Plant Growth Regulation (2021) 95:65–82

https://doi.org/10.1007/s10725-021-00726-4

ORIGINAL PAPER

Proteomic analysis of salt‑responsive proteins in the leaves of two

contrasting Tunisian barley landraces
R. Jardak1 · J. Riahi1 · W. Dallagi1 · S. Planchon2 · H. Boubakri3 · B. Bouamama1 · A. Bouagila1 · R. Nefissi1 ·
S. Mejri1 · J. Renaut2 · H. P. Mock4 · A. Ghorbel1

Received: 26 June 2020 / Accepted: 7 June 2021 / Published online: 17 June 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Salinity is a brutal environmental factor that severely affects barley growth and development. In this context, local landraces,
commonly cultivated under stressful conditions, could represent important reservoirs of valuable traits in barley breeding
programs. Therefore, understanding salt-tolerance mechanisms in such genotypes is of great interest. Here, based on a
2D-PAGE comparative proteomic study, salt-induced proteome changes were explored in the seedling leaves of two con-
trasting Tunisian landraces, namely Boulifa (tolerant) and Testour (sensitive). The analysis showed that 11 salt-responsive
proteins were differentially accumulated in both accessions under salt stress and 43 were genotype-specific (18 in Boulifa
and 25 in Testour). Using mass spectrometry identification and annotation, 11 function categories revealed being involved in
salt-stress response, specifically the defense/cell wall related metabolism. In fact, a chitinase, was up-regulated in the tolerant
accession and down-regulated in the sensitive one in addition to a ricin B-like lectin R40G3 as well as a predicted BSP that
were up-regulated in the tolerant one. Then, two other chitinases, PR10, glucan endo-1.3-β-glucosidase, were down-regulated
in Testour, while still unchanged in the tolerant accession Boulifa. In the latter, signaling, redox/polyamine catabolism and
the energy metabolism were found as part of the biochemical pathways underlying salt-tolerance. These results suggest that
Boulifa may alleviate salt stress by activation of specific defense responses, and adjustment of both redox/polyamine catabo-
lism and energy metabolism processes. Our findings represent a basis that would assist selection of candidates as markers
in improving barley salt tolerance and elite genotypes creation.

Keywords Barley landraces · Comparative proteomics · Leaves · Seedling stage · Salinity tolerance

Abbreviations BSP Basic Secretory Proteins

ATP Adenosine triphosphates related to defense
ATPE ATP synthase CF1 epsilon CHCA Alpha-cyano-4-hydroxycin-
subunit namic acid
GCSH Glycine cleavage system H
Communicated by Zhong-Hua Chen. JA Jasmonic acid
SA Salicylic acid
* R. Jardak JIP23 23 KDa jasmonate-induced
rjardak@yahoo.fr protein
1
Laboratory of Plant Molecular Physiology, 28 KDa RNP 28 KDa ribonucleoproteins
Centre of Biotechnology of Borj-Cedria, BP 901, MDH Malate dehydrogenase
2050 Hammam‑lif, Tunisia PAO Polyamine oxidase
2
“Environmental Research and Innovation” (ERIN) PAP9 Plastid-lipid-associated 9
Department, Luxembourg Institute of Science protein
and Technology, 5 Avenue des Hauts‑Fourneaux, PPase Inorganic pyrophosphatase
4362 Esch‑sur‑Alzette, Luxembourg
PR10 Pathogenesis-related protein 10
3
Laboratory of Leguminous, Centre of Biotechnology ROS Reactive oxygen species
of Borj-Cedria, BP 901, 2050 Hammam‑lif, Tunisia
4
Leibniz Institute of Plant Genetics and Crop Plant Research
(IPK), Corrensstrasse 3, 06466 Gatersleben, Germany

