Large-scale gene expression profiling data for the model moss Physcomitrella patens aid understanding of developmental progression, culture and stress conditions.

The moss Physcomitrella patens is an important model organism for studying plant evolution, development, physiology and biotechnology. Here we have generated microarray gene expression data covering the principal developmental stages, culture forms and some environmental/stress conditions. Example analyses of developmental stages and growth conditions as well as abiotic stress treatments demonstrate that (i) growth stage is dominant over culture conditions, (ii) liquid culture is not stressful for the plant, (iii) low pH might aid protoplastation by reduced expression of cell wall structure genes, (iv) largely the same gene pool mediates response to dehydration and rehydration, and (v) AP2/EREBP transcription factors play important roles in stress response reactions. With regard to the AP2 gene family, phylogenetic analysis and comparison with Arabidopsis thaliana shows commonalities as well as uniquely expressed family members under drought, light perturbations and protoplastation. Gene expression profiles for P. patens are available for the scientific community via the easy-to-use tool at https://www.genevestigator.com. By providing large-scale expression profiles, the usability of this model organism is further enhanced, for example by enabling selection of control genes for quantitative real-time PCR. Now, gene expression levels across a broad range of conditions can be accessed online for P. patens.

Co-option of a default secretory pathway for plant immune responses.

Cell-autonomous immunity is widespread in plant-fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1-SNAP33-VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

Dual function of Arabidopsis glucan synthase-like genes GSL8 and GSL10 in male gametophyte development and plant growth.

Members of the glucan synthase-like (GSL) family are believed to be involved in synthesis of the cell-wall component callose in specialized locations throughout the plant. We identified two members of the Arabidopsis GSL gene family, GSL8 and GSL10, that are independently required for male gametophyte development and plant growth. Analysis of gsl8 and gsl10 mutant pollen during development revealed specific malfunctions associated with asymmetric microspore division. GSL8 and GSL10 are not essential for normal microspore growth and polarity, but play a role in entry of microspores into mitosis. Impaired function of GSL10 also leads to perturbation of microspore division symmetry, irregular callose deposition and failure of generative-cell engulfment by the cytoplasm of the vegetative cell. Silencing of GSL8 or GSL10 in transgenic lines expressing gene-specific dsRNAi constructs resulted in a dwarfed growth habit, thereby revealing additional and independent wild-type gene functions for normal plant growth.

Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens.

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

Long-term protein intake and dietary potential renal acid load are associated with bone modeling and remodeling at the proximal radius in healthy children.

BACKGROUND: Protein and alkalizing minerals are increasingly described as playing a major role in influencing bone status, not only in the elderly but also in children and adolescents. OBJECTIVE: We examined whether the long-term dietary protein intake and diet net acid load are associated with bone status in children. DESIGN: In a prospective study design in 229 healthy children and adolescents aged 6-18 y, long-term dietary intakes were calculated from 3-d weighed dietary records that were collected yearly over the 4-y period before a one-time bone analysis. Dietary acid load was characterized as potential renal acid load (PRAL) by using an algorithm including dietary protein, phosphorus, magnesium, and potassium. Proximal forearm bone variables were measured by peripheral quantitative computed tomography. RESULTS: After adjustment for age, sex, and energy intake and control for forearm muscularity, BMI, growth velocity, and pubertal development, we observed that long-term dietary protein intake was significantly positively associated with periosteal circumference (P < 0.01), which reflected bone modeling, and with cortical area (P < 0.001), bone mineral content (P < 0.01), and polar strength strain index (P < 0.0001), which reflected a combination of modeling and remodeling. Children with a higher dietary PRAL had significantly less cortical area (P < 0.05) and bone mineral content (P < 0.01). Long-term calcium intake had no significant effect on any bone variable. CONCLUSIONS: Long-term dietary protein intake appears to act anabolically on diaphyseal bone strength during growth, and this may be negated, at least partly, if dietary PRAL is high, ie, if the intake of alkalizing minerals is low.

Bone mineral content per muscle cross-sectional area as an index of the functional muscle-bone unit.

