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

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

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.

Proteins from the FLOWERING LOCUS T-like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco.

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine-binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case-by-case basis. We isolated four novel FT-like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild-type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short-day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT-like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long-day conditions, suggesting that all four tobacco FT-like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT-like clade has led to the evolution of antagonistic regulators that may help to fine-tune floral initiation in response to environmental cues.

Native and artificial forisomes: functions and applications.

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca(2+), thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future.

Molecular and phylogenetic characterization of the sieve element occlusion gene family in Fabaceae and non-Fabaceae plants.

BACKGROUND: The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae. RESULTS: We performed a comprehensive genome-wide comparative analysis by screening the M. truncatula, Glycine max, Arabidopsis thaliana, Vitis vinifera and Solanum phureja genomes, and a Malus domestica EST library for homologs of MtSEO1, MtSEO2 and MtSEO3 and identified numerous novel SEO genes in Fabaceae and even non-Fabaceae plants, which do not possess forisomes. Even in Fabaceae some SEO genes appear to not encode forisome components. All SEO genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with Fabaceae-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. SEO genes are organized in genomic clusters and the conserved synteny allowed us to identify several M. truncatula vs G. max orthologs as well as paralogs within the G. max genome. CONCLUSIONS: The unexpected occurrence of forisome-like genes in non-Fabaceae plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.

Recombinant artificial forisomes provide ample quantities of smart biomaterials for use in technical devices.

Forisomes are mechanoproteins that undergo ATP-independent contraction-expansion cycles triggered by divalent cations, pH changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4) can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes. These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts and used as components of microscale and nanoscale devices.

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.

Characteristics of artificial forisomes from plants and yeast.

Forisomes are protein bodies found exclusively in the phloem of the Fabaceae (legumes). In response to wounding, the influx of Ca ( 2+) induces a conformational change from a condensed to a dispersed state which plugs the sieve tubes and prevents the loss of photoassimilates. This reversible, ATP-independent reaction can be replicated with purified forisomes in vitro by adding divalent cations or electrically inducing changes in pH, making forisomes ideal components of technical devices. Although native forisomes comprise several subunits, we recently showed that functional homomeric forisomes with distinct properties can be expressed in plants and yeast, providing an abundant supply of forisomes with tailored properties. Forisome subunits MtSEO-F1 and MtSEO-F4 can each assemble into homomeric artificial forisomes, which indicates functional redundancy. However, we provide further evidence that both proteins are subunits of the native heteromeric forisome body in planta. We also show that the properties of artificial forisomes can be modified by immobilization, which is a prerequisite for their incorporation into technical devices.

The sieve element occlusion gene family in dicotyledonous plants.

Sieve element occlusion (SEO) genes encoding forisome subunits have been identified in Medicago truncatula and other legumes. Forisomes are structural phloem proteins uniquely found in Fabaceae sieve elements. They undergo a reversible conformational change after wounding, from a condensed to a dispersed state, thereby blocking sieve tube translocation and preventing the loss of photoassimilates. Recently, we identified SEO genes in several non-Fabaceae plants (lacking forisomes) and concluded that they most probably encode conventional non-forisome P-proteins. Molecular and phylogenetic analysis of the SEO gene family has identified domains that are characteristic for SEO proteins. Here, we extended our phylogenetic analysis by including additional SEO genes from several diverse species based on recently published genomic data. Our results strengthen the original assumption that SEO genes seem to be widespread in dicotyledonous angiosperms, and further underline the divergent evolution of SEO genes within the Fabaceae.

Spatial and temporal regulation of the forisome gene for1 in the phloem during plant development.

Forisomes are protein aggregates found uniquely in the sieve elements of Fabaceaen plants. Upon wounding they undergo a reversible, calcium-dependent conformational switch which enables them to act as cellular stopcocks. Forisomes begin to form in young sieve elements at an early stage of metaphloem differentiation. Genes encoding forisome components could therefore be useful as markers of early sieve element development. Here we present a comprehensive analysis of the developmental expression profile of for1, which encodes such a forisome component. The for1 gene is highly conserved among Fabaceaen species and appears to be unique to this phylogenetic lineage since no orthologous genes have been found in other plants, including Arabidopsis and rice. Even so, transgenic tobacco plants expressing reporter genes under the control of the for1 promoter display reporter activity exclusively in immature sieve elements. This suggests that the regulation of sieve element development is highly conserved even in plants where mature forisomes have not been detected. The promoter system could therefore provide a powerful tool for the detailed analysis of differentiation in metaphloem sieve elements in an unexpectedly broad range of plant species.