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 et
al. , 2011). Our results showed that the only treatment that affected
plant (root and shoot) dry weight (DW) (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 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
CO2 impact in LMA, but there is less knowledge about how
LMA responds to stresses like salinity (Poorter 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 et al. , 2022; Poorteret al. , 2009). Our results indicate that only salinity treatment
increased LMA (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
(Figure 2 ). 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 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 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 inVitis vinifera were its stomatal conductance changed after 14
days of NaCl treatments applications (Gunes et al. , 2006), in the
same way it had been reported that B toxicity reduced stomatal
conductance in Arabidopsis plants (Macho-Rivero et al. , 2017).
As shown in 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 (Figure 2 ), as B is commonly transported through
the xylem (Yermiyahu et al. , 2008). This hypothesis has been
previously tested in other plants, including wheat (Holloway and Alston,
1992), melon (Edelstein 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+ movement within the plant is
closely related to K+, the salinity treatment also
impacted K+ levels, reducing them compared to control
plants. Interestingly, B deficiency also reduced K+levels in the leaves of B-deficient plants compared to the control.
Although the decrease in K+ 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+, but its deficiency somehow lowered
K+ levels. However, it had been shown that B supply
does not influence the K+ content to a major extent,
as it is not involved in the K+ uptake pathway (Wu and
Wei-Hua, 2013).
It is well known that Ca2+ 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 (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+/Na+ balance at membranes (Mohamedet 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 Ca2+ transport (Bastías 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+ competition and high salinity scenarios has been
also described to inhibit Mg2+ (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 et al. , 2008). Similarly,
boron toxicity can also reduce stomatal conductance and limit the uptake
of boron in plants (Barzana 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 et al. , 2002).
Based on Figure 4 , it was observed that only B (-) treatment
had a significant impact on the PIP2 group, resulting in decreased
levels of PIP2-1 , PIP2-2/3 , and PIP2-7 transcripts.
B (+) treatment also led to a lower presence of PIP2-1transcripts 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 (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 et
al. , 2016), while AQPs inactivate to prevent B leakage though these
channels. Additionally, the salinity treatment up-regulated the
expression of 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 PIP1-4 , PIP2-3 ,PIP2-4 , and PIP2-2 were over-expressed after 3 and 6 days
of the treatment application (Kumawat et al. , 2021). In contrast
to the Brassica rapa PIP genes, which were first up-regulated
during salt stress and them down-regulated (Kayum 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 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 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 HKT1and SOS1 , transporters that contribute to Na+efflux and K+ absorption, respectively, (Horieet al. , 2009), to improve tolerance to salt stress and maintain
cell ion homeostasis in salinity-stressed transgenic plants (Chenet 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
(Figure 6F ). Even though, no changes in PIP2-7expression 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 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 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 (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 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 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 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 et al. ,
2001), salt-tolerant wheat (Salama et al. , 2007), and the
halophyte 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 et al. , 2019).