The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

Focus on Water: Leaf Shrinkage with Dehydration: Coordination with Hydraulic Vulnerability and Drought Tolerance

Leaf shrinkage with dehydration has attracted attention for over 100 years, especially as it becomes visibly extreme during drought. However, little has been known of its correlation with physiology. Computer simulations of the leaf hydraulic system showed that a reduction of hydraulic conductance of the mesophyll pathways outside the xylem would cause a strong decline of leaf hydraulic conductance (K_leaf). For 14 diverse species, we tested the hypothesis that shrinkage during dehydration (i.e. in whole leaf, cell and airspace thickness, and leaf area) is associated with reduction in K_leaf at declining leaf water potential (Ψ_leaf). We tested hypotheses for the linkage of leaf shrinkage with structural and physiological water relations parameters, including modulus of elasticity, osmotic pressure at full turgor, turgor loss point (TLP), and cuticular conductance. Species originating from moist habitats showed substantial shrinkage during dehydration before reaching TLP, in contrast with species originating from dry habitats. Across species, the decline of K_leaf with mild dehydration (i.e. the initial slope of the K_leaf versus Ψ_leaf curve) correlated with the decline of leaf thickness (the slope of the leaf thickness versus Ψ_leaf curve), as expected based on predictions from computer simulations. Leaf thickness shrinkage before TLP correlated across species with lower modulus of elasticity and with less negative osmotic pressure at full turgor, as did leaf area shrinkage between full turgor and oven desiccation. These findings point to a role for leaf shrinkage in hydraulic decline during mild dehydration, with potential impacts on drought adaptation for cells and leaves, influencing plant ecological distributions.As leaves open their stomata to capture CO₂ for photosynthesis, water is lost to transpiration, which needs to be replaced by flow through the hydraulic system. The leaf hydraulic system has two components, which act essentially in series: the pathways for water movement through the xylem from the petiole to leaf minor veins, and those through the living bundle sheath and mesophyll cells to the sites of evaporation (Tyree and Zimmermann, 2002; Sack et al., 2004; Sack and Holbrook, 2006). The decline in leaf hydraulic conductance (K_leaf) with dehydration may thus depend on both components. The importance of the xylem component is well established. Vein xylem embolism and cell collapse have been observed in dehydrating leaves (Salleo et al., 2001; Cochard et al., 2004a; Johnson et al., 2009), and computer modeling and experimental work showed that species with high major vein length per leaf area (VLA; i.e. for the first three vein-branching orders) were more resistant to hydraulic decline, providing more pathways around embolisms (Scoffoni et al., 2011). However, the physical impacts of dehydration on the extraxylem pathways have not been studied, even though in turgid leaves these pathways account for 26% to 88% of leaf hydraulic resistance (i.e. of 1/K_leaf), depending on species (Sack et al., 2003a; Cochard et al., 2004b). The aim of this study was to determine whether leaf shrinkage during dehydration relates to the decline of K_leaf as well as the structural determinants of leaf shrinkage.The shrinkage of leaves with dehydration has drawn attention for over 100 years. Leaves shrink in their area (Bogue, 1892; Gardner and Ehlig, 1965; Jones, 1973; Tang and Boyer, 2007; Blonder et al., 2012) and, considered in relative terms, even more strongly in their thickness (Fig. 1; Meidner, 1952; Gardner and Ehlig, 1965; Downey and Miller, 1971; Syvertsen and Levy, 1982; Saini and Rathore, 1983; Burquez, 1987; McBurney, 1992; Sancho-Knapik et al., 2010, 2011). Leaves fluctuate in thickness daily and seasonally according to transpiration (Kadoya et al., 1975; Tyree and Cameron, 1977; Fensom and Donald, 1982; Rozema et al., 1987; Ogaya and Peñuelas, 2006; Seelig et al., 2012). Indeed, the relation of leaf thickness to water status is so tight that using leaf thickness to guide irrigation has led to water savings of up to 45% (Seelig et al., 2012).Open in a separate windowFigure 1.Sketches of a fully turgid leaf (A) versus a strongly dehydrated leaf (B; drawings based on leaf cross sections of sunflower in Fellows and Boyer, 1978). Note the strong reduction in leaf thickness, cell thickness, and intercellular airspaces in the dehydrated leaf. Epidermal cells are shrunk in the dehydrated leaf, inducing whole-leaf area shrinkage. Note that this sketch represents shrinkage for a typical drought-sensitive species. Many species such as oaks (Quercus spp.) will experience less thickness shrinkage and an increase in intercellular airspace (see “Discussion”). [See online article for color version of this figure.]Previous studies of leaf shrinkage with progressive dehydration have tended to focus on single or few species. These studies showed that thickness declines with water status in two phases. Before the bulk leaf turgor loss point (TLP; leaf water potential [Ψ_leaf] at TLP) is reached, the slope of leaf thickness versus Ψ_leaf or relative water content (RWC) is shallower than past TLP for most species (Meidner, 1955, Kennedy and Booth, 1958, Burquez, 1987, McBurney, 1992, Sancho-Knapik et al., 2010, 2011). This is because before TLP, declining Ψ_leaf is strongly driven by declines in turgor pressure, which have a relatively low impact on cell and airspace volume, whereas past the TLP, declining Ψ_leaf depends only on solute concentration, which increases in inverse proportion as cell water volume declines while airspaces may shrink or expand (Tyree and Hammel, 1972, Sancho-Knapik et al., 2011). However, the steepness of the slope of leaf thickness versus Ψ_leaf before TLP seems to vary strongly across species (Meidner, 1955; Kennedy and Booth, 1958; Fellows and Boyer, 1978; Burquez, 1987; Colpitts and Coleman, 1997; Sancho-Knapik et al., 2010).A high leaf cell volume and turgor is crucial to physiological processes (Boyer, 1968; Lawlor and Cornic, 2002). Shrinkage may affect cell connectivity and water transport (Sancho-Knapik et al., 2011). However, no studies have tested for a possible relationship of leaf shrinkage with the decline of K_leaf during dehydration. Such an association would arise if, across species, shrinkage occurred simultaneously with vein xylem embolism or if tissue shrinkage led to declines in the extraxylem hydraulic conductance.To refine our hypotheses, we modified a computer model of the leaf hydraulic system (Cochard et al., 2004b; McKown et al., 2010; Scoffoni et al., 2011) to predict the impact of losses of xylem and extraxylem conductance on the response of K_leaf to dehydration. We characterized the degree of leaf shrinkage in thickness, in the thickness of cells and airspaces within the leaf, and in leaf area for 14 species diverse in phylogeny, leaf traits, and drought tolerance. We hypothesized that loss of extraxylem hydraulic conductance should have a greater impact on K_leaf at less negative water potentials when xylem tensions are too weak to trigger embolism and induce dramatic declines in K_leaf. We hypothesized that species with greater degrees of shrinkage before TLP would experience greater loss of K_leaf. Furthermore, we hypothesized that species from moist habitats would have greater degrees of shrinkage.For insight into the mechanisms and consequences of leaf shrinkage, we also investigated the relationships of 18 indices of leaf shrinkage with a wide range of aspects of leaf structure and composition, including gross morphology, leaf venation architecture, parameters of pressure-volume curves, and leaf water storage. We hypothesized that, across species, shrinkage in whole leaf, cell, and intercellular airspace thickness would be lower for species with greater allocation to structural rigidity and osmotic concentration, and thus shrinkage would be positively correlated with a lower modulus of elasticity (ε), less negative osmotic pressure at full turgor (π_o), lower leaf mass per area (LMA), and lower leaf density. Additionally, we tested the longstanding hypothesis that species with higher major VLA and/or minor VLA (i.e. the fourth and higher vein-branching orders) would shrink less in area and/or thickness with dehydration (Gardner and Ehlig, 1965). Finally, we tested the ability of dehydrated leaves to recover in size with rehydration. We hypothesized that recovery would be greater for mildly than for strongly dehydrated leaves and that species with greater leaf shrinkage would be better able to recover from shrinkage.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome

Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol β-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening.Terpenoids, a class of secondary metabolites, are involved in interactions between plants and insect herbivores or pollinators and are implicated in general defense and stress responses (Gershenzon and Dudareva, 2007). The C10 and C15 members of this family were also found to affect the flavor profiles of most fruits and the scent of flowers at varying levels (Lund and Bohlmann, 2006; Schwab et al., 2008). The biosynthetic pathway and molecular genetics of terpenoids have been intensively and widely studied in different species (Loza-Tavera, 1999; Trapp and Croteau, 2001; Mahmoud and Croteau, 2002; Dudareva et al., 2004, Pichersky et al., 2006). Recent analyses of the grapevine (Vitis vinifera) genome (Jaillon et al., 2007; Velasco et al., 2007; Martin et al., 2010) revealed a large family of terpenoid synthases, many of which have been shown to produce fragrant monoterpenols (Lücker et al., 2004; Martin and Bohlmann, 2004; Martin et al., 2010, 2012).Monoterpenes are one major class of positive aroma compounds in traditional grapevine varieties, as they impart floral and citrus notes to some white wines (Mateo and Jiménez, 2000; Ebeler, 2001). The quality and quantity of terpenes vary strongly among grapevine varieties and even show differences between lines (clones) of the same variety. Berries of grapes such as cv Muscat of Alexandria and cv Gewurztraminer contain various and large amounts of C10 terpenoids, which are essential for the typical cv Muscat-like aroma. The characteristic aroma of cv Muscat, the most terpene-laden of all grapevine varieties, is primarily determined by a combination of just three terpenoid alcohols: geraniol, 3S-linalool, and nerol. Geraniol is considered to be the most crucial (Fig. 1). The same terpenes are also essential to the varietal aroma of cv Riesling, in addition to which α-terpineol, 3S-citronellol, and 3S-hotrienol are deemed equally important (Bayrak, 1994; Luan et al., 2005). Among the odoriferous monoterpenes, the cyclic ether rose oxide is a potent odorant in cv Scheurebe and cv Gewurztraminer (Guth, 1996, 1997). Terpenes are found mainly in the exocarp of grape berries, and the concentration of many terpenes accumulates as the grape ripens (Martin et al., 2012). The absolute levels of terpenes are highly affected by environmental conditions. This makes a systematic search for clones with preferable phenotypes difficult (Rivoal et al., 2010).Open in a separate windowFigure 1.Structural formulae of selected monoterpenes and their glycosylated conjugates found in grapes and wines.In the early 1980s, it was discovered that most terpenes found in aromatic grapevine varieties are oxidized to polyhydroxylated derivatives or chemically bound to sugars in the form of O-β-d-glucopyranosides, 6-O-α-l-rhamnopyranosyl-β-d-glucopyranosides, 6-O-α-l-arabinofuranosyl-β-d-glucopyranosides, and 6-O-β-d-apiofuranosyl-β-d-glucopyranosides, making them odorless (Fig. 1; Williams et al., 1982). Glycosylation is one of the most widespread modifications of plant secondary metabolites, and merely 17% to 23% of the terpenes found in cv Riesling grapes are present in their free (unglycosylated) form (Gunata et al., 1985b; Razungles et al., 1993; Maicas and Mateo, 2005). The temporal accumulation of monoterpenol glycosides has been investigated in two different grapevine varieties (Luan et al., 2005, 2006a, 2006b). The high proportion of bound monoterpenes is considered as a wine’s “hidden aromatic potential” (Gunata et al., 1985a), since only free terpenes contribute to the aroma of a wine. Some of the bound terpenes are converted to their free form by either acid or enzymatic hydrolysis, usually both, during must fermentation and wine maturation. However, not only positive aroma contributors are released but also musty-smelling phenols are liberated that also occur as glycosides (Rocha et al., 2004).Glycosides are formed by the action of glycosyltransferases (GTs), which are a ubiquitous group of enzymes that catalyze the transfer of a sugar moiety from an activated sugar donor, usually UDP-Glc, to acceptor molecules (Ross et al., 2001; Bowles et al., 2006). Sugar conjugation results in increased stability and water solubility. It has also been considered to control the compartmentalization of metabolites (Zhao et al., 2011). Over the past few years, the completion of several plant genome sequencing projects has unraveled unsuspected complexity within modifying enzymes such as GTs. These enzymes are encoded by large multigene families comprising several hundred genes. Thousands of GTs (http://www.cazy.org/) have been proposed and classified into 91 families to date (Coutinho et al., 2003). GTs in class 1, which recognize small-molecule scaffolds, are encoded by 120, 165, and 210 genes in Arabidopsis (Arabidopsis thaliana), Medicago truncatula (Gachon et al., 2005), and grapevine, respectively (http://genomes.cribi.unipd.it/grape/). The encoded proteins that contain a conserved plant secondary product GT motif toward the N terminus belong to the GT family 1 of the classification and typically use UDP-α-d-Glc as sugar donor (UGT). Heterologous expression of all Arabidopsis secondary metabolism UGTs (AtUGTs) in Escherichia coli and in vitro testing of the substrate specificity led to the identification of enzymes capable of catalyzing key glycosylation reactions (Lim et al., 2001, 2002; Paquette et al., 2003). It has also been shown that 27 AtUGTs glycosylate a diversity of monoterpenes, sesquiterpenes, and diterpenes such as geraniol, linalool, terpineol, and citronellol (Caputi et al., 2008). Although only linaloyl glucoside has been tentatively identified in Arabidopsis (Aharoni et al., 2003), this plant seems to have the capacity to glycosylate a variety of terpenes, comprising monoterpenols that are key flavor compounds in grape.Using protein extracts prepared from leaves and berries of grape cultivars, it has been shown that glucosyltransferase activities can be detected against a wide range of substrates (Ford and Høj, 1998). Several classes of phenylpropanoids, including flavonols, anthocyanidins, flavanones, flavones, isoflavones, a stilbene, simple phenols, and monoterpenols, were among the substrates glucosylated. Total soluble leaf proteins separated into fractions with differing glucosyltransferase activities. Furthermore, it was demonstrated that glycosylation proceeds with a clearly detectable enantiodiscrimination for linalool and its hydroxylated derivatives in cv Morio-Muscat and cv Muscat Ottonel using enantioselective gas chromatography (GC)-mass spectrometry (MS; Luan et al., 2004). These findings show that monoterpene glycosylation in grape has a big impact on the resulting wine flavor. However, to date, only an anthocyanidine 3-O-glucosyltransferase (VvGT1; Ford et al., 1998) and 5-O-glucosyltransferase (VvB12H3_5-GT; Jánváry et al., 2009), a resveratrol/hydroxyl cinnamic acid O-glucosyltransferase from Concord grape (Vitis labrusca [VlRSGT]; Hall and DeLuca 2007), three phenolic acid O-glucosyltransferases (Vitis vinifera galloyl glucosyl transferase1 [VvgGT1]–VvgGT3; Khater et al., 2012), a flavonol 3-O-glucuronosyltransferase (VvGT5), and a bifunctional 3-O-glucosyltransferase/galactosyltransferase (VvGT6; Ono et al., 2010) have been cloned from grapes and functionally characterized. The substrate specificities of their encoded proteins are restricted to phenolics. On the other hand, recombinant GT proteins encoded by genes from Arabidopsis, Eucalyptus perriniana, Rauvolfia serpentine, Sorghum bicolor, and M. truncatula are multisubstrate enzymes that glucosylate monoterpenols, although with much lower efficiency than their assumed in planta substrates (Hefner et al., 2002; Hansen et al., 2003; Nagashima et al., 2004; He et al., 2006; Caputi et al., 2008).Studies on the improvement of grape aroma are mainly focused on the enhancement of monoterpene biosynthesis. However, high levels of terpene glycosides (up to 80% of total terpenes) show that the reduction of terpene metabolism toward aroma-inactive glycosides could be an alternative approach, as higher levels of aroma-active terpenes would be present.The now publicly available genome database of the grapevine variety cv Pinot Noir provides the opportunity to systematically identify monoterpenol GT genes in Vitis spp. (Jaillon et al., 2007; Velasco et al., 2007). In this study, we used 27 GT sequences of Arabidopsis that encode putative terpene alcohol GTs (Caputi et al., 2008) as a basis for the functional characterization of similar genes in the Vitis spp. genome. Metabolite profiling analyses in combination with gene expression analyses in different varieties and biochemical characterization of recombinant proteins resulted in the identification of a nerol/geraniol glucosyltransferase. Detection of functional and nonfunctional allelic forms of the enzyme followed by site-directed mutagenesis led to the identification of three amino acid residues that affect GT activity.     

