Accepted Manuscript

Title: Saline and osmotic stresses stimulate

PLD/diacylglycerol kinase activities and increase the level of
phosphatidic acid and proline in barley roots

Author: Maria V. Meringer Ana L. Villasuso Micaela Peppino

Margutti Javier Usorach Susana J. Pasquaré Norma M. Giusto
Estela E. Machado Graciela E. Racagni

PII: S0098-8472(16)30057-0
DOI: http://dx.doi.org/doi:10.1016/j.envexpbot.2016.03.011
Reference: EEB 3047

To appear in: Environmental and Experimental Botany

Received date: 14-12-2015

Revised date: 28-3-2016
Accepted date: 28-3-2016

Please cite this article as: Meringer, Maria V., Villasuso, Ana L., Margutti,
Micaela Peppino, Usorach, Javier, Pasquaré, Susana J., Giusto, Norma M.,
Machado, Estela E., Racagni, Graciela E., Saline and osmotic stresses stimulate
PLD/diacylglycerol kinase activities and increase the level of phosphatidic
acid and proline in barley roots.Environmental and Experimental Botany
http://dx.doi.org/10.1016/j.envexpbot.2016.03.011

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Saline and osmotic stresses stimulate PLD / diacylglycerol kinase
activities and increase the level of phosphatidic acid and proline in
barley roots

Maria V. Meringera, Ana L. Villasusoa*, Micaela Peppino Marguttia, Javier Usoracha, Susana J.
Pasquaréb, Norma M. Giustob, Estela E. Machadoa, Graciela E. Racagnia

a
Química Biológica, FCEFQN, Universidad Nacional de Río Cuarto, X5804BYA Río Cuarto,
Córdoba and
b
INIBIBB (CONICET), Universidad Nacional del Sur, Bahía Blanca, Argentina.

Corresponding author: Ana Laura Villasuso, Dpto. Biología Molecular, FCEFQN, Universidad
Nacional de Río Cuarto, X5804BYA Río Cuarto, Córdoba, Argentina. Fax: (54)-358-4676232
E-mail: lvillasuso@exa.unrc.edu.ar

1
 Highlights
 In response to saline and osmotic stresses, PA increased by PLD and diacylglycerol
kinase activities.
 NaCl and mannitol stresses increase the proline accumulation.
 In response to saline and osmotic stresses, the roots exhibited a reduced growth.
 Saline and osmotic stresses decrease the GA3 and ABA levels but increase the SA and
JA endogenous amounts.

Abstract
Soil salinity is one of the major abiotic stresses that affect the crop productivity.
Understanding the mechanisms by which plants transmit the signals to cellular machinery to trigger
adaptive responses is essential to develop more stress tolerant crops. Barley (Hordeum vulgare L.)
is an important cereal and its production is affected by increasing dryland salinity. This severely
limits growth and reduces yields. In barley seedling, NaCl and mannitol stresses modulated the
level of phosphatidic acid (PA), the proline accumulation and the reduced root length. PA is a well-
known lipid signal and an intermediary in the lipid synthesis. However, little is known about their
role during the saline and osmotic stresses in barley roots. PA increased by phospholipase D (PLD,
E.C. 3.1.4.4) and by diacylglycerol kinase activities (DAG-k, EC 2.7.1.107) and its conversion to
DGPP suggested that they are part of stress responses to salinity in barley. In contrast, saline stress
decreased the activity of the Mg2+-independent, NEM-insensitive form of phosphatidate
phosphohydrolase (PAP2, E.C. 3.1.3.4), keeping the PA levels. The application of 1-butanol also
stimulated proline accumulation while the DGK-inhibitor treatment decreased proline levels.
Endogenous phytohormone levels measured by liquid chromatography-tandem mass spectrometry
revealed that, under stress, the barley roots decreased the GA3 and ABA levels and increased the SA
and JA endogenous amounts. The results presented here suggest that PA may modulate the cellular
signal of barley roots by differentially affecting components of the abiotic stress - response cascade.

Abbreviations: ABA, abscisic acid; CL, cardiolipin; DAG, diacylglycerol; DAG-k, diacylglycerol
kinase; DGPP, diacylglycerol pyrophosphate; DGPPase, diacylglycerol pyrophosphate phosphatase;
IAA, indole-3-acetic acid; IP3, inositol 1,4,5-trisphosphate; GA3, gibberellic acid; GPL,
glycerophospholipids; JA, jasmonic acid; LPPs, lipid phosphate phosphatases; PA, phosphatidic
acid; PA-k, phosphatidate kinase; PAP2, phosphatidate phosphohydrolase type 2; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;PI,
phosphatidylinositol; PI4-k, phosphatidylinositol 4-kinase; PI4P, phosphatidylinositol 4-phosphate;

2
PI-k, phosphatidylinositol kinases; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase
C; PLD, phospholipase D; SA, salicylic acid; TLC, thin layer chromatography.

