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FITC‐dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves
Author(s) -
Hoffmann Bernd,
Kosegarten Harald
Publication year - 1995
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1995.tb00846.x
Subject(s) - apoplast , symplast , xylem , helianthus annuus , biophysics , chemistry , sunflower , transpiration stream , botany , cell wall , biochemistry , biology , transpiration , horticulture , photosynthesis
The liquid in the free space of leaf cell walls, the apoplast, is in direct contact with the plasma membrane and its nutrient uptake systems. Therefore, the pH of the apoplast is of utmost interest. We have elaborated a non‐destructive method by which excised sunflower leaves ( Helianthus annuus cv. Erika) were perfused with fluorescein isothiocyanate‐dextran (FITC‐dextran) (4 000 Da) via the transpiration stream. We showed that leaf apoplast pH can be measured by using the fluorescence ratio technique together in conjunction with this dye. Evidence is provided that FITC‐dextran does not penetrate the plasma membrane over a period of ca 17 h from the beginning of dye perfusion. Dye enrichment in the leaf apoplast did not cause an ‘inner filter effect’ and thus the fluorescence ratio was only dependent on pH. In vivo calibration yielded a pKa of 5.92, which was virtually identical to the pKa of 5.93 calculated for dye solutions. Hence, FITC‐dextran can be detected in complex environments and covers a pH range prevailing in the leaf apoplast. Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells.