© The Authors, 2024, Published by the Universidad del Zulia*Corresponding author:raaniaahmed@arc.sci.eg
Keywords:
Grape
Salinity
Recovery
Genotypes
Adaptive Mechanisms During the Recovery of Tolerant and Sensitive Local Grape Genotypes
Subjected to Salt Stress
Mecanismos adaptativos durante la recuperación de genotipos de uvas locales tolerantes y sensibles
sometidos a estrés salino
Mecanismos adaptativos durante a recuperação de genótipos de uva locais tolerantes e sensíveis
submetidos ao estresse salino
Rania Mahmoud
1*
Fouad Ashry
2
Roqaya Shallan
2
Rev. Fac. Agron. (LUZ). 2024, 41(3): e244124
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v41.n3.IV
Crop production
Associate editor: Dr. Jorge Vilchez-Perozo
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Deciduous Fruits Research Department, Biotechnology
Central Lab, Horticulture Research Institute (HRI),
Agricultural Research Center (ARC), Egypt.
2
Faculty of Biotechnology, October University for Modern
Sciences and Arts, Egypt.
Received: 23-05-2024
Accepted: 22-07-2024
Published: 06-08-2024
Abstract
Utilization of distinct genetic resources is an auspicious
prospective strategy to combat adverse impacts of salinity, which
is expected to get worse under climate change conditions, for
maintaining grape production and quality. This research aims to
study the adaptive mechanisms during the recovery of tolerant
and sensitive salt-stressed local grape genotypes on the bases
of biochemical, anatomical and gene expression responses.
Transplants of three Egyptian grapes (Vitis vinifera); Baltim Eswid,
Romy Ahmer and Romy Abiad, were exposed to sodium chloride-
induced salt stress of 2.28 and 3.75 mS compared to 695 µS water-
irrigated control for two months, then all plants were irrigated
with tap water for additional one month for recovery. Recovered
Baltim Eswid cultivar following the highest saline treatment
gave maximum survival percentage (100 %), while Romy Abiad
recorded the lowest rate (40 %). Suggested adaptive mechanisms
include: damage reduction caused by salinity-related oxidative
stress, osmotic adjustment, and perform structural modications
that allow protection. It was concluded that, Blatim Eswid is a
superior salt-tolerant local grape genotype, while Romy Abiad is
the most sensitive as aected mostly by oxidative stress represented
by a signicant increment of hydrogen peroxide content.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2024, 41(3): e244124 July-September. ISSN 2477-9407.
2-6 |
Resumen
La utilización de distintos recursos genéticos es una estrategia
prospectiva auspiciosa para contrarestar los impactos adversos de
la salinidad, que se espera que empeore bajo las condiciones del
cambio climático, para mantener la producción y la calidad de la
uva. Esta investigación tiene como objetivo estudiar los mecanismos
adaptativos durante la recuperación de genotipos de uva locales
tolerantes y sensibles al estrés salino sobre bases de respuestas
bioquímicas, anatómicas y de expresión genética. Trasplantes de tres
uvas egipcias (Vitis vinifera); Baltim Eswid, Romy Ahmer y Romy
Abiad fueron expuestos a un estrés salino inducido por cloruro de
sodio de 2,28 y 3,75 mS en comparación con el control regado con
agua a 695 µS durante dos meses, luego todas las plantas se irrigaron
con agua del grifo durante un mes adicional para su recuperación. El
cultivar Baltim Eswid recuperado después del tratamiento salino más
alto dio el porcentaje máximo de supervivencia (100 %), mientras
que Romy Abiad registró la tasa más baja (40 %). Los mecanismos
adaptativos sugeridos incluyen: reducción de daños causados por
estrés oxidativo relacionado con la salinidad, ajuste osmótico y
realizar modicaciones estructurales que permitan la protección.
Se concluyó que Blatim Eswid es un genotipo de uva local superior
tolerante a la sal, mientras que Romy Abiad es el más sensible ya que
se ve afectado principalmente por el estrés oxidativo representado por
un incremento signicativo del contenido de peróxido de hidrógeno.
