Skeletal muscle wasting causes both morbidity and mortality of cancer patients. Accumulating evidence suggests that the markers of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways are increased in skeletal muscle under multiple catabolic conditions, including cancer. However, the signaling mechanisms and the role of individual arms of the UPR in the regulation of skeletal muscle mass remain largely unknown. In the present study, we demonstrated that gene expression of Toll-like receptors (TLRs) and myeloid differentiation primary response gene 88 (MyD88) was increased in skeletal muscle in a Lewis lung carcinoma (LLC) model of cancer cachexia. Targeted ablation of MyD88 inhibits the loss of skeletal muscle mass and strength in LLC tumor-bearing mice. Inhibition of MyD88 attenuates the LLC-induced activation of the UPR in skeletal muscle of mice. Moreover, muscle-specific deletion of X-box binding protein 1 (XBP1), a major downstream target of IRE1α arm of the UPR, ameliorates muscle wasting in LLC tumor-bearing mice. Our results also demonstrate that overexpression of an active form of XBP1 caused atrophy in cultured myotubes. In contrast, knockdown of XBP1 inhibits myotube atrophy in response to LLC or C26 adenocarcinoma cell conditioned medium. Collectively, our results demonstrate that TLR/MyD88-mediated activation of XBP1 causes skeletal muscle wasting in LLC tumor-bearing mice.

Skeletal muscle mass is regulated by a complex array of signaling pathways. TGF-beta-activated kinase 1 (TAK1) is an important signaling protein, which regulates context-dependent activation of multiple intracellular pathways. However, the role of TAK1 in the regulation of skeletal muscle mass remains unknown. Here, we report that inducible inactivation of TAK1 causes severe muscle wasting, leading to kyphosis, in both young and adult mice.. Inactivation of TAK1 inhibits protein synthesis and induces proteolysis, potentially through upregulating the activity of the ubiquitin-proteasome system and autophagy. Phosphorylation and enzymatic activity of AMPK are increased, whereas levels of phosphorylated mTOR and p38 MAPK are diminished upon inducible inactivation of TAK1 in skeletal muscle. In addition, targeted inactivation of TAK1 leads to the accumulation of dysfunctional mitochondria and oxidative stress in skeletal muscle of adult mice. Inhibition of TAK1 does not attenuate denervation-induced muscle wasting in adult mice. Finally, TAK1 activity is highly upregulated during overload-induced skeletal muscle growth, and inactivation of TAK1 prevents myofiber hypertrophy in response to functional overload. Overall, our study demonstrates that TAK1 is a key regulator of skeletal muscle mass and oxidative metabolism.

Mutants affected in the Arabidopsis TBL29/ESK1 xylan O-acetyltransferase display a strong reduction in total wall O-acetylation accompanied by a dwarfed plant stature, collapsed xylem morphology, and enhanced freezing tolerance. A newly identified tbl29/esk1 suppressor mutation reduces the expression of the MAX4 gene, affecting the biosynthesis of methyl carlactonoate (MeCLA), an active strigolactone (SL). Genetic and biochemical evidence suggests that blocking the biosynthesis of this SL is sufficient to recover all developmental and stress-related defects associated with the TBL29/ESK1 loss of function without affecting its direct effect-reduced wall O-acetylation. Altered levels of the MAX4 SL biosynthetic gene, reduced branch number, and higher levels of MeCLA, were also found in tbl29/esk1 plants consistent with a constitutive activation of the SL pathway. These results suggest that the reduction in O-acetyl substituents in xylan is not directly responsible for the observed tbl29/esk1 phenotypes. Alternatively, plants may perceive defects in the structure of wall polymers and/or wall architecture activating the SL hormonal pathway as a compensatory mechanism.

