.65 1.21 3.81 1.64 3.36
0.5 13.75 21.55 113.28 165.27 0.47 2.25 80.54 43.73 0.87 2.75 2.36 4.83
1 17.91 28.07 129.61 189.10 0.65 3.13 84.39 45.83 0.68 2.14 2.54 5.22
1.5 23.80 37.31 149.60 218.27 0.92 4.39 76.72 41.66 0.70 2.21 2.79 5.73
2 28.22 44.21 182.67 266.52 0.80 3.87 80.38 43.65 0.62 1.94 2.43 4.99
2.5 30.69 48.10 226.07 329.84 0.68 3.28 80.54 43.73 1.13 3.56 2.17 4.46
'TT1' 0 11.45 17.94 91.14 132.97 0.22 1.04 84.39 45.83 0.82 2.58 1.71 3.51
0.5 10.69 16.76 91.88 134.05 0.23 1.10 76.72 41.66 0.66 2.08 1.65 3.39
1 11.95 18.73 92.39 134.79 0.21 0.99 80.38 43.65 1.26 3.96 1.50 3.07
1.5 10.46 16.40 92.62 135.12 0.22 1.07 80.54 43.73 0.95 2.99 1.43 2.93
2 10.52 16.49 90.61 132.20 0.23 1.09 84.39 45.83 0.71 2.23 1.33 2.73
2.5 10.98 17.21 92.62 135.13 0.23 1.11 76.72 41.66 1.10 3.45 1.15 2.36
'Chandler' 0 10.79 16.91 94.71 138.19 0.23 1.10 80.38 43.65 0.79 2.50 1.82 3.74
0.5 13.73 21.53 106.49 155.36 0.45 2.18 80.54 43.73 0.77 2.43 2.36 4.83
1 17.89 28.04 121.84 177.76 0.63 3.02 84.39 45.83 1.05 3.31 2.54 5.33
1.5 23.77 37.26 140.63 205.18 0.89 4.25 77.25 41.94 0.92 2.90 2.79 5.73
2 28.18 44.16 171.72 250.54 0.78 3.74 80.94 43.95 0.69 2.16 2.43 4.99
2.5 30.66 48.04 212.51 310.06 0.66 3.17 82.66 44.88 1.11 3.50 2.17 4.46
LSD 1.78 2.80 9.89 14.43 0.06 0.30 6.42 3.48 0.15 0.45 0.18 0.52

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1%
level.

genotype and the interaction between these two factors (Table 6). In
sum, the TPC contents in tolerant genotypes ('ZM1′, 'Haward' and
'Chandler') multiplied by 1.85 and 1.97 in the radicles and plumules,
respectively.
3.5. Phytohormones status
Significant increases were observed in the JA content under osmotic
stress, compared with the control. This trend of increase was especially
highlighted in the genotypes 'ZM1′, 'Haward' and 'Chandler'. The max-
imum amount of JA was achieved at -2.5 MPa. Its content in the plu-
mule was higher than in the radicles among all genotypes (Table 5).
Between 'TT2', 'TT1′ and 'SS2', there were no significant variations in
the content of JA by the different levels of osmotic stress. The JA
concentration in the radicles and Plumules multiplied by 2.84 and 2.87,
respectively, under PEG treatment, compared to the control (Table 5).
Variations in the SA content at different concentrations of PEG were
similar to the variations in JA. Also, a similar trend was observed in the
radicles and Plumules of the same genotypes. The SA concentrations in
the radicles and Plumules multiplied by 2.23 and 2.74 under the PEG
treatment, as compared to the control. The ABA content increased at a
gradual pace, parallel to the increase in the PEG concentration in the
culture medium of seeds. This increase was especially noticeable in the
genotypes 'ZM1′, 'Haward' and 'Chandler'. In 'TT2', 'TT1′ and 'SS2',
Variations in the ABA content were not significant between the 'TT2',
'TT1′ and 'SS2' as caused by the different levels of osmotic stress, and
these genotypes showed the same trends of change in the ABA content
(Table 5). There were similar changes in the GA3 content of radicles
among all genotypes, and there was no significant variation among the
GA3 levels as affected by the different levels of osmotic stress and by the
genotypes.
Also, the GA3 content in the plumules was similar to that of the
radicles. Contrary to the JA and SA, there was a decrease in IAA, par-
allel to the increase in the severity of osmotic stress. These trends were
observed among all genotypes in their radicles and Plumules, as the IAA
content multiplied by 0.34 and 0.36, respectively, thereby showing a
declining trend compared to the control seedlings (Table 5). Also, the
IAA level in the plumule was higher than in the radicles among all
genotypes. The CK content increased significantly in the genotypes
'ZM1′, 'Haward' and 'Chandler', especially when the value of Ψs reached
−1 MPa and thereafter remained constant. In other genotypes, there
was no significant variation in the CK level at different levels of osmotic
stress (Table 5). Under the PEG treatment, the CK concentration in the
radicles and plumules multiplied by 1.53 and 1.69, respectively, as

336
 N. Lotfi, et al.

Table 6
- The effect of PEG-induced osmotic stress and genotype interaction on the phenolic compounds of Radicle (R.) and Plumule (P.) of walnut genotypes.
Genotype Stress level (MPa) Juglone Gallic acid Chlorogenic phenolic compounds content (mg/g DW) Rutin Cinnamic acid Epicathechin Vanillic acid

