A simple method for mitochondrial respiration and calcium uptake assessment in pollen tubes

Graphical abstract


Method details
Background Pollen tubes require active mitochondrial respiration and copious amounts of calcium to achieve efficient growth [1]. However, the role of substrate-specific mitochondrial respiration and calcium transport for plant and pollen tube metabolism has remained less studied. Recent reports have successfully measured Mitochondrial Calcium Uniporter-dependent calcium uptake activity either using electrophysiology of reconstituted MCU or through heterologous expression of MCU in yeast [2,3]. Here we present methodological details to assess in situ mitochondrial respiration and calcium uptake in permeabilized pollen tubes [4]. This method could be applied -in principle-to pollen tubes from any plant provided adequate germination and assay buffers are used throughout the process. Pollen tube germination 5 Close the Eppendorf tube and gently stir at 60 rpm in an orbital shaker at 28 C for 2 h. 6 Place 20mL of the suspension into the hematocytometer sample port and determine cell count by multiplying by the chamber predetermined factor.

Materials and reagents
7 Wash pollen tubes twice in 1 mL PTA buffer by spinning at 3000 rpm for 20 s. Oxygen consumption assay 9 Calibrate the Liquid-Phase Hansatech Oxygraph Plus System according to the manufacturer instructions.
10 Resuspend pollen tubes in a final volume of 0.5 mL containing 1 Â10 6 pollen tubes into the Oxygraphs water-jacketed chamber containing PTA buffer with 10 mM sodium succinate, 0.01% digitonin and 200 mM ADP.
11 Close the chamber with the included plug and make sure no air bubbles remain inside. 12 Allow five minutes for efficient pollen tube permeabilization and baseline obtention. 13 Measure oxygen consumption rate for 1-2 min (state 3).
15 Calculate the Respiratory Control Ratio (RCR) by dividing State 3 by State 4.
16 Add 100 mM KCN plus 500 mM SHAM to inhibit mitochondrial respiration.
18 Place pollen tube suspension in a cuvette inside the fluorimeter with mild stirring at 60 rpm. IMPORTANT: Make sure the cuvette port is located at the center of the magnetic stirrer to ensure constant and laminar (non-turbulent) stirring.
19 Turn on and connect the mini fluorimeter either though Bluetooth1 or with a USB cable. 20 Open Logger Pro and select '500 nm fluorescence mode' under the sensor SpectroVis plus configuration icon.
21 Select the 530 nm acquisition box and 'Absorbance versus time' under the spectrophotometer configuration icon. IMPORTANT: Make sure data collection is set to 1 sample/second and select the appropriate acquisition time intervals for each experiment. A value of 2000s is a good start.
22 Start recording baseline fluorescence for 500 s.
23 Add desired amount of calcium (a pulse around 50 mM is usually a good starting point).
24 Measure calcium transport for at least 2000s and save data. 25 Start a new trace and measure calcium transport in the presence of freshly made 100 nM ruthenium red (RuR) as negative control. Important: A 100 mM RuR stock solution must be freshly made.
26 Perform required biological replicates and quantify calcium transport rates (fold changes) in arbitrary units. This can be easily done under Vernier's Logger Pro 1 or Excel 1 .

Method validation
In order to test the method, pollen grains were isolated from mature flowers (Fig. 1A) hydrated in PG buffer and incubated for 2 h. As expected, pollen tubes emerged and were counted in the Neubauer chamber (Fig. 1B). Cell number was determined and adjusted either for oxygen consumption experiments or calcium transport assessment. Samples were then washed twice in PTA buffer in order to remove excess calcium. Pollen tubes were then permeabilized with 0.01% digitonin or 0.5% DMSO (control) for 5 min and stained with 0.1% Evans Blue solution. As expected, permeabilized pollen tubes were stained with Evans Blue (Fig. 1C). In parallel, a separate batch of pollen tubes were treated with Fig. 1. Pollen tube germination (A,B) and plasmalemma permeabilization assessment (C). Pollen tubes were allowed to grow for 2 h, the cells were counted and permeabilized with 0.01% digitonin or 0.5% DMSO. Plasma membrane permeabilization was then assessed by staining with 0.1% Evans Blue. Representative experiments n = 4. 0.01% digitonin for five minutes inside the oxygraphs chamber and ADP was added to obtain state 3 respiration (Fig. 2). Oligomycin addition resulted in a transient decrease in respiration typically associated with ADP phosphorylation blockade ( Fig. 2A). Furthermore, addition of 100 mM KCN significantly decreased state 3 respiration whereas subsequent addition of 500 mM SHAM resulted in no significant changes when compared with respiration in the presence of cyanide (Fig. 2B).
Once mitochondrial coupling was determined, mitochondrial calcium uptake was assessed by placing pollen tubes in a mini fluorimeter with constant stirring in the presence of 10 mM succinate and 2mM calcium green. A single pulse of 100mM CaCl 2 was then added and fluorescence spiked to a maximum value followed by a constant fluorescence decrease indicating calcium transport (Fig. 2C). Under the same conditions, preincubation with 100 nM RuR significantly inhibited such fluorescence Fig. 2. In situ monitoring of oligomycin-sensitive respiration (A), CN-and SHAM-sensitive respiration (B) and RuR-sensitive calcium transport activity in permeabilized pollen tubes (C). Permeabilized pollen tubes were resuspended inside the oxygraph's reaction chamber and state 3 respiration was measured. State 4 respiration was then assessed by adding 2 mg oligomycin (A). Respiration in the absence or presence of cytochrome oxidase inhibitor (cyanide) or alternative oxidase inhibitor (SHAM) was then assessed (B). Data are presented as mean + S.E.M. Statistical evaluation between groups was performed by unpaired t-tests and a P value < 0.05 was considered as criteria of statistical significance against control conditions, as denoted with an asterisk. In (C) calcium transport was assessed either in the absence (control) or presence of 100 nM freshly made RuR. Representative experiments n = 4. decrease, indicating calcium transport blockade. These results indicate the in-situ method is also suitable for assessing mitochondrial respiration and calcium transport in permeabilized pollen tubes.