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\begin{document}

\title{Deciphering the broccoli plants response to salinity and boron stress on
with a focus on membranes and aquaporins}



\author[1]{Micaela Carvajal}%
\author[1]{Juan Nicolas-Espinosa}%
\author[1]{Lucia Yepes}%
\author[1]{Fuensanta Martinez-Bernal}%
\author[1]{Miriam Fernandez-Pozurama}%
\affil[1]{Centro de Edafologia y Biologia Aplicada del Segura}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Abiotic stresses as salinity, and boron toxicity, and deficiency are
commonly found in arid and semi-arid areas were broccoli is abundantly
grown. In this work, the physiological response of broccoli leaves
(including growth, relative water content, stomatal conductance, and
mineral concentration) was studied under salinity and boron stresses
(deficiency and toxicity), individually or in combination. Also, the
molecular study of PIP aquaporins were studied in relation to their
presence in plasma membrane PIP presence and their membrane lipid
environment. The results showed that only the combination of salinity
and boron deficiency decreased plant biomass, suggesting good adaptation
to the other treatments. Changes in stomatal conductance and mineral
nutrients suggest that the adaptation was related to water and boron
transport through leaves, involving aquaporins since avoidance strategy
was observed. Furthermore, changes in aquaporins PIP expression revealed
that each individual aquaporin is involved in each treatment, either
alone or in combination. However, the discrepancy between the presence
of aquaporins in the plasma membrane and microsomal fraction pointed
towards the regulation of trafficking and membrane composition (lipids
and proteins) should be highly present in this plant under stress.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The \emph{Brassicaceae} family includes broccoli (\emph{Brassica
oleracea} L. var \emph{italica} ), kale, cauliflower, radish, and
others, being a widely cultivated crop group. Of all brassicas, broccoli
is especially economically significant, with 26,000 kt produced
worldwide in 2020 from approximately 1.36 million hectares of harvested
land (\emph{http://data.un.org/}). Broccoli is also highly valued for
its health benefits, thanks to its rich content of bioactive compounds
such as glucosinolates, polyphenols, and vitamin C. Additionally, the
byproducts of broccoli cultivation serve as source of different
bioactive molecules, such as peptides, for cosmeceutical applications
(Picchi et al., 2012; Nicolas-Espinosa et al., 2022).

Abiotic stresses, including drought, waterlogging, salinity, nutrient
deficiency, and temperature, pose challenges to broccoli cultivation
(Beacham \emph{et al.} , 2017). Salinity stress is one of the major
abiotic stress that affects crops globally, particularly in arid and
semi-arid regions (Parida and Das, 2005; Yadav S. P. \emph{et al.} ,
2019). Approximately 20\% of irrigated lands and 6\% of the total global
land are impacted by salinization, resulting in a reduction of crop
yields by a maximum of 70\% (El-Badri \emph{et al.} , 2021). Soil
salinity results from the accumulation of salts in the soil, which can
occur due to a variety of factors, including evaporation, leaching, and
irrigation practices. Salinity stress interferes with plant growth and
productivity by disrupting water and nutrient uptake, altering osmotic
balance, and damaging plant cell tissues (Ma \emph{et al.} , 2020).

In the same way, boron (B) stress, being more relevant B toxicity, is a
significant challenge for plant growth and productivity worldwide,
especially in region with high soil B content. B is an essential
micronutrient for plants, but high levels of B can lead to toxicity
symptoms, including stunted growth, reduced crop yields, and even death
of the plant (Landi \emph{et al.} , 2019). The toxicity symptoms result
from the interference of excess B with various physiological processes,
including cell division and differentiation, protein synthesis, and
membrane stability. In this sense, B toxicity can disrupt the transport
of water and nutrients within the plant, further exacerbating the
negative effects of the stressor (Wimmer and Eichert, 2013).

The simultaneous occurrence of B stress and soil salinity is a common
phenomenon in semiarid and arid regions, as high soil salinity
concentrations often result in limited leaching and this the
accumulation of B in the form of sodium salts. This combination of
stress factors is associated with irrigations practices that use water
containing high levels of B and salts. The use of desalinated water
often contains varying levels of B, leading to increased levels of soil
B and further exacerbation of B toxicity in crops (Hilal \emph{et al.} ,
2011). Additionally, the use of desalinated water for irrigation could
also exacerbate soil salinity, as the desalination process can be
limited in its ability to remove all salts, particularly those that are
present in high concentrations (Darre and Toor, 2018). This can result
in the remaining water still containing high levels of salts. The plasma
membrane (PM) of plant cells plays a crucial role in regulation
communication between the cell and the environment. As a selective
barrier, the PM acts as the main receptor and transducer of external
signals, and is critical in maintaining plant homeostasis, providing
cellular nutrition, enabling endocytosis, and responding to biotic and
abiotic stresses (Gronnier \emph{et al.} , 2018; Morel \emph{et al.} ,
2006). The PM is particularly important in responding to salinity stress
as it is the first line of defence for the cell. Lipid and transport
proteins within the PM play a significant role in regulating membrane
permeability and fluidity, triggering responses to salinity
(Yepes-Molina \emph{et al.} , 2020). In this scenario, aquaporins (AQPs)
have a crucial role in the stress response mechanisms of the plant,
acting as key components of the PM and carrying out vital functions
within the plant cell. AQPs are integral membrane proteins (MIPs) that
are responsible for the regulated transport of water across cell
membranes. The AQPs family can be classified into different subfamilies
based on their sequence homology and membrane location. Among them, the
PIP subfamily is one of the most relevant for maintaining water
homeostasis in plants and plays an important role in their ability to
cope with environmental stress conditions (Barzana \emph{et al.} ,
2021). The PIP subfamily can be further subdivided into two groups, PIP1
and PIP2. This subfamily is primarily located in the PM and acts as
water channels, especially the PIP2 group. Additionally, they allow the
transport of other neutral molecules such as nitrogenous compounds
(e.g., urea and NH\textsubscript{3}), boric acid,
H\textsubscript{2}O\textsubscript{2}, and
CO\textsubscript{2}(Nicolas-Espinosa and Carvajal, 2022). It is well
known that AQPs are involved in both stresses, salinity and boron; under
saline conditions, the concentration of ions in the soil solution
increases which leads to an increase in osmotic potential. This causes a
reduction in water uptake by the plant, leading to dehydration and
ultimately, plant stress (Mart\selectlanguage{ngerman}ínez-Ballesta \emph{et al.} , 2006). AQPs
are involved in mitigating this stress by allowing the plant to control
water uptake, reducing the amount of water taken up under saline
conditions (Barzana \emph{et al.} , 2021). The presence of a diverse
array of plant AQPs suggest that different isoforms may serve distinct
functions in various cell types, as their regulation is influenced by
specific physiological contexts. Salinity has been shown to alter the
expression of AQPs, suggesting that these proteins may play a role in
the physiological response that maintains homeostasis under stress. The
transcripts levels of PIPshave been observed to decrease under saline
stress in Arabidopsis, barley, among others (Boursiac \emph{et al.} ,
2005; Horie \emph{et al.} , 2011; Katsuhara \emph{et al.} , 2011).
However, in some cases, such as radish seedlings, the mRNA and protein
levels of PIPs and TIPs remain unchanged (Suga \emph{et al.} , 2002).
Conversely, an increase in PIP certain isoforms expression has been
observed under saline stress in Arabidopsis (Jang \emph{et al.} , 2004;
Sutka \emph{et al.} , 2011), \emph{Brassica juncea} (Srivastava \emph{et
al.} , 2010), and \emph{Brassica rapa}(Kayum \emph{et al.} ,
2017)\emph{.} Similarly, many AQPs have been described to be able to
transport boric acid, playing a key role in stress conditions, such as
AtNIP7;1 and AtNIP5;1 in Arabidopsis (Li\emph{et al.} , 2011), but also
AtPIP2;2 and AtPIP2;7 were permeable to boric acid (Groszmann \emph{et
al.} , 2023).

