Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

Relative nutritional quality of C3 and C4 grasses for a graminivorous lepidopteran,Paratrytone melane (Hesperiidae)

Summary We tested the hypothesis that C₄ grasses are inferior to C₃ grasses as host plants for herbivorous insects by measuring the relative performance of larvae of a graminivorous lepidopteran, Paratrytone melane (Hesperiidae), fed C₃ and C₄ grasses. Relative growth rates and final weights were higher in larvae fed a C₃ grass in Experiment I. However, in two additional experiments, relative growth rates and final weights were not significantly different in larvae fed C₃ and C₄ grasses. We examined two factors which are believed to cause C₄ grasses to be of lower nutritional value than C₃ grasses: foliar nutrient levels and nutrient digestibility. In general, foliar nutrient levels were higher in C₃ grasses. In Experiment I, protein and soluble carbohydrates were digested from a C₃ and a C₄ grass with equivalent efficiencies. Therefore, differences in larval performance are best explained by higher nutrient levels in the C₃ grass in this experiment. In Experiment II, soluble carbohydrates were digested with similar efficiencies from C₃ and C₄ grasses but protein was digested with greater efficiency from the C₃ grasses. We conclude (1) that the bundle sheath anatomy of C₄ grasses is not a barrier to soluble carbohydrate digestion and does not have a nutritionally significant effect on protein digestion and (2) that P. melane may consume C₄ grasses at compensatory rates.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Photosynthesis pathways,ecological characteristics,and the geographical distribution of the Cyperaceae in Japan

Summary The nature of the photosynthetic pathways of Cyperaceae found in Japan were investigated on the basis of Kranz anatomy, the CO₂ compensation concentration and previously reported data. Among 301 species (96% of all cyperaceous species recorded in the region), 58 species were classified as being C₄ plants. These C₄ species were scattered among the tribes Fimbristylideae, Lipocarpheae, Cypereae and Rhynchosporeae in the subfamily Cyperoideae. The genera Cyperus, Eleocharis and Rhynchospora included, in Japan, both C₃ and C₄ species within a single genus. Using these data, an analysis was made of the ecological characteristics and geographical distribution of the C₃ and C₄ species in Japan. Although cyperaceous species grow in markedly different environments, the majority were found in wet and aquatic areas (61%) or shaded areas, such as forest floors (20%). Most of the C₃ species were also hygrophytes (58%) and forest-living species (25%), and C₃ species growing in mesic and dry areas were relatively rare. The C₄ species inhabited wet and aquatic (75%), mesic (13%) and dry areas (6%) and showed marked ecological characteristics with respect to soil-moisture conditions, unlike other C₄ plants, although they were absent from shaded habitats. In order to determine the climatic factors that influence the relative floristic abundance of C₃ and C₄ members of the Cyperaceae in Japan, the ratios of number of C₄ species to the total number of members of Cyperaceae (C₄ percentage) in 16 representative locales were examined in terms of various climatic variables. There were strong positive correlations between the C₄ percentage and temperature. Among the C₃ groups of three subfamilies, there were different distributional trends for various temperature regimes. The C₃ subfamily Caricoideae increased its relative contribution to the cyperaceous flora with a decrease in mean annual temperature, while the C₃ subfamily Sclerioideae exhibited the opposite pattern. The C₃ group of the subfamily Cyperoideae did not show any marked change in pattern along temperature gradients, unlike the two other C₃ subfamilies, and seemed to be heterogeneous in terms of its response to temperature. The relationships between the C₄ biochemical subtypes and ecological characteristics are also discussed.     

Presence and Importance of Connexin43 During Myogenesis

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C₂C₁₂cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C₂C₁₂myoblasts in the presence or absence of the gap junction blocker 18β-glycyrrhetinic acid (β-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with β-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in β-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43^Cre-ER(T)/fltransgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.     

