RESUMEN
The phloem is a highly specialized vascular tissue that forms a fundamentally important transport and signaling pathway in plants. It is therefore a system worth protecting. The main function of the phloem is to transport the products of photosynthesis throughout the whole plant, but it also transports soluble signaling molecules and propagates electrophysiological signals. The phloem is constantly threatened by mechanical injuries, phloem-sucking pests and parasites, and the spread of pathogens, which has led to the evolution of efficient defense mechanisms. One such mechanism involves structural phloem proteins, which are thought to facilitate sieve element occlusion following injury and to defend the plant against pathogens. In leguminous plants, specialized structural phloem proteins known as forisomes form unique mechanoproteins via sophisticated molecular interaction and assembly mechanisms, thus enabling reversible sieve element occlusion. By understanding the structure and function of forisomes and other structural phloem proteins, we can develop a toolbox for biotechnological applications in material science and medicine. Furthermore, understanding the involvement of structural phloem proteins in plant defense mechanisms will allow phloem engineering as a new strategy for the development of crop varieties that are resistant to pests, pathogens and parasites.
Asunto(s)
Fabaceae , Floema , Floema/metabolismo , Fabaceae/fisiología , Plantas/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
The production of biopharmaceutical proteins in mammalian cells by transient expression or stable transformation requires robust and viable cells. Cell line engineering must therefore balance improved cell growth and viability with high productivity. We tested the ability of nonmammalian phosphatidylethanolamine-binding proteins to enhance cell proliferation in monolayers and suspension cultures. The tobacco protein NtFT4 improved the proliferation of multiple human cell lines. Viable cell density is usually impaired by efficient transfection, but we found that the number of HEK-293TNtFT4 cells at the peak of protein expression was twice that of standard HEK-293T cells, and the antibody yield increased by approximately one-third. Improved growth and viability were observed in different cell lines, in different culture media, and also after transient transfection, suggesting the beneficial trait is consistent and transferable. Additional modifications could boost the productivity of high-density HEK-293TNtFT4 cells even further as we showed for a fluorescent marker protein and recombinant antibody expressed in monolayer cultures. The HEK-293TNtFT4 cell line provides a new human model platform that increases cell proliferation, also achieving a fundamental improvement in recombinant protein expression.
Asunto(s)
Técnicas de Cultivo de Célula , Nicotiana/genética , Proteínas de Unión a Fosfatidiletanolamina , Proteínas de Plantas , Supervivencia Celular , Células HEK293 , Humanos , Células MCF-7 , Proteínas de Unión a Fosfatidiletanolamina/biosíntesis , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Tipificación Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedades de las Plantas/virología , Tubérculos de la Planta/virología , ARN Viral/genética , Solanum tuberosum/virología , Colorimetría/instrumentación , Colorimetría/métodos , Humanos , Técnicas de Diagnóstico Molecular/instrumentación , Tipificación Molecular/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Virus de Plantas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Photoperiod is an important external stimulus governing the precise timing of the floral transition in plants. Members of the FLOWERING LOCUS T (FT)-like clade of phosphatidylethanolamine-binding proteins induce this developmental process in numerous species by forming regulatory protein complexes with FD-like bZIP transcription factors. We identified several thus far unknown FT-like and FD-like genes in the genus Nicotiana and found that, even in the day-neutral species Nicotiana tabacum, floral initiation requires the photoperiod-dependent expression of several FT-like genes. Furthermore, floral promotion under long-day (LD) and short-day (SD) conditions is mediated by an FT-like protein (NtFT5) that originates from the genome of the paternal, facultative SD ancestor Nicotiana tomentosiformis. In contrast, its ortholog of the maternal LD ancestor Nicotiana sylvestris is not present in the genome of N. tabacum cv. SR1. Expression profiling in N. tabacum and its ancestors confirmed the relevance of these FT and FD orthologs in the context of polyploidization. We also found that floral inhibition by tobacco FT-like proteins is not restricted to SD conditions, highlighting the coincident expression of tobacco FT-like genes encoding floral activators and floral inhibitors. Multicolor bimolecular fluorescence complementation analysis revealed the preferential formation of FT/FD complexes that promote rather than inhibit flowering, which in concert with the regulation of NtFT and NtFD expression could explain how floral promotion overcomes floral repression during the floral transition in tobacco.
