Morphological, biochemical and physiological traits of upper and lower canopy leaves of European beech tend to converge with increasing altitude

The present work has explored for the first time acclimation of upper versus lower canopy leaves along an altitudinal gradient. We tested the hypothesis that restrictive climatic conditions associated with high altitudes reduce within-canopy variations of leaf traits. The investigated beech (Fagus sylvatica L.) forest is located on the southern slope of the Hrubý Jeseník Mountains (Czech Republic). All measurements were taken on leaves from upper and lower parts of the canopy of mature trees (>85 years old) growing at low (400 m above sea level, a.s.l.), middle (720 m a.s.l.) and high (1100 m a.s.l.) altitudes. Compared with trees at higher altitudes, those growing at low altitudes had lower stomatal conductance, slightly lower CO2 assimilation rate (Amax) and leaf mass per area (LMA), and higher photochemical reflectance index, water-use efficiency and Rubisco content. Given similar stand densities at all altitudes, the different growth conditions result in a more open canopy and higher penetration of light into lower canopy with increasing altitude. Even though strong vertical gradients in light intensity occurred across the canopy at all altitudes, lower canopy leaves at high altitudes tended to acquire the same morphological, biochemical and physiological traits as did upper leaves. While elevation had no significant effect on nitrogen (N) and carbon (C) contents per unit leaf area, LMA, or total content of chlorophylls and epidermal flavonoids in upper leaves, these increased significantly in lower leaves at higher altitudes. The increases in N content of lower leaves were coupled with similar changes in Amax. Moreover, a high N content coincided with high Rubisco concentrations in lower but not in upper canopy leaves. Our results show that the limiting role of light in lower parts of the canopy is reduced at high altitudes. A great capacity of trees to adjust the entire canopy is thus demonstrated

Morphological, biochemical and physiological traits of upper and lower canopy leaves of European beech tend to converge with increasing altitude

The present work has explored for the first time acclimation of upper versus lower canopy leaves along an altitudinal gradient. We tested the hypothesis that restrictive climatic conditions associated with high altitudes reduce within-canopy variations of leaf traits. The investigated beech (Fagus sylvatica L.) forest is located on the southern slope of the Hrubý Jeseník Mountains (Czech Republic). All measurements were taken on leaves from upper and lower parts of the canopy of mature trees (>85 years old) growing at low (400 m above sea level, a.s.l.), middle (720 m a.s.l.) and high (1100 m a.s.l.) altitudes. Compared with trees at higher altitudes, those growing at low altitudes had lower stomatal conductance, slightly lower CO2 assimilation rate (Amax) and leaf mass per area (LMA), and higher photochemical reflectance index, water-use efficiency and Rubisco content. Given similar stand densities at all altitudes, the different growth conditions result in a more open canopy and higher penetration of light into lower canopy with increasing altitude. Even though strong vertical gradients in light intensity occurred across the canopy at all altitudes, lower canopy leaves at high altitudes tended to acquire the same morphological, biochemical and physiological traits as did upper leaves. While elevation had no significant effect on nitrogen (N) and carbon (C) contents per unit leaf area, LMA, or total content of chlorophylls and epidermal flavonoids in upper leaves, these increased significantly in lower leaves at higher altitudes. The increases in N content of lower leaves were coupled with similar changes in Amax. Moreover, a high N content coincided with high Rubisco concentrations in lower but not in upper canopy leaves. Our results show that the limiting role of light in lower parts of the canopy is reduced at high altitudes. A great capacity of trees to adjust the entire canopy is thus demonstrated

Conductance.

<p>Conductance of <b>DR5047</b> (<b>A</b>) and <b>DR5026</b> (<b>B</b>) single pores measured in 1M KCl, 10 mM Tris, pH 7.4 at membrane potential of 45 mV. The histograms of different conductance states were fitted with Gaussian functions. <b>C</b>. Representative single channel recordings of <b>DR5047</b> and <b>DR5026</b> in planar lipid membranes.</p

TEM pictures of <i>B</i>. <i>subtilis</i> cells.

<p>0.25% phosphotungstic acid at pH 7.3 was used for staining. <b>A.</b> Untreated. <b>B</b>. Treated with 10 mg/L of <b>DR5026</b> for 15 min. <b>C</b>. Treated with 10 mg/L of <b>DR5026</b> for 30 min. <b>D</b>. Treated with 20 mg/L of <b>DR5026</b> for 15 min. <b>E.</b> Treated with 20 mg/L of <b>DR5026</b> for 30 min. The scale bars in the right-hand corners of the pictures represent 500 nm.</p

The effect of LPPOs on the biosynthesis of selected macromolecules.

<p>In all panels, <b>DR5026</b> is shown with black circles, <b>DR5047</b> grey circles, and control (no compound added) empty circles. The red symbols depict the effect of a known inhibitor. The amount of the radiolabeled material incorporated at the time of inhibitor addition (shown with arrows) was set as 1. <b>A.</b> The effect on RNA synthesis. Rif, rifampicin. <b>B.</b> The effect on protein synthesis. Cm, chloramphenicol. <b>C</b>. The effect on DNA synthesis. <b>D</b>. The effect on lipid synthesis. Cer, cerulenin. <b>E</b>. The effect on cell wall synthesis. Amp, ampicillin. The experiments were conducted in three biological replicates. Representative experiments are shown. The error was below 10%.</p

<i>B</i>. <i>subtilis</i> 168 develops resistance against rifampicin (rif) but not against DR5026.

<p><i>B</i>. <i>subtilis</i> was incubated with subcytotoxic concentrations of rif (starting at 0.01 mg/L; MIC 0.06 mg/L) and <b>DR5026</b> (0.5 mg/L MIC ~ 3 mg/L) and grown for 24 h. Then, aliquots of the cultures were transferred to new tubes with fresh medium and a two-fold increased concentration of the active compound. A binary representation is shown; 1 indicates growth of the cells, i. e. their resistance to the respective compound; 0 represents lack of growth—the cells were sensitive to the compound and no resistant cell appeared within the time frame of the experiment. The experiment was conducted in three biological replicates with the same results.</p

Permeabilization of cytoplasmic membrane induced by DR5026 and DR5047.

<p>At time 0 s permeabilizing agents were added to the cells at final concentrations of 10 mg/L or 10 μM for LPPOs and melittin, respectively. Increase in fluorescence intensity signifies that the membrane impermeant dye PI entered the cells.</p

Localization of DR5026 in <i>B</i>. <i>subtilis</i> cells.

<p><b>A</b>. A scheme of the experiment. SN, supernatant; P, pellet. <b>B.</b> HPLC data of supernatant after cell sedimentation, SN1. <b>C.</b> HPLC analysis of cell debris and remaining non-lysed cells, P2. <b>D.</b> HPLC analysis of cell cytoplasm, SN3. <b>E.</b> HPLC analysis of the plasma membrane fraction, P3. The dotted red line: <b>DR5026</b> treated cells; the blue line: mock-treated cells. The arrows indicate where <b>DR5026</b> eluted from the column. The identity of <b>DR5026</b> was confirmed by MS detection.</p

A model of the interaction of DR5026 with a bacterial membrane.

<p>The final state of MD simulation after 55 ns shows <b>DR5026</b> molecules penetrated into the phospholipid bilayer. The nitrogen atoms from the iminosugar modules of <b>DR5026</b> are highlighted as blue spheres. Phosphorus atoms of the phospholipid bilayer (PB) are depicted as yellow/red spheres. For clarity, almost all atoms of the PB are hidden.</p