13
Vol.:(0123456789)
66
RuBisCO large sub BP a 1,5‐Bisphosphate carboxylase/
oxygenase large subunit-bind-
ing protein subunit alpha
SSP Standard Spots number
TCA​ Trichloroacetic acid
TCTP Translationally-controlled
tumor protein
TFA Trifluoroacetic acid
Introduction
Salinity is one of the major abiotic factors limiting crop
production. In fact, according to FAO, around 800 million
hectares of land are affected by salinity worldwide (Machado
and Serralheiro 2017; Hernández 2019). Moreover, salini-
zation is expanding and more than 50% of arable lands
are devoted to be saline by 2050 mainly due to unsuitable
farming and water management (Butcher et al. 2016). To
cope with this constraint, the most promising solution is
the selection and development of crop cultivars with high
salt tolerance (Fita et al. 2015). Within glycophytes, salin-
ity tolerance is differing and among cereals, barley (Hor-
deum vulgare L.), ranking fourth in terms of cultivation area
worldwide (Thormann et al. 2016), is the most tolerant one
(Colmer et al. 2005, 2006; Munns et al. 2006). It is therefore
an important reservoir of key tolerance genes (Witzel et al.
2010; Nguyen and Sticklen 2013) and a model plant used in
various studies dealing with the mechanisms of salt stress
tolerance (Munns and Tester 2008). In this context, toler-
ant barley cultivars exhibited different physiological strat-
egies, including sodium exclusion (Tavakkoli et al. 2011;
Bezouwa et al. 2019), vacuolar compartmentalization (Mian
et al. 2011), development regulation (Tilbrook et al. 2017;
Bousorra et al. 2019) and growth sustaining/salt-shielding
(Ho et al. 2020). The mechanisms involved in tolerance are
distinct throughout the barley life cycle (Angessa et al. 2017;
Saade et al. 2020). Besides, different studies reported a high
adaptive ability to salt stress in several wild and cultivated
barley genotypes (Wu et al. 2011; Ben Chikha et al. 2016;
Shen et al. 2016a; Shen et al. 2017; Bousorra et al. 2019;
Hammami et al. 2020; Ho et al. 2020). However, yield losses
due to salinity are still significant for most other genotypes
(Mahmood 2011; Bezouwa et al. 2019). Therefore, examin-
ing salt tolerance mechanisms remains important for barley
breeding programs and improvement (Mian et al. 2011; Zhu
et al. 2020a, b).
To understand molecular mechanisms governing salin-
ity tolerance, specific genomic knowledge associated with
transcriptomic and metabolomic approaches has made
large progress (Widodo et al. 2009; Shelden and Roessner
2013; Nguyen and Sticklen (2013); Huang et al. 2019; Ho
et al. 2020; Zhu et al. 2020a, b). Nevertheless, due to the
13
Plant Growth Regulation (2021) 95:65–82
post-transcriptional and -translational modifications, prot-
eomics approach and recent improvements in mass spec-
trometry and bioinformatics are becoming powerful tools
for identifying/characterizing key candidates (Aslam et al.
2017; Spoel 2018; Bittremieux et al. 2018). Consequently,
different key proteins related to plant stress tolerance and
involved in several biochemical pathways including signal-
ing transduction, protein synthesis, reactive oxygen species
(ROS) scavenging, photosynthesis and regulation of cell
development were annotated (Zhang et al. 2012; Kosová et
al. 2013; Ghosh and Xu 2014; Ahmad et al. 2016; Zhu et al.
2020a, b). Within barley, proteome of different tissues was
studied with respect to salt stress tolerance. In the seedling
root proteome, several biochemical pathways involved in
salinity tolerance were emphasized including ROS detoxi-
fication (e.g. lactoylglutathione lyase, L-ascorbate per-
oxidase; monodehydroascorbate reductase; peroxidases;
glutathione-S-transferases) (Witzel et al. 2014; Shen et al.
2017; Zhu et al. 2020a, b), secondary metabolism (Witzel
et al. 2009), defense (e.g. germin-like and pathogenesis-
related proteins) (Witzel et al. 2014), signal transduction
[e.g. Annexin and translationally-controlled tumor protein
(TCTP)], protection (e.g. HSP) (Mostek et al. 2015) and ion
homeostasis (e.g. H ­ +-ATPase) (Wu et al. 2014). Concern-
ing shoot proteome, reported studies have shown distinct
responsive proteins and biochemical pathways. In fact, a
comparative analysis between Afzal, a salt-tolerant geno-
type and L-527, a salt-sensitive one, led to the identification
of some proteins involved in the antioxidant system (e.g.
glutathione S-transferase), signal transduction (magnesium
chelatase), protein biosynthesis (cysteine synthase), ATP
generation (e.g. triosephosphate isomerase and FBP aldo-
lase) and photosynthesis (e.g. phosphoribulokinase and
sedoheptulose 1,7-bisphosphatase) (Rasoulnia et al. 2011).
Within the barleys with contrasted salinity tolerance XZ26
(tolerant) and XZ169 (sensitive), the results of Shen et al.
(2016a) highlighted a better shoot growth of the tolerant and
proved significant reduction in glycolysis and elevated level
of the tricarboxylic acid (TCA) cycle. In the wild XZ113
barley, other proteins related to salt tolerance were identified
by Shen et al. (2016b) including phosphoglycerate kinase,
sedoheptulose-1,7-bisphosphatase associated with photosyn-
thesis and shoot related metabolisms. Moreover, shoot barley
tolerance mechanisms were related to a higher expression
of chloroplastic ATPase, psbp2 and germin-like protein,
associated with photosynthesis, metabolic process, and ion
homeostasis (Wu et al. 2014). Regarding the tillering stage,
signal transduction (TCTP), ROS scavenging [e.g. polyam-
ine oxidase (PAO)], protein processing, photosynthesis (e.g.
PsbP family protein) and ATP metabolism (e.g. ATP syn-
thase) related proteins were observed in the tolerant barley
(Fatehi et al. 2012; Zhu et al. 2020a, b). Besides these stud-
ies on cultivated barley, proteomics for exploring tolerance
Plant Growth Regulation (2021) 95:65–82
strategy to salinity was performed for the halophytic wild
barley Hordeum marinum in comparison to Hordeum vul-
gare. In fact, tolerance of H. marinum to high salinity was
associated mainly to enhanced levels of stress-responsive
transcription factors (i.e. basic leucine zipper and nascent
polypeptide-associated complex families) and over abun-
dance of proteins related to energy metabolism (Maršálová
et al. 2016).Therefore, multiple and complex pathways seem
to be involved in barley tolerance mechanisms to salt stress.
Nevertheless, investigating specific biochemical mechanisms
could provide additional insights on salinity response and
help to identify novel key proteins for breeding programs
(Zhu et al. 2020a, b).
In Tunisia, barley landraces are considered as reservoir
of tolerance markers (Hamza et al. 2004; Zoghlami et al.
2011; Ben Naceur et al. 2012; Ben Romdhane et al. 2017;
Hammami et al. 2017; Ben Romdhane et al. 2018; Boussora
et al. 2019; Youssef et al. 2020). In the framework of bar-
ley improvement programs for food and feed, barleys with
contrasted salinity tolerance were selected based on a deep
physiological characterization (Ben Chikha et al. 2016). In
our previous studies, proteomic comparative analyses of two
contrasted barley accessions (Boulifa, salt-tolerant and Tes-
tour, salt-sensitive) allowed the identification of genotype-
specific and abundant proteins in albumin/globulin grain
proteome fraction (Riahi et al. 2019). To better understand
the molecular mechanisms and explore the proteome of
these barley landraces, we investigated in the present study
the proteome changes in the first seedling leaves under salt
stress conditions. This aims particularly to discern key can-
didates that can be valuable for an efficient selection and for
creating elite genotypes with high salinity tolerance.
Material and methods
Plant material and exposure to salinity
under hydroponics
The grains of two Tunisian winter barley landraces (Hor-
deum vulgare L.), namely Boulifa and Testour, were col-
lected from two different sites in El Kef and Beja districts,
respectively (Ben Chikha et al. 2016; Riahi et al. 2019).
Both landraces were previously characterized as genetically
distinct (Zoghlami et al. 2011; Ben Romdhane et al. 2017)
and with a contrasting salinity tolerance (Ben Chikha et al.
2016; Riahi et al. 2019).
Mature grains of Boulifa and Testour were germinated
and the seedlings were transferred, after seven days (d),
into bacs filled with a half strength nutrient solution of
Hewitt (1966) under a 16 h photoperiod and a 20–23 °C
temperature range for a 2 days acclimation phase. Thereafter,
seedlings were subjected to a further acclimation step on
67
a full-strength nutrient solution under aerated hydroponics
for 3 days. The experiment was carried out under a light
intensity of 200 µmol ­m−2/s and 70% relative humidity, with
a continuously aerated nutrient solution. The solutions were
refreshed every 2 days. Salt stress was imposed gradually
with the addition of 50 mM NaCl in all bacs, except those
holding the control plants. After 1 day, NaCl concentration
was raised up to 100 mM then up to 200 mM (13 days-
old seedlings). Salt stress assays were carried out in trip-
licate (Fig. S1). The leaves were harvested within 4 days
after exposure to 200 mM NaCl (17 days-old seedlings) for
growth parameter monitoring and protein extraction and
analyses.
Plant growth and mineral element content
determination
Measurements of both length and fresh weight of the sec-
ond leaf were conducted on 10 individual plants of both
genotypes (Boulifa and Testour) under control and salt stress
conditions. The leaves were dried at 70 °C for 24 h and incu-
bated in 0.5% ­HNO3 for 48 h before sodium (Na), potassium
(K) and calcium (Ca) content analysis using atomic absorp-
tion spectrophotometry.
Relative water content, osmotic potential and sugar
content determination
At the end of salt application; the 2nd leaf was picked in
the morning on stressed and control plants of both geno-
types and used for relative water content (RWC) assess-
ment according to Turner (1981). The RWC was calculated
based on the formula: RWC (%) = (fresh weight − dry
weight) × 100/(turgid weight − dry weight).
Tissue osmotic potential (Ψs) was measured using a
vapor pressure osmometer (Osmomat 3000 Type D-10553,
Berlin, Germany) and calculated using the formula: Ψs
(MPa) = − m × R × T, where m is the osmolality, R is the
universal gas constant, and T is the temperature (K).
For soluble sugar content evaluation, the leaves of control
and stressed seedlings were analyzed according to Morris
(1948). Absorbance values were recorded at 640 nm using
a spectrophotometer (Ultrospec 200, Pharmacia). Sugar
concentrations (µg/g DW) were determined according to
a standard curve previously established based on a set of
glucose solutions.
Determination of ­H2O2 and MDA contents
H2O2 content in the leaves of control and stressed bar-
ley seedlings were determined using the method of Wolff
(1994) with some modification. Fresh material (500 mg)
