Isolation of guard cell protoplasts from mechanically prepared epidermis of Vicia faba leaves

Journal Article · Tue Aug 01 04:00:00 EDT 1989 · Plant Physiology; (USA)

DOI:https://doi.org/10.1104/pp.90.4.1382· OSTI ID:6874031

Kruse, T; Tallman, G; Zeiger, E ^[1]

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1. Pepperdine Univ., Malibu, CA (USA) Univ. of California, Santa Cruz (USA)

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl{sub 2}, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl{sub 2}, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl{sub 2}, PVP, pepstatin A, cellulase Onozuka RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained {le}0.017% mesophyll protoplasts, {le}0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 {times} 10{sup 4} protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl{sub 2}. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the M peak usually associated with a functional photosynthetic carbon reduction pathway.

• OSTI ID: 6874031
• Journal Information: Plant Physiology; (USA), Journal Name: Plant Physiology; (USA) Vol. 90:4; ISSN 0032-0889; ISSN PLPHA
• Country of Publication: United States
• Language: English

Related Subjects

550300* -- Cytology
59 BASIC BIOLOGICAL SCIENCES
ANIMAL TISSUES
BODY
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHLOROPLASTS
CYTOLOGICAL TECHNIQUES
DYES
ELECTRON TRANSFER
EPIDERMIS
EPITHELIUM
FLUORESCENCE
LEGUMINOSAE
LUMINESCENCE
MAGNOLIOPHYTA
MAGNOLIOPSIDA
ORGANS
PHOTOCHEMICAL REACTIONS
PHOTOSYNTHESIS
PLANTS
REMOVAL
SAMPLE PREPARATION
SKIN
SYNTHESIS
TISSUES
VICIA