The double life of Irs

The liver plays a central role in lipid and glucose metabolism. Two studies in this issue (Kubota et al., 2008; Dong et al., 2008) on the insulin-signaling adaptors Irs1 and Irs2 prompt a critical reappraisal of the physiology of fasting and of the integrated control of hepatic insulin action.     

Neuronal glucose sensing: still a physiological orphan?

Although hypothalamic glucose sensing is a long-established phenomenon, its physiological role remains unclear. New studies (Parton et al., 2007; Claret et al., 2007) disrupting glucose sensing in pro-opiomelanocortin neurons via differing methods have yielded disparate energy and glucose homeostasis phenotypes, suggesting that neuronal glucose sensing is not critical for these processes.     

Combined Effects of CO2 and Light on the N2-Fixing Cyanobacterium Trichodesmium IMS101: Physiological Responses

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum (IMS101) showed that increasing CO₂ partial pressure (pCO₂) enhances N₂ fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO₂, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses of Trichodesmium IMS101 grown under a matrix of low and high levels of pCO₂ (150 and 900 μatm) and irradiance (50 and 200 μmol photons m⁻² s⁻¹). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO₂ and light levels in the cultures. The pCO₂-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO₂ stimulated rates of N₂ fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO₂. HCO₃⁻ was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO₂ uptake was enhanced under high pCO₂. A comparison between carbon fluxes in vivo and those derived from ¹³C fractionation indicates high internal carbon cycling, especially in the low-pCO₂ treatment under high light. Light-dependent oxygen uptake was only detected under low pCO₂ combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO₂ effect on N₂ fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO₂. These findings are supported by a complementary study looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.Human-induced climate change will significantly alter the marine environment within the next century and beyond. Future scenarios predict an increase from currently approximately 380 to about 750 to 1,000 μatm CO₂ partial pressure (pCO₂) in the atmosphere until the end of this century (Raven et al., 2005; Raupach et al., 2007). As the ocean takes up this anthropogenic CO₂, dissolved inorganic carbon (DIC) in the surface ocean increases while the pH decreases (Wolf-Gladrow et al., 1999). Rising global temperatures will increase surface ocean stratification, which may affect the light regime in the upper mixed layer as well as nutrient input from deeper waters (Doney, 2006). Uncertainties remain regarding both the magnitude of the physicochemical changes and the biological responses of organisms, including species and populations of the oceanic primary producers at the basis of the food webs.In view of potential ecological implications and feedbacks on climate, several studies have examined pCO₂ sensitivity in phytoplankton key species (Burkhardt and Riebesell, 1997; Riebesell et al., 2000; Rost et al., 2003; Tortell et al., 2008). Pronounced responses to elevated pCO₂ were observed in N₂-fixing cyanobacteria (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007; Fu et al., 2008; Kranz et al., 2009), which play a vital role in marine ecosystems by providing a new source of biologically available nitrogen species to otherwise nitrogen-limited regions. Recent studies focused on the impact of different environmental factors on the filamentous Trichodesmium species, which is known for high abundance and the formation of massive blooms in tropical and subtropical areas (Capone et al., 2005; Mahaffey et al., 2005). Higher pCO₂ levels stimulated growth rates, biomass production, and N₂ fixation (Hutchins et al., 2007; Levitan et al., 2007; Kranz et al., 2009) and affected inorganic carbon acquisition of the cells (Kranz et al., 2009). While elevated sea surface temperatures are predicted to shift the spatial distribution of Trichodesmium species toward higher latitudes (Breitbarth et al., 2007), the combined effects of pCO₂ and temperature may favor this species and extend its niche even farther (Hutchins et al., 2007; Levitan et al., 2010a). An increase in the average light intensity, caused by the predicted shoaling of the upper mixed layer, may further stimulate photosynthesis and thus growth and N₂ fixation of Trichodesmium (Breitbarth et al., 2008). To our knowledge, the combined effects of light and pCO₂ have not been studied yet, although these environmental factors are likely to influence photosynthesis and other key processes in Trichodesmium.To understand the responses of an organism to changes in environmental conditions, metabolic processes must be studied. In Trichodesmium, photosynthetically generated energy (ATP and NADPH) is primarily used for the fixation of CO₂ in the Calvin-Benson cycle. A large proportion of this energy, however, is also required for the process of N₂ fixation via nitrogenase and for the operation of a CO₂-concentrating mechanism (CCM). The latter involves active uptake of inorganic carbon, which functions to increase the rate of carboxylation reaction mediated by Rubisco. This ancient and highly conserved enzyme is characterized by low affinities for its substrate CO₂ and a susceptibility to a competing reaction with oxygen (O₂) as substrate (Badger et al., 1998); the latter initiates photorespiration. As cyanobacterial Rubisco possesses one of the lowest CO₂ affinities among phytoplankton (Badger et al., 1998), a considerable amount of resources have to be invested to achieve sufficient rates of carbon fixation and to avoid photorespiration. A first step toward a mechanistic understanding of responses in Trichodesmium has been taken by Levitan et al. (2007), focusing on pCO₂ dependency of nitrogenase activity and photosynthesis. Subsequently, Kranz et al. (2009) described variations in CCM efficiency with pCO₂ and suggested that the observed plasticity in CCM regulation allowed energy reallocation under high pCO₂, which may explain the observed pCO₂-dependent changes in nitrogenase activity, growth, and elemental composition (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007).In this study, we measured growth responses as well as metabolic key processes in Trichodesmium erythraeum (IMS101) under environmental conditions that likely alter the energy budget and/or energy allocation of the cell. Cultures were acclimated to a matrix of low and high pCO₂ (150 and 900 μatm) at two different light intensities (50 and 200 μmol photons m⁻² s⁻¹). For each of the four treatments, changes in growth rates, elemental ratios, and the accumulation of particulate carbon and nitrogen were measured. Metabolic processes (gross photosynthesis, CCM activity, and O₂ uptake) were obtained by means of membrane-inlet mass spectrometry (MIMS), while N₂ fixation was detected by gas chromatography. As these processes may vary over the diurnal cycle in Trichodesmium (Berman-Frank et al., 2001; Kranz et al., 2009), measurements were performed in the morning and around midday. The results on metabolic processes were accompanied by measurements of the fluorescence of PSII, ratios of the photosynthetic units (PSI:PSII), and pool sizes of key proteins involved in carbon and nitrogen fixation as well as assimilation (Levitan et al., 2010b).     

Decreased Mitochondrial Activities of Malate Dehydrogenase and Fumarase in Tomato Lead to Altered Root Growth and Architecture via Diverse Mechanisms

Transgenic tomato (Solanum lycopersicum) plants in which either mitochondrial malate dehydrogenase or fumarase was antisense inhibited have previously been characterized to exhibit altered photosynthetic metabolism. Here, we demonstrate that these manipulations also resulted in differences in root growth, with both transgenics being characterized by a dramatic reduction of root dry matter deposition and respiratory activity but opposite changes with respect to root area. A range of physiological, molecular, and biochemical experiments were carried out in order to determine whether changes in root morphology were due to altered metabolism within the root itself, alterations in the nature of the transformants'' root exudation, consequences of alteration in the efficiency of photoassimilate delivery to the root, or a combination of these factors. Grafting experiments in which the transformants were reciprocally grafted to wild-type controls suggested that root length and area were determined by the aerial part of the plant but that biomass was not. Despite the transgenic roots displaying alteration in the expression of phytohormone-associated genes, evaluation of the levels of the hormones themselves revealed that, with the exception of gibberellins, they were largely unaltered. When taken together, these combined experiments suggest that root biomass and growth are retarded by root-specific alterations in metabolism and gibberellin contents. These data are discussed in the context of current models of root growth and biomass partitioning.The structure of the plant tricarboxylic acid (TCA) cycle has been established for decades (Beevers, 1961), and in vitro studies have established regulatory properties of many of its component enzymes (Budde and Randall, 1990; Millar and Leaver, 2000; Studart-Guimarães et al., 2005). That said, relatively little is known, as yet, regarding how this important pathway is regulated in vivo (Fernie et al., 2004a; Sweetlove et al., 2007). Indeed, even fundamental questions concerning the degree to which this pathway operates in illuminated leaves (Tcherkez et al., 2005; Nunes-Nesi et al., 2007a) and the influence it has on organic acid levels in fruits (Burger et al., 2003) remain contentious. Furthermore, in contrast to many other pathways of primary metabolism, the TCA cycle has been subjected to relatively few molecular physiological studies. To date, the functions of pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, succinyl-CoA ligase, fumarase, and malate dehydrogenase have been studied via this approach (Landschütze et al., 1995; Carrari et al., 2003; Yui et al., 2003; Nunes-Nesi et al., 2005, 2007a; Lemaitre et al., 2007; Studart-Guimarães et al., 2007); however, several of these studies were relatively cursory. Despite this fact, they generally corroborate one another, with at least two studies providing clear evidence for an important role of the TCA cycle in flower development (Landschütze et al., 1995; Yui et al., 2003) or in the coordination of photosynthetic and respiratory metabolisms of the illuminated leaf (Carrari et al., 2003; Nunes-Nesi et al., 2005, 2007a).In our own studies on tomato (Solanum lycopersicum), we have observed that modulation of fumarase and mitochondrial malate dehydrogenase activities leads to contrasting shoot phenotypes, with the former displaying stunted growth while the later exhibited an enhanced photosynthetic performance (Nunes-Nesi et al., 2005, 2007a). We were able to demonstrate that the stunted-growth phenotype observed in aerial parts of the fumarase plants was a consequence of altered stomatal function (Nunes-Nesi et al., 2007a), whereas the increased photosynthetic performance of the mitochondrial malate dehydrogenase seems likely to be mediated by the alterations in ascorbate metabolism exhibited by these plants (Nunes-Nesi et al., 2005; Urbanczyk-Wochniak et al., 2006). In keeping with the altered rates of photosynthesis in these antisense plants, the fruit yield of fumarase and mitochondrial malate dehydrogenase plants was decreased and increased, respectively. However, the root biomass of both transgenics was significantly reduced (Nunes-Nesi et al., 2005, 2007a). These observations were somewhat surprising given that it is estimated that 30% to 60% of net photosynthate is transported to root organs (Merckx et al., 1986; Nguyen et al., 1999; Singer et al., 2003). When taken together, these results suggest that the root phenotype must result from either an impairment of translocation or a root-specific effect. Neither of these explanations is without precedence, with inhibition of the expression of Suc transporters (Riesmeier et al., 1993; Gottwald et al., 2000) resulting in dramatically impaired root growth while organic acid exudation itself has been implicated in a wide range of root organ functions, including nutrient acquisition (de la Fuente et al., 1997; Imas et al., 1997; Neumann and Römheld, 1999; López-Bucio et al., 2000; Anoop et al., 2003; Delhaize et al., 2004), metal sequestration (Gillooly et al., 1983; de la Fuente et al., 1997; Cramer and Titus, 2001), and microbial proliferation in the rhizosphere (Lugtenberg et al., 1999; Weisskopf et al., 2005). In addition to the putative mechanisms listed above, the TCA cycle could be anticipated to play a vital role in meeting the high energy demands of nitrogen fixation and polymer biosynthesis associated with rapidly growing heterotrophic organs (Pradet and Raymond, 1983; Dieuaide-Noubhani et al., 1997; Stasolla et al., 2003; Deuschle et al., 2006). In keeping with this theory, alteration of the energy status of roots and other heterotrophic tissue has been documented to positively correlate with elevated biomass production (Anekonda, 2001; Regierer et al., 2002; Carrari et al., 2003; Lovas et al., 2003; Geigenberger et al., 2005). Here, we performed a detailed physiological, molecular, and biochemical evaluation of whole plant and root metabolism of the mitochondrial malate dehydrogenase and fumarate antisense tomato lines. In this manner, we broadly assessed biochemical changes in the root, including the levels of several major phytohormones, as well as dissected which characteristics were influenced by aerial parts of the plant. The results obtained are discussed both with respect to the regulation of the TCA cycle per se and within the context of the determination of root morphology and growth.     

