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Salt-excluder rootstock improves physio-biochemical responses of grafted

grapevine plants subjected to salinity stress

Article in Current Plant Biology · March 2024

DOI: 10.1016/j.cpb.2023.100316

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 Current Plant Biology 37 (2024) 100316

Contents lists available at ScienceDirect

Current Plant Biology

journal homepage: www.elsevier.com/locate/cpb

Salt-excluder rootstock improves physio-biochemical responses of grafted

grapevine plants subjected to salinity stress
Elania Freire da Silva a, Hugo Rafael Bentzen Santos a, Jean Pierre Henry Balbaud Ometto b,
Alexandre Maniçoba da Rosa Ferraz Jardim c, *, Thieres George Freire da Silva a, Pedro
José Hermínio a, Adriano Nascimento Simões a, Eduardo Souza a, Sérgio Luiz Ferreira-Silva a, *
a
Postgraduate Program in Plant Production, Academic Unit of Serra Talhada, Federal Rural University of Pernambuco, Serra Talhada, Pernambuco, Brazil
b
National Institute for Space Research—INPE, São José dos Campos, São Paulo, Brazil
c
Department of Biodiversity, Institute of Biosciences, São Paulo State University (UNESP), Rio Claro, São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: This study tests the hypothesis that a more salt-excluder rootstock can attenuate salt stress in grapevine plants by
Oxidative disturbances enhancing photosynthesis and providing ionic and oxidative protection. Plants of ‘BRS Vitória’ variety, grafted
Photosynthesis with the rootstocks IAC 313 (salt-excluder) and SO4, were subjected to salinity by adding NaCl (0, 50, and 100
Rootstock
mM) for 30 days. Plants with SO4 showed more severe salt toxicity symptoms in leaves and lower chlorophyll
Salinity
content under salinity. Conversely, plants with IAC 313 showed improved photosynthesis and stomatal
conductance, along with higher carboxylation efficiency under salt compared to SO4. Under salinity, plants with
SO4 showed higher losses of K+ in stems, roots, and petioles, as well as increased accumulation of Na+ in these
organs, relative to IAC 313. Furthermore, plants with IAC 313 had lower leaf Na+ content under salinity and
reduced leaf Cl− content at 50 mM NaCl, a response associated with a higher Na+ allocation in petioles of IAC
313. At 50 mM, IAC 313 exhibited better photochemical activity, as indicated by electron transport rate and non-
photochemical quenching. However, at 100 mM, both rootstocks showed similar trends, suggesting that the
photosynthetic restriction was primarily due to stomatal disturbances. Plants with IAC 313 showed better APX
activity and ascorbate balance under salinity. IAC 313 showed more salt-resistance traits than SO4, although the
growth was similarly affected in both rootstocks. This response could be due to the reduced time of salt treatment
(30 days). In summary, our data indicate that IAC 313 rootstock possesses better salt tolerance traits than SO4.

1. Introduction which is crucial as it can confer greater salinity tolerance to grafted

Salt stress is an abiotic condition that represents one of the most
critical challenges in 21st-century agriculture worldwide [26,60]. In
agricultural areas, the high salt concentration in the soil is a threat, as it
limits the growth and development of plants, in addition to affecting
biochemical pathways and causing damage to the photosynthetic
apparatus [1,60,71]. These disturbances are exacerbated in semi-arid
regions since edaphoclimatic conditions linked to inadequate tech­
niques for soil management and irrigation with high saline water levels
trigger the environmental degradation processes [14,61]. In this
context, grafting techniques are employed to mitigate salt’s effects on
some plant species. This technique helps identify rootstock genotypes
with characteristics of exclusion of toxic saline ions (e.g., Na+ and Cl− ),
plants, although studies on this topic are still insufficient.
Ionic instability is related to the excessive accumulation of sodium
(Na+) and chloride (Cl− ) in the plant tissues that affect ionic homeo­
stasis, causing toxicity and nutritional disturbances in the short and long
term [1,74]. These ions are the main toxic components for the plants
grown under salinity conditions, as they negatively affect normal
physiological processes, causing damage to membranes, nutrient
imbalance, changes in growth regulator levels, enzymatic inhibition,
metabolic dysfunction, and decreased photosynthesis, which can
compromise biomass production and crop yield [1,41,42]. Furthermore,
the imbalance of osmotic and ionic components according to plant
species, developmental stage, and responses to adaptation mechanisms
can result in severe oxidative stress [63].

* Corresponding authors.
E-mail addresses: elania.freire23@gmail.com (E.F. Silva), alexandremrfj@gmail.com (A.M.R.F. Jardim), sergio.luiz@ufrpe.br (S.L. Ferreira-Silva).

