Root and vascular tissue-specific expression of glycine-rich protein AtGRP9 and its interaction with AtCAD5, a cinnamyl alcohol dehydrogenase,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The vascular tissue of roots performs essential roles in the physical support and transport of water, nutrients, and signaling molecules in higher plants. The molecular mechanisms underlying the function of root vascular tissue are poorly understood. In this study, we analyzed the expression pattern of AtGRP9, a salt stress-responsive gene encoding a glycine-rich protein, and its interacting partner, in Arabidopsis thaliana. Analysis of GUS or GFP expression under the control of the AtGRP9 promoter showed that AtGRP9 was expressed in the vascular tissue of the root; subcellular localization analysis further demonstrated that AtGRP9 proteins were localized in the cell wall and in the cytoplasm. Yeast two-hybrid analysis revealed that AtGRP9 interacted with AtCAD5, a major cinnamyl alcohol dehydrogenase (CAD) involved in lignin biosynthesis, for which tissue-specific distribution was comparable with that of AtGRP9. These results suggest that AtGRP9 may be involved in lignin synthesis in response to salt stress as a result of its interaction with AtCAD5 in A. thaliana.     

Greenhouse gas emissions from a constructed wetland in southern Sweden

This paper investigates the greenhouse gas emissions from a Swedish wetland, constructed to decrease nutrient content in sewage treatment water. To evaluate the effect of the construction in terms of greenhouse gas emissions we carried out ecosystem-atmosphere flux measurements of CO₂, CH₄ and N₂O using a closed chamber technique. To evaluate the importance of vascular plant species composition to gas emissions we distributed the measurement plots over the three dominating plant species at the field site, i.e., Typha latifolia, Phragmites australis and Juncus effusus. The fluxes of CO₂ (total respiration), CH₄ and N₂O from vegetated plots ranged from 1.39 to 77.5 (g m⁻² day⁻¹), −377 to 1387 and −13.9 to 31.5 (mg m⁻² day⁻¹) for CO₂, CH₄ and N₂O, respectively. Presence of vascular plants lead as expected to significantly higher total respiration rates compared with un-vegetated control plots. Furthermore, we found that the emission rates of N₂O and CH₄ was affected by presence of vascular plants and tended to be species-specific. We assessed the integrated greenhouse warming effect of the emissions using a Global Warming Potential over a 100-year horizon (GWP₁₀₀) and it corresponded to 431 kg CO₂ equivalents m⁻² day⁻¹. Assuming a 7-month season with conditions similar to the study period this is equal to 90 tonnes of CO₂ equivalents annually. N₂O emissions were responsible for one third of the estimated total greenhouse forcing. Furthermore, we estimated that the emission from the forested bog that was the precursor land to Magle constructed wetland amounted to 18.6 tonnes of CO₂ equivalents annually. Hence, the constructed wetland has increased annual greenhouse gas emissions by 71.4 tonnes of CO₂ equivalents for the whole area. Our findings indicate that management processes in relation to wetland construction projects must consider the primary function of the wetland in decreasing eutrophication, in relation to other positive aspects on for instance plant and animal life and recreation as well as possible negative climatic aspects of increased emissions of CH₄ and N₂O.     

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Long-term effects of nitrogen deposition on vegetation in a deciduous forest near Munich,Germany

Question: What are the main reasons for changes in the spatial distribution of vegetation types during the last four decades? Location: Isolated small deciduous forest; surrounded by farmland in the northeast of Munich (Germany). Methods: Based on sequential vegetation mapping from the last four decades the spatial development of the vegetation was analysed. Additionally, environmental parameters (soil parameters, PAR, N-deposition) have been analysed to describe the different vegetation types. Results: By linking the vegetation types to environmental parameters, it was possible to identify N-deposition as the most important factor for the changes. In the 1960s to 1980s the replacement of vegetation types adapted to N-poor conditions by N-rich vegetation was very fast. A vegetation type containing species signifying soil impoverishment vanished totally, another vegetation type indicating nutrient poor conditions decreased dramatically. However, since 1985 up to now the decrease of N-poor vegetation types has slowed, but is still ongoing. As a reason for the decreased rate of replacement, we stressed changes in the vertical structure: From 1961 to 1985 both N-deposition as well as changes in vertical vegetation structure seem to be important. Since 1985 up to now only minor changes in vertical structure could be found; changes are mainly due to N-availability. Conclusion: In this paper, the limitations of different methods to detect vegetation changes are discussed. We focus on the potentials of historical vegetation data and vegetation maps. It is shown that valuable information on N-induced vegetation changes can be retrieved from historical vegetation data.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Specific Activation of Insulin-like Growth Factor-1 Receptor by Ginsenoside Rg5 Promotes Angiogenesis and Vasorelaxation

Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE^−/− mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and G_i-mediated phospholipase C/Ca²⁺/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC₅₀ of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.     

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (k_cat/K_M^app) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.     

The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell Migration

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an “outside-in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.