To fully comprehend the impact of multiple stress factors on plant
growth and development, it is essential to evaluate the interactions
between different stressors, including how the plant responds to
different stress combinations, and the extent to which each stress
factor affects the other. This requires a comprehensive approach that
considers the molecular, physiological, and biochemical changes that
occur in the plant in response to these stress combinations
(Kissoudis\emph{et al.} , 2014).

Consequently, the aim of this study was to assess the physiological
(growth, relative water content, stomatal conductance, and mineral
concentration) and molecular (aquaporins) impacts of salinity and boron
stresses (deficiency and toxicity) on broccoli leaves. The evaluation
has been carried out individually and in combination, in order to
identify molecular markers among aquaporins PIP and their membrane lipid
environment that could indicate stress resistance coping with
water/boron uptake and transport.

\textbf{Material and methods}

\textbf{Plant growth and treatments application}

\emph{Brassica oleracea} L. var. \emph{italica} cv. Parthenon (broccoli)
seeds were germinated and cultivated as reported in Muries et al.,
(2011) with some modifications. Firstly, a pre-hydration with deionized
water was performed for 24 h. Then, seeds were germinated in dark with
vermiculite at 28°C for two days and after, the sprouts were transferred
to hydroponic conditions in a controlled-environment chamber with a 16-h
light and 8-h dark cycle, with air temperatures of 25 and 20 °C,
day/night. The relative humidity (RH) was 60--80\% and the
photosynthetically active radiation (PAR) was 400 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2} s\textsuperscript{-1}, provided by a Pacific LED,
WT 470C, LED8OS/840 PSD WB L1600, Philips. After 15 days of growth,
treatments were applied consisting in: control conditions (Hoagland, 6
mM KNO\textsubscript{3}, 4 mM Ca(NO\textsubscript{3})\textsubscript{2},
2 mM NH\textsubscript{4}H\textsubscript{2}PO\textsubscript{4}, 1 mM
MgSO\textsubscript{4}, 25 \selectlanguage{greek}µ\selectlanguage{english}M H\textsubscript{3}BO\textsubscript{3}, 4 \selectlanguage{greek}µ\selectlanguage{english}M
MnSO\textsubscript{4}, 4 \selectlanguage{greek}µ\selectlanguage{english}M ZnSO\textsubscript{4}, 1 \selectlanguage{greek}µ\selectlanguage{english}M
CuSO\textsubscript{4}, 0.13 \selectlanguage{greek}µ\selectlanguage{english}M
(NH\textsubscript{4})\textsubscript{6}Mo\textsubscript{7}O\textsubscript{24},
and 40 \selectlanguage{greek}µ\selectlanguage{english}M Fe\textsubscript{3}\textsuperscript{+}-EDDHA), salinity (80 mM
NaCl), B starvation (no B was added), B excess (250 \selectlanguage{greek}µ\selectlanguage{english}M of B), and the
combination of both salinity and B starvation and salinity and B excess.
Growth medium was renewed after 1 week and plants were harvested 15 days
after treatments applications. Finally, when harvested plant growth was
measured by determining dry and fresh weight, and leaf area for
calculating the leaf per mass area (LMA).

\textbf{Analysis of Mineral Nutrients}

Leaves and roots were collected, dried, and ground into a fine powder
dust. Then samples were digested in a microwave oven (CEM Mars Xpress,
Stallings, NC, USA), by HNO\textsubscript{3}:HClO\textsubscript{4} (2:1)
digestion. The different elements were detected by a coupled plasma
(ICP) analysis (Optima 3000, PerkinElmer, Waltham, MA, USA).

\textbf{Stomatal conductance}

Stomatal conductance was measured every 3-4 days after treatments
application. Four leaves were measured in three plants for each
treatment. Measures were made with AP4 Delta-T Devices, Cambridge UK.

\textbf{Microsomal fraction and plasma membrane isolation}

A pool of leaves from five different plants of the same treatment was
used for four microsomal fraction extractions in order to obtain enough
protein and lipid amounts for further analysis. Leaves plasma membranes
were purified from microsomal fractions using the two-phase aqueous
polymer technique first described by Larsson \emph{et al.} , (1987) and
modified by Casado-Vela \emph{et al.} , (2010).

Leaves (50 g) were cut before vacuum infiltrated with 0.5 g of PVP and
160 ml of extraction buffer containing 0.5 M sucrose, 10\% glycerol, 20
mM Na\textsubscript{2}EDTA, 20 mM EGTA, 50 mM NaF, 5 mM
\selectlanguage{greek}β-\selectlanguage{english}glycerophosphate, 1 mM 1,10-phenanthroline, 1 mM
Na\textsubscript{3}VO\textsubscript{4}, 0.6\% PVP, 5 mM ascorbic acid, 5
mM DTT, and 0.5 mg/L leupeptin in 50 mM Tris-MES, pH 8.0. After buffer
infiltration, tissues were homogenized using a blender and filtered
through a nylon mesh (pore diameter of 100 \selectlanguage{greek}µ\selectlanguage{english}m). The homogenate was
centrifuged at 10,000 \emph{x g} for 40 min at 4\selectlanguage{ngerman}°C. The supernatant was
collected and centrifuged at 100,000 \emph{x g} for 45 min at 4°C. The
pellet was suspended in buffer (FAB) containing 330 mM sucrose, 2 mM
DTT, 10 mM NaF, and 5 mM phosphate buffer (pH 7.8). Plasma membranes
were purified from microsomal fraction by partitioning in a two
phase-system mixture with a final concentration of PEG-3350
(Sigma)/Dextran-T500 (GE Healthcare), 6.0\% (w/w), in the presence of
330 mM sucrose, 5 mM KCl, 5 mM potassium phosphate (pH 7.8). The
two-phase system was centrifuged for 5 min at 4000 \emph{x g} at 4°C.
The upper phase, enriched plasma membranes, was recollected and washed
in 300 mM sucrose, 9 mM KCl, 6 mM Na\textsubscript{2}EDTA, 6 mM EGTA, 60
mM NaF, and Tris-borate, pH 8.3, and centrifuged at 100,000 \emph{x} g
for 45 min, 4°C. The resulting pellet was suspended in resuspension
buffer (FAB). The protein concentration of both, microsomal fraction and
plasma membrane-enriched fractions, was determined with Protein assay
dye reagent (BioRad), with BSA as standard.