was homogenized in an ice bath in 5 mL of trichloroacetic
13
68
acid TCA (5%) and filtered using Wattman paper. The fil-
trate was incubated with the FOX1 (ferrous oxidation)
reagent (100 µM Xylenol orange, 250 µM ammonium sul-
fate, 100 mM sorbitol and 25 mM H ­ 2SO4) for 30 min. The
absorbance was then recorded at 560 nm. A ­H2O2 stand-
ard curve was used for H­ 2O2 content determination in the
samples.
Malondialdehyde (MDA) content as extent of lipid perox-
idation was determined according to the method of Hernán-
dez and Almansa (2002). An amount of 500 mg of the frozen
samples, was homogenized in 5 mL of 5% (w/v) TCA and
centrifuged for 15 min at 12,000g and 4 °C. The supernatant
(0.5 mL) was mixed with 0.5 mL of 0.5% (w/v) thiobarbi-
turic acid (TBA) in 20% (w/v) TCA and heated at 95 °C for
30 min. after which, the reaction was quickly cooled using
ice. The mixture was then centrifuged at 10,000g for 10 min.
The supernatant absorbance was measured at 532 nm and
600 nm.
Leaf protein extraction and quantification
The total proteins of the harvested leaves were extracted
from each genotype, under control and stress conditions
from the three independent assays (Fig. S1) (60 plants of
each genotype, for each treatment in each assay). The protein
extraction was performed according to Fatehi et al. (2012)
with some modifications. Fresh leaves stored at − 80 °C
were ground in liquid nitrogen, and the powder (1 g) was
suspended in 10 mL ice-cold solution of 10% (w/v) trichlo-
roacetic acid (TCA) in acetone with 0.07% w/v DTT for at
least 1 h at − 20 °C, prior to be centrifuged for 20 min at
39,000g at 4 °C. The pellets were rinsed twice with ace-
tone including 0.07% w/v DTT for 1 h at − 20 °C and then
dried under Speed Vac Concentrator SAVANT SPD1010
(Thermo). The resulting pellet was solubilized in lysis buffer
as previously described by Riahi et al. (2019). The extracts
were, then, aliquoted and stored at − 80 °C for 2-DE elec-
trophoresis. The protein concentrations were determined
according to the Bradford method using BSA as standard
(Ramagli and Rodriguez 1985).
2‑DE gel electrophoresis
Isoeletric focusing (IEF) was performed with 500 μg protein,
loaded by active rehydration to a 17 cm immobilized pH
gradient (IPG) strip using pH gradients of 4–7 (Bio-Rad).
Two independent separations (two technical replicates) of
each sample (control and stressed) from the three extractions
were performed to ensure technical reproducibility (Fig. S1).
Following the procedures described by Riahi et al. (2019),
the IEF was carried out at 20 °C with PROTEAN i12TM
IEF Cell from Bio-Rad and the separation in the second
dimension was performed using PROTEAN II xi Multi-Cell
13
Plant Growth Regulation (2021) 95:65–82
(Bio-Rad). The strips were loaded on top of 12% SDS-PAGE
and covered with 0.5% agarose. The SDS PAGE low range
(Bio-Rad, Hercules, CA) was used as marker.
Protein staining and image analyses of the 2‑DE gel
patterns
The gels were stained according to Riahi et al. (2019) before
to be scanned using a ChemiDoc MP Imaging System (Bio-
Rad) and then analyzed by PDQuest software (ver. 8.0.1,
Bio-Rad). Comparison of the mean value volume of each
spot between control and stress conditions was considered
for Boulifa and Testour genotypes. Differentially abundant
spots for each genotype were selected when difference
between salt-treated versus control plants is significant
according to Student’s t-test with P < 0.01. Combined results
from both genotypes were assessed to deduce differentially
accumulated spots. The ratio of the spot volume varia-
tion was calculated to determine the rate of the abundance
change after salt treatment.
Mass spectrometry
Selected spots presenting differential accumulation were
manually excised from gels and treated as previously
described by Riahi et al. (2019). Leaf spots were digested,
extracted and spotted onto MALDI plate by Tecan pipetting
platform EVO2 (Tecan Trading AG, Männedorf, Switzer-
land). Spot washing and tryptic digestion was performed by
incubations in 50 mM N ­ H4HCO3 in 50% MeOH and 100%
Acetonitrile (ACN) followed by 40 ng Trypsin (Promega,
Madison, WI, USA) in 20 mM ­NH4HCO3 (5 ng/μL), respec-
tively. A 0.1% trifluoroacetic acid (TFA) in 50% (v/v) ACN
and 7 mg/mL alpha-cyano-4-hydroxycinnamic acid (CHCA)
in 50% (v/v) CAN 0.1% (v/v) TFA solutions were used for
peptide extraction and spotting, respectively. MALDI TOF/
TOF analysis was performed with a TOF/TOF™ 5800 (AB
SCIEX, Redwood City, CA, USA) mass spectrometer in MS
and MS/MS mode. For each spot, the 10 most intense peaks
of the MS spectrum were selected for MS/MS acquisition.
Database interrogation was carried out over on an in house
Mascot server version 2.6.1 (Matrix Science Ltd., London,
UK) through Protein Pilot v4.5 (AB Sciex). Spectra were
searched against three databases, Triticeae-NCBI, Hordeum-
JGI and NCBIprot, downloaded on 25th January 2017,
restricted to the taxonomy Other Proteobacteria (36,675,354
sequences). The Triticeae-NCBI database contains all Trit-
iceae (txid147389) protein sequences from NCBI on 16th
August 2017 (213,862 sequences). The Hordeum-JGI data-
base was downloaded from phytozome.jgi.doe.gov (Hor-
deum vulgare r1 (Mascher et al. 2017; Beier et al. 2017),
(248,180 sequences). Trypsin was used as enzyme allowing
two missed cleavages. The mass tolerances were 100 ppm
Plant Growth Regulation (2021) 95:65–82
for precursor ions and 0.5 Da for fragment ions. The vari-
able modifications allowed were Tryptophan oxidation and
dioxidation, Methionine oxidation and Trp to Kynurenin
(W). Carbamidomethyl cysteine was set as fixed modifica-
tion. Each result had been manually validated. In order to
obtain gene ontology (GO), all identified protein sequences
were subjected to Blast search against Arabidopsis thaliana
proteome and to Interproscan using Blast2GO software.
Real‑time quantitative polymerase chain reaction
Real-time quantitative polymerase chain reaction (RT qPCR)
experiments were conducted to determine the expression
patterns of five selected genes corresponding to key candi-
date proteins (according to the proteomic data). These genes
included, TCTP, bpao, R40G3, PR10 and rbcL encoding for
translationally-controlled tumor protein homolog, flavin con-
taining polyamine oxidase, ricin B-like lectin R40G3, patho-
genesis related protein 10, and ribulose-1.5-bisphosphate
carboxylase/oxygenase large subunit, respectively. Leaves
from control and stressed seedlings were collected at the
end of the salt assay and used for total RNA extraction using
the RNeasy Plant Mini Kit (Qiagen). The examined genes
and primer sequences are listed in Table S1. To check the
specificity of the PCR product, the presence of a single band
of expected size for each primer pair was checked in agarose
gels (1% w/v). Real-time PCR reactions were carried out
according to the instructions manual of Brilliant II SYBR®
Green QRT-PCR Master Mix Kit, 1-Step (Agilent Technolo-
gies). QRT-PCR (1-Step) conditions started with 30 min at
50 °C and 10 min at 95 °C for denaturation followed by 40
cycles, each consisting of a step 30 s at 95 °C and 1 min
at 60 °C for hybridization and annealing. The expression
of each gene in different samples was normalized with the
expression of an internal control gene, HvTUB2 encoding for
the tubiline protein. The amplifications were performed on
an AriaMx Real-time PCR System (Agilent) and the relative
expression was calculated by ­2−∆∆CT method, according to
descriptions of Livak and Schmittgen (2001).
Statistical analysis
Statistical analysis concerned the data from three biologi-
cal replicates for physiological, biochemical and RT-qPCR
experiments. STATISTICA software (Statistica 2004-Ver-
sion 6) was used for data analysis and comparisons were
done using the Least Significant Difference (LSD; P < 0.05)
tests. For proteomic analysis, differentially abundant spots
were selected when difference between salt-treated versus
control plants is statistically significant according to Stu-
dent’s t-tests with P < 0.01. Venn diagram (https://​bioin​
fogp.​cnb.​csic.​es/​tools/​venny/​index.​html) was used to repre-
sent the number of protein spots with significant abundance
69
changes upon salt stress in both genotypes. Hierarchical
cluster analysis of the identified protein abundance was
performed by Expander version 6 software (http://​acgt.​cs.​
tau.​ac.​il/​expan​der/) by means of the log-transformed fold
change values (ratios corresponding to protein expression of
both studied genotypes under control and stress conditions;
B-C/T-C; B-ST/B-C; T-ST/T-C; B-ST/-T-ST). Euclidean
distances and Ward’s minimum criteria were used.
Results
Assessment of plant growth under salinity
The contrasting response of Boulifa and Testour to salinity
was assessed at the early seedling stage under 200 mM NaCl
for 4 days using hydroponic culture system. The develop-
ment of the third leaf was observed only in Boulifa, while,
Testour leaves were bland (Fig. S2a). Measurement of the
length of the second leaves showed a significant reduc-
tion upon salt stress for Boulifa compared to those of the
controls (22 cm ± 0.76 vs 19.53 cm ± 0.44 for control and
stressed leaves, respectively). Whereas, Testour seedlings
exhibited a significant larger decrease (19.06 cm ± 0.91 vs
14.39 cm ± 1.01 for controls and stressed leaves, respec-
tively), circa twofold (24.5%) in comparison with that of
Boulifa (11.21%) (Fig. S2b). Regarding the second leaf fresh
biomass, significant differences between studied genotypes
were noticed under control conditions (0.245 g ± 0.018 and
0.170 g ± 0.014 for Boulifa and Testour second leaf, respec-
tively). Additionally, under salt stress, a non-significant
change in the biomass of Boulifa was noted (13.59%), while,
the growth of the second leaf within Testour was signifi-
cantly reduced by 27.49% (Fig. S2c).This data consolidates
the contrasting behavior of both studied genotypes when
grown under 200 mM NaCl concentration that was consid-
ered as appropriate for proteome response analyses.
Changes of element contents in leaves in response
to salt stress
In absence of salt, no significant difference was found
between Boulifa and Testour. While under stress conditions,
the leaf Na contents increased significantly under salt stress
to about 10 and 19-fold in Boulifa and Testour, respectively.
Whereas, K contents significantly decreased by 34.9% and
49.7% in both genotypes, respectively. The contents of both
elements were significantly different under stress. These
deductions were also noted for the K ­ +/Na+ ratio, with a
higher value in Boulifa than Testour. Regarding Ca content,
the reductions and the difference between both genotypes
were significant under salinity conditions (29.3% and 45.5%
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70 Plant Growth Regulation (2021) 95:65–82

