Introduction
The Brassicaceae family includes broccoli (Brassica oleracea L. var 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 (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 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. 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 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 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 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 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 et al. , 2018; Morel 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 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 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 NH3), boric acid, H2O2, and CO2(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 (Martínez-Ballesta 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 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 et al. , 2005; Horie et al. , 2011; Katsuhara et al. , 2011). However, in some cases, such as radish seedlings, the mRNA and protein levels of PIPs and TIPs remain unchanged (Suga et al. , 2002). Conversely, an increase in PIP certain isoforms expression has been observed under saline stress in Arabidopsis (Jang et al. , 2004; Sutka et al. , 2011), Brassica juncea (Srivastava et al. , 2010), and Brassica rapa(Kayum et al. , 2017). 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 (Liet al. , 2011), but also AtPIP2;2 and AtPIP2;7 were permeable to boric acid (Groszmann 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 (Kissoudiset 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.