Pyrocatechin Catechin Syringic acid

R. P. R. P. R. P. R. P. R. P. R. P. R. P. R. P. R. P. R. P.

*
'ZM1' 0 220.34 240.65 256.04 310.56 112.43 135.79 834.64 956.20 1446.70 1643.08 258.49 327.90 485.80 582.05 8.59 11.80 856.90 945.86 15.79 19.48
0.5 234.89 253.04 264.27 3442.04 123.79 148.28 862.06 981.37 1516.83 1712.05 317.07 376.94 504.74 640.61 9.20 14.67 897.53 994.78 19.05 21.94
1 259.04 274.65 289.65 384.03 146.62 163.12 891.67 1036.07 1596.21 1793.04 356.02 419.53 546.27 692.04 12.73 16.93 943.80 1024.68 23.85 25.82
1.5 258.34 262.04 327.94 404.94 184.04 181.95 943.64 1126.69 1682.57 1834.50 394.23 467.39 591.04 751.78 15.84 18.50 981.79 1112.78 29.27 34.70
2 230.54 252.16 284.03 365.93 130.72 147.03 873.04 1025.76 1562.03 1773.05 347.41 394.02 521.05 695.63 13.07 15.39 924.80 1034.69 24.07 30.79
2.5 224.95 246.68 264.37 315.05 125.38 132.56 837.19 972.63 1501.47 1710.58 284.38 345.03 493.01 614.60 11.43 12.37 875.62 973.70 20.51 26.48
'SS2' 0 191.03 208.64 221.99 278.56 108.45 117.73 723.63 829.03 1254.29 1446.49 232.80 284.97 431.79 504.64 6.45 10.23 742.93 820.06 10.67 16.89
0.5 196.45 212.57 219.59 287.56 107.33 115.47 721.43 830.51 1246.98 1438.26 227.50 282.70 426.08 501.74 5.35 10.03 753.79 824.30 11.50 14.09
1 194.70 203.69 220.45 268.09 110.46 120.73 714.08 823.09 1254.79 1442.89 219.59 294.50 432.73 497.21 6.47 9.64 737.08 813.06 10.35 15.37
1.5 197.05 206.67 226.70 273.63 102.79 114.70 720.65 826.03 1247.09 1437.80 225.62 281.08 428.80 490.24 5.27 10.61 733.70 815.74 9.37 13.05
2 193.60 211.08 224.80 284.08 108.57 115.05 721.69 824.12 1236.37 1432.80 228.51 284.62 436.58 485.42 6.06 9.78 741.70 810.91 10.59 14.59
2.5 195.04 210.45 223.79 273.15 110.46 114.93 715.80 824.69 1246.08 1414.70 224.10 279.41 435.04 496.70 6.57 10.72 745.83 816.45 9.61 15.48
'TT2' 0 209.76 229.09 243.74 305.86 119.08 129.27 794.55 910.27 1377.21 1588.25 255.61 312.90 474.11 554.09 7.08 11.23 815.74 900.43 11.72 18.54
0.5 215.70 233.40 241.11 315.74 117.84 126.79 792.13 911.90 1369.18 1579.21 249.80 310.40 467.84 550.91 5.87 11.01 827.66 905.08 12.63 15.47
1 213.78 223.65 242.05 294.36 121.29 132.56 784.06 903.75 1377.76 1584.29 241.11 323.36 475.14 545.94 7.10 10.58 809.31 892.74 11.36 16.88
1.5 216.36 226.92 248.92 300.45 112.86 125.94 791.27 906.98 1369.30 1578.70 247.73 308.63 470.82 538.28 5.79 11.65 805.60 895.68 10.29 14.33