https://doi.org/10.1016/j.cpb.2023.100316
Received 3 November 2023; Received in revised form 11 December 2023; Accepted 26 December 2023
Available online 7 January 2024
2214-6628/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
E.F. Silva et al.
Oxidative stress caused by salinity is responsible for increased
reactive oxygen species (ROS) levels, due to the electron transport chain
being suppressed and causing leakages in electron transport [43]. When
these ROS generation exceed the plant’s antioxidant protection capacity,
they also trigger the activity of some ROS-scavenging enzymes such as
superoxide dismutase (SOD), catalase (CAT) and other peroxidases
(POD). This activation may provide relief from saline stress and
contribute to mitigating cellular ionic toxicity [3,30]. Once affected,
ionic disturbances in the chloroplast can lead to the dissociation of
proteins supporting the function of photosystem II (PSII) and photo­
system I (PSI), intensifying damage to light-harvesting complexes and
pigment oxidation [38,54,62,65]. Yan et al. [72] reported that PSII
photoinhibition that restricts electron donation to PSI could be effective
in preventing PSI photoinhibition by reducing ROS generation via the
Mehler reaction on the acceptor side. However, electron donation and
efficiency in ROS production and removal differs between in glycophyte
and halophyte species.
The grapevine (Vitis spp.) belongs to the Vitaceae family and is
moderately sensitive to salt stress. In addition, it is a widely cultivated
crop, covering an area of cultivation greater than 7.5 million hectares,
and stands as the major fruit crop worldwide due to its economic and
nutritional value [11,20,66,73]. It is usually grown as a scion grafted
onto a rootstock (i.e., it forms part of the trunk and root system), and
rootstock selection is considered the most promising method for
achieving high-stress tolerance levels. In the past, this practice aimed to
solve problems caused by grape phylloxera (Daktulosphaira vitifoliae
Fitch) in most vineyards worldwide [17]. However, interest in selecting
new rootstocks with greater salinity tolerance has increased signifi­
cantly. This approach seeks genotypes with salt-resistance attributes for
physiological and biochemical processes, such as best biomass accu­
mulation, ionic homeostasis, vigor, and fruit quality. These character­
istics can be achieved through grafting with more salt-tolerant
rootstocks [3,11,45,66].
Although the grapevine is an agricultural species widely cultivated in
the Brazilian semi-arid region and in the world as grafted plants, there
are still no known salinity-tolerant genotypes that can be used as
metabolically more efficient rootstocks. Recently, we demonstrated that
among grapevine rootstocks commonly grown in semi-arid Brazilian,
the IAC 313 presents higher salt tolerance through the best K+/Na+
selectivity and photosynthetic performance [3,64]. These studies
demonstrated that IAC 313 rootstock presents the best leaf salt-exclude
characteristics under salinity relative to other evaluated genotypes.
However, components involved with the cell and tissue oxidative pro­
tection did not were accessed. Thus, to better understand the salt
tolerance mechanism, we investigated rootstocks’ potential in attenu­
ating damages relative to photosynthesis, ionic homeostasis, and
oxidative protection in grapevine plants under salinity. This study tested
the hypothesis that the IAC 313 rootstock can attenuate salt stress in
grafted plants with the cultivar ‘BRS Vitória’, thereby improving the
photosynthetic performance associated with higher ionic and oxidative
protection.
2. Materials and methods
2.1. Experiment site, plant material and treatments
The experiment was carried out in a greenhouse at the Federal Rural
University of Pernambuco – Academic Unit of Serra Talhada (UFRPE/
UAST), located in the municipality of Serra Talhada, state of Pernam­
buco, Brazil (07º59’31″ S, 38º17’54″ W and 444 m above sea level). We
used grafted plants of grapevine (Vitis spp.) from the combination of the
cultivar ‘BRS Vitória’ (scion) grafted onto the rootstocks IAC 313
[’Golia’ (V. riparia × V. rupestris) × V. cinerea] (hereafter called IAC 313)
and Selection Oppenheim 4 ’SO4’ (V. berlandieri × V. riparia) (hereafter
called SO4). The plants, in the post-grafting stage (~30 days) with
uniform growth, were transferred to 5-liter polypropylene pots filled
2
Current Plant Biology 37 (2024) 100316
with a 1:1 mixture (volume: volume) of sand and vermiculite. They were
acclimatized for 20 days in the greenhouse under environmental con­
ditions [air temperature: 35/24ºC ± 1ºC (day/night), relative humidity:
64/76% (day/night), light intensity: 1100 µmol m− 2 s− 1, and photope­
riod: 12 h light] (Fig. 1). In the first week, we applied a half-strength
Hoagland nutrient solution to the plants [29], and then a full concen­
trated solution during the experiment, with applications every three
days. Subsequently, the plants were submitted to treatments with NaCl
(non-salt control, 50 mM NaCl, and 100 mM NaCl) diluted in the
concentrated nutrient solution (pH 6.0) for 30 days, with applications
every three days (Fig. 1). Before applying saline treatments, the grape­
vines received a gradual increase in salt concentration at 25 mM NaCl
until reaching levels of 50 and 100 mM NaCl. In the interval between
irrigation with saline solution, we used distilled water to prevent the
substrate from concentrating in excess salt.
During the experimental period, measurements of photochemical
parameters and gas exchange in plants (non-destructive measurements)
were carried out. In the final stage of the experiment, we collected and
separated the constituent parts of grapevine plants into roots, stems, and
leaves to determine both fresh mass (FM) and dry mass (DM). Triplicate
leaves subsamples were frozen in liquid nitrogen (N2) and stored at
− 80ºC for use in biochemical analyses involving measurement of the
content of oxidative damage indicators (i.e., thiobarbituric acid reactive
substances—TBARS) and oxidative protection (enzymatic and non-
enzymatic antioxidants). At the end of the experiment, after 30-day of
saline treatment, substrate saturation waters were collected to quantify
the electrical conductivity of each NaCl concentration. The water sam­
ples collected from the 0, 50, and 100 mM NaCl treatments showed
electrical conductivity values of 1.70, 16.33, and 30.55 mS cm− 1,
respectively.
2.2. Experimental design
The experiment was conducted in a completely randomized design
with a factorial arrangement (2 × 3) and three replications, totaling 18
experimental units. The treatments were consisted of a combination of
two factors: two combinations of grapevine scion/rootstock (i.e., ‘BRS
Vitória’/IAC 313 and ‘BRS Vitória’/SO4) and three concentrations of
NaCl [non-salt control (0 mM NaCl), 50 mM NaCl, and 100 mM NaCl].
In addition, all pots were rotated from one place to another every two
days in order to ensure better use and distribution of sunlight on the
plants. Each plot consisted of a pot containing one grafted plant. Data
referring to the measured variables were expressed as mean (n = 3)
± standard deviation (SD).
2.3. Physiological and biochemical analyses
2.3.1. Fluorescence parameters: light and induction curves
In this study, we measured the parameters indicating the photo­
chemical efficiency of PSII, aiming to monitor damage to the light en­
ergy capture apparatus in response to salinity. Each chlorophyll
fluorescence measurement was performed on physiologically mature
leaves of the middle third of the plant using a portable chlorophyll
fluorimeter (MINI-PAM-II, Heinz Walz, Germany). The estimated
photochemical parameters were (i) maximum quantum efficiency of
PSII [Fv/Fm = (Fm − Fo/Fm)], (ii) effective quantum yield of PSII [ɸPSII
= (Fm’ − Fs)/Fm’], (iii) electron transport rate [ETR = (ɸPSII × PPFD ×
0.5 × 0.84)], (iv) photochemical quenching coefficient [qP = (Fm’ −
Fs)/(Fm’ − Fo’)] and (v) non-photochemical quenching coefficient [NPQ
= (Fm − Fm’/Fm’)]. The parameters Fm, Fo, and Fv respectively represent
the maximum, minimum, and variable fluorescence after leaf adaptation
to 50 min of darkness; Fm’, Fo’, and Fs respectively represent maximum,
minimum, and steady-state fluorescence in the presence of light; and
PPFD is the photosynthetic photon flux density. To perform the light
curves, the readings were taken on leaves pre-adapted to the dark for
50 min with the aid of tweezers. This dark adaptation promotes the
E.F. Silva et al.
Fig. 1. Schematic overview of the application of treatments to grapevine plants (IAC 313 and SO4) under salinity stress (0, 50, and 100 mM NaCl) in greenhouse.
complete opening of the PSII reaction centers. After this period, the
leaves were irradiated by PAM fluorimeter light ranging from 0 to
1500 µmol photons m− 2 s− 1, in an interval of 5 min. In addition, we
performed measurements of induction curves, with dark-adapted leaves
for 50 min and measurements made in the presence of light (270 µmol
photons m− 2 s− 1) provided through the device’s optical fiber for 220 s.
2.3.2. Gas exchange measurements
To assess the impact of salinity, we performed measurements of net
CO2 assimilation (PN), transpiration rate (E), stomatal conductance (gS),
and intercellular CO2 concentration (Ci) using a Walz GFS-3000 gas-
exchange system (Heinz Walz GmbH, Effeltrich, Germany) on physio­
logically mature leaves sampled in the middle third of the plant. These
measurements allowed us to estimated the water use efficiency (WUE)
parameters through the PN/E ratio and the instantaneous carboxylation
efficiency (PN/Ci ratio). In addition, gas exchange measurements were
performed once at the beginning of the experiment when the grapevines
had 10 days of treatment application. Before the readings, the infrared
gas analyzer (IRGA) was calibrated, and we standardized at a PPFD of
1000 µmol photons m− 2 s− 1, CO2 flow controller set at 400 µmol CO2
mol− 1, and temperature of 28ºC in the chamber.
2.3.3. Measurements of photosynthetic pigments
To obtain chlorophyll (Chl) data, 0.2 g of FM from the leaves of each
plant were weighed. The material was solubilized in 80% acetone using
10 mL test tubes. All tubes were stored in a refrigerated environment
(10ºC) and in the dark. After 48 h, we performed the readings of the
chlorophyll extract using UV-Vis spectrophotometry at 647 and 663 nm
for chlorophyll a (Chla) and chlorophyll b (Chlb), respectively [47].
Chla, Chlb, and total chlorophyll (Chlt) contents were calculated using
the following expressions: Chla = [(12.25 × A663 – 2.79 × A647)]; Chlb
