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.     

Oxytocin: the neuropeptide of love reveals some of its secrets

The neuropeptide oxytocin is synthesized in the brain and released from neurohypophyseal terminals into the blood and within defined brain regions that regulate emotional, cognitive, and social behaviors. A recent study of CD38-/- mice (Jin et al., 2007) has demonstrated an essential role for the transmembrane receptor CD38 in secretion of oxytocin into the blood.     

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

A lot of evidence testifies that aromatase is expressed in the central nervous system where it has been detected not only in hypothalamic and limbic regions but also in the cerebral cortex and spinal cord. In physiological conditions, aromatase is expressed exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than those from males, suggesting a protective role of estradiol. The aim of this study was to evaluate changes in aromatase expression in response to 3-nitro-L-tyrosine, a marker of oxidative stress, in primary neuronal cell cultures from brains of 60-day old sheep fetuses. Cells were identified as neurons by using class III β-tubulin, a marker of neuronal cells. Two morphological types were consistently recognizable: i) bipolar cells with an oval cell body; ii) multipolar cells whose processes formed a wide net with those of adjacent cells. In situ hybridization technique performed on 60-day old fetal neurons revealed that in baseline conditions aromatase gene expression occurs. Importantly, cells exposed to 360 µM 3-nitro-L-tyrosine were fewer and showed more globular shape and shorter cytoplasmic processes in comparison to control cells. The immunocytochemical study with anti-aromatase antibody revealed that cells exposed to 360 µM 3-nitro-L-tyrosine were significantly more immunoreactive than control cells. Thus, it can be postulated that the oxidant effects of the amino acid analogue 3-nitro-L-tyrosine could be counterbalanced by an increase in aromatase expression that in turn can lead to the formation of neuroprotective estradiol via aromatization of testosterone.Key words: 3-nitro-L-tyrosine, aromatase, oxidative injury, neuroprotection, neuronal cell cultures, sheep.The brain is an important site of steroid synthesis in vertebrates (Baulieu, 1997). Neuroendocrine tissue is capable of converting androgens into estrogens by the enzyme P450 aromatase (Naftolin et al., 1971). Estradiol, through its specific receptors, promotes many crucial regulatory effects on various processes, such as viability and survival of neurons in rat primary cultures (Chowen et al., 1992), neural differentiation and plasticity as well as sexual behavior. Aromatase modulates synaptic plasticity in the hippocampus and other brain regions related to cognition.The enzyme may also influence synaptic development and plasticity in other non-reproductive regions of the central nervous system. For instance, Purkinje cells in aromatase-knockout mice show decreased dendritic growth and impairment of formation of dendritic spines and synapses (Sasahara et al., 2007). In addition, numerous studies have shown expression, activity and distribution of aromatase in the central nervous system of rats (Shinoda et al., 1994) and humans (Yague et al., 2006). In the brain, aromatase is predominantly expressed in hypothalamic and limbic regions, but also other structures such as the cerebral cortex, midbrain and spinal cord reveal aromatase activity and immunoreactivity.It has been demonstrated that aromatase is neuroprotective in the central nervous system. For instance, treatment with the neurotoxin kainic acid resulted in significant neuronal loss in the hippocampus of rats treated with the aromatase inhibitor fadrozole (Azcoitia et al., 2001). Under baseline conditions, aromatase is expressed in the central nervous system of mammals exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals (Naftolin et al., 1996). Since aromatase is expressed in several cellular compartments, it can be supposed that it leads to the formation of estrogen that acts not only through the classical receptors but also by direct and rapid effects at neuronal membranes (Roselli, 2007).Aromatase-expressing astrocytes have been observed in rats after stressful conditions such as serum deprivation or ischemia (Azcoitia et al., 2003, Roselli, 2007). The increased expression of aromatase in injured brain areas suggests that the enzyme may be involved in the protection of nervous tissue by increasing levels of local estrogens. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than males, suggesting estradiol has a protective role (Liu et al., 2007). In particular, these Authors demonstrated that astrocytes isolated from neonatal cortex exhibit marked sex differences in the sensitivity to oxygen-glucose deprivation and oxidant cell death since female cells exhibited enhanced aromatization and estradiol formation.The present investigation describes for the first time changes in aromatase expression in response to 3-nitro-L-tyrosine - a marker of oxidative stress - in primary neuronal cultures from fetal sheep brain.     