337
2 212.57 231.77 246.83 311.92 119.21 126.32 792.42 904.88 1357.53 1573.21 250.90 312.51 479.36 532.99 6.65 10.74 814.39 890.38 11.63 16.02
2.5 214.15 231.07 245.72 299.92 121.29 126.19 785.95 905.51 1368.20 1553.34 246.06 306.79 477.67 545.38 7.21 11.78 818.92 896.46 10.55 17.00
'Haward' 0 254.87 278.36 296.16 359.22 130.05 157.07 965.43 1106.04 1673.40 1900.55 299.00 379.28 561.92 673.26 9.94 13.65 991.18 1094.08 18.26 22.53
0.5 271.70 292.69 305.68 395.64 143.19 171.52 997.14 1135.15 1754.52 1980.33 366.75 436.01 583.83 740.99 10.64 16.97 1038.17 1150.66 22.04 25.38
1 299.63 317.69 335.04 444.21 169.60 188.68 1031.39 1198.42 1846.34 2074.01 411.81 485.27 631.87 800.48 14.72 19.58 1091.69 1185.25 27.59 29.87
1.5 298.82 303.10 379.33 468.39 212.88 210.46 1091.51 1303.24 1946.23 2121.97 456.01 540.63 683.66 869.58 18.32 21.40 1135.64 1287.15 33.86 40.14
2 266.67 291.67 328.54 423.27 151.20 170.07 1009.85 1186.50 1806.80 2050.89 401.85 455.76 602.70 804.64 15.12 17.80 1069.72 1196.83 27.84 35.61
2.5 260.20 285.33 305.80 364.42 145.03 153.33 968.38 1125.04 1736.75 1978.63 328.94 399.10 570.26 710.91 13.22 14.31 1012.83 1126.28 23.72 30.63
'TT1' 0 176.90 193.20 205.56 257.95 100.42 109.02 670.08 767.68 1161.47 1339.45 215.57 263.88 399.84 467.29 5.97 9.47 687.96 759.38 9.88 15.64
0.5 181.91 196.84 203.34 266.28 99.38 106.93 668.04 769.05 1154.70 1331.83 210.67 261.78 394.55 464.61 4.95 9.29 698.01 763.30 10.65 13.05
1 180.29 188.62 204.14 248.25 102.29 111.80 661.24 762.18 1161.94 1336.12 203.34 272.71 400.71 460.42 5.99 8.93 682.54 752.89 9.58 14.23
1.5 182.47 191.38 209.92 253.38 95.18 106.21 667.32 764.90 1154.81 1331.40 208.92 260.28 397.07 453.96 4.88 9.82 679.41 755.38 8.68 12.08
2 179.27 195.46 208.16 263.06 100.54 106.54 668.28 763.14 1144.88 1326.77 211.60 263.56 404.27 449.50 5.61 9.06 686.81 750.90 9.81 13.51
2.5 180.61 194.88 207.23 252.94 102.29 106.42 662.83 763.66 1153.87 1310.01 207.52 258.73 402.85 459.94 6.08 9.93 690.64 756.03 8.90 14.33
'Chandler' 0 245.95 268.62 285.80 346.65 125.50 151.57 931.64 1067.33 1614.83 1834.03 288.53 366.01 542.26 649.69 9.59 13.17 956.49 1055.78 17.63 21.74
0.5 262.19 282.45 294.98 381.79 138.18 165.51 962.24 1095.42 1693.11 1911.02 353.92 420.75 563.40 715.06 10.27 16.37 1001.84 1110.39 21.26 24.49
1 289.14 306.57 323.31 428.66 163.66 182.08 995.30 1156.48 1781.71 2001.42 397.40 468.29 609.76 772.47 14.21 18.90 1053.48 1143.76 26.62 28.82
1.5 288.36 292.49 366.05 452.00 205.43 203.10 1053.31 1257.63 1878.11 2047.70 440.05 521.71 659.73 839.15 17.68 20.65 1095.89 1242.10 32.67 38.73
2 257.33 281.46 317.04 408.46 145.91 164.12 974.50 1144.97 1743.56 1979.11 387.78 439.81 581.60 776.47 14.59 17.18 1032.28 1154.94 26.87 34.37
2.5 251.09 275.35 295.09 351.66 139.95 147.97 934.48 1085.66 1675.96 1909.38 317.43 385.13 550.31 686.03 12.76 13.81 977.38 1086.86 22.89 29.56
LSD 4.87 3.59 2.63 8.93 3.78 3.57 7.13 16.78 34.52 23.75 14.46 12.62 16.37 17.52 0.24 1.16 8.37 15.73 1.48 1.08

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1% level.
Scientia Horticulturae 250 (2019) 329–343
N. Lotfi, et al. Scientia Horticulturae 250 (2019) 329–343

Table 7
The effect of PEG-induced osmotic stress and genotype interaction on the polyamine compounds (mg/g DW) of radicles and Plumule of walnut genotypes.
Genotype Stress level (MPa) Polyamine Putrescine Spermidine Spermine

radicle Plumule radicle Plumule radicle Plumule radicle Plumule

*
'ZM1' 0 958.91 1321.38 326.99 450.58 328.04 452.04 303.88 418.74
0.5 1052.56 1450.43 358.92 494.59 360.08 496.19 333.55 459.64
1 1448.50 1996.03 493.94 384.03 495.53 682.84 459.03 632.54
1.5 1893.67 2609.48 645.74 404.94 647.82 892.70 600.10 826.94
2 1754.11 2417.17 598.15 365.93 600.08 826.91 555.87 766
2.5 1695.33 2336.16 578.10 315.05 579.97 799.20 537.24 740.32
'SS2' 0 960.06 1322.97 327.38 278.56 328.43 452.58 304.24 419.25
0.5 964.03 1328.45 328.73 287.56 329.79 454.46 305.50 420.98
1 961.77 1325.33 327.96 268.09 329.02 453.39 304.78 419.99
1.5 954.64 1315.50 325.53 273.63 326.58 450.03 302.52 416.88
2 960.00 1322.89 327.35 284.08 328.41 452.56 304.22 419.22
2.5 964.14 1328.59 328.77 273.15 329.83 454.51 305.53 421.03
'TT2' 0 1022.47 1408.96 348.66 305.86 349.78 482.00 324.02 446.49
0.5 1026.70 1414.79 350.10 315.74 351.23 484.00 325.36 448.35
1 1024.30 1411.48 349.28 294.36 350.41 482.86 324.59 447.29
1.5 1016.69 1401.01 346.69 300.45 347.81 479.28 322.19 443.97
2 1022.41 1408.88 348.63 311.92 349.76 481.97 324.00 446.47
2.5 1026.81 1414.95 350.14 299.92 351.27 484.05 325.39 448.39
'Haward' 0 1021.24 1407.27 348.24 359.22 349.36 481.42 323.63 445.96
0.5 1120.98 1544.71 382.25 395.64 383.48 528.44 355.23 489.51
1 1542.65 2125.78 526.04 444.21 527.74 727.23 488.86 673.65
1.5 2016.76 2779.09 687.71 468.39 689.93 950.72 639.11 880.69
2 1868.13 2574.28 637.03 423.27 639.08 880.66 592.01 815.79
2.5 1805.52 2488.01 615.68 364.42 617.76 851.14 572.17 788.45
'TT1' 0 958.62 1320.99 326.89 257.95 327.94 451.90 303.78 418.62
0.5 962.59 1326.45 328.24 266.28 329.30 453.78 305.04 420.35
1 960.33 1323.34 327.47 248.25 328.53 452.71 304.33 419.36
1.5 953.21 1313.52 325.04 253.38 326.07 449.35 302.07 416.25
2 958.56 1320.90 326.87 263.06 327.92 451.87 303.77 418.59
2.5 962.69 1326.60 328.28 252.94 329.34 453.82 305.07 420.39
'Chandler' 0 957.47 1319.40 326.49 346.65 327.55 451.36 303.42 418.11
0.5 1050.98 1448.25 358.38 381.79 359.53 495.44 333.05 458.95
1 1446.33 1993.04 493.19 428.66 494.78 681.61 458.34 631.59
1.5 1890.3 2605.56 644.77 452.00 646.5 891.36 599.20 825.70
2 1751.48 2413.54 597.25 408.46 599.18 825.67 555.04 764.85
2.5 1692.79 2332.66 577.24 351.66 579.10 798.00 536.44 739.21
LSD 61.95 85.37 21.12 8.93 21.19 29.20 19.63 27.05

