Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Protein binding and functional characterization of plakophilin 2. Evidence for its diverse roles in desmosomes and beta -catenin signaling.

Plakophilins are a subfamily of p120-related arm-repeat proteins that can be found in both desmosomes and the nucleus. Among the three known plakophilin members, plakophilin 1 has been linked to a genetic skin disorder and shown to play important roles in desmosome assembly and organization. However, little is known about the binding partners and functions of the most widely expressed member, plakophilin 2. To better understand the cellular functions of plakophilin 2, we have examined its protein interactions with other junctional molecules using co-immunoprecipitation and yeast two-hybrid assays. Here we show that plakophilin 2 can interact directly with several desmosomal components, including desmoplakin, plakoglobin, desmoglein 1 and 2, and desmocollin 1a and 2a. The head domain of plakophilin 2 is critical for most of these interactions and is sufficient to direct plakophilin 2 to cell borders. In addition, plakophilin 2 is less efficient than plakophilin 1 in localizing to the nucleus and enhancing the recruitment of excess desmoplakin to cell borders in transiently transfected COS cells. Furthermore, plakophilin 2 is able to associate with beta-catenin through its head domain, and the expression of plakophilin 2 in SW480 cells up-regulates the endogenous beta-catenin/T cell factor-signaling activity. This up-regulation by plakophilin 2 is abolished by ectopic expression of E-cadherin, suggesting that these proteins compete for the same pool of signaling active beta-catenin. Our results demonstrate that plakophilin 2 interacts with a broader repertoire of desmosomal components than plakophilin 1 and provide new insight into the possible roles of plakophilin 2 in regulating the signaling activity of beta-catenin.     

Interaction between Erbin and a Catenin-related protein in epithelial cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.     

Evaluation system for management measures of invasive alien species

The introduction and spread of invasive alien species is a serious threat to ecosystem structures, functions and services worldwide. At enormous cost and effort, attempts are being made to stop and reduce the ongoing appearance of alien species in ecosystems to protect the regional and native biodiversity. Still, management of invasive alien species in particular is often not successful. One reason is a lack of robust toolkits for the management of these species. Guidance on measures which are efficient and effective and which have no negative impact on the environment and human health is urgently needed for decision-making. As a result of an expert workshop, we present a set of three evaluation criteria: effectiveness, ecological impact, and impact on human health. These criteria allow an evaluation of management measures for the prevention, eradication, control and utilisation/disposal of invasive alien species for nature conservation purposes. We applied this evaluation system for 168 invasive and potentially invasive alien species of five taxonomic groups (fungi, non-vascular plants, vascular plants, invertebrates and vertebrates) involving 3619 different management measures. The evaluation of management measures and resulting recommendations for their application are a necessary basis for appropriate, ecologically necessary and professionally sound management. This new approach provides an essential framework for future decisions on management measures, for example in accordance with the provisions of the new Regulation (EU) No 1143/2014 of the European Parliament and of the Council of 22 October 2014 on the prevention and management of the introduction and spread of invasive alien species.     

Plakophilins: Multifunctional proteins or just regulators of desmosomal adhesion?

Plakophilins 1-3 are members of the p120(ctn) family of armadillo-related proteins. The plakophilins have been characterized as desmosomal proteins, whereas p120(ctn) and the closely related delta-catenin, ARVCF and p0071 associate with adherens junctions and play essential roles in stabilizing cadherin mediated adhesion. Recent evidence suggests that plakophilins are essential components of the desmosomal plaque where they interact with desmosomal cadherins as well as the cytoskeletal linker protein desmoplakin. Plakophilins stabilize desmosomal proteins at the plasma membrane and therefore may function in a manner similar to p120(ctn) in the adherens junctions. The three plakophilins reveal distinct expression patterns, and although partially redundant in their function, mediate distinct effects on desmosomal adhesion. Besides a structural role, a function in signaling has been postulated in analogy to other armadillo proteins such as beta-catenin. At least plakophilins 1 and 2 are also localized in the nucleus, and all three proteins occur in a cytoplasmic pool. This review aims to summarize the current knowledge of plakophilin function in the context of cell adhesion, signaling and their putative role in diseases.     

Transporters for uptake and allocation of organic nitrogen compounds in plants

Nitrogen is an essential macronutrient for plant growth. Following uptake from the soil or assimilation within the plant, organic nitrogen compounds are transported between organelles, from cell to cell and over long distances in support of plant metabolism and development. These translocation processes require the function of integral membrane transporters. The review summarizes our current understanding of the molecular mechanisms of organic nitrogen transport processes, with a focus on amino acid, ureide and peptide transporters.     

Fesselin is a natively unfolded protein

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.