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Plant and Cell Physiology Advance Access originally published online on September 10, 2008
Plant and Cell Physiology 2008 49(11):1699-1710; doi:10.1093/pcp/pcn141
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
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GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

Hélène C. Pélissier1, Winfried S. Peters2, Ray Collier1, Aart J. E. van Bel3 and Michael Knoblauch1,*

1 School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
2 Indiana/Purdue University Fort Wayne, Department of Biology, 2101 East Coliseum Boulevard, Fort Wayne, IN 46805-1499, USA
3 Institut für Allgemeine Botanik, Justus-Liebig-Universität, Senckenbergstr. 17–21, D-35390 Gießen, Germany

*Corresponding author: E-mail, Knoblauch{at}wsu.edu; Fax, +1-509-335-4848.


  Abstract

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

Keywords: Ca2+-dependent contractility - Composite plant - Fabaceae - Forisome - Phloem-specific protein - Sieve element occlusion (SEO) protein

Abbreviations: BAC, bacterial artificial chromosome; CC, companion cell; CD, conserved domain; FOR, forisome-related protein; GFP, green fluorescent protein; NRX, nucleoredoxin; ORF, open reading frame; PP, phloem-specific protein; RT–PCR, reverse transcription–PCR; SE, sieve element; SEO, sieve element occlusion; TryX, tryparedoxin.

The nucleotide sequences reported in this paper have been submitted to GenBank data libraries under accession numbers: EU938018 [GenBank] (MtSEO1), EU938017 [GenBank] (MtSEO2) and EU938016 [GenBank] (MtSEO3).

(Received August 4, 2008; Accepted September 6, 2008)
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