* These parameters are for mean comparison of interaction effect. Values show the real values for parameters. Mean separation within columns by LSD, at 1%
level.

compared to the control.
Higher PEG concentrations caused significant increases in the con-
tents of free Spd and Put contents among the germinating seeds
(Table 7). These incremental trends were substantially dependent on
the genotype. The 'ZM1′, 'Haward' and 'Chandler' were more efficient in
tolerating osmotic stress, which corresponds with the higher amounts of
free Spd in their seedlings, as compared to the susceptible genotypes in
this study ('TT2', 'TT1′ and 'SS2') when recording the contents on the
thirtieth day after treatment. Changes in the content of free Put in
seeds, as induced by osmotic stress, varied more than the extent of
variations observed in the content of free Spd. Significantly higher le-
vels of free Put were observed in the seedlings of genotypes 'ZM1′,
'Haward' and 'Chandler', as compared to the control seedlings. The
maximum amount of free Put was achieved in seedlings exposed to
-1.5 MPa. In contrast, however, the susceptible genotypes did not ex-
hibit significant amounts of free Put in their seeds at different osmotic
levels, as compared to the control seedlings. The contents of Spd and
Spm showed trends of change that were similar to the trend observed
for the Put. In summary, higher PEG levels caused the tolerant geno-
types to undergo notable increases in their contents of Put, Spd and
Spm, and their amounts in the plumules were higher than in the ra-
dicles, among all genotypes, and at all levels of osmotic stress.
4. Discussion
Polyethylene glycol, as a solution, was used for simulating drought
stress during the seed germination test. In this experiment, the per-
centage of germination corresponded with a significant reduction in
water potential, thereby observing values less than -1.5 MPa. The main
reason for this was the decrease in the amount of water absorbed by
plant tissues in the studied genotypes, especially in the susceptible
genotypes ('TT2', 'TT1′ and 'SS2'). With regard to the decrease in the
FGP, the results of this study are in agreement with the results obtained
by Lotfi et al. (2009) and Vahdati et al. (2009). According to Miransari
and Smith (2014), this reduction in FGP could be due to the incapable
vigor of seeds among the genotypes, and the lack of sufficient energy
which is required to start the germination. As the cellular respiration is
intensified, the consumption of energy usually increases when the
process of water absorption begins in plants. Then, in response to a
decrease in the water potential, the rate of water absorption decreases.
Under conditions of limited water availability in this experiment, the
final seed germination rate and the water potential were reduced si-
multaneously, and the effects of water deficiency on seeds were sub-
stantial when the water potential was less than 1.5 MPa. The responses
to different levels of drought stress were not similar among the geno-
types, as the 'ZM1′, 'Haward' and 'Chandler' showed higher levels of
tolerance to drought stress compared to the other three genotypes (i.e.
'TT2', 'TT1′ and 'SS2'). During drought stress, seed germination was
observed when the water potential of the medium was ≥ −2 MPa, and
it was only observed in tolerant genotypes, namely, 'ZM1′, 'Haward' and
'Chandler'. This observation generally indicates that walnuts can be
categorized as a species susceptible to drought stress, and the response

