Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Intact barley (Hordeum vulgare L.) roots have been shown to take up the highly fluorescent dye Lucifer Yellow CH (LYCH) into their cell vacuoles. In the apical 1 cm of root tip, differentiating and dividing cells showed a prolific uptake of LYCH into their provacuoles. The LYCH was retained during fixation, apparently becoming bound to electron-dense material in the vacuoles. The dye freely entered the apoplast of roots in which the Casparian band was not developed, being taken up into the vacuoles of cells in both the cortex and stele. However, when LYCH was applied to a 1-cm zone approx. 6 cm behind the root tip the Casparian band on the radial walls of the endodermis completely prevented the dye from entering the cells of the stele, only the cell walls and vacuoles of the cortical cells taking up the dye. The inability of LYCH to cross the plasmalemma of the endodermal cells and enter the stele via the symplast substantiates previous claims that the dye is unable to cross the plasmalemma of plant cells. The results are discussed in the light of recent demonstrations that LYCH is a particularly effective marker for fluid-phase endocytosis in animal and yeast cells. A calculation of the energetic requirements for LYCH uptake into barley roots supports the contention that LYCH is taken up into the vacuoles of plant cells by fluid-phase endocytosis.Abbreviation LYCH Lucifer Yellow CH     

A simplified paraffin embedding method for small botanical samples

Abstract

We present a simplified paraffin embedding method suitable for unsuberized or unlignified small botanical samples (diameter < 0.3 cm). Only 2 h are required to yield plant tissues embedded in paraffin for anatomical observation and molecular analysis. Our method achieved morphological preservation of cell structures and conservation of nucleic acids that were equivalent to the traditional protocol. Fourier transform infrared spectrometry showed that the degree of degradation of the cytoplasmic components (e.g., protein) resulting from our simplified protocol was similar to that of the traditional protocol. The DNA samples embedded using the simplified method was extractable and could be used for PCR analysis. The DNA quality was equivalent to that embedded using the traditional method.     

Movement of Lucifer Yellow CH in potato tuber storage tissues: A comparison of symplastic and apoplastic transport

The fluorescent dye Lucifer Yellow CH (LYCH) was introduced directly into the symplast of potato (Solanum tuberosum L.) tuber storage parenchyma by microinjection and also into the apoplast through cuts made in the stolon cortex. Microinjected LYCH moved away rapidly from a single storage cell and spread radially via the symplast. When the microinjected tissue was subsequently fixed in glutaraldehyde and sectioned the dye was seen clearly to be localised in the cytoplasm but not in the vacuole. In comparison, when LYCH was introduced into cuts made in the stolon cortex the dye entered the tuber by the xylem and subsequently spread apoplastically. No movement of dye was observed in the phloem. In glutaraldehyde-fixed tissues, in which LYCH was introduced to the apoplast, the dye was found within xylem vessels, in the cell walls and in intercellular spaces. Wall regions, possibly associated with plasmodesmata, became stained by the dye as it moved through the apoplast. Three hours after introduction of the dye to the stolon, intense deposits of LYCH were found in the vacuoles of all cells in the tuber, many aligned along the tonoplast. Differentiating vascular parenchyma elements contained large amounts of dye within enlarging vacuoles. However, with the exception of plasmolysed and-or damaged cells, LYCH was absent from the cytoplasm following its introduction to the plasmalemma it is suggested that the most likely pathway from the cell wall to the vacuole was by endocytosis, the dye being transported across the cytoplasm in membrane-bound vesicles. Clathrin-coated vesicles were abundant in the storage cells, providing a possible endocytotic pathway for dye movement. The significance of these observations is discussed in relation to the movement of LYCH in plant tissues and to the movement of solutes within and between storage cells of the tuber.Abbreviation LYCH Lucifer Yellow CH     

An alginate matrix double-embedding method for paraffin sectioning of minute specimens

A method has been devised for placing minute specimens in viscous aqueous sodium alginate solution, which is polymerized in 0.25 molar calcium chloride. The resulting matrix can then be dehydrated, embedded, and sectioned, maintaining the orientation of the specimens. The sectioned alginate can be dissolved in 0.1 molar EDTA if desired. Tissue sections respond to normal staining procedures.