De Novo Synthesis and Degradation of Lx and V Cycle Pigments during Shade and Sun Acclimation in Avocado Leaves

The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect α- and β-carotene pools on the first day, but during long-term acclimation α-carotene pools changed noticeably. Nonetheless, the total change in α- and β-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the α- and β-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.It has long been recognized that photosynthesis in plants must resolve two conflicting requirements, the need to ramp up maximum light-harvesting efficiency in dim light and to wind back to lower efficiency when light is in excess, in order to maintain high rates of growth and productivity in varying light environments (Björkman, 1981; Pearcy, 1990). A wealth of research has established that plants adjust through an array of morphological and molecular events that confer photoprotection, mitigate and repair photoinactivation of PSII, and facilitate acclimation of the photosynthetic apparatus over different time scales in response to variable light regimes in wild plants, crops, and algae (Osmond et al., 1999; Demmig-Adams et al., 2006). In the context of the light reactions, low light acclimation optimizes light harvesting and energy transfer to the photosystems, particularly PSII, via enlarged functional antennae, accumulation of accessory light-harvesting pigments, and down-regulation of unnecessary competing photoprotective processes. High light acclimation involves increased photoprotection and photorepair, downsized antennae, fewer photosystems, and sometimes changes in the PSI to PSII stoichiometry (Osmond et al., 1999; Förster et al., 2005). Along with their function in energy transfer to the photosynthetic reaction centers as accessory pigments to chlorophylls, the xanthophyll pigments violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) play a central role in these transformations of the photosynthetic apparatus, especially in thermal energy dissipation and detoxification of reactive oxygen species.Two xanthophyll cycles are now known in terrestrial plants, the lutein epoxide cycle (Lx cycle) based on interconversions of lutein-5,6-epoxide (Lx) and lutein (L) synthesized from α-carotene (α-C), and the violaxanthin cycle (V cycle) based on the interconversions of V and A+Z synthesized from β-carotene (β-C; García-Plazaola et al., 2007). Presumably, both cycles are catalyzed by the same enzymes, violaxanthin epoxidase (VDE) for deepoxidation in high light and zeaxanthin epoxidase (ZE) for the reverse reactions in low light or darkness (Latowski et al., 2004). Rediscovery of the Lx cycle in the parasitic angiosperm Cuscuta reflexa (Bungard et al., 1999) has led to growing interest in differing manifestations of this cycle in terrestrial plants and its relationships to the apparently universal V cycle (Demmig-Adams, 1998). A complete Lx cycle seems to function on a daily basis in both C. reflexa and the mistletoe Amyema miquelii (Matsubara et al., 2001), even though Lx conversion to L is sometimes slower than V to A+Z and dark recovery of Lx is usually slower than that of V. Intriguingly, in shade leaves of Inga sapindoides, high concentrations of Lx were seemingly irreversibly converted to L on exposure to strong light, in marked contrast to the co-occurring, fully reversible V cycle (Matsubara et al., 2005). Similar responses have been found in other woody plants with long-lived leaves in deeply shaded canopies, including Mediterranean oaks (Quercus spp.; García-Plazaola et al., 2003), sweet bay laurel (Laurus nobilis), and avocado (Persea americana; Esteban et al., 2007, 2008). This response type is known as a truncated Lx cycle (García-Plazaola et al., 2007).The functions attributed to the Lx cycle were initially based on structural analogies between Lx and A and between L and Z (Bungard et al., 1999; Pogson and Rissler, 2000; Matsubara et al., 2001). With increased evidence for the possible role of L in photoprotection (Pogson et al., 1996, 1998; Lokstein et al., 2002; Dall''Osto et al., 2006), additional functional analogies emerged. Furthermore, recent in vitro reconstitution studies with light-harvesting complex proteins and purified pigments also support a spatial overlap of the cycles, as some pigment-binding sites can be occupied by either α- or β-xanthophylls (Matsubara et al., 2007). An attractive hypothesis is that photoconversion of Lx to L might sustain or enhance photoprotection associated with the V cycle (Demmig-Adams and Adams, 1992; Niyogi, 2000). In support of this view, it has been demonstrated in leaves of Quercus rubra and in leaflets of Inga marginata that increasing amounts of photoconverted L, which persist even when A and Z are epoxidized to V, were associated with faster engagement and higher levels of nonphotochemical quenching (NPQ) of chlorophyll fluorescence (García-Plazaola et al., 2003; Matsubara et al., 2008). Furthermore, evidence from mammalian eye research as well as from plants suggests that L also acts as a highly efficient reactive oxygen species scavenger (Kim et al., 2006; Johnson et al., 2007).Broader issues, such as the roles of short-term dynamics of the two cycles in relation to long-term processes of shade and sun acclimation and in relation to leaf development and age, are poorly understood. Nonfruiting shoots of avocado trees constitute a very suitable model system in which to address these issues. Long-lived leaves of shade-grown avocado contain some of the highest levels of Lx thus far recorded (Esteban et al., 2007; García-Plazaola et al., 2007) and have two to four flushes of leaf initiation per year that exhibit a form of delayed greening in which leaf expansion precedes increases in stomatal conductance, chlorophyll content, and CO₂ assimilation. Expanding leaves remain sinks for up to 1 month until they reach about 70% to 80% of full expansion (Schaffer et al., 1991), and stomata do not become fully functional until leaves attain 90% of full expansion (Scholefield and Kriedemann, 1979). However, shoots also retain old leaves through several flushes, and leaves from the previous season contribute significantly to total plant carbon gain (Liu et al., 2002), with photosynthesis rates up to 50% of those in new, fully expanded leaves (Heath et al., 2005). These properties offer an array of opportunities for new research into the concurrent operation of the two xanthophyll cycles.Since there have been very few studies of these complex responses, we carried out a series of short- and long-term light treatments that are likely to reflect what leaves may experience in natural environments, with the aim to gain further insight into the physiological relevance of the Lx and V cycles under those circumstances. Four types of acclimation experiments were undertaken in this study. First, short-term acclimation from shade to sun addressed fast responses to a drastic increase in the light environment, simulating a prolonged sun fleck in shaded mature leaves or exposure to a bright sunny day in young leaves that had emerged during a prolonged overcast (shaded) growth period. These experiments revealed an unexpected loss of L prior to deepoxidation of Lx and V and a reverse Lx cycle in young leaves. Second, long-term acclimation of sun leaves to prolonged shade simulated normal processes of shading by further growth of outer canopy leaves. These treatments established the very slow accumulation of Lx in avocado leaves. Third, sequential sun exposures of mature leaves over several days, followed by continuous shade, were applied to simulate successive prolonged sun flecks, mimicking stochastic canopy disturbance during severe weather events, which confirmed many responses in the above experiments, particularly the very slow epoxidation of L to Lx in prolonged shade. Fourth, long-term acclimation of young and mature leaves to sun was examined. These experiments simulated sudden changes to canopy architecture as experienced during pruning and extended our understanding of the comparative rates and magnitude of Lx and V cycle engagement. We discuss the short-and long-term kinetics of both cycles in avocado leaves of different ages during acclimation, with particular attention to the stoichiometric relationships between xanthophyll and carotenoid pools and changing PSII efficiency.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

The α-Subunit of the Arabidopsis Heterotrimeric G Protein,GPA1, Is a Regulator of Transpiration Efficiency