The Stromal Chloroplast Deg7 Protease Participates in the Repair of Photosystem II after Photoinhibition in Arabidopsis

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).Chloroplasts of higher plants carry out one of the most important biochemical reactions: the capture of light energy and its conversion into chemical energy. Although light is the ultimate source of energy for photosynthesis, it can also be harmful to plants. Light-induced loss of photosynthetic efficiency, which is generally termed as photoinhibition, limits plant growth and lowers productivity, especially when combined with other abiotic stresses.The main target of photoinhibition is PSII, which catalyzes the light-dependent water oxidation concomitantly with oxygen production (for review, see Prasil et al., 1992; Aro et al., 1993; Adir et al., 2003). In higher plants, PSII consists of more than 20 subunits, including the reaction center D1 and D2 proteins, cytochrome (Cyt) b₅₅₉, the light-harvesting chlorophyll a-binding proteins CP47 and CP43, the oxygen-evolving 33-kD protein (PsbO), and several low molecular mass proteins (Nelson and Yocum, 2006). The PSII reaction center D1 protein has been identified among PSII proteins as the primary target of light-induced damage (Kyle et al., 1984; Mattoo et al., 1984; Ohad et al., 1984; Adir et al., 1990), but several studies have shown that the D2, CP47, and CP43 proteins are degraded under photoinhibitory conditions (Schuster et al., 1988; Yamamoto and Akasaka, 1995; Jansen et al., 1999; Adir et al., 2003). Moreover, several small PSII subunits, such as PsbH, PsbW, and Cyt b₅₅₉, were also found to be frequently replaced within PSII (Hagman et al., 1997; Ortega et al., 1999; Bergantino et al., 2003). Evidence for the involvement of two families of proteases, FtsH and Deg, in the degradation of the D1 protein in thylakoids of higher plants has been recently described (Lindahl et al., 1996, 2000; Bailey et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; Kapri-Pardes et al., 2007; Sun et al., 2007a, 2007b). However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases.DegP (or HtrA) proteases were initially identified based on the fact that they are required for the survival of Escherichia coli at high temperatures and for the degradation of abnormal periplasmic proteins (Lipinska et al., 1988; Strauch and Beckwith, 1988). DegP is an ATP-independent Ser endopeptidase, and it contains a trypsin-like protease domain at the N terminus, followed by two PDZ domains (Gottesman, 1996; Pallen and Wren, 1997; Clausen et al., 2002). PDZ domains appear to be important for complex assembly and substrate binding through three or four residues in the C terminus of their target proteins (Doyle et al., 1996; Harris and Lim, 2001). DegP switches between chaperone and protease functions in a temperature-dependent manner. The chaperone function dominates at low temperatures, and DegP becomes proteolytically active at elevated temperatures (Spiess et al., 1999). Crystal structures of different members of the DegP protein family (Krojer et al., 2002; Li et al., 2002; Kim et al., 2003; Wilken et al., 2004) have revealed the structure-function relationship of these PDZ-containing proteases. Trimeric DegP is the functional unit, and the hexameric DegP is formed via the staggered association of trimers (Clausen et al., 2002; Kim and Kim, 2005). At normal growth temperatures, the active site of the protease is located within the chamber of hexameric DegP, which is not accessible to the substrates. However, at high temperatures, conformational changes induce the activation of the protease function (Krojer et al., 2002). Recent studies have shed light on the substrate binding-induced formation of larger oligomeric complexes of DegP (Jiang et al., 2008; Krojer et al., 2008).In Arabidopsis (Arabidopsis thaliana), 16 genes coding for DegP-like proteases have been identified, and at least seven gene products are predicted to be located in chloroplasts (Kieselbach and Funk, 2003; Huesgen et al., 2005; Adam et al., 2006; Sakamoto, 2006; Kato and Sakamoto, 2009). Based on proteomic data, four Deg proteases have been shown to be localized to the chloroplast (Peltier et al., 2002; Schubert