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(NicolasâEspinosa 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
(MartĂnez-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.