Palabras clave: uva, salinidad, recuperación, genotipos.
Resumo
A utilização de recursos genéticos distintos é uma estratégia
prospectiva auspiciosa para combater os impactos adversos da
salinidade, que deverá piorar sob as condições das alterações
climáticas, para manter a produção e a qualidade da uva. A pesquisa
atual visa estudar os mecanismos adaptativos durante a recuperação
de genótipos de uva locais tolerantes e sensíveis ao estresse salino
com base em respostas bioquímicas, anatômicas e de expressão
gênica. Transplantes de raiz própria de três uvas egípcias (Vitis
vinifera); Baltim Eswid, Romy Ahmer e Romy Abiad, foram
expostos ao estresse salino induzido por cloreto de sódio de 2,28 e
3,75 mS em comparação com o controle irrigado com água de 695 µS
por dois meses, depois todas as plantas foram irrigadas com água da
torneira por mais um mês para recuperação. A cultivar Baltim Eswid
recuperada após o tratamento salino mais elevado proporcionou a
percentagem máxima de sobrevivência (100 %), enquanto Romy
Abiad registou a taxa mais baixa (40 %). Os mecanismos adaptativos
sugeridos incluem: redução de danos causados pelo estresse oxidativo
relacionado à salinidade, ajuste osmótico e realização de modicações
estruturais que permitam proteção. Concluiu-se que Blatim Eswid é
um genótipo de uva local tolerante ao sal superior, enquanto Romy
Abiad é o mais sensível, pois é afetado principalmente pelo estresse
oxidativo representado por um aumento signicativo no teor de
peróxido de hidrogênio.
Palavras-chave: uva, salinidade, recuperação, genótipos.
Introduction
Grapes (Vitis vinifera L.) are one of the most widespread fruit
crops worldwide. In 2017, Mirás-Avalos and Intrigliolo noted that
climate change imposes constraints on grape production, which are
expected to worsen with rising temperatures, less precipitation, and
more frequent heatwaves. Anyway, the most insistent challenges
facing the grapevine, especially in arid and semi-arid regions, are the
increased risk of drought and salinity due to high evaporation rate and
water scarcity. Considering the fact that, diversity of Vitis spp. genetic
resources is certainly a main factor that reinforced the historical
prevalence of grapevine cultivation around the world, so selecting
the most proper plant materials is the principle underlying long-term
resilience strategies for viticulture under the inuence of a changing
climate (Pastore et al., 2022). There is a limited number of publications
that have focused on evaluating comparative salt tolerance and
recovery traits among diverse grape genotypes, especially local ones,
which is important both for adequate cultivars selection, as well as
for improving and developing superior grapevines through breeding
eorts. Accordingly, current research aims to study the adaptive
mechanisms during recovery of tolerant and sensitive salt-stressed
local grape genotypes on bases of biochemical, anatomical and gene
expression responses.
Materials and methods
Plant materials and experimental design
A pot culture experiment was done under shade net house
conditions at Horticulture Research Institute experimental orchard.
The experiment was repeated twice during 2022 and 2023 successive
seasons to conrm the results obtained. Three Egyptian genotypes of
grapes (Vitis vinifera L.) were selected for evaluation: Baltim Eswid
which is a blue-black grape traditionally grown on the hillsides of
Egypt’s northern coast under rainfed conditions, Romy Ahmer red
grape as one of the most common local cultivars, and Romy Abiad
white-green grape that is classied as salt sensitive genotype based
on morpho-physiological responses (Mahmoud et al., 2023). Six
months old own rooted healthy transplants of the three tested
genotypes, planted in polyethylene bags lled by 1:2 (v/v) mixture
of peatmoss and washed sand, were exposed to sodium chloride-
induced salt stress of 2.28 mS (low salt treatment) and 3.75 mS (high
salt treatment) compared to 695 µS tap water-irrigated control for
two months (Mahmoud et al., 2023), then all plants were irrigated
with tap water (695 µS) for another month to recover. A complete
randomized blocks design was applied for arranging experimental
treatments. Three replicates were sampled from each treatment, and
each replicate included four symmetrical transplants. Survival rate
(%) was calculated as a percent of survivors count versus the total
number of treated plants, thereafter, leaf samples were collected
to perform the required analysis. Leaf samples of seedlings that
recovered after being exposed to the highest level of salt stress were
subjected to anatomical and gene expression analysis compared to the
corresponding control for each genotype.
Biochemical analysis
Content of total phenols was measured with Folin-Ciocalteu’s
reagent (Singleton & Rossi, 1965). The ferric reducing antioxidant
power (FRAP) assay (Quisumbing, 1978) was applied for
determination of total antioxidant activity using ascorbic acid as
reference standard for calculation. Fresh leaf samples were subjected
to hydrogen peroxide (H
2
O
2
) quantication as described by Velikova
et al. (2000). The reducing sugars content was determined using the
3,5-dinitrosalicylic acid (DNSA) method (Miller, 1959). Quantitative
amino acids content was determined using the ninhydrin reaction
(Kowalska et al., 2022).
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Mahmoud et al. Rev. Fac. Agron. (LUZ). 2024 41(3): e244124
3-6 |
Leaves anatomical structure
Specimens were separated from the midrib region of the 4
th
upper
leaf on the main stem (Mahmoud et al., 2023). Anatomical analysis
following microtechnique procedures described by Nassar and El-
Sahhar (1998) were carried out at the Cairo University Research Park,
Faculty of Agriculture.
Gene expression analysis
Collected leaf samples were immediately frozen in liquid
nitrogen, then kept at -80°C for extraction of total RNA as explained
by Mahmoud et al. (2023). Dierential expression of two targeted
genes; VvChS & VvEDS1, and VvEF1-α as a reference gene
(Mohammadkhani et al., 2016) was conrmed by qPCR. Reverse
transcription was performed with “Thermo Scientic” RevertAid
First Strand cDNA Synthesis Kit. “Willowfort” HERA
PLUS
SYBR
®
Green qPCR Kit was used for all assays. Thermal prole was set as;
95 °C for 10 min, forty repeated cycles; 95 °C for 15 s, 58 °C for 1
min, and 72 °C for 20 s, nally a climb in increments of 0.05 °C from
58 to 95 °C for the high-resolution melting curve (Buesa et al., 2022).
Statistical analysis
Data were ANOVA statistically analysed as factorial randomized
block design and signicant variations were specied by obtained
values of L.S.D. at 0.05 (Snedecor & Cochran, 1989). The results
listed represent mean values of the two conducted experiments where
data of collected samples for both seasons were remarkably similar.
Results and discussion
Survivability
Low salt pretreatment (2.28 mS) had no eect on survival
percentage of grape seedlings during recovery (gure 1). The high
salt pretreatment (3.75 mS) signicantly decreased recovered grape
seedlings survivability of Romy Ahmer and Romy Abiad recording 60
and 40 %, respectively, while Baltim Eswid maintained the maximum
survivability of 100 %.
Often the response of growth to salinity is considered a basic
assessment of how tolerant genotype is, which reects on its
performance during recovery. Mahmoud et al. (2023) evaluated
nine grapevine varieties against salinity where Romy Abiad was
categorized as the most sensitive variety. Based on morpho-
physiological responses of the three tested local grape genotypes
under stress conditions, Romy Abiad was the least regarding leaves
count, moisture and relative water contents, while Romy Ahmer
recorded the highest root electrolyte leakage as aected by salt, with
no signicant eect on survivability up to 2,000 ppm NaCl for all
cultivars (Mahmoud et al., 2023). However, survival percentage
during recovery notably varied according to the cultivars under study
indicating limited ability of Romy Ahmer to recover after being
exposed to salt stress comparing to Baltim Eswid genotype which
is the most relatively salt tolerant. Baltim Eswid cultivar is a local
grapevine, that is being planted long time ago on the hill side of
the north coast of Egypt specically in Baltim city, Kafr El-Sheikh
governorate, which is now on the verge of extinction.