In-situ immunofluorescent localization of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylases in leaves of C3, C4, and C3−C4 intermediatePanicum species

The in-situ inter- and intracellular localization patterns of phosphoenolpyruvate (PEP) and ribulose 1,5-bisphosphate (RuBP) carboxylases in green leaves of severalPanicum species were investigated using an indirect immunofluorescence technique. Four species were examined and compared:P. miliaceum (C₄),P. bisulcatum (C₃), andP. decipiens andP. milioides (C₃–C₄ intermediates which have Kranz-like leaf anatomy and reduced photorespiration). In the C₄ Panicum, PEP carboxylase was located in the cytosol of the mesophyll cells and RuBP carboxylase was restricted to the bundle-sheath chloroplasts. In contrast, in the C₃ Panicum species, PEP carboxylase was found throughout the leaf chlorenchyma, in both the cytosol and chloroplasts, and RuBP carboxylase was located in the chloroplasts. For the C₃–C₄ intermediate plants, the patterns depended on the species examined. ForP. decipiens, the in-situ localization of both carboxylases was similar to that described forP. bisulcatum and other C₃ plants. However, inP. milioides, PEP carboxylase was found exclusively in the cytosol of the mesophyll cells, as inP. miliaceum and other C₄ species, whereas RuBP carboxylase was distributed in both the mesophyll and bundle-sheath chloroplasts.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

Photosynthetic Pathway Types of Forage Species Along Grazing Gradient from the Songnen Grassland,Northeastern China

Photosynthetic pathway types (C₃ and C₄ species) and their dynamics along grazing gradient were determined for 42 plant species in 30 genera and 13 families from the Songnen grassland, Northeastern China. Of the total, 10 species in 9 genera and 4 families had C₄ photosynthesis; 32 species in 21 genera and 12 families had C₃ photosynthesis. The proportion of C₄ species in total plants and C₄/C₃ increased with grazing intensity, and peaked in overgrazed plot. Most of the increased C₄ species (6 of 10) along the grazed gradient were annual grasses and halophytes. This indicated that the C₄ species had greater capacity to tolerate environmental stresses (e.g. drought and saline) caused by animal grazing in the Songnen grassland, Northeastern China.     

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species

The leafless amphibious sedge Eleocharis vivipara develops culms with C₄ traits and Kranz anatomy under terrestrial conditions, but develops culms with C₃ traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C₄ enzyme activities, while those of the submerged form have decreased C₄ enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C₃ pathway in the submerged form. To verify the presence of the C₃ cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C₄ control), but by only 20% in potato (C₃ control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C₄ and C₃ plants, at 58–81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO₂ by Rubisco and probably the C₃ cycle in the MC. These features are reminiscent of those in C₄-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C₃ activity in the MC. This revised version was published online in June 2006 with corrections to the Cover Date.     

A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Neuregulin-dependent protein synthesis in C2C12 myotubes and rat diaphragm muscle

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [³H]phenylalanine into the protein pool) were investigated in differentiated C₂C₁₂ myotubes and rat diaphragm muscle (DIAm). In C₂C₁₂ myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased 30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [³H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K. Akt; ErbB; heregulin; protein biosynthesis; skeletal muscle     

Photosynthetic and morphological functional types for native species from mixed prairie in Southern Saskatchewan, Canada

Photosynthetic pathways (e.g. C₃, C₄) and morphological functional types (e.g. trees, shrubs, high perennial grasses, perennial forbs) were identified for the native species from the Saskatchewan mixed prairie, using the data from references published between 1950 and 2003. Of the total 219 identified species in 145 genera and 45 families, 208 species in 137 genera and 44 families were found with C₃ photosynthesis, and most of these species are dominants (e.g. Agropyron dasystachyum Hook. and Stipa spartea var. curtiseta Hitchc.). 11 species in 10 genera and 3 families were identified with C₄ photosynthesis (e.g. Atriplex argentea Nutt., Andropogon scoparius Michx., Boutelou gracilis Lag., Calamovilfa longifolia Hook.). The amount of total identified C₄ species in the region is much less than that from the South Dakota mixed prairie (27 species). Gramineae is the leading family with C₄ photosynthesis (8 species), Chenopodiaceae ranks the second (2 species). Relatively less forb types [50 % perennial forbs (PEF) and 12 % annual forbs (ANF)] and more graminoid types (25 %) composition suggested that the rangelands in the region are relatively stable. Lacking of the knowledge on the optimal traits for PFTs classification in the region, further studies (e.g. C₃ and C₄ plant identification and optimal trait selection) are needed to explore the relationships between PFTs and vegetation variations, as well as land-use and climate changes.     