et al., 2002) and functionally characterized. Deg1, Deg5, and Deg8 are located in thylakoid lumen, and Deg2 is peripherally associated with the stromal side of thylakoid membranes (Itzhaki et al., 1998; Haußühl et al., 2001; Sun et al., 2007a). Recombinant DegP1, now renamed Deg1, has been shown to be proteolytically active toward thylakoid lumen proteins such as plastocyanin and PsbO of PSII in vitro (Chassin et al., 2002). A 5.2-kD C-terminal fragment of the D1 protein was detected in vitro after incubation of recombinant Deg1 with inside-out thylakoid membranes. In transgenic plants with reduced levels of Deg1, fewer of its 16- and 5.2-kD degradation products were observed (Kapri-Pardes et al., 2007). Deg5 and Deg8 form a dodecameric complex in the thylakoid lumen, and recombinant Deg8 is able to degrade the photodamaged D1 protein of PSII in an in vitro assay (Sun et al., 2007a). The 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in a deg5 deg8 double mutant after high-light treatment. The deg5 deg8 double mutant showed increased sensitivity to high light and high temperature in terms of growth and PSII activity compared with the single mutants deg5 and deg8, suggesting that Deg5 and Deg8 have overlapping functions in the primary cleavage of the CD loop of the D1 protein (Sun et al., 2007a, 2007b). In vitro analysis has demonstrated that recombinant stroma-localized Deg2 was also shown to be involved in the primary cleavage of the DE loop of the D1 protein (Haußühl et al., 2001). However, analysis of a mutant lacking Deg2 suggested that Deg2 may not be involved in D1 degradation in vivo (Huesgen et al., 2006).Here, we have expressed and purified a recombinant DegP protease, His-Deg7. In vitro experiments showed that His-Deg7 is proteolytically active toward the PSII proteins D1, D2, CP43, and CP47. In vivo analyses of a deg7 mutant revealed that the mutant is more sensitive to high light stress than the wild-type plants. We demonstrated that Deg7 is a chloroplast stroma protein associated with the thylakoid membranes and that it interacts with PSII, which suggests that it can cleave the stroma-exposed region of substrate proteins. Our results also provide evidence that Deg7 is important for maintaining PSII function.     

H3K27 demethylases, at long last

Methylation of lysine 27 on histone H3 (H3K27me) by the Polycomb complex (PRC2) proteins is associated with gene silencing in many developmental processes. A cluster of recent papers (Agger et al., 2007; De Santa et al., 2007; Lan et al., 2007; Lee et al., 2007) identify the JmjC-domain proteins UTX and JMJD3 as H3K27-specific demethylases that remove this methyl mark, enabling the activation of genes involved in animal body patterning and the inflammatory response.     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

Mighty Piwis defend the germline against genome intruders

Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007; Gunawardane et al., 2007; Houwing et al., 2007). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007).     

After hours keeps clock researchers CRYing Overtime

Three recent reports, including one in this issue of Cell, reveal that the circadian regulator CRY is targeted for degradation by the F box E3 ubiquitin ligase FBXL3 (Siepka et al., 2007; Busino et al., 2007; Godinho et al., 2007). These studies confirm the importance of targeted protein degradation as a key design feature of the mammalian circadian clock.     

Bone remodeling, energy metabolism, and the molecular clock

The adult skeleton is constantly renewed through bone remodeling. Four recent papers (Baldock et al., 2007; Lee et al., 2007; Lundberg et al., 2007; Sato et al., 2007) provide new insights into central and peripheral control of this remodeling sequence. Two of the studies add to our knowledge of the complex hypothalamic modulation of bone turnover mediated by NMU and NPY via the sympathetic nervous system, while the other two focus on the peripheral neural target, the osteoblast, and its regulation by neuropeptides and osteocalcin. These findings support a new paradigm concerning the regulation of bone remodeling and provide a foundation for novel approaches to preventing osteoporosis.     

Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.Plant defense against pathogens often involves the induction of mechanisms after pathogen recognition, including defense signaling, cell wall strengthening, and localized cell death, but plants also have preformed chemical and structural defense barriers. Fungal pathogens that penetrate the plant tissue directly through the outer surface, rather than via natural plant openings or wounds, must pass through the plant cuticle and epidermal cell wall. Penetration of the host surface happens either by physical means (i.e. by a highly localized pressure in the appressorium) or by chemical means (i.e. by the release of hydrolyzing enzymes). Necrotrophic plant pathogens like Botrytis cinerea typically use the latter strategy. During penetration, they produce cutinases and pectinolytic enzymes such as pectin methylesterases, endopolygalacturonases, and exopolygalacturonases (van Kan, 2006).The cuticle is a hydrophobic barrier that covers the aerial surfaces of the plant. It is mainly composed of cutin, a polyester matrix, and soluble waxes, a complex mixture of hydrophobic material containing very-long-chain fatty acids and their derivatives, embedded into and deposited onto the cutin matrix. It plays an important role in organ development and protection against water loss (Yephremov et al., 1999; Sieber et al., 2000; Kurata et al., 2003; Jung et al., 2006). The cuticle is generally considered as a mere passive physical barrier against pathogen invasion, but it has also been recognized as a potential source of signaling and elicitor molecules (Jenks et al., 1994; Reina-Pinto and Yephremov, 2009). Plant cutin monomers trigger cutinase secretion in pathogenic fungi (Woloshuk and Kolattukudy, 1986), and cutin and wax components initiate appressorium formation and penetration in appressorium-forming pathogens (Kolattukudy et al., 1995; Francis et al., 1996; Gilbert et al., 1996; Fauth et al., 1998; Dickman et al., 2003). In plants, cutin monomers induce pathogenesis-related gene expression and elicit hydrogen peroxide (H₂O₂) synthesis (Fauth et al., 1998; Kim et al., 2008; Park et al., 2008). Transgenic tomato (Solanum lycopersicum) plants expressing the yeast Δ-9 desaturase gene had high levels of cutin monomers that inhibited powdery mildew (Erysiphe polygoni) spore germination, leading to enhanced resistance (Wang et al., 2000). Arabidopsis (Arabidopsis thaliana) plants expressing a fungal cutinase or mutants with a defective cuticle, such as long-chain acyl-CoA synthetase2 and bodyguard, are generally more susceptible to bacteria and equally susceptible to biotrophic fungi but are surprisingly resistant to B. cinerea (Bessire et al., 2007; Chassot et al., 2007; Tang et al., 2007). It has been postulated that a defective or thin cuticle encourages these plants to constitutively express defense-related mechanisms and to secrete antifungal compounds to the plant surface, thereby inhibiting B. cinerea growth (Bessire et al., 2007; Chassot et al., 2007). In addition, cuticle metabolic pathways might directly modulate plant-pathogen interactions by interacting with hormonally regulated defense pathways (Fiebig et al., 2000; Garbay et al., 2007; Mang et al., 2009) or with complex lipid signaling pathways leading to hypersensitive cell death (Raffaele et al., 2008).Once plant pathogens have penetrated the cuticle, they secrete hydrolases that target the plant cell wall (ten Have et al., 1998; Oeser et al., 2002; Vogel et al., 2002; Jakob et al., 2007) that is mainly composed of cellulose, hemicellulose, and pectin (35% of total dry weight). Pectin consists mainly of the polysaccharides homogalacturonan and rhamnogalacturonan I and II. Homogalacturonans are linear chains of α-(1–4)-linked d-GalA residues that can be methylesterified at C-6. Rhamnogalacturonan I and II are more complex, branched polysaccharides. B. cinerea is typically regarded as a pectinolytic pathogen because it possesses an efficient pectinolytic machinery, including a variety of polygalacturonases and pectin methylesterases (PMEs), some of which are important virulence factors (ten Have et al., 1998, 2001; Valette-Collet et al., 2003; Kars et al., 2005). Pectins are a rich source of oligogalacturonides (OGAs), biologically active signaling molecules that can activate plant defense mechanisms (Hahn et al., 1981; Côté and Hahn, 1994; Messiaen and Van Cutsem, 1994; Ridley et al., 2001). The eliciting capacity of the OGAs was shown to depend on their size, which in turn is influenced by the methylesterification pattern of the homogalacturonan fraction (Mathieu et al., 1991; Messiaen and Van Cutsem, 1994). To counteract the activity of fungal pectinases, many plants express polygalacturonase-inhibiting proteins and PME inhibitors, which are localized in the cell wall. The role of these proteins in plant defense against B. cinerea has been extensively demonstrated (Powell et al., 2000; Ferrari et al., 2003; Sicilia et al., 2005; Joubert et al., 2006, 2007; Lionetti et al., 2007). The interaction with the inhibitors not only limits the destructive potential of polygalacturonases but also leads to the accumulation of elicitor-active OGAs (De Lorenzo and Ferrari, 2002). How OGAs are perceived by the plant is still unclear, but in view of the diversity of biological activities and structure requirements, they are thought to be recognized through different proteins, including receptor-like kinases, wall-associated kinases, arabinogalactan proteins, and Pro-rich proteins (Côté and Hahn, 1994; Showalter, 2001; Humphrey et al., 2007).Over the past years, the role of abscisic acid (ABA) in plant-pathogen interactions has gained increased attention. ABA is mostly negatively correlated with resistance against phytopathogens through down-regulation of defense responses orchestrated by salicylic acid, jasmonic acid, and ethylene (Mohr and Cahill, 2001; Audenaert et al., 2002; Mauch-Mani and Mauch, 2005; Asselbergh et al., 2008). In tomato, the ABA-deficient mutant sitiens has an enhanced resistance to B. cinerea (Audenaert et al., 2002) that depends on a timely, localized oxidative burst leading to rapid epidermal cell wall fortification and a faster and higher induction of defense-related gene expression upon infection compared with the wild type (Asselbergh et al., 2007). Moreover, basal defense gene expression is higher in this mutant than in the wild type. As this early response is of vital importance for the resistant reaction of tomato against B. cinerea, we investigated whether alterations in cuticle and/or cell wall, which form the first barrier to the invading pathogen, affect resistance. We demonstrate that the sitiens cuticle is more permeable and that permeability is positively correlated with resistance to B. cinerea. Furthermore, differences in pectin composition and rate of methylesterification occur. Together, these data hint at an unanticipated role for extracellular matrix components in the resistance of tomato against B. cinerea and thus shed new light on the largely unexplored interrelationship between the extracellular matrix and plant-pathogen interactions.     