Biochemical responses
Antioxidant potential
Antioxidant potential of leaves, represented in content of total
phenols and quantied overall antioxidant activity, was reduced after
recovery following high salt treatment with the lowest mean values
of 8.64 and 7.25 mg.g
-1
, respectively (table 1). Generally, recovered
Romy Abiad genotype scored relatively lowest values of both total
phenols content and total antioxidant activity as compared with other
tested cultivars.
One of the key components associated with salt stress and eventual
damage is reactive oxygen species (ROS). Hence, synthesis of
antioxidants is another focal point of salt tolerance, which is a crucial
mechanism for ROS detoxication in grapevine (Mohammadkhani
& Abbaspour, 2017). Many phenolic compounds are stress-induced
metabolites in plants. Excessive accumulation of phenolics has been
reported to prevents cellular oxidative rupture by improving radical
scavenging activity. Soluble phenols contain electron-donating
mediators that provide their antioxidant capabilities, thus mitigating
additional ROS accumulation. This overproduction is likely stimulated
by promoting the phenylpropanoid pathway and enhancing phenyl-
aminolyase gene expression (El-Banna et al., 2022).
Oxidative stress indicator (H
2
O
2
)
Leaf H
2
O
2
content of recovered seedlings increased gradually
as aected by increasing level of salt stress pretreatment (gure 2).
Romy Abiad genotype scored signicantly highest content of H
2
O
2
(2.67 µg.g
-1
in average). Hydrogen peroxide (H
2
O
2
) is a relatively
long-lived molecule of reactive oxygen species (ROS) that is
enhanced in response to osmotic and ionic stresses. Mohammadkhani
et al. (2016) demonstrated that, low ROS level acts as an elicitor or
hormone-like substance with easy diusion properties that plays a
signicant role in signal transduction pathways stimulating systemic
responses in plants to salt stress which may aid salinity tolerance,
while an excessive ROS content, as for Romy Abiad genotype, could
cause oxidative damage and degradation of biopolymers including
cell wall polysaccharides and nucleic acids.
LSD 0.05 : Cultivars & Treatments = 8.81 , Interaction = 15.26
Figure 1. Survivability (%) of recovered local grape genotypes
following salt treatments.
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Rev. Fac. Agron. (LUZ). 2024, 41(3): e244124 July-September. ISSN 2477-9407.
4-6 |
LSD 0.05 : Cultivars & Treatments = 0.27 , Interaction = 0.47
Figure 2. Leaf hydrogen peroxide content (µg.g
-1
) of recovered
local grape genotypes following salt treatments.
Osmotic adjustment
Osmotic adjustment was expressed in leaf contents of reducing
sugars and total free amino acids (table 2). Upon recovery, the highest
leaf reducing sugars content mean value (2.73 g.100 g
-1
) was recorded
as aected by low salt pretreatment, while the lowest leaf total free
amino acids content mean value (0.09 g.100 g
-1
) was observed as
aected by high salt pretreatment. However, Romy Abiad genotype
attained lowest contents of both reducing sugars and total free amino
acids (2.36 and 0.10 g.100 g
-1
in average, respectively).
Table 2. Leaf osmolytes content (g.100 g
-1
) of recovered local grape genotypes following salt treatments.
Reducing sugars content Total free amino acids content
Control Low salt High salt Mean Control Low salt High salt Mean
Baltim Eswid
2.61 2.87 2.58 2.68 0.12 0.13 0.10 0.12
Romy Ahmer
2.55 2.85 2.12 2.51 0.13 0.13 0.09 0.12
Romy Abiad
2.25 2.47 2.35 2.36 0.11 0.11 0.08 0.10
Mean 2.47 2.73 2.35 0.12 0.12 0.09
LSD 0.05 Cultivars & Treatments = 0.15
Interaction = 0.27
Cultivars & Treatments = 0.01
Interaction = 0.02
Table 1. Leaf antioxidant capacity (mg.g
-1
) of recovered local grape genotypes following salt treatments.