Table 1  Leaf element contents Genotypes Na (mg/g DW) K (mg/g DW) K/Na Ca (mg/g DW)
under control and stress
conditions (200 mM NaCl) Boulifa Control 2.17 ± 0.09 c
93.18 ± 2.48a
43.06 ± 3.09a
6.94 ± 0.46a
200 mM NaCl 23.38 ± 1.46b 38.66 ± 2.08b 1.78 ± 0.12b 4.91 ± 0.30b
Testour Control 1.95 ± 0.15c 94.79 ± 1.77a 43.75 ± 4.05a 6.37 ± 0.98a
200 mM NaCl 37.36 ± 0.58a 29.85 ± 1.73c 0.79 ± 0.1c 3.47 ± 0.32c

Data are mean ± SE of three replicates. The least significant differences (LSD) were calculated. The differ-
ent letters indicate significant differences according to LSD test (p ≤ 0.05)

Table 2  Relative water content (RWC), osmotic potential (Ψs) and Table 3  Comparison of ­H2O2 and MDA contents in leaves of Boulifa
total soluble sugar contents in leaves of control and stressed seedlings and Testour at 4 days of 200 mM NaCl treatment
Genotype RWC (%) Ψs (Mpa) Sugar (µg/g DW) Genotype H2O2 (µM/g FW) MDA (nM/g FW)
a a c a
Boulifa Control 88.8 − 1.42 219.33 Boulifa Control 3.03 ± 0.08 4.61 ± 0.32a
200 mM NaCl 82.9b − 2.42b 376.54a 200 mM NaCl 7.12 ± 0.5c 6.39 ± 0.16b
Testour Control 87.47a − 1.44a 193.07c Testour Control 3.71 ± 0.4ab 5.05 ± 0.05a
200 mM NaCl 77.37c − 2.37b 252.21b 200 mM NaCl 13.26 ± 0.66d 13.33 ± 0. ­5c

Data are mean ± SE of three replicates. The least significant differ- Data are mean ± SE of three replicates. The least significant differ-
ences (LSD) were calculated. The different letters indicate significant ences (LSD) were calculated. The different letters indicate significant
differences according to LSD test (p ≤ 0.05) differences according to LSD test (p ≤ 0.05)

decrease in Boulifa and Testour, respectively). In Boulifa,
the reduction of Ca content was less than that of Testour
(Table 1).
Osmotic status evaluation
The osmoregulatory ability was examined at the end of salt
application (200 mM NaCl) through Ψs, RWC, and sugar
content (Table 2). RWC in the leaves were almost similar
in both genotypes under control conditions with an average
value of 88.3%. After 4 days of 200 mM NaCl application,
a significant RWC decline was recorded within Boulifa and
Testour compared to corresponding controls. Nevertheless,
RWC decline in the tolerant Boulifa was lower (6.64%) than
that recorded in Testour (11.57%). Regarding Ψs, similar
values were registered under control conditions. When
subjected to 200 mM NaCl application, Ψs decreased sig-
nificantly in both genotypes after 4 days NaCl application
without significant variation between both genotypes. On the
other hand, soluble sugar contents increased in both geno-
types. Nevertheless, Boulifa exhibited a significantly higher
accumulation of soluble sugar than Testour (1.72-fold and
1.3-fold, respectively). The results indicated an increased
solute concentration and a possible occurrence of an osmotic
adjustment. The ability of Boulifa to exhibit this behavior
was more apparent.
The accumulation of ­H2O2 and MDA after salinity
treatment
Changes of H ­ 2O 2 and MDA contents in the leaves in
response to NaCl treatments are shown in Table 3. The
contents of H­ 2O2 and MDA increased significantly in both
tolerant and sensitive genotypes after salinity treatment.
While, the leaves in the sensitive barley accumulated higher
amounts compared with those of the tolerant one. In Boulifa,
the increase in ­H2O2 level was 2.3-fold. However, in Testour,
a significantly higher accumulation by 3.58-fold compared
to controls was recorded. Regarding the MDA, it increased
by 1.38-fold in Boulifa, and 2.63-fold in Testour compared
to controls. The significant variation of H
­ 2O2 and MDA leaf
contents between stressed genotypes revealed a contrasting
behavior of the tolerant and the sensitive genotype.
Comparative proteome analysis and statistical
assessment of the expression profiles
Barley leaves of Boulifa and Testour genotypes either
grown under control (0 mM NaCl:C) or salt treatment
(200 mM NaCl:ST) were used for the proteomic analysis.
Protein concentration averages ranged from 7.67 ± 0.48 µg/
µL to 8.83 ± 1.04 µg/µL (Table S2). For the proteomic
analyses, twenty-four 2-D gels from three biological and
two technical replicates (Fig. S1) were analyzed to exam-
ine specific proteins related to genotype or induced in
response to NaCl treatment. In total, 252 common spots
were matched between groups of all technical replicates
(B-C and T-C; Fig. 1a) (Table S3). Comparison between

