Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

Localization of Reactive Cysteine Residues by Maleidoyl Undecagold in the Mitochondrial Creatine Kinase Octamer

Octamers of mitochondrial creatine kinase (Mi-CK) wore modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryoconditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view, Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the notamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis.     

Forward Modeling of Fluctuating Dietary 13C Signals to Validate 13C Turnover Models of Milk and Milk Components from a Diet-Switch Experiment

Isotopic variation of food stuffs propagates through trophic systems. But, this variation is dampened in each trophic step, due to buffering effects of metabolic and storage pools. Thus, understanding of isotopic variation in trophic systems requires knowledge of isotopic turnover. In animals, turnover is usually quantified in diet-switch experiments in controlled conditions. Such experiments usually involve changes in diet chemical composition, which may affect turnover. Furthermore, it is uncertain if diet-switch based turnover models are applicable under conditions with randomly fluctuating dietary input signals. Here, we investigate if turnover information derived from diet-switch experiments with dairy cows can predict the isotopic composition of metabolic products (milk, milk components and feces) under natural fluctuations of dietary isotope and chemical composition. First, a diet-switch from a C₃-grass/maize diet to a pure C₃-grass diet was used to quantify carbon turnover in whole milk, lactose, casein, milk fat and feces. Data were analyzed with a compartmental mixed effects model, which allowed for multiple pools and intra-population variability, and included a delay between feed ingestion and first tracer appearance in outputs. The delay for milk components and whole milk was ∼12 h, and that of feces ∼20 h. The half-life (t_½) for carbon in the feces was 9 h, while lactose, casein and milk fat had a t_½ of 10, 18 and 19 h. The ¹³C kinetics of whole milk revealed two pools, a fast pool with a t_½ of 10 h (likely representing lactose), and a slower pool with a t_½ of 21 h (likely including casein and milk fat). The diet-switch based turnover information provided a precise prediction (RMSE ∼0.2 ‰) of the natural ¹³C fluctuations in outputs during a 30 days-long period when cows ingested a pure C3 grass with naturally fluctuating isotope composition.     

Nitrogen Stress Affects the Turnover and Size of Nitrogen Pools Supplying Leaf Growth in a Grass