Total phenolic content Total antioxidant activity
Control Low salt High salt Mean Control Low salt High salt Mean
Baltim Eswid
9.47 10.56 10.16 10.06 7.53 9.33 8.41 8.42
Romy Ahmer
10.38 9.57 8.52 9.49 10.13 9.67 7.22 9.01
Romy Abiad
8.83 8.69 7.25 8.26 7.66 7.52 6.13 7.10
Mean 9.56 9.61 8.64 8.44 8.84 7.25
LSD 0.05
Cultivars & Treatments = 0.70
Interaction = 1.21
Cultivars & Treatments = 0.97
Interaction = 1.68
The mechanism of osmotic adjustment is a strategy that could
help plants to avoid ion toxicity and maintain water uptake under
saline conditions. To preserve osmotic homeostasis, plants secrete
osmolytes and osmoprotectants as essential abiotic stress alleviators
to encounter harsh environmental conditions by lowering or
balancing the osmotic potential of intracellular and extracellular ions.
There are some low-molecular-weight, nontoxic compounds that
accumulate in plants in response to drought and salinity stress without
interfering with normal metabolism. Soluble sugars such as sucrose,
trehalose, and sugar alcohols and other osmolytes such as glycine
betaine and proline amino acid act as the osmoprotectants. Soluble
sugars play an important role in maintaining cellular organization
and photosynthesis eciency, and detoxication of reactive oxygen
species (ROS) by acting as metabolic signals in response to several
abiotic stresses. Combined together, they protect plants by exercising
many physiological responses, such as strengthening membrane
integrity, regulating antioxidant enzymatic activity, and fullling
water requirements under stress conditions (Roychoudhury &
Tripathi, 2020).
Leaves anatomical structure
Most measured leaf anatomical characteristics of recovered Romy
Abiad genotype were adversely aected by severe salt stress, except
for thickness of palisade chlorenchyma tissue which is the principal
site of photosynthesis as it contains numerous chloroplasts (table 3 &
gures 3, 4).
Aside from biochemical responses, plants can modify leaf’s
anatomical structure along with altering morphology to adapt under
saline conditions (Mahmoud et al., 2023). Since preventing water loss
is a crucial adaptive mechanism versus salt stress, thick epidermis is a
characteristic of many plant varieties that are tolerant to salt, helping
to avoid dehydration associated with salinity (Ashraf et al., 2010).
In accordance, the most tolerant genotype “Baltim Eswid” could
maintain thicky of both lower and upper epidermis under conditions
of high salt stress, while both Romy Ahmer and Romy Abiad got
thinner lower epidermis.
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Table 3. Relative changes (%) in measured leaf anatomical traits
of recovered local grape genotypes compared to control.
Anatomical features
Baltim
Eswid
Romy
Ahmer
Romy
Abiad
Upper Epidermis thickness +46.3 +49.6 -4.7
Lower Epidermis thickness +20.4 -23.4 -15.8
Palisade Mesophyll thickness +42.2 +25.5 +44.3
Spongy Mesophyll thickness +40.3 +32.1 -15.4
Xylem Tissue thickness +17.9 +84.9 -3.6
Phloem Tissue thickness +43.8 +22.7 -22.4
Midvein thickness +27.6 +44.0 -2.14
Xylem Vessel diameter +18.1 0 -11.9
Figure 3. Transverse sections through the leaf blade of recovered
local grape genotypes comparing to control. In the
gure: 1: Upper Epidermis; 2: Lower Epidermis; 3:
Palisade Mesophyll; 4: Spongy Mesophyll.
Figure 4. Xylem vessels transverse sections through the leaf
blade of recovered local grape genotypes comparing to
control.
Also, diameter of conductive xylem vessels was enhanced in
Baltim Eswid during recovery while maintained stable in Romy
Ahmer, which is functionally related to holding of various elements.
A reduction in xylem vessel diameter and xylem tissue thickness
was observed in the recovered Romy Abiad genotype, indicating
that there was no considerable increase in xylem vessel density as
well. On the other hand, xylem tissue thickness was increased in the
recovered Romy Ahmer genotype which indicates enhanced xylem
vessel density where constant xylem vessel diameter was observed.