Skeletal muscle mass is regulated by the coordinated activation of several anabolic and catabolic pathways. The endoplasmic reticulum (ER) is a major site of protein folding and a reservoir for calcium ions. Accretion of misfolded proteins or depletion in calcium concentration causes stress in the ER, which leads to the activation of a signaling network known as the unfolded protein response (UPR). In the present study, we investigated the role of the protein kinase R-like endoplasmic reticulum kinase (PERK) arm of the UPR in the regulation of skeletal muscle mass and function in naive conditions and in a mouse model of cancer cachexia. Our results demonstrate that the targeted inducible deletion of PERK reduces skeletal muscle mass, strength, and force production during isometric contractions. Deletion of PERK also causes a slow-to-fast fiber type transition in skeletal muscle. Furthermore, short hairpin RNA-mediated knockdown or pharmacologic inhibition of PERK leads to atrophy in cultured myotubes. While increasing the rate of protein synthesis, the targeted deletion of PERK leads to the increased expression of components of the ubiquitin-proteasome system and autophagy in skeletal muscle. Ablation of PERK also increases the activation of calpains and deregulates the gene expression of the members of the FGF19 subfamily. Furthermore, the targeted deletion of PERK increases muscle wasting in Lewis lung carcinoma tumor-bearing mice. Our findings suggest that the PERK arm of the UPR is essential for the maintenance of skeletal muscle mass and function in adult mice.Gallot, Y. S., Bohnert, K. R., Straughn, A. R., Xiong, G., Hindi, S. M., Kumar, A. PERK regulates skeletal muscle mass and contractile function in adult mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, leads to severe muscle wasting and eventual death of the afflicted individuals, primarily due to respiratory failure. Deficit in myofiber regeneration, potentially due to an exhaustion of satellite cells, is one of the major pathological features of DMD. Myeloid differentiation primary response 88 (MyD88) is an adaptor protein that mediates activation of various inflammatory pathways in response to signaling from Toll-like receptors and interleukin-1 receptor. MyD88 also regulates cellular survival, proliferation and differentiation in a cell-autonomous manner. However, the role of MyD88 in satellite stem cell homeostasis and function in dystrophic muscle remains unknown. In this study, we demonstrate that tamoxifen-inducible deletion of MyD88 in satellite cells causes loss of skeletal muscle mass and strength in the mdx mouse model of DMD. Satellite cell-specific deletion of MyD88 inhibits myofiber regeneration and stimulates fibrogenesis in dystrophic muscle of mdx mice. Deletion of MyD88 also reduces the number of satellite cells and inhibits their fusion with injured myofibers in dystrophic muscle of mdx mice. Ablation of MyD88 in satellite cells increases the markers of M2 macrophages without having any significant effect on M1 macrophages and expression of inflammatory cytokines. Finally, we found that satellite cell-specific deletion of MyD88 leads to aberrant activation of Notch and Wnt signaling in skeletal muscle of mdx mice. Collectively, our results demonstrate that MyD88-mediated signaling in satellite cells is essential for the regeneration of injured myofibers in dystrophic muscle of mdx mice.

Skeletal muscle regeneration in adults is attributed to the presence of satellite stem cells that proliferate, differentiate, and eventually fuse with injured myofibers. However, the signaling mechanisms that regulate satellite cell homeostasis and function remain less understood. While IKKβ-mediated canonical NF-κB signaling has been implicated in the regulation of myogenesis and skeletal muscle mass, its role in the regulation of satellite cell function during muscle regeneration has not been fully elucidated. Here, we report that canonical NF-κB signaling is induced in skeletal muscle upon injury. Satellite cell-specific inducible ablation of IKKβ attenuates skeletal muscle regeneration in adult mice. Targeted ablation of IKKβ also reduces the number of satellite cells in injured skeletal muscle of adult mice, potentially through inhibiting their proliferation and survival. We also demonstrate that the inhibition of specific components of the canonical NF-κB pathway causes precocious differentiation of cultured satellite cells both ex vivo and in vitro. Finally, our results highlight that the constitutive activation of canonical NF-κB signaling in satellite cells also attenuates skeletal muscle regeneration following injury in adult mice. Collectively, our study demonstrates that the proper regulation of canonical NF-κB signaling is important for the regeneration of adult skeletal muscle.