Key words: barley, lipid kinases, phospholipase D, proline, saline stress.

1. Introduction
Salinity is one of the most severe environmental stresses that cause crop yield loss. Plants
have developed different mechanisms to adapt to saline stress involving complex physiological and
biochemical changes (Hasegawa et al., 2000;Widodo et al., 2009). Salinity causes ionic stress,
osmotic stress, and secondary stresses including nutritional imbalance and oxidative stress (Zhu,
2002). High concentrations of Na disturb the osmotic balance causing “physiological drought”,
which prevents plant water uptake. To survive to the harmful effects of salt stress, plants have
developed a series of biochemical and molecular mechanisms, mainly those including selective
build up or exclusion of salt ions, control of ion uptake by roots and transport into leaves, ion
compartmentalization, synthesis of compatible osmolytes, and induction of antioxidative enzymes
(Shabala and Lew, 2002;Chen et al., 2007a;Cuin and Shabala, 2008;Rodriguez-Rosales et al.,
2008;Munns and Gilliham, 2015). The amino acid proline is an osmolyte that accumulates in a wide
range of plant species in response to stress (Szabados and Savoure, 2010). The exact role of proline
accumulation in stress tolerance is still unknown, although, several studies have suggested that it
may play other roles to limit damage stress (Shabala and Lew, 2002;Chen et al., 2007a;Shabala,
2013;Munns and Gilliham, 2015). Consequently, understanding the plant’s mechanisms to salt
tolerance will provide effective means to breed or genetically engineer salt tolerant crops. Barley
(Hordeum vulgare L.) is a valuable cereal, grown primarily for animal feed and beer production.
Barley production is affected by increasing dryland salinity, which severely limits growth and
reduces yields (Rengasamy et al., 2003). Although some of the late responses to saline and osmotic
stresses in barley are relatively well studied (Chen et al., 2007a;Widodo et al., 2009), the role of
phospholipids in the signalling pathways is still unknown. During osmotic stress, several
phospholipid-based signalling pathways in plants are rapidly activated. They include phospholipase
D (PLD) and phospholipase C (PLC) coupled with diacylglycerol kinase (DAG-k) pathways that
result in the increase of phosphatidic acid (PA) (Munnik et al., 1998;Munnik et al., 2000;Arisz et
al., 2009;Li et al., 2009;Kolesnikov et al., 2012;Pokotylo et al., 2014). PA is converted into
diacylglycerol pyrophosphate (DGPP) by a phosphatidate kinase (PA-k) (Wissing and Behrbohm,
1993;van Schooten et al., 2006;Racagni et al., 2008). Thus, the enzymes that metabolize PA/DGPP
play important roles in switching the PA/DGPP signal on/off (Villasuso et al., 2013).

3
 PA is the glycerophospholipid with the simplest chemical structure in biological
membranes. Its behavior is crucial for cell survival since it is a phospholipid involved in the
synthesis of phospholipids and triacylglycerols, thus playing a central role in cell signaling
(Athenstaedt and Daum, 1999). PA signalling acts by binding effector proteins and recruiting them
to a membrane, which regulates the proteins' activity in cellular pathways (Testerink and Munnik,
2011). Binding is mainly dependent on the concentration of the lipid in the bilayer and it depends
on nonspecific electrostatic interactions between clusters of positively charged amino acids in the
protein and the negatively charged phosphomoester headgroup of PA (Shin and Loewen, 2011).
The formation of PA is an integral part of the adaptation of plants to saline environments
(Hou et al., 2015;Julkowska and Testerink, 2015) and its levels increase when plants are exposed to
salinity (Munnik et al., 2000;Yu et al., 2010). Recently, it has been shown that PA mediates
important adaptive mechanisms including the maintenance of root architecture and cytoskeletal
organization. PA and DGPP were shown to bind to glyceraldehyde-3-phosphate dehydrogenase and
modulate its activity, a key glycolysis enzyme, in response to salt stress in roots (Kim et al.,
2013;McLoughlin et al., 2013;Astorquiza et al., 2016). PA is implicated in controlling the growth of
the primary root (Kim et al., 2013). Besides, members of the sucrose non-fermenting 1-related
protein kinase 2 (SnRK2s) family with PA-binding affinities were modulated by saline stress
(Testerink et al., 2004;McLoughlin et al., 2012). McLoughlin et al. (2012) suggests an involvement
of PA in membrane trafficking and cellular re-organization during salt stress. The PLD-induced PA
under saline conditions also affects the organization of the cytoskeleton (Lee et al., 2003;Zhang et
al., 2012). The activity of the microtubule-associated protein MAP65-1, that bundles and stabilises
adjacent microtubules, was increased after PA binding in response to salinity, while mutants with
low PA concentrations (PLDα1, PLDα3, PLDδ and PLDε) were more sensitive to salt stress (Zhang
et al., 2012). Furthermore, the plants have developed other survival strategies, including also the
synthesis of stress-related hormones like abscisic acid (ABA) and salicylic acid (SA) to protect
themselves from the detrimental surroundings. Although hormones are likely to play important
roles in root growth regulation under water-stressed conditions, the involvement of most of these
compounds has not been still elucidated in barley roots. The aim of this work was to study the lipid-
signalling pathway to better understand the physiological and biochemical responses induced by the
saline and osmotic stresses in barley roots.