Another window into disordered protein function

Multiple crystal structures of the same proteins often have specific regions that switch between structure and disorder. In this issue of Structure, Zhang et al. (2007) show that these "dual personality fragments" are distinct from both structured and disordered protein and are functionally important.     

miRNAs play a tune

Two new studies describe functionally relevant interactions between microRNAs (miRNAs) and their targets in the immune system and the brain (Xiao et al., 2007; Karres et al., 2007). Furthermore, these studies illustrate the involvement of miRNAs in tuning the expression of target genes to physiologically relevant levels.     

Molecules to remember

"Working memory" is used for the transient storage of information in the brain. In this issue of Cell, Wang et al. (2007) now reveal how a series of molecular events involving alpha2A-adrenoceptors and a class of ion channels gated by cAMP tune the responses of neural circuits that function in working memory in mammals.     

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.     

Circadian control of neurogenesis

The life-long addition of new neurons has been documented in many regions of the vertebrate and invertebrate brain, including the hippocampus of mammals (Altman and Das, 1965; Eriksson et al., 1998; Jacobs et al., 2000), song control nuclei of birds (Alvarez-Buylla et al., 1990), and olfactory pathway of rodents (Lois and Alvarez-Buylla, 1994), insects (Cayre et al., 1996) and crustaceans (Harzsch and Dawirs, 1996; Sandeman et al., 1998; Harzsch et al., 1999; Schmidt, 2001). The possibility of persistent neurogenesis in the neocortex of primates is also being widely discussed (Gould et al., 1999; Kornack and Rakic, 2001). In these systems, an effort is underway to understand the regulatory mechanisms that control the timing and rate of neurogenesis. Hormonal cycles (Rasika et al., 1994; Harrison et al., 2001), serotonin (Gould, 1999; Brezun and Daszuta, 2000; Beltz et al., 2001), physical activity (Van Praag et al., 1999) and living conditions (Kemperman and Gage, 1999; Sandeman and Sandeman, 2000) influence the rate of neuronal proliferation and survival in a variety of organisms, suggesting that mechanisms controlling life-long neurogenesis are conserved across a range of vertebrate and invertebrate species. The present article extends these findings by demonstrating circadian control of neurogenesis. Data show a diurnal rhythm of neurogenesis among the olfactory projection neurons in the crustacean brain, with peak proliferation during the hours surrounding dusk, the most active period for lobsters. These data raise the possibility that light-controlled rhythms are a primary regulator of neuronal proliferation, and that previously-demonstrated hormonal and activity-driven influences over neurogenesis may be secondary events in a complex circadian control pathway.     

Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.     