= [(21.5 × A647 – 5.10 × A663)]; and Chlt = [(Chla) + (Chlb)], as well
as the ratio of Chla to Chlb (Chla/b ratio). In each equation, A is the
absorbance at specific wavelengths. All data were determined in mg g− 1
FM.
2.4. Daily dry mass accumulation rate
The dry mass accumulation rate (DMAR) of the root, stem, leaf, and
3
Current Plant Biology 37 (2024) 100316
whole plant was estimated using as a reference the dry mass contents
measured at the beginning of the saline treatment in the respective or­
gans of the plants. For this purpose, plants (3 replicates) of the two
grapevine cultivars were collected at the beginning and end of the NaCl
application, subsequently separated into roots, stems, and leaves. The
plant materials collected were individually identified, packed in paper
bags, and dried in an oven at 65ºC for 72 h. After drying, the samples
were weighed to quantify the dry mass (DM) weight of the parts (i.e.,
root, stem, and leaf) and of the entire plant. With the sample weights, we
determined the DMAR using the following expression: DMAR = [(DMf –
DMi)/(T)], where DMf is the DM content at the final of the experiment;
DMi is the DM content at the initial of the experiment; and T is the time
(days) of exposure to the salt treatment [56]. Results were expressed as
mg g− 1 DM day− 1. The DMAR reflects the accumulated average daily
amount of DM that the whole plant or different plant organs obtained
during the evaluation period, which this parameter (i.e., DMAR) may
indicate stability, increments, and reduction. Using the biomass data, we
calculated the shoot/root ratio by dividing the shoot’s dry mass by the
roots’ dry mass.
2.5. Plant water status
We measured predawn leaf water potential (Ψpd) using a Scholander-
type pressure chamber (model 1505D, PMS Instrument Company,
Albany, OR, USA) at the end of the experiment after 30 days of appli­
cation of the saline treatment. Measurements were taken between 4:30
and 5:30 h, just before sunrise. One day before carrying out the leaf
water potential readings, all plants were irrigated with the same water
depth. The leaves used for measurements were fully expanded, mature,
and located in the middle third of the plants. The potential of each leaf
was evaluated immediately after it was detached from the plant. During
the readings, the pressure in the chamber was slowly increased, and
pressure data were recorded when a meniscus of water began forming on
the cut branch’s surface.
2.6. Sodium, potassium and chloride content
The contents of sodium (Na+) and potassium (K+) ions were deter­
mined by flame photometry [59]. Briefly, the plant material (tissues of
E.F. Silva et al.
leaves, petioles, stems and roots) was dried in an oven, ground sepa­
rately in a knife mill, and then passed through a 1 mm sieve. We
collected 0.5 g samples of the different tissues crushed for extraction by
incubation in test tubes with screw caps, each tube containing 10 mL of
ultrapure water (Milli-Q), and they were boiled in a water bath at 100ºC
for 1 h. The extracts were filtered and used in a flame photometer for
Na+ and K+ readings (Micronal®, model B462, São Paulo, Brazil). Na+
and K+ contents were estimated based on a standard curve (0 to
1000 µM) of NaCl and KCl, respectively, and the results were expressed
in µmol g− 1 DM. The chloride (Cl− ) content was established according to
Ferreira-Silva et al. [15]. In this analysis, 0.2 g samples of ground tissues
were subjected to extraction by incubation in test tubes with screw caps.
The ground sample was added to 25 mL of ultrapure water and boiled in
a water bath at 100ºC for 1 h. The extracts were filtered, and 20 mL were
collected for chloride titration using silver nitrate (AgNO3; 28 mM
concentration) and a magnetic stirrer. In addition, we used potassium
chromate (K2CrO4) at a concentration of 5% (w/v) as a colorimetric
indicator and the results were expressed in µmol g− 1 DM.
2.7. Daily ion accumulation rate
To calculate the daily ion accumulation rate (DIAR) in the different
organs (root, stem, and leaf) of the plant, the concentration of Na+ and
K+ at the beginning of the saline treatment and their accumulation over
time was considered. From each treatment, three plants of the two
grapevine cultivars were sampled over time, and the Na+ and K+ levels
in roots, stems, petioles, and leaves were quantified using flame
photometry. For each studied ion, we calculated the DIAR according to
the following equation: DIAR(ion) = [(ICf – ICi)/(T)], where ICf and ICi
refer to the ion concentration (either K+ or Na+) final and initial of the
experiment (µmol g− 1 DM) [28]. Results were expressed in µmol g− 1 DM
day− 1. In this case, the ion accumulation rate reflects the accumulated
average daily amount of each ion (K+ or Na+), and this may indicate
stability, increments, and ionic reduction in tissues. Furthermore, when
it presents negative values may indicate ion losses relative to concen­
trations initially present in the plants (before the salt application).
2.8. Protein extraction and enzymatic activities
Lyophilized leaf samples (0.2 g FM) were homogenized in 100 mM
Tris-HCl buffer (pH 8.0) containing 20% glycerol, polyethylene glycol
(3%), and Triton X-100 at 0.1%. The buffer was bubbled in CO2 to
prevent extract oxidation. After extraction, the extract was centrifuged
at 14,000 × g for 30 min at 4ºC. Protein content was quantified ac­
cording to Bradford [9]. Soluble protein content was estimated from a
standard curve using bovine serum albumin (BSA), and results were
expressed as μg protein g− 1 DM.
For the measurement of ascorbate peroxidase—APX (EC 1.11.1.11)
activity was used methodology described by Nakano and Asada [53].
For specific extraction of this protein was added ascorbate 1 mM in the
extraction buffer to avoid APX denaturation. In this test, the reagents
used were kept in a dry bath to maintain a temperature of 27ºC. In
addition, 12.5 μL aliquots of the protein extract were added to 850 μL of
50 mM potassium phosphate buffer (pH 6.0) + 37.5 μL of extraction
buffer + 50 μL of ascorbic acid at 5 mM (added at the time of the assay)
and 50 μL of hydrogen peroxide (H2O2) at 2 mM. Spectrophotometric
readings were performed at the absorbance of 290 nm for 3 min at in­
tervals of 15 s, and the molar extinction coefficient used was 2.8 mM− 1
cm− 1. APX specific activity was expressed in nmol ASC μg− 1 protein
min− 1. The activity of catalases—CAT (EC 1.11.1.6) was performed
according to the method of Havir and McHale [25]. Briefly, 12.5 μL
aliquots of protein extract were added to 900 μL of 50 mM potassium
phosphate buffer (pH 7.0), 37.5 μL of extraction buffer, and 50 μL of
H2O2. Readings were taken at an absorbance of 240 nm for 3 min
(15-second intervals). Enzymatic activity was measured by the H2O2
molar extinction coefficient (36 mM− 1 cm− 1), and the results were
4
Current Plant Biology 37 (2024) 100316
1
expressed in µmol H2O2 µg− protein min− 1.
2.9. Ascorbate and glutathione content
To determine reduced ascorbate levels, samples of fresh grapevine
leaves (0.2 g) were macerated in liquid N2 with a mortar and pestle and
homogenized with 1.5 mL of a 6% trichloroacetic acid (TCA) solution
[37]. Subsequently, the extract was transferred to Eppendorf tubes,
identified, and centrifuged at 12,000 × g for 15 min at 4ºC. We removed
100 μL aliquots of the supernatant and added them to a reaction medium
containing 300 μL of 200 mM potassium phosphate buffer (pH 7.4),
100 μL of deionized water, 500 μL of TCA (10%), 400 μL of phosphoric
acid (45%), 400 μL of bipyridyl (4%) and 200 μL of FeCl3 (3%). The
reaction was submitted to a water bath at 40ºC for 30 min. Absorbance
readings were performed at 525 nm, with the content expressed in μmol
g− 1 FM, estimated based on a standard curve.
The reduced glutathione content was determined following the
method proposed by Griffith [21]. Briefly, fresh leaf tissues (0.2 g) were
macerated in liquid nitrogen and homogenized in 1.5 mL of 6% TCA at
extraction. Furthermore, the extract was centrifuged at 12,000 × g for
15 min at 4ºC. In this assay, 200 μL of the supernatant was added to the
reaction medium containing 2600 μL of sodium phosphate buffer
(150 mM, pH 7.4), 1000 μL of 100 mM sodium phosphate buffer (pH
7.0), and 200 μL of 30 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)
in 100 mM potassium phosphate buffer (pH 7.0). Then, the reaction was
maintained for 10 min in a water bath at 30ºC. Readings were performed
with absorbance at 412 nm and content estimated based on the standard
curve, expressed in μmol g− 1 FM.
2.10. Determination of lipid peroxidation
We determined lipid peroxidation by the method described by Heath
and Packer [27], which analyzes the content of thiobarbituric acid
reactive substances (TBARS), such as malondialdehyde (MDA). Fresh
leaf samples (0.2 g) were macerated in liquid nitrogen and homogenized
with 1.5 mL of TCA (6%). The homogenate was transferred to Eppendorf
tubes, which were identified and centrifuged at 12,000 × g for 15 min at
4ºC. In the collected supernatant (500 μL of extract), 20% TCA and 0.5%
thiobarbituric acid (TBA) were added to 2 mL of the reaction solution.
The solutions were heated in a water bath at 95ºC for 1 h and cooled on
ice. Readings were taken with absorbance at 532 nm (reaction readings)
and 600 nm to review any non-specific turbidity. Furthermore, we used
an extinction coefficient of 155 mM− 1 cm− 1 for the calculations and
expressed the results in nmol MDA g− 1 FM.
2.11. Visual symptoms of salt toxicity
Leaf toxicity symptoms were assessed immediately before leaf sam­
pling using a visual scale ranging from 0 to 5 for each plant. In this scale,
0—indicates the absence of symptoms (no foliar damage), 1—signifies
symptoms such as leaf tip burn in less than 25% of the leaves,
2—represents burn symptoms covering 25 to 50% of the leaves,
3—indicates burn symptoms spanning 50 to 75% of the leaves,
4—denotes symptoms present in more than 75% of the leaves, and
5—signifies the presence of dead plants [12].
2.12. Statistical analysis
All data were tested for normality (Shapiro-Wilk test) and homoge­
neity of variance (Bartlett’s test). No data transformation was required.
Subsequently, the data were submitted to one-way analysis of variance
(ANOVA). We used multiple comparisons of means by Tukey’s test, and
the significance limit was set at a probability level of 0.05 (P < 0.05).
Finally, to examine the interrelationships of biotic and abiotic factors in
the characteristics of grapevine rootstock genotypes, a principal
component analysis (PCA) was applied. To unify the different
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