A backup DNA repair pathway moves to the forefront

Chromosomal translocations between antigen receptor loci and oncogenes are a hallmark of lymphoid cancers. Several new studies now reveal that programmed DNA breaks created during assembly of antigen receptor genes can be channeled into an alternative DNA end-joining pathway that is implicated in the chromosomal translocations of lymphoid cancers (Corneo et al., 2007; Soulas-Sprauel et al., 2007; Yan et al., 2007).     

The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b₆/f (Cyt b₆/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b₆/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700⁺ indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b₆/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.The Antarctic psychrophilic green alga Chlamydomonas sp. UWO 241 (UWO 241) originates from the lowest trophic zone of Lake Bonney, which is characterized by an extremely stable environment of low temperatures (4°C–6°C), low irradiance (less than 50 µmol photons m⁻² s⁻¹), high salt concentrations (700 mm), and a narrow spectral distribution enriched in the blue-green region (Lizotte and Priscu, 1992; Morgan-Kiss et al., 2006). Adaptation of UWO 241 to this unique natural aquatic environment has resulted in the evolution of a structurally and functionally distinct photosynthetic apparatus relative to the mesophilic strains Chlamydomonas raudensis SAG 49.72 (SAG 49.72; Pocock et al., 2004) and the model green alga Chlamydomonas reinhardtii (Morgan et al., 1998; Morgan-Kiss et al., 2006). UWO 241 is a halotolerant psychrophile (Morgan-Kiss et al., 2006; Takizawa et al., 2009) that dies at temperatures of 20°C or higher (Possmayer et al., 2011). This is consistent with the fact that temperature-response curves for light-saturated rates of CO₂-saturated oxygen evolution indicate that UWO 241 photosynthesizes maximally at 8°C at rates that are comparable to rates of the mesophile, C. reinhardtii, grown and measured at 29°C (Pocock et al., 2007). Although UWO 241 exhibits a low quantum requirement for photoinhibition and the degradation of the PSII reaction center polypeptide D1 (PsbA), this is complemented by a rapid, light-dependent recovery of PSII photochemistry associated with the de novo biosynthesis of D1 at low temperature (Pocock et al., 2007). Thus, this psychrophile appears to be photosynthetically adapted to growth at low temperature (Pocock et al., 2007).UWO 241 exhibits significantly enhanced fatty acid unsaturation associated with all of the major thylakoid lipid classes (monogalactosyldiacylglyceride, digalactosyldiacylglyceride, sulfoquinovosyldiacylglyceride, and phosphatidyldiacylglyceride) as well as a 2- to 10-fold increase in the unique, unsaturated fatty acid 16:4, depending on the specific thylakoid lipid species (Morgan-Kiss et al., 2002a). Consequently, the biophysical determination of the critical temperature for thylakoid membrane destabilization for UWO 241 (40°C) was significantly lower than that for C. reinhardtii (50°C), which is consistent with the adaptation of UWO 241 to low temperature (Morgan-Kiss et al., 2002a).Biochemical analyses of the chlorophyll-protein complexes coupled with immunoblots of their constituent polypeptides indicate that UWO 241 exhibits abundant PSII light-harvesting complex (LHCII) associated with a low chlorophyll a/b (Chl a/b) ratio (1.8–2) relative to the mesophiles, SAG 49.72 and C. reinhardtii (Chl a/b ratio = 3). In addition, UWO 241 exhibits an unusually low level of PSI such that the stoichiometry of PSI/PSII was estimated to be about 0.5 in UWO 241, whereas the mesophiles, SAG 49.72 and C. reinhardtii, grown under optimal growth conditions, exhibited a PSI/PSII of about 1. These biochemical data were confirmed by measurements of P700 photooxidation (Morgan-Kiss et al., 2002b; Szyszka et al., 2007), which indicated that UWO 241 exhibits high rates of PSI cyclic electron flow (CEF; Morgan-Kiss et al., 2002b).Recently, we reported that acclimation of UWO 241 to low temperature and low growth irradiance results in alterations in the partitioning of excess excitation energy to maintain cellular energy balance compared with the mesophile, SAG 49.72 (Szyszka et al., 2007). While SAG 49.72 favors energy partitioning for photoprotection through the induction of the xanthophyll cycle, the psychrophilic strain, UWO 241, favors energy partitioning for photoprotection through constitutive quenching processes involved in energy dissipation, even though UWO 241 exhibits an active xanthophyll cycle (Pocock et al., 2007; Szyszka et al., 2007). Although the molecular basis of the constitutive quenching process for photoprotection has not been elucidated unequivocally, this may reflect the differences in the predisposition for energy dissipation through either the Q2 or the Q1 site in PSII-LHCII supercomplexes (Jahns and Holzwarth 2012; Derks et al., 2015) or, alternatively, it may indicate quenching through PSII reaction centers, as suggested previously (Hüner et al., 2006; Sane et al., 2012). Regardless of the mechanism, one consequence of this enhanced energy-quenching capacity of UWO 241 is that the psychrophile