13
Plant Growth Regulation (2021) 95:65–82
Fig. 1  a Representative 2-DE gels of Boulifa and Testour leaf pro-
teomes from the control plants of Boulifa (B) and Testour (T) geno-
types showing the position of the 54 differentially accumulated spots.
Identified spots are showed in red. b Venn diagram showing the 54
protein spots with significant abundance changes under salt stress
control proteomes indicated a distinctiveness between gen-
otypes (p < 0.01). A number of 15 protein spots revealed
to be genotype-dependent in absence of any stress, with
13 spots in total that were more accumulated in the leaves
of the tolerant Boulifa genotype and two spots were
more abundant in those of the sensitive Testour genotype
(Table 4). Overall, the contrast was obvious in terms of
proteome profiles (Table 4) between control and salinity-
stressed samples. In fact, when comparing Testour profiles
under control and stress conditions (T-C vs T-ST), 24 spots
with reduced accumulation (1.43-fold to 333.3-fold) were
71
detected in Boulifa and Testour. Bold numbers, up-regulated protein
spots, normal numbers, down-regulated protein spots. Among these
spots, 11 common spots were altered in both genotypes. Significant
differences were evaluated by PDQuest software via Student’s t-test at
0.01 level. (Color figure online)
noticed. However, in Boulifa proteomes (B-C vs B-ST),
only 12 spots with reduced abundance and low change fold
(1.3-fold to 2.6-fold) were observed. Thus, the number of
proteins affected by salinity was higher in the sensitive
genotype than the tolerant one (Table 4).
The combined comparative 2-D proteomes of both gen-
otypes (Fig. 1a) allowed the identification of 54 differen-
tially abundant spots that were detected based on the vol-
ume spot fold change upon salt treatment (B-ST/B-C and
T-ST/T-C) by PDQuest software using Student’s t-test at
0.01 level (Table S4). Among these spots, 11 common spots
13
72 Plant Growth Regulation (2021) 95:65–82

were altered in both genotypes including one spot with an

increased accumulation in Boulifa and lower abundance in
Testour, seven spots with a higher accumulation and three
spots with a reduced abundance in both genotypes. The num-
ber of protein spots, with a significant differential abundance
under salt stress, was illustrated by a Venn diagram for both

Higher accumulation Lower accumulation

genotypes (Fig. 1b).

Grouping of protein abundance patterns

Table 4  Number of differentially accumulated spots between Boulifa and Testour genotypes in image proteome analyses under both control and stress conditions

The analyses of all differentially abundant spots identified

24
seven groups (Tables S4 and S5). Hence, we found that
group n#3, representing spots, which are unchanged in Bou-
Control versus stress

lifa and with a reduced accumulation in Testour, was the

major one (20 spots). The remaining groups, include Group
n#1: spots with higher accumulation in Boulifa and lower
Testour

accumulation in Testour (one spot), Group n#2: spots with

enhanced accumulation in Boulifa and unchanged in Testour
12
36

(nine spots), Group n#4: spots with increased accumulation

in Boulifa and in Testour (seven spots), Group n#5: spots
with invariable abundance in Boulifa and exhibiting higher
accumulation in Testour (five spots), Group n#6: spots show-
ing lower accumulation in Boulifa and unchanged in Testour
Differentially abundant spots under salt stress

(nine spots), and Group n#7: spots exhibiting reduced accu-

Higher accumulation Lower accumulation

mulation in both Boulifa and Testour (three spots).

Identification and gene ontology analysis of salt

responsive proteins
12

The 54 spots showing differential abundance between the

Control versus stress

leaf proteome profiles were subjected to MALDI TOF/TOF

MS analysis. Therefore, 48 proteins were successfully identi-
fied by MASCOT search engine over Triticeae-NCBI, Hor-
deum-JGI and NCBIprot databases with high confidence.
Boulifa

However, 19 spots identified to be two or three different

17
29

proteins with high Mascot scores (as 2-D electrophoresis has

limitation in distinguishing proteins with similar isoelectric
points and molecular weights) were excluded from the analy-
Differences were evaluated by Student’s t-test at 0.01 level
Differentially abundant spots between Boulifa and Testour

sis and 29 identified proteins were considered (Table S6).

The spots which were identified in at least two proteins on
the same gel, such as chitinase and RuBisCO large subunit-
Abundance in Testour

binding protein subunit alpha, could be attributed to the

presence of different isoforms of the same protein or by
modification or post-translational degradation.
The gel migration points corresponding to the 54 differ-
entially abundant spots are shown in Fig. 1a and the results
of the comparative proteome analysis are presented in Tables
2
under control conditions

S5 and S6.
The 29 identified proteins were found to be involved in
11 different functional categories including defense/cell wall
Abundance in