dimensions of the sample data, before applying the PCA, we performed a rootstock IAC 313 under 50 mM NaCl, while SO4 reduced only 5%, in
standardization of the data (i.e., zero mean and unit variance) [32]. relation to the respective controls (P > 0.05). In leaf DM accumulation
Furthermore, Kaiser’s criterion was applied to reduce the number of rate, the IAC 313 rootstock was not affected; however, reductions of
components. This criterion consists of using any principal component 34% and 94% were observed for treatments with 50 and 100 mM NaCl,
(PC) that exhibits an eigenvalue greater than 1.0 [33,36]. All statistical respectively, compared to control plants. There was an increase in the
analyzes were performed using R software [58]. biomass accumulation rate of the shoot/root ratio, although there was
no significant effect (P > 0.05).
3. Results Fig. 2 shows the results of dry mass relative allocation in IAC 313 and
SO4 grapevines. Regardless of treatment, IAC 313 showed the lowest
3.1. Dry mass accumulation and partitioning results for leaf biomass allocation, averaging 16.74%. On the other
hand, on average in the treatments (0, 50, and 100 mM NaCl), the
The salinity stress affected biomass production in the entire plant, relative allocation of stem and root was superior to the SO4 rootstock by
and this effect was independent of the genotype of the rootstock used for 33.42%. Exposure of SO4 to a concentration of 50 mM NaCl increased
grafting (Table 1). Root DM was similarly reduced on both rootstocks the aerial part (i.e., leaf + stem) by 69.94%, 1.53% greater than the IAC
under salinity of 50 mM NaCl and more severely under 100 mM NaCl 313. In both rootstocks, the 100 mM NaCl treatment reduced root and
(about 48%) when compared to the respective control treatments (0 mM leaf dry mass and increased stem biomass.
NaCl). In both rootstocks, there was a low DM reduction in the stem. The
leaves DM did not show difference for IAC 313 to NaCl levels submitted, 3.2. Visual symptoms of salinity toxicity and changes in chlorophyll
but it was significantly reduced (55%) in plants with the rootstock SO4
under highest salinity level compared to the control plants. For total Our phenotypic analysis results show typical foliar symptoms of
plant DM a progressive reduction was verified as the salinity level toxicity caused by the osmotic/ionic effects of salt stress in plants. For
increased, decreasing 37% (IAC 313) and 35% (SO4) when exposed to example, there was growth restriction, chlorosis, and necrosis associated
100 mM NaCl compared to the control plants. IAC 313 significantly with leaf senescence (Fig. 3). Specifically, IAC 313 showed leaf brown­
increased its shoot/root biomass ratio between saline treatments. Both ing with necrotic spots in the central part of the leaf when 50 and
IAC 313 and SO4 had increases in their shoot/root ratios but showed no 100 mM NaCl were applied (visual score of 1, indicating leaf burn in less
difference for grapevines at the same salinity level. than 25%). Likewise, IAC 313 and SO4 under 100 mM NaCl showed a
In general, the daily rate of total dry mass accumulation showed lower intensity of green coloring on leaves (Fig. 3A–B). An increased leaf
greater damage in both rootstocks exposed to salt across all plant parts sensitivity was observed in SO4, with drying and necrosis initiating at
(i.e., root, stem and leaf) and in the plant’s total biomass (Table 1). IAC the margins and central region of the leaves when exposed to 50 and
313 and SO4 showed a significant reduction in root dry mass accumu­ 100 mM NaCl (Fig. 3B), earning visual scores of 1 (i.e., leaf burn in less
lation rate under salinity treatments. The stem reduced 33% of DM in the than 25%) and 2 (i.e., burn symptoms at 25–50%), respectively. Addi­
tionally, all plants subjected to saline stress exhibited toxicity symptoms,
Table 1 predominantly affecting the older leaves located in the basal part of the
Effect of salt stress on dry mass partitioning, and dry mass accumulation rates in plants.
grapevine plants grafted on IAC 313 and SO4, 30 days after treatment with NaCl Chla content significantly decreased in both rootstocks from 50 mM
(0, 50 and 100 mM). NaCl treatment (Fig. 3C). On the other hand, Chlb was lower in SO4 as
Rootstock NaCl Root Stem Leaf Plant Shoot/ saline levels increased (Fig. 3D). Furthermore, the total chlorophyll
(mM) root content was significantly altered in SO4 with increasing salinity
Dry mass partitioning (g plant− 1) (Fig. 3E). The Chla/b ratio reached its highest value with the application
of 50 mM NaCl in both grapevines, whereas the lowest chlorophyll ratio
IAC 313 0 16.97 18.46 Aa 6.16 Ba 41.60 1.45 Ab
Aa Aa was observed when exposed to 100 mM NaCl (Fig. 3F).
50 9.98 Ab 15.79 Aa 5.77 Ba 31.55 2.17 Aa
Bb 3.3. Gas exchange, photochemical parameters and water potential
100 7.17 Ab 14.55 Aa 4.54 Aa 26.27 2.66 Aa
Ab
Gas exchange parameters showed a severe effect of salt stress on
SO4 0 16.31 19.51 Aa 12.49 Aa 48.32 1.97 Aa stomatal conductance associated with a strong restriction of carbon
Aa Aa
50 12.17 18.49 Aa 10.00 Aa 40.66 2.37 Aa
Ab Aab
100 8.85 Ac 16.79 Aa 5.62 Ab 31.27 2.54 Aa
Ab
1
Accumulation rates (mg g− DM day− 1)