Bone densitometric data often are difficult to interpret in children and adolescents because of large inter- and intraindividual variations in bone size. Here, we propose a functional approach to bone densitometry that addresses two questions: Is bone strength normally adapted to the largest physiological loads, that is, muscle force? Is muscle force adequate for body size? To implement this approach, forearm muscle cross-sectional area (CSA) and bone mineral content (BMC) of the radial diaphysis were measured in 349 healthy subjects from 6 to 19 years of age (183 girls), using peripheral quantitative computed tomography (pQCT). Reference data were established for height-dependent muscle CSA and for the variation with age in the BMC/muscle CSA ratio. These reference data were used to evaluate results from three pediatric patient groups: children who had sustained multiple fractures without adequate trauma (n = 11), children with preterminal chronic renal failure (n = 11), and renal transplant recipients (n = 15). In all three groups mean height, muscle CSA, and BMC were low for age, but muscle CSA was normal for height. In the multiple fracture group and in renal transplant recipients the BMC/muscle CSA ratio was decreased (p <. 0.05), suggesting that bone strength was not adapted adequately to muscle force. In contrast, chronic renal failure patients had a normal BMC/muscle CSA ratio, suggesting that their musculoskeletal system was adapted normally to their (decreased) body size. This functional approach to pediatric bone densitometric data should be adaptable to a variety of densitometric techniques.

Adrenarche and bone modeling and remodeling at the proximal radius: weak androgens make stronger cortical bone in healthy children.

UNLABELLED: Adrenarche, the physiological increase in adrenal androgen secretion, may contribute to better bone status. Proximal radial bone and 24-h urinary steroid hormones were analyzed cross-sectionally in 205 healthy children and adolescents. Positive adrenarchal effects on radial diaphyseal bone were observed. Obviously, adrenarche is one determinant of bone mineral status in children. INTRODUCTION: Increased bone mass has been reported in several conditions with supraphysiological adrenal androgen secretion during growth. However, no data are available for normal children. Therefore, our aim was to examine whether adrenal androgens within their physiological ranges may be involved in the strengthening of diaphyseal bone during growth. METHODS: Periosteal circumference (PC), cortical density, cortical area, bone mineral content, bone strength strain index (SSI), and forearm cross-sectional muscle area were determined with peripheral quantitative computed tomography (pQCT) at the proximal radial diaphysis in healthy children and adolescents. All subjects, aged 6-18 years, who collected a 24-h urine sample around the time of their pQCT analysis (100 boys, 105 girls), were included in the present study, and major urinary glucocorticoid (C21) and androgen (C19) metabolites were quantified using gas chromatography-mass spectrometry. RESULTS AND CONCLUSIONS: We found a significant influence of muscularity, but not of hormones, on periosteal modeling (PC) before the appearance of pubic hair (prepubarche). Similarly, no influence of total cortisol secretion (C21) was seen on the other bone variables. However, positive effects of C19 on cortical density (p < 0.01), cortical area (p < 0.001), bone mineral content (p < 0.001), and SSI (p < 0.001)--reflecting, at least in part, reduction in intracortical remodeling-were observed in prepubarchal children after muscularity or age had been adjusted for. This early adrenarchal contribution to proximal radial diaphyseal bone strength was further confirmed for all cortical variables (except PC) when, instead of C19 and C21, specific dehydroepiandrosterone metabolites were included as independent variables in the multiple regression model. During development of pubic hair (pubarche), muscularity and pubertal stage rather than adrenarchal hormones seemed to influence bone variables. Our study shows that especially the prepubarchal increase in adrenal androgen secretion plays an independent role in the accretion of proximal radial diaphyseal bone strength in healthy children.

Cytological and molecular analysis of the Hordeum vulgare-Puccinia triticina nonhost interaction.