Land plants must balance CO₂ assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the α-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO₂ concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO₂ concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::β-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.Land plants, in particular plants that utilize C₃ photosynthesis, must balance CO₂ acquisition with water loss in order to maximize fitness. The water loss cost per unit of biomass acquired can be expressed as transpiration efficiency (TE; also referred to as water-use efficiency), the ratio of CO₂ assimilation (A) to transpiration. TE strongly correlates with the δ¹³C of plant tissue, the ratio of ¹³C to ¹²C relative to a standard (Farquhar et al., 1982, 1989; Dawson et al., 2002). The physiological basis of this correlation is that in plants there is diffusional and biochemical discrimination against ¹³C, the heavier and less abundant stable isotope of carbon. Discrimination against ¹³C decreases with decreasing internal CO₂ concentration (C_i), which can result from either increased A or reduced stomatal conductance (g_s; Farquhar et al., 1982). While it is known that g_s (a main factor controlling transpiration) correlates with A (Wong et al., 1979), genetic variation for TE and/or δ¹³C has been documented in a number of species (Farquhar and Richards, 1984; Virgona et al., 1990; Ehleringer et al., 1991; Comstock and Ehleringer, 1992; Hammer et al., 1997; Lambrides et al., 2004). In Arabidopsis (Arabidopsis thaliana), multiple quantitative trait loci associated with TE have been identified, indicating that TE is under genetic control (Juenger et al., 2005; Masle et al., 2005; McKay et al., 2008). However, only one gene, ERECTA, has been specifically identified as a regulator of TE (Masle et al., 2005). ERECTA encodes a Leu-rich repeat receptor-like kinase (Torii et al., 1996) and regulates TE via the control of stomatal density, g_s, mesophyll cell proliferation, and photosynthetic capacity (Masle et al., 2005).Heterotrimeric G proteins are GTP-binding proteins that function in the transduction of extracellular signals into intracellular responses. In its inactive state, the G protein classically exists as a trimer consisting of an α-subunit (Gα) bound to GDP, a β-subunit (Gβ), and a γ-subunit (Gγ). When a ligand binds to a G protein-coupled receptor (GPCR), a conformational change occurs in the G protein, resulting in the exchange of GDP for GTP and the dissociation of Gα-GTP from the Gβγ dimer. The G protein subunits remain active until the intrinsic GTPase activity of Gα results in the hydrolysis of GTP to GDP and the reassociation of the inactive trimer. The Arabidopsis genome contains canonical Gα and Gβ genes, GPA1 and AGB1, and two genes known to encode Gγs, AGG1 and AGG2 (Assmann, 2002). One likely GPCR, GCR1, has been functionally characterized (Pandey and Assmann, 2004), and additional GPCRs have been predicted using bioinformatics (Moriyama et al., 2006; Gookin et al., 2008) and interaction with GPA1 in yeast-based protein-protein interaction assays (Gookin et al., 2008). Recently, a new class of G proteins, GPCR-type G proteins (GTG1 and GTG2), have been identified in Arabidopsis that also serve as one class of abscisic acid (ABA) receptors (Pandey et al., 2009).Despite the paucity of heterotrimeric G protein subunit genes in the Arabidopsis genome as compared with mammalian systems, functional studies of heterotrimeric G protein mutants suggest that G protein function is diverse in Arabidopsis. G proteins have been shown to function in developmental processes and hormonal and environmental signaling, including stomatal aperture regulation (Perfus-Barbeoch et al., 2004; Joo et al., 2005; Chen et al., 2006; Pandey et al., 2006; Trusov et al., 2006; Warpeha et al., 2007; Fan et al., 2008; Zhang et al., 2008a, 2008b). In response to drought stress, ABA concentration increases in the leaves (Davies and Zhang, 1991; Davies et al., 2005), where it promotes stomatal closure and inhibits stomatal opening (Schroeder et al., 2001). The G protein α- and β-subunit mutants, gpa1 and agb1, respectively, are hyposensitive to ABA inhibition of stomatal opening while displaying wild-type ABA promotion of stomatal closure (Wang et al., 2001; Fan et al., 2008). ABA inhibits stomatal opening in part by inhibiting inward-rectifying K⁺ channels, reducing K⁺ influx and therefore water entry into the cell (Schroeder et al., 2001). ABA inhibition of inward K⁺ channel activity is reduced in both gpa1 and agb1 mutants (Wang et al., 2001; Fan et al., 2008). agg1 and agg2 mutants show no altered regulation of ABA-induced stomatal movements or ion channel activities, suggesting that the genome contains additional unknown Gγ(s) or that heterotrimeric G protein signaling in plants does not always operate according to the mammalian paradigm (Trusov et al., 2008). gcr1 mutants are hypersensitive to both ABA inhibition of opening and ABA promotion of stomatal closure (Pandey et al., 2006). gtg1 gtg2 double mutants show a wild-type response for ABA inhibition of stomatal opening and are hyposensitive in ABA promotion of stomatal closure (Pandey et al., 2009).While the altered stomatal sensitivities of the G protein mutants to ABA suggest that heterotrimeric G proteins may function in the regulation of whole plant water status, few experiments have been performed at the whole leaf or whole plant level. gpa1 mutants in the Wassilewskija background display increased water loss from excised leaves (Wang et al., 2001); however, there are no published reports of experiments assessing whole plant water status in gpa1 or agb1 mutants. gcr1 mutants show reduced water loss from excised leaves, drought tolerance, and improved recovery following the cessation of drought stress (Pandey and Assmann, 2004). In addition to their altered guard cell sensitivities to ABA, gpa1, agb1, and gcr1 mutants are hypersensitive to ABA inhibition of root and seedling development (Pandey et al., 2006), which could have impacts on whole plant water status. Finally, it has been recently reported that gpa1 and agb1 mutants have reduced and increased stomatal densities, respectively, in cotyledons (Zhang et al., 2008a). While stomatal density of leaves can be an important component of whole plant water status, the study by Zhang et al. (2008a) was performed on cotyledons only, whose developmental programs are often independent from those of true leaves (Chandler, 2008). Therefore, it is difficult to infer how this cotyledon phenotype will affect water relations at the whole plant level. Taken together, the stomatal aperture, electrophysiology, and tissue-specific ABA phenotypes of the G protein mutants, in addition to the possibility for altered stomatal density in the G protein mutant leaves, make it difficult to predict how G proteins contribute to the regulation of whole-plant TE. For example, the ABA-hyposensitive stomatal phenotype of gpa1 could result in increased transpiration, possibly reducing TE under certain conditions. Conversely, if gpa1 mutant leaves have reduced stomatal density, transpiration may be reduced, which could enhance TE under a range of conditions. Previous attempts to address the contributions of G proteins to whole plant transpiration, TE, and drought response using excised leaf/rosette assays to measure water loss are not sufficient, because both transpiration and A must be taken into account. Therefore, we investigated the role of GPA1 in regulating TE under ample water and drought stress conditions and in the presence of ABA. We have identified GPA1 as a negative regulator of TE in Arabidopsis via the control of g_s and stomatal proliferation.     

Alpha-Synuclein affects neurite morphology,autophagy, vesicle transport and axonal degeneration in CNS neurons

Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.Growing evidence suggests that Parkinson''s disease (PD) pathology starts at the presynaptic terminals and the distal axons and is then propagated back to the soma in a ''dying back'' pattern.^{1, 2} Accordingly, at the time of clinical onset, there is only a 30% loss of total substantia nigra pars compacta neurons but a far more severe loss of striatal dopaminergic markers (70–80%), suggesting that axonal terminals of the nigrostriatal pathway are affected earlier.¹ It is thus essential to understand the pathomechanisms specifically affecting the axon in PD in order to interfere with early disease progression.Neurodegeneration in PD is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).³ Interestingly, also LB pathology is initially found in the distal axons before becoming evident in the neuronal somata, and dystrophic neurites, so called ''Lewy neurites'', outnumber LBs in the early stages of PD.^{2, 4, 5} A main component of LBs is the protein alpha-synuclein (αSyn) that is not only widely used as a histopathological marker for PD but is also believed to have a major role in PD pathogenesis.^{6, 7} The importance of αSyn is further underlined by the discovery of αSyn point mutations (e.g. Ala53Thr (A53T), Ala30Pro (A30P)) and multiplications of the αSyn gene, all of which cause autosomal dominant forms of PD.^{8, 9, 10} However, neither the physiological functions nor the pathogenetic mechanisms of αSyn are well understood.⁷The biological effects of αSyn expression strongly depend on the model system. Wild-type (WT) human αSyn does not lead to major clinical or histological abnormalities when expressed in transgenic mice,^{11, 12} but its overexpression mediated by adeno-associated viral vectors (AAV) results in severe neurodegeneration, suggesting a dose-dependent toxic effect.^{13, 14} Different human αSyn-A30P and -A53T transgenic mouse lines develop severe motor impairments, partly resembling symptoms of human PD, accompanied by a degeneration of the nigrostriatal neuronal system and LB-like pathology.^{11, 12, 15} In line with the pathological findings in human PD, the axonal compartment is affected early and most prominently in these animal models.Different putative pathomechanisms of αSyn toxicity have been explored. For example, the cytoskeleton is an important molecular target of αSyn. Multimeric forms of αSyn were shown to impair the polymerization of tubulin and microtubule formation.^{16, 17} Overexpression of αSyn increased actin instability and induced actin bundling in cultured hippocampal neurons.¹⁸ There are, however, divergent data on the resulting effects of αSyn overexpression on neurite outgrowth and integrity in different model systems.^{19, 20, 21, 22}Moreover, a dysregulation of autophagy has been implicated in PD pathology. Aberrant αSyn is normally degraded by autophagy and only to a negligible degree by the proteasome.²³ Several studies have shown that the inhibition of autophagy results in an accumulation and increased toxicity of αSyn, whereas the activation of autophagy has therapeutic effects in PD models.^{23, 24, 25, 26} However, the direct effects of αSyn and its mutants on autophagy seem to rely strongly on the model system and the published data are highly controversial.^{24, 26, 27, 28, 29, 30, 31, 32}Given the central role of axonal degeneration in PD, it is likely that disturbances of axonal transport are involved.³³ In support of this proposition, the motor protein kinesin was shown to be decreased early and stage-dependently in PD patients, preceding the loss of substantia nigra neurons.³⁴ αSyn itself is actively transported along the axons, mainly by the slow component of axonal transport, but the role of αSyn in axonal vesicle transport is unclear.³⁵Here, we present a comprehensive analysis of the effects of αSyn on neurite morphology and examine important pathomechanisms.     