\textbf{RNA extraction and expression studies, real time qPCR}

\textbf{Primer design}

For primer design, general primers for various aquaporin isoforms was
followed since the aquaporin family is very extended in broccoli plants,
counting with 65 members (Nicolas\selectlanguage{english}-Espinosa and Carvajal, 2022), primer
design was focused in evaluate the expression of the different
subfamilies within PIP1 and PIP2. The primer design was carried out
using Geneious prime 2020.1.2 software
(\emph{https://www.geneious.com/}), focused in conserved sites of the
consensus sequences of PIP1-1, PIP1-2, PIP1-4, PIP1-5, PIP2-1, PIP2-2/3,
PIP2-4, PIP2-5, and PIP2-7.

\textbf{Table 1} . Primer set for aquaporin expression analyses.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Gene name} & \textbf{Forward (3'-5')} & \textbf{Forward (3'-5')}
& \textbf{Reverse (3`-5')} & \textbf{Reverse (3`-5')} &\tabularnewline
\midrule
\endhead
BoiPIP1-1a & BoiPIP1-1a & ACAGCTTCCGGGCTAAGAAC & ACAGCTTCCGGGCTAAGAAC &
TTTCGAGCCTGGTGAGCTTT & TTTCGAGCCTGGTGAGCTTT\tabularnewline
BoiPIP1-2 & BoiPIP1-2 & GCTCGTGACTCTCATGTTCC & GCTCGTGACTCTCATGTTCC &
AGCGTTGTCCTTGTTGAAGATG & AGCGTTGTCCTTGTTGAAGATG\tabularnewline
BoiPIP1-4b & BoiPIP1-4b & CCACAACATTCGCTCCTCCA & CCACAACATTCGCTCCTCCA &
ATGGGTTGCCTCTCAGGGAA & ATGGGTTGCCTCTCAGGGAA\tabularnewline
BoiPIP1-5 & BoiPIP1-5 & TAAGGATCATGCTTGGGGCG & TAAGGATCATGCTTGGGGCG &
GGTACAGAGCAGCAAGAGCA & GGTACAGAGCAGCAAGAGCA\tabularnewline
BoiPIP2-1 & BoiPIP2-1 & AAGCTGTTTCCGGAGAAGGG & AAGCTGTTTCCGGAGAAGGG &
CAGCTCCTCCGGATCAAACA & CAGCTCCTCCGGATCAAACA\tabularnewline
BoiPIP2-2/3 & BoiPIP2-2/3 & GACGTTCGGCTTGTTCTTGG & GACGTTCGGCTTGTTCTTGG
& CCAACTCCACAAATCGCACC & CCAACTCCACAAATCGCACC\tabularnewline
BoiPIP2-4 & BoiPIP2-4 & CAACCAAGAAAAGGCCTGGG & CAACCAAGAAAAGGCCTGGG &
CCAGCCGCTCTTAGAACAAA & CCAGCCGCTCTTAGAACAAA\tabularnewline
BoiPIP2-5 & BoiPIP2-5 & AGCTTGGGACCATCATTGGA & AGCTTGGGACCATCATTGGA &
ACCAGCCCTCAACACAAACT & ACCAGCCCTCAACACAAACT\tabularnewline
BoiPIP2-6 & BoiPIP2-6 & GCGGTGGATCTTGGTAGTCC & GCGGTGGATCTTGGTAGTCC &
CTCAGCTACAGACCCCAAGC & CTCAGCTACAGACCCCAAGC\tabularnewline
BoiPIP2-7 & BoiPIP2-7 & TGGATTCGCTGTGTTCATGG & TGGATTCGCTGTGTTCATGG &
ACAGCAGCACCAAAGCTTCT & ACAGCAGCACCAAAGCTTCT\tabularnewline
Boi18-S-RNA * & Boi18-S-RNA * & CCTGCGGCTTAATTTGACTC &
CCTGCGGCTTAATTTGACTC & AGACAAATCGCTCCACCAAC &
AGACAAATCGCTCCACCAAC\tabularnewline
\bottomrule
\end{longtable}

*(Muries \emph{et al.} , 2011)

\textbf{Real time qPCR}

To assess and compare the expression of aquaporins across various
cultivars and treatments, RT-qPCR was conducted using 2 \selectlanguage{greek}μ\selectlanguage{english}L of 1:10
diluted cDNA samples in an 8 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture containing 600 nM
gene-group primers (\textbf{Table 1} ) and 5 \selectlanguage{greek}μ\selectlanguage{english}L of SYBR Green Master Mix
2X (Applied Biosystems) in an Applied Biosystems 7500 Real-Time PCR
system. Amplification was performed in a two-step process, consisting of
denaturation at 95\selectlanguage{ngerman}°C for 10 min, followed by 40 cycles of 15 s of
denaturation at 95°C and 1 min of annealing and extension at 60°C. A
dissociation stage was then carried out. Data collection was performed
at the end of each cycle's step 2. These conditions were used for both
target and reference genes. The reactions were conducted in triplicate
for each sample (technical replicates) on a 96-well plate, and four
independent samples were tested for each treatment (biological
replicates). Transcript levels were calculated using the 2--\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method
(Livak and Schmittgen, 2001) for both target and reference genes.

\textbf{Western Blot analyses (Gel electrophoresis and immunoblotting)}

Protein (5 \selectlanguage{greek}µ\selectlanguage{english}g per lane in plasma membrane fractions and 15 \selectlanguage{greek}µ\selectlanguage{english}g for
microsomal fractions) was loaded for 12\% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel
(Yepes-Molina\emph{et al.} , 2020). The antibodies used were against
\emph{Arabidopsis thaliana} PIP1 group, PIP2 (PIP2-1, PIP2-2, and
PIP2-3) and PIP2-7 (Agrisera, V\selectlanguage{ngerman}ännäs, Sweden). After checking predicted
reactivity against broccoli proteins, the fact that PIP1 antibody had a
reactivity against BoiPIP1-1a, BoiPIP1-2a, BoiPIP1-2b, BoiPIP1-2c,
BoiPIP1-3a, BoiPIP1-4a, BoiPIP1-4b, and BoiPIP1-5a were determined,
whereas PIP2 antibody had a predicted reactivity against (BoiPIP2-1a,
BoiPIP2-1b, BoiPIP2-2a, BoiPIP2-3a, BoiPIP2-3b, BoiPIP2-4a, BoiPIP2-4b,
BoiPIP2-4c, BoiPIP2-5a, PIP2-5b), while PIP2-7 was specific against
BoiPIP2-7 isoforms. Goat anti-rabbit IgG coupled to horseradish
peroxidase was used as the secondary antibody. A chemiluminescent signal
was developed using SuperSignal West Pico PLUS substrate
(Thermoscientific., Rockford, IL, USA). The intensity of each band was
determined by ImageJ software (Schneider \emph{et al.} , 2012).