Asunto(s)
Flores/genética , Nicotiana/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Fotoperiodo , Flores/fisiología , Flores/efectos de la radiación , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/fisiología , Nicotiana/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Cucurbita/genética , Fabaceae/genética , Nicotiana/genética , Floema/genética , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Evolución Molecular , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Luz , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Datos de Secuencia Molecular , Floema/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARNRESUMEN
P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.
Asunto(s)
Glycine max/genética , Proteínas de Plantas/genética , Familia de Multigenes , Filogenia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/metabolismo , Transporte de Proteínas , Glycine max/metabolismo , Nicotiana/genéticaRESUMEN
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.
Asunto(s)
Fabaceae/genética , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestructura , Secuencia de Aminoácidos , Biología Computacional , Fabaceae/química , Fabaceae/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Nicotiana/crecimiento & desarrollo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Secuencia de Bases , Flores/genética , Flores/fisiología , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Luz , Datos de Secuencia Molecular , Fenotipo , Floema/genética , Floema/crecimiento & desarrollo , Floema/fisiología , Floema/efectos de la radiación , Proteínas de Unión a Fosfatidiletanolamina/genética , Fotoperiodo , Filogenia , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Tiempo , Nicotiana/genética , Nicotiana/fisiología , Nicotiana/efectos de la radiaciónRESUMEN
Introduction: Plants are sessile organisms that maximize reproductive success by adapting to their environment. One of the key steps in the reproductive phase of angiosperms is flower development, requiring the perception of multiple endogenous and exogenous signals integrated via a complex regulatory network. Key floral regulators, including the main transcription factor of the photoperiodic pathway (CONSTANS, CO) and the central floral pathway integrator (FLOWERING LOCUS T, FT), are known in many species. Methods and results: We identified several CO-like (COL) proteins in tobacco (Nicotiana tabacum). The NtCOL2a/b proteins in the day-neutral plant N. tabacum were most closely related to Arabidopsis CO. We characterized the diurnal expression profiles of corresponding genes in leaves under short-day (SD) and long-day (LD) conditions and confirmed their expression in phloem companion cells. Furthermore, we analyzed the orthologs of NtCOL2a/b in the maternal LD ancestor (N. sylvestris) and paternal, facultative SD ancestor (N. tomentosiformis) of N. tabacum and found that they were expressed in the same diurnal manner. NtCOL2a/b overexpression or knock-out using the CRISPR/Cas9 system did not support a substantial role for the CO homologs in the control of floral transition in N. tabacum. However, NsCOL2 overexpression induced flowering in N. sylvestris under typically non-inductive SD conditions, correlating with the upregulation of the endogenous NsFTd gene. Discussion: Our results suggest that NsFTd is transcriptionally regulated by NsCOL2 and that this COL2-dependent photoperiodic floral induction seems to be lost in N. tabacum, providing insight into the diverse genetics of photoperiod-dependent flowering in different Nicotiana species.
RESUMEN
Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.
Asunto(s)
Proteínas de Plantas/química , Agrobacterium tumefaciens/química , Células Epidérmicas , Epidermis/química , Epidermis/metabolismo , Medicago truncatula/química , Membranas Artificiales , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Moleculares , Proteínas de Plantas/biosíntesis , Nicotiana/química , Nicotiana/citologíaRESUMEN
Forisomes are giant self-assembling mechanoproteins that undergo reversible structural changes in response to Ca2+ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO-F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca2+ association of MtSEO-F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO-F1 dimers neither associate with Ca2+ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO-F1 dimers and fully-assembled forisomes associate with Ca2+ , allowing the hydration of poorly-hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca2+ interacts with negatively-charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca2+ response of forisome polymers.
Asunto(s)
Proteínas de Plantas , Proteínas de Plantas/química , PolimerizacionRESUMEN
Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.
