Molecular and ultrastructural analysis of forisome subunits reveals the principles of forisome assembly.
OBJECTIVE
Forisomes are specialized structural phloem proteins that mediate sieve element occlusion after wounding exclusively in papilionoid legumes, but most studies of forisome structure and function have focused on the Old World clade rather than the early lineages. A comprehensive phylogenetic, molecular, structural and functional analysis of forisomes from species covering a broad spectrum of the papilionoid legumes was therefore carried out, including the first analysis of Dipteryx panamensis forisomes, representing the earliest branch of the Papilionoideae lineage. The aim was to study the molecular, structural and functional conservation among forisomes from different tribes and to establish the roles of individual forisome subunits.


METHODS
Sequence analysis and bioinformatics were combined with structural and functional analysis of native forisomes and artificial forisome-like protein bodies, the latter produced by expressing forisome genes from different legumes in a heterologous background. The structure of these bodies was analysed using a combination of confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the function of individual subunits was examined by combinatorial expression, micromanipulation and light microscopy.


RESULTS
Dipteryx panamensis native forisomes and homomeric protein bodies assembled from the single sieve element occlusion by forisome (SEO-F) subunit identified in this species were structurally and functionally similar to forisomes from the Old World clade. In contrast, homomeric protein bodies assembled from individual SEO-F subunits from Old World species yielded artificial forisomes differing in proportion to their native counterparts, suggesting that multiple SEO-F proteins are required for forisome assembly in these plants. Structural differences between Medicago truncatula native forisomes, homomeric protein bodies and heteromeric bodies containing all possible subunit combinations suggested that combinations of SEO-F proteins may fine-tune the geometric proportions and reactivity of forisomes.


CONCLUSIONS
It is concluded that forisome structure and function have been strongly conserved during evolution and that species-dependent subsets of SEO-F proteins may have evolved to fine-tune the structure of native forisomes.
Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, 48143 Münster, Germany.
Boje
B
Müller
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schlossplatz 8, 48143 Münster, Germany.
Sira
S
Groscurth
Fraunhofer Institute for Mechanics and Materials IWM, Walter-Hülse-Strasse 1, 06120 Halle, Germany.
Matthias
M
Menzel
Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, 48143 Münster, Germany.
Boris A
BA
Rüping
TRM Ltd, PO Box 93, York YO43 3WE, UK.
Richard M
RM
Twyman
Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, 48143 Münster, Germany Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schlossplatz 8, 48143 Münster, Germany dpruefer@uni-muenster.de.
Dirk
D
Prüfer
Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, 48143 Münster, Germany.
Gundula A
GA
Noll
D000595:Amino Acid Sequence; D019295:Computational Biology; D007887:Fabaceae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D016415:Sequence Alignment
D010940:Plant Proteins
Canavalia gladiata; Dipteryx panamensis; Fabaceae; Forisome evolution; Lotus japonicus; Medicago truncatula; Papilionoideae; Pisum sativum; SEO; Vicia faba; macromolecular assembly; papilionoid legumes; sieve element occlusion gene family; structural phloem protein