Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.     

Effect of unilateral partial facial paralysis on periosteal growth at the muscle-bone interface of facial muscles and facial bones

In a previous study, the influence of the midfacial musculature upon growth and development of the maxilla and mandible was established macroscopically. Dry skull measurements revealed a reduced premaxillary, maxillary, mandibular, and anterior corpus length with a simultaneous increase in mandibular ramal height on the paralyzed side. It was demonstrated that these reduced premaxillary and maxillary lengths were among others the result of reduced nasofrontal growth, whereas the increased ramal height was accompanied by condylar growth alterations. This study investigated whether the growth alterations at the mandibular corpus region could be explained by altered periosteal growth at the muscle-bone interface of the zygomatico-auricular muscle and the mandibular corpus, caused by altered muscle activity acting upon the periosteal sleeve. Fifty-six 12-day-old New Zealand White rabbits were randomly assigned to either a control or an experimental group. In the experimental group, left-sided partial facial paralysis was induced surgically when the animals were 12 days old. To study the muscle-bone interface, seven follow-up time intervals were defined between 3.5 and 60 days following the surgery. At these time intervals, four randomly selected control animals and four randomly selected experimental animals were killed. The anterior mandibular corpus region with the muscle-bone interface of the left control hemimandible and the left and right experimental hemimandibles was processed for undecalcified tissue preparation. Quantitative analysis of the total bone area at the muscle-bone interface revealed no significant differences between the left control hemimandible and the left and right experimental hemimandibles. Also, qualitative study of the histologic sections showed no major changes in the appearance or development of the trabecular pattern between the groups. However, slight differences in the distribution pattern of osteoblasts and osteoclasts along the bony surface were found between the left control hemimandible and the left and right experimental hemimandibles, which seemed to explain the alterations in mandibular corpus shape between these groups. It was suggested that these changes in the distribution pattern of osteoblasts and osteoclasts were the result of changes in the loading distribution pattern acting upon the mandible, caused by an altered neuromuscular recruitment pattern of the remaining functionally intact, mandibularly attached muscles. The latter was probably the result of adaptive mandibular positioning in response to an altered occlusal relationship, which was induced by the abnormal maxillary growth as a result of the unilateral partial facial paralysis.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast

Mitochondrial fusion and fission play important roles for mitochondrial morphology and function. We identified Mdm30 as a novel component required for maintenance of fusion-competent mitochondria in yeast. The Mdm30 sequence contains an F-box motif that is commonly found in subunits of Skp1-Cdc53-F-box protein ubiquitin ligases. A fraction of Mdm30 is associated with mitochondria. Cells lacking Mdm30 contain highly aggregated or fragmented mitochondria instead of the branched tubular network seen in wild-type cells. Deltamdm30 cells lose mitochondrial DNA at elevated temperature and fail to fuse mitochondria in zygotes at all temperatures. These defects are rescued by deletion of DNM1, a gene encoding a component of the mitochondrial division machinery. The protein level of Fzo1, a key component of the mitochondrial fusion machinery, is regulated by Mdm30. Elevated Fzo1 levels in cells lacking Mdm30 or in cells overexpressing Fzo1 from a heterologous promoter induce mitochondrial aggregation in a similar manner. Our results suggest that Mdm30 controls mitochondrial shape by regulating the steady-state level of Fzo1 and point to a connection of the ubiquitin/26S proteasome system and mitochondria.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

Differential Impact of Lipoxygenase 2 and Jasmonates on Natural and Stress-Induced Senescence in Arabidopsis

