D. Brauer et al., SUBCELLULAR COMPARTMENTATION OF DIFFERENT LIPOPHILIC FLUORESCEIN DERIVATIVES IN MAIZE ROOT EPIDERMAL-CELLS, Protoplasma, 192(1-2), 1996, pp. 70-79
Fluorescence microscopy offers some distinct advantages over other tec
hniques for studying ion transport processes in situ with plant cells.
However, the use of this technology in plant cells has been limited b
y our lack of understanding the mechanisms that influence the subcellu
lar distribution of dyes after loading with the lipophilic precursors.
In this study, the subcellular distribution of 5-(and 6-)carboxydichl
orofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compar
ed to that of 2',7'-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein
(BCECF) after incubation of maize roots with their respective lipophil
ic precursors. Previously, we reported that incubation of roots with B
CECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this
dye. Similar results were found when roots were incubated with CDCF-d
iacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the
cytoplasm based on the distribution of fluorescence and the excitation
spectra of the dye in situ. On the other hand, incubation of roots wi
th carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence througho
ut the cell. When the cytoplasm of epidermal cells was loaded with the
BCECF acid by incubation at pH 4 in the absence of external Ca, the d
ye was retained in the cytoplasm at least 3 h after the loading period
. This result indicated that vacuolar accumulation of BCECF during loa
ding of BCECF-AM was not due to transport of BCECF from cytoplasm to v
acuole. The esterase activities responsible for the production of eith
er carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor
by extracts of roots were compared. The characterization of esterase
activities was consistent with the subcellular distribution of these d
yes in root cells. The results of these experiments suggest that in ma
ize root epidermal cells the subcellular distribution of these fluores
cein dyes may be determined by the characteristics of the esterase act
ivities responsible for hydrolysis of the lipophilic precursor.