Myoblast fusion is an indispensable step for skeletal muscle development, postnatal growth, and regeneration. Myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein that mediates Toll-like receptors and interleukin-1 receptor signaling. Here we report a cell-autonomous role of MyD88 in the regulation of myoblast fusion. MyD88 protein levels are increased during in vitro myogenesis and in conditions that promote skeletal muscle growth in vivo. Deletion of MyD88 impairs fusion of myoblasts without affecting their survival, proliferation, or differentiation. MyD88 regulates non-canonical NF-κB and canonical Wnt signaling during myogenesis and promotes skeletal muscle growth and overload-induced myofiber hypertrophy in mice. Ablation of MyD88 reduces myofiber size during muscle regeneration, whereas its overexpression promotes fusion of exogenous myoblasts to injured myofibers. Our study shows that MyD88 modulates myoblast fusion and suggests that augmenting its levels may be a therapeutic approach to improve skeletal muscle formation in degenerative muscle disorders.

Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

Chronic low-grade inflammation, adipocyte hypertrophy, and glucose intolerance are common features of obesity and a risk factor for cancer. Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an adaptor protein that also possesses a non-conventional E3 ubiquitin ligase activity. In response to receptor-mediated events, TRAF6 activates transforming growth factor-activated kinase 1 (TAK1), which leads to activation of the MAPK and nuclear factor-kappa B (NF-κB) signaling pathways. However, the roles of TRAF6 and TAK1 in the regulation of adipocyte function remain less understood. Here, we demonstrate that adipocyte-specific deletion of TAK1, but not TRAF6, in mice reduces the survival of adipocytes and abundance of white adipose tissue (WAT). Adipocyte-specific ablation of TAK1, but not TRAF6, increases the expression for markers of beige/brown fat in WAT. Deletion of TAK1 in WAT increases phosphorylation of AMPK, abundance of PGC-1α, non-canonical NF-κB signaling, markers of M2 macrophages, and diminishes phosphorylation of JNK and canonical NF-κB signaling. Levels of TRAF6 and enzymatic activity of TAK1 are increased in WAT of mice fed with high-fat diet (HFD). Our results demonstrate that ablation of TAK1 drastically reduces HFD-induced obesity and improves energy expenditure and glucose metabolism. In contrast, adipocyte-specific ablation of TRAF6 has a minimal effect on HFD-induced obesity. Collectively, our results suggest that even though TRAF6 is an upstream activator of TAK1 in many signaling cascades, inactivation of TAK1, but not TRAF6, regulates adipocyte survival, energy expenditure, and HFD-induced obesity in mice.

ELISA absorbance values for RG-I fractions from NbPAGR-silenced and virus-infected, control N. benthamiana plants as well as 35S::PAGR-YFP and wild type Arabidopsis plants probed with a diverse array of plant cell wall glycan-directed monoclonal antibodies. (XLSX 21 kb)

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

36 37 Plant cell walls are important barriers against microbial pathogens. Cell walls of 38 Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, 39 various hemicelluloses and pectins. UDP-D-galacturonic acid, the key building block of 40 pectins, is produced from the precursor UDP-D-glucuronic acid by the action of 41 glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv. maculicola ES4326 42 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity 43 to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants 44 had brittle leaves and cell walls of leaves had less galacturonic acid (GalA). Resistance 45 to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant 46 plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in 47 gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, 48 macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released 49 less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from 50 wild-type Col-0. Although both OGs and macerozyme induced immunity to B. cinerea in 51 Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important 52 contributor to plant immunity, and this is due at least in part to the induction of immune 53 responses by soluble pectin, likely OGs, that are released during plant-pathogen 54 interactions. 55

Background: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth.Results: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1: 1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content.Conclusions: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Cachexia is a devastating syndrome that causes morbidity and mortality in a large number of patients with cancer. However, the mechanisms of cancer cachexia remain poorly understood. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes stress. The ER responds to this stress through activating certain pathways commonly known as the unfolding protein response (UPR). The main function of UPR is to restore homeostasis, but excessive or prolonged activation of UPR can lead to pathologic conditions. In this study, we examined the role of ER stress and UPR in regulation of skeletal muscle mass in naive conditions and during cancer cachexia. Our results demonstrate that multiple markers of ER stress are highly activated in skeletal muscle of Lewis lung carcinoma (LLC) and Apc(Min/+) mouse models of cancer cachexia. Treatment of mice with 4-phenylbutyrate (4-PBA), a chemical chaperon and a potent inhibitor of ER stress, significantly reduced skeletal muscle strength and mass in both control and LLC-bearing mice. Blocking the UPR also increased the proportion of fast-type fibers in soleus muscle of both control and LLC-bearing mice. Inhibition of UPR reduced the activity of Akt/mTOR pathway and increased the expression of the components of the ubiquitin-proteasome system and autophagy in LLC-bearing mice. Moreover, we found that the inhibition of UPR causes severe atrophy in cultured myotubes. Our study provides initial evidence that ER stress and UPR pathways are essential for maintaining skeletal muscle mass and strength and for protection against cancer cachexia.Bohnert, K. R., Gallot, Y. S., Sato, S., Xiong, G., Hindi, S. M., Kumar, A. Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia.

TGFβ-activated kinase 1 (TAK1), a member of the MEK kinase (MAP3K) family, has been demonstrated to be a component of signaling pathways leading to the activation of NF-kB and activator protein-1 (AP-1) in response to cytokines and growth factors. Our previous study has shown that TAK1 is a critical regulator for the proliferation and differentiation of cultured C2C12 myoblasts. However, the role of TAK1 in regulation of skeletal muscle mass has not yet been investigated. PURPOSE: To determine if skeletal muscle-specific deletion of TAK1 alters the skeletal muscle mass and function in adult mice. METHODS: Skeletal muscle-specific TAK1 knockout (TAK1mko, n=5) mice were generated by crossing HSA-cre mice with floxed TAK1 (i.e. TAK1f/f) mice. Tamoxifen was injected intraperitoneally to inactivate TAK1 and the mice were fed with tamoxifen-containing standard chow for the entire duration of the experiment. TAK1f/f mice (n=6) were used as control. All mice were sacrificed at approximately 3 months of age and the tibialis anterior (TA) and soleus (SOL) muscles were excised for further analysis. RESULTS: TAK1mko mice were found to have reduced body weight (14%) and TA muscle weight (10%) compared to TAK1f/f mice. Mean fiber-cross sectional area (CSA) was decreased in TAK1mko mice by 42% and 39% in TA and SOL muscles, respectively. TAK1mko mice showed lower grip strength compared to control mice by 39%. Genetic ablation of TAK1 in skeletal muscle significantly reduced the levels of specific muscle proteins whereas the levels of atrogin-1, poly-ubiquitinated proteins, and proteasome activity were increased. CONCLUSION: These data demonstrate that TAK1 is essential for maintenance of skeletal muscle in adult animals. Supported by NIH Grant AR059810 and AG029623 to AK.