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2. Materials and Methods
2.1. Plant materials, growth conditions, and separation roots
Barley seeds (Hordeum vulgare, cv. Carla INTA) were surface sterilized and soaked in
sterilized water for 4 days in the dark at 25 ºC. Seedlings were obtained from whole grains, surface
sterilized, grown in a growth chamber on disks of filter paper moistened with sterilized water for
control seedling and with 100 mM NaCl or 200 mM mannitol solutions for seedling under stress, in
Petri dishes (10-cm diameter), for 4 days in the dark at 25 ºC, and then harvested. Roots were
separated and kept frozen in liquid nitrogen at -80 ºC until use. When seedlings were treated with 1-
butanol, R59949 (DAG-k inhibitor type I) or R59022 (DAG-k inhibitor type I), these inhibitors
were added to the other treatment

2.2. Preparation of membranes

Control and stressed roots prepared as above were thawed and homogenized in 10 volumes of
50 mM HEPES (pH 7.4) containing 0.25 M sucrose, 5 mM KCl, 1 mM EDTA, and protease
inhibitors (1 mg mL-1 leupeptin, 1 mM phenyl methane sulfonyl fluoride (PMSF), 1 mg mL-1
aprotinin). The homogenate was centrifuged at 1,000 x g for 15 min at 4 ºC to remove unbroken
cells and cell debris, and the resulting supernatant was further centrifuged at 105,000 x g for 60 min
at 4 ºC. The supernatant was eliminated, and the pellet was resuspended in 50 mM HEPES (pH 7.4)
and used as crude membrane fraction. Protein concentration of samples was measured using
Bradford reagent with BSA as standard (Bradford, 1976).

2.3. Lipid kinase activity and phospholipid extraction and separation

The membrane fraction isolated as above (60 g protein) was added to thermally equilibrated
(30 ºC) 50 mM HEPES buffer (pH 7.4), 0.1 mM EDTA, 0.5 mM DTE, 10 mM MgCl2, 0.1 mM
sodium orthovanadate, 1 mM Mg2+-ATP, and [γ-32P]ATP (370 MBq). Lipid kinase activities were
assayed simultaneously using endogenous lipids as substrates. Lipid phosphorylation was allowed
to proceed for 2 min at 30 ºC in a final volume of 100 L, and reaction was stopped by addition of
1.5 mL chloroform/methanol (1:2, v/v). Lipids were extracted from membranes and phospholipids
were separated by TLC as described by Racagni et al. (2008). Plates were developed with
chloroform/methanol/acetone/acetic acid/water (40:14:15:12:7, v/v/v/v/v) in a plate of 20 cm.
Positions of radiolabeled lipids were determined by autoradiography on Kodak film.

5
2.4. [32P]Pi phospholipid labelling, extraction and separation
Roots (6 tips root) were incubated in 1 mL label medium (20 mM CaCl 2 and 20 mM
sodium-succinate, pH 6.5) containing 50 mCi carrier-free [32P]orthophosphate, abbreviated as
[32P]Pi. Treatments were stopped at specified times by adding 250 L of 25 % v/v perchloric acid
vortexing for 5 min, and maintaining sample-containing tubes for 30 min at room temperature.
After discarding perchloric acid, root lipids were extracted by adding 400 L
chloroform/methanol/hydrochloric acid (50:100:1, v/v/v) and freezing and thawing the mixture by
means of liquid nitrogen. After 5 min of vigorous mixing, lipid extracts were transferred to clean
tubes and 400 L chloroform and 214 L 0.9 % (v/v) NaCl were added to produce a two-phase
system. After vortexing (1 min) and centrifugation (1,000 x g, 5 min), the upper phase was removed
and the lower phase washed with 500 L chloroform/methanol/1M hydrochloric acid (3:48:47,
v/v/v). Lipid extracts were dried by vacuum centrifugation, dissolved in a suitable volume of
chloroform, and immediately subjected to TLC analysis. An alkaline solvent system,
chloroform/methanol/25% ammonium hydroxide/water (45:35:2:8, v/v/v/v), was used to separate
labelled GPL as described by Villasuso et al. (2013).

