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Direct covalent linkage of fluorescent probes to the plant protoplast surface

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Summary

Methods for fluorescently labeling the surface of live plant protoplasts are presented and characterized. Two classes of chemical reactions were exploited to accomplish direct covalent attachment of reactive fluorophores to plasma membrane components. One class of reactions involved the derivatization of existing amino-groups on the protoplast surface. The other class of reactions involved a two-step procedure to derivatize surface carbohydrates. In the first step, aldehyde groups were generated by oxidation of surface carbohydrates with either periodate or galactose oxidase. The second step involved formation of a Schiff base linkage between the aldehydes and a hydrazide or aromatic amine on a fluorophore. Both classes of reactions were demonstrated with several fluorophores including fluorescein, rhodamine, and Lucifer yellow. The reactions were carried out under conditions that allowed good protoplast survival while producing a high ratio of surface fluorescence to cytoplasmic fluorescence. The reaction products were characterized to the extent of determining whether the fluorophores were incorporated into proteins or into lipids. These fluorescence-labeling procedures should prove useful in a variety of cell biological and biochemical applications.

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Abbreviations

FITC:

fluorescein-5-isothiocyanate

TexR:

Texas Red

LRB-SC:

Lissamine rhodamine B sulfonyl chloride

DTAF:

dichlorotriazinylaminofluorescein

XRITC:

substituted rhodamine isothiocyanate

RBITC:

rhodamine B isothiocyanate

LY-VS:

Lucifer yellow VS

FTSC:

fluorescein-5-thiosemicarbazide

LY-CH:

Lucifer yellow CH

TRH:

Texas red hydrazide

FA:

fluorescein amine, isomer II

FLUOR:

fluorophore

PPLST:

protoplast

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Author notes

  1. Vesna Furtula

    Present address: Institute of Physical Chemistry, University of Belgrade, P.O Box 550, 11001, Beograd, Yugoslavia

Authors and Affiliations

  1. Department of Botany and Plant Sciences, University of California, 92521, Riverside, CA, USA

    Vesna Furtula, R. M. Walko & E. A. Nothnagel

Authors
  1. Vesna Furtula
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  2. R. M. Walko
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  3. E. A. Nothnagel
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Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.

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Furtula, V., Walko, R.M. & Nothnagel, E.A. Direct covalent linkage of fluorescent probes to the plant protoplast surface. Protoplasma 139, 117–129 (1987). https://doi.org/10.1007/BF01282282

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  • DOI: https://doi.org/10.1007/BF01282282

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