(461) Endocytic Uptake of Quantum Dots and Endocytic Markers into `Sweet Lime' Juice Cells

Abstract

To determine whether the mechanisms of sucrose accumulation into the low acid `Sweet Lime' (Citrus limmetioides Blanco) juice cells are consistent with those previously reported for the more acidic cultivars, we followed similar developmental changes in determinants of sink strength. In addition, we followed the incorporation and distribution of quantum dots and fluorescent endocytic probes into the cell with time of incubation. As in other citrus fruits, sucrose levels, sucrose synthase, sucrose phosphate synthase, and sucrose phosphate phosphatase increased throughout fruit development. The pH however, was much higher than in the more acidic cultivars. Sucrose uptake into energized plasmalemma vesicles was inhibited by gramicidin, in accordance with the presence of an active symport mechanism of sucrose from the apoplast into the cytosol. On the contrary, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. In conformity with recent findings showing the occurrence of an endocytic mechanism in `Murcott' mandarin, `Sweet Lime' juice cells were shown to incorporate membrane-impermeable dyes into their vacuoles in the presence of sucrose. High-definition confocal microscopy revealed the co-localization of membrane-impermeable markers in cytoplasmic vesicles, in membrane-bound intermediate structures such as the endosome and multi-vesicular body, and the eventual distribution of such fluorescent particles. The data provide strong evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole and for the visualization of intermediate distribution and cargo centers in the cell.