Plant cell walls or lignocellulosics are considered an important renewable resource for the generation of bioenergy and other commodity chemicals. Wall materials are composed of cellulose, lignin, and various non-cellulosic polysaccharides. Most of the non-cellulosic polysaccharides contain O-acetyl substituents. The acetate residues are an impediment to biorefining as these substituents hinder the enzymatic saccharification of wall polymers and, upon processing, the released acetate represents a potent inhibitor of yeast fermentation of the sugars (Gille and Pauly, 2012Gille S. Pauly M. O-acetylation of plant cell wall polysaccharides.Front. Plant Sci. 2012; 3: 12Crossref PubMed Scopus (159) Google Scholar). The dominant source of acetate in dicot and grass walls is the hemicellulose xylan, where 40%–80% of the backbone xylosyl residues can be O-acetylated (Pauly et al., 2013Pauly M. Gille S. Liu L. Mansoori N. de Souza A. Schultink A. Xiong G. Hemicellulose biosynthesis.Planta. 2013; 238: 627-642Crossref PubMed Scopus (241) Google Scholar). Recently, a member of the Arabidopsis Trichome Birefringence-Like (TBL) gene family TBL29 has been identified as a xylan O-acetyltransferase based on a detailed wall analysis of the corresponding knockout mutant (Xiong et al., 2013Xiong G. Cheng K. Pauly M. Xylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the Arabidopsis mutant tbl29.Mol. Plant. 2013; 6: 1373-1375Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Yuan et al., 2013Yuan Y. Teng Q. Zhong R. Ye Z.H. The Arabidopsis DUF231 domain-containing protein ESK1 mediates 2-O and 3-O-acetylation of xylosyl residues in xylan.Plant Cell Physiol. 2013; 54: 1186-1199Crossref PubMed Scopus (97) Google Scholar) and in vitro activity of the recombinant TBL29 protein, which leads to the transfer of O-acetyl-groups from acetyl-CoA onto xylo-oligosaccharides (Urbanowicz et al., 2014Urbanowicz B.R. Pena M.J. Moniz H.A. Moremen K.W. York W.S. Two Arabidopsis proteins synthesize acetylated xylan in vitro.Plant J. 2014; 80: 197-206Crossref PubMed Scopus (140) Google Scholar). The Arabidopsis tbl29 knockout mutant exhibits a nearly 60% reduction in acetate on xylan, thus lowering this unfavorable substituent in the corresponding biomass. However, the loss of TBL29 leads to a typical irregular xylem phenotype exhibiting decreased cellulose content, severely retarded growth, and a loss of plant biomass. Similar phenotypes have been observed in the Altered XYloglucan 9 (axy9) mutant, which lacks a Golgi-membrane localized component of the wall polysaccharide acetylation machinery resulting in collapsed xylem cells with a concomitant 30%–80% reduction of xylan acetylation (Schultink et al., 2015Schultink A. Naylor D. Dama M. Pauly M. The role of the plant specific AXY9 gene in plant cell wall polysaccharide O-acetylation.Plant Physiol. 2015; https://doi.org/10.1104/pp.114.256479Crossref PubMed Scopus (64) Google Scholar), and in the quadruple mutant rwa1/2/3/4 of Reduced Wall Acetylation genes (Manabe et al., 2013Manabe Y. Verhertbruggen Y. Gille S. Harbolt J. Chong S.L. Pawar P. Mellerowicz E. Tenkanen M. Cheng K. Pauly M. Scheller H.V. RWA proteins play vital and distinct roles in cell wall O-acetylation in Arabidopsis thaliana.Plant Physiol. 2013; 163: 1107-1117Crossref PubMed Scopus (62) Google Scholar). Mutations in the latter putative Golgi acetyl-CoA transporters resulted in a 40% decrease in xylan acetylation also accompanied by collapsed xylem and stunted growth. In the wall, especially the secondary wall, xylan is closely associated with cellulose. Acetyl and other substituents not only make xylan more water soluble (Saha, 2003Saha B.C. Hemicellulose bioconversion.J. Ind. Microbiol. Biot. 2003; 30: 279-291Crossref PubMed Scopus (1541) Google Scholar); molecular dynamic simulation predicts that when xylan is O-acetylated or glycosylated at regular intervals, the conformation of the polymer leads to a stable interaction with cellulose (Busse-Wicher et al., 2014Busse-Wicher M. Gomes T.C.F. Tryfona T. Nikolovski N. Stott K. Grantham N.J. Bolam D.N. Skaf M.S. Dupree P. The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.Plant J. 2014; 79: 492-506Crossref PubMed Scopus (198) Google Scholar) stabilizing a functional cellulose-xylan network. Reducing the degree of xylan O-acetylation in tbl29 thus likely leads to an abnormal cellulose-xylan network compromising cell wall strength and consequentially resulting in a collapsed xylem that cannot properly transport water. In addition to O-acetylation, the xylan backbone can also be substituted with occasional glucuronic acid (GlcA) and/or 4-O-methyl-glucuronic acid (MeGlcA). Glucuronic acid residues are added to the xylan backbone by xylan:GlcA transferase(GUX) genes (Mortimer et al., 2010Mortimer J.C. Miles G.P. Brown D.M. Zhang Z. Segura M.P. Weimar T. Yu X. Seffen K.A. Stephens E. Turner S.R. et al.Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.Proc. Natl. Acad. Sci. USA. 2010; 107: 17409-17414Crossref PubMed Scopus (227) Google Scholar). The gux1 gux2 gux3 triple mutant led to a complete loss of GlcA and MeGlcA substitution on xylan resulting again in collapsed vessels and dwarfism (Lee et al., 2012Lee C. Teng Q. Zhong R. Ye Z.H. Arabidopsis GUX proteins are glucuronyltransferases responsible for the addition of glucuronic acid side chains onto xylan.Plant Cell Physiol. 2012; 53: 1204-1216Crossref PubMed Scopus (83) Google Scholar) underpinning the functional significance of xylan substituents. In the walls of grasses, the xylan backbone can be decorated with arabinose (Ara), the transfer of which is mediated by xylan arabinofuranosyl transferases (XATs). Heterologous overexpression of wheat and rice XATs was reported to successfully lead to arabinosylated xylan in Arabidopsis albeit only in low quantities (Anders et al., 2012Anders N. Wilkinson M.D. Lovegrove A. Freeman J. Tryfona T. Pellny T.K. Weimar T. Mortimer J.C. Stott K. Baker J.M. et al.Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.Proc. Natl. Acad. Sci. USA. 2012; 109: 989-993Crossref PubMed Scopus (178) Google Scholar). Since the favorable reduction in O-acetylation in tbl29 wall materials is accompanied by an undesired significant reduction in plant biomass, we wanted to test if missing acetyl substituents can be replaced by glycosyl residues such as GlcA or Ara by overexpression of the corresponding glycosyltransferases, GUX or XAT, in the tbl29 mutant. When OsXAT2, OsXAT3, and AtGUX1 but also AtTBL29 itself were overexpressed in the Arabidopsis tbl29 mutant utilizing the promoter EntCUP2 for ubiquitous expression, no changes compared with the untransformed tbl29 mutant in terms of growth phenotype and wall characteristics were observed (Supplemental Figures 1 and 2) rendering the use of this promotor ineffective. Therefore, the native TBL29 promotor was used instead. pTBL29::OsXAT2 and pTBL29::OsXAT3 were successfully transformed into tbl29 as expression of the two exogenous genes in stem tissue could be confirmed by quantitative RT–PCR (Supplemental Figure 3A). However, the transformants did not exhibit any significant increase in wall Ara content or any noticeable improvement in the dwarfed growth phenotype. Apparently, these rice XATs were not able to arabinosylate the partially de-acetylated xylan in tbl29. Reasons might include improper folding/post-translational modification/degradation of the XAT protein and/or incompatibility of the protein with the xylan biosynthesis machinery in Arabidopsis. However, arabinosylation of xylan by overexpressing OsXAT in wild-type Arabidopsis has been reported previously (Anders et al., 2012Anders N. Wilkinson M.D. Lovegrove A. Freeman J. Tryfona T. Pellny T.K. Weimar T. Mortimer J.C. Stott K. Baker J.M. et al.Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.Proc. Natl. Acad. Sci. USA. 2012; 109: 989-993Crossref PubMed Scopus (178) Google Scholar), but only to a very low extent. Therefore, there remains a possibility that only trace amounts of arabinose were transferred onto xylan in the tbl29 mutants, which did not affect the overall wall arabinose content. In any case, expression of rice XATs was not effective in improving the tbl29 growth phenotype. Using the same complementation approach, AtGUX1 was expressed under the control of the TBL29 native promoter in tbl29 plants. In the pTBL29::AtGUX1-tbl29 plants, expression of AtGUX1 doubled compared with the intrinsic AtGUX1 level in elongating stem tissue (Supplemental Figure 3B). A concomitant 70%–80% increase in wall GlcA content was detected in mature stem wall material (Figure 1C and Supplemental Figure 4B). Based on a more detailed xylan analysis by NMR of ball-milled dissolved whole walls, it became evident that the (Me-)GlcA xylan substitution is not altered in the tbl29 mutant, but increased by ∼40% when AtGUX1 is expressed in the tbl29 mutants (Figure 1D and Supplemental Figure 5). At the same time, the wall acetate content remained low (Figure 1C and Supplemental Figure 4B), and the growth phenotype and dry biomass weight of tbl29 was restored to wild-type levels (Figure 1A and 1B). The reduced cellulose content in tbl29 walls, which is typical among irregular xylem mutants, was also fully restored in the transformants. Interestingly, the wall xylose content is lower in the AtGUX1 complemented tbl29 transformants than in wild-type plants, inferring that overall xylan synthesis might be affected due to hitherto unknown reasons. However, this apparent decrease in xylan did not affect cellulose deposition or the growth phenotype (Figure 1C and Supplemental Figure 4B). When AtGUX1 is overexpressed in wild-type Arabidopsis plants utilizing a secondary wall-specific IRX9 promotor, only a ∼20% increase in wall GlcA is observed (Bromley et al., 2013Bromley J.R. Busse-Wicher M. Tryfona T. Mortimer J.C. Zhang Z. Brown D.M. Dupree P. GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns.Plant J. 2013; 74: 423-434Crossref PubMed Scopus (123) Google Scholar). The larger increase of 40% of xylan (Me) GlcA substitution in the pTBL29::AtGUX1-tbl29 transformants suggests that the AtGUX1 glycosyltransferase can take advantage of the open positions of the xylan backbone freed from acetyl substituents in tbl29 to increase xylan GlcA substitution levels. Since the growth phenotype of the tbl29 mutant is restored, glucuronosylation can thus be considered functionally equivalent to O-acetylation in vivo. The recalcitrance of the wall material to saccharification was investigated in a standard saccharification measurement and the glucose released from pTBL29::AtGUX1-tbl29 is comparable with both wild-type and tbl29 mutant. Hence, altering xylan substitution as presented here does not affect saccharification, providing an encouraging genetic strategy to improve energy crops for biofuel production. This work was funded by the Energy Bioscience Institute.