IAC 313 0 47.13 270.44 78.77 Ba 80.87 7.39 Aa

Aa Aa Aa
50 23.84 181.44 65.76 Ba 47.38 10.42 Aa
Ab Aa Bb
100 14.45 140.33 24.63 Aa 29.77 11.35 Aa
Ab Aa Ab

SO4 0 39.76 324.00 242.22 94.87 14.62 Aa

Aa Aa Aa Aa
50 25.94 290.22 158.93 69.34 21.45 Aa
Aab Aa Ab Aa
100 14.90 233.44 12.89 Ac 38.02 27.75 Aa
Ab Aa Ab

Data are presented as means of three replications (n = 3). For each parameter, Fig. 2. Changes in the relative biomass allocation to root, stem, and leaf
different uppercase letters compare the two rootstocks at each salt concentra­ fractions in two grapevine rootstocks (IAC 313 and SO4) after 30 days of
tion, and different lowercase letters compare the same rootstock at all salt treatment with 0, 50, and 100 mM NaCl. The error bars represent mean
concentrations indicating significant differences by Tukey’s test (P < 0.05). ± standard deviation (SD) of three replicates for all treatments.

5
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

Fig. 3. Phenotypic analysis and chlorophyll content (mg g− 1 FM) in grapevine plants in response to salt stress (0, 50, and 100 mM NaCl). Phenotypic observations of
‘BRS Vitória’ grapevine plants grafted on IAC 313 (A) and SO4 (B) rootstocks. Content of chlorophyll a (C), chlorophyll b (D), total chlorophyll (E), and the ratio of
Chla to Chlb [Chla/b ratio] (F) in leaves of grapevine during 30 days in greenhouse. Scale bar = 10 cm. Error bars show the mean ± SD of three replicates. Means
followed by different letters are significantly different (Tukey’s test, P < 0.05). The uppercase letters compare the means of two rootstocks at each salt concentration,
whereas the lowercase letters compare the same rootstock at all salt concentrations.

assimilation when compared to plants grown in the absence of salt
(Fig. 4). Plants grown in the absence of salt showed significant difference
in net CO2 assimilation (PN), with values of 9.8 and 8.2 µmol CO2 m− 2
s− 1 for IAC 313 and SO4, respectively (Fig. 4A). IAC 313 did not reduce
PN when subjected to 50 mM NaCl, while the SO4 plants presented a
reduction in PN of approximately 50% in relation to the control plants.
Likewise, when we applied 100 mM NaCl, the PN reduced to approxi­
mately 60% and 85% in IAC 313 and SO4 plants, respectively, in
contrast to their respective control plants. The transpiration rate (E) and
stomatal conductance (gS) showed a significant difference between IAC
313 and SO4 in the presence and absence of salinity (Fig. 4B–C).
Interestingly, plants subjected to salinity stress showed a similar trend in
E and gS observed for PN, with those grafted onto IAC 313 being less
affected by salinity. The intercellular CO2 concentration (Ci) differed
between grapevines when grown in the absence of salt (Fig. 4D). At 50
and 100 mM NaCl, IAC 313 reduced Ci by 20.8% and 33.4%, respec­
tively, compared to control plants (0 mM NaCl). SO4 was reduced by
20.9% (50 mM NaCl) and 24% (100 mM NaCl) compared to control
plants.
Water use efficiency (WUE) and instantaneous carboxylation effi­
ciency (PN/Ci) showed significant changes (P < 0.05) between salinity
levels and the two rootstocks (Fig. 4E–F). IAC 313 plants showed a slight

6
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

Fig. 4. Net CO2 assimilation [PN, μmol (CO2) m− 2 s− 1] (A), transpiration rate [E, mmol (H2O) m− 2 s− 1] (B), stomatal conductance [gS, mmol (H2O) m− 2 s− 1] (C),
intercellular CO2 concentration [Ci, μmol mol− 1] (D), water use efficiency [WUE, µmol (CO2) mmol− 1 (H2O)] (E), instantaneous carboxylation efficiency [PN/Ci] (F)
and predawn leaf water potential [Ψpd, MPa] (G) in grapevine plants of the ‘BRS Vitória’ cultivar grafted on the rootstocks IAC 313 and SO4 grown in the absence
(0 mM NaCl) and presence of NaCl (50 and 100 mM) for 30 days under greenhouse conditions. Error bars show the mean ± SD of three replicates. Means followed by
different letters are significantly different (Tukey’s test, P < 0.05). The uppercase letters compare the means of two rootstocks at each salt concentration, whereas the
lowercase letters compare the same rootstock at all salt concentrations.

Fig. 5. Rapid light curves of the effective quantum yield of PSII [ɸPSII] (A), photochemical quenching [qP] (B), electron transport rate [ETR, μmol m− 2 s− 1] (C), and
non-photochemical quenching [NPQ] (D) in grapevine plants of the ‘BRS Vitória’ cultivar grafted on the rootstocks IAC 313 and SO4 grown in the absence (0 mM
NaCl) and presence of NaCl (50 and 100 mM) for 30 days in greenhouse conditions. Data are presented as mean ± standard deviations (n = 3). PPFD refer to the
photosynthetic photon flux density.