The effect of nitrogen (N) stress on the pool system supplying currently assimilated and (re)mobilized N for leaf growth of a grass was explored by dynamic ¹⁵N labeling, assessment of total and labeled N import into leaf growth zones, and compartmental analysis of the label import data. Perennial ryegrass (Lolium perenne) plants, grown with low or high levels of N fertilization, were labeled with ¹⁵NO₃⁻/¹⁴NO₃⁻ from 2 h to more than 20 d. In both treatments, the tracer time course in N imported into the growth zones fitted a two-pool model (r² > 0.99). This consisted of a “substrate pool,” which received N from current uptake and supplied the growth zone, and a recycling/mobilizing “store,” which exchanged with the substrate pool. N deficiency halved the leaf elongation rate, decreased N import into the growth zone, lengthened the delay between tracer uptake and its arrival in the growth zone (2.2 h versus 0.9 h), slowed the turnover of the substrate pool (half-life of 3.2 h versus 0.6 h), and increased its size (12.4 μg versus 5.9 μg). The store contained the equivalent of approximately 10 times (low N) and approximately five times (high N) the total daily N import into the growth zone. Its turnover agreed with that of protein turnover. Remarkably, the relative contribution of mobilization to leaf growth was large and similar (approximately 45%) in both treatments. We conclude that turnover and size of the substrate pool are related to the sink strength of the growth zone, whereas the contribution of the store is influenced by partitioning between sinks.This article examines the nitrogen (N) supply system of growing grass leaves, and it investigates how functional and kinetic properties of this system are affected by N stress. The N supply of growing leaves is a dominant target of whole-plant N metabolism. This is primarily related to the high N demand of the photosynthetic apparatus and the related metabolic machinery of new leaves (Evans, 1989; Makino and Osmond, 1991; Grindlay, 1997; Lemaire, 1997; Wright et al., 2004; Johnson et al., 2010; Maire et al., 2012). The N supply system, as defined here, is an integral part of the whole plant: it includes all N compounds that supply leaf growth. Hence, it integrates all events between the uptake of N from the environment (source), intermediate uses in other processes of plant N metabolism, and the eventual delivery to the leaf growth zone (sink; Fig. 1). N that does not ultimately serve leaf growth is not included in this system; all N that serves leaf growth is included, irrespective of its localization in the plant. Conceptually, two distinct sources supply N for leaf growth: N from current uptake and assimilation that is directly transferred to the growing leaf (“directly transferred N”) and N from turnover/redistribution of organic compounds (“mobilized N”).Open in a separate windowFigure 1.Schematic representation of N fluxes in the leaf growth zone and in the N supply system of leaf growth in a grass plant. A, Scheme of a growing leaf, with its growth zone (including zones of cell division, expansion, and maturation) and recently produced tissue (RPT). N import (I; μg h⁻¹) into the growth zone is mostly in the form of amino acids. Inside the growth zone, the nitrogenous substrate is used in new tissue construction. Then, N export (E; μg h⁻¹) is in the form of newly formed, fully expanded nitrogenous tissue (tissue-bound export with RPT) and is calculated as leaf elongation rate (L_ER; mm h⁻¹) times the lineal density of N in RPT (ρ; μg mm⁻¹): E = L_ER × ρ (Lattanzi et al., 2004). In a physiological steady state, import equals export (I = E) and the N content of the growth zone (G; μg [not shown]) is constant. Labeled N import into the growth zone (I_lab) commences shortly after labeling of the nutrient solution with ¹⁵N. The labeled N content of the growth zone (G_lab; μg) increases over time (dG_lab/dt) until it eventually reaches isotopic saturation (Fig. 2B). Similarly, the lineal density of labeled N in RPT (ρ_lab) increases until it approaches ρ. At any time, the export of labeled N in RPT (E_lab) equals the concurrent ρ_lab × L_ER. The import of labeled N is obtained as I_lab = E_lab + dG_lab/dt (Lattanzi et al., 2005) and considers the increasing label content in the growth zone during labeling. The fraction of labeled N in the import flux (f_{lab I}) is calculated as f_{lab I} = I_lab/I. The time course of f_{lab I} (Fig. 3) reflects the kinetic properties of the N supply system of leaf growth (C). B, Scheme of a vegetative grass