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.

Satellite cells are resident adult stem cells that are required for regeneration of skeletal muscle. However, signalling mechanisms that regulate satellite cell function are less understood. Here we demonstrate that transforming growth factor-β-activated kinase 1 (TAK1) is important in satellite stem cell homeostasis and function. Inactivation of TAK1 in satellite cells inhibits muscle regeneration in adult mice. TAK1 is essential for satellite cell proliferation and its inactivation causes precocious differentiation. Moreover, TAK1-deficient satellite cells exhibit increased oxidative stress and undergo spontaneous cell death, primarily through necroptosis. TAK1 is required for the activation of NF-κB and JNK in satellite cells. Forced activation of NF-κB improves survival and proliferation of TAK1-deficient satellite cells. Furthermore, TAK1-mediated activation of JNK is essential to prevent oxidative stress and precocious differentiation of satellite cells. Collectively, our study suggests that TAK1 is required for maintaining the pool of satellite stem cells and for regenerative myogenesis.

The large yellow croaker (Larimichthys crocea) is one of the largest marine net-cage cultured species in the oceans around China. In the present study, we isolated and characterized 13 polymorphic microsatellite markers from genomic libraries of L. crocea. Loci were screened for 10 wild specimens from 2 sites in southeast of China. All loci were polymorphic. The number of alleles per locus ranged from 2 to 21. The expected heterozygosity ranged from 0.233 to 0.838 and observed heterozygosity ranged from 0.527 to 0.935. Eleven loci were highly informative (polymorphic information content >0.5). Significant deviation from Hardy-Weinberg equilibrium was observed at 3 loci after Bonferroni's correction. The microsatellite loci may be valuable tools for studying the genetic diversity and genetic structure for conservation planning of the fish.