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).