Nicotiana attenuata SIPK,WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.Jasmonic acid (JA) and some of its precursors and derivatives are signal molecules that function as essential mediators of the plant''s wound, antiherbivore, and antipathogen responses, as well as in growth and development (Farmer, 1994; Creelman and Mullet, 1997; Turner et al., 2002). In unelicited mature leaves, JA is maintained at very low levels, however, upon specific stimulations, its biosynthesis is induced within a few minutes (Glauser et al., 2008). This rapid biosynthetic response must result from the activation of constitutively expressed JA biosynthesis enzymes in unelicited tissue by substrate availability and/or posttranslational modifications. At present, little is known about the molecular mechanisms that activate JA biosynthetic enzymes.According to the canonical mechanism for JA biosynthesis (Vick and Zimmerman, 1983), free α-linolenic acid (18:3^Δ9,12,15, 18:3) forms 13(S)-hydroperoxyoctadecatrienoic acid [13S-(OOH)-18:3] by the action of 13-lipoxygenase (13-LOX) in plastids. 13S-(OOH)-18:3 is converted by allene oxide synthase (AOS) into a highly unstable allene oxide intermediate that is processed by allene oxide cyclase (AOC) to yield (9S,13S)-12-oxo-phytodienoic acid (OPDA). OPDA is transported from the plastid into the peroxisome where it is reduced by the action of OPDA reductase 3 (OPR3) and after three cycles of β-oxidation, (3R,7S)-JA is formed. Due to the large number of enzymes and different cellular compartments involved in JA biosynthesis, it is expected that the pathway is regulated at multiple steps. Resolution of the structures of the tomato (Solanum lycopersicum) OPR3 and Arabidopsis (Arabidopsis thaliana) AOC2 and ACX1 has provided insights into potential regulatory mechanisms for these enzymes (e.g. oligomerization and phosphorylation; Pedersen and Henriksen, 2005; Breithaupt et al., 2006; Hofmann et al., 2006).The identification of two Arabidopsis plastidial glycerolipases, DAD1 and DGL (Ishiguro et al., 2001; Hyun et al., 2008), has provided genetic evidence for the importance of the release of trienoic fatty acids (FAs) from plastidial lipids in the activation of JA biosynthesis. Recently, some oxylipins have been found esterified to galactolipids in Arabidopsis leaves and hence it is possible that in this species preformed precursors could also supply the JA biosynthesis pathway after their release from lipids (Stelmach et al., 2001; Hisamatsu et al., 2003; Buseman et al., 2006). However, lipid-bound oxylipins are not formed in the leaves of all plant families (Böttcher and Weiler, 2007).In Nicotiana attenuata, wound-induced JA production is amplified by the application of lepidopteran larvae (e.g. Manduca sexta) oral secretions (OS) to mechanical wounds. Major elicitors of the OS-mediated response are FA-amino acid conjugates (FACs) that are sufficient to enhance JA production in leaves of this plant species (Halitschke et al., 2001). Recently, several regulatory factors with a potential function upstream of JA biosynthesis have been identified (Ludwig et al., 2005; Takabatake et al., 2006; Schweighofer et al., 2007; Takahashi et al., 2007); however, how these regulators affect JA biosynthesis is at present unknown. For example, wounding and herbivory in Nicotina spp. and tomato activate the mitogen-activated protein kinases salicylate-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK; Seo et al., 1999; Kandoth et al., 2007; Wu et al., 2007). When SIPK and WIPK expression is silenced in tobacco (Nicotiana tabacum), the plants accumulate 60% to 70% less JA than wild type after wounding or OS elicitation (Seo et al., 2007; Wu et al., 2007). Another regulatory component that affects JA production in N. attenuata is Nonexpressor of PR-1 (NPR1), an essential component of the salicylic acid (SA) signal transduction pathway first identified in Arabidopsis (Cao et al., 1994). N. attenuata NPR1-silenced plants accumulate 60% to 70% lower JA levels after elicitation than wild type (Rayapuram and Baldwin, 2007). NPR1 interacts with the JA and ethylene signaling cascades, and a cytosolic role for this factor in the regulation of JA-dependent responses/biosynthesis has been proposed (Spoel et al., 2003).In contrast to the mechanisms acting upstream of JA biosynthesis, the mechanisms mediating downstream JA responses are better characterized (Kazan and Manners, 2008; Browse, 2009). Among the best-characterized regulators of these responses is CORONATIVE INSENSITIVE1 (COI1), a gene that participates in jasmonate perception (Xie et al., 1998) and regulates gene expression through its interaction with the JASMONATE ZIM-DOMAIN repressors (Chini et al., 2007; Thines et al., 2007).To understand the early processes regulating the activation of JA biosynthesis by wounding and FAC elicitation in N. attenuata leaves, we quantified the initial rates of accumulation of plastid-derived JA precursors after these stimuli in wild type and four JA-deficient genotypes previously described: ir-sipk, ir-wipk, ir-npr1, and ir-coi1 (Rayapuram and Baldwin, 2007; Paschold et al., 2008; Meldau et al., 2009). We show that SIPK, WIPK, NPR1, and FACs contribute to the activation of de novo JA biosynthesis by affecting diverse early enzymatic steps in this pathway. The identification of a plastidial glycerolipase A₁ type I family protein (GLA1) essential for JA biosynthesis pointed to this enzyme as one potential target of some of these activating mechanisms.     

The Ecological Complexities of Biological Control: Trophic Cascades, Spatial Heterogeneity, and Behavioral Ecology

Biological control can be considered an intentional induction of a trophic cascade, whereby the addition of herbivores’ natural enemies or other habitat manipulations effectively enhance natural enemy populations, lead to reduced herbivore populations or feeding damage, and indirectly improve or protect plant health, agricultural yield, or the condition of some other biotic population or community of interest to man. The following set of papers (Denno et al., 2008; Ram et al., 2008; Stuart and Duncan, 2008; Spence et al. 2008) offer insights into the broad- and fine-scale factors that ultimately contribute to the success of biological control efforts. Many of the ideas herein were presented and discussed during a special session at the 2007 Annual Meeting of the Society of Nematologists. The goal of this session was to examine explicitly the ramifications of spatial and temporal heterogeneity in the context of effective biological control. The biological focus was primarily on interactions involving entomopathogenic nematodes (EPN), although many of the authors’ conclusions are applicable to other types of nematodes, soil fauna and natural enemies in general.     

A Host RNA Helicase-Like Protein,AtRH8, Interacts with the Potyviral Genome-Linked Protein,VPg, Associates with the Virus Accumulation Complex,and Is Essential for Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.Plant viruses are obligate intracellular parasites that infect many agriculturally important crops and cause severe losses each year. One of the common characteristics of plant viruses is their relatively small genome that encodes a limited number of viral proteins, making them dependent on host factors to fulfill their infection cycles (Maule et al., 2002; Whitham and Wang, 2004; Nelson and Citovsky, 2005; Decroocq et al., 2006). In order to establish a successful infection, the invading virus must recruit an array of host proteins (host factors) to translate and replicate its genome and to move locally from cell to cell via the plasmodesmata and systemically via the vascular system. It has been suggested that down-regulation or mutation of some of the required host factors may result in recessively inherited resistance to viruses (Kang et al., 2005b).Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, constitute the largest group of plant viruses (Rajamäki et al., 2004). Potyviruses have a single positive-strand RNA genome approximately 10 kb in length, with a viral genome-linked protein (VPg) covalently attached to the 5′ end and a poly(A) tail at the 3′ end (Urcuqui-Inchima et al., 2001; Rajamäki et al., 2004). The viral genome contains a single open reading frame (ORF) that translates into a polypeptide with a molecular mass of approximately 350 kD, which is cleaved into 10 mature proteins by viral proteases (Urcuqui-Inchima et al., 2001). Recently, a novel viral protein resulting from a frameshift in the P3 cistron has been reported (Chung et al., 2008). Of the 11 viral proteins, VPg is a multifunctional protein and the only other viral protein present in the viral particles (virions) besides the coat protein and the cylindrical inclusion protein (CI; Oruetxebarria et al., 2001; Puustinen et al., 2002; Gabrenaite-Verkhovskaya et al., 2008). The nonstructural protein is linked to the viral RNA by a phosphodiester bond between the 5′ terminal uridine residue of the RNA and the O⁴-hydroxyl group of amino acid Tyr (Murphy et al., 1996; Oruetxebarria et al., 2001; Puustinen et al., 2002). Mutation of the Tyr residue that links VPg to the viral RNA abolishes virus infectivity completely (Murphy et al., 1996). In infected cells, VPg and its precursor NIa are present in the nucleus and in the membrane-associated virus replication vesicles in the cytoplasm (Carrington et al., 1993; Rajamäki and Valkonen, 2003; Cotton et al., 2009). As a component of the replication complex, VPg may serve as a primer for viral RNA replication (Puustinen and Mäkinen, 2004) and as an analog of the m⁷G cap of mRNAs for the viral genome to recruit the translation complex for translation (Michon et al., 2006; Beauchemin et al., 2007; Khan et al., 2008). Furthermore, VPg has been suggested to be an avirulence factor for recessive resistance genes in diverse plant species (Moury et al., 2004; Kang et al., 2005b; Bruun-Rasmussen et al., 2007). Thus, VPg plays a pivotal role in the virus infection process. The molecular identification of VPg-interacting host proteins and the subsequent functional characterization of such interactions may advance knowledge of the intricate virus replication mechanisms and help develop novel antiviral strategies.Previous studies have shown that VPg and its precursor NIa interact with several host proteins, including three essential components of the host protein translation apparatus (Thivierge et al., 2008). The first protein is the cellular translation initiation factor eIF4E or its isoform eIF(iso)4E, identified through a yeast two-hybrid screen using VPg as a bait (Wittmann et al., 1997; Schaad et al., 2000). The protein complex of VPg and eIF4E is an essential component for virus infectivity (Robaglia and Caranta, 2006). Mutations and knockout of eIF4E or eIF(iso)4E confer resistance to infection (Lellis et al., 2002; Ruffel et al., 2002; Nicaise et al., 2003; Gao et al., 2004; Kang et al., 2005a; Ruffel et al., 2005; Decroocq et al., 2006; Bruun-Rasmussen et al., 2007). It is well known that potyviruses recruit selectively one of the eIF4E isoforms, depending on specific virus-host combinations (German-Retana et al., 2008). For instance, in Arabidopsis (Arabidopsis thaliana), eIF(iso)4E is required for infection by Turnip mosaic virus (TuMV), Plum pox virus (PPV), and Lettuce mosaic virus, while eIF4E is indispensable for infection by Clover yellow vein virus (Duprat et al., 2002; Lellis et al., 2002; Sato et al., 2005; Decroocq et al., 2006). The second cellular protein interacting with VPg is another translation initiation factor, eIF4G. Analysis of Arabidopsis knockout mutants for eIF4G or its isomers eIF(iso)4G1 and eIF(iso)4G2 has yielded results supporting the idea that the recruitment of eIF4G for potyvirus infection is also isoform dependent (Nicaise et al., 2007). Recently, poly(A)-binding protein (PABP), the translation initiation factor that bridges the 5′ and 3′ termini of the mRNA into proximity, has been proposed to be essential for efficient multiplication of TuMV (Dufresne et al., 2008). PABP was previously documented to interact with NIa, a VPg precursor containing both VPg and the proteinase NIa-Pro (Léonard et al., 2004). As the translation factors eIF(iso)4E and PABP have been found to be internalized in virus-induced vesicles, it has been suggested that the interactions between VPg and these translation factors are crucial for viral RNA translation and/or replication (Beauchemin and Laliberté, 2007; Beauchemin et al., 2007; Cotton et al., 2009). Besides these three translation factors, a Cys-rich plant protein, potyvirus VPg-interaction protein, was also found to associate with VPg (Dunoyer et al., 2004). This plant-specific VPg-interacting host protein contains a PHD finger domain and acts as an ancillary factor to support potyvirus infection and movement (Dunoyer et al., 2004).In this study, we describe the identification of an Arabidopsis DEAD-box RNA helicase (DDX), AtRH8, and a peach (Prunus persica) DDX-like protein, PpDDXL, both interacting with the potyviral VPg protein. Using the atrh8 mutant, we demonstrate that AtRH8 is not required for plant growth and development in Arabidopsis but is necessary for infection by two plant potyviruses, PPV and TuMV. Furthermore, we present evidence that AtRH8 colocalizes with the virus accumulation complex in potyvirus-infected leaf tissues, which reveals a possible role of AtRH8 in virus infection. Finally, we have identified the VPg-binding region (VPg-BR) of AtRH8 and PpDDX and show that overexpression of the VPg-BR either from AtRH8 or PpDDXL suppresses virus accumulation.     