2.5. Lipid phosphate phosphatase activity assays

For determination of phosphatidate phosphohydrolase type 2 (PAP2) activities, the assay
mixture consisted of 50 mM Tris-maleate buffer, pH 6.5, 1 mM DTT, 1 mM EDTA plus 1 mM
EGTA, 4.2 mM NEM, and 100 g of membrane protein in a volume of 0.1 mL. The reaction was
started by addition of 0.6 mM [2-3H]phosphatidate, continued for 30 min at 37 ºC, and stopped by
addition of chloroform/ methanol (2:1, v/v). PAP activity product 1,2-diacyl[3H]glycerol was
isolated and measured. Radiolabeled PA was obtained from [2-3H]phosphatidylcholine, which was
synthesized as described by Pasquare de Garcia and Giusto (1986). PAP activity was expressed as
the sum of nmol ([3H]diacylglycerol and [3H]monoacylglycerol) x (h x mg protein) -1.
DGPP phosphatase activity was assayed as described by Meringer et al. (2012), based on
release of water-soluble [32P]Pi from chloroform-soluble [β-32P]DGPP (2,000 cpm/ pmol). The
reaction mixture contained 50 mM citrate buffer (pH 5.0), 0.1 mM DGPP, 2 mM Triton X-100, 10
mM 2-mercaptoethanol, and enzyme protein, in a total volume of 0.1 mL. DGPP was synthesized
from PA using enriched-membrane fraction H. vulgare PA kinase.

2.6. Protein gel blot analysis

Barley roots homogenates were prepared as described above for lipid kinase assay. Extract
samples were separated by 12.5% SDS-PAGE, and proteins were electrotransferred to

6
nitrocellulose membranes. Protein blots were blocked with 5% skim milk powder in phosphate-
buffered saline (4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl).
Antibodies directed to C-terminal DGPP phosphatase (a generous gift from Dr. George Carman)
were added at 1:1000 dilution and incubated for 2 h. Secondary antibody coupled to horseradish
peroxidase was used to visualize target proteins, in combination with chromogenic substrates.

2.7. Phospholipase D activity

PLD activity was measured by production of phosphatidyl butanol (PBut), mainly as
described by Villasuso et al. (2013). After labelling lipids with [32P]Pi, controls and stresses roots
were incubated in the presence of 0.75 % v/v n-butanol for 30 min at 24 °C. Reactions were stopped
and lipids were extracted for separation of PA and PBut of the other phospholipids, which was
achieved by using the organic upper phase of the biphasic system, ethyl acetate/ isooctane/ formic
acid/ water (13:2:3:10, v/v/v/v) as the TLC solvent. Radiolabelled lipids were located by
autoradiography on Kodak film. Spots were scraped off the plates, and fractions were counted in a
liquid scintillation counter.

2.8. Phytohormone analysis

Levels of GA3, ABA, IAA, SA, and JA were analyzed simultaneously by electrospray
ionization/ tandem mass spectrometry (LC-ESI-MS-MS), essentially as described by Durgbanshi et
al. (2005). Briefly, tissues samples (200 mg) were ground in liquid nitrogen, extracted with acetone/
water/acetic acid (80:19:1, v/v/v), added with 5 μL of a mixture of internal standards (50 ng/ sample
of 2H5-IAA, 2H6-ABA, 2H6-JA, and 2H4-SA, and 100 ng 2H2-GA3,), and centrifuged at 500 x g for
15 min. The resulting supernatant was collected and evaporated, and the solid residue was dissolved
in 500 μL of methanol and evaporated. The resulting residue was dissolved in methanol/ 1% acetic
acid (99:1, v/v), and then passed through a DEAE Sephadex A-25 column. Aliquots of the resulting
solution were injected directly into the LC-ESI-MS-MS system. MS/MS experiments were
performed on a Micromass Quatro UltimaTM Pt double quadrupole mass spectrometer (Micromass,
Manchester City, UK). The conditions used for a MS/MS were the same as those in Meringer et al.
(2012).

2.9. Proline accumulation

Proline was determined from roots as described by Bates et al. (1973). Briefly, 500 mg of
roots were homogenized in 10 mL of 3 % aqueous sulfosalicylic acid and the homogenate filtered
through filter paper. Two mL of homogenate filtrated reacted with 2 mL acid ninhydrin and 2 mL

7
glacial acid acetic in the tube at 100 °C, and the reaction was terminated in ice bath. The reaction
mixture was extracted with 4 mL toluene, and mixed vigorously with a tube stirrer for 15-20 sec.
The chromophore containing toluene was aspirated from aqueous phase, warmed to room
temperature and the absorbance read at 520 nm using toluene for a blank. The proline concentration
was determined from a standard curve and calculated on a fresh weight.