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.It is now clear that chronic inflammation is a hallmark of type I and type II diabetes, affecting both β-cell mass and insulin secretion.¹ Type I diabetes is characterized by drastic decreases in β-cell mass and insulin secretion, in part mediated by proinflammatory cytokines produced following autoimmune activation.¹ Proinflammatory cytokines, particularly interleukin-1β (IL-1β), in combination with interferon-γ (IFN-γ) and/or tumor necrosis factor-α (TNF-α), promote death by apoptosis and decrease function of differentiated β-cells, leading to β-cell destruction.¹ Pancreatic islet transplantation is a promising alternative therapy for some type I diabetic patients.² However, clinical outcome is not always achieved because of significant loss of islet mass during and after transplantation.³ Up to 80% of transplanted islets can die during the post-transplantation period as a result of apoptosis because of several mechanisms, notably the instant blood-mediated inflammatory response (IBMIR) and the release of a mix of cytokines including IL-1β, TNF-α and IFN-γ.⁴Immune-modulatory strategies for type I diabetes therapy and improvement of islet transplantation outcomes have emerged, targeting a single specific cytokine, such as IL-1β or TNF-α.^{2, 5} However, these strategies may only target inflammation partially.² Indeed, multiple cytokines, originating from surrounding immune cells and/or β-cells themselves, are more likely to be present simultaneously^{4, 6} and act synergistically to induce β-cell death and dysfunction.^{7, 8, 9} Preclinical and clinical studies demonstrated that glucagon-like peptide-1 (GLP-1) analogs, in addition to regulating glucose homeostasis in vivo, contribute to the restoration of normoglycemia after islet transplantation.^{10, 11, 12, 13} GLP-1 receptor (GLP-1R) analogs protect β-cell survival and function from proinflammatory cytokine attack.^{12, 14, 15} However, some studies have shown only modest and short-term anti-inflammatory effects of GLP-1 analogs when used alone.^{11, 13, 16}Mitogen-activated protein kinases (MAPKs) (i.e., extracellular-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK) play important roles in cytokine-induced β-cell dysfunction and death.¹ Conversely, ERK1/2 are involved in the beneficial effects of glucose and GLP-1 analogs.^{17, 18, 19} In this context, upstream protein kinases that specifically control the activation of MAPK in response to a combination of inflammatory cytokines (IL-1β, TNF-α and IFN-γ), rather than a single cytokine, may be useful targets for therapeutic interventions against pancreatic β-cell failure.The serine/threonine kinase tumor progression locus 2 (Tpl2) (also known as COT (Cancer Osaka Thyroid) in humans) is a member of the MAP3K family (the MAP3K8) whose activation stimulates primarily the ERK1/2 pathway, but also JNK and/or p38 MAPK in some cell types, specifically in response to various inflammatory stimuli.^{20, 21, 22} Dysregulation of Tpl2 expression and signaling is associated with acute and chronic inflammatory diseases,^{20, 21, 22} and several studies highlight a critical function of Tpl2 in the control of inflammatory responses and survival in adipocytes, fibroblasts and immune and epithelial cells.^{21, 22, 23, 24}However, there is currently nothing known about the effects of Tpl2 in β-cells. The aim of this study was to determine whether Tpl2 may be a new key inflammatory regulator in β-cells or islets. We demonstrate that Tpl2 contributes to cytokine-induced β-cell apoptosis and dysfunction, and suggest that Tpl2 inhibition, either alone or combined with a GLP-1 receptor agonist, represents a potential new therapeutic strategy for the treatment of diabetes.     

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype.Breast cancer is a clinically heterogeneous disease and both intra and inter-tumor heterogeneities provide great challenges for developing successful therapies. Expressions (or absence thereof) of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2)/neu are widely used to clinically classify breast tumors into multiple therapeutic groups.¹ The ER/PR-positive and the HER2-positive breast cancer patients could be benefited from endocrine and HER2-targeted therapies.¹ However, triple-negative breast cancers (TNBCs), which represent ∼12–17% of all breast cancer,² lack ER, PR and HER2/neu expressions² and are not responsive to therapies targeting these receptors. Therefore, the only systemic therapy available for TNBC is chemotherapy.³ Furthermore, TNBC is associated with aggressive pathologic features like higher histology grade and mitotic index⁴ and often found to be associated with higher rate of metastasis and recurrence leading to limited clinical outcome.^{5, 6, 7, 8} Recurrence of TNBC tends to recur within a few years after successful initial treatment^{6, 9} and often develops metastasis to the bone, brain and lungs with poor prognosis.^{2, 6} Thus, identification of signaling pathways that regulate malignant progression of breast cancer subtypes, especially TNBCs, would be therapeutically important.In recent years, PKC signaling has been implicated in modulating invasion and metastasis of multiple tumors.^{10, 11} The PKC family consists of multiple serine/threonine kinases and the relative contribution of individual PKC isoforms during cancer progression varies due to pleiotropism.¹² PKC isoforms regulate diverse cellular functions such as cell-cycle regulation, cellular survival, cell–cell communications and apoptosis.¹³ In particular, atypical PKC isoforms, PKCζ and atypical protein kinase C lamda/iota (PKCλ/ι), are known to be important for chemotaxis, cell polarity, migration and wound healing processes.^{14, 15} Aberrations in all these processes are manifested in tumor progression and metastasis.¹⁴ Consistent with these notions, recent studies indicated that atypical PKCs are associated with various human cancers.^{10, 11} Importantly, the PKCλ/ι gene is located at the 3q26.2 genomic region, which is most frequently amplified in human cancer^{16, 17}, and overexpression of PKCλ/ι has been implicated in cancer development in multiple tissues including the lung,^{18, 19} pancreas,²⁰ stomach,²¹ colon,²² esophagus,²³ liver,²⁴ bile duct,²⁵ ovary,¹⁷ prostate²⁶ and brain.²⁷ Recently, few studies have been reported higher expression of PKCλ/ι in ER/PR- and HER-positive breast cancer and also in lymph node metastases.^{28, 29} Kojima et. al.²⁸ showed that PKCλ/ι expression is highly induced in the ER/PR- and HER2-positive IDCs compared with ductal carcinoma in situ (DCIS) and normal breast. PKCλ/ι forms apical-junctional complexes (AJCs) with other polarity proteins such as partitioning defective 3 homolog (PAR3) and partitioning defective 6 homolog (PAR6),^{30, 31, 32, 33} and invasiveness of breast tumor cells was shown to be associated with loss of PKCλ/ι localization from their apical domains.²⁸ In addition, predominant nuclear localization of PKCλ/ι in both normal and atypical ductal hyperplasia (ADH) lesions prompted the concept that PKCλ/ι might be in an inactive state in these lesions.²⁸ However, expression and activation of PKCλ/ι in TNBCs and the functional importance of PKCλ/ι signaling in relation to invasive breast cancer progression and metastasis are very poorly understood.^{10, 11}Here, we studied PKCλ/ι signaling during invasive progression of TNBC. We utilized expression evaluations in triple-negative IDCs as well as metastatic breast cancers of human patients, in vitro and in vivo functional assays, and global gene expression analysis of human patient samples. We concluded that PKCλ/ι signaling is an important regulator for invasion and metastatic progression of human breast cancers including triple-negative subtypes.     