Asunto(s)
Proteínas de Drosophila , Longevidad , Envejecimiento/metabolismo , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Longevidad/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteoma/metabolismo , Proteostasis/genética , NicotianaRESUMEN
Cucurbits have been used as phloem research models for many decades because their exudates can be accessed with ease. However, cucurbit plants possess two distinct phloem systems known as the fascicular phloem (FP) and extrafascicular phloem (EFP). Therefore, the molecular composition and function of certain exudates can be misinterpreted due to their unclear origin. To characterize the anatomy and function of the different phloem systems more clearly, we generated specific antibodies against marker proteins (PP1 homologs) allowing the clear identification of the EFP at the organ, tissue and cellular levels by immunological staining. We also used detailed microscopy to determine common and unique anatomical features of the FP and EFP sieve elements (SEs) in cucumber (Cucumis sativus). The comparison of exudation rates and the dynamic viscosity, density and sugar content of the exudates from plants grown in the light and dark revealed the consistent composition and behavior of the EFP exudate even when photosynthesis was prevented, thus differing from the properties of the FP exudate. Furthermore, the analysis of phloem transport using a fluorescein disodium salt showed only wound-induced exudation of dye from the EFP, indicating the absence of transport in this tissue. Our results show that it is important to distinguish between the EFP and FP in cucurbits, particularly their differing behaviors in response to wounding.
Asunto(s)
Cucumis sativus , Cucurbita , Transporte Biológico , Cucumis sativus/metabolismo , Cucurbita/anatomía & histología , Cucurbita/metabolismo , Floema/metabolismo , FotosíntesisRESUMEN
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.
Asunto(s)
Biotecnología/métodos , Fabaceae/química , Complejos Multiproteicos/metabolismo , Floema/química , Proteínas de Plantas/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Complejos Multiproteicos/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.
Asunto(s)
Cisteína/química , Proteínas de Plantas/química , Plantas/química , Alquilantes/química , Disulfuros/química , Oxidación-Reducción , Compuestos de Sulfhidrilo/químicaRESUMEN
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.
Asunto(s)
Fabaceae/genética , Familia de Multigenes , Proteínas de Plantas/genética , Sintenía , Hibridación Genómica Comparativa , Biología Computacional , Fabaceae/metabolismo , Perfilación de la Expresión Génica , Biblioteca de Genes , Genes de Plantas , Cadenas de Markov , Floema/genética , Floema/metabolismo , Filogenia , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras GenéticasRESUMEN
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.
Asunto(s)
Fusión Artificial Génica , Materiales Biocompatibles/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Contráctiles/metabolismo , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/metabolismo , Medicago truncatula/metabolismo , Complejos Multiproteicos/genética , Proteínas de Plantas/genética , Multimerización de Proteína , Saccharomyces cerevisiae/metabolismo , Nicotiana/metabolismoRESUMEN
Forisomes are large mechanoprotein complexes found solely in legumes such as Medicago truncatula. They comprise several "sieve element occlusion by forisome" (SEO-F) subunits, with MtSEO-F1 as the major structure-forming component. SEO-F proteins possess three conserved domains -an N-terminal domain (SEO-NTD), a potential thioredoxin fold, and a C-terminal domain (SEO-CTD)- but structural and biochemical data are scarce and little is known about the contribution of these domains to forisome assembly. To identify key amino acids involved in MtSEO-F1 dimerization and complex formation, we investigated protein-protein interactions by bimolecular fluorescence complementation and the analysis of yeast two-hybrid and random mutagenesis libraries. We identified a SEO-NTD core region as the major dimerization site, with abundant hydrophobic residues and rare charged residues suggesting dimerization is driven by the hydrophobic effect. We also found that ~45% of the full-length MtSEO-F1 sequence must be conserved for higher-order protein assembly, indicating that large interaction surfaces facilitate stable interactions, contributing to the high resilience of forisome bodies. Interestingly, the removal of 62 amino acids from the C-terminus did not disrupt forisome assembly. This is the first study unraveling interaction sites and mechanisms within the MtSEO-F1 protein at the level of dimerization and complex formation.