Relationships between Growth,Growth Response to Nutrient Supply,and Ion Content Using a Recombinant Inbred Line Population in Arabidopsis

Growth is an integrative trait that responds to environmental factors and is crucial for plant fitness. A major environmental factor influencing plant growth is nutrient supply. In order to explore this relationship further, we quantified growth-related traits, ion content, and other biochemical traits (protein, hexose, and chlorophyll contents) of a recombinant inbred line population of Arabidopsis (Arabidopsis thaliana) grown on different levels of potassium and phosphate. Performing an all subsets multiple regression analyses revealed a link between growth-related traits and mineral nutrient content. Based on our results, up to 85% of growth variation can be explained by variation in ion content, highlighting the importance of ionomics for a broader understanding of plant growth. In addition, quantitative trait loci (QTLs) were detected for growth-related traits, ion content, further biochemical traits, and their responses to reduced supplies of potassium or phosphate. Colocalization of these QTLs is explored, and candidate genes are discussed. A QTL for rosette weight response to reduced potassium supply was identified on the bottom of chromosome 5, and its effects were validated using selected near isogenic lines. These lines retained over 20% more rosette weight in reduced potassium supply, accompanied by an increase in potassium content in their leaves.Plants in natural environments face abiotic constraints limiting growth and ultimately affecting their fitness. In response to such constraints, flowering time (Korves et al., 2007) and seed dormancy (Donohue et al., 2005) as well as vegetative growth (Barto and Cipollini, 2005; Milla et al., 2009) are the main traits controlling fitness (for review, see Alonso-Blanco et al., 2009). These traits are under the control of complex networks integrating genetic (G) and environmental (E) factors as well as their interaction (G × E). Due to the implications for food and renewable energy sources, dissecting the genetic architecture that underlies plant growth is becoming a priority for plant science (Rengel and Damon, 2008; Carroll and Somerville, 2009; Gilbert, 2009).Plant growth is highly dependent on mineral nutrient uptake (Clarkson, 1980; Sinclair, 1992). Minerals can be distinguished into two categories based on the amount required by plants: micronutrients, which are found in relatively small amounts in the plant (such as copper and iron), and macronutrients, which constitute between 1,000 and 15,000 μg g⁻¹ plant dry weight (such as potassium and phosphate; Marschner, 1995, Buchanan et al., 2002). Phosphate is an important structural and signaling molecule with an essential role in photosynthesis, energy conservation, and carbon metabolism. Its deficiency leads to a reduction of growth and an increase of pathogen susceptibility (Marschner, 1995; Williamson et al., 2001; Abel et al., 2002; López-Bucio et al., 2005; Poirier and Bucher, 2008; Vijayraghavan and Soole, 2010). Potassium is not incorporated into any organic substances but acts as the major osmoticum of the cell, controlling cell expansion, plasma membrane potential and transport, pH value, and many other catalytic processes (Maathuis and Sanders, 1996; Armengaud et al., 2004; Christian et al., 2006; Di Cera, 2006). Potassium deficiency leads to reduced plant growth, a loss of turgor, increased susceptibility to cold stress and pathogens, and the development of chlorosis and necrosis (Marschner, 1995; Véry and Sentenac, 2003; Ashley et al., 2006; Amtmann et al., 2008). To cope with changes in nutrient availability, plants have evolved different mechanisms of adaptation, such as changes in ion transporter expression and activity (Ashley et al., 2006; Jung et al., 2009), morphological