7
E.F. Silva et al.
increase in water use efficiency in response to salinity (20.1%). This
increase in WUE was also seen in SO4 (10.8%) (Fig. 4E). While IAC 313
plants did not show a reduction in carboxylation efficiency (PN/Ci ratio)
under 50 mM NaCl, SO4 plants showed a significant reduction (24.6%)
(Fig. 4F). In plants subjected to 100 mM NaCl, the PN/Ci ratio was
reduced by 43.5% in IAC 313, while in plants under SO4 rootstock, this
reduction was 76% compared to the respective control plants.
Salt application caused significant reductions in Ψpd in both grape­
vines (IAC 313 and SO4) (Fig. 4G). However, both grapevine control
plants (0 mM NaCl) showed similar Ψpd (− 0.14 MPa). In the treatment
with 50 mM NaCl, we observed a significant reduction of − 0.49 MPa
(equivalent to 313.2%) in the Ψpd of IAC 313 and − 0.82 MPa (690.4%)
in SO4, when compared to the respective control treatments. Notably,
grapevines treated with 100 mM NaCl showed a higher (more negative)
reduction of Ψpd, being significantly lower (− 1.16 MPa) in SO4.
In the present study, salinity stress on grapevine rootstocks reduced
photochemical parameters (Fig. 5). In general, photochemical responses
sloped more steeply in the 100 mM NaCl condition. We observed a
marked reduction in ɸPSII in all treatments when exposed to 190 µmol
photons m− 2 s− 1. In this same PPFD, SO4 with 100 mM NaCl presented
ɸPSII of 0.20, being 25% lower than IAC 313 in the same condition.
Furthermore, SO4 (50 mM NaCl) and IAC 313 (100 mM NaCl) results for
ɸPSII were similar (0.27). The qP decreased with increasing PPFD for all
treatments. The most pronounced difference in the qP curve was after a
PPFD of 90 µmol m− 2 s− 1, and initially, the IAC 313 showed a higher qP
(>0.7) when the PPFD was between 0 and 90 µmol m− 2 s− 1 (Fig. 5A–B).
It is noteworthy that this range (intensity) of light (800 µmol photons
m− 2 s− 1) is compatible with light saturation for CO2 assimilation in
grapevine. The ETR of the two rootstocks at the same salinity (i.e., 0, 50,
and 100 mM NaCl) showed no difference. On average, there was a
Fig. 6. Photosynthetic induction curves of the effective quantum yield of PSII [ɸPSII] (A), photochemical quenching [qP] (B), electron transport rate [ETR, μmol m− 2
s− 1] (C) and non-photochemical quenching [NPQ] (D) in plants of grapevine cultivar ‘BRS Vitória’ grafted on the rootstocks IAC 313 and SO4 grown in the absence
(0 mM NaCl) and presence of NaCl (50 and 100 mM) during 30 days in greenhouse conditions. Data are presented as mean ± standard deviations (n = 3).
8
Current Plant Biology 37 (2024) 100316
reduction of 19.2% (50 mM NaCl) and 44.8% (100 mM NaCl) in the ETR
of the rootstocks when compared to the control plants (Fig. 5C). Non-
photochemical quenching (NPQ) increased significantly in SO4 with
100 mM NaCl and lower in IAC 313 plants with 100 mM (increased
photochemical quenching). However, the control treatment of IAC 313
was slightly similar when 50 mM NaCl was applied (Fig. 5D).
The dynamics of photochemical efficiency parameters revealed
limiting effects of salinity on the step of light reactions of rootstock-
dependent photosynthesis (Fig. 6). Control plants on SO4 rootstock
showed greater induction of ɸPSII, qP, and ETR parameters during the
initial induction of photosynthesis, followed by the grafted plant on IAC
313 (Fig. 6). Under salinity of 50 and 100 mM, SO4 plants showed a
strong reduction in ɸPSII, qP, and ETR, while IAC 313 was slightly lower
in these parameters when 100 mM NaCl was applied (Fig. 6A–C).
Furthermore, the NPQ of both grapevines exposed to 100 mM NaCl was
significantly higher than the control and 50 mM NaCl (Fig. 6D). Inter­
estingly, there was a decline in NPQ in all treatments except for SO4
with 50 mM NaCl after 120 s. In the same way, during the time from 120
to 220 s, the SO4 with 0 mM NaCl reduced on average 15.5% when
compared to the 50 mM NaCl plants and 32.7% in contrast to the
100 mM NaCl plants. Overall, our results imply that salt treatment
substantially decreases the photochemical efficiency and increases the
NPQ of the grapevines.
3.4. Na+, K+, Cl− contents and Na+ and K+ accumulation rate
When we compared the IAC 313 and SO4 grapevines at the same
salinity level, the Na+ (stem), K+ (petiole and stem), and Cl− (stem)
contents did not show significant differences. However, both grapevines
under salinities of 50 and 100 mM showed a gradual and slightly similar
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

increase in Na+ content in root and shoot (i.e., stems, petioles, and significant difference between IAC 313, and SO4 treated with 50 mM
leaves) (Table 2). As expected, the Na+ and K+ contents increased and NaCl in the petiole. On the other hand, IAC 313 was significantly su­
decreased, respectively, as the NaCl concentration increased. Likewise, perior (16%) to SO4 under 100 mM NaCl for Na+ accumulation in the
Cl− in all plant structures increased as the NaCl concentration increased. petiole.
Interestingly, the petiole had a higher Na+ content, and IAC 313 with The lowest accumulated levels of Na+ in grapevines were observed in
100 mM NaCl was 11.3% higher than SO4 in the same treatment. The the stem (Fig. 7F), and different salinity levels provided a significant
salinity (50 and 100 mM NaCl) considerably reduced the K+ of the SO4 difference between the grapevines. There was no difference in 50 mM
root (33.8% and 24.4%) compared to the IAC 313, and there was an NaCl in the stem of IAC 313 and SO4 (P > 0.05). However, the appli­
increase of the ion in the leaf, being 20.3% (50 mM NaCl) and 42.3% cation of 100 mM NaCl increased the Na+ accumulation rate of IAC 313,
(100 mM NaCl). which was 15.7% higher than that of SO4. The root was the second
The rate of K+ accumulation in grapevine leaves ranged from 0.75 to organ with the highest sodium accumulation in grapevines (Fig. 7H).
14.10 μmol g− 1 DM day− 1 (Fig. 7A). The salinity levels of 50 and The 50 mM NaCl-treated plants significantly increased sodium accu­
100 mM NaCl caused restrictions in the K+ rates of the root, stem, and mulation (P < 0.05). In the present study, with increments of salt
petiole of IAC 313 and SO4 (Fig. 7C, E, and G). The grapevines that application, SO4 significantly showed lower sodium accumulation
showed negative accumulation rates, for example, in the petiole, stem, (17.4%).
and root of SO4 with 50 and 100 mM NaCl, and the root of IAC 313
(100 mM NaCl) indicate loss of K+ in the plants over the days when 3.5. Enzymatic, non-enzymatic activity and lipid peroxidation
compared to the plants collected at the beginning of the experiment.
In the present study, the daily Na+ accumulation rate in the leaf, Salinity gradually reduced the soluble protein content in IAC 313 and
petiole, and root showed an intense ion accumulation during the SO4 grapevines (Fig. 8A). There was no significant difference between
experiment in the plants on both rootstocks when exposed to 50 and IAC 313 and SO4 in protein content under 50 mM NaCl and CAT activity
100 mM NaCl (Fig. 7B, D and H). In addition, IAC 313 exhibited a higher in control plants (Fig. 8A–B). In IAC 313, salinity inhibited protein by an
sodium accumulation rate in the root, gradually increasing each average of 25.4% and 22.4% in SO4. Protein content at 100 mM NaCl
grapevine when salinity was increased. Under moderate (50 mM NaCl) increased by 19.6% in SO4 plants compared to IAC 313. The highest
and high (100 mM NaCl) salinity, SO4 showed greater Na+ accumula­ activities of CAT and APX enzymes were more pronounced at 100 mM
tion in the leaves, 118.1% higher than IAC 313, with 50 mM NaCl. NaCl than their respective corresponding values for the control and
Similarly, for SO4 with 100 mM NaCl, the accumulation was 41.4% 50 mM NaCl treatments (Fig. 8B–C). We observed a significant differ­
higher than IAC 313 (Fig. 7B). Interestingly, despite the petiole being ence in CAT activity under 50 and 100 mM NaCl, being greater in SO4.
part of the leaf structure, in the different treatments, there were varia­ The highest level of salinity increased CAT activity (Fig. 8B) by 13.5% in
tions and an increase in sodium accumulation (Fig. 7D). The results SO4 compared to IAC 313 (P < 0.05). APX activity increased signifi­
showed that the petiole of the plants has an increase in the Na+ accu­ cantly when plants were exposed to salinity (Fig. 8C), being 187.2% and
mulation capacity with the increase in salinity. However, the application 217.1% with 50 and 100 mM NaCl for IAC 313 compared to its control
of Na+ had a more expressive effect, especially on IAC 313. There was no plants, respectively. In SO4, it was 189.1% (50 mM NaCl) and 322.4%
(100 mM NaCl) compared to the control activity. When compared be­
tween the grapevines with 50 mM NaCl, the APX activity in IAC 313 was
Table 2 superior to SO4 in 56.9%. This was similar to the 100 mM NaCl treat­
Sodium (Na+), potassium (K+) and chloride (Cl− ) contents in roots, stems, pet­ ment, with an 18.6% increase in APX activity for IAC 313.
ioles and leaves of ‘BRS Vitória’ grapevine cultivar grafted on IAC 313 and SO4 There was a significant effect of salinity on lipid peroxidation,
rootstocks subjected to salinity (0, 50 and 100 mM NaCl) during 30 days grown
expressed in MDA content, where the salinity of 100 mM NaCl increased
in greenhouse.
peroxidation by 57.2% of IAC 313 compared to SO4 in the same con­
Rootstock NaCl Root Stem Petiole Leaf dition (Fig. 8D). Furthermore, there was no difference between salinity
(mM)
Na+ (µmol g− 1
DM) levels for SO4, whereas IAC 313 was significantly elevated. Similar to
IAC 313 0 89.14 Ac 57.30 Ac 191.02 Bc 41.38 Ac
the results of lipid peroxidation, our findings indicate an increase in GSH
50 595.35 Ab 130.53 Ab 1400.83 Ab 238.77 Bb levels with NaCl application (Fig. 8E). SO4 with 50 and 100 mM NaCl
100 815.02 Aa 203.75 Aa 1572.75 Aa 445.71 Ba showed the highest GSH values, being 33.9% and 102.6%, respectively,
SO4 0 82.77 Ac 50.94 Ac 324.74 Ab 22.28 Ac higher than IAC 313 under the same conditions. Clearly, the grapevines
50 334.29 Bb 136.89 Ab 1432.66 Aa 445.71 Ab are different, as there was a difference between them in the control
100 646.28 Ba 219.67 Aa 1413.56 Ba 573.06 Aa treatment (P < 0.05).
K+ (µmol g− 1
DM) Total ascorbate content levels showed no difference between salinity
IAC 313 0 523.26 Aa 203.49 Aa 1098.19 Aa 510.34 Ab
levels and the two grapevines (Table 3). However, reduced ascorbate
50 478.04 Aab 148.58 Ab 729.97 Ab 684.75 Ba (ASC) in grapevine leaves increased with salinity. Despite the high ASC
100 423.13 Ab 184.11 Aab 562.02 Ac 633.07 Ba values in IAC 313 with 50 and 100 mM NaCl, there was no significant
SO4 0 419.90 Ba 232.56 Aa 1201.55 Aa 520.03 Ab difference. On the other hand, when comparing IAC 313 with SO4 in
50 316.54 Bb 145.35 Ab 729.87 Ab 823.64 Aa 100 mM NaCl, IAC 313 was 70.4% higher in ASC content. The oxidized
100 319.77 Bb 164.73 Ab 600.77 Ab 901.16 Aa ascorbate (DHA) values were reduced with saline stress, varying be­
Cl− (µmol g− 1
DM) tween 5.47 and 11.74 µmol g− 1 FW and showing no significant differ­
ence between the grapevines at the same salinity level. The ascorbate
IAC 313 0 204.16 Ac 29.16 Ab 128.33 Ac 11.66 Ac
50 877.91 Ab 110.83 Ab 840.00 Bb 542.50 Bb redox state [ASC/(ASC + DHA)] increased with salinity in IAC 313 and
100 1137.50 Aa 221.66 Aa 1295.00 Aa 1120.00 Aa SO4, with the highest value reached in IAC 313 at 100 mM NaCl
SO4 0 350.00 Ab 23.33 Ab 210.00 Ab 29.16 Ac (43.53%). Interestingly, SO4 under 100 mM NaCl was reduced by 20.8%
50 775.83 Aa 189.58 Aa 1114.16 Aa 950.83 Ab compared to 50 mM NaCl.
100 974.16 Ba 192.50 Aa 1295.00 Aa 1201.66 Aa

Data are presented as means of three replications (n = 3). For each parameter, 3.6. Multivariate statistical analysis of experimental parameters
different uppercase letters compare the two rootstocks at each salt concentra­
tion, and different lowercase letters compare the same rootstock at all salt Fig. 9 shows the principal components 1 and 2 (i.e., PC1 and PC2,
concentrations indicating significant differences by Tukey’s test (P < 0.05). respectively) obtained from the two rootstocks IAC 313 and SO4

9
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

Fig. 7. Changes in the K+ and Na+ accumulation rate (μmol g− 1 DM day− 1) in different grapevine (IAC 313 and SO4) parts under salinity stress (0, 50, and 100 mM
NaCl). Error bars show the mean ± SD of three replicates. Means followed by different letters are significantly different (Tukey’s test, P < 0.05). The uppercase letters
compare the means of two rootstocks at each salt concentration, whereas the lowercase letters compare the same rootstock at all salt concentrations.

Fig. 8. Effect of NaCl stress (0, 50 and 100 mM) on soluble protein content [μg g− 1 DM] (A), activity of catalase [CAT, μmol H2O2 μg− 1 protein min− 1] (B), ascorbate
peroxidase [APX, nmol ASC μg− 1 protein min− 1] (C), malondialdehyde [MDA, nmol g− 1 FW] (D) and reduced glutathione [GSH, μmol g− 1 FW] (E) in plants of
grapevine cultivar ‘BRS Vitória’ on the rootstocks IAC 313 and SO4 during 30 days in greenhouse conditions. Error bars show the mean ± SD of three replicates.
Means followed by different letters are significantly different (Tukey’s test, P < 0.05). The uppercase letters compare the means of two rootstocks at each salt
concentration, whereas the lowercase letters compare the same rootstock at all salt concentrations.

subjected to three levels of salinity (0, 50 and 100 mM NaCl). The
dataset with 31 variables resulted in four principal components (PC)
with eigenvalues greater than 1.0. Here, we have summarized the
analysis into just two principal components due to the biplot’s presen­
tation and the results’ relevance (Fig. 9A–B). The first two principal
components explained 87.91% of the total data variance (Fig. 9A),
where PC1 was responsible for 74.15%, and PC2 explained 13.76%. Our
PCA results indicate that there was a clear separation between IAC 313
(lower negative/positive quadrants) and SO4 (upper positive/negative
quadrants). This ordering of the PCA characterizes and separates the
variables in terms of salinity levels and rootstock type in the sampling
period. Inserted in PC1, treatments with 100 mM NaCl for IAC 313 and
SO4 grapevines showed higher positive scores, with values of 1.56 and
1.47, respectively. Both grapevine cultivars in their control treatments

10
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

Table 3 metabolic processes to cope with adverse factors. In this study, the effect
Contents of reduced (ASC) and oxidized (DHA) forms of ascorbate and the of salinity stress resulted in lower growth and development of both the
ascorbate redox state [ASC/(ASC + DHA) ratio] in leaves of grapevine plants of shoot and the roots of the grapevines, which is consistent with changes
‘BRS Vitória’ cultivar grafted on the IAC 313 and SO4 rootstocks grown at three in the analyzed photosynthetic parameters [50,64]. Photosynthesis
NaCl salinity levels (0, 50, and 100 mM) for 30 days in the greenhouse. limitations can affect plant growth and the yield of crops; this occurs
Rootstock NaCl Total ascorbate ASC DHA Redox state mainly due to stomatal and non-stomatal disturbances [1,69]. The salts
(mM) (ASC þ DHA) present in the soil solution can cause a negative effect due to toxic ions
(µmol g− 1 FW) (%)
that lead to the interference of root function in the absorption of water,
0 12.32 Aa 1.23 Ab 11.09 Aa 9.98 Ac as well as nutrients. The roots, for example, are responsible for con­
IAC 313 50 11.23 Aa 3.08 Aa 8.15 Aab 27.43 Ab
trolling the absorption and translocation of nutrients throughout the
100 9.11 Aa 3.64 Aa 5.47 Ab 39.96 Aa
0 12.92 Aa 1.18 Ab 11.74 Aa 9.13 Ab plant’s life cycle. However, studies have shown that, despite direct
SO4 50 10.63 Aa 2.60 Aa 8.02 Aa 24.46 Aa exposure to the saline environment, the roots are less vulnerable to
100 11.05 Aa 2.14 Ba 8.91 Aa 19.37 Bab growth when compared to the aerial parts of the plant (e.g., [4,16,44]).
Data are presented as means of three replications (n = 3). For each parameter, Biomass allocation and plant growth were reduced due to the use of
different uppercase letters compare the two rootstocks at each salt concentra­ water with different saline concentrations; this behavior is considered
tion, and different lowercase letters compare the same rootstock at all salt one of the most sensitive physiological responses manifested by plants.
concentrations indicating significant differences by Tukey’s test (P < 0.05). These results are similar to the studies by Martin et al. [51] and Liu et al.
[48], who submitted grapevine plants to saline irrigation water signifi­
showed a high negative score in PC1 (Fig. 9A). High positive loading cantly inhibited their biomass and growth. Sivritepe et al. [67] inves­
vectors (>0.5) are observed for APX, AsA/DHAR, DHAR, stem dry tigated the biomass of grapevines under salinity stress (2.7 and 5.45 dS
weight, Na+ in root, and Cl− in stem and root of IAC 313 under 100 mM m− 1) and found a reduction in biomass and partitions (shoots and roots)
NaCl. Likewise, the variables associated with treatments with SO4 (50 due to salinity.
and 100 mM NaCl) were CAT, Cl− , and Na+ in the leaf (loadings > 0.6) In plants subjected to salt stress, leaf damage is recurrent, affecting
and K+ in the leaf with a loading of 0.5. their production and quality. Excess salts in the soil solution can cause
In addition, IAC 313 (0 mM NaCl) had a higher negative PCA score dehydration in the leaves, leading to the appearance of chlorosis
(− 1.97). This shows higher results for K+ in the root, Ci, E, PN, gS, root symptoms, necrosis, and premature senescence [7,75]. In addition,
dry weight, and K+ in the petiole of these plants, with their respective excess sodium can interfere with the absorption of other nutrients, such
loading values, − 0.40, − 0.53, − 0.55, − 0.56, − 0.56, − 0.57 and as potassium and magnesium, which results in a reduction in chlorophyll
− 0.62 (Fig. 9B). On the other hand, SO4 in the same saline condition production and, consequently, in photosynthetic efficiency [1,64]. In
presented a score of − 1.86 (PC1) and high factor loadings, being this work, with the increase in salinity levels, the presence of foliar
grouped by the variables DHA (− 0.55), AsA (− 0.56), Chlb (− 0.59), damage was observed in the plants through visual diagnosis carried out,
protein (− 0.59), Chlt (− 0.60) and Chla (− 0.60). Clearly, plants with where we verified the occurrence of toxicity since the plants presented
high WUE have lower K+ in the roots (inverse relationship between dryness and foliar necrosis, in addition to burning on the edges of the
vectors) and higher K+ content in the leaves, as observed in SO4 in leaves (Fig. 3).
contrast to IAC 313 when exposed to salinity. Plants under high salinity Our results support findings previously reported by Hannachi et al.
increased leaf K+, and this caused an inverse relationship between [24], who found symptoms of chlorosis and foliar toxicity with the
vectors (variables) of gas exchange. application of NaCl at 40, 80, and 160 mM in two cultivars of Solanum
melongena (L.). They reported severe leaf damage of 70% and 100% at
4. Discussion 15 and 21 days, respectively, after applying 80 and 160 mM NaCl. These
characteristics were also reported by Sohrabi et al. [68], evaluating six
Under abiotic stress conditions, plants allocate energy in various types of grapevines with salinity levels ranging from 0 to 100 mM. In