2.10. Root Na and K contents

Barley roots (1 g) were homogenized with liquid nitrogen and then they were centrifuged to
40,000 x g, during 60 min. The Na+ and K+ contents were determined in the supernatant using a
flame photometer.

2.11. Statistical analysis

To determine the statistical difference between at least one pair of means, analysis of variance
test (ANOVA) was used. When the assumptions of homogeneity of variance (Levene test) and
normality (Shapiroe Wilk test) were not checked, corresponding transformations were performed
using the appropriate functions. To determine significant differences between treatments, Tukey’s
Test was applied, with a significance level of 0.05 (P < 0.05). The statistical program used was
InfoStat (2015e version, Grupo InfoStat, Facultad de Ciencias Agropecuarias, Universidad Nacional
de Córdoba, Argentina)

3. Results
Saline stress triggers a wide range of plant responses, from altered gene expression and
cellular metabolism to change in growth rates and crop yields. To evaluate the effect of saline stress
on barley seedling, a growth analysis for the characterization of plant’s response was carried out.
Figure 1A shows that the root length (cm) of stressed seedlings decreased after 96 h of imbibition,
compared to that of control seedling. The effect was more significant in the saline than in the
osmotic stress (Fig. 1B). Reduction on the length of the roots of 40.5 ± 10.06% with 100 mM NaCl
and 18.98 ± 4.77% with 200 mM mannitol was observed. Likewise, both 100 mM NaCl and 200
mM mannitol reduced the percentage of germination, although the effect induced by mannitol was
lower than that evoked by NaCl (data not shown). This difference in growth was also reflected on
root Na+ and K+ content (Fig. 2). In absence of salt, the Na+ content was around 50 mol/g FW.
Treatment with 100 mM NaCl resulted in a significant 6-fold increase in the Na+ content in seedling
roots (Fig. 2A). In contrast, the K+ content did not change significantly as result of NaCl or
mannitol stress (Fig. 2B).

8
 It is well known that accumulation of compatible solutes, as the amino acid proline, is a
shared stress signal by plants. To investigate this response, the proline level was measured in roots
obtained from control and stress seedlings. Figure 3 shows that proline content in roots increased
significantly 3- fold by 100 mM NaCl. Similarly, the root treated with 200 mM mannitol increased
2-fold the proline level.
Lipid signalling associated to accumulation of PA is a rapid response to saline stress in plants.
To investigate the possible role of PA signalling, the lipid kinase activities in NaCl and mannitol
treated barley roots were assayed. A sequential measurement system was used, with both
endogenous substrates and DAG-k and PA-k present in the same membrane fraction, prepared by
centrifugation at 105,000 x g. Phospholipids present in this fraction were identified by
autoradiography as PA and DGPP (Fig. 4A). Saline and osmotic stresses induced changes on
phosphorylated products of barley lipid kinase activities (Fig. 4B). An increase in the PA (150 ±
5%; n = 5) formation was observed after 100 mM NaCl and 200 mM manitol treatment, with
respect to unstimulated control (100%). In contrast, NaCl treated root displayed lower PA-k
activities, and higher activities in response to mannitol. This reduced activity of PA-k may be
correlated with increased activities of the enzymes that regulate PA and DGPP levels. PAP2 activity
(measured with [3H]PA as substrate) and DGPPase activity (measured with [β-32P]DGPP as
substrate) of root tissues, expressed as lipid phosphate phosphatase activities relative to values in
unstimulated barley root, are shown in Figure 5A and B.
DGPPase activity was 20% higher, whereas PAP2 activity was 25% lower in stimulated root
tissues relative to control roots. In vitro phosphatase activities decreased in stressed roots compared
to control. For this reason, a protein blotting was performed to determine whether protein levels
were different. Protein fraction isolated from barley tissues was separated by SDS-PAGE and
immunoblotted. Antibody directed to C-terminus of S. cerevisiae DGPPase, used for detection of
DGPPase protein levels, showed band with 30 kDa. Signal levels were significantly different from
stressed roots compared to control (Fig. 5C). DGPPase signal levels were 0.41  0.1 and 0.62  0.1
(n =4, P < 0.05) in NaCl and mannitol treated root compared to control.
PA levels can also be increased through the hydrolysis of PC/PE catalysed by PLD. In order
to know the PLD’s contribution to PA signal, in vivo transphosphatidylation activity of PLD was
measured. Barley roots were labelled O/N and then treated with salt or mannitol in the presence of
0.5% (v/v) 1-butanol. After 30 min, reactions were stopped, and lipids were extracted and separated
by ethyl acetate TLC to monitor the PLD-catalyzed phosphatidylbutanol (PBut) formation by liquid
scintillation. As shown in Figure 6, increased PBut levels were found after NaCl and manitol
treatment. PLD activity was 0.5-fold higher in presence of salt (Fig. 6A) and 0.25-fold higher in