plant (reduced to a rooted tiller with three leaves) with leaf growth zone. N import into the growth zone (I) originates from (1) N taken up from the nutrient solution that is transferred directly to the growth zone following assimilation (directly transferred N) and (2) N derived from turnover/redistribution of stores (mobilized N). The store potentially includes proteins in all mature and senescing tissue in the shoot and root of the entire plant. As xylem, phloem, and associated transfer cells/tissue provide for a vascular network that connects all parts of the plant, the mobilized N may principally originate from any plant tissue that exhibits N turnover/mobilization. The fraction of total N uptake that is allocated to the N supply system of the growth zone equals U (see model in C). The fraction of total mobilized N allocated to the growth zone equals M (see model in C). C, Compartmental model of the source-sink system supplying N to the leaf growth zone, as shown by Lattanzi et al. (2005) and used here. Newly absorbed N (U; μg h⁻¹) enters a substrate pool (Q₁); from there, the N is either imported directly into the growth zone (I) or exchanged with a store (Q₂). Q₁ integrates the steps of transport and assimilation that precede the translocation to the growth zone. Q₂ includes all proteins that supply N for leaf growth during their turnover and mobilization. The parameters of the model, including the (relative) size and turnover of pools Q₁ and Q₂, the deposition into the store (D; μg h⁻¹), and the mobilization from the store (M; μg h⁻¹), and the contribution of direct transfer relative to mobilization to the N supply of the growth zone are obtained by fitting the compartmental model to the f_{lab I} data (A) obtained in dynamic ¹⁵N labeling experiments (for details, see “Materials and Methods”). During physiological steady state, the sizes of Q₁ and Q₂ are constant, I = U, and M = D. [See online article for color version of this figure.]Amino acids are the predominant form in which N is supplied for leaf growth in grasses, and incorporation in new leaf tissue occurs mainly in the leaf growth zone (Gastal and Nelson, 1994; Amiard et al., 2004). This is a heterotrophic piece of tissue that includes the zones of cell division and elongation, is located at the base of the leaf, and is encircled by the sheath of the next older leaf (Volenec and Nelson, 1981; MacAdam et al., 1989; Schnyder et al., 1990; Kavanová et al., 2008). As most N is taken up in the form of nitrate but supplied to the growth zone in the form of amino acids, the path of directly transferred N includes a series of metabolic and transport steps. These include transfer to and loading into the xylem, xylem transport and unloading, reduction and ammonium assimilation, cycling through photorespiratory N pools, amino acid synthesis, loading into the phloem, and transport to the growth zone (Hirel and Lea, 2001; Novitskaya et al., 2002; Stitt et al., 2002; Lalonde et al., 2003; Dechorgnat et al., 2011). The time taken to pass through this sequence is unknown at present, as is the effect of N deficiency on that time. Also, it is not known how much N is contained in, and moving through, the different compartments that supply leaf growth with currently assimilated N.At the level of mature organs, mainly leaves, there is considerable knowledge about N turnover and redistribution. Much less is known about the fate of the mobilized N and its actual use in sink tissues like the leaf growth zone. The processes in mature organs are associated with the maintenance metabolism of proteins, organ senescence, and adjustments in leaf protein levels to decreasing irradiance inside growing canopies when leaves become shaded by overtopping newer ones (Evans, 1993; Vierstra, 1993; Hikosaka et al., 1994; Anten et al., 1995; Hirel et al., 2007; Jansson and Thomas, 2008; Moreau et al., 2012). N mobilization in shaded leaves supports the optimization of photosynthetic N use efficiency at plant and canopy scale (Field, 1983; Evans, 1993; Anten et al., 1995), it reduces the respiratory burden of protein maintenance costs (Dewar et al., 1998; Amthor, 2000; Cannell and Thornley, 2000), and it provides a mechanism for the conservation of the most frequently growth-limiting nutrient (Aerts, 1996). Mobilization of N involves protein turnover and net degradation (Huffaker and Peterson, 1974), redistribution in the form of amino acids (Simpson and Dalling, 1981; Simpson et al., 1983; Hörtensteiner and Feller, 2002), and (at least) some of the mobilized N is