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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.

The transition of the Russian dandelion Taraxacum koksaghyz (Asteraceae) to a profitable, alternative crop producing natural rubber and inulin requires the optimization of several agronomic traits, cultivation conditions and harvesting procedures to improve the yield. However, efficient breeding is hindered by the obligatory sexual outcrossing of this species. Several other asters have been investigated to determine the mechanism of self-incompatibility, but the underlying molecular basis remains unclear. We therefore investigated the self-pollination and cross-pollination of two compatible T. koksaghyz varieties (TkMS2 and TkMS3) by microscopy and transcriptomic analysis to shed light on the pollination process. Self-pollination showed typical sporophytic self-incompatibility characteristics, with the rare pollen swelling at the pollen tube apex. In contrast, cross-pollination was characterized by pollen germination and penetration of the stigma by the growing pollen tubes. RNA-Seq was used to profile gene expression in the floret tissue during self-pollination and cross-pollination, and the differentially expressed genes were identified. This revealed three candidates for the early regulation of pollination in T. koksaghyz, which can be used to examine self-incompatibility mechanisms in more detail and to facilitate breeding programs.

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.

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. �� 2018

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.

Taraxacum koksaghyz has been identified as one of the most promising alternative rubber crops. Its high-quality rubber is produced in the latex of laticifers, a specialized cell type that is organized in a network of elongated tubules throughout the entire plant body. In order to gain insights into the physiological role(s) of latex and hence laticifer biology, we examine the effects of barnase-induced latex RNA degradation on the metabolite and protein compositions in the roots. We established high-quality datasets that enabled precise discrimination between cellular and physiological processes in laticifers and non-laticifer cell types of roots at different vegetative stages. We identified numerous latex-specific proteins, including a perilipin-like protein that has not been studied in plants yet. The barnase-expressing plants revealed a phenotype that did not exude latex, which may provide a valuable genetic basis for future studies of plant-environment interactions concerning latex and also help to clarify the evolution and arbitrary distribution of latex throughout the plant kingdom. The overview of temporal changes in composition and protein abundance provided by our data opens the way for a deeper understanding of the molecular interactions, reactions, and network relationships that underlie the different metabolic pathways in the roots of this potential rubber crop.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

The Russian dandelion Taraxacum koksaghyz produces high‐value isoprenoids such as pentacyclic triterpenes and natural rubber in the latex of specialized cells known as laticifers. Squalene synthase (SQS) and squalene epoxidase (SQE) catalyze key steps in the biosynthesis of cyclic terpenoids, but neither enzyme has yet been characterized in T. koksaghyz. Genomic analysis revealed the presence of two genes (TkSQS1 and TkSQS2) encoding isoforms of SQS, and four genes (TkSQE1-4) encoding isoforms of SQE. Spatial expression analysis in different T. koksaghyz tissues confirmed that TkSQS1 and TkSQE1 are the latex‐predominant isoforms, with highly similar mRNA expression profiles. The TkSQS1 and TkSQE1 proteins colocalized in the endoplasmic reticulum membrane and their enzymatic functions were confirmed by in vitro activity assays and yeast complementation studies, respectively. The functions of TkSQS1 and TkSQE1 were further characterized in the latex of T. koksaghyz plants with depleted TkSQS1 or TkSQE1 mRNA levels, produced by RNA interference. Comprehensive expression analysis revealed the coregulation of TkSQS1 and TkSQE1, along with a downstream gene in the triterpene biosynthesis pathway encoding the oxidosqualene cyclase TkOSC1. This indicates that the coregulation of TkSQS1, TkSQE1, and TkOSC1 could be used to optimize the flux toward specific terpenoids during development.

Symptomatic patients with chronic thromboembolic disease (CTED) without pulmonary hypertension often show an excessive increase in mean pulmonary arterial pressure (MPAP) during exercise.We report on the impact of pulmonary endarterectomy (PEA) on pulmonary haemodynamics in a prospective series of 32 consecutive CTED patients who underwent PEA. All patients had a comprehensive diagnostic work-up including right heart catheterisation at baseline and 12 months after PEA. Furthermore, in 12 patients exercise right heart catheterisation was performed before and after PEA.After PEA, MPAP was lower at rest (20±3versus17±3 mmHg; p=0.008) and during maximal exercise (39±8versus31±6 mmHg; p=0.016). The mean total pulmonary resistance (TPR) decreased from 3.6±0.8 Wood Units (WU) pre-operatively to 2.7±0.7 WU 1 year after PEA (p=0.004) and the mean slope of the MPAP/cardiac output (CO) relationship decreased from 3.6±1.0 to 2.3±0.8 WU (p=0.002). Peak oxygen uptake increased from 1.2±0.4 to 1.5±0.3 L·min−1(p=0.014) and ventilatory equivalents of carbon dioxide decreased from 39±2 to 30±2 (p=0.002). There was a significant improvement in quality of life assessed by the Cambridge Pulmonary Hypertension Outcome Review questionnaire.In CTED patients, PEA resulted in haemodynamic and clinical improvements. The means of TPR and MPAP/CO slopes decreased to

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1-5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1-5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3-5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.