\textbf{Lipid analysis}

Sterol and fatty acids were determined as described by Chalbi \emph{et
al.} , (2015). A mixture of chloroform-methanol (1:2, 0.75 mL) was added
in an Eppendorf tube to both, microsomal and plasma membrane fractions
(0.5 mL), along with \selectlanguage{greek}β-\selectlanguage{english}cholestanol (20 \selectlanguage{greek}µ\selectlanguage{english}L, 0.1 mg
mL\textsuperscript{-1}) used as internal standard for sterol analysis.
Chloroform (CHCl\textsubscript{3}; 0.25 mL) was added and the mixture
was shaken and centrifuged at 10,000 \emph{x} g for 6 min. The
CHCl\textsubscript{3} layer was retained, evaporated to dryness under
N\textsubscript{2} and made up to 100 \selectlanguage{greek}µ\selectlanguage{english}L with CHCl\textsubscript{3}. For
sterol analysis, 20 \selectlanguage{greek}µ\selectlanguage{english}L of the CHCl\textsubscript{3} extract was placed
in a glass vial (2 mL), evaporated to dryness under
N\textsubscript{2}and acetylated using pyridine (50 \selectlanguage{greek}µ\selectlanguage{english}L) and
Ac\textsubscript{2}O (100 \selectlanguage{greek}µ\selectlanguage{english}L). After 2h, the solvents were evaporated
under N\textsubscript{2}, ethyl acetate was added and the sterols
analyzed by GC using an HP5-bonded capillary column (30 m x 0.25 mm x
0.25 \selectlanguage{greek}µ\selectlanguage{english}m) coupled to a flame ionization detector (FID), with
H\textsubscript{2} as carrier (1mL min\textsuperscript{-1}) and a
temperature programmed of 120-260\selectlanguage{ngerman}°C at 5°C min\textsuperscript{-1}, then
260-280°C at 2°C min\textsuperscript{-1}, and finally 280-300°C at 6°C
min\textsuperscript{-1}. The injector and detector temperatures were 150
and 320°C, respectively. Bound fatty acids were determined by using
20-II portions of the CHCl\textsubscript{3} extract; evaporating them to
dryness under N\textsubscript{2}, transmethylating with sodium methoxide
(0.5 N) in methanol (0.5 mL) and heating at 30°C for 7 min. The
resultant fatty acids methyl esters were extracted with hexane (1 mL),
evaporated under N\textsubscript{2}, dissolved in ethyl acetate (20 \selectlanguage{greek}µ\selectlanguage{english}L)
and analyzed by GC using an HP5-bonded capillary column (30 m x 0.25 mm
x 0.25 \selectlanguage{greek}µ\selectlanguage{english}m), with FID, He as carrier (1 mL min-1) and a temperature
programmed of 150-195\selectlanguage{ngerman}°C at 3°C min\textsuperscript{-1}, then 195-220°C
at 2°C min\textsuperscript{-1}, and finally 220-300°C at 6°C
min\textsuperscript{-1}. The injector and detector temperatures were 280
and 300°C, respectively.

\textbf{Microscopy, TEM and particle size}

Microsomal and plasma membrane enriched fractions from broccoli leaves
were pelleted at 100,000 \emph{x} g\emph{.} For chemical fixation,
pelleted vesicles were sequentially fixed with glutaraldehyde (2.5\% in
100 mM phosphate buffer, 2 h at 4°C), osmium tetroxide (1\% buffered, 2
h at 4°C), and tannic acid (1\% in deonized water, 30 min at 22°C). The
pellets were then thoroughly rinsed with water and covered with 2\% low
melting point agarose, then dehydrated with ethanol and epoxypropane at
22°C and embedded in Epon. Blocks were sectioned on a Leica EM UC6
ultramicrotome, collected on Formvar-coated copper grids and stained
with uranyl acetate followed by lead citrate. Sections were examined
using a JEOL 1011 transmission electron microscope with digital camera
GATAN ORIUS SC200. For each treatment, an average of 5-10 ultrathin
sections were examined.

\textbf{Vesicle size}

The average size of the two fractions of vesicles was analyzed using
light-scattering technology; through intensity measurements with a
Malvern ZetaSizer Nano XL machine (Malvern Instruments Ltd., Orsay,
France), as previously described by Barrajón-Catalán \emph{et al.} ,
(2010). This allowed the measurement of the particles with a size range
from 1 nm to 3 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Data analysis}

The different statistical analyses were performed using SPSS software
(v.26; Chicago, IL). Statistical differences between the experimental
groups were determined via \emph{post hoc} analyses, Duncan test was
used for parametric data and non-parametric data were analyzed via
Kruskal-Wallis analysis of variance test. In all the analyses,
\emph{p}\textless{} 0.05 was selected to represent statistically
significant differences.

\textbf{Results}

\textbf{Physiological analysis}

After measuring the dry weight of the upper part and roots of plants
that were treated for 15 days (\textbf{Figure 1A} ), a decrease in the
upper part dry weight (DW) was observed in salinity plus B-deprivation
(Comb (-)) treatment compared to control plants. The salinity (NaCl) and
Comb (-) treatments were lower and statistically different upper part
values from plants treated with B excess (B (+)), which had the highest
upper part total dry mass. Additionally, NaCl and Comb (-) treated
plants presented lower root DW than the control plants. The analysis of
relative water content showed no differences of any of the treatments
with the control plants. However, while B (-) and B (+) showed no
significant differences, when they were combined with salinity, Comb (-)
were significantly lower than Comb (+). The Leaf per Mass Area (LMA)
data (\textbf{Figure 1B} ) showed that only the salinity leaves were
significantly different from the control plants, providing higher
values.

We analyzed stomatal conductance (\textbf{Figure 2} ) every 3-4 days
from the beginning to the end of the experiment. The control and B (-)
plants showed the highest stomatal conductance that were maintained
during all the time of the experiment. Plants treated with B (+)
maintained similar values than control until the 3\textsuperscript{rd}
day of treatment showing from that day a significant decrease that
remain stable from 10\textsuperscript{th} to 13\textsuperscript{th} day
of treatment. Salinity (NaCl), Comb (-), and Comb (+) treatments had the
lowest stomatal conductance that decreased at first day of treatment,
about half that of the control plants, being the lowest those plants
treated with NaCl. During the experiment, the stomatal conductance of B
(+) treated plants reached similar stomatal conductance to the
salinity-related treatments at 10\textsuperscript{th} day of treatment.

The mineral analysis of leaves (\textbf{Figure 3A} ) showed a clear
division between salinity-related treatments and non-salinity treatments
along the horizontal axis and between B (-) and B (+) treatments
(including Comb (-) and Comb (+) respectively) along the vertical axis,
as summarized by a Principal Component Analysis (PCA). The interactions
between Na and K, as well as between B and Mn, were negative. However,
Cu and Zn demonstrated a positive interaction with NaCl and Comb (-)
treatments, but a negative interaction with treatments related to excess
B (B (+) and Comb (+)). \textbf{Figure 3B-D} illustrates individual
plots of B, Na, and K concentrations as mass per DW represented as box
plots. The control plants had around 50 mg mg kg\textsuperscript{-1} DW
of B, while NaCl, B (-), and Comb (-) treatments had lower
concentrations, being Comb (-) leaves the one that presented the lowest
concentration of B among all treated plants with around 10 mg
kg\textsuperscript{-1} DW. Alternatively, B (+) treated plants had the
highest amount of B at 109 mg kg\textsuperscript{-1} DW, doubling the
control B concentration, followed by Comb (+) at 87 mg
kg\textsuperscript{-1} DW. The three salinity-related treatments (NaCl
and the two combinations) had 3.0-3.5 g of Na per 100 g DW, while the
rest of the plants had marginal concentrations. In contrast, K levels
were lower these salinity treatments, along with B (-), which also had a
reduction in K presence compared to control plant leaves, but it was
still higher than the salinity treatments.

\textbf{Aquaporins and lipids molecular analysis}

The expression analysis of integral membrane proteins such as AQPs
(\textbf{Figure 4} ) showed that B deficiency had the greatest effect on
the expression of these proteins in broccoli leaves. The B (-) treated
plants leaf presented generally lower levels of mRNA transcripts
compared to control leaves, particularly in PIP2 subfamily isoforms,
PIP2-1, PIP2-2/3, and PIP2-7. Similarly, B (+) leaves also resulted in
lower expression levels of PIP2-1. Out of the other treatments, only
salinity had an impact on the expression of PIP1-2, which showed an
almost threefold increase in mRNA transcript levels compared to control.

The vesicles obtained from microsomal fraction and plasma membrane
isolation were analyzed to characterize their size and shape
(see\textbf{Figure 5} ), as well as their polydispersity index (PDI).
Overall, no differences were observed among the different samples with
respect to the treatments applied. However, differences were found
between the types of samples. Specifically, the plasma membrane vesicles
were more size-stable with a PDI ranging from 0.06 to 0.18 and a mean
size around 240-280 nm, while the microsomal fraction vesicles were
larger (830 nm) and more polydisperse (PDI of 0.7-0.9).

The immunoblotting analysis (\textbf{Figure 6} ) displayed the
quantification of PIP1, PIP2 subfamilies, and PIP2-7 isoforms in
different samples of microsomal fraction and plasma membrane from
broccoli leaves of various treatments. In the case of microsomal
fractions, changes were only observed in the membrane extractions of the
Comb (+) treated plant leaves for PIP1 abundance (\textbf{Figure 6A} ).
However, the levels of PIP1 in plasma membrane increased in the NaCl and
B (-) treatments compared to the control, but not in the combination of
these two, Comb (-). There was a general increase in PIP2 subfamily
abundance compared to control, but only the Comb (-) treatment had the
same levels of PIP2 as the control leaves. The levels of PIP2-7
decreased in the plasma membranes of the two combinations, Comb (-) and
Comb (+), while they remained stable in other treatments compared to the
control leaves (\textbf{Figure 6D-F} ).

Finally, during lipids analysis, no changes were observed in fatty acid
composition (data not shown), but changes were found in sterol content
(\textbf{Table 2} ). For instance, lower levels of campesterol were
observed in all treatments except B (+) when compared to the control in
the microsomal fraction samples. Also, in the same fraction, lower
levels of stigmasterol and sitosterol were found in salinity and B (-)
treated samples. When analyzed the total sterol content of MF, only the
combination treatment presented differences, with lower concentration in
total sterol content. In plasma membrane sterols, changes were mainly
seen in campesterol abundance, with lower concentrations in B (-), Comb
(-), and Comb (+) when compared to control leaves. All treatments,
except salinity, showed a reduction in sitosterol content, although no
changes were observed in stigmasterol. Overall, the total presence of
sterols was reduced in all treatment but salinity when compared to
control. However, no changes were observed in the
stigmasterol/sitosterol ratio in either the plasma membrane or
microsomal fraction sample leaves among all treatments and control.

\textbf{Figure 1} . (\textbf{A} ) Graphical representation of dry weight
(g) root and upper part with the scale on the left and relative water
content of the plants with the scale on the right represented with
diamonds. Each measure is represented as the mean \selectlanguage{ngerman}± SE (n =6).
(\textbf{B} ) Leaf per mass area (LMA) (g m\textsuperscript{-2}) of
treated plants after 15 days of treatment application. Data are
represented as box plot. Different letters show statistical differences,
the data corresponding both dry weight measures resulted in
non-parametric data and \emph{Kluscal wallis post hoc} was selected, on
the case of relative water content and LMA statistical differences were
evaluated with one-way ANOVA using Duncan test as \emph{post hoc} . Both
analyses were conducted with \emph{p} \textless{} 0.05.

\textbf{Figure 2.} Stomatal conductance (mmol
m\textsuperscript{-2}s\textsuperscript{-1}) of leaves from each
treatment measured each 3-4 days starting the day of treatment
application. Each measure is represented as means ± SE (n = 4).
Different letters in lower case represent the statistical differences
between treatments each separate day, statistical differences were
calculated with one-way ANOVA using as\emph{post hoc} Duncan test when
the data were parametric and non-parametric data was evaluated via
\emph{Kluscal wallis} test as\emph{post hoc} . Legend of the right
represent statistical differences in capital letters of each treatment
using repeated measures ANOVA. All analyses were conducted with \emph{p}
\textless{}0.05.

\textbf{Figure 3.} Analysis of mineral nutrients of broccoli leaves,
(\textbf{A} ) PCA analysis of micro and macronutrients, (\textbf{B} )
concentration of B, (\textbf{C} ) Na, (\textbf{D} ) and K in in treated
plants leaves. Data is represented as boxes (25-75\%), error bars
represent range within 1.5 quartile, and median line (n = 4). Different
letters mean statistical differences of one-way ANOVA with Duncan test
as \emph{post hoc} , \emph{p} \textless{} 0.05.

\textbf{Figure 4.} Gene expression of different aquaporins in broccoli
leaves expressed as fold change (F. C) respect the control of isoforms
of PIP1 group (\textbf{A-D} ) and PIP2 group (\textbf{E-I} ). Data is
represented as means ± SE (n = 4). Different letters show statistical
differences one-way ANOVA and Duncan test as \emph{post hoc} .
(\textbf{E} ) PIP2-1 and (\textbf{F} ) PIP2-2/3 statistical analyses for
non-parametric data, Kruskal--Wallis. \emph{p} \textless{} 0.05 in all
cases.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Transmission electronic microscopy images of membrane
isolation from broccoli leaves. (\textbf{A} ) microsomal fraction (MF)
with scale bar of 2 \selectlanguage{greek}µ\selectlanguage{english}m, (\textbf{B} ) MF, scale bar of 1 \selectlanguage{greek}µ\selectlanguage{english}m, (\textbf{C}
) plasma membrane (PM) with scale bar of 1 \selectlanguage{greek}µ\selectlanguage{english}m, and (\textbf{D} ) PM,
scale bar of 500 nm, isolated from leaves of broccoli plants.

\textbf{Figure 6} . Broccoli leaves aquaporin protein relative
quantification via western-blot analysis of microsomal fraction (MF)
(\textbf{A-C} ), and plasma membrane (PM), \textbf{(D-F} ). PIP1
(\textbf{A} and \textbf{D} ), PIP2 (\textbf{B} and \textbf{E} ), and
PIP2-7 (\textbf{C} and \textbf{F} ) were analyzed in all treated plants
representing control as 100\%. Data is represented as means \selectlanguage{ngerman}± SE (n =
4). Different letters show statistical differences one-way ANOVA and
Duncan test as \emph{post hoc (p \textless{}} 0.05\emph{)} .