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C₁₃ apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.In plants, the synthesis of carotenoids is plastid localized, with the plastid type determining their function (Ruiz-Sola and Rodríguez-Concepción, 2012; Nisar et al., 2015). In nonphotosynthetic chromoplasts, carotenoids and their volatile derivatives attract pollinating insects or zoochoric animals. Here, carotenoids are sequestered in diverse suborganellar structures, which can be tubulous, globulous, membranous, or crystalline (Sitte et al., 1980; Egea et al., 2010). In chloroplasts, carotenoids are present in light-harvesting complex proteins and photosynthetic reaction centers. They extend the light spectrum used for photosynthetic energy transformation and act photoprotectively because of their ability to quench excitation energy from singlet- or triplet-state chlorophylls, thereby decreasing the risk that singlet oxygen forms (Niyogi, 1999; Demmig-Adams and Adams, 2002). Furthermore, the regulated epoxidation and deepoxidation of zeaxanthin in the xanthophyll cycle contribute to the nonphotochemical quenching of energy (Niyogi, 1999; Ballottari et al., 2014). In contrast to these processes, which maintain carotenoid integrity, carotenoids are also capable of chemically quenching singlet oxygen by their own oxidation, which is accompanied by the release of various carotenoid degradation products (Ramel et al., 2012a, 2013).The various functions of carotenoids require their dynamic qualitative and quantitative tuning in response to environmental conditions to attain and maintain adequate steady-state concentrations. These include both the regulation of their synthesis and the formation, release, or disposal of their breakdown products. The synthesis of carotenoids is initiated by the condensation of two molecules of geranylgeranyl diphosphate to form phytoene catalyzed by the enzyme phytoene synthase (PSY), which is considered as the rate-limiting enzyme (von Lintig et al., 1997; Li et al., 2008; Rodríguez-Villalón et al., 2009; Welsch et al., 2010; Zhou et al., 2015). In plants, two desaturases, phytoene desaturase and ζ-carotene desaturase, and two carotene cis-trans-isomerases convert the colorless phytoene into the red-colored all-trans-lycopene (Isaacson et al., 2002; Park et al., 2002; Chen et al., 2010; Yu et al., 2011). Two lycopene cyclases introduce either β- or ε-ionone rings, yielding α-(ε,β-)-carotene and β-(β,β)-carotene. In Arabidopsis (Arabidopsis thaliana), four enzymes hydroxylate carotenes with partially overlapping substrate specificity (Kim et al., 2009). Two nonheme iron-dependent β-carotene hydroxylases (BCH), BCH1 and BCH2, convert β-carotene into zeaxanthin. The second set of hydroxylases, cytochrome P450 (CYP)97A3 and CYP97C1, prefers α-carotene and produces zeinoxanthin and lutein, respectively. Absence of each cytochrome P450 hydroxylase constitutes a distinct phenotype, named lutein deficient5 (lut5) for CYP97A3 deficiency and lut1 for CYP97C1 deficiency, characterized by altered pigment compositions and the accumulation of monohydroxylated intermediates, whereas deficiency in BCH1 and BCH2 does not affect the pigment composition.In green tissues, photooxidative destruction seemingly predominates and consumes carotenoids (Simkin et al., 2003). Moreover, ¹⁴CO₂ pulse-chase experiments with Arabidopsis leaves identified α- and β-carotene as the main targets for photooxidation, whereas xanthophylls were less affected (Beisel et al., 2010). Oxidation assays with β-carotene showed epoxy- and peroxy-derivatives as the main primary products, which however, undergo additional reactions, yielding more stable degradation products that are, in part, the same apocarotenals/ones as those being produced enzymatically (Ramel et al., 2012a, 2013).In Arabidopsis, genes coding for carotenoid cleaving enzymes (carotenoid cleavage dioxygenases [CCDs]) form a small gene family comprising nine members, five of which are attributed to the synthesis of abscisic acid (ABA; nine-cis-epoxycarotenoid cleavage dioxygenases [NCEDs]; AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9; Iuchi et al., 2001; Tan et al., 2003), whereas two are committed to strigolactone biosynthesis (CCD7/MORE AXILLARY GROWTH3 [MAX3] and CCD8/MAX4; Alder et al., 2012; Bruno et al., 2014). Orthologs of CCD1 are involved in the generation of volatile apocarotenoids contributing to flower scents and aroma production (e.g. saffron [Crocus sativus; Rubio et al., 2008; Frusciante et al., 2014] and tomato [Solanum lycopersicum; Simkin et al., 2004]), whereas CCD4 enzymes are involved in citrus peel and chrysanthemum (Chrysanthemum morifolium) petal coloration (Ohmiya et al., 2006; Rodrigo et al., 2013). Recent analysis of Arabidopsis mutants revealed a major function of CCD4 in regulating seed carotenoid content with only a minor contribution of CCD1 (Gonzalez-Jorge et al., 2013). Moreover, CCD4 activity was required for the synthesis of an apocarotenoid-derived signaling molecule involved in leaf development and retrograde gene expression (Avendaño-Vázquez et al., 2014).Elevated carotenoid pathway flux caused by PSY overexpression increases carotenoid accumulation in various nongreen tissues, such as tomato fruits, canola (Brassica napus) seeds, cassava (Manihot esculenta) roots, and rice (Oryza sativa) endosperm (Shewmaker et al., 1999; Ye et al., 2000; Fraser et al., 2002; Welsch et al., 2010). Similarly, the constitutive overexpression of PSY in Arabidopsis results in dramatically increased carotenoid amounts accumulating as crystals in nongreen tissues, such as roots and callus, yielding β-carotene as the main product (Maass et al., 2009). However, leaves of the very same plants do not show altered pigment composition, and phytoene or other pathway intermediates are not detected. Similarly, increased levels of active PSY protein achieved though overexpression of the ORANGE protein exclusively affect carotenoid amounts in roots but not in leaves (Zhou et al., 2015). Leaves from constitutively PSY-overexpressing tomato and tobacco (Nicotiana tabacum) plants are also reported to show only slightly increased carotenoid levels compared with the wild-type control (Fray et al., 1995; Busch et al., 2002). These contrasting responses of leaves versus nongreen tissues to elevated pathway flux suggest fundamental differences in the modes of carotenoid formation and/or degradation.In this work, we identified xanthophyll-derived apocarotenoid glycosides (AGs) in Arabidopsis leaves that increase upon higher pathway flux. This suggests that apocarotenoid glycosylation functions as a valve regulating carotenoid steady-state levels in leaves. The analysis of Arabidopsis mutants enabled us to conclude on potential precursor carotenoids and assess the contribution of carotenoid cleavage enzymes on their formation. Moreover, apocarotenoids but not the identified glycosides were increased in carotenoid-hyperaccumulating roots, indicating tissue-specific different modes of carotenoid turnover regulation.     

Exploring the Ultrastructural Localization and Biosynthesis of β(1,4)-Galactan in Pinus radiata Compression Wood

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of β(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of β(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-β(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. β(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, β(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the β(1,4)-galactan backbone in pine. A β(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a β(1,4)-specific endogalactanase confirmed the production of β(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20°C, respectively. The β(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring β(1,4)-galactan in pine.The ability to respond to gravitropic stimuli is important for the survival of most terrestrial plants. Arborescent angiosperm and gymnosperm species generate wood with modified properties, called reaction wood, in response to gravitropic stimuli (Timell, 1969, 1986; Du and Yamamoto, 2007). The formation of reaction wood enables the return of bent stems to a vertical orientation. Interestingly, the location and type of the reaction wood deposited in woody gymnosperms and angiosperms generally differs significantly. Gymnosperms respond to gravitropic stimuli by compression wood formation on the underside of leaning stems (Timell, 1986), and arboreal angiosperms generate reaction wood primarily in the form of tension wood on the upper side of inclined stems (Timell, 1969).Compression wood in conifers differs significantly from normal wood in its anatomical, chemical, and physical properties. Typical anatomical features of severe compression wood are short, rounded, and thick-walled tracheids with a prominent band of lignin in the outer S2 layer of the cell wall as well as spiral checks and the absence of an S3 layer (Timell, 1986). Biochemically, compression wood is characterized by high levels of lignin, rich in condensed p-hydroxyphenyl units, as well as reduced cellulose and galactoglucomannan relative to normal wood (Timell, 1986; Nanayakkara et al., 2005; Yeh et al., 2006). Most striking, though, is that β(1,4)-galactan can constitute more than 10% (w/w) of the cell wall material in severe compression wood but is virtually absent in normal wood (Nanayakkara et al., 2005; Yeh et al., 2006). Recent work suggests that β(1,4)-galactan biosynthesis represents an early step in compression wood formation and confirms that its presence is diagnostic for this wood type (Altaner et al., 2007). However, the molecular signal cascades in conifers that lead to the deposition of β(1,4)-galactan are currently not well understood.Immunological studies in conifer species using the monoclonal anti-β(1,4)-galactan LM5 antibody (Jones et al., 1997) indicate that β(1,4)-galactan in compression wood is located in the S1 and outer S2 layers of mature tracheids but is virtually absent from the primary cell walls (Schmitt et al., 2006; Altaner et al., 2007; Möller and Singh, 2007). Instead of β(1,4)-galactan, most conifers contain small amounts of arabinogalactan, a polysaccharide characterized by a highly branched β(1,3)-galactan backbone (Vikkula et al., 1997; Willför et al., 2002; Laine et al., 2004) in their primary cell walls. The ultrastructural distribution of β(1,4)-galactan in compression wood appears to be largely consistent with highly lignified cell wall layers (Möller and Singh, 2007), which might explain the involvement of β(1,4)-galactan in the formation of lignin-carbohydrate complexes (Mukoyoshi et al., 1981; Minor, 1982; Timell, 1986; Laine et al., 2004).The investigation of β(1,4)-galactan structure in preparations from Pinus sylvestris (Laine et al., 2004) and Pinus radiata (Nanayakkara 2007) revealed a linear polymer. In Pinus densiflora Siebold & Zucc., β(1,4)-galactan was found to be slightly branched at positions C2, C3, and C6 (Mukoyoshi et al., 1981). β(1,4)-Galactan in conifers display a high degree of polymerization (DP), which was originally estimated to be in the range of 200 to 300 (Timell, 1986). More recent studies with P. radiata compression wood found the native polysaccharide to have a DP of approximately 380 (Nanayakkara 2007).β(1,4)-Galactan is a very good biochemical marker for compression wood (Altaner et al., 2007), but its physiological role is currently not well understood. Various functions for β(1,4)-galactan in compression wood have been proposed, such as strengthening of the secondary cell wall, absorption of mechanical stresses, and generation of compressive forces (Möller and Singh, 2007). Furthermore, β(1,4)-galactan is also found in tension wood, with a proposed role in cross-linking cellulose microfibrils (Arend, 2008). However, all of those hypotheses on the molecular function of β(1,4)-galactan in reaction wood await experimental verification.Despite substantial efforts to characterize the biosynthesis of this polymer, β(1,4)-galactan biosynthetic enzymes and their corresponding genes are currently unknown (Peugnet et al., 2001; Geshi et al., 2002, 2004; Abdel-Massih et al., 2003; Kato et al., 2003; Ishii et al., 2004; Konishi et al., 2004, 2007; Gorshkova and Morvan, 2006). However, based on other cell wall polysaccharide biosynthetic enzymes, it is likely that the enzymes involved in the biosynthesis of β(1,4)-galactan are either Golgi localized or pass through the Golgi in transit to the apoplastic space (Reyes and Orellana, 2008).To better understand β(1,4)-galactan synthesis in compression wood formation, we sampled both normal wood and severe compression wood from two 6-year-old P. radiata trees, which displayed stark differences in lignin and carbohydrate content and composition. Using these wood samples, new insights into the subcellular localization of β(1,4)-galactan in pine were generated using confocal laser fluorescence microscopy and transmission electron microscopy. An enzyme assay was developed, based on 2-aminopyridine (2AP)-labeled galacto-oligosaccharides as acceptor molecules, which we used to identify and partially purify a robust, microsome-associated, UDP-Gal-dependent β(1,4)-galactosyltransferase (GalT) activity in compression wood that was virtually undetectable in normal wood. Assays of the partially purified GalT revealed that this enzyme has some properties similar to those of previously characterized pectic GalTs, but a marked difference was observed in the size distribution of the enzymatic products.     

Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer''s disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.In Alzheimer''s disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.¹ Oligomers of the amyloid-β (Aβ) peptide appear to induce synaptic failure as an early event in the etiology of AD.^{2, 3, 4} However, despite its well-established synapse-impairing effects in rodent models,^{5, 6, 7} the synaptotoxic actions of Aβ most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of Aβ in cultured rodent neurons and in transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic Aβ actions result in the loss of functional (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,^{8, 9, 10} involve long-term depression-like mechanisms,^{9, 11, 12} and lead to the degradation of the entire postsynapse (dendritic spines).^{9, 11, 13} In addition, several distinct presynaptic Aβ actions on the synaptic vesicle cycle have been described.^{10, 14} Furthermore, Aβ-induced impairments of axonal transport regulation and Aβ-induced axon degeneration have been found in rodent neurons.^{15, 16, 17} This puzzling diversity of Aβ-induced synapse-related defects raises the question whether all of them are involved in the early pathomechanisms of human AD.In addition to well-established animal systems, the modelling of human neurological disease pathologies by human induced pluripotent stem cell (hiPSC) technology¹⁸ has been proposed as an innovative approach.^{19, 20, 21} The in vitro differentiation of hiPSCs to excitable neurons has been reported using a variety of protocols.^{22, 23, 24} However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.^{19, 25, 26} In addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the in vitro differentiation of patient-derived iPSCs has been used to model complex neurodevelopmental and neurodegenerative diseases.^{19, 27, 28} Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of Aβ upon in vitro neuronal differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.^{21, 29, 30, 31, 32, 33} Here we describe a hiPSC-based, carefully optimized in vitro differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of Aβ on human cortical neurons and on human synapses.     

Continuous Turnover of Carotenes and Chlorophyll a in Mature Leaves of Arabidopsis Revealed by 14CO2 Pulse-Chase Labeling

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by ¹⁴CO₂ pulse-chase labeling under control-light (CL; 130 μmol photons m⁻² s⁻¹) and high-light (HL; 1,000 μmol photons m⁻² s⁻¹) conditions. Following a 30-min ¹⁴CO₂ administration, photosynthetically fixed ¹⁴C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, ¹⁴C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the ¹⁴C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced ¹⁴C labeling of Chl a but not β-C. Consequently, the maximal ¹⁴C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing α-carotene (α-C) together with reduced amounts of β-C, remarkably high ¹⁴C labeling was found for α-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum ¹⁴C ratios between carotenes and Chl a were 1:2 for α-C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of α-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.Carotenoids are classified as accessory pigments in photosynthesis because they augment light harvesting in the blue spectral region by transferring the absorbed light energy to chlorophyll (Chl). However, the universal occurrence of carotenoids in photosynthetic cells, from bacteria to higher plants, indicates their essential roles, rather than mere accessory roles, in photosynthesis. Under excess light, carotenoids provide protection against photooxidative damage by facilitating dissipation of excitation energy from singlet- or triplet-state Chl and scavenging highly reactive singlet oxygen, which is generated through interaction between triplet excited Chl and oxygen (Demmig-Adams, 1990; Müller et al., 2001). These photoprotective functions make carotenoids indispensable for oxygenic photosynthesis, as demonstrated by lethal effects of inhibitors of carotenoid biosynthesis in plants (Bramley, 1993). Regulation of light harvesting and photoprotection by carotenoids requires their close proximity as well as the proper orientation to Chl molecules in pigment-protein complexes of PSI and PSII. Furthermore, a small fraction of non-protein-bound carotenoids serves as antioxidants in the lipid phase of photosynthetic membranes (Havaux and Niyogi, 1999; Havaux et al., 2004) and influences the structure and fluidity of the lipid bilayer (Gruszecki and Strzałka, 2005). Despite these and other lines of defense, the PSII reaction center polypeptide D1, and to a lesser extent also D2, undergo frequent photooxidative damage and repair in the light (Melis, 1999; Baena-González and Aro, 2002). When the repair process cannot keep up with the rate of photodamage, the overall quantum yield of PSII declines.Carotenoids are derived from isoprenoid precursors in plastids (for reviews on carotenoid biosynthesis in plants, see Lichtenthaler, 1999; Hirschberg, 2001; DellaPenna and Pogson, 2006; Giuliano et al., 2008; Tanaka et al., 2008; Cazzonelli and Pogson, 2010). Following the formation of linear C₄₀ lycopene, the pathway splits into two branches of major cyclic carotenoids: the β,β-branch gives rise to β-carotene (β-C) having two β-rings, whereas the β,ϵ-branch leads to formation of α-carotene (α-C) having one β-ring and one ϵ-ring. Hydroxylation of β-C and α-C produces the xanthophylls zeaxanthin (Z) and lutein (L), respectively. In the β,β-branch, epoxidation of the β-rings of Z results in successive synthesis of antheraxanthin (A) and violaxanthin (V); subsequently, V can be converted to neoxanthin (N), the last carotenoid product of the β,β-branch. Except for some species (García-Plazaola et al., 2007), L does not undergo β-ring epoxidation and the β,ϵ-branch thus stops with L, the most abundant carotenoid in leaves.Each of these carotenoids occupies specific binding sites in the photosynthetic apparatus to fulfill distinct roles. In both PSI and PSII, carotenes (α-C and β-C) are generally bound in core complexes, which also harbor Chl a molecules, while the majority of xanthophylls (L, Z, A, V, and N) are bound in light-harvesting antenna complexes together with Chl a and Chl b molecules (Bassi et al., 1993; Lee and Thornber, 1995). Accumulation of β-C in core complexes is a common feature of diverse photosynthetic organisms, whereas the occurrence of α-C in addition to β-C is restricted to certain taxa. For higher plants, α-C has been found in leaves of some, but not all, shade-tolerant species (Thayer and Björkman, 1990; Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on this photoacclimatory behavior, it has been proposed that α-C may function as a light-harvesting pigment while β-C may contribute to photoprotection (Krause et al., 2001), presumably by scavenging singlet oxygen and mediating a cyclic electron transfer around PSII (Tracewell et al., 2001; Telfer, 2005).Pronounced light-dependent changes are also observed for xanthophyll composition in light-harvesting antenna complexes. In a short term (minutes to hours), operation of the xanthophyll cycle, involving Z, A, and V, modulates levels of Z in a light-dependent manner. It is widely accepted that Z is able to enhance the dissipation of excess light energy from singlet excited Chl while V is not (Demmig-Adams, 1990; Müller et al., 2001). Long-term acclimation (days) to strong irradiance typically results in an increased pool size of the xanthophyll-cycle pigments (V + A + Z) and downsizing of PSII antenna, as indicated by a greater Chl a-to-Chl b ratio (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on the observed changes in steady-state amounts of xanthophylls and carotenes following irradiance shifts, alterations in the balance between biosynthesis and degradation, or turnover, have been implicated as a mechanism for long-term adjustment of carotenoid levels in leaves (Förster et al., 2009). However, just how much biosynthesis and degradation of different carotenoids occurs in photosynthesizing green leaves is an open question to date.In order to gain insight into carotenoid turnover of mature leaves, we conducted ¹⁴CO₂ pulse-chase labeling experiments with Arabidopsis (Arabidopsis thaliana) plants. Carotenoid turnover has been studied in algae in the past by applying [¹⁴C]bicarbonate (Blass et al., 1959; Grumbach et al., 1978); for example, no more than 1% to 2% of the photosynthetically incorporated ¹⁴C was allocated to the lipophilic fraction containing Chl and carotenoid in Chlorella pyrenoidosa after a 2-h pulse application (Grumbach et al., 1978). Even lower labeling efficiency is expected for photosynthetic pigments in nongrowing green leaves, in which pigment turnover takes place almost exclusively as part of the maintenance and acclimation of photosynthetic membranes. To overcome this intrinsic but anticipated difficulty, a ¹⁴CO₂ application setup was established for efficient and reproducible ¹⁴CO₂ incorporation in detached leaves of Arabidopsis during a short (30-min) pulse period. Moreover, a method of pigment separation was developed for ¹⁴C detection in concentrated leaf pigment extracts using a radio-HPLC system. Because carotenoid composition exhibits marked sun-shade responses in leaves (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009), ¹⁴CO₂ labeling patterns were studied in three different sets of Arabidopsis plants: (1) plants grown under 130 μmol photons m⁻² s⁻¹ (control light [CL]) and exposed to CL during a chase period of up to 10 h (CL plants); (2) plants acclimated to 1,000 μmol photons m⁻² s⁻¹ (high light [HL]) for 2 weeks and exposed to HL during the chase period (HL plants); and (3) plants grown under CL but exposed to HL during the chase period (CL→HL plants). These treatments simulated short-term (CL→HL) and long-term (CL or HL) responses to irradiance. Finally, as ¹⁴C was found to be rapidly incorporated in β-C and Chl a molecules in leaves of wild-type plants, in which β-C represents the only carotene species, ¹⁴C labeling experiments were also conducted with leaves of lut5 mutants containing both α-C and β-C (Fiore et al., 2006; Kim and DellaPenna, 2006) to compare turnover of the two carotenes.     