338
N. Lotfi, et al.
of the plant during germination depends on the degree of drought stress
and the genotype.
A decrease in water potential caused wider differences in the MGT,
compared to the control. According to previous results reported by
Willenborb et al. (2004), it was demonstrated that drought stress not
only affects seed germination but also increases the average amount of
time required for germination in plants (Brassica napus L.). Further-
more, similar results were reported on wheat (Yang et al., 2017) and
ornamental sunflower (Toscano et al., 2017). The germination index
(GI) is another factor which can be taken as a reliable index when
studying seed germination (Salehzade et al., 2009; Sadeghi et al.,
2011). Germination is usually characterized by a speed which is com-
monly considered in relation to the amount of time that seeds need to
complete their germination, and this definition is considered as a GI
indicator. Accordingly, seeds that germinate faster are more likely to
have higher values of the GI (Yang et al., 2017). The highest values of
GI were recorded in 'ZM1′, 'Haward' and 'Chandler'. This confirms the
faster rate of germination in these genotypes, even when osmotic stress
levels prevail. However, when the water potential decreased, the GI
exhibited a downward trend, especially in susceptible genotypes. This
point indicates that walnut genotypes of 'SS2', 'TT1′ and 'TT2' are sus-
ceptible to drought stress, parallel to greater severities of stress at the
time of germination. Similarly, Silva et al. (2001) proved that GI indices
in the seeds of Bowdichia virgilioides declined with the decrease in water
potentials, and that germination was suppressed at -0.9 MPa, thereby
suggesting a varied threshold of water potential for the tissues in the
seedlings of B. virgilioides when the seeds germinated in a specific en-
vironment (between -0.7 and -0.9 MPa). In the current work, the
threshold of water potential in walnuts ranged from -1 to -1.5 MPa, and
this confirms the findings of a previous study on relevant evaluations of
walnut (Lotfi et al., 2009; Vahdati et al., 2009).
The occurrence of germination on a daily basis and the evaluation of
its frequency are defined as the GRI index. Higher values of GRI in-
dicate that these seeds can germinate more and faster. This parameter
can be a measure of germination vigor (Emmerich and Hardegree,
1990). This study revealed that the GRI showed various ranges in re-
sponse to the variations in water potential. The relationship between
germination rate and water potential was confirmed at this point, with
respect to different osmotic levels (Table 2). Our results demonstrate
that the GRI index can be used for categorizing and screening the
genotypes for their tolerance to drought stress.
Lower values of GSI were observed when the stress intensified. In
previous cases of study, the identification of drought tolerance in plants
could be assisted by the GSI index (Ahmad et al., 2009). Also, this index
can indicate the degree of resistance against drought stress, as staged by
the genotypes, and the quality of seeds as mentioned previously in the
scientific literature. In wheat and triticale genotypes, there were high
values of GSI (Yang et al., 2016b). This state can only be achieved in
genotypes with greater tolerance to drought, which is in agreement
with our results. It seems that the screening and selection of genotypes
for drought tolerance can be helped by the “germination stress toler-
ance index” which makes researchers arrive at a better criterion when
dealing with conditions of severe drought stress. However, it seems that
germination and its factors cannot be considered merely as a means of
reflecting the tolerance in drought-tolerant plants, but can be used for
assessing the quality of germination and the occurrence of differences
between genotypes. However, these criteria may not be comprehensive
for the description of sustainability among plants that grow under field
conditions (Ashraf et al., 1996).
Our results showed that plumule growth was suppressed in seeds
that germinated at lower levels of water potential (-1.5 MPa). In our
study, similar conditions were observed in the fresh and dry weights of
seedlings which showed a downward trend in response to the stress, as
the drought progressively reduced the water potential. In drought-tol-
erant genotypes, there were no changes in dry weight, indicating that
an increase in the severity of drought can induce a substantial decrease
339
Scientia Horticulturae 250 (2019) 329–343
in the water content of seedlings. Nonetheless, this decrease did not
affect the accumulation of biomass in seedlings. According to the re-
sults, the fresh and dry weights of seedlings were least affected by
drought stress in the genotypes 'ZM1′, 'Haward' and 'Chandler'. A no-
ticeable decrease was observed in the germination ability of six walnut
genotypes when the water potential ranged between -1 and -2.5 MPa. In
other words, seed germination was affected negatively when the water
deficit resembled osmotic potentials that were more or equal to
−1 MPa. A greater tolerance to drought stress was observed among the
genotypes 'ZM1′, 'Haward' and 'Chandler'.
ROS, like H2O2 and O2–, is produced in response to oxidative stress.
This phenomenon has been widely reported in plants by many scientists
(Abbasi et al., 2015; Fahad et al., 2015; Ozgur et al., 2013). Nucleic
acids of cells and proteins are the main components of essential mem-
brane lipids that could be damaged by the ROS. A major index that can
be used for quantifying lipid peroxidation in the membrane is MDA, and
its concentration can also be used for assessing the degree of damage to
membrane lipids (Zhuang et al., 2010). The production of H2O2 can
substantially increase the amount of O–2, the concentration of MDA and
the permeability of the membrane, as observed under different levels of
osmotic stress (Ψs ≥−1 MPa). Consequently, this causes oxidative
bursts and thus facilitates cellular damage. According to our results,
drought stress generally causes the increase in O·–2 and H2O2 contents,
followed by a decrease in the MDA concentration and a decline in the
permeability of cellular membranes in 'ZM1′, 'Haward' and 'Chandler'. It
appears that the mechanisms of tolerance can partly reduce the con-