Comparison of Belowground Biomass in C3- and C4-Dominated Mixed Communities in a Chesapeake Bay Brackish Marsh

Belowground biomass is a critical factor regulating ecosystem functions of coastal marshes, including soil organic matter (SOM) accumulation and the ability of these systems to keep pace with sea-level rise. Nevertheless, belowground biomass responses to environmental and vegetation changes have been given little emphasis marsh studies. Here we present a method using stable carbon isotopes and color to identify root and rhizomes of Schoenoplectus americanus (Pers.) Volk. ex Schinz and R. Keller (C₃) and Spartina patens (Ait.) Muhl. (C₄) occurring in C₃− and C₄-dominated communities in a Chesapeake Bay brackish marsh. The functional significance of the biomass classes we identified is underscored by differences in their chemistry, depth profiles, and variation in biomass and profiles relative to abiotic and biotic factors. C₃ rhizomes had the lowest concentrations of cellulose (29.19%) and lignin (14.43%) and the lowest C:N (46.97) and lignin:N (0.16) ratios. We distinguished two types of C₃ roots, and of these, the dark red C₃ roots had anomalously high C:N (195.35) and lignin:N (1.14) ratios, compared with other root and rhizome classes examined here and with previously published values. The C₄-dominated community had significantly greater belowground biomass (4119.1 g m⁻²) than the C₃-dominated community (3256.9 g m⁻²), due to greater total root biomass and a 3.6-fold higher C₃-root:rhizome ratio in the C₄-dominated community. C₃ rhizomes were distributed significantly shallower in the C₄-dominated community, while C₃ roots were significantly deeper. Variability in C₃ rhizome depth distributions was explained primarily by C₄ biomass, and C₃ roots were explained primarily by water table height. Our results suggest that belowground biomass in this system is sensitive to slight variations in water table height (across an 8 cm range), and that the reduced overlap between C₃ and C₄ root profiles in the C₄-dominated community may account for the greater total root biomass observed in that community. Given that future elevated atmospheric CO₂ and accelerated sea-level rise are likely to increase C₃ abundance in Atlantic and Gulf coast marshes, investigations that quantify how patterns of C₃ and C₄ belowground biomass respond to environmental and biological factors stand to improve our understanding of ecosystem-wide impacts of global changes on coastal wetlands.     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

Metabolic regulation of carbon flux during C4 photosynthesis

The potential for glycolate and glycine metabolism and the mechanism of refixation of photorespiratory CO₂ in leaves of C₄ plants were studied by parallel inhibitor experiments with thin leaf slices, different leaf cell types and isolated mitochondria of C₃ and C₄ Panicum species. CO₂ evolution by leaf slices of P. bisulcatum, a C₃ species, fed glycolate or glycine was light-independent and O₂-sensitive. The C₄ P. maximum and P. miliaceum leaf slices fed glycolate or glycine evolved CO₂ in the dark but not in the light. In C₄ species, dark CO₂ evolution was abolished by the addition of phosphoenolpyruvate (PEP)⁴. The addition of maleate, a PEP carboxylase inhibitor, resulted in photorespiratory CO₂ efflux by C₄ leaf slices in the light also. However, PEP and maleate had no effect on either glycolate-dependent O₂ uptake by the C₄ leaf slices or on glycolate and glycine metabolism in C₃ leaf slices. The rate of photorespiratory CO₂ evolution in the C₃ Panicum species was 3 times higher than that observed with the C₄ species. The ratio of glycolate-dependent CO₂ evolution to O₂ uptake in both groups was 1:2. Isolated C₄ mesophyll protoplasts or their mitochondria did not metabolize glycolate or glycine. However, both C₃ mesophyll protoplasts and C₄ bundle sheath strands readily metabolized glycolate and glycine in a light-independent, O₂-sensitive manner, and the addition of PEP or maleate had no effect. C₄ bundle sheath- and C₃-mitochondria were capable of oxidizing glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. C₄ bundle sheath- and C₃-mitochondrial glycine decarboxylation was stimulated by oxaloacetate and NAD had no effect. In marked contrast, mitochondria isolated from C₄ mesophyll cells were incapable of oxidizing or decarboxylating added glycine. The results suggest that in leaves of C₄ plants bundle sheath cells are the primary site of O₂-sensitive photorespiratory CO₂ evolution and the PEP carboxylase present in the mesophyll cells has the Potential for efficiently refixing CO₂ before it escapes out of the leaf. The relative role of the PEP carboxylase mediated CO₂ pump and reassimilation of photorespiratory CO₂ are discussed in relation to the apparent lack of photorespiration in leaves of C₄ species.Abbreviations BSA bovine serum albumin - Chl chlorophyll - PEP phosphoenolpyruvate - Rbu-P ₂ ribulose 1,5-bisphosphate - Rib-5-P ribose-5-phosphate - Ru-5-P ribuluse-5-phosphate - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone Journal Series Paper, New Jersey Agricultural Experiment Station