Cultivated barley, Hordeum vulgare L., is considered to be a nonhost or intermediate host species for the wheat leaf rust fungus Puccinia triticina. Here, we have investigated, at the microscopic and molecular levels, the reaction of barley cultivars to wheat leaf rust infection. In the nonhost resistant cultivar Cebada Capa, abortion of fungal growth occurred at both pre- and posthaustorial stages, suggesting that defense genes are expressed throughout the development of the inappropriate fungus during the nonhost resistance reaction. In the two barley lines L94 and Bowman, a low level of prehaustorial resistance to P. triticina was observed and susceptibility was comparable to that of wheat control plants. Suppression subtractive hybridization was used to identify genes that are differentially expressed during the nonhost resistance reaction in Cebada Capa as well as during the successful establishment of the inappropriate wheat leaf rust fungus in L94. Northern analysis indicated that two candidate genes, including a barley ortholog of the rice resistance gene Xa21, are putatively involved in nonhost and non-race-specific resistance reactions. In addition, a new gene that is specifically induced during the successful development of the inappropriate fungus P. triticina in barley has been identified.

Anthropometric assessment of muscularity during growth: estimating fat-free mass with 2 skinfold-thickness measurements is superior to measuring midupper arm muscle area in healthy prepubertal children.

BACKGROUND: Anthropometric measurements are widely used to determine body composition, especially in children. OBJECTIVE: Our aim was to compare 2 of the simplest anthropometry-based equations available for determining nutritional status and muscularity in children and adolescents, examined in relation to other methodologically independent muscle variables. DESIGN: Midupper arm muscle area (UAMA) and fat-free mass (FFM) according to the equations of Slaughter et al (Hum Biol 1988;60:709-23), as well as separate biochemical, physical, and radiologic muscle variables, were determined cross-sectionally in 91 males and 91 females aged 6-18 y. The ability of UAMA and FFM to estimate muscularity, as measured by 24-h creatinine excretion, grip force, and peripheral quantitative computer tomography analysis of forearm muscle, was compared after dividing the study population into prepubertal and pubertal groups. RESULTS: Before puberty, correlations of all 3 muscularity variables were higher with FFM than with UAMA in both males and females. Multiple regression analyses confirmed FFM to be the predominant predictor, with partial R(2) >/= 0.68 (P < 0.001). However, in puberty, FFM did not consistently show this major influence. Only before puberty did FFM provide a significantly better fit (P < 0.05) than did UAMA for 2 of the 3 muscularity variables in each sex. CONCLUSIONS: The FFM estimate proved to be the better predictor for muscularity in healthy prepubertal children and is on a par with UAMA during puberty. FFM can be recommended as a simple anthropometric method to assess nutritional status before puberty, at least in healthy children.

Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity.

Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.

Genetic mapping of the Lr20-Pm1 resistance locus reveals suppressed recombination on chromosome arm 7AL in hexaploid wheat.

The Lr20-Sr15-Pm1 resistance locus in hexaploid wheat confers resistance to three different fungal wheat pathogens (leaf rust, stem rust, and powdery mildew). It was previously localized in the distal region of chromosome arm 7AL. As a first step towards the isolation of this complex locus, we performed molecular mapping of the Lr20 and Pm1 genes in three F2 populations. In two populations, a cluster of 8 and 12 markers, respectively, cosegregated with the resistance genes. In a third population based on a cross between a susceptible lr20 mutant and a resistant cultivar, all clustered markers were monomorphic. However, in this population the recombination frequency proximal to the Lr20 gene was up to 60 times higher, indicating that the complete genetic linkage of the clustered markers is not due to a close physical linkage of the probes but is caused by suppressed recombination. This was supported by the analysis of Triticum monococcum BAC clones where no physical linkage between cosegregating probes was observed. Suppressed recombination at the Lr20-Pm1 locus is likely the result of an alien introgression of chromatin from an unidentified wild relative species or is due to chromosomal rearrangements.

Muscle analysis by measurement of maximal isometric grip force: new reference data and clinical applications in pediatrics.

Skeletal muscle development is one of the key features of childhood and adolescence. Determining maximal isometric grip force (MIGF) using a hand-held Jamar dynamometer is a simple method to quantify one aspect of muscle function. Presently available reference data present MIGF as a function of chronological age. However, muscle force is largely determined by body size, and many children undergoing muscle performance tests in the clinical setting suffer from growth retardation secondary to a chronic disorder. Reference data were established from simple regressions between age or log height and log MIGF in a population of 315 healthy children and adolescents aged 6 to 19 y (157 girls). These data were used to calculate age- or height-dependent SD scores (SDS) for MIGF in three pediatric patient groups. In renal graft recipients (n = 14), the age-dependent MIGF SDS was markedly decreased (-2.5 +/- 1.9; mean +/- SD). However, these patients had short stature (height SDS, -2.5 +/- 1.2), and the height-dependent MIGF SDS was close to normal (-0.4 +/- 1.5). Similarly, in cystic fibrosis patients (n = 13) age-dependent MIGF SDS was -1.6 +/- 1.6, but height-dependent MIGF SDS was -0.5 +/- 1.1. Children with epilepsy who were taking anticonvulsant therapy (n = 34) had normal stature, and consequently age- and height-dependent MIGF SDS were similar (0.4 +/- 1.0 and 0.4 +/- 0.8, respectively). In conclusion, MIGF determination provides information on an important aspect of physical development. Height should be taken into account to avoid misinterpretation.

Adrenal steroid hormones and metaphyseal bone in children.

BACKGROUND/OBJECTIVES: The responses of metaphyseal bone tissue to physiological variations of endogenous adrenal steroid hormones during childhood are unclear. Therefore, we studied potential hormonal influences in children before the appearance of pubic hair (onset of pubarche). METHODS: Excretions of major glucocorticoid metabolites (C21), cortisol, sum of adrenarchal dehydroepiandrosterone and its immediate 16-hydroxylated metabolites (DHEA&M), and 5-androstene-3beta,17beta-diol (hermaphrodiol) were analyzed in a cross-sectional study in 24-hour urine samples of 109 healthy boys and girls, aged 6-13 years, using steroid profiling by gas chromatography-mass spectrometry. Total and trabecular volumetric bone mineral densities, bone mineral content (BMC) and bone strength strain index were determined with peripheral quantitative computed tomography at the distal forearm. RESULTS: In multiple regression analyses significant associations with the metaphyseal radius were seen for grip force, age, or BMI depending on gender and bone variable analyzed. DHEA&M did not contribute to the explanation of the variance of any bone variable. However, hermaphrodiol positively explained a significant part of variation of bone mineral densities, and BMC (p < 0.01) in girls. Significantly negative associations with all bone variables were seen in boys for cortisol. CONCLUSIONS: The steroid hormones, cortisol and hermaphrodiol, in their physiological ranges, but not the adrenarche marker DHEA&M, appear to associate with metaphyseal bone in a sex-dependent manner during childhood.

Collagen markers deoxypyridinoline and hydroxylysine glycosides: pediatric reference data and use for growth prediction in growth hormone-deficient children.

BACKGROUND: In children and adolescents, markers of bone and collagen metabolism reflect the dynamics of skeletal growth and development. The aim of this study was to assess the relationship of the urinary collagen markers deoxypyridinoline (DPD) and hydroxylysine (Hyl) and its glycosides [galactosyl-Hyl (Gal-Hyl) and glucosyl-Gal-Hyl] with growth. METHODS: Urine samples from 240 apparently healthy children and adolescents (6-19 years; 124 girls) and from 51 prepubertal children with growth hormone (GH) deficiency (3-14 years; 14 girls) were analyzed. Urinary Hyl and its glycosides were quantified by HPLC, and DPD was assessed by chemiluminescence assay. Urinary concentrations of all markers were related to urinary creatinine. RESULTS: Multiple regression analysis revealed that only age and height velocity were independently associated with these markers in healthy children. In GH-deficient patients, the urinary excretion of both analytes after 4 weeks of GH therapy correlated significantly with the height increase during the first treatment year (r = 0.79 for Gal-Hyl; r = 0.70 for DPD; P <0.001 each). In a multivariate linear regression model using Gal-Hyl concentrations at 4 weeks, baseline concentrations of insulin-like growth factor 1 and height velocity after 3 months accounted for 80% of the variability in height gain during the first treatment year. A model using DPD concentrations at 4 weeks, in place of Gal-Hyl concentrations, as well as baseline concentrations of insulin-like growth factor 1 and height velocity after 3 months accounted for 83% of the variability. CONCLUSIONS: These urinary bone and collagen markers give some early indication of growth response, but the prediction of an individual marker is too imprecise to serve as a basis for clinical decisions. Markers of bone and collagen metabolism might be more useful as components of multivariate growth prediction models.