Kim A. Kristiansen

Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

Arabidopsis protein kinase PKS5 inhibits the plasma membrane H +-ATPase by preventing interaction with 14-3-3 protein

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM H+-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM H+-ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+-ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+-ATPase regulation.

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.

Neurogenic inflammation: a study of rat trigeminal ganglion

Calcitonin gene-related peptide (CGRP) is linked to neurogenic inflammation and to migraine. Activation of the trigeminovascular system plays a prominent role during migraine attacks with the release of CGRP. The trigeminal ganglion (TG) contains three main cell types: neurons, satellite glial cells (SGC) and Schwann cells; the first two have before been studied in vitro separately. Culture of rat TG provides a method to induce inflammation and the possibility to evaluate the different cell types in the TG simultaneously. We investigated expression levels of various inflammatory cytokines on mRNA level using RT-PCR arrays and qRT-PCR; furthermore expression at protein level was studied using immunohistochemistry. We report that (1) organ culture of the TG is possible with preserved morphology, (2) organ culture is associated with enhanced expression of cytokines and mitogen-activated protein kinases (MAPKs) primarily in neurons, (3) CGRP can induce expression of some cytokines and (4) cytokine expression is still upregulated following MAPK pathway inhibition by MEK inhibitor U0126 and pp38 inhibitor SB202192, but the cytokine expression is abolished when co-incubating with the JNK inhibitor SP600125. This method may be of value to examine local TG inflammation, putatively involved in the pathophysiology of some forms of primary headaches.

Seedlings of Neuwiedia (Orchidaceae subfamily Apostasioideae) have typical orchidaceous mycotrophic protocorms

Naturally occurring seedlings of Neuwiedia veratrifolia were found in three localities in Sabah, Borneo, Malaysia. Seedlings consisted of an irregular oblong protocorm and a terminal leafy rooted shoot. Protocorms contained mycotrophic tissue of the kind typical of orchid mycorrhiza (tolypophagy). This finding demonstrates an important synapomorphy between Neuwiedia and other orchids and strongly supports the monophyly of Orchidaceae in the broad sense, including apostasiod orchids.

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ETB) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM-0.3 μM) and ET-1 (1 pM-0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.