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.

Natural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink-to-source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum and functionally characterized the key enzyme fructan 1-exohydrolase (1-FEH), which catalyses the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1-FEH almost doubled the rubber content in the roots of two dandelion species without any trade-offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber-enriched varieties for industrial rubber production.

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.

High-molecular-mass natural rubber is a valuable plant-derived poly(cis-1,4-isoprene) with many industrial and medical applications. It is synthesized by a rubber cis-prenyltransferase (CPT) complex on the surface of rubber particles in specialized latex-producing cells known as laticifers. Here we show that Taraxacum brevicorniculatum rubber transferase activator (TbRTA), a dandelion homologue of the human Nogo-B receptor, is an essential component of the rubber transferase complex which interacts with rubber CPTs on the surface of rubber particles. The knockdown of TbRTA by RNA interference eliminated rubber biosynthesis, without affecting dolichol accumulation or protein glycosylation in the latex. We also found that TbRTA is localized on the endoplasmic reticulum membrane, supporting the current favoured model of rubber particle biogenesis. We therefore propose that TbRTA acts as a rubber CPT-binding protein that is necessary for the formation of an active rubber transferase complex.

Plant cytosolic lipid droplets are storage organelles that accumulate hydrophobic molecules. They are found in many tissues and their general structure includes an outer lipid monolayer with integral and associated proteins surrounding a hydrophobic core. Two distinct types can be distinguished, which we define here as oleosin-based lipid droplets (OLDs) and non-oleosin-based lipid droplets (NOLDs). OLDs are the best characterized lipid droplets in plants. They are primarily restricted to seeds and other germinative tissues, their surface is covered with oleosin-family proteins to maintain stability, they store triacylglycerols (TAGs) and they are used as a source of energy (and possibly signaling molecules) during the germination of seeds and pollen. Less is known about NOLDs. They are more abundant than OLDs and are distributed in many tissues, they accumulate not only TAGs but also other hydrophobic molecules such as natural rubber, and the structural proteins that stabilize them are unrelated to oleosins. In many species these proteins are members of the rubber elongation factor superfamily. NOLDs are not typically used for energy storage but instead accumulate hydrophobic compounds required for environmental interactions such as pathogen defense. There are many potential applications of NOLDs including the engineering of lipid production in plants and the generation of artificial oil bodies.

Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis-prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF-silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal s tability of rubber particles isolated from TbREF-silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production. Significance Statement Natural rubber is used in thousands of products but little is known about its synthesis. Russian dandelion plants produce natural rubber and can be used to characterize the biosynthesis steps and to determine how other rubber-producing plants such as the rubber tree can be induced to produce larger amounts. We present a comprehensive analysis of a unique rubber elongation factor family protein that is necessary for efficient rubber biosynthesis in the dandelion root latex.

Because of the substantial risk of thromboembolism early after valve replacement, perioperative initiation of anticoagulation is necessary, despite the increased risk for bleeding. Anticoagulation should be initiated within 24 h after the procedure with unfractionated heparin or low-molecular-weight heparin (LMWH). Subcutaneous LMWH appears more beneficial than intravenous heparin therapy, but this approach requires further evaluation. Oral anticoagulants, preferably at low dosage, are added following the removal of chest tubes. Heparin anticoagulation is monitored by checking the activated partial thromboplastin time or anti-Xa activity, and the International Normalized Ratio (INR) is used to measure the effects of oral anticoagulants. Heparin treatment should be continued until the INR is stable in the therapeutic range in order to avoid hypercoagulable conditions caused by varying degrees of decay in coagulation factors.

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.

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.

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.

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.