Fig. 9. Principal component analysis (PCA) of the measured variables of two rootstocks (IAC 313 and SO4) grown at three salinity levels (0, 50 and 100 mM NaCl).
Biplots with PCA scores (A) and loadings of the first two principal components (B).

11
E.F. Silva et al.
addition, another factor contributing to leaf damage is the reduction in
the activity of enzymes involved in photosynthesis, which can decrease
the production of ATP and NADPH, which are important for maintaining
leaf integrity. According to Capitulino et al. [10], salt stress can interfere
with protein synthesis, and in grapevines, the harmful effects of salinity
can trigger several symptoms [68].
Photosynthesis is a complex process in plants, which allows energy
production through light absorption. However, photosynthesis can be
significantly affected due to the duration and intensity of salt stress. In
this study, long-term exposure of plants to salinity (i.e., 30 days)
impacted the reduction of net photosynthesis in IAC 313 and SO4
grapevines (Fig. 4). In the short term (i.e., 15 days) of salt stress (80 mM
NaCl), Silva et al. [64] found no difference in net photosynthesis results
compared to the control treatment in IAC 313 grapevine plants
(10.45 µmol m− 2 s− 1). This demonstrated that exposure time and
salinity level are crucial in understanding the photosynthetic perfor­
mance of grapevine since, in 30 days, the PN of IAC 313 was
9.08 µmol m− 2 s− 1 (50 mM NaCl) and 3.75 µmol m− 2 s− 1 (100 mM
NaCl). These results are consistent with other findings previously re­
ported in grapevine cultivars [16,48,49,57]. Furthermore, high salt
concentrations in soil and plant tissues can cause osmotic stress, ionic
toxicity, and oxidative damage, which leads to inhibition of photosyn­
thesis and reduced plant growth. These stressors can trigger the pro­
duction of ROS in chloroplasts, causing damage to the membrane
through the breakage of double bonds in unsaturated fatty acids. This
damage, in turn, results in the release of chlorophyll from thylakoids.
The photosynthetic pigments in the leaves referring to chlorophylls
a, b, and total are in some cases capable of limiting photosynthesis rates
in grapevine [39]. This could be due to the photodynamic action of
chlorophylls when they are not efficiently and regularly degraded. Here,
we show that stress influenced the reduction of Chla content by 22.73%
and 40.36% in IAC 313 and SO4, respectively (Fig. 3). This decrease is
associated with oxidative damage to chloroplast membranes, chloro­
plast metabolism, and leaf anatomy due to salinity or increased chlor­
ophyllase enzyme activity [13,73]. Similar behavior was reported in the
study by Zahedi et al. [73] when investigating the chlorophyll content in
grapevines under 0, 50, and 100 mM NaCl salinity. They reported a
significant reduction of 42.2% in Chla content in ‘Sultana’ grapevines.
Our results for chlorophylls (i.e., Chla, Chlb, and total Chl content) are
also in agreement with those reported by Liu et al. [48], who found
values of 0.88–1.37 mg g− 1 FW (Chla), 0.34–0.55 mg g− 1 FW (Chlb) and
1.21–1.91 mg g− 1 FW (total Chl) using saline water in ‘Summer Black’
grapevines.
The gas exchanges parameters of grapevines were negatively
affected by the accumulation of toxic ions in the roots and leaves (see
Fig. 4 and Table 3). There is increasing evidence that PN and gS in
grapevines are not only compromised by stomatal closure but also by
lower leaf K+ content [3,51]. This hypothesis confirms our findings. In
more detail, we observed that grapevine plants accumulate a higher K+
content in the petiole (0 mM NaCl), which is a constituent of the entire
leaf (leaf blade and petiole/midrib tissue). However, when exposed to
salt stress, K+ levels in the petiole decreases while increasing in the leaf
(Fig. 9). Although this K+ increase gradually increases in the leaf,
photosynthesis abruptly declined with the application of 100 mM NaCl.
We believe that salinity had a synergistic effect on the accumulation of
Na+ and Cl− in the plant, which compromised photosynthesis [3].
Elevated Cl− levels in the plant were more associated with salt-treated
plants, and the high concentration of Cl− in cell organelles hindered
essential plant metabolic processes [34]. Net photosynthesis, transpi­
ration rate, stomatal conductance, and intercellular CO2 concentration
of grapevine plants were affected by increasing NaCl [3,48,57].
It is well known that salinity stress can reduce the soil water potential
and compromise the osmotic potential of the plant, which leads to the
accumulation of toxic Na+ and Cl− ions in apoplast of root cells, as well
as in the aerial part. Under these conditions, the roots lose their ability to
uptake water and nutrients, leading to the plants’ inability to meet their
12
Current Plant Biology 37 (2024) 100316
transpiration demands. In our study, plants grown under salinity of 50
and 100 mM showed variations in water potential between − 0.6 and
− 1.1 MPa. Other studies have also reported similar predawn leaf water
potential results with grapevines in response to salt treatment, being
greater than − 0.8 MPa [64] to − 0.93 MPa [4] and rising after noon
from − 1.1 MPa [51] to − 1.5 MPa [52]. To survive these adverse
conditions, plants develop salt tolerance controls, such as the ability to
absorb ions from the environment and store them in specific cells (e.g.,
vacuoles) to minimize their negative effects. However, further in­
vestigations are needed to elucidate the relationship between specialist
cells to circumvent salinity damage in grapevines.
In the face of the challenges posed by environmental stresses, certain
measures can elucidate the response of plants. That said, photochemical
efficiency parameters such as chlorophyll fluorescence and electron
transport rate (ETR) are considered reliable parameters for assessing
plant photosynthesis and heat dissipation (non-photochemistry).
Furthermore, alterations in the ETR can negatively affect the availability
of electron acceptors, such as NADP+, and the utilization of ADP,
resulting in limitations in the regeneration of ribulose-1,5-bisphosphate.
In the primary processes of photosynthesis, the proper ionic balance in
the cytosol and chloroplasts is a crucial factor in regulating the
biochemical machinery [54]. In this study, we observed that the
photochemical parameters showed severe damage in extreme saline
treatment (see Fig. 4 and Fig. 5). The decrease in chlorophyll fluores­
cence under salinity occurs due to decreased photosynthetic efficiency
[35,70]. This was clearly seen in the gradual reduction of photochemical
quenching (qP), which is indicative of the efficiency of photosynthesis.
Similar results were reported in grapevines with salt stress, exhibiting
more expressive reductions in qP and ETR due to salt [3,55].
The decline in these photosynthetic parameters may be linked to
stomatal and diffusion limitations of CO2 influx to the active site of
Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase); this fact
enables saturation and imbalance of the electron transport chain, which
causes excessive production of ROS [2]. The NPQ values showed a
higher effect (maximum of 4.03) on the SO4 rootstock when subjected to
severe saline stress (100 mM NaCl), while at the same salinity level, the
IAC 313 was lower (maximum of 2.83). Numerous studies have shown
that NPQ can increase or decrease under stress conditions (e.g., [5,46]).
An increase in NPQ reflects the self-protection of the photosynthetic
machinery and is used as an indicator of excess dissipation of light en­
ergy in the form of heat in the antenna complex of PSII [46]. In contrast,
the reduction of the NPQ in adverse conditions refers to the decline in
the heat dissipation capacity, which indicates that the amount of light
energy is not being absorbed due to the imbalance in the antenna
complex. Askri et al. [5] reported a 181% increase in NPQ for
salinity-sensitive grapevines (150 mM NaCl) and reductions of approx­
imately 27% and 65% in NPQ for tolerant grapevines.
The accumulation of Na+ and Cl− in the various grapevine tissues is
influenced by choice of rootstocks [51]. Furthermore, these ions in
plants under salt stress may adversely affect water uptake and chloro­
phyll production. When there is an excess of Na+ in the soil, potassium
entry into the roots can be blocked, which affects the activity of enzymes
involved in photosynthesis, damaging cell membranes [23]. Our study
showed a significant increase in Na+ content in roots, stems, petioles and
leaves in all scion/rootstock combinations in response to increased
salinity (see Table 3 and Fig. 7). This reflected greater sensitivity due to
the apparent lack of Na+ restriction and allocation in grapevine plants.
The excessive increase of toxic ions such as Na+ occurs due to ionic
imbalances caused by salinity, so the K+/Na+ ratio decreases [3,64].
Despite the compartmentalization of toxic ions, Na+ accumulation can
lead to plant cell dehydration and interfere with sap conduction, which
can result in irreparable damage to leaves and plant growth. On the
other hand, potassium is crucial for maintaining cellular homeostasis
and efficient photosynthesis. Our results suggest that grapevine petiole
cells have the ability to help control salinity, although this hypothesis
needs further investigation in the future.
E.F. Silva et al.
Under salinity conditions, the reduction in protein content may be
due to the inhibition of protein synthesis, as well as the denaturation of
enzymes. In plants, when high levels of H2O2 are detected under saline
stress, the species can trigger lipid peroxidation and subsequently
damage the membrane. Salinity promotes increased CAT activity be­
tween 0.33 and 1.04 μmol H2O2 μg− 1 protein min− 1 (Fig. 8). Gohari
et al. [19] also reported increased CAT activity in grapevines with
100 mM NaCl (0.1 to 0.15 U g− 1 FW). A key role of antioxidant enzymes
is to rid free radicals present in plant cells, which can cause loss of
photosynthetic yield and biomass. In addition, APX is closely linked to
eliminating reactive oxygen species. In the chloroplast region, APX plays
a crucial role in eliminating reactive oxygen species, mainly due to the
absence of CAT in the chloroplast. APX also eliminates H2O2 in the
ascorbate-glutathione cycle, which is necessary for cell detoxification.
Comparing the activity of the two enzymes APX and CAT, it was
observed that CAT has substantially greater activity in sequestering
H2O2. These results corroborate the findings by Bari et al. [6], who
investigated grapevine rootstocks under salinity treatments of 0, 25, 50
and 100 mM NaCl.
When high levels of H2O2 are detected under saline stress, plants can
trigger lipid peroxidation and damage the membrane. Products pro­
duced by lipid peroxidation, such as those reacting with thiobarbituric
acid, are important parameters for monitoring damage caused by reac­
tive oxygen species. This study observed an increase in MDA content
with increasing treatment with a saline solution (Fig. 8D). These results
imply oxidative damage caused by excess salt, mainly to the membrane
through lipid peroxidation, measured by the MDA content, which varied
between 531.62–901.16 nmol g− 1 FW (IAC 313) and 562.38–573.18
nmol g− 1 FW (SO4). Such findings were previously confirmed by
Amorim et al. [3]. In this way, the accumulation and imbalance of ROS
production in plant tissues can lead to these and other physiological,
metabolic and biochemical changes [18]. In saline conditions, as seen in
our results, the increase in reduced glutathione may be due to the better
performance of plants in tolerating stress because reduced glutathione is
important in protecting ROS in plants under stress conditions [40].
Ascorbate is an important non-enzymatic antioxidant for plants,
acting as a cofactor for many enzymes, and it also interacts (enzymati­
cally and non-enzymatically) in the detoxification of reactive oxygen
species. In plants, a key process for maintaining cell redox status is the
regeneration of ascorbate (ASC) and glutathione (GSH) contents in the
ascorbate-glutathione cycle, also known as the Foyer-Halliwell-Asada
pathway. For the removal of intracellular H2O2, APX utilizes ascorbate
as an electron donor. In conjunction with ascorbate, APX, along with the
enzymes glutathione reductase (GR), dehydroascorbate reductase
(DHAR), and monodehydroascorbate reductase (MDHAR), can maxi­
mize H2O2 removal through efficient regeneration of reduced ascorbate.
Our results indicate that grapevine genotypes have broad changes in
redox balance behavior (Table 3). Ascorbate stores can be depleted by
oxidative stress. Furthermore, oxidized ascorbate can be accumulated
from monodehydroascorbate (MDHA) disproportion or oxidation of
reduced ascorbate. Salt stress affected reduced ascorbate levels under
high salinity. This effect resulted in an increase in the ascorbate redox
state in plants subjected to high salinity levels. In grapevine, Ikbal et al.
[31] observed changes in ascorbate contents when salinity was
increased; another key point is that ascorbate contents can change not
only by salt application but also depending on the structural part of the
plant [8].
Our PCA results clearly showed a negative effect of salinity on
chlorophyll content, leaf and root weight, photosynthesis and gas ex­
change in grapevines. Although we showed a positive linear relationship
with the Na+ and Cl− variables in IAC 313 with 100 mM NaCl, this
grapevine under salinity has better photosynthetic activity under
stressful conditions (Fig. 9). The positive correlation of APX with plant
structures with higher levels of Na+ and Cl− confirms the better effi­
ciency of IAC 313 in controlling ROS. Amorim et al. [3] also reported
better physiological and photochemical efficiency in IAC 313 under
13
Current Plant Biology 37 (2024) 100316
salinity. We believe that this rootstock has less damage to the photo­
synthetic electron transport chain, which does not significantly
compromise the Calvin cycle reactions. Utilizing PCA, Grzesiak et al.
[22] observed that tolerant genotypes were plotted in the positive
quadrant next to promising stress traits, as seen in IAC 313 in the present
study. They concluded that physiological parameters linked to the use of
PCA generate adequate information to study the complex resistance
mechanisms of plants.
5. Conclusion
The data from the present study indicate that the rootstocks showed
different physiological behaviors under salt stress, revealing the
complexity of physiological interactions between scion and rootstock
genotypes. In general, it can be concluded that the ‘BRS Vitória’/IAC
313 combination showed better growth capacity, dry mass accumula­
tion, associated to lower Na+ accumulation in leaf tissues, and higher
Na+ content in stem and root tissues. This clarified that this rootstock
has retention/exclusion mechanisms for toxic ions, which is one of the
salt tolerance mechanisms. The grafted plant combination most salt-
sensitive was ‘BRS Vitória’/SO4. We observed that the grapevines
exhibited considerable variations in Na+ and K+ contents, root and shoot
growth performance, as well as osmotic potential and gas exchange.
After 30 days of salinity exposure, IAC 313 and SO4 grapevines
exhibited an increase in WUE due to stomatal regulation and a decline in
transpiration. Furthermore, increased lipid peroxidation indicated
oxidative damage in grapevine leaves subjected to salt stress. According
to the results of ‘BRS Vitória’/IAC 313, the higher APX activity and,
consequently, the increased GSH content was directly influenced by the
Ascorbate/Glutathione cycle. Future research with an increase in the
salinity level and time of exposure to stress in ‘BRS Vitória’/IAC 313
grapevines is necessary to understand the high level of tolerance of the
crop and its physiological responses. Additionally, we highly encourage
the application of climate models and biophysical parameters for a
comprehensive analysis of climate change within grapevine-producing
regions that face arid and semi-arid conditions. This urgency arises
from the realization that climate change may hasten the onset of soil
salinity issues and pose a significant threat to crop productivity in such
areas.
CRediT authorship contribution statement
Elania Freire da Silva: Formal analysis, Investigation, Performed
the experiment, Validation, Visualization, Writing – original draft. Hugo
Rafael Bentzen Santos: Methodology, Writing – review & editing,
Validation. Jean Pierre Henry Balbaud Ometto: Funding acquisition,
Project administration, Conceptualization, Review. Alexandre Man­
içoba da Rosa Ferraz Jardim: Data curation, Investigation, Validation,
Visualization, Writing – original draft. Thieres George Freire da Silva:
Methodology, Review and editing. Pedro José Hermínio: Methodol­
ogy, Performed the experiment, Writing – review & editing. Adriano
Nascimento Simões: Methodology, Writing – review & editing.
Eduardo Souza: Methodology, Writing – review & editing. Sérgio Luiz
Ferreira-Silva: Conceptualization, Writing – review & editing, Funding
acquisition, Project administration, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
E.F. Silva et al. Current Plant Biology 37 (2024) 100316

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