\textbf{Table 2} . Broccoli leaves sterol content of microsomal fraction
(MF) and plasma membrane (PM) (in 125 \selectlanguage{greek}μ\selectlanguage{english}g of protein) from broccoli
leaves under different stress treatments. Data is represented as means \selectlanguage{ngerman}±
SE (n = 4). Different letters show statistical differences \emph{p
\textless{}} 0.05 with Duncan test as \emph{post hoc} .\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& & \selectlanguage{greek}\textbf{Campesterol (µg)}\selectlanguage{english} & \selectlanguage{greek}\textbf{Stigmasterol (µg)}\selectlanguage{english} &
\selectlanguage{greek}\textbf{Sitosterol (µg)}\selectlanguage{english} & \textbf{Ratio Stigma/ Sitosterol} &
\selectlanguage{greek}\textbf{Total Sterol content (µg)}\selectlanguage{english}\tabularnewline
& \textbf{C} & 2.7 \selectlanguage{ngerman}± 0.39 c & 2.65 \selectlanguage{ngerman}± 0.37 c & 7.93 \selectlanguage{ngerman}± 2.02 c & 0.383 \selectlanguage{ngerman}±
0.08 ab & 13.28 \selectlanguage{ngerman}± 2.62 c\tabularnewline
& \textbf{NaCl} & 1.78 \selectlanguage{ngerman}± 0.13 ab & 1.79 \selectlanguage{ngerman}± 0.28 ab & 6.25 \selectlanguage{ngerman}± 0.45 bc &
0.285 \selectlanguage{ngerman}± 0.043 a & 9.82 \selectlanguage{ngerman}± 0.61 bc\tabularnewline
\textbf{MF} & \textbf{B (-)} & 1.81 \selectlanguage{ngerman}± 0.17 ab & 1.92 \selectlanguage{ngerman}± 0.55 ab & 5.85 \selectlanguage{ngerman}±
0.73 abc & 0.329 \selectlanguage{ngerman}± 0.083 a & 9.58 \selectlanguage{ngerman}± 1.13 bc\tabularnewline
& \textbf{B (+)} & 2.41 \selectlanguage{ngerman}± 0.3 bc & 2.69 \selectlanguage{ngerman}± 0.48 c & 5.38 \selectlanguage{ngerman}± 0.96 abc &
0.502 \selectlanguage{ngerman}± 0.008 b & 10.49 \selectlanguage{ngerman}± 1.71 bc\tabularnewline
& \textbf{Comb (-)} & 1.89 \selectlanguage{ngerman}± 0.22 ab & 0.93 \selectlanguage{ngerman}± 0.1 a & 3.85 \selectlanguage{ngerman}± 0.41 ab &
0.242 \selectlanguage{ngerman}± 0.011 a & 6.67 \selectlanguage{ngerman}± 0.63 ab\tabularnewline
& \textbf{Comb (+)} & 1.52 \selectlanguage{ngerman}± 0.09 a & 0.83 \selectlanguage{ngerman}± 0.06 a & 2.82 \selectlanguage{ngerman}± 0.18 a &
0.301 \selectlanguage{ngerman}± 0.038 a & 5.17 \selectlanguage{ngerman}± 0.24 a\tabularnewline
& \textbf{C} & 7.54 \selectlanguage{ngerman}± 0.35 c & 4.87 \selectlanguage{ngerman}± 1.17 a & 20.39 \selectlanguage{ngerman}± 2.21 b & 0.247 \selectlanguage{ngerman}±
0.076 ab & 32.79 \selectlanguage{ngerman}± 2.79 b\tabularnewline
& \textbf{NaCl} & 7.85 \selectlanguage{ngerman}± 0.24 c & 4.02 \selectlanguage{ngerman}± 0.84 a & 24.04 \selectlanguage{ngerman}± 3.42 b & 0.184
\selectlanguage{ngerman}± 0.06 a & 35.91 \selectlanguage{ngerman}± 2.81 b\tabularnewline
\textbf{PM} & \textbf{B (-)} & 3.78 \selectlanguage{ngerman}± 0.45 a & 4.41 \selectlanguage{ngerman}± 1.33 a & 9.46 \selectlanguage{ngerman}±
2.28 a & 0.437 \selectlanguage{ngerman}± 0.088 b & 17.64 \selectlanguage{ngerman}± 3.91 a\tabularnewline
& \textbf{B (+)} & 6.37 \selectlanguage{ngerman}± 1.16 bc & 3.57 \selectlanguage{ngerman}± 0.69 a & 12.66 \selectlanguage{ngerman}± 0.64 a &
0.288 \selectlanguage{ngerman}± 0.061 ab & 22.6 \selectlanguage{ngerman}± 1.58 a\tabularnewline
& \textbf{Comb (-)} & 5.27 \selectlanguage{ngerman}± 0.44 ab & 1.37 \selectlanguage{ngerman}± 0.12 a & 12.84 \selectlanguage{ngerman}± 1.05 a &
0.111 \selectlanguage{ngerman}± 0.019 a & 19.48 \selectlanguage{ngerman}± 1.37 a\tabularnewline
& \textbf{Comb (+)} & 4.38 \selectlanguage{ngerman}± 0.16 a & 1.23 \selectlanguage{ngerman}± 0.17 a & 8.48 \selectlanguage{ngerman}± 0.33 a &
0.146 \selectlanguage{ngerman}± 0.022 a & 14.09 \selectlanguage{ngerman}± 0.25 a\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Discussion}

Salinity and boron levels have significant impact in crop yield, leading
to high economic losses, particularly in semi-arid regions where low
quality water is used as the main source of irrigation (Díaz \emph{et
al.} , 2011). Our results showed that the only treatment that affected
plant (root and shoot) dry weight (DW) (\textbf{Figure 1} ) was the
combination of salinity and boron deficiency (Comb (-)). These results
contradicts previous findings that stated that salinity is the stress
that produced main impact in plant growth, provoking crop growth
inhibition lowering the productivity (Munns and Tester, 2008). In this
way, the low impact of the salinity and B stresses, separately, on our
broccoli plant growth could be due to the short duration of the
experiment (15 days of stress application in 15-day-old plants), since
the salt concentration applied (80 mM) has been reported to
significantly reduced shoot and root growth (López-Berenguer \emph{et
al.} , 2008). The LMA, which represents the relationship between leaf
structure and function, is an important factor in determining
photosynthetic capacity of the plant and water use efficiency. It is
well understood how other stressors such as light and
CO\textsubscript{2} impact in LMA, but there is less knowledge about how
LMA responds to stresses like salinity (Poorter \emph{et al.} , 2009).
Previous studies has shown that plants tend to increase their LMA under
salinity conditions, as the increase in LMA was associated with the
production of thicker and stronger cell walls that provide structural
support and help preserve water (Westoby \emph{et al.} , 2022;
Poorter\emph{et al.} , 2009). Our results indicate that only salinity
treatment increased LMA (\textbf{Figure 1B} ), leading to a reduction in
water use efficiency by the increased rigidity of the cell walls and
elasticity of the plasma membranes.