centrations of H2O2, O·–2 and MDA, while also preventing their accu-
mulation and upholding the membrane permeability. Previous studies
found that drought stress tends to increase the amounts of O·–2 and
H2O2 in the leaves and petals of marigold (Tagetes erecta L.). Similar
results were observed by (Tian et al., 2012) and by an experiment on
long-term salinity stress (Garg and Bhandari, 2016).
The RWC values of radicles and plumules were reduced significantly
by the application of PEG when the water potential reached low levels
(i.e. -1 ≤ MPa), especially in genotypes that are susceptible to drought.
The exposure of seedlings to PEG, especially among drought-tolerant
genotypes, caused an increase in the value of RWC. This trend appeared
to be consistent with the decrease in morphological qualities and traits.
These observations can also be explained by an increase in the ABA
content which can reduce the dry mass, fresh biomass, radicle and
Plumule length, and the thickness of these tissues (Wang et al., 2009;
Ahmed et al., 2012).
In this study, the accumulation of proline and soluble sugars, along
with changes in their contents, were moderated under high con-
centrations of PEG solutions. These solutions altered the germination
traits, even in tolerant walnut seeds. In the present study, drought stress
significantly increased the amounts of total soluble sugars and proline
in seedlings, parallel to higher concentrations of PEG, especially in the
genotypes 'ZM1′, 'Haward' and 'Chandler'. Our results are in agreement
with previous reports which indicated that drought stress causes an
increase in the amounts of free proline and sugar content in plants,
thereby increasing the plant growth and the qualities of germination in
certain circumstances under drought stress (Lotfi et al., 2010; Krasensky
and Jonak, 2012). Seed germination is usually inhibited or delayed by
drought stress, and water uptake is prevented due to the decrease in
water potential (Kaya et al., 2006). However, the accumulation of
proline and soluble sugars in plants can usually regulate osmotic
pressure (Farhad et al., 2011; Liu et al., 2011; Moaveni, 2011). The
destabilization of biochemical equilibrium in plants, as induced by
drought, may also prevent compatible solutes from replacing the mo-
lecules of water around other essential components. The scavenging of
free radicals and the regulation of osmotic potential can take place by
compatible osmolytes such as proline (Krasensky et al., 2012).
AsA and GSH are two components which can partially protect cells
from stress-induced damages. These compounds host non-enzymatic
components in their structures, but their modes of action may mimic
N. Lotfi, et al.
those of antioxidant enzymes. They can be used in order to control
harmful levels of ROS in plants. The over-production of ROS can cause
oxidative stress and may damage membrane structures (Fazeli et al.,
2007). It has been observed that AsA levels decrease under salt-stress in
wild-type plants (Koffler et al., 2015). In this research, the AsA content
in walnut genotypes increased significantly when Ψs ≥−1 MPa.
However, it decreased when Ψs ≤−1 MPa in susceptible genotypes,
namely, 'TT2', 'TT1′ and 'SS2'. In the available literature, drought and
salinity stresses are known to reduce the activity of GR during the initial
days of stress. This reduction is transcribed into the low capability of
plants (as in Tagetes erecta L. and Arabidopsis) in reducing the oxidized
glutathione and converting it into GSH (Montilla-Bascon et al., 2017).
In this study, there was an increase in the GSH content, and then a high
GR activity in tolerant genotypes under drought stress. This revealed an
increased capability of these plants in converting the GSSG to GSH (Zhu
and Gong, 2014). The addition of PEG prompted a sharp increase in the
GSH content when plants were under drought stress. This became
parallel to an upward trend of ROS scavenging capacity, thereby low-
ering the damages done to lipids in plasma membranes. The application
of PEG corresponded with intensifications in the activities of GR and
APX (Table 4) in seedlings under drought stress. This denotes that low
concentrations of PEG (i.e. a moderate level of stress) can improve the
tolerance of plants to water deficiency, possibly through an increase in
the cyclic conversions and actions of AsA and GSH. However, similar
studies reported that the addition of Si had no significant effect on the
content of AsA, indicating that low levels of AsA could be compensated
by the accumulation of GSH in peroxisomes and chloroplasts.
The conversion of O·–2 to H2O2 is the first layer of a defense system
in plants, and it emanates from activities by superoxide dismutase
(SOD). Accordingly, the H2O2 is converted to H2O by activities of
peroxidase (POD) and catalase (CAT) (Ahmad et al., 2015). According
to the results of this study, the increase in PEG concentration sub-
stantially affected the SOD activity under moderate and intense levels
of osmotic stress, as compared to the changes observed in the POD and
CAT activities. Polyethylene glycol increases the activity of antioxidant
enzymes under the negative effects of drought stress, which is due to
fluctuations in the composition or production of proteins, their per-
formance, and changes in the expression of genes associated with en-
zymes (Wu et al., 2014; Zhang et al., 2015). Yang et al. (2009) reported
that the amounts of CAT, POD, SOD, APX and glutathione reductase
(GR) increase under drought stress, compared with the control group.
Likewise, our study confirmed that POD and SOD activities in 'ZM1′,
'Haward' and 'Chandler' seedlings increase significantly, parallel to the
intensification of drought stress, compared with the control. Further-
more, the APX and CAT activities increased sharply under moderate
and severe drought stress.
Phytohormones are key factors that can regulate the growth of
plants and their development. Moreover, phytohormones are char-
acterized by their actions of mediating mechanisms that trigger specific
responses to various abiotic stresses in plants (Sreenivasulu et al.,
2012). This study confirmed that drought stress can induce the bio-
synthesis of ABA in both radicles and Plumules. The observation was