does not exhibit any pigment change in response to photoacclimation (Morgan-Kiss et al., 2006), typically observed for other mesophilic green algae such as C. reinhardtii, Dunaliella tertiolecta (Escoubas et al., 1995), Dunaliella salina (Smith et al., 1990; Maxwell et al., 1995), and Chlorella vulgaris (Maxwell et al., 1995; Wilson et al., 2003). In addition, maximum growth rates of UWO 241 are sensitive to light quality, since rates of growth and photosynthesis are inhibited under red light, which results in increased excitation pressure in the psychrophile (Morgan-Kiss et al., 2005).However, the most unusual feature of UWO 241 is that it represents a natural variant that is deficient in state transitions (Morgan-Kiss et al., 2002b; Takizawa et al., 2009). State transitions have been well documented as a short-term mechanism for photoacclimation employed by algae and plants to balance light excitation between PSII and PSI (Allen et al., 1981; Allen, 2003; Eberhard et al., 2008; Rochaix, 2011, 2014). Overexcitation of PSII relative to PSI results in the phosphorylation of several peripheral Chl a/b-binding LHCII proteins, which causes their dissociation from the PSII core and subsequent association with PSI (Eberhard et al., 2008; Rochaix, 2011). As a result, excitation energy is redistributed in favor of PSI at the expense of PSII. Phosphorylation of LHCII polypeptides is essential in the regulation of state transitions and energy distribution between the two photosystems (Allen, 2003; Eberhard et al., 2008; Kargul and Barber, 2008; Rochaix, 2011, 2014). LHCII phosphorylation is initiated by modulation of the redox state of the plastoquinone (PQ) pool, which is sensed through the preferential binding of plastoquinol to the quinone-binding site of the cytochrome b₆/f (Cyt b₆/f) complex. As a consequence, the thylakoid protein kinases STT7 in C. reinhardtii and its ortholog, STN7, in Arabidopsis (Arabidopsis thaliana) are activated and LHCII is phosphorylated (Rochaix, 2011, 2014; Wunder et al., 2013). Similar to all other photosynthetic organisms, the LHCII polypeptides represent the major phosphorylated polypeptides detected in thylakoids of the mesophile, SAG 49.72 (Szyszka et al., 2007). Consistent with a deficiency in state transitions, UWO 241 does not phosphorylate the major LHCII polypeptides in response to changes in either growth irradiance or growth temperature (Morgan-Kiss et al., 2002b; Szyszka et al., 2007; Takizawa et al., 2009). In fact, UWO 241 exhibits a unique thylakoid membrane phosphorylation profile compared with either SAG 49.72 or C. reinhardtii (Morgan-Kiss et al., 2005; Szyszka et al., 2007; Takizawa et al., 2009). Rather than phosphorylation of LHCII polypeptides, UWO 241 preferentially phosphorylates several novel high-molecular-mass polypeptides (greater than 70 kD; Morgan-Kiss et al., 2002b; Szyszka et al., 2007).The Cyt b₆/f complex of the photosynthetic intersystem electron transport chain is essential in the regulation of state transitions and the activation of the STT7 kinase (Rochaix, 2011, 2014). The Cyt b₆/f complex of UWO 241 exhibits a unique cytochrome f (Cyt f) that is 7 kD smaller than the expected molecular mass of 41 kD exhibited by C. reinhardtii based on SDS-PAGE (Morgan-Kiss et al., 2006; Gudynaite-Savitch et al., 2006, 2007). No other differences in the structure and composition of the Cyt b₆/f complex are apparent. Sequencing of the entire Cytochrome f gene (petA) from UWO 241 indicated that the amino acid sequence of Cyt f from UWO 241 exhibited 79% identity to that of C. reinhardtii. Through domain swapping between petA of UWO 241 and that of C. reinhardtii and subsequent transformation of a ΔpetA mutant of C. reinhardtii with the chimeric gene constructs, we reported that the apparent differences in molecular masses observed for petA in UWO 241 are due to differences in the amino acid sequences of the small domain of Cyt f. However, complementation of the ΔpetA mutant of C. reinhardtii with the entire petA from either UWO 241 or C. reinhardtii completely restored the capacity for state transitions in the ΔpetA mutant. Thus, we concluded that the changes in the amino acid sequence of the small domain of Cyt f of UWO 241 cannot account for the inability of UWO 241 to undergo state transitions (Gudynaite-Savitch et al., 2006, 2007).Since state transitions are inhibited in UWO 241, we hypothesized that the unique protein phosphorylation pattern observed in UWO 241 reflects an alternative mechanism to regulate energy flow within the photosynthetic apparatus of this Antarctic psychrophile. Thus, the objective of this research was to identify and characterize the high-molecular-mass polypeptides phosphorylated in the psychrophile, UWO 241. We report that UWO 241 preferentially phosphorylates specific polypeptides associated with a PSI-Cyt b₆/f supercomplex. The role of the PSI-Cyt b₆/f supercomplex and its phosphorylation status in the regulation of PSI cyclic electron transport in UWO 241 are discussed. We suggest that adaptation of UWO 241 to its unique low-temperature and low-light environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of LHCII proteins.     