supplied to new leaf growth (Lattanzi et al., 2005).N fertilizer supply has multiple direct and indirect effects on plant N metabolism (Stitt et al., 2002; Schlüter et al., 2012). In particular, it modifies the N content of newly produced leaves, leaf longevity/senescence, and the dynamics of light distribution inside expanding canopies (Evans, 1983, 1989; Lötscher et al., 2003; Moreau et al., 2012). Thus, N fertilization influences the availability of recyclable N. At the same time, it augments the availability of directly transferable N to leaf growth. The net effect of these factors on the importance of mobilized versus directly transferred N substrate for leaf growth is not known. Also, it is unknown how N fertilization influences the functional characteristics of the N supply system, such as the size and turnover of its component pools.The assessment of the importance of directly transferred versus mobilized N for leaf growth requires studies at the sink end of the system (i.e. investigations of the N import flux into the leaf growth zone). Directly transferred N and mobilized N can be distinguished on the basis of their residence time in the plant, the time between uptake from the environment and import into the leaf growth zone: direct transfer involves a short residence time (fast transfer), whereas mobilized N resides much longer in the plant before it is delivered to the growth zone (slow transfer; De Visser et al., 1997; Lattanzi et al., 2005). Such studies require dynamic labeling of the N taken up by the plant (Schnyder and de Visser, 1999) and monitoring of the rate and isotopic composition/label content of N import into the leaf growth zone (Lattanzi et al., 2005). For grass plants in a physiological steady state, N import and the isotopic composition of the imported N are calculated from the leaf elongation rate and the lineal density of N in newly formed tissue (Fig. 1A; Lattanzi et al., 2004) and the change of tracer content in the leaf growth zone and recently produced leaf tissue over time (Lattanzi et al., 2005). Such data reveal the temporal change of the fraction of labeled N in the N import flux (f_{lab I}), which then can be used to characterize the N supply system of leaf growth via compartmental modeling. So far, there is only one study that has partially characterized this system (Lattanzi et al., 2005): this work was conducted with a C₃ grass, perennial ryegrass (Lolium perenne), and a C₄ grass, Paspalum dilatatum, growing in mixed stands and indicated that two interconnected N pools supplied the leaf growth zone in both species: a “substrate pool” (Q₁), which provided a direct route for newly absorbed and assimilated N import into the leaf growth zone (directly transferred N), and a mobilizing “store” (Q₂), which supplied N to the leaf growth zone via the substrate pool (Fig. 1C). The relative contribution of mobilization from the store was least important in the fast-growing, dominant individuals and most important in subordinate, shaded individuals. That work did not address the role of N deficiency, and the limited short-term resolution of the study (labeling intervals of 24 h or greater) precluded an analysis of the fast-moving parts of the system.Accordingly, this work addresses the following questions. How does N deficiency influence the substrate supply system of the leaf growth sink in terms of the number, size, and turnover (half-life) of its kinetically distinct pools? How does N deficiency affect the relationship between directly transferred and mobilized N for leaf growth? And what additional insight on the compartmental structure of the supply system is obtained when the short-term resolution of the analysis is increased by 1 order of magnitude? The work was performed with vegetative plants of perennial ryegrass grown in constant conditions with either a low (1.0 mm; termed low N) or high (7.5 mm; high N) nitrate concentration in the nutrient solution. In both treatments, a large number of plants were dynamically labeled with ¹⁵N over a wide range of time intervals (2 h to more than 20 d). The import of total N and ¹⁵N tracer into growth zones was estimated at the end of each labeling interval. Tracer data were analyzed with compartmental models following principles detailed by Lattanzi et al. (2005, 2012) and Lehmeier et al. (2008) to address the specific questions. Previous articles reported on root and shoot respiration (Lehmeier et al., 2010) and cell division and expansion in leaf growth zones (Kavanová et al., 2008) in the same experiment.     