Modications of vessel size are likely to be a common mechanism of
response to water stress in a large-vessel species such as the grapevine
(Lovisolo & Schubert, 1998). Reduced development of xylem vessels
in grapevines exposed to moderate water stress may contribute to
controlling water ow and reducing vulnerability to xylem embolism,
which helps maintain hydraulic conductivity at low water potential,
thus increasing the integrity of xylem if compensated by an increase
in vessel density.
Gene expression analysis
Both VvChS and EDS1 genes were over-expressed in leaves of
Romy Ahmer and Romy Abiad genotypes during recovery following
the highest saline treatment compared to control, while down-
regulated in Baltim Eswid cultivar (gure 5).
The reduced thickness of epidermis may be due to restricted cell
division with increased salt level (Parida et al., 2016). Otherwise,
thicker epidermis may aid better adaptation under elevated salt stress
by keeping a rate of transpiration that maintains moisture of the
mesophyll tissue and improve the eciency water usage, also oers
extra area for ecacious segregation of sodium in the epidermis of
leaves. Additionally, enhanced thickness of the spongy mesophyll
in recovered Baltim Eswid and Romy Ahmer genotypes may aid
maintain moisture and turgor of leaves (Parida et al., 2016).
Figure 5. Changes in expression level of targeted genes for
recovered local grape genotypes relatively to
control. In the gure: ChS: Chalcone synthase; EDS1:
ENHANCED DISEASE SUSCEPTIBILITY 1.
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A key step to interpret genome function in response to various
environmental signals is to determine the pattern of how gene
expression is regulated. Upon exposure to stress, plants can express
several genes as a defense response. Synthase of chalcone (ChS),
that is a main enzyme in biosynthesis of avonoids which intrinsic
in the pathway of phenylpropanoid, is commonly induced in dierent
species of plants under abiotic stress conditions (Mohammadkhani et
al., 2016). It is possibly that, salt-aected Romy Ahmer and Romy
Abiad genotypes that suer during recovery attempt to stimulate
the mechanisms of defense by rising the VvChS transcription. For
VvEDS1, more studies should be done to specify its exact function
in grapevine defense mechanism, in which agrees with Chong et al.
(2008). EDS1 is a key regulator of cell death in the hypersensitive
response to stress signals. Plants responses to stress can be induced
by ROS perception as a signal which promotes the program of genetic
response to stress, where protein of EDS1 appears to be related to
controlling the singlet-oxygen–mediated visible responses to stress
(Mohammadkhani et al., 2016). It was hypothesized that, EDS1
may regulate plant recovery after being exposed to ecological stress
conditions.
Conclusion
Recovery is one of stress tolerance mechanisms in plants
indicating the ability to restore metabolism after cessation of severe
stress conditions. Basic studies on stress tolerance in plants have
generally focused on their responses under dierent applied stress
treatments, but very little is known about the equally important stress
recovery mechanisms, which are essential to ensure sustainable crop
production under intermittent stress events. The objectives of this
research were to evaluate the salt tolerance and recovery attributes
of three native grape genotypes (Vitis vinifera); Baltim Eswid, Romy
Ahmer and Romy Abiad, on the bases of biochemical, anatomical
and gene expression responses. Suggested adaptive mechanisms
include: damage reduction caused by salinity-related oxidative stress,
osmotic adjustment, and perform structural modications that allow
protection. It was concluded that, Blatim Eswid is a superior salt-
tolerant local grape genotype, while Romy Abiad is the most sensitive
as aected mostly by oxidative stress. The study highlighted the
paramount importance of preserving Egyptian table grape germplasm,
especially those that are threatened with extinction, as they adapt to
harsh ecological conditions and neglected cultural managements,
which can aid countering the expected constraints associated with
climate change on viticulture and adapt under adverse conditions to
ensure maintaining productivity and quality of grapes.
Funding source
Presented supplementary experiment is a part of the guidance
and training activities within the framework of the project
“FRUIT CROPS RESILIENCE TO CLIMATE CHANGE IN THE
MEDITERRANEAN BASIN (FREECLIMB)”, funded by “Science,
Technology & Innovation Funding Authority (STDF)” with grant
number “PRIMA”, and was accomplished in cooperation with
Faculty of Biotechnology, October University for Modern Sciences
and Arts (MSA).
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