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.Senescence is an important, highly regulated process at the end of development. Senescence is characterized by breakdown of organelles and molecules, export and transport of these nutrients to other organs/parts of the organism, and finally programmed cell death of the senescing organ.The process of senescence has been intensively studied in leaves, and morphological as well as molecular changes in senescing leaves have been described. Yellowing as a consequence of chlorophyll and chloroplast degradation is the most obvious process during natural leaf senescence. In addition, gene expression changes dramatically during senescence. Some senescence-associated genes (SAG, SEN) have been reported that are induced during this process, and several of the encoded proteins function in macromolecule degradation, detoxification and defense metabolism, or signal transduction (Gepstein et al., 2003). Based on the degradation of chloroplasts and macromolecules, leaf metabolism changes from carbon assimilation to catabolism (Lim et al., 2007).The initiation and progression of senescence is regulated by endogenous as well as exogenous factors. Among the endogenous factors, the developmental status of the organ and of the whole plant (e.g. age and progress in flowering and seed production) has a great impact on the process of senescence. Different stress factors such as pathogen attack, drought, osmotic stress, heat, cold, ozone, UV light, and shading can induce or accelerate senescence (Quirino et al., 2000). Phytohormones are very important regulators that integrate information about the developmental status and the environmental factors. Cytokinins are antagonistic signals and delay senescence. Endogenous levels of cytokinins decrease during senescence, and exogenous application and transgenic approaches, enhancing endogenous levels of these compounds, lead to delayed senescence (Gan and Amasino, 1995). In contrast, the gaseous phytohormone ethylene is known to induce and accelerate senescence (John et al., 1995). There are also several indications that abscisic acid modulates senescence (van der Graaff et al., 2006). The roles of other phytohormones/signaling compounds such as auxin, salicylic acid, and jasmonates are less clear (Lim et al., 2007).Jasmonates are oxylipin signaling molecules derived from linolenic acid. The term jasmonates comprises 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives such as the methyl ester and amino acid conjugates of JA. One of the first biological activities described for these compounds was the promotion of senescence in oat (Avena sativa) leaves by methyl jasmonate (MeJa) isolated from Artemisia absinthium (Ueda and Kato, 1980). Later on, the induction of senescence-like phenotypes by exogenous application of MeJa was also found in other plant species (Ueda and Kato, 1980; Weidhase et al., 1987a; He et al., 2002). On the molecular level, this senescence-promoting effect of MeJa is accompanied by chlorophyll loss and decreases in Rubisco and photosynthesis (Weidhase et al., 1987a, 1987b). In addition, expression of some senescence-up-regulated genes is also responsive to JA; examples are SEN1, SEN4, SEN5, SAG12, SAG14, and SAG15 (Park et al., 1998; Schenk et al., 2000; He et al., 2002). Due to the results described above, jasmonates have been described for decades as compounds with senescence-promoting activities, while the function of these compounds in natural senescence in planta was critically discussed (Parthier, 1990; Sembdner and Parthier, 1993; Creelman and Mullet, 1997; Wasternack, 2007; Balbi and Devoto, 2008; Reinbothe et al., 2009). Additional indications for a role of jasmonates in regulating senescence are the transient up-regulation of expression of some enzymes involved in JA biosynthesis, such as allene oxide synthase (AOS) and OPDA reductase 3 (OPR3), and the increase in JA levels during natural senescence (He et al., 2002; van der Graaff et al., 2006). Furthermore, alterations in natural and induced senescence have been reported for some mutants with defects in the JA pathway. The mutant coi1, which is impaired in JA signaling, exhibited delayed chlorophyll loss upon dark incubation of detached leaves (Castillo and Leon, 2008). Plants with reduced expression of the 3-ketoacyl-CoA-thiolase KAT2, which is involved in β-oxidation and JA production, showed delayed yellowing during natural senescence and upon dark incubation of detached leaves (Castillo and Leon, 2008).However, there are also several reports that cast doubt on an important function of JA in senescence. For most mutants in JA biosynthesis or signaling, no differences in natural senescence are apparent (He et al., 2002; Schommer et al., 2008). In addition, mutants defective in the expression of AOS or OPR3 do not show altered senescence-like phenotypes upon dark treatment (Schommer et al., 2008; Kunz et al., 2009). It has to be taken into consideration that the knockout in these mutants has pleiotrophic effects during whole plant development. For example, the leaves of plants with reduced expression of the lipase DGL or of OPR3 are larger (Hyun et al., 2008). In addition, several knockout mutants defective in JA biosynthesis or signaling do not produce fertile flowers (Feys et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; von Malek et al., 2002). These changes in development might affect other developmental processes such as senescence.To investigate the function of jasmonates in senescence in more detail, we compared the oxylipin profile of wild-type leaves during natural senescence and upon stress induction of senescence-like phenotypes. The analysis of lipoxygenase 2 (LOX2)-RNA interference (RNAi) plants, which produce low basal levels of oxylipins but are impaired in the accumulation of OPDA and JA during senescence or in response to stress, indicates that 13-LOX products are not necessary for natural senescence or dark-induced chlorophyll loss but are involved in the response to sorbitol.     