The Dok-3/Grb2 Protein Signal Module Attenuates Lyn Kinase-dependent Activation of Syk Kinase in B Cell Antigen Receptor Microclusters

Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5′ phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.     

Last-Century Increases in Intrinsic Water-Use Efficiency of Grassland Communities Have Occurred over a Wide Range of Vegetation Composition,Nutrient Inputs,and Soil pH

Last-century climate change has led to variable increases of the intrinsic water-use efficiency (W_i; the ratio of net CO₂ assimilation to stomatal conductance for water vapor) of trees and C₃ grassland ecosystems, but the causes of the variability are not well understood. Here, we address putative drivers underlying variable W_i responses in a wide range of grassland communities. W_i was estimated from carbon isotope discrimination in archived herbage samples from 16 contrasting fertilizer treatments in the Park Grass Experiment, Rothamsted, England, for the 1915 to 1929 and 1995 to 2009 periods. Changes in W_i were analyzed in relation to nitrogen input, soil pH, species richness, and functional group composition. Treatments included liming as well as phosphorus and potassium additions with or without ammonium or nitrate fertilizer applications at three levels. W_i increased between 11% and 25% (P < 0.001) in the different treatments between the two periods. None of the fertilizers had a direct effect on the change of W_i (ΔW_i). However, soil pH (P < 0.05), species richness (P < 0.01), and percentage grass content (P < 0.01) were significantly related to ΔW_i. Grass-dominated, species-poor plots on acidic soils showed the largest ΔW_i (+14.7 μmol mol⁻¹). The ΔW_i response of these acidic plots was probably related to drought effects resulting from aluminum toxicity on root growth. Our results from the Park Grass Experiment show that W_i in grassland communities consistently increased over a wide range of nutrient inputs, soil pH, and plant community compositions during the last century.The intrinsic water-use efficiency (W_i) of plants is controlled by photosynthetic carbon assimilation and stomatal conductance via the leaf-level coupling of CO₂ and water fluxes. A general, but variable, increase of W_i under rising atmospheric CO₂ has been observed in long-term studies (Peñuelas et al., 2011; Franks et al., 2013; Saurer et al., 2014), but little is known about other environmental or ecosystem factors, which may interact with the effect of increasing CO₂ on W_i. An improved understanding of putative interactive mechanisms is important because changes in W_i may have significant effects on the global terrestrial carbon and water cycles (Gedney et al., 2006; Betts et al., 2007). This study explores the interactive effects of the increase in atmospheric CO₂ (observed over the last century), nutrient loading, and soil pH together with other related effects on plant species richness and functional group composition on the coupling of plant CO₂ and water fluxes in a seminatural grassland in southeastern England.W_i is a leaf-level efficiency that has also been termed potential water-use efficiency or physiological water-use efficiency, as it excludes the direct influence of vapor pressure deficit (VPD), a parameter determined by environmental conditions, on leaf-level water-use efficiency (Farquhar et al., 1989; Franks et al., 2013). W_i reports the relationship between net CO₂ assimilation rate (A_n) and stomatal conductance for water vapor (g_H2O):(1)According to the first law of Fick, A_n can be given as the product of the stomatal conductance for CO₂ (g_CO2) and the concentration gradient between the atmosphere (c_a) and the leaf internal gas space (c_i): A_n= g_CO2 (c_a – c_i). Using g_CO2 (c_a – c_i) instead of A_n in Equation 1, replacement of g_H2O/g_CO2 by the numerical value of g_H2O/g_CO2 (1.6) and rearrangement yields the following alternative expression of W_i:(2)Equation 2 reveals that past changes of W_i must have been controlled by two parameters: the change of c_a and the concurrent change of 1 – c_i/c_a, the relative gradient for CO₂ diffusion into the leaf (Franks et al., 2013). A change in the relative gradient is determined by the changes in A_n relative to g_H2O, as leaves respond to changing c_a and other environmental factors. In particular, Equation 2 shows that any variation in the climate change response of W_i is determined by the c_i/c_a response, if the comparison is made for vegetation at the same location and in the same period of time.Studies with C₃ vegetation, including trees/forests and C₃ grasslands, have revealed a general increase of W_i in the last century (Bert et al., 1997; Duquesnay et al., 1998; Feng, 1999; Arneth et al., 2002; Saurer et al., 2004; Barbosa et al., 2010; Köhler et al., 2010; Andreu-Hayles et al., 2011). In many cases, c_i/c_a, estimated by ¹³C discrimination (Farquhar et al., 1989), varied relatively little. Indeed, it has been suggested, based on theoretical grounds and empirical evidence from studies over geological/evolutionary to short time scales, that adaptive feedback responses will tend to maintain c_i/c_a approximately constant (Ehleringer and Cerling, 1995; Franks et al., 2013), as plants optimize carbon gain with respect to water loss (Cowan and Farquhar, 1977). Yet, c_i/c_a-dependent variation in the W_i response to climate change has also been noted (Peñuelas et al., 2011; Köhler et al., 2012) over the last century, indicating that additional factors, perhaps including other global change drivers, can modify the W_i response over this time scale, at least transiently. A meta-analysis by Peñuelas et al. (2011) reports c_i/c_a-dependent increases of W_i for different forests between 6% and 36% from the early 1960s to 2000s. A recent study by Saurer et al. (2014) on European forest trees found increases in W_i ranging from 1% to 53% during the last century. The strongest increase of W_i was recorded in regions where summer soil-water availability decreased in the last century. For different grassland communities, the c_i/c_a-dependent increases of W_i varied between 13% and 28% at one site (Köhler et al., 2012) from 1915 to 2009. Evidently, such variation can have important repercussions for the coupling of terrestrial CO₂ and water fluxes. Yet, little is known about the mechanism(s) underlying the variation.At the Park Grass Experiment (PGE) at Rothamsted, England, Köhler et al. (2012) observed a nitrogen supply-dependent enhancement of the W_i response on plots receiving nitrate fertilizer and maintained at a near-neutral soil pH by liming. However, the actual relationship between nitrogen supply and W_i response did not hold when the unlimed control (soil pH approximately 5.2) was included in the comparison. Remarkably, however, there was a significant positive relationship between the grass content of the community and the W_i response of the experimental plots in the investigation. These results suggested that the effect of nutrient supply on the W_i response of the grassland communities was indirect, perhaps working via effects on soil pH and/or vegetation composition (plant species richness or functional group composition).The PGE provides a unique opportunity to study century-scale variation in the c_i/c_a-dependent variation of W_i for a wide range of diverse grassland communities. Much of the extant ecosystem-scale variability of plant species richness and soil pH in temperate grasslands of Europe (Ceulemans et al., 2014) is included in the range of plot-scale plant species richness and soil pH at the PGE (which is reported in this investigation). The different long-term applications of fertilizer and lime over the past century have resulted in substantial changes in soil pH, species richness, and grass content on the experimental plots, but in most cases, within-plot changes over the study period considered here (1915–2009) were comparatively small (Crawley et al., 2005; Silvertown et al., 2006). All experimental plots are located at the same site and are exposed to the same weather conditions. Consequently, trends in climate as a direct driver for differences in W_i between plots can be ruled out.Here, we explore putative mechanisms underlying eventual c_i/c_a-dependent variation of W_i during the last century at the PGE by, first, quantifying the sustained effect of a wide range of contrasting fertilizer treatments (n = 16) on the change of W_i during the last century and, second, analyzing the relationships between the observed W_i response of treatments and the respective nutrient status, soil pH, plant species richness, and plant functional group composition of the grassland communities.     