related metabolisms (10), photosynthesis (5), ATP metabo-

Boulifa

lism (4), signaling (3), RNA regulation (2), photorespira-

tion (1), protein synthesis (1), protein regulation (1), TCA
15

13

13
Plant Growth Regulation (2021) 95:65–82
Fig. 2  a Functional pathways associated to all identified proteins in
both genotypes. b, c Functional classification of the identified pro-
teins after the comparative proteome analysis in Testour and Boulifa,
respectively. Annotation was achieved using the gene ontology analy-
sis by submission of the identified sequences to Interproscan and to
Blast against Arabidopsis thaliana genome using Blast2Go. d Hier-
archical clustering analysis of the 29 identified proteins showing dif-
ferential accumulation patterns under salt stress (200 mM NaCl for
4 days) in barley seedling leaves. The rows represent change rates
of proteins: both genotypes under control conditions (B-C/T-C);
the genotypes under stress and their respective controls (B-ST/B-C;
T-ST/T-C) and between stressed genotypes (B-ST/T-ST). The protein
73
rates that increased and decreased in abundance are indicated in red
and green, respectively. The intensity of the colors increases as the
differences in rates increase, as shown in the bar. ATPE ATP synthase
CF1 epsilon subunit, PAP9 plastid-lipid-associated 9 protein, Rubisco
large sub BP a: 1,5‐bisphosphate carboxylase/oxygenase large subu-
nit-binding protein subunit alpha, MDH malate dehydrogenase, TCTP
translationally-controlled tumor protein, 28 KDa RNP 28 KDa ribo-
nucleoprotein, JIP23 23 kDa jasmonate-induced protein, PPase inor-
ganic pyrophosphatase, PR10 pathogenesis-related protein 10, PAO
polyamine oxidase, GCSH glycine cleavage system H. (Color figure
online)
13
74
cycle (1) and redox/polyamine catabolism like (1) (Fig. 2a).
Proteins associated defense/cell wall related metabolisms,
photosynthesis, ATP metabolism and signaling were the
most commonly annotated functions. When examining
each genotype, these functions represented also the larg-
est groups and protein synthesis was involved in salt stress
response within both. Nevertheless, in Testour (Fig. 2b),
besides defense, which was more affected compared to
Boulifa (Fig. 2c), additional functions including the RNA
regulation (2), photorespiration (1) and protein regulation
(1) were altered. In Boulifa, TCA cycle and redox/polyamine
catabolism functions seem to be also involved in salt stress
response (Fig. 2c).
Hierarchical clustering analysis
To select key candidate proteins, the identified salt-respon-
sive proteins were subjected to a hierarchical clustering
analysis, in order to group proteins with similar abun-
dance profiles either under control and/or salt stress condi-
tions, as follows: both genotypes under control conditions
(B-C/T-C); each genotype under stress and its respective
control (B-ST/B-C; T-ST/T-C) and both genotypes under
stress conditions (B-ST/T-ST). Indeed, four main clusters
were noted (Fig. 2d). The first included two proteins with
an accumulation only in Boulifa in response to stress. The
accumulation remains higher compared to stressed Testour
(B-ST/T-ST). The second incorporated 13 proteins that were
down-regulated in majority only in the sensitive genotype
and with a higher abundance in stressed Boulifa compared
to stressed Testour. The third class encompassed six proteins
that exhibited a lower accumulation in stressed Boulifa com-
pared to controls (B-ST/B-C) but with unchanged abundance
in Testour under stress (T-ST/T-C). These proteins showed
a slight abundance in stressed Boulifa compared to stressed
Testour (B-ST/T-ST). Finally, the fourth class covered eight
proteins with a high accumulation in stressed Testour and
Boulifa (mostly) compared to controls but did not show a
higher abundance in the latter compared to stressed Testour
(B-ST/T-ST). To select key candidates from the 15 proteins
representing the first and the second clusters that are consid-
ered as the most important ones in terms of salt-responsive
protein accumulation in the tolerant Boulifa, we performed
t-test at 99% for B-ST/T-ST rates (Table S4). Indeed, five
proteins did not show a significant difference (the ankyrin,
GCSH, RNA-binding, beta 1–3 glucanase and PPase 6),
while ten key proteins were interestingly found to be sig-
nificantly more accumulated in salt-treated Boulifa leaves
than in those of Testour. Among these key candidates, seven
were found to be related to defense/cell wall metabolisms
including, 3 chitinases [Standard Spot Number (SSP) SSP
2204 (Fig. S3a), SSP 4203 and SSP 1102], PR10 (SSP 0003;
Fig. S3b); Glucan ß 1,3 glucosidase (SSP 0405; Fig. S3c);
13
Plant Growth Regulation (2021) 95:65–82
Ricin B-like lectin R40G3 (SSP 6408; Fig. S3d); and the
predicted protein (SSP0205/BSPs; Basic Secretory Proteins
related to defense). The three remaining ones among the ten
key proteins were found to be involved in signaling such as
JIP23 (SSP 6101) and TCTP (SSP 0108) and to redox/poly-
amine catabolism via flavin containing PAO (SSP 2308).
Validation of some key candidate proteins
To confirm the expression of candidates at both transcrip-
tomic and proteomic levels, we chose, among the selected
key candidate proteins, five genes encoding for TCTP, PAO,
Ricin B-like lectin R40G, PR and ribulose 1.5-bisphosphate
carboxylase/oxygenase large subunit proteins. The obtained
results, based on the comparison of fold expression changes
between Boulifa and Testour upon salt treatment, showed
consistent expression trends with protein profiles (Fig. 3a–e
corresponding to TCTP, bpao, R40G3, PR10 and rbcL genes,
respectively). Based on the annotated functions of the pro-
teins encoded by these five genes, a related contribution in
salt tolerance mechanisms of Boulifa could be suggested.
These genes are associated to signaling, redox/polyamine
catabolism, defense and energy metabolism.
Discussion
In the present study, the response of two Tunisian barley lan-
draces with contrasting salinity tolerance was investigated
based on a proteomic approach to elucidate salt tolerance
mechanisms. Contrasting tolerance towards constraints is
an effective way to gain insights at the OMICs levels (Gao
et al. 2013). As records related to leaf proteome for these
local germplasm are missing, the differential proteome was
explored in their leaf tissues at the early seedling stage, a
very sensitive stage to salt stress (Ibrahim 2016). At the
physiological level, a contrasting behavior between the two
genotypes at the seedling stage was noticed. Within the
tolerant Boulifa, the maintenance of leaf length and bio-
mass may be key factors governing salinity tolerance (Shen
et al. 2016a, b). In addition, regarding mineral contents, our
results indicate that Boulifa is a salt tolerant genotype with
low leaf Na accumulation, slighter loss of K and higher K ­ +/
+
Na ratio compared to Testour. This is in concordance with
the response characterizing salinity tolerance (Abbasi et al.
2015). In addition, it is well known that preserving a high
cytosolic ­K+/Na+ ratio of around 1.0, as found in Boulifa,
is usually considered as the main trait of salt tolerance in
plants (Shabala and Cuin 2008). Consequently, low sodium
concentration does not induce a significant inhibition of the
activities of metabolisms (Munns and Rawson 1999). Simi-
lar results were found in salt-tolerant barleys (Fatehi et al.
2012; Wu et al. 2014; Shen et al. 2016a; Zhu et al. 2020a,
Plant Growth Regulation (2021) 95:65–82
Fig. 3  Differential expression of genes chosen from key candidates
of the two studied genotypes contrasting in their salinity tolerance,
Boulifa (tolerant) and Testour (sensitive). These genes are a TCTP, b
bpao, c R40G3, d PR10, e rbcL. Leaves were harvested within 4 days
b). On the other hand, the significant decrease of ψs was
accompanied by an enhanced accumulation of sugars, previ-
ously described as osmoprotectant and signaling molecules.
This deduced behavior in Boulifa would compensate the ­Na+
accumulation (Garthwaite et al. 2005). Concerning the con-
tent of Ca, which is a crucial indicator of cell growth protec-
tion (Thor 2019), a less decrease was observed in Boulifa
than in Testour. Its involvement in signal transduction and
protein phosphorylation has been previously reported (Sei-
fikalhor et al. 2019). On the other hand, the least increase of
­H2O2 and MDA in the tolerant Boulifa under stress would
reflect its capacity to control redox balance and gene acti-
vation (Hung et al. 2005; Tagnon and Simeon 2017).This
behavior was also described for tolerant barley genotypes
(Kiani et al. 2017; Zhu et al. 2020a, b). ­H2O2, in addition
to being a toxicant, has been considered as a signaling
molecule and a regulator of the expression of some genes
in cells (Hung et al. 2005). It serves as an indicator of the
genotype ability to remove ROS. On the other hand, MDA
which implies the degree of lipid peroxidation, has been
widely designated as a good indicator of salt stress tolerance