Stomatal conductance showed reductions with all salinity treatments
(NaCl and the two combinations) throughout the experiment duration
(\textbf{Figure 2} )\textbf{.} It is well established that salinity
affects water transport and transpiration, and the reduction in stomatal
conductance serves as an indicator of stress in plants (Gao \emph{et
al.} , 2002). Despite the overall lack of change in plant DW, the
reduction in stomatal conductance suggests a decrease in transpiration
for all treatments except for B (-). In the case of salinity treatments,
both salinity itself and in combination with boron deficiency and
excess, decreased the stomatal conductance dramatically from the first
day of treatment and remained lower throughout the experiment. The lower
stomatal conductance in response to salinity could be the strategy of
the broccoli plants to reduce sodium uptake, since its presence
generates toxicity (Kronzucker \emph{et al.} , 2013). For B (+),
stomatal conductance decreased at the middle of treatment application
(after 7 d), reaching the same levels of the salinity-related
treatments. This could be attributed to the fact that plants reached
toxic levels of boron in their tissues and tried to minimize boron
uptake by reducing stomatal conductance. Similar strategies have been
reported in\emph{Vitis vinifera} were its stomatal conductance changed
after 14 days of NaCl treatments applications (Gunes \emph{et al.} ,
2006), in the same way it had been reported that B toxicity reduced
stomatal conductance in Arabidopsis plants (Macho-Rivero \emph{et al.} ,
2017).

As shown in \textbf{Figure 3A} , ion profiles clearly divided the
salinity and non-salinity treatments with B (-) and B (+) stresses. High
levels of B were found in B (+) and Comb (+) treated plant leaves, while
salinity also affected B uptake, lowering levels of B as seen in B (-),
with a negative correlation in both combinations. As it can be seen in
Comb (+) the plants presented lower presence of B in leaves when
compared with B (+), and also, in Comb (-) being the plants with the
lowest B concentration. This negative relation between B excess and
salinity in B uptake could be attributed to the lower stomatal
conductance presented when salinity is applied, trying to reduce the
uptake of B and thus increasing its deficiency in Comb (-) plants. The
observed decrease in B accumulation in broccoli leaves under salinity
stress may be attributed also to lower stomatal conductance and thus
transpiration (\textbf{Figure 2} ), as B is commonly transported through
the xylem (Yermiyahu \emph{et al.} , 2008). This hypothesis has been
previously tested in other plants, including wheat (Holloway and Alston,
1992), melon (Edelstein \emph{et al.} , 2005), and tomatoes (Ben-Gal and
Shani, 2002), where exposure to combined B-salinity resulted in altered
B levels in leaves. In this sense, in cases where the concentration of
salt is below the tolerance threshold of each crop, the combined
stresses of B and salinity may alleviate B toxicity.

Moreover, only salinity treated plants were found to have high levels of
Na when analyzed. As Na\textsuperscript{+} movement within the plant is
closely related to K\textsuperscript{+}, the salinity treatment also
impacted K\textsuperscript{+} levels, reducing them compared to control
plants. Interestingly, B deficiency also reduced
K\textsuperscript{+}levels in the leaves of B-deficient plants compared
to the control. Although the decrease in K\textsuperscript{+} was not as
pronounced as with salinity, the reduction was statistically different
from the control and B (+). It appears that high presence of B did not
affect K\textsuperscript{+}, but its deficiency somehow lowered
K\textsuperscript{+} levels. However, it had been shown that B supply
does not influence the K\textsuperscript{+} content to a major extent,
as it is not involved in the K\textsuperscript{+} uptake pathway (Wu and
Wei-Hua, 2013).

It is well known that Ca\textsuperscript{2+} ion concentrations in
plants play a major role in the effect of salinity on B accumulation, as
Ca helps maintain the integrity of cell membranes (Cramer, 2006). When
analyzing the Ca concentration in the control and the treated plant
leaves, a reduction in Ca was observed in the two combined treatments,
with the Comb (+) treatment having the lowest Ca presence. On the other
hand, salinity treatment by itself did not reduce Ca levels in plant
leaves, but B starvation led to an increase of Ca levels compared to
control plant leaves (\textbf{Figure 3A} ). It is reported that salinity
stress causes Ca deficiency in plants by disrupting its distribution and
reducing its uptake by the plant through disturbance of the
K\textsuperscript{+}/Na\textsuperscript{+} balance at membranes
(Mohamed\emph{et al.} , 2016), thus inhibiting Ca movement from the root
to the xylem and its translocation to upper parts of the plant, like
leaves (Läuchli and Grattan, 2007). Although high levels of B are known
to positively affect Ca\textsuperscript{2+} transport (Bastías \emph{et
al.} , 2010; Läuchli and Grattan, 2007) in this case the opposite
occurred, exacerbating the reduction in Ca concentration with B excess
and acting as an enhancer of Ca uptake with B starvation.
Na\textsuperscript{+} competition and high salinity scenarios has been
also described to inhibit Mg\textsuperscript{2+} (Syvertsen and
Garcia-Sanchez, 2014), but in our case, differences were observed with
lower levels in Comb (+), but no differences appeared in the other
salinity treatments.

The decrease in stomatal conductance caused by salinity stress and B
toxicity reduces the uptake and transport of B in plants, which turn
mitigates its potential toxicity. Salinity stress has been reported to
affect water relations and reduce transpiration which can limit the
transport of B in plants (Yermiyahu \emph{et al.} , 2008). Similarly,
boron toxicity can also reduce stomatal conductance and limit the uptake
of boron in plants (Barzana \emph{et al.} , 2021). When both stresses
are present, their combined effect can further decrease stomatal
conductance and water uptake, leading to an even greater reduction in
boron uptake and transport. In this scenario, the role of plasma
membrane transporters is crucial in determining how plants cope with
combined stresses.

The study of aquaporins, which have been shown to transport mainly water
but also B, in addition to other neutral solutes, is important in this
context of abiotic stresses as salinity (Tyerman \emph{et al.} , 2002).
Based on \textbf{Figure 4} , it was observed that only B (-) treatment
had a significant impact on the PIP2 group, resulting in decreased
levels of \emph{PIP2-1} , \emph{PIP2-2/3} , and \emph{PIP2-7}
transcripts. B (+) treatment also led to a lower presence of
\emph{PIP2-1}transcripts in plant leaves. However, when the presence of
aquaporins in plasma membrane were analysed, an increase of expression
of PIP2 group was found in both B (-) and B (+) treatments
(\textbf{Figure 5A and B} ). This discrepancy in expression and protein
presence could be attributed to the response of plants to B starvation,
which modifies AQPs expression to prevent passive transport of B. Thus,
the decrease in the expression of several AQPs during B starvation can
be interpreted as a strategy to prevent passive transport of B, as the
other concentration of B should be lower than the intracellular
concentration. Since AQPs act as passive channels, driven by the
concentration gradient (Martinez-Ballesta and Carvajal, 2014), this
decrease in gene expression helps prevent B leakage through these
channels. Furthermore, boric acid tends to easily pass though cellular
membranes. In cases of B deficiency, B transporters, formed principally
by BOR family, activate to transport boric acid against concentration
gradient (Princi \emph{et al.} , 2016), while AQPs inactivate to prevent
B leakage though these channels. Additionally, the salinity treatment
up-regulated the expression of \emph{PIP1-2} compared to the control
after 15 days of the experiment started. Similar results were reported
in pomegranate leaves subjected to salinity stress, where \emph{PIP1-4}
, \emph{PIP2-3} ,\emph{PIP2-4} , and \emph{PIP2-2} were over-expressed
after 3 and 6 days of the treatment application (Kumawat \emph{et al.} ,
2021). In contrast to the \emph{Brassica rapa} PIP genes, which were
first up-regulated during salt stress and them down-regulated (Kayum
\emph{et al.} , 2017), the lower expression of certain AQPs contrasts
with the findings on the plasma membrane presence of these proteins,
where a higher signal was observed in the PIP1 group of NaCl and B (-)
treated plants. The differences between gene and protein results in
broccoli plants under salinity leaded to conclude that mRNA synthesis
could be inhibited by the accumulation of the corresponding encoded
protein (Muries \emph{et al.} , 2011). However, the regulation at the
level of trafficking must be reconsidered and deeply studied.

