COL2-dependent photoperiodic floral induction in Nicotiana sylvestris seems to be lost in the N. sylvestris × N. tomentosiformis hybrid N. tabacum.

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.

Guardians of the phloem - forisomes and beyond.

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.

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network.

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.

So similar yet so different: The distinct contributions of extrafascicular and fascicular phloem to transport and exudation in cucumber plants.

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.

The Ca2+ response of a smart forisome protein is dependent on polymerization.

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.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

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.

The tobacco phosphatidylethanolamine-binding protein NtFT4 simultaneously improves vitality, growth, and protein yield in human cells.

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.

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification.

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.

Identification and molecular analysis of interaction sites in the MtSEO-F1 protein involved in forisome assembly.

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.

The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement.

The FLOWERING LOCUS T (FT)-like gene family encodes key regulators of flower induction that affect the timing of reproduction in many angiosperm species. Agricultural research has therefore focused on such genes to improve the success of breeding programs and enhance agronomic traits. We recently identified a novel FT-like gene (NtFT5) that encodes a day-neutral floral activator in the model tobacco crop Nicotiana tabacum. However, further characterization is necessary to determine its value as a target for breeding programs. We therefore investigated the function of NtFT5 by expression analysis and mutagenesis. Expression analysis revealed that NtFT5 is transcribed in phloem companion cells, as is typical for FT-like genes. However, high levels of NtFT5 mRNA accumulated not only in the leaves but also in the stem. Loss-of-function mutants (generated using CRISPR/Cas9) were unable to switch to reproductive growth under long-day conditions, indicating that NtFT5 is an indispensable major floral activator during long-days. Backcrossing was achieved by grafting the mutant scions onto wild-type rootstock, allowing the restoration of flowering and pollination by a wild-type donor. The resulting heterozygous Ntft5- /NtFT5+ plants flowered with a mean delay of only ~2 days, demonstrating that one functional allele is sufficient for near-normal reproductive timing. However, this minor extension of the vegetative growth phase also conferred beneficial agronomic traits, including a >10% increase in vegetative leaf biomass on the main shoot and the production of more seeds. The agronomic benefits of the heterozygous plants persisted under various abiotic stress conditions, confirming that NtFT5 is a promising target for crop improvement to address the effects of climate change.

The FT/FD-dependent initiation of flowering under long-day conditions in the day-neutral species Nicotiana tabacum originates from the facultative short-day ancestor Nicotiana tomentosiformis.

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.

Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes.

The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins, forisomes and forisome tails.

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.

Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

BACKGROUND: Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. METHODOLOGY/PRINCIPAL FINDINGS: We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. CONCLUSIONS/SIGNIFICANCE: We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

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.

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast.

Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.

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.

P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing.

Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing.