Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

Elżbieta Kuta,Przemysław Malec,Ulf Göransson,Blazej Slazak,Małgorzata Kapusta,Jerzy Bohdanowicz,Sohaib Malik

doi:10.1007/s00425-016-2562-y

Elżbieta Kuta, Przemysław Malec + Show 5 more

Open Access

https://doi.org/10.1007/s00425-016-2562-y

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Abstract

Main conclusion The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique’s resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense—relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2562-y) contains supplementary material, which is available to authorized users.

Highlights

Cyclotides are head-to-tail cyclic plant peptides consisting of approximately 30 amino acid residues with a characteristic motif known as the cyclic cystine knot (Craik et al 1999; Goransson and Craik 2003)
Antibodies bind to bracelet type cyclotides with high specificity and efficiency Polyclonal antibodies were raised in rabbits using a mixture of cycloviolacin O2 (cyO2) and cyO2 conjugated to the adjuvant keyhole limpet hemocyanin (KLH)
Antibodies bound with high efficiency to the cycloviolacin cyclotides in general, and cyO2 in particular

Summary

Introduction

Cyclotides are head-to-tail cyclic plant peptides consisting of approximately 30 amino acid residues with a characteristic motif known as the cyclic cystine knot (Craik et al 1999; Goransson and Craik 2003). Two main subfamilies of cyclotides can be distinguished based on their amino acid sequences: the Mobius and the bracelets (Fig. 1a). The Violaceae, is rich in cyclotides, which appear to be expressed in all members of this plant family (Nguyen et al 2011; Poth et al 2012; Gerlach et al 2013; Nguyen et al 2013; Burman et al 2014, 2015; Hellinger et al 2015). While the details of the biosynthesis and functions of these peptides are gradually being revealed, we still lack fundamental knowledge about their localization and distribution in planta

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