9
presence of manitol (Fig. 6B) in relation to that of the control values. Under these conditions, 1-
butanol did lead to the formation of PBut instead of PA. Accordingly, in Figure 6C we show that
the stress-induced PA levels in the presence of 1-butanol are below the level obtained in the absence
of 1-butanol. These results indicate that the increase of PA, in response to saline and osmotic
stresses, is also as consequence of PLD activation.
Saline and osmotic stresses could trigger changes in the phospholipid patterns. In order to
analyse whether stress treatment can produce a change in the PLs turnover (either structural or
minor PLs), studies on the lipid profiles were carried out (Fig. 7). First, barley seedlings were
grown-up for 4 d with NaCl or mannitol and then labelled with [32P]Pi for 18 h (isotopic
equilibrium). In control roots, it was determined that structural PLs had a high percentage of
[32P]Pi-incorporation, being PE ones of the most abundant phospholipids and representing 60% of
total PLs, followed by CL, PG and PC, with values of 18, 9 and 4%, respectively. The rest of the
phospholipids had a percentage of [32P]Pi-incorporation lower than structural lipid, being all similar
around less than 5%. As shown in Figure 7B, differences on the lipid profiles in presence of NaCl
or mannitol were observed. In NaCl treatment, several PLs i.e. PI, DGPP and PIP, showed higher
percentages of [32P]Pi-incorporation than the control treated with water. Regarding structural lipids,
the lipid turnover was similar to that of the control for PC, PE and LPE, with the exception of CL,
which showed a significant decrease in the lipid turnover, while PA showed a lower [32P]Pi-
incorporation compared to the control.
We found that the formation of PA during saline and osmotic stresses is a consequence of
both, PLD action and DAG-k activity. This fact could be related to proline accumulation.
Consequently, the possibility that DGK and PLD inhibition (by using 1-butanol and DAG-k
inhibitors) may modulate the proline levels during stress responses was tested. Figure 8 shows that
in response to 4-d of treatments with either 100 mM NaCl or 200 mM mannitol, proline was
accumulated to 3- and 2-fold, respectively, compared to control seedlings. In presence of 1-butanol,
proline increased to 4-and 3 fold during treatments with either 100 mM NaCl or 200 mM mannitol,
respectively. On the contrary, in presence of DAG-k inhibitors the proline accumulation was
abolished.
Changes in phytohormone concentrations in plants mediate a wide range of developmental
processes, many of which involve interactions with phospholipid metabolism. Endogenous
phytohormone concentrations in barley roots, expressed as ng per g dry weight, are summarized in
Table 1. GA3 level was 25% lower in stressed roots than in control tissue. ABA level was 50%
lower in stressed than in control roots. IAA content was similar among the three tissues. SA level

10
was 0.8-fold higher in mannitol treated roots. Similarly, JA level was 4-fold higher in plants
treated with mannitol.

4. Discussion
In our present work, the effects of NaCl and mannitol on barley roots were investigated in
relation to lipid signalling. In response to NaCl, the barley root accumulated Na, although K
content did not change significantly. This result could be explained through the barley root’s ability
to retain K+. It was also observed that NaCl and mannitol treatment evoked a reduction on the
length of the primary roots. Adaptation to salinity is related to cell's ability to maintain the optimal
cytosolic K/Na ratio and this fact involves orchestrated regulation of a large number of Na+ and K+
transporters (Shabala and Lew, 2002). In presence of salt, the cytosolic K+/Na+ ratio decreases due
to excessive Na+ accumulation in the cytosol and enhanced K+ leakage. This salinity-induced K+
loss from cells (Cuin and Shabala, 2005;Shabala et al., 2006;Chen et al., 2007b) is a result of NaCl-
induced membrane depolarization, leading to the activation of depolarization-activated outward-
rectifying K+ channels (Shabala et al., 2006;Shabala et al., 2016).
Studies on barley have suggested that the magnitude of this NaCl-induced efflux from the
roots of young seedlings has a negative correlation with salt tolerance (Chen et al., 2007a;Chen et
al., 2007b). Therefore, the root’s ability to retain K+ is significant to salt tolerance (Cuin et al.,
2008). We have also observed that NaCl and mannitol treatment in barley seedlings evoked a
reduction on the length of the primary roots and an increased development of new roots (data not
shown). This event could be due to a process of salt-induced programmed cell death (PCD) of
primary roots as an adaptive response and they could also be related to ionic homeostasis
(Affenzeller et al., 2009;Shabala, 2009). The elimination of a primary root that makes the plant
susceptible to a saline medium, and its replacement with roots that can facilitate nutrient and water
acquisition, may modulate a plant's capacity to regulate nutrients and water uptake. Likewise,
accumulation of compatible solutes is often regarded as a basic strategy for the protection and
survival of plants under abiotic stress (Cuin and Shabala, 2005;Shabala, 2013;Munns and Gilliham,
2015). In barley seedling roots, we have observed an increased proline accumulation in response to
salt and mannitol. Compatible solutes are suggested to act as low- molecular-weight chaperones,
stabilizing the photosystem II complex, protecting the structure of enzymes and proteins,
maintaining membrane integrity and scavenging ROS (Szabados and Savoure, 2010). Recently, it
was also shown that some of these compatible solutes are very efficient in reducing the extent of K +
loss in response to both salinity and oxidative stress in barley and Arabidopsis roots (Cuin and
Shabala, 2005; 2008). However, signalling cascades modulating proline accumulation are still

