Chitosan-Modified Polyethyleneimine Nanoparticles for Enhancing the Carboxylation Reaction and Plants' CO2 Uptake

Abstract

Increasing plants' photosynthetic efficiency is a major challenge that must be addressed in order to cover the food demands of the growing population in the changing climate. Photosynthesis is greatly limited at the initial carboxylation reaction, where CO₂ is converted to the organic acid 3-PGA, catalyzed by the RuBisCO enzyme. RuBisCO has poor affinity for CO₂, but also the CO₂ concentration at the RuBisCO site is limited by the diffusion of atmospheric CO₂ through the various leaf compartments to the reaction site. Beyond genetic engineering, nanotechnology can offer a materials-based approach for enhancing photosynthesis, and yet, it has mostly been explored for the light-dependent reactions. In this work, we developed polyethyleneimine-based nanoparticles for enhancing the carboxylation reaction. We demonstrate that the nanoparticles can capture CO₂ in the form of bicarbonate and increase the CO₂ that reacts with the RuBisCO enzyme, enhancing the 3-PGA production in in vitro assays by 20%. The nanoparticles can be introduced to the plant via leaf infiltration and, because of the functionalization with chitosan oligomers, they do not induce any toxic effect to the plant. In the leaves, the nanoparticles localize in the apoplastic space but also spontaneously reach the chloroplasts where photosynthetic activity takes place. Their CO₂ loading-dependent fluorescence verifies that, in vivo, they maintain their ability to capture CO₂ and can be therefore reloaded with atmospheric CO₂ while in planta. Our results contribute to the development of a nanomaterials-based CO₂-concentrating mechanism in plants that can potentially increase photosynthetic efficiency and overall plants' CO₂ storage.

Keywords: CO2 capture; chitosan; nanoparticles; photosynthesis; polyethyleneimine.

Figure 1

Infiltration of PEI-Chi in Nicotiana tabacum (tobacco) plants and evaluation of its phytotoxicity (A) Functionalization of PEI with oligochitosan by reductive amination. (B) Scheme of the infiltration method: a solution contained in a needleless syringe is infiltrated in the abaxial part of a leaf by gently applying pressure. The solution is pushed in the intercellular space existing between the cells’ membrane called apoplast (created with BioRender.com). (C) Comparison of the effects of the infiltration of PEI (above) and PEI-Chi (below) on the tobacco leaf. PEI infiltration leads to a quick damage in the infiltrated area and around it while the infiltration of PEI-Chi does not show any visible sign of toxicity even one month after infiltration (Figure S14). The insets on the right show fluorescent imaging obtained after staining of the samples with propidium iodide, a cationic dye that does not cross intact membranes but binds to cell walls, forming an outline of living cells. Scale bar: 50 μm.

Figure 2

Functionalization of PEI-Chi for fluorescent detection in vivo and CO₂ uptake emission dependence. (A) Scheme of the ammonium bicarbonate formation from the reaction between amine and CO₂ in aqueous environment (PEI contains primary, secondary, and tertiary amines and all three kinds can react with CO₂. We here used primary amines only as a showcase). (B) APT NMR spectra of PEI-Chi in D₂O before and after bubbling carbon dioxide. After bubbling, strong peaks corresponding to hydrogen carbonate and CO₂ appear. (C) Absorption and (D) excitation and emission spectra of gPEI-Chi (0.05 mg/mL) in water before and after saturating the solution with carbon dioxide.

Figure 3

Evaluation of 3-phosphoglycerate production using gPEI-Chi as a CO₂-giver to activate RuBisCO. (A) Simplified Calvin cycle showing the importance of RuBisCO as catalyst for the conversion of RuBP to 3-PGA using CO₂ (created with BioRender.com). (B) Either CO₂ gas or N₂ gas, to remove CO₂, was bubbled for 30 min in 10 mL of either 1 mg/mL of gPEI-Chi or distilled water. The solutions loaded with CO₂ were then used as substrates for the carboxylation reaction between RuBP and CO₂ catalyzed by RuBisCO and compared to their equivalent without CO₂ treatment and to NaHCO₃ (2 mM) as a carbon source for positive control. The amount of 3-PGA produced by the reaction was then analyzed by LC-MS. Bars indicate the standard errors (n = 3). Treatments not labeled by the same letter are significantly different. Tukey-Kramer HSD, P-value < 0.001. JMP Pro software was used to run the analysis.

Figure 4

Confocal microscopy imaging to evaluate the chloroplast accessibility to gPEI-Chi-CO₂. Imaging of column (A) a control tobacco leaf, column (B) and (C) a leaf infiltrated with gPEI-Chi-CO₂. The top red panel (i) shows the autofluorescence of the chlorophyll, the middle green panel (ii) shows the gPEI-Chi-CO₂ fluorescence, and the panel (iii) is a merging of the two previous. The graphs (A-iv) and (B-iv) are emission spectra of the circles in the merged images, obtained with a monochromator, and serve to evaluate the spatial distribution of the gPEI-Chi-CO₂ nanoparticles in respect to the chlorophyll. The schematic (C-iv) represents the structure of a chloroplasts containing thylakoids forming stacks of disks (grana), which are the sites of photosynthetic reactions and contain the chlorophyll (created with BioRender.com). Scale bar: 10 μm.

Figure 5

In vivo CO₂ uptake of gPEI-Chi. (A) Scheme of the experimental setup where a leaf, still attached to its plant, is isolated in a CO₂ incubator and imaged with fluorescence microscopy (created with BioRender.com). (B) Fluorescence microscopy of Nicotiana tabacum (tobacco) leaves infiltrated with gPEI-Chi, with or without incubation in a CO₂-rich environment for 2 h, (n = 4 plants in each condition). Scale bar: 50 μm. (C) Fluorescence intensity evolution under atmospheric CO₂ conditions or in a CO₂-rich environment. Bars indicate the standard errors (n = 4 plants).