changes, such as an increase in root growth to explore more soil volume (Marschner, 1995; Shirvani et al., 2001; Jiang et al., 2007; Jordan-Meille and Pellerin, 2008), or acidification of the surrounding soil in order to mobilize more mineral nutrients (for review, see Ryan et al., 2001). Although these adaptations are well known, the mechanisms involved in sensing and signaling low mineral nutrient status are less well understood, despite significant progress in this area being made (Doerner, 2008; Jung et al., 2009; Luan et al., 2009; Wang and Wu, 2010).One approach to identify genes that are involved in plant responses to environmental factors is to perform a quantitative trait locus (QTL) analysis on a mapping population grown in contrasting environments, allowing the identification of QTL-environment (QTL × E) interactions. Some QTLs for growth-related traits in response to environmental changes were cloned already. For example, the differential response of root growth of some Arabidopsis (Arabidopsis thaliana) accessions to phosphate starvation led to the identification of allelic differences responsible for this phenotype (Reymond et al., 2006; Svistoonoff et al., 2007). Other studies have identified QTLs for shoot dry matter under changing nitrogen supply (Rauh et al., 2002; Loudet et al., 2003). In parallel to natural variation for growth, natural variation for ion content has also been reported. In Arabidopsis, considerable variation in the content of mineral nutrients exists both in seeds (Vreugdenhil et al., 2004; Waters and Grusak, 2008) and in leaves (Harada and Leigh, 2006; Rus et al., 2006; Baxter et al., 2008a; Morrissey et al., 2009). Furthermore, changes in mineral nutrient homeostasis have also been reported to be associated with characteristic multivariate changes in the leaf ionome, the mineral nutrient and trace element composition of an organism or an organ (Baxter et al., 2008b). Due to higher throughput and lower costs, such “omics” analyses examining alterations of large numbers of certain molecules at once have recently become available for mapping purposes. Some QTL studies have linked the variations of these omics data to variation of growth or other physiological traits. For instance, Meyer et al. (2007) and Schauer et al. (2008) linked plant growth or morphological traits to a synergistic network of metabolomic compounds in Arabidopsis and tomato (Solanum lycopersicum), respectively. In addition, Sulpice et al. (2009) associated differences in growth with starch content using a set of Arabidopsis accessions. Compiling the importance of ions in the process of cell division (Lai et al., 2007; Sano et al., 2007) or cell expansion (Philippar et al., 1999; Elumalai et al., 2002), ionomics appears to be a major unexplored field for understanding growth.In this study, we focus on variation in plant growth, the root and leaf ionomes, and their response to varying supplies of potassium and phosphate. Studying variations for these traits among recombinant inbred lines (RILs) in Arabidopsis enabled us to detect QTL and QTL × E interactions for all of these traits. To understand the observed variation in plant growth, predictors that explained a high percentage of variation of growth-related traits have been selected especially among the root and leaf ionomes. The colocalization between growth-related trait QTLs and QTLs for their predictors allowed us to point out genetic regions of possible causality. In addition, the effect of a growth-response QTL on reduced potassium supply was validated with selected near isogenic lines (NILs) that maintained a higher rosette weight when grown in reduced potassium supply. This growth advantage went along with significant changes in ion contents that further emphasize the impact of the ionome in plant growth variations.     

Is Huntington disease a developmental disorder?

