GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

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.

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

“…Most of these herbivores have distinct geographical distributions, resulting in divergent herbivore communities across latitudinal (Züst et al, 2012), longitudinal (Kozlov, 2008), and altitudinal gradients (Pélissier et al, 2008). The pronounced geographical heterogeneity of herbivore communities coincides with considerable variation in herbivore resistance and expression of defensive traits in many plants (Johnson, 2011).…”

Sieve element occlusion(SEO) genes encode structural phloem proteins involved in wound sealing of the phloem

“…Thus far, four sieve element occlusion (SEO) gene family members have been identified in Medicago truncatula (MtSEO-F1-F4) and Glycine max (GmSEO-F1-F4) and characterized as encoding forisome subunits, which assemble into a special type of crystalloid P-protein found uniquely in Fabaceae (23)(24)(25)(26)(27)(28). Forisomes differ from conventional P-proteins because they can undergo a reversible switch between condensed spindle-shaped and dispersed plug-forming states, triggered by a calcium influx (29,30).…”

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…Light and confocal laser scanning microscopy studies have demonstrated that forisomes, crystalloid P-proteins found in Vicia faba and other faboid legumes, control the conductivity of sieve tubes (Knoblauch et al, 2001(Knoblauch et al, , 2003. The components of the forisomes have been identified and appear to be proteins unique to faboid legumes, although some of the motifs they contain have been identified in proteins from other plant taxa (Noll et al, 2007;Pelissier et al, 2008). These findings demonstrate a role of forisome-type P-proteins in the occlusion of sieve tubes in the Fabaceae, but the function of the P-proteins present in other species remains unclear.…”