Molecular and ultrastructural analysis of forisome subunits reveals the principles of forisome assembly.

BACKGROUND AND AIMS: 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. KEY 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.

Uncertain role of MtSEO-F3 in assembly of Medicago truncatula forisomes.

Forisomes are specialized multimeric protein complexes found only in the papilionoid legumes. They undergo a reversible conformational change in response to phloem injury to enable the occlusion of sieve tubes, thus preventing the loss of photoassimilates. The individual subunits are designated by the letters SEO-F (sieve element occlusion by forisomes) and are part of the larger SEO protein family, which also includes the typical P-proteins found in most dicots and some monocots. When specific SEO-F subunits from different species are expressed in a heterologous background, they self-assemble into fully-functional artificial forisomes. However, with the exception of basal species such as Dipteryx panamensis, the geometry of these artificial forisomes differs from that of their native counterparts. Studies involving SEO-F proteins from the model legume Medicago truncatula have shown that a combination of 3 of the 4 subunits can fine-tune the geometry of artificial forisomes. However, MtSEO-F3 was excluded from these studies because it was not incorporated into either the native or artificial forisomes in our original experiments. In this addendum, we present further data concerning the interactive properties of the SEO-F proteins and confirm that all 4 MtSEO-F proteins interact in all possible pairwise combinations. These data indicate that the exclusion of MtSEO-F3 from the compact forisome may reflect the steric hindrance of binding sites rather than an inability to interact with other forisome subunits.