In addition, the PIP2 group was also found to be present in plasma
membrane at higher levels than in the control in almost all treated
plants, except for Comb (-). Previous studies have shown that
overexpression of PIP AQPs could improve tolerance to salinity in
transgenic tobacco (Chen \emph{et al.} , 2022). The increased levels of
AQPs may be associated with adaptation to water stress. Studies have
also demonstrated that overexpression of PIPs can increase
\emph{HKT1}and \emph{SOS1} , transporters that contribute to
Na\textsuperscript{+}efflux and K\textsuperscript{+} absorption,
respectively, (Horie\emph{et al.} , 2009), to improve tolerance to salt
stress and maintain cell ion homeostasis in salinity-stressed transgenic
plants (Chen\emph{et al.} , 2022). Overall, overexpression of AQPs has
been observed to result in better cell membrane integrity under salt
stress. Alternatively, the only treatments that showed a lower presence
of AQPs in plasma membrane were the two combinations, specifically in
PIP2-7 (\textbf{Figure 6F} ). Even though, no changes in
\emph{PIP2-7}expression were found for the two combinations, this
reduction was only observed in the plasma membrane. It has been shown
that a cargo receptor, Tryptophan-rich Sensory Protein (TPSO), that is a
heme-binding protein induced by abiotic stress (Vanhee \emph{et al.} ,
2011), interacts with the intracellular part of PIP2-7, triggering its
degradation though the autophagic pathway, downregulating it in the
cell, and modulating the osmotic water permeability (Hachez \emph{et
al.} , 2014). This recruitment of PIP2-7 into the phagosomes could
explain the lower levels of PIP2-7 in the two combined treatments in the
plasma membrane while maintaining the protein levels in all membranes
together (\textbf{Figure 6C} ).

The efficiency of B absorption via passive diffusion may depend on the
sensitivity of the plant to salt stress, which is influenced by the
functionality of AQPs (Bastías \emph{et al.} , 2004). Furthermore, not
only AQPs could influence the transport capabilities of the cell, but
plants also have the ability to remodel membrane lipids, in addition to
protein composition, in plasma membrane to adapt to abiotic stress
scenarios (Rawat \emph{et al.} , 2021). In salinity, an increase in
sterol content is expected, but in this case, only a reduction in total
sterol concentration was observed in microsomal fraction and plasma
membrane, particularly under salinity plus B toxicity stress. More
changes were observed in the plasma membrane fraction, with a general
decrease in sterol content, possibly due to a relocation of sterols to
lower compartments of the plant cell. Under saline conditions, the
permeability of the plasma membrane has been observed to increase in
numerous plant species, such as barley, broccoli, and tomato. This rise
in permeability leads to an elevated leakage of electrolytes being a
consequence of a reduction in the total lipid content, ultimately
resulting in membrane damage (Guo \emph{et al.} , 2019). Salt stress is
known to enhance the processes of lipolysis and lipid peroxidation,
while also inhibiting lipid biosynthesis pathways, which collectively
decrease the overall lipid content in salt-sensitive cultivars.
Alternatively, increased in total sterol content induced by NaCl
treatment were found in salt-adapted tomato calli (Kerkeb \emph{et al.}
, 2001), salt-tolerant wheat (Salama \emph{et al.} , 2007), and the
halophyte \emph{Kosteletzkya virginica} (Blits and Gallagher, 1990). In
contrast, non-tolerant species/genotypes, such as sensitive wheat
cultivar, showed a significant reduction in the amount of sterol lipids
reduced (Salama and Mansour, 2015). Based on this evidence, it has been
proposed that the ability to increase total sterol content under salt
stress may be an important adaptive mechanism in salt-tolerant
species/genotypes (Salama and Mansour, 2015). In this way, as no studies
have been performed with boron, we could indicate that that maintaining
a constant level of sterols in the membrane is essential for plant
tolerance (Salama and Mansour, 2015; Guo \emph{et al.} , 2019)\textbf{.}

\textbf{Conclusion}

Understanding the plant response to salinity and boron stresses is
crucial in developing strategies to overcome these abiotic stresses with
resilient cultivars. In our experiments, we observed that the
combination of boron deficiency and high salinity has a different impact
on plant growth than each stress alone, providing evidence that salinity
is not the predominant stress in our conditions. Additionally, the fact
that some fewer damaging effects were found under combinations leads us
to think that the three stresses applied have strong impact on broccoli
plant physiology, triggering adaptation mechanisms in different
directions than individually. In this way, water pass, in relation to
aquaporins, could be targeted as an avoidance strategy since mineral
nutrients did not appear to change in an avoidance direction. Therefore,
the broccoli plant appeared to modulate aquaporins expression towards
allowing increase water uptake in case of salinity, for reducing B
uptake in case of toxicity and for increasing B uptake in case of
deficiency. However, the individual aquaporin involved in each treatment
differed, pointing to a difficulty of identifying a marker aquaporin
gene since each aquaporin expression changed depending on the individual
or combination treatment, demonstrating the enormous complexity of
aquaporins response.

As the results can be interpreted as a strategy to prevent the stresses
applied, the fact that there are still several gaps in the knowledge
constitute a challenge for further investigation. However, shutting down
or upregulating the transcription of the gene seems not be enough, since
the broccoli leave cells has to deal with trafficking of proteins in
route to the plasma membrane and those already active in the plasma
membrane. In this sense, the regulation of aquaporins in each membrane
fraction need to be addressed, along with the functionality of the
aquaporins according to the lipid and other proteins environment.

\textbf{Data Availability statement}

The authors confirm that all data referred to here are available in the
body of this article and its supplementary materials.

\textbf{Acknowledgments}

This work was supported by the Spanish Ministry of Science and
Innovation with a grant for Juan Nicolas-Espinosa (FPU20/03823) and
Lucia Yepes-Molina (FPU17/02261).

\textbf{Author contributions}

Juan Nicolas-Espinosa: conceptualization, investigation, methodology,
writing-original draft. Lucia Yepes-Molina: conceptualization,
investigation, methodology, writing-original draft. Fuensanta
Martinez-Bernal: investigation. Miriam Fernandez-Pozurama:
investigation. Micaela Carvajal: conceptualization, funding acquisition,
methodology, supervision, validation, writing-original draft.

\textbf{Conflict of interest}

The authors declare that there is no conflict of interest.

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