Nanofertilization is postulated as a new technology to deal with the environmental problems caused by the intensive use of traditional fertilizers. One of the aims of this new technology is to improve foliar fertilization, which has many environmental advantages but currently there are numerous factors that limit its efficiency. In this research, the objective was to study the potential of membrane vesicles derived from plant material as nanofertilizers of iron (Fe) and boron (B) for foliar application in almond trees (Prunus dulcis L.). The results show that the application of vesicles caused invaginations in the plasma membrane of the leaf cells. Also, the increase in leaf B and Fe was greater when these elements were applied in an encapsulated form rather than in a non-encapsulated form. The distribution of these elements in leaf tissues indicated the existence of an intracellular element transport pathway and accumulation areas providing greater element entrance and mobility.

1 Experiment under controlled conditions

Clones of the Prunus dulcis L. variety Avijor were acquired from Jodar nurseries S.L. in Murcia (Spain). The plants were grown for 15 days in a substrate. They were then transferred to 12-L boxes (5 plants in each of 8 boxes, = 40 plants) filled with Hoagland solution, pH 5.5. The solution was continuously aerated and was changed every week. The composition of the solution was: 6 KNO₃, 4 Ca(NO₃)₂, 1 KH₂PO₄, and 1 MgSO₄ (mM), and 25 H₃BO₃, 2 MnSO₄, 2 ZnSO₄, 0.5 CuSO₄, 0.5 (NH₄)₆Mo₇O₂₄, and 20 Fe-EDDHA (μM). The plants were grown in a culture chamber with controlled conditions; a cycle of 16 h of light and 8 h of darkness, with a temperature of 25 and 20 °C and relative humidity of 70% and 80%, respectively. The photosynthetically active radiation (PAR) was 400 μmol m⁻² s^-1, provided by fluorescent tubes (Philips TLD 36 W/83, Jena, Germany and Sylvania F36 W/GRO, Manchester, NH, USA) and metal halide lamps (Osram HQI, T 400W, Berlin, Germany). After 10 days, deficiency treatments were applied: 2 boxes continued with full nutrient solution, 3 boxes had Fe deficiency (0 μM Fe added), and 3 boxes had B deficiency (0 μM B added). The plants were grown under these conditions until deficiency symptoms appeared progressively in the young leaves.

Once the deficiency symptoms had appeared, treatments were applied to young leaves growing under deficiency conditions, as far as the second leaves under the meristematic apex (that were considered not totally developed). Foliar applications were carried out for leaves that had expanded under B or Fe deficiency, avoiding the meristematic area. The treatments were applied approximately 2 h after the onset of the light period in the growth chamber. The experiment had a completely randomized design and was repeated three times. The solutions applied were: (i) 0.02% Fe (from FeSO₄), nanoencapsulated, (ii) 0.02% free Fe (from FeSO₄), (iii) 0.04% B (from H₃BO₃), nanoencapsulated, (iv) 0.04% free B (from H₃BO₃). In the control treatment, water was applied. All the treatments were applied with 0.1% of surfactant [27]. The treatments were applied to the abaxial side of the leaves of each plant using a sprayer. Leaf samples were taken for mineral and microscopic analysis 60 min and 24 h after the application.

2 Experiment under field conditions

Trees (15) from three different experimental fields - between 400 and 534 m above sea level and subjected to a Mediterranean climate - were selected for this experiment. The foliar treatments applied were: (i) 0.02% Fe (from FeSO₄), nanoencapsulated, (ii) 0.02% free Fe (from FeSO₄), (iii) 0.04% B (from H₃BO₃), nanoencapsulated, (iv) 0.04% free B (from H₃BO₃). In the control treatment, water was applied. All the treatments were applied with 0.1% of surfactant. To randomize the treatments, 5 branches of each tree were selected around all the cardinal points. Each of the 5 treatments was applied randomly to each of the selected branches of each tree (3 trees per treatment). The orientation of each applied treatment was varied in each tree, to avoid the cardinal effect. These applications were performed in the early morning, by spraying the fertilizers on leaves until they were completely wet. Leaf samples were taken for mineral analyses 24 h after the application.

3 Vesicles isolation

Leaves (100 g) were cut into small pieces before vacuum filtering with 0.5 g of PVP and 160 ml of buffer containing 0.5 M sucrose, 1 mM DTT, 50 mM HEPES, and 1.37 mM ascorbic acid, at pH 7.5. Then, the sample was homogenized using a blender and filtered through a nylon mesh with a pore diameter of 100 μm. The filtrate was centrifuged at 10,000 x g for 30 min, at 4 ºC. The supernatant was recovered and centrifuged for 35 min at 100,000 x g, at 4 ºC. Afterwards, the pellet obtained was suspended in 500 μl of 5 mM phosphate buffer and 0.25 M sucrose, at pH 6.5. The protein concentration in the vesicles fraction was determined with an RC DC Protein Assay kit (BioRad, California, USA), using BSA as the standard.

4 Encapsulation

The Fe (as FeSO₄) and B (as H₃BO₃) were dissolved at concentrations of 2% and 4%, respectively, in distilled water. These solutions were vortexed for 30 s with the vesicles fraction (1:1 v:v) and were reconstituted for 10 min.

The percentage of each element encapsulated in the vesicles was determined by the direct measurement of the Fe or B concentration in the original solution and in the supernatant after centrifugation of the vesicles of the nanofertilizer.

5 Size and charge of vesicles

The average size (nm), polydispersity index (0 – 1), and Z potential (mV) of the vesicles containing Fe or B were checked using Dynamic Light Scattering (DLS), through intensity measurements with a Malvern ZetaSizer Nano XL machine (Malvern Instruments Ltd., Orsay, France). This allowed the analysis of particles with a size range from 1 nm to 3 μm.

6 Osmotic water permeability (Pf)

This parameter was measured as the velocity of the volume adjustment of the plasma membrane vesicles after changing the osmotic potential of the surrounding medium. The volume of the vesicles was followed by 90° light scattering at λex = 515 nm. The samples used were vesicles with encapsulated Fe or B. The measurements were carried out at 20°C in a PiStar-180 Spectrometer (Applied Photophysics, Leatherhead, UK), as described previously by [28].

7 Ion concentrations

For ion analysis, finely-ground samples of lyophilized leaves were digested in a microwave oven (CEM Mars Xpress, North Carolina, USA), by HNO₃:HClO₄ (2:1) digestion. The elements were detected by inductively coupled plasma (ICP) analysis (Optima 3000, PerkinElmer).

The concentrations of ions in the vesicles were determined after centrifugation of the vesicle preparations. The pellets obtained were dried and digested and the ions were determined by ICP, as described previously.

11 Statistical analysis

The statistical analysis of the ion concentrations measured in the leaves of plants grown under controlled conditions was carried out with 105 values (5 plants x 7 treatments x 3 analytical replicates). For the field conditions experiment, the analysis involved 180 values (15 plants × 4 treatments x 3 analytical replicates). All values were analyzed by one-way analysis of variance (ANOVA), at the 95% confidence level, using the software SPSS Release 18 for Windows (SPSS Inc., Chicago, IL, USA). The statistical significance was considered as: *P < 0.05; **P < 0.01; ***P < 0.001; and n.s. - not significant. Also, Duncan’s test at P ≤ 0.05 was chosen to determine the significance of differences between treatments. The values presented are the means ± SE.