Laticifers are highly specialized plant cells that produce latex enriched with secondary metabolites. The articulated laticifers of Taraxacum brevicorniculatum synthesize natural rubber, an industrially-valuable composite biopolymer comprising >95% high-molecular-weight (HMW) poly(cis-1,4-isoprene). Here we present a proof-of-concept approach for the cultivation of cell suspension cultures exclusively containing laticifers. We transformed T. brevicorniculatum plants with a plasmid conferring laticifer-specific hygromycin resistance. Transgenic callus tissue was used to induce a cell suspension culture under antibiotic selection to favor laticifer growth. The cultured cells appeared laticiferous in terms of morphology and expressed laticifer-specific genes. Confocal laser scanning microscopy revealed intracellular lipid accumulation in vesicle-like structures. Nuclear magnetic resonance and diffusion ordered spectroscopy indicated the presence of mid-length poly(cis-1,4 -isoprene) chains but no high HMW natural rubber in the cells. Precursor feeding with mevalonolactone increased the accumulation of poly(cis-1,4-isoprene) by 17-fold, reaching a concentration of 2.7 mg/g dry weight. Our approach could lead to the development of a production platform for the efficient conversion of isopentenyl diphosphate into poly(cis-1,4-isoprene) in an optimized cell suspension culture system. The apparent absence of HMW natural rubber is discussed in terms of our current knowledge of rubber biosynthesis.

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.

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.

Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and beta-gincuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins.

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P-proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

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.

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Forisomes are protein bodies found exclusively in the phloem of the Fabaceae (legumes). In response to wounding, the influx of Ca2+ 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

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.

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 Ca2+, 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 appl ications. 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.

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.

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.

alpha-Hederin, a natural triterpene saponin and its derivative kalopanaxsaponin I (ksI) exhibit cytotoxicity against various cancer cell lines and in vivo tumors. We studied the genetic variants contributing to the activity of these two anticancer compounds. Cell lines derived from 30 trios of European descent (Centre d'Etude du Polymor-phisme Human, CEPH; CEU) and 30 trios of African descent (Yoruban, YRI) were used. Cytotoxicity was determined as inhibition of cell growth at increasing concentrations of alpha-hederin or ksI for 24 h. In comparison to the European, the Yoruban populations revealed a higher sensitivity to alpha-hederin and to ksI that can be attributed to several unique SNPs. These SNPs are located near 111 and 130 genes in the European and the Yoruban populations, respectively, raising the possibility that some of these genes contribute to the differential sensitivity to these compounds.

Forisomes are mechanoproteins that function as valves in the phloem sieve tubes of the Fabaceae. Changes in the Ca2+ level that occur after wounding trigger an instantaneous switch between the condensed and dispersed conformations, allowing forisomes to seal the sieve plate pores of injured sieve elements. Recently, three genes encoding forisome components from Medicago truncatula were identified [Medicago truncatula sieve element occlusion 1 (MtSEO1), MtSEO2 and MtSEO3] and MtSEO1 expression was shown to be restricted to immature sieve elements. Here, we present a detailed molecular analysis of the MtSEO2 and MtSEO3 promoters and show, through reverse transcriptase-polymerase chain reaction and reporter-transgenic experiments in M. truncatula roots and tobacco plants, that their expression profiles are comparable to MtSEO1. The impact of these data on the likelihood of MtSEO gene co-regulation by common transcription factors is discussed.

Background Ischaemia-reperfusion injury is still a major problem after lung transplantation. Several reports describe the benefits of controlled graft reperfusion. In this study the role of length of the initial pressure-controlled reperfusion (PCR) was evaluated in a model of isolated, buffer-perfused rabbit lungs. Methods Heart-lung blocks of 25 New Zealand white rabbits were used. After measurement of baseline values (haemodynamics and gas exchange) the lungs were exposed to 120 minutes of hypoxic warm ischaemia followed by repeated measurements during reperfusion. Group A was immediately reperfused using a flow of 100 ml/min whereas groups B, C and D were initially reperfused with a maximum pressure of 5 mmHg for 5, 15 or 30 minutes, respectively. The control group had no period of ischaemia or PCR. Results Uncontrolled reperfusion (group A) caused a significant pulmonary injury with increased pulmonary artery pressures (PAP) and pulmonary vascular resistance and a decrease in oxygen partial pressure (PO2), tidal volume and in lung compliance. All groups with PCR had a significantly higher PO2 for 5 to 90 min after start of reperfusion. At 120 min there was also a significant difference between group B (264 ± 91 mmHg) compared to groups C and D (436 ± 87 mmHg; 562 ± 20 mmHg, p < 0.01). All PCR groups showed a significant decrease of PAP compared to group A. Conclusion Uncontrolled reperfusion results in a severe lung injury with rapid oedema formation. PCR preserves pulmonary haemodynamics and gas exchange after ischaemia and might allows for recovery of the impaired endothelial function. 30 minutes of PCR provide superior results compared to 5 or 15 minutes of PCR.