11
poorly characterized in barley. We also found that PA is rapidly and transiently formed during
saline and osmotic stresses. PA may be produced by multiple enzymes: (a) PLD, acting
hydrolytically on membrane phospho- lipids; (b) DGK, phosphorylating DAG; (c) acyl transferase,
adding a fatty acid to lysoPA; and (d) the enzymes of the de novo pathway from glyceraldehyde 3-
phosphate. Available data suggest that PLD and DGK are the two principal routes that produce
signalling PA. These enzymes are subject to complex and tight regulation. In barley, the two main
pathways involved in PA levels during the response to abiotic stress are PLD and DAG-k.
Therefore, we evaluated whether inhibiting these enzymes could affect the proline accumulation.
Application of 1-butanol stimulated proline increased, indicating a negative correlation between
proline and PLD/PA (Thiery et al., 2004). Nevertheless, PA pool triggered by DAG-k activity could
be related to the proline synthesis, since the DGK-inhibitors abolished proline accumulation. In
barley, cellular PA level is highly dynamic and its production and removal are mediated by several
complex families of enzymes (Meringer et al., 2012;Villasuso et al., 2013). PA formed in response
to salt stress has been suggested to function as a signalling molecule directing the plant’s
acclimation responses to salt stress. In Arabidopsis, PA can bind and affect the activity of various
signalling proteins, including protein kinases and phosphatases. The protein kinase SnRK2 was
found in a proteomic screen for PA targets (Testerink et al., 2004) and it has been shown to be
activating in response to salt stress (Boudsocq et al., 2004). In barley, PA binds and modulates the
glyceraldehyde 3-phosphate dehydrogenase activity (Astorquiza et al., 2016). However, the
specificity in which molecular species of PA transduce saline stress signals remains largely
unknown. Here, we suggest that the different PA pools could modulate the ability to sense and
response stress condition.
Cardiolipin (CL), a minor structural phospholipid that is predominantly present in
mitochondria, was found to decrease in response to salt stress. This fact seems to be associated with
the response to saline stress in crops like in rice (Darwish et al., 2009). CL is a key component of
both prokaryotic and eukaryotic membranes, with unique structure and functions (Lewis and
McElhaney, 2009). It is an anionic phospholipid with a dimeric structure and contains a triple
glycerol backbone and four acyl groups, most of which are highly unsaturated. In barley and rice, it
is still unclear what the decrease in CL in response to salt stress means (Darwish et al., 2009);
although we speculate that CL could be a substrate of PLD and produce PA and PG. However,
under our condition PG increase was not observed. Considering this, more experiments are
necessary to solve this point.
The adaptive response of salt-stressed plants is controlled by chemical signals that
compensate adjustment of growth and development in response to such unfavourable conditions.

12
Some of these signals play a dual role; they can act as signals triggering adaptation or they
accompany stress-related damage (Hazman et al., 2015). In barley, endogenous ABA
concentrations were found to decrease in roots upon 4-d treatment with salt. In contrast, in maize
roots, ABA increased in response to saline stress. We consider that in barley root samples, ABA
could be released into NaCl solutions (Jia et al., 2002). Conversely, JA only accumulated in
response to mannitol stress. Presumably, a decrease in turgor could have generated membrane
damage and hence, triggered the release of the lipid precursors for jasmonate synthesis in roots
exposed to salt stress. JA has been reported to accumulate in response to saline stress in tomato
(Pedranzani et al., 2003) and rice (Moons et al., 1997). Whether this accumulation is a signal
triggering adaptation is not very clear. However, the fact that a salt-tolerant cultivar of rice shows
higher endogenous JA contents as compared to a salt-sensitive cultivar indicates a function for JAs
in salt adaptation (Kang et al., 2005). Signalling triggered by JA is complex, because this signal has
been found to interact with the signalling triggered by other plant hormones known to be involved
in the adaptation to salt stress such as ABA. These interactions, often referred to as ‘hormonal
cross-talk’, need more investigations, especially in crops of economic importance (Hazman et al.,
2015). Most of our knowledge about stress signalling has been learnt through the study of A.
thaliana, although our understanding about cereals as barley is quite incomplete.
Finally, we hope that the future studies will reveal the role of PA and its relation to others
partners in the lipid cascade signalling as part of a mechanism by which barley responds and adapts
to salt stress.

Acknowledgements
This work was supported by SECyT-UNRC, Río Cuarto, (grant number: 18/C426); PPI-
CONICET ( PPI 2012-2015, grant numbers: 4541/12 and 3646/14) Argentina. Ing JC Tomaso of
INTA-Bordenave generously provided seeds. ALV is a Career Investigator of CONICET, MVM
and MPM are fellowship of CONICET.

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Legends of figures

Figure 1: (A) Effect of saline and osmotic stresses on root length of barley. Results are expressed in
centimetres (cm) and shown as mean ± S.D., n=30, different lowercase letters indicate significance
at P < 0.05. (B) Image of 4 d plant treated with 100 mM NaCl or 200 mM mannitol.

Figure 2: Determination of root Na+ (A) and K+ (B) contents. Barley roots (1 g) were homogenized
with liquid nitrogen and then, they were centrifuged to 40,000 x g, during 60 min. The Na+ and K+
contents were determined in the supernatant using a flame photometer. Results are expressed in
µmol g FW-1 and represent the mean ± S.D., (n=3). Different lowercase letters indicate significance
at P < 0.05.

Figure 3: NaCl and mannitol treatment induce proline accumulation in barley roots. The

18
determination of proline concentration was performed according to Bates (1973). Results are
expressed in µmol g FW-1 and represent the mean ± S.D., n=3, different lowercase letters indicate
significance at P < 0.05.

Figure 4: Effect of saline and osmotic stresses on DAG-k and PA-k activities in barley roots. (A)
Representative autoradiography of lipid products from barley roots treated with 100 mM NaCl or
200 mM mannitol. Phosphorylation assay of endogenous phospholipids was carried out with the
105,000 x g membrane fraction in the presence of 370 MBq [γ-32P]ATP for 2 min at 30 ºC. Labelled
lipids were extracted and separated by 1-dimensional TLC with chloroform/ methanol/ acetone/
acetic acid/ water (40:14:15:12:7, v/v). (B) Results are expressed as specific activity (pmol min-1
mg protein-1) and shown as mean ± S.D., n=3, different lowercase letters indicate significance at P
< 0.05.

Figure 5: Effect of saline and osmotic stresses on phosphatidate phosphohydrolase and

diacylglycerol pyrophosphate phosphatase. Enzymatic activities were assayed in the 105,000 x g
membrane fraction obtained from barley roots. PAP2 activity was determined using [ 3H]-PA (0.6
mM) as substrate. DGPP phosphatase activity was measured by the release of water-soluble [32P]Pi
using chloroform-soluble [β-32P]DGPP. Results are expressed as specific activity and shown as
mean ± S.D., n=3, different lowercase letters indicate significance at P < 0.05.

Figure 6: Effect of saline and osmotic stresses on PLD activity. (A) Representative TLC, with lipids
separated with the solvent containing ethyl acetate. Barley roots were incubated in labelling buffer
in presence of [32P]Pi overnight, and then treated with 0.5% (v/v) 1-butanol. Lipids were extracted,
separated by TLC and detected by autoradiography. Results are expressed as 32P-PtdBut (B) or 32P-
PA (C) formation percentage over the total 32P-(PC-PtdBut-PA). Results are shown as mean values
± S.D., n=3, different lowercase letters indicate significance at P < 0.05.

Figure 7: Effect of saline and osmotic stresses on phospholipid turnover in barley roots. (A)
Autoradiography of products from lipids radiolabelled with [32P]Pi. Barley roots were incubated in
presence of [32P]Pi, extracted and separated by TLC. (B) Results are expressed as percentage
distribution of each lipid class and shown as means ± S.D., n=3, different lowercase letters indicate
significance at P < 0.05.

Figure 8: Effect of 1-butanol and DGK inhibitors (R59022 and R59949) on proline accumulation in

19
barley roots. Seedling were incubated with 0.5% v/v 1-butanol, 100 µM R59022 or 150 µM
R59949. The determination of proline concentration was performed as described in materials and
methods. Results are expressed in µmol g FW-1 and represent the mean ± S.D., n=4, different
lowercase letters indicate significance at P < 0.05.

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