cFLIP is critical for oligodendrocyte protection from inflammation

Neuroinflammation associated with degenerative central nervous system disease and injury frequently results in oligodendrocyte death. While promoting oligodendrocyte viability is a major therapeutic goal, little is known about protective signaling strategies. We report that in highly purified rat oligodendrocytes, interferon gamma (IFNγ) activates a signaling pathway that protects these cells from tumor necrosis factor alpha (TNFα)-induced cytotoxicity. IFNγ protection requires Jak (Janus kinase) activation, components of the integrated stress response and NF-κB activation. Although NF-κB activation also occurred transiently in the absence of IFNγ and presence of TNFα, this activation was not sufficient to prevent induction of the TNFα-responsive cell death pathway. Genetic inhibition of NF-κB translocation to the nucleus abrogated IFNγ-mediated protection and did not change the cell death induced by TNFα, suggesting that NF-κB activation via IFNγ induces a different set of responses than activation of NF-κB via TNFα. A promising candidate is the NF-κB target cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which is protease-deficient caspase homolog that inhibits caspase-3 activation. We show that IFNγ-mediated protection led to upregulation of cFLIP. Overexpression of cFLIP was sufficient for oligodendrocyte protection from TNFα and short hairpin RNA knockdown of cFLIP-abrogated IFNγ -mediated protection. To determine the relevance of our in vitro finding to the more complex in vivo situation, we determined the impact on oligodendrocyte death of regional cFLIP loss of function in a murine model of neuroinflammation. Our data show that downregulation of cFLIP during inflammation leads to death of oligodendrocytes and decrease of myelin in vivo. Taken together, we show that IFNγ-mediated induction of cFLIP expression provides a new mechanism by which this cytokine can protect oligodendrocytes from TNFα-induced cell death.Interferon gamma (IFN-γ), the only type-II class IFN, has a paradoxical role in modulating cell function. It is critical for innate and adaptive immunity, but has multiple other functions. In the central nervous system (CNS), IFNγ has contrasting effects on the oligodendrocyte progenitor cells (O-2A/OPCs) that generate myelin-producing oligodendrocytes. O-2A/OPCs show suppressed division when exposed to IFNγ.^{1, 2, 3} However, when O-2A/OPCs differentiate into oligodendrocytes, IFNγ becomes pro-apoptotic.^{4, 5, 6, 7} Although IFNγ has a critical role in the pathogenesis of immune-mediated demyelinating disease;^{8, 9} the response of committed oligodendrocytes to IFNγ is more complex. For example, tumor necrosis factor alpha (TNFα) can show enhanced cytotoxicity in oligodendrocytes and transformed human neural cell lines when co-exposed with IFNγ.^{3, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}In contrast with reported toxic effects of IFNγ on oligodendrocytes, other studies did not see negative effects on mature oligodendrocytes^{5, 9, 20} or saw protection of glial lineage cells. IFNγ protects the Oli-neu oligodendrocyte-like cell line from reactive oxygen and nitrogen species,²¹ and overexpression of IFNγ before the induction of experimental autoimmune encephalomyelitis (EAE) protected oligodendrocytes from immune-mediated damage.⁹ The mechanism of such protection remains elusive.We now report that IFNγ protects purified, committed oligodendrocytes from TNFα-mediated apoptosis via Janus kinase (Jak)-mediated activation of the stress kinase PKR (double-stranded RNA-dependent protein kinase) and NF-κB-induced expression of cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which inhibits caspase activation. Moreover, gain-of-function and loss-of-function experiments show that cFLIP is necessary and sufficient for oligodendrocyte protection from TNFα. These results demonstrate induction of cFLIP in a stress response and NF-κB-dependent manner, leading to inhibition of caspase-mediated apoptosis, and reveal an important role for cFLIP in oligodendrocyte protection in vivo.     

The Green Microalga Chlamydomonas reinhardtii Has a Single ω-3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.Research on lipid metabolism in microalgae has flourished in recent years due to their potential as a rich source of ω-3 fatty acids (Guschina and Harwood, 2006; Khozin-Goldberg et al., 2011) and as a feedstock for biodiesel (Hu et al., 2008b; Rosenberg et al., 2008; Beer et al., 2009; Radakovits et al., 2010; Wijffels and Barbosa, 2010; Merchant et al., 2012; Work et al., 2012). Oils produced by microalgae resemble that of plants (Hu et al., 2008b), with the exception that they contain higher proportions of polyunsaturated fatty acid (PUFA) species (Harwood and Guschina, 2009). Desaturation of acyl groups in glycerolipids is catalyzed by fatty acid desaturases (FADs), which insert a C=C bond at a specifically defined position of an acyl chain (Shanklin and Cahoon, 1998). The degree of unsaturation of fatty acid components largely determines the chemical property and thus the utility of the oils produced. FADs have been one of the major tools for the genetic engineering of oil composition in land crops (Shanklin and Cahoon, 1998; Napier et al., 1999). In view of biodiesel applications, low PUFA content is advantageous in algal oil because of oxidation issues (Frankel, 1991).With the suites of sophisticated molecular genetic and genomic tools developed in the green microalga Chlamydomonas reinhardtii and the existence of substantial literature related to its cell biology, physiology, and biochemistry, this organism has emerged as a major model for research on algal oil (Radakovits et al., 2010; Merchant et al., 2012; Liu and Benning, 2013). Although the understanding of lipid metabolism in C. reinhardtii largely relies on sequence homologies to other models (Riekhof et al., 2005) and is still rather limited compared with the model plant Arabidopsis (Arabidopsis thaliana; Li-Beisson et al., 2010), functional studies based on mutants have started to provide important insights into the biosynthesis and turnover of membrane and storage lipids in this model alga (Riekhof et al., 2005; Work et al., 2010; Fan et al., 2011; Goodson et al., 2011; Boyle et al., 2012; Li et al., 2012a, 2012b; Yoon et al., 2012).In C. reinhardtii, C16 and C18 PUFAs (ω-3 + ω-6) make up to 60 mol% of total membrane fatty acids, of which more than 80% are ω-3 species (Giroud and Eichenberger, 1988; Siaut et al., 2011). Biochemical evidence for lipid-linked desaturation of fatty acyl chains has been established in C. reinhardtii over 20 years (Giroud and Eichenberger, 1989), but only two C. reinhardtii mutants affected in fatty acid desaturation have been described to date. These are crfad6 (hf-9), an insertional mutant for the plastidial ω-6 desaturase FAD6 (Sato et al., 1995), and microRNA-based silenced lines for the Δ4 desaturase CrΔ4FAD (Zäuner et al., 2012). The putative microsomal Δ12 desaturase FAD2 (Chi et al., 2008) and front-end ω-13 desaturase (Kajikawa et al., 2006) have been characterized by heterologous expression in the methylotrophic yeast Pichia pastoris, but no mutant is available. Moreover, although ω-3 PUFA is the most abundant fatty acid class in C. reinhardtii, the ω-3 desaturase remains uncharacterized, and no mutant with specific reduction in ω-3 content has been isolated so far.In Arabidopsis and C. reinhardtii, ω-3 PUFAs are present in both plastidic and extraplastidic lipids such as monogalactosyldiacylglycerol (MGDG) and phosphatidylethanolamine (PtdEtn), respectively (Mendiola-Morgenthaler et al., 1985; Giroud et al., 1988). While in plants there are distinct genes for plastidial and extraplastidial ω-3 FADs (Wallis and Browse, 2002), only one putative ω-3 desaturase seems encoded in the C. reinhardtii genome (version 5.0; Merchant et al., 2007). This raises several intriguing possibilities, including the existence of a mechanism to export ω-3 acyls from their site of biogenesis to other membranes or a dual localization of the ω-3 desaturase homolog (plastid and endoplasmic reticulum [ER]). In this study, we report the identification and characterization of a C. reinhardtii mutant defective in the promoter region of the putative ω-3 FAD encoded by the Cre01.g038600 locus. We show that while this enzyme is localized to plastids, impairment in its expression leads to a reduction of ω-3 fatty acids acylated to both plastidial and ER lipids. Additionally, using plastidial transformation of the mutant, it is demonstrated that the location of this desaturase in the plastid alone is sufficient to ensure normal ω-3 fatty acid content in extraplastidic lipids. Possible acyl desaturation and trafficking mechanisms implied by these findings are discussed.