Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness. (16th July 2020)

• Record Type: Journal Article
• Authors: Liu, Yan; Peters, Winfried S; Froelich, Daniel R; Howell, Alexander H; Mooney, Sutton; Evans, James E; Hellmann, Hanjo A; Knoblauch, Michael
• Is Part Of: Plant & cell physiology. Volume 61:Number 10(2020)
• Page Start: 1699
• Page End: 1710
• Publication Date: 2020-07-16
• Subjects: Ca2+-dependent contractility -- Forisome -- Phloem -- SEO protein -- SEOR protein -- Sieve element; Plant physiology -- Periodicals; Microbiology -- Periodicals; Cytology -- Periodicals; Cell Physiology -- Periodicals; Plant Physiological Phenomena -- Periodicals; Cytology; Microbiology; Plant physiology; Periodicals; 571.205
• DOI: 10.1093/pcp/pcaa093
• Languages: English
• ISSNs: 0032-0781

Abstract

Abstract: Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca 2+ -controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca 2+ -dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca 2+ -binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca 2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast ( Saccharomyces cerevisiae ) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca 2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca 2+ -controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca 2+ -dependent contractility.