especially noticeable in tolerant genotypes, namely, 'ZM1′, 'Haward'
and 'Chandler', particularly in their radicle tissues. Also, the biosynth-
esis and degradation of ABA was enhanced by osmotic stress in sus-
ceptible genotypes, namely, 'TT2', 'TT1′ and 'SS2'. This pattern has also
been shown in many earlier studies, where the accumulation of ABA in
leaves can occur in response to drought stress, especially in tolerant
genotypes (Zorb et al., 2013). In this study, the ABA concentration in-
creased in the leaves only, which is probably due to the redistribution of
ABA and its migration from the radicles to the leaves in walnut seed-
lings as affected by drought stress.
In addition to the previous factors, JA and methyl jasmonate are
mechanisms for biochemical regulation in plants under biotic and
abiotic stress, and they play an active role in osmotic regulation
(Kupper et al., 2009; Koo et al., 2009; Wasternack and Hause, 2013).
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Scientia Horticulturae 250 (2019) 329–343
Furthermore, the responses of plants to drought stress may not be si-
milar, and any specific response could depend on the content of JA and
its related compounds (Pedranzani et al., 2003). Based on our results,
the 'ZM1′, 'Haward' and 'Chandler' showed a significant increase in the
JA and SA contents of the radicles and plumules at low and medium
levels of stress. Nonetheless, the amount of JA decreased slightly in
both tissues when Ψs was less than or equal to −2 MPa. Our results are
in agreement with a previous report by Wasternack and Hause. (2013),
where the JA content remained stable in drought-tolerant cultivars of
tomato, but increased in the susceptible cultivars, especially in their
radicles. It has been revealed that promising walnut genotypes parti-
cipate in the JA and SA signaling pathway in response to drought and
salinity stress, thereby counterbalancing the conditions of stress.
However, different tissues (i.e. the leaves and radicles) may exhibit
different JA pathways in the regulation of drought stress, and their
mechanisms of tolerance may be different (Wasternack and Hause,
2013; Ahmad et al., 2015).
Here, tolerant walnut genotypes synthesized higher amounts of IAA,
especially in their plumules. The IAA content and its fluctuation in
tolerant genotypes occurred at low levels in the radicles, compared to
the plumules. This study indicated that the biosynthesis of IAA im-
proved in six tolerant genotypes, especially in their radicles and plu-
mules. In this regard, one hypothesis can suggest that the FW and DW of
leaves are possibly affected by a decrease in the changes to the con-
centration of IAA and its redistribution. Our results are in agreement
with a previous study which concluded that the redistribution of IAA
and its maximum extent of formation in plant tissues occur when these
mechanisms operate in correlation with and in response to suppressed
levels of plant growth (Ryu and Cho, 2015).
Radicle tips and emerging seeds are the main tissues were CKs are
produced in plants, and their translocation is performed by the xylem,
taking them from the radicles to the plumules where plant development
and growth processes are regulated by this hormone (Wang et al.,
2015). Abiotic stress, especially in the radicle, inhibits the biosynthesis
of CKs and, consequently, the plumules receive less CKs in response to
moderate levels of stress (Tran et al., 2010; Nishiyama et al., 2011,
2012). In our experiment, the ZR and CKs existed endogenously.
Through time, the radicles and leaves acquired lower amounts of these
compounds, especially in the three susceptible genotypes after being
treated with the PEG solution. Among the studied hormones, the GA3
content underwent no significant change in the six genotypes. This
could imply that the GA3 content in walnut seedlings might have a
negligible role in response to drought stress.
Drought stress is associated and correlated with PAs for plant re-
sistance (Muscolo et al., 2014). PAS metabolism, the external applica-
tion of PAS and the manipulation of the endogenous PAS are a few
options to induce drought resistance in crops (Farooq et al., 2009;
Muscolo et al., 2014). According to our results, a significant difference
was observed in the free Spd, Spm and Put contents in the seeds of
different genotypes during their germination under normal conditions
and osmotic stress levels. However, the tolerant genotypes 'ZM1′, 'Ha-
ward' and 'Chandler' showed contents of free Spd that were significantly
higher than the contents found in drought-susceptible genotypes,
namely, 'TT2', 'TT1′ and 'SS2'. As a result of drought stress, the free Put
content in seeds showed a similar trend of change in drought-tolerant
genotypes, compared to drought-susceptible genotypes that were al-
most constant during germination. These results suggest that, under
drought stress, seed germination in walnuts is supported by the accu-
mulation of free Spd. In addition, it can be assisted by insoluble-con-
jugated Put in seeds during their different phases of germination.
However, the accumulation of free Put appeared to increase the func-
tionality of osmoprotectants which assist the regulation of an electro-
chemical gradient for the phases of seed germination in walnuts. Yang
et al. (2016a) reported that the pre-germination soaking of rice affected
the Put content and caused inhibitory effects on the seed germination,
the coleoptile growth and the radicle development. The existence of Put
N. Lotfi, et al.
as a primary ingredient in the production of Spd can be applied exo-
genously, and thus contribute to the relative balance of these three
compounds in the plant. These results are contradicted by some studies
about polyamines in other plants (Gracia et al., 2017). Put has a more
stimulating and indirect role in most plant processes. This compound
can increase tolerance to stress by adjusting the levels of ABA hormones
(Cuevas et al., 2008), activating the antioxidant system, and inducing
the action of phenylalanine ammonium lysis, as one of the key enzymes
in the synthesis of flavonoids. Putrescine acts as a buffer and osmolyte,
and causes an increase in proline content which helps maintain the leaf
water status under stress conditions (Hao et al., 2012). Exogenous pu-
