Two-dimensional, high-resolution imaging of pH dynamics in the phyllosphere of submerged macrophyte using a new...

The phyllosphere pH helps shape the plant microbiome and strongly influences aboveground interactions in plant canopies. Yet little is known about the distribution of pH at a microscale within the macrophyte phyllosphere and the factors promoting them because achieving high-resolution quantitative imaging of phyllosphere pH is a great challenge. Here, new ratiometric pH nano-optodes were prepared by firstly encapsulating the self-synthesized lipophilic dyes (8-acetoxypyrene-N¹, N³, N⁶-trioctadecyl-1, 3, 6-tri-trisulfonamide) to poly(1-vinylpyrrolidone-co-styrene) nanoparticles, and then immobilizing the resulting nanoparticles in polyurethane hydrogel on transparent foils. The nano-optodes presented reversible and fast response (t₉₅ < 80 s) to the pH range from 7.0 to 11.0, with merits of good spatial resolution, photobleaching/leaching resistance and negligible cross-sensitives toward temperature, O₂ and ionic strength (< 100 mM). The nano-optodes together with a self-designed phyllosphere chamber were further applied to directly measure the pH distributions at a microscale around single leaves of V. spiralis grown in natural sediment. The pronounced pH microheterogeneity and leaf basification within the V. spiralis phyllosphere were quantitatively visualized. We also provided direct empirical evidence that the dynamic of the phyllosphere pH at high resolution was significantly controlled by the shifting light intensity and temperature. Implementation of the nano-optodes holds great potential for various laboratory applications, which will provide an in-depth insight into phyllosphere activities on the microscale.