The recently discovered roles of huntingtin in non-differentiated cells indicate that it is a key molecule in brain development. Humbert argues that the haploinsufficiency of wild-type huntingtin in Huntington disease might lead to various cellular alterations well before the onset of symptoms and ultimately cause disease.The construction of an organism is a complex process that involves a developmental challenge: the orchestrated proliferation, migration and differentiation of cells, leading to the assembly of organs. Huntingtin—the protein that is mutated in the neurodegenerative disorder Huntington disease—is widely expressed in the early developing mouse embryo, in which it has an essential role. The most compelling proof that huntingtin is essential for early development is that inactivation of the murine gene results in defects in extra-embryonic tissues and embryonic death at embryonic day 7.5 (Dragatsis et al, 1998). My group has recently directly linked a cellular function of huntingtin to brain development (Godin et al, 2010a). Indeed, huntingtin regulates cortical neurogenesis through, at least in part, its role during spindle pole orientation.The growing evidence that huntingtin functions during development opens the door to viewing Huntington disease as a developmental disorder. Development could be abnormal in carriers of the mutant protein and precede the manifestation of the disease by decades. Changes during development might not have phenotypical consequences until the mature cells are required to function later in life. Indeed, a given protein will not function in the same context during development and adulthood, and the resulting phenotypes of these functions will not be the same. Furthermore, compensatory mechanisms that respond to abnormal development might be overwhelmed when the organism is ageing. We have not yet identified all of the neurodevelopmental defects—both functional and morphological—involved in Huntington disease. However, there are changes in the brain before the onset of disease, including a smaller intracranial adult brain volume in pre-manifest Huntington disease carriers (Nopoulos et al, 2010). It is tempting to consider that this might be a consequence of altered brain development.A complex molecular picture of the biology of huntingtin is emerging, suggesting that it is a scaffold protein that could couple many cellular events. Huntingtin regulates the assembly of the dynein–dynactin complex for axonal transport and Golgi apparatus organization (Caviston et al, 2007; Gauthier et al, 2004). During cell division, this role extends to a complex that also contains NuMA, a component that is essential for the organization of microtubules at the spindle pole (Godin et al, 2010a). Furthermore, NuMA and the Goloco-containing protein LGN form a complex that regulates the interaction between astral microtubules and the cell cortex (Du & Macara, 2004). Therefore, huntingtin could also participate in the distribution of the dynein–dynactin complex at the cell cortex and, as a consequence, regulate mitosis at several points. Similarly, huntingtin could regulate the assembly of other supramolecular complexes that are involved in various cell pathways, as suggested by the diverse nature of its interactors (Kaltenbach et al, 2007). For example, huntingtin interacts with the β-catenin destruction complex and thus participates in the tight regulation of the steady-state levels of β-catenin (Godin et al, 2010b; Kaltenbach et al, 2007). This might be crucial to regulate the Wingless/Wnt signalling pathway, known for its central role during development and adulthood. Thus, the functions attributed to huntingtin so far are important cellular processes in the early stages of development and adulthood, and contribute as initial or secondary disease mechanisms to several neurodegenerative disorders. This might not be a coincidence!Finally, the scaffold nature of huntingtin might be important for histogenesis in general and explain the widespread abnormalities observed in Huntington disease. A high level of huntingtin is found in the testes and one of the peripheral manifestations of the disease is testicular pathology, with a reduced number of germ cells and abnormal seminiferous tubule morphology (van Raamsdonk et al, 2007). Testes require functional intracellular transport for their normal development, and asymmetrical division is particularly important for germline stem-cell maintenance. Thus, an abnormal developmental programme induced by defective huntingtin function would alter cells and, thereby, the homeostasis of the tissues expressing this protein.Several other disorders are caused by the toxic presence of an abormal polyglutamine expansion. These ‘polyglutamine diseases'' have this mutation in common; however, the mutated proteins are unrelated and the disorders are phenotypically distinct, affecting different brain regions and neurons. A defect in the function of the mutated proteins explains the separate mechanisms of neuronal degeneration observed. Huntington disease is a dominant disorder, but the vision of it as a gain-of-function disease with loss-of-function manifestations could be outdated. The situation seems to be more complicated, and most patients express not only one copy of the mutant huntingtin, but also half the amount of the wild-type protein. It is time to rethink the idea that studying huntingtin protein is irrelevant to Huntington disease. In the light of recent advances in the understanding of its function, one might even suggest that taking developmental biology into account could provide new insight into the pathological mechanisms of this so far incurable adult-onset disorder.     

CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase,Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008; Wang et al., 2012; Pauly et al., 2013). Each of these polysaccharides is composed of a β-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of α-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the β-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Somerville, 2000; Liepman et al., 2005, 2007; Pauly et al., 2013). In Arabidopsis (Arabidopsis thaliana), nine CSLA genes have been identified; different CSLAs are responsible for the synthesis of different mannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro (Liepman et al., 2005) and has been shown to synthesize stem glucomannan in vivo (Goubet et al., 2009). Disrupting the CSLA7 gene results in defective pollen growth and embryo lethality phenotypes in Arabidopsis, indicating structural or signaling functions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results in the inhibition of Agrobacterium tumefaciens-mediated root transformation in the rat4 mutant (Zhu et al., 2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in the biosynthesis of stem glucomannans, although mutations in CSLA2, CSLA3, or CSLA9 have no effect on stem development or strength (Goubet et al., 2009). All of the Arabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2005, 2007), although the precise physiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded to envelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two different mucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is loosely attached and can be easily extracted by shaking seeds in water. Compositional and linkage analyses suggest that this layer is almost exclusively composed of unbranched rhamnogalacturonan I (RG-I) (>80% to 90%), with small amounts of branched RG-I, arabinoxylan, and high methylesterified homogalacturonan (HG). By contrast, the inner, adherent mucilage layer is tightly attached to the seed and can only be removed by strong acid or base treatment, or by enzymatic digestion (Macquet et al., 2007a; Huang et al., 2011; Walker et al., 2011). As with the nonadherent layer, adherent mucilage is also mainly composed of unbranched RG-I, but with small numbers of arabinan and galactan ramifications (Penfield et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet et al., 2007a, 2007b; Arsovski et al., 2009; Haughn and Western, 2012). There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al., 2001; Macquet et al., 2007a; Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilage contains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilage onto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).Screening for altered seed coat mucilage has led to the identification of several genes encoding enzymes that are involved in the biosynthesis or modification of mucilage components. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesis of UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 can potentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5 appears to function in the regulation of the final size of the mucilage RG-I (Kong et al., 2011, 2013). Mutant seeds defective in these genes display reduced thickness of the extruded mucilage layer compared with wild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coat epidermal cells appears to be synthesized in a branched form that is subsequently modified by enzymes in the apoplast. MUM2 encodes a β-galactosidase that removes Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes an α-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydration properties and affect the extrusion of mucilage. Furthermore, correct methylesterification of mucilage HG is also required for mucilage extrusion. HG is secreted into the wall in a high methylesterified form that can then be enzymatically demethylesterified by pectin methylesterases (PMEs; Bosch and Hepler, 2005). PECTIN METHYLESTERASE INHIBITOR6 (PMEI6) inhibits PME activities (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease (SBT1.7) can activate other PME inhibitors, but not PMEI6 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Disruption of either PMEI6 or SBT1.7 results in the delay of mucilage release.Although cellulose is present at low levels in adherent mucilage, it plays an important adhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. The orientation and amount of pectin associated with the cellulose network is largely determined by cellulose conformation properties (Macquet et al., 2007a; Haughn and Western, 2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) is required for the production of seed mucilage cellulose and the adherent mucilage in the cesa5 mutant can be easily extracted with water (Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011).Despite all of these discoveries, large gaps remain in the current knowledge of the biosynthesis and functions of mucilage polysaccharides in seed coats. In this study, we show that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, we show that CSLA2 functions in the maintenance of the normal structure of the adherent mucilage layer through modifying the mucilage cellulose ultrastructure.     

Statistical Significance of Precisely Repeated Intracellular Synaptic Patterns

Can neuronal networks produce patterns of activity with millisecond accuracy? It may seem unlikely, considering the probabilistic nature of synaptic transmission. However, some theories of brain function predict that such precision is feasible and can emerge from the non-linearity of the action potential generation in circuits of connected neurons. Several studies have presented evidence for and against this hypothesis. Our earlier work supported the precision hypothesis, based on results demonstrating that precise patterns of synaptic inputs could be found in intracellular recordings from neurons in brain slices and in vivo. To test this hypothesis, we devised a method for finding precise repeats of activity and compared repeats found in the data to those found in surrogate datasets made by shuffling the original data. Because more repeats were found in the original data than in the surrogate data sets, we argued that repeats were not due to chance occurrence. Mokeichev et al. (2007) challenged these conclusions, arguing that the generation of surrogate data was insufficiently rigorous. We have now reanalyzed our previous data with the methods introduced from Mokeichev et al. (2007). Our reanalysis reveals that repeats are statistically significant, thus supporting our earlier conclusions, while also supporting many conclusions that Mokeichev et al. (2007) drew from their recent in vivo recordings. Moreover, we also show that the conditions under which the membrane potential is recorded contributes significantly to the ability to detect repeats and may explain conflicting results. In conclusion, our reevaluation resolves the methodological contradictions between Ikegaya et al. (2004) and Mokeichev et al. (2007), but demonstrates the validity of our previous conclusion that spontaneous network activity is non-randomly organized.