Measuring the osmotic water permeability coefficient (Pf) of spherical cells: isolated plant protoplasts as an example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P(f)) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P(f)

The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection

Effect of major peaks resulting from HPLC fractionation of sweet potato active fraction (SPAF) on <i>Rhizopus delemar</i> spore swelling and germination.

<p>The pH of each HPLC fraction was modified to 4.7 or 7. The peaks contained: 1 –a mixture of organic and amino acids, 2 –sucrose, 3- glucose, 4 –fructose, or the combination of 1 and 3. SPAF and water served as controls. Values are means ± SE (n = 500).</p

The role of the outer cell His residues in pH sensing.

<p>(a) Predicted three-dimensional structure of RdAQP1 showing two His residues (His85 and His275) positioned on the loops of the protein and facing the outside of the cell. (b) <i>P</i>_f values of GFP::WT (wild type), GFP::RdAQP1 and GFP::RdAQP1 mutant,. Different lowercase letters above the bars denote significant differences for each pH level between fungal types (<i>P</i> < 0.05).</p

Effect of HgCl₂, an inhibitor of AQPs function on swelling of spore of <i>Rhizopus delemar</i>.

<p>Treatment of <i>R</i>. <i>delemar</i> spores with 40 μM HgCl₂ for 5 min inhibited spore swelling and germination. Additional treatment (5 min) with 5 μM of the reducing agent 2-β-mercaptoethanol (2ME) fully reversed the inhibition effect. Pictures were taken 15 min and 3 h, in the upper and lower rows, respectively, after incubation in water or sweet potato active fraction (SPAF). Bar = 100 μm.</p

Effect of pH on <i>Rhizopus delemar</i> spore germination.

<p>Percentage of <i>R</i>. <i>delemar</i> spores germinated under different pH conditions. Spores were incubated in SPAF solution (20 mg/ml, 42°C) and scored at 6 h. Values are means ± SE (n = 500).</p

The effect of pH on <i>Rhizopus delemar</i> aquaporin (AQP) water permeability.

<p>The protoplast osmotic water permeability coefficient (<i>P</i>_f) determined in <i>Arabidopsis thaliana</i> protoplasts transiently expressing GFP::AQP (GFP, green fluorescent protein) and in control protoplasts transiently expressing GFP. Values are means ± SE (n = 15). Different lowercase letters above the bars denote significant differences for each pH level in each transformed gene (<i>P</i> < 0.05).</p

Phylogenetic tree of 231 fungal major intrinsic proteins (MIPs).

<p>The MIPs clustered into four distinct groups: Cluster I—putative water channels MIPs (represented by ADC55259|<i>Saccharomyces cerevisiae</i> [Black circle] and JF491353|<i>Terfezia claveryi</i> [black square]), cluster II—putative aquaglyceroporins MIPs that preferentially transporting small neutral molecules (represented by Lacbi2|671860|<i>Laccaria bicolor</i> [open triangle]), cluster III—MIPs that putatively act as water and small neutral molecule transport channels (represented by GAA23030|<i>S</i>. <i>cerevisiae</i> [black rhombus]), and cluster IV—putative fungal X intrinsic proteins (XIPs) (represented by TmeAQP2|<i>Tuber melanosporum</i> [black triangle]). Both <i>RdAQP1</i> and <i>RdAQP2</i> (open circles) are located in Cluster III, pointing on their potential capability to act as a water channels. Bar represents 0.2 changes.</p