in plants (Kong et al. 2016). Therefore, the role of these
compounds as damagers or protectors depends upon produc-
tion, scavenging, and signaling modulation and regulation of
75
post-treatment with NaCl (200 mM) for RNA extraction and RT-
qPCR analysis. The data represent means of three independent exper-
iments. The different letters indicate significant differences according
to LSD test (p ≤ 0.05)
different stress-responsive gene including transcription fac-
tors (Khedia et al. 2019; Morales and Munné-Bosch 2019).
Through the annotation of the 29 identified proteins
associated, we evoked here for barley the contribution of
ATPE (ATP synthase cF1 epsilon subunit; Chloroplast),
thylakoid lumenal 17.4 kDa protein chloroplastic-like, and
a probable plastid-lipid-associated nine protein (chloroplas-
tic) in the response to salinity. The atpE was up-regulated
in the tolerant Boulifa (twofold change), while not affected
in Testour. Within Barley, different subunits including a
vacuolar one (Shen et al. 2016a), a delta chain (Wu et al.
2014), a mitochondrial precursor (Maršálová et al. 2016)
and five alpha and gamma subunits (Shen et al. 2016b) were
detected in shoot response to salinity. However, in the root,
an alpha (Shen et al. 2017) and beta subunits (Witzel et al.
2014; Mostek et al. 2015) were identified. Amongst five
ATP synthase subunits, the ε one (Rühle et al. 2014; Hahn
et al. 2018) would prevent wasteful ATP hydrolysis by the
enzyme (Cruz et al. 1997). The contribution of ε subunit
in Tunisian barley landraces response to salt stress would
underline the importance of ATP synthases. Regarding the
thylakoid lumenal 17.4 kDa protein chloroplastic-like, with
1.8-fold accumulation only in the sensitive genotype, its role
in photosynthesis regulation was reported but its function
is not yet well characterized (Järvi et al. 2013). In addition,
13
76
the probable plastid-lipid-associated nine protein (PAPs,
chloroplastic) which up-regulated in both genotypes under
salinity (1.6 and 1.8-fold change respectively, in Boulifa and
Testour) is known for involvement in chromoplastogenesis
and stress response (Leitner-Dagan et al. 2006). In total,
protein annotation led to the identification of 11 functional
categories related to different biochemical pathways. The
most prominent category was the defense/cell wall-related
metabolisms which is in concordance with our previous
studies on Boulifa and Testour grain proteome, revealing
specific and abundant proteins related to defense including
heat shock (HSP) and late embryogenesis abundant (LEA)
proteins (Riahi et al. 2019). In Testour leaves, defense/cell
wall-related metabolisms were found to be more altered
compared to those of Boulifa. On the other hand, hierarchi-
cal clustering analysis led to the selection of 10 key pro-
teins, which exhibited a significant higher accumulation
in the treated tolerant genotype compared to the treated
sensitive one. These proteins were found to be involved in
majority in defense/cell wall metabolisms (seven proteins:
3 chitinases, PR10, Glucan ß 1,3 glucosidase; Ricin B-like
lectin R40G3 and the predicted protein BSPs); besides, sign-
aling (JIP23; TCTP) and redox/polyamine catabolism (fla-
vin containing PAO). These selected proteins would be key
players in salinity tolerance mechanisms in Boulifa barley
landrace at the early seedling stage. A part from these key
candidates, photosynthesis and ATP metabolism (energy
metabolism) were amongst the common important function
categories. The contribution of related proteins showing dif-
ferential abundance between control and stressed plants in
barley salinity tolerance mechanisms would be supposed.
These proteins are represented by the rubisco (RuBisCOs),
the malate dehydrogenase protein (MDH) and the ATPE.
The importance of photosynthesis in barley salinity toler-
ance at the seedling and the tillering stages has been also
reported (Rasoulnia et al. 2011; Fatehi et al. 2012; Shen
et al. 2016a, b). Moreover, Shen et al. (2016a) and Marsa-
lova et al. (2016) reported that the ATP synthases, in differ-
ent subunits, might be pioneers for tolerance development
at an early stress exposure stage. Additional reports showed
that photosynthesis, energy metabolism and signal transduc-
tion were the most common metabolic pathways associated
with barley salt tolerance (Rasoulnia et al. 2011; Fatehi et al.
2012; Shen et al. 2016a, b). In the present study, defense/cell
wall metabolisms beside the energy metabolism, signaling
and redox/polyamine catabolism seem to be key pathways
for salinity tolerance in barley at an early stage.
13
Plant Growth Regulation (2021) 95:65–82
Potential roles of the key identified proteins
in salinity tolerance with respect to their functional
classification
Among the differentially accumulated proteins, ten key
candidates were found to be highly accumulated in Bou-
lifa treated-plants compared to Testour ones, which led to
deduce their corresponding functional categories.
Defense and cell wall related metabolisms
In defense and cell wall related metabolisms, 3 chitinases
(hydrolytic enzymes targeting chitin) known to play a key
role against abiotic stresses like in seed adaptation to desic-
cation (Dana et al. 2006; Alikhani et al. 2013) were identi-
fied and thus qualified as proteins that support plant growth
by improving their endurance towards stresses (Kumar et al.
2018). In our case, chitinase amounts were either unchanged
or increased in the tolerant treated plants compared to the
sensitive ones. In contrast, in Tibetan wild barley, their
induction by salt stress was observed in shoots regardless
of the tolerance degree (Shen et al. 2016a). On the other
hand, glucan endo-1,3-β-glucosidase which was accumu-
lated in stressed Boulifa plants and down regulated in Tes-
tour, is known to be implicated in the hydrolysis of 1,3-β-D-
glucosidic linkages in 1,3-beta-D-glucans and involved in
cell wall formation via regulation of carbohydrate metabo-
lism and defense response (Song et al. 2011). The decreased
accumulation within Testour (1.43-fold decrease) would be
associated to carbohydrate metabolism and cell wall com-
position alteration leading probably to cell growth cessa-
tion and down regulation of cell-wall remodeling processes
(Kosová et al. 2013). The increased abundance of this pro-
tein was reported in Hordeum vulgare response to drought
(Abebe et al. 2010) and in the roots in response to salinity
(Shen et al. 2016b). Otherwise, different hydrolases includ-
ing glucan exohydrolase from roots (Mostek et al. 2015),
glucanase precursor from both leaves (Shen et al. 2016a)
and roots (Witzel et al. 2009, 2014) as well as in Hordeum
marinum shoots (Maršálová et al. 2016) were evoked. This
would mark our results showing its association to salinity
tolerance in the leaves.
Regarding PR10 showing down-regulation only in Tes-
tour (142.8-fold decrease), previous reports have described
that this protein family is inducible under abiotic stresses
and phytohormone treatment such as jasmonic acid, ethyl-
ene and salicylic acid (SA) (Dana et al. 2006). PR10 was
described as a good candidate associated with salt tolerance
in peanuts (Jain et al. 2006), Vitis (Jellouli et al. 2008) and
potato (El-Banna et al. 2010). In barley, PR10 involvement
in salinity response (Witzel et al. 2014; Wu et al. 2014) and
tolerance (Sugimoto and Takeda 2009) was reported only in
Plant Growth Regulation (2021) 95:65–82
the roots. We show here the contribution of this protein in
salinity tolerance of barley leaves.
Ricin B-like lectin R40G3 protein accumulated only in
Boulifa leaves, suggesting its possible involvement in the
tolerance mechanisms. The R-type lectins are members of
a superfamily of proteins containing a carbohydrate-recog-
nition domain structurally similar to that of ricin. Ricin, as
the first discovered lectin, represents the prototypical lec-
tin in this category. Plant lectins play an important role in
protein-saccharide interactions and stress signaling (Kosová
et al. 2013) as well as in cell wall structure, development
and defense (Jiang et al. 2010; Esch and Schaffrath 2017).
Amongst defense-related nucleocytoplasmatic lectins, the
ricin-B lectin is the most important one (Yang et al. 2016).
Within barley, a root-specific lectin was characterized
(Lerner and Raikhel 1989). Moreover, abundance of ricin
B lectin was observed in the leaf upon salt (Maršálová et al.
2016) and cold (Hlavácková et al. 2013) treatments but its
accumulation was correlated to susceptibility to drought
(Vítámvás et al. 2015). In our case, the accumulation of ricin
B lectins in Boulifa suggests its association with salinity
tolerance through possible saccharide signaling and defense
processes.
The predicted protein (SSP 0205) with an up-regulation
only in Boulifa (1.97 fold-change) was found to be associ-
ated to BSP-related defense. These proteins belong to PRs
protein family that is regulated by ABA (Kuwabara et al.
1999). In barley, defensive roles of PRs are known against