Phosphorus nutrition and mycorrhiza effects on grass leaf growth. P status- and size-mediated effects on growth zone kinematics

This study tested whether leaf elongation rate (LER, mm h(-1)) and its components--average relative elemental growth rate (REGRavg, mm mm(-1) h(-1)) and leaf growth zone length (L(LGZ), mm)--are related to phosphorus (P) concentration in the growth zone (P(LGZ) mg P g(-1) tissue water) of Lolium perenne L. cv. Condesa and whether such relationships are modified by the arbuscular mycorrhizal fungus (AMF) Glomus hoi. Mycorrhizal and non-mycorrhizal plants were grown at a range of P supply rates and analysed at either the same plant age or the same tiller size (defined by the length of the sheath of the youngest fully expanded leaf). Both improved P supply (up to 95%) and AMF (up to 21%) strongly increased LER. In tillers of even-aged plants, this was due to increased REGRavg and L(LGZ). In even-sized tillers, it was exclusively due to increased REGRavg. REGRavg was strictly related to P(LGZ) (r2 = 0.95) and independent of tiller size. Conversely, L(LGZ) strictly depended on tiller size (r2 = 0.88) and not on P(LGZ). Hence, P status affected leaf growth directly only through effects on relative tissue expansion rates. Symbiosis with AMF did not modify these relationships. Thus, no evidence for P status-independent effects of AMF on LER was found.