Lipoxygenase6-Dependent Oxylipin Synthesis in Roots Is Required for Abiotic and Biotic Stress Resistance of Arabidopsis

Jasmonates are oxylipin signals that play important roles in the development of fertile flowers and in defense against pathogens and herbivores in leaves. The aim of this work was to understand the synthesis and function of jasmonates in roots. Grafting experiments with a jasmonate-deficient mutant demonstrated that roots produce jasmonates independently of leaves, despite low expression of biosynthetic enzymes. Levels of 12-oxo-phytodienoic acid, jasmonic acid, and its isoleucine derivative increased in roots upon osmotic and drought stress. Wounding resulted in a decrease of preformed 12-oxo-phytodienoic acid concomitant with an increase of jasmonic acid and jasmonoyl-isoleucine. 13-Lipoxygenases catalyze the first step of lipid oxidation leading to jasmonate production. Analysis of 13-lipoxygenase-deficient mutant lines showed that only one of the four 13-lipoxygenases, LOX6, is responsible and essential for stress-induced jasmonate accumulation in roots. In addition, LOX6 was required for production of basal 12-oxo-phytodienoic acid in leaves and roots. Loss-of-function mutants of LOX6 were more attractive to a detritivorous crustacean and more sensitive to drought, indicating that LOX6-derived oxylipins are important for the responses to abiotic and biotic factors.Oxylipins are ubiquitous signaling molecules that are derived from polyunsaturated fatty acids by enzymatic and nonenzymatic processes. In plants, the biosynthesis and function of oxylipins of the jasmonate family in aboveground tissues has been investigated in detail. Jasmonates comprise 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives of JA. In leaves, jasmonates accumulate in response to abiotic factors such as wounding, drought, osmotic stress, darkness, and ozone and during interactions with organisms such as herbivores, pathogens, and mutualistic organisms (Wasternack, 2007). The relevance of jasmonates in wound response, ozone tolerance, and the defense against herbivores and necrotrophic pathogens in leaves has been well investigated using mutants in JA biosynthesis and signaling (Browse, 2009a). In addition, jasmonates play an important role in flower development, and Arabidopsis (Arabidopsis thaliana) mutants in the JA pathway are male sterile (Browse, 2009b). The first step in jasmonate biosynthesis is catalyzed by 13-lipoxygenases (LOXs). The resulting 13(S)-hydroperoxyoctadecatrienoic acid (13-HPOTE) is converted by allene oxide synthase (AOS) and allene oxide cyclase to OPDA (Wasternack, 2007). These enzymatic steps are located in plastids. OPDA is transported to peroxisomes and converted to JA. JA can be further metabolized to different derivatives that take place mainly in the cytosol. The conjugation of JA with Ile is an important step because jasmonoyl-Ile (JA-Ile) has been identified as a biologically active jasmonate (Staswick and Tiryaki, 2004). OPDA is also biologically active without conversion to JA derivatives. In contrast to all other jasmonates, the OPDA structure contains an electrophilic α,β-unsaturated carbonyl group that renders OPDA more reactive than JA. Therefore, OPDA is classified as a reactive electrophile species with unique signaling properties different from other jasmonates (Farmer and Davoine, 2007).Of the six lipoxygenase genes present in Arabidopsis, four genes encode 13-LOX. For the respective enzymes LOX2, LOX3, LOX4, and LOX6, it was shown that linolenic acid is the preferred substrate and that 13-HPOTE is formed in vitro (Bannenberg et al., 2009). All four enzymes are proposed to be located in plastids. LOX2 is highly expressed in leaves; expression is up-regulated by jasmonates and stress treatments such as wounding and osmotic stress (Bell and Mullet, 1993; Seltmann et al., 2010a). LOX2 was shown to contribute the majority of jasmonate synthesis upon wounding and osmotic stress and during senescence in leaves (Bell et al., 1995; Glauser et al., 2009). LOX2 is also responsible for the accumulation of arabidopsides (Glauser et al., 2009), which are galactolipids containing esterified OPDA in plastids by direct oxidation of galactolipids (Zoeller et al., 2012). LOX3 and LOX4 are required for the development of fertile flowers (Caldelari et al., 2011). LOX6 shows overall low expression (Bannenberg et al., 2009). Recently, it was reported that LOX6 contributes to the fast accumulation of JA and JA-Ile in wounded leaves and is required for the fast increase of JA and JA-Ile in distal leaves after wounding (Chauvin et al., 2013).In contrast to leaves and flowers, little is known on jasmonate biosynthesis and function in roots. Expression of the plastid-localized enzymes of jasmonate synthesis LOX2, AOS, and allene oxide cyclase2 is very low in roots (Zimmermann et al., 2004). By contrast, enzymes such as 9-LOX and α-dioxygenase1 are strongly expressed in roots. These enzymes are involved in the biosynthesis of oxylipins different from jasmonates, and 9-LOX products have been shown to regulate lateral root development because mutants in LOX1 and LOX5 produce more lateral roots (Vellosillo et al., 2007). However, jasmonate function in roots is still obscure. Here, we analyzed jasmonate accumulation in roots upon different stress treatments and show that mutants defective in LOX6 are impaired in stress-induced jasmonate synthesis and are more susceptible to drought and detritivore feeding.