An ABC Transporter Mutation Alters Root Exudation of Phytochemicals That Provoke an Overhaul of Natural Soil Microbiota

Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.The diversity of the microbial (bacterial and fungal) communities in soil is extraordinary; 1 g of soil contains more than 10 billion microorganisms belonging to thousands of different species (Roselló-Mora and Amann, 2001). Soil microbial populations are involved in a framework of interactions known to affect key environmental processes like biogeochemical cycling of nutrients, plant health, and soil quality (Pace, 1997; Barea et al., 2004; Giri et al., 2005). Most of the dynamic soil microbial interactions happen near the plant roots and root soil interface, an area called the rhizosphere (Lynch, 1990; Barea et al., 2002; Bais et al., 2006; Prithiviraj et al., 2007). Rhizosphere microbial communities differ between plant species (Priha et al., 1999; Innes et al., 2004; Batten et al., 2006), between ecotypes/chemotypes within species (Kowalchuk et al., 2006; Micallef et al., 2009), between different developmental stages of a given plant (Mougel et al., 2006; Weisskopf et al., 2006), and from those present in bulk soil (Broz et al., 2007). Different root types can also cultivate specific microbes (Lilijeroth et al., 1991; Yang and Crowley, 2000; Baudoin et al., 2002), a response that has generally been attributed to the microenvironments surrounding a root and the varying ability of specific root types to uptake nutrients from soils and secrete exudates. Recent evidence suggests that specific plant species support a highly coevolved soil fungal community, and this process is mediated by root-secreted compounds (Broeckling et al., 2008). Rhizosphere interactions are initiated by the release of compounds from different organisms, and it is believed that carbon compounds secreted by roots act as substrates for certain species of microbes in the rhizospshere (Morgan et al., 2005).Root exudates are released into the rhizosphere by three major pathways: diffusion, ion channel, and vesicle transport (Bertin et al., 2003). Recent evidence has implicated ATP-binding cassette (ABC) transporters in the secretion of phytochemicals present in the root exudates of Arabidopsis (Arabidopsis thaliana) and other plants (Loyola-Vargas et al., 2007; Sugiyama et al., 2007; Badri et al., 2008; Badri and Vivanco, 2009). ABC transporters are the largest family of membrane transport proteins found in all organisms from bacteria to humans (Higgins, 1992). These transmembrane proteins use the energy of ATP to pump a wide variety of substrates across the membranes, including peptides, carbohydrates, lipids, heavy metal chelates, inorganic acids, steroids, and xenobiotics (Goossens et al., 2003). ABC transporters are also involved in plant disease resistance at the leaf level (Kobae et al., 2006; Stein et al., 2006).There is accumulating evidence that root exudates play a role in establishing specific interactions with particular microbes in the rhizosphere (legume''s symbiotic interaction with rhizobia, interaction of plants with mycorrhizae, and plant-growth-promoting rhizobacteria [PGPR]; Nagahashi and Douds, 2000; Bais et al., 2006, 2008; Prithiviraj et al., 2007; Rudrappa et al., 2008). However, how root exudation processes that result in large-scale changes to the surrounding soil microbial community compared to individual microbes have not been determined, although some recent reviews have referred to it as a biological frontier (O''Connell et al., 1996; Kuiper et al., 2004; Ryan et al., 2009). In contrast, gene deletions and overexpression of specific genes in plants have been shown to attract or deter specific microbes (Widmer, 2007), herbivores, or their predators (Baldwin et al., 2006; Pandey and Baldwin, 2007; Mitra and Baldwin, 2008), and recently it has been shown that mutations in nonpigment floral chemistry genes affect flower visitation by native pollinators (Kessler et al., 2008). Thus, it is possible that gene expression manipulation leading to an altered spectrum of root exudates can influence the widespread community of soil organisms surrounding a plant. Using all available information described above, we present the most comprehensive study on the effect of a single gene mutation in an ABC transporter involved in root secretion of phytochemicals by Arabidopsis on the natural and coevolved soil microbial composition. We further determine the compounds that are likely to have an effect on moderating the microbial composition and characterized specific and natural microbes that interact with Arabidopsis in the soil by employing pyrosequencing technology.