biotic and abiotic stresses (Zhang et al. 2012). This family
of proteins has a similar sequence and motif of one of the
two zinc-dependent metallopeptidases (Ko et al. 2018). Until
now, 1354 sequences of 772 species were reported to belong
to the BSP family (http://​pfam.​xfam.​org/​family/​PF044​50#​
tabvi​ew=​tab7). Among them, 85 sequences were known to
be from the Poaceae family, including T. aestivum (pfam.
xfam.org/family/PF04450).
Signaling
The JIP23 accumulation was more decreased in Testour than
in Boulifa (5.88 vs 1.75-fold changes, respectively) under
salt stress. The reduction of JIP23 abundance was reported
only in salt-treated roots of barley contrasting genotypes
(Witzel et al. 2009, 2010). Here, in barley leaves, we noted
its association to tolerance. Even in other species, both jas-
monic acid (JA) and its volatile methyl-ester, (Me-JA) regu-
late gene expression based on a positive regulation of JIPs
and a negative regulation of photosynthetic proteins (Wast-
ernack 2007). This finding would corroborate our results
showing in Boulifa, but not in Testour, a slight JIP23 reduc-
tion and a lower RuBisCO accumulation under stress. The
association of JIP23 to translational control might indicate
the implication of JA-signaling pathway in Boulifa tolerance
77
mechanisms. Indeed, Walia et al. (2007) demonstrated the
up-regulation of genes related to JA biosynthesis and salt
stress response in barley seedling including JIP23 and 1,5‐
bisphosphate carboxylase/oxygenase activase.
The TCTP, which is a conserved protein associated with
plant signaling and development and cell death processes
(Betsch et al. 2017), was found to be down-regulated under
salt stress in the leaves of both studied genotypes (< 1.5 fold-
change). It was also reported to be involved in the protection
against various stresses including cold, salt, drought, alu-
minum, and pathogen infection (Hoepflinger et al. 2013). In
response to salt stress, an up-regulation of TCTP (Cao et al.
2010) via calcium binding-signaling pathway (Gong et al.
2001) was reported. In barley, lower TCTP abundance was
revealed in the sensitive grain proteome compared to the
tolerant one during germination (Witzel et al. 2010). In salt-
treated barley plants, an increased accumulation of TCTP
was noted in the roots (Mostek et al. 2015) as well as in the
leaves (Fatehi et al. 2012; Maršálová et al. 2016). Here, the
TCTP protein abundance decreased in both genotypes upon
stress application but remained more abundant in the toler-
ant compared to the sensitive, suggesting its association to
salinity tolerance mechanisms.
Redox and polyamine catabolism
In our work, the flavin-containing polyamine oxidase (PAO),
which is an oxidoreductase, was found to be accumulated in
salt-treated Boulifa leaves. In previous report, PAO exhibited
the highest induction factor in the tolerant barley compared
to the sensitive one (Fatehi et al. 2012). Polyamines can be
oxidized by copper-containing diamine oxidases (CuAOs)
and flavin-containing polyamine oxidases (PAOs) (Yu et al.
2019). In barley plants, the apoplastic PAOs (Cervelli et al.
2004) was described to be involved in the catabolism of PA
terminal oxidation which lead to the production of hydrogen
peroxide ­(H2O2). These molecules are important signaling
elements regulating the expression of numerous genes under
stress conditions (Wang et al. 2019; Yu et al. 2019). The
PAO is involved in plant growth and development, and in
adaptation to environmental stresses through direct functions
including protection of macromolecules, cellular pH mainte-
nance, ROS scavenging, stabilisation of native protein struc-
tures as well as by an indirect role that is manifested during
plant abiotic stress tolerance by participating in osmolyte
synthesis (Sengupta et al. 2016). In addition, PAO activity
led to H­ 2O2 generation and mediation of systemic signals
(Gilroy et al. 2016) to regulate key gene expression in salt
tolerance mechanisms (Enoki and Suzuki 2016; Armijo et al.
2013).
13
78
Additional possible contributions of the identified proteins
Besides the selected key candidates exhibiting a significant
accumulation in stressed Boulifa plants, we evoked the
RuBisCOs, MDH, ATPE and PPase 6 that are involved in the
energy metabolism. The increased accumulation of ribulose
1.5-bisphosphate carboxylase/oxygenase large subunit in the
sensitive Testour might be due to a high level of protein
degradation (Wendelboe-Nelson and Morris 2012; Chaves
et al. 2002), while, their lower accumulation in Boulifa can
be related to JIP (Wasternack 2007) and ROS regulation
(Wendelboe-Nelson and Morris 2012). On the other hand,
the decreased amount of the MDH in Boulifa would be asso-
ciated to the TCA cycle and redox control of the mitochon-
drial matrix (Tomaz et al. 2010; Lindénet al. 2016). In this
context, Maršálová et al. (2016) detected MDH in control
plants but not in salt-treated ones, suggesting an implication
of TCA cycle regulation in salinity tolerance mechanisms.
The expression pattern of the genes encoding for TCTP,
PAO, PR10, Ricin B-like lectin R40G and ribulose 1.5-bis-
phosphate carboxylase/oxygenase large subunit was coherent
with protein profiles as shown by RT-qPCR analysis. Even
though, the different variation between transcriptional and
Fig. 4  Possible mechanisms and schematic summary of pathways
involving key candidate salt responsive proteins leading to salinity
tolerance in Boulifa at the early seedling stage. PA polyamine; PAO
polyamine oxidase; SA salicylic acid; JA jasmonic acid; JIP jas-
13
Plant Growth Regulation (2021) 95:65–82
proteomic levels would suggest possible post-translational
modifications for plant adaptation (Adem et al. 2014). Our
results consolidate the contribution of these proteins in the
tolerance mechanisms.
Taken together, our findings suggest that the described
functional categories may act in a highly sophisticated
network ensuring salinity tolerance in Boulifa (Fig. 4). In
response to abiotic stresses, the implication of PAO in the
generation of ­H2O2 and mediation of the whole-plant sys-
temic signals was reported (Gilroy et al. 2016). Addition-
ally, ­H2O2 and calcium, both represent key molecules for
rapid systemic signaling in plants (Evans et al. 2016). In
stressed plants, H
­ 2O2, as a signaling molecule, may interact
with both salicylic acid (SA) (Wrzaczek et al. 2013) and
jasmonic acid (Ahmed et al. 2015) signaling pathways.
Besides, the importance of ­H2O2 consists in the activation
of ­Ca2+ channels to regulate ­Ca2+ cytoplasmic concentra-
tion which triggers downstream events and enhances the
accumulation of defense proteins regulated by SA pathway
including Glucan 1,3-beta-glucosidase, PR10 and the pre-
dicted protein (BSP) (Enoki and Suzuki 2016). Amongst
the protein associated to JA pathway, chitinases and the JIPs
are involved (Enoki and Suzuki 2016). On the other hand,
monate induced protein; TCTP translationally-controlled tumor pro-
tein; PR pathogenesis related protein; BSP Basic Secretory Proteins
related to defense; RuBisCO 1,5‐bisphosphate carboxylase/oxyge-
naselarge subunit; MDH malate deshydrogenase
Plant Growth Regulation (2021) 95:65–82
the protectant proteins such as TCTP, which is known to be
regulated by calcium, would contribute to ­Ca2+ homeostasis,
and ROS regulation (Betsch et al. 2017). Regarding the ricin
B-like lectin R40G3, its contribution in defense (Esch and
Schaffrath 2017) would be through saccharide signaling and
development regulation (Hlavácková et al. 2013).
Conclusion
The comparative analysis of leaf proteomes of studied con-
trasting barley landraces in response to salinity, revealed
significant differences between control and salt-treated
plants as well as between stressed plants of both geno-
types, at the early seedling stage. Based on selected pro-
teins exhibiting a higher accumulation in Boulifa treated-
plants compared to those of Testour, salt tolerance of this
landrace may be attributed to a less affected signaling
pathway, an enhanced redox/PAO catabolism, a more sta-
ble defense/cell wall related metabolisms, an activated
ATP metabolism and photosynthesis homoeostasis.
The key candidates involved in these pathways will
be subjected to a further characterization to explore
their accurate function in salt tolerance in Tunisian bar-
ley landraces. They would be therefore valuable in sup-
porting novel elite barley creation and marker-assisted
selection that can be useful for marginal and saline land
management.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10725-0​ 21-0​ 0726-4.
Acknowledgements This work was supported by the Tunisian-Ger-
man Joint Research Programme TUNGER 019-0004-06619 and the
MHESR (Tunisia). The authors thank Dr. Farhat Chibani and Dr. Was-
sim Azri (CBBC) for fruitful discussion.
Declaration
Conflict of interest The authors declare that there are no conflict of
interest regarding the publication of this paper.
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