Aquaporins (AQPs) are intrinsic membrane proteins responsible for facilitating water transport across biological membranes. AQPs found in plant membrane vesicles (MV) have been related to the functionality and stability of the vesicles. In this study, we focused on AQPs obtained from Brassica oleracea var. L. italica (broccoli) by the great potential for different biotechnological applications. To gain further insight into the role of AQPs in MV and advance the biotechnological applications of AQPs, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in the purification of both AQPs with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2, respectively). We reconstituted purified AQPs in liposomes to study their functionality, showing no changes in size compared to liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4oC and 25ºC but not at 37oC, whereas BoPIP1;2 did not enhance water transport across the proteoliposome membrane. Additionally, BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract in proteoliposome vesicles, resulting in increased entrapment efficiency compared to conventional liposomes. Molecular docking identified potential binding sites for resveratrol in PIP2s, highlighting the role of AQPs in the improved entrapment efficiency of resveratrol. Moreover, a modelling study was conducted, demonstrating interactions between a plant AQP and human integrin, which may be a benefit to increase contact and internalization by the human target cells. Thus, our results suggest that AQPs-based alternative encapsulation systems can be used in specifically target biotechnological applications.

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.