trescine can increase the phospholipase D activity, which has a role in
the mitigation of drought stress injury in the early stages of drought
treatment (An et al., 2012). It is also known that exogenous putrescine
can enhance the transcript levels of a heat shock protein gene and heat
shock proteins (HSP17) during heat shock. This response was found to
be much more pronounced in thermotolerant than in susceptible cul-
tivars (Kumar et al., 2012). A primary substance for the synthesis of Spd
is the Put, even though Spd and Put have different effects on plant
growth and development (Yang et al., 2008; Liu et al., 2013; Yang et al.,
2016a). In previous studies, Spd has been described as a substance that
counters the inhibitory effects of drought on plant growth. Multiple
environmental stresses like drought can be alleviated by the over-
expression and activity of Spd synthase. This has been observed pre-
viously regarding stress-regulated genes in transgenic Arabidopsis
thaliana (Kasukabe et al., 2004).
Variations in endogenous hormones can be partly due to the
amounts of Spd or Spm, since they both seem to be involved in the
germination of walnut seeds, as the results of the present study suggest.
This fact can be transcribed into the understanding that exogenous
applications of Spd and Spm may significantly increase the content of
endogenous hormones such as Z + ZR, GA3 and IAA contents. In fact,
this hypothesis has been proven in wheat grains during their germi-
nation (Yang et al., 2016a,b). Their coleoptile and radicle growth were
promoted under moderate levels of drought stress during the phases of
germination, and this confirms the present findings regarding hormones
in walnut seeds.
The TPC of walnut seedlings was significantly affected by PEG, the
genotype and their interaction. These compounds were found more
abundantly in the plumules than in the radicles of all genotypes, and
different levels of osmotic stress were also a source for variation in this
regard. Among the compounds, the cinnamic acid contents of the ra-
dicles and plumules were found to be in their lowest amounts, but in-
creased in response to high levels of PEG. From this study, it was found
that tolerant genotypes have high phenolic contents, and that they
contain the highest levels of catechin, vanillic acid, rutin and juglone
among the examined genotypes. The inhibition of polyphenol oxidase
activity and, consequently, the varied availability of phenol compounds
suggest that, in certain circumstances, drought stress can improve the
antioxidant action of phenols in walnuts. If the phenolic components
are to be ranked quantitatively in this study, vanillic acid would be the
first, followed by catechin, pyrocatechol, epicatechin, rutin, syringin
acid, gallic acid, juglone, and cinnamic acid. The scientific literature
contains scanty amounts of information on the phenolic contents of
walnut tissues. Nonetheless, it has been reported that Persian walnut
genotypes in Iran have higher levels of phenolic compounds compared
to those in Serbia, France and the USA (Bujdoso et al., 2014). The
reason for this phenomenon may be the high adaptation of Persian
walnuts to different climatic conditions which vary in comparison with
their source of origin. In this study, the high content of TPC in drought-
tolerant genotypes was found to be consistent with high contents of
CKS. The CKs are partially involved in the energy status of plants, and
they serve as determinants of growth and photosynthesis in adult
leaves, besides moderating the effects of biotic or abiotic stresses and
their interactions with plants (Argueso et al., 2012; Kieber and Schaller,
2013; Bahaji et al., 2015).
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Scientia Horticulturae 250 (2019) 329–343
5. Conclusion
In general, this study can be summarized by the finding that 'ZM1′,
'Haward' and 'Chandler' are more tolerant than the other three geno-
types when exposed to drought stress. The differences among genotypes
in their tolerance could be attributed to the capability of plants in in-
ducing different defensive mechanisms. Their ability to acclimate under
progressive drought stress can also cause variations in tolerance. In
order to tolerate osmotic stress, the Ψw gradient is known to exhibit
protective measures taken from the radicle to the plumule, and this is
collectively defined as osmotic adjustment. These were important me-
chanisms whereby a lower osmotic potential occurred in the plumules,
as compared to the radicles. Other mechanisms that build the pathways
of tolerance to drought in walnuts are defensive systems characterized
by enzymatic and non-enzymatic antioxidant activities, as these two
parameters varied greatly among the studied genotypes. The response
of walnuts to water deficiency showed that different hormones have
different effects on the plants, and that synergistic or antagonistic in-
teractions may be observed among them if exogenous growth regulators
are used together. In drought-tolerant genotypes, ('ZM1′, 'Haward' and
'Chandler'), it was observed that ABA plays a major role in response to
drought stress. On the other hand, drought-susceptible genotypes ('TT2',
'TT1′ and 'SS2') suffered from the drought stress because JA is the key
hormone which acts against stress in susceptible genotypes. In addition,
walnut growth receives additional regulation by the actions of IAA and
CKS. These effects were clearer when low levels of water potential
prevailed (i.e. -1≥ MPa Ψs). Ultimately, it can be suggested that im-
provements in drought tolerance can substantially assist future studies
if the right biochemical factors are analyzed and their roles are iden-
tified in accordance with the plants responses to drought. Promising
walnut genotypes can be a primary material for further genetic and
molecular analyses in making provisions for a more detailed under-
standing of their horticultural specifications.
Acknowledgements
We are grateful to the University of Zanjan and the Atatürk
University of Turkey for their supports. We also thank Dr. Parisa
Mohammadi for her scientific and laboratory support at Atatürk
University of Turkey. We acknowledge the University of Urmia for
providing greenhouse supplies during practical stages of research.
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