Nanocarrier foliar uptake pathways affect delivery of active agents and plant physiological response

Abstract

Layered double hydroxide (LDH) nanoparticles enable foliar delivery of genetic material, herbicides, and nutrients to promote plant growth and yield. Understanding the foliar uptake route of nanoparticles is needed to maximize their effectiveness and avoid unwanted negative effects. In this study, we investigated how delivering layered double hydroxide (d = 37 ± 1.5 nm) through the adaxial (upper) or abaxial (lower) side of leaves affects particle uptake, nutrient delivery, and photosynthesis in tomato plants. LDH applied on the adaxial side was embedded in the cuticle and accumulated at the anticlinal pegs between epidermal cells. On the abaxial side, LDH particles penetrated the cuticle less, but the presence of the stomata enables penetration to deeper leaf layers. Accordingly, the average penetration levels of LDH relative to the cuticle were 2.47 ± 0.07, 1.25 ± 0.13, and 0.75 ± 0.1 μm for adaxial, abaxial with stomata, and abaxial without stomata leaf segments, respectively. In addition, the colocalization of LDH with the cuticle was ∼2.3 times lower for the adaxial application, indicating the ability to penetrate the cuticle. Despite the low adaxial stomata density, LDH-mediated delivery of magnesium (Mg) from leaves to roots was 46% higher for the adaxial than abaxial application. In addition, adaxial application leads to ∼24% higher leaf CO₂ assimilation rate and higher biomass accumulation. The lower efficiency from the abaxial side was, at least partially, a result of interference with the stomata functionality which reduced stomatal conductance and evapotranspiration by 28% and 25%, respectively, limiting plant photosynthesis. This study elucidates how foliar delivery pathways through different sides of the leaves affect their ability to deliver active agents into plants and consequently affect the plants' physiological response. That knowledge enables a more efficient use of nanocarriers for agricultural applications.

Fig. 1. S-LDH and L-LDH characterization and surface modification. (a) Size distribution by DLS measurements. (b) XRD spectra. TEM images of (c) L-LDH and (d) S-LDH. (e) Metal ratio description of S-LDH obtained by single particle ICP-TOF-MS. The red square represents the value obtained by ICP-MS. (f) ATR spectrum of S-LDH-ssDNA-Cy3 in comparison to the S-LDH.

Fig. 2. Attachment of LDH with tomato abaxial and adaxial leaf surfaces. (a) Comparison between L-LDH and Tb³⁺ salt applied from either abaxial or adaxial sides. Statistical analysis was done using one-way ANOVA followed by Tukey test, significance level for * p < 0.01, n = 4. (b) S-LDH distribution on leaf surface from adaxial and abaxial sides obtained by confocal microscopy.

Fig. 3. LDH internalization pathways into tomato leaf. (a and b) Orthogonal display of the z-stack imaging by confocal microscopy, blue, red, and green represent the cuticle, S-LDH, and chloroplast, respectively. (a) Abaxial application- cuticle section (upper) and stomata section (lower, white arrow points to S-LDH that internalized through the stomata). (b) Adaxial application-cuticle section (upper, yellow arrows point to the anticlinal pegs) and stomata section (lower), scale bar = 10 μm. (c and d) Parameters describe the localization of S-LDH in relation to the cuticle as obtained by 3D volume rendering. (c) The distribution of the relative overlapped volume of S-LDH particles within the cuticle (ratio of 0–0.5 related to particles located mostly below the cuticle). (d) The distribution of the shortest distance of S-LDH particles in relation to the cuticle (negative to zero values represent particles embedded in the cuticle, while positive values represent the penetration depth compared to the cuticle). (e) An illustration of S-LDH penetration through the cuticle of the adaxial side and accumulation in the anticlinal pegs as suggested by the confocal images.

Fig. 4. Effect of adaxial and abaxial applications of S-LDH (2.5 g L⁻¹) on (a) weight accumulation. (b) An illustration of the plant sections that were tested. (c) Mg concentration in selected organs under conditions of Mg deficiency. Statistical analysis for graphs a and c was done using one-way ANOVA followed by the Tukey test. For graph a, significance level for * p < 0.05, and ** p < 0.1, n = 5. For graph c, * p < 0.07, n = 5.

Fig. 5. Effect of S-LDH application on leaf gas exchange and photosynthesis parameters. (a) Carbon dioxide assimilation rate, (b) transpiration rate, (c) and stomatal conductance compared between adaxial and abaxial application of S-LDH at 0.15 g L⁻¹, light intensity of 1200 μmol m⁻² s⁻¹ (d) CO₂ assimilation rate as a function of light intensity (PAR) and S-LDH concentration, application from the abaxial side. Statistical analysis was done using a t-test for graph a–c and using one-way ANOVA followed by the Fisher LSD test for graph d (comparison to the control). Significance level for * p < 0.01, ** p < 0.05 *** p < 0.07, n = 4 or 5, and errors represent SD.