4 Hydraulics
4.1 Leaf turgor loss point, \(\pi_{tlp}\)
We assessed the leaf turgor loss point, \(\pi_{tlp}\) in MPa, from a previously established relationship with the osmotic potential at full hydration, \(\pi_{osm}\) in MPa. \(\pi_{osm}\) is linked to the equilibrium solute concentration value \(C_0\) (in mmol.kg^{-1}) directly measured with a vapor pressure osmometer (Vapro 5600, Wescor, Logan, UT). This is referred as the osmometer method (Bartlett et al. 2012a; Maréchaux et al. 2016).
4.1.1 Materials
- Vapor pressure osmometer (Vapro 5520, Wescor, Logan, UT)
- Vapro software (Vapro Lab Report)
- Fridge
- Liquid Nitrogen
- Ziplock bag
- Paper towel
- Distilled water
- Metal tea ball
- Tin foil
- Needle
- Liquid nitrogen gloves + goggles
- Liquid nitrogen contenant
- 2 Tweezers
- Cork borer
4.1.2 Methods
4.1.2.1 Installing Vapro for measurements
- Turn on Vapro the day before for the thermocouple’s stability
- Test Water Quality cf Vapro_cheatsheet
- Clean
- Calibration cf Vapro_cheatsheet
- Control tests cf Vapro_cheatsheet
- Verify temperature
- Always have the black diamond at the center of the display
Used daily: * clean beforehand * select automatic mode (10 runs)
4.1.2.2 Sampling on the field
- Collect at least 3 healthy mature leaves on branch
- Place them in sample ziplock bag with:
- wet paper towel
- Exhale in bag to saturate in CO2
- Annotate bag with sample information
- Zip bag and stock in cooler
4.1.2.3 Lab measurements
4.1.2.3.1 Field day
- Recut branch under water
- Replace in ziplock bag with wet paper towel
- Put 24h in fridge to hydrate overnight
4.1.2.3.2 N+1 Field day
Vapro:
- check distilled water in vapro reservoir
- clean
- select automatic mode (10 runs)
- make sure vapro software is on
Sample measurement:
- Sample from a leaf a 5 mm disc with a cork borer: avoid 1st and 2nd order veins to avoid apoplastic dilution that would lead to less negative osmometer values
- Wrap disc in tin foil
- Immerse in liquid nitrogen for at least 2 min using metal tea ball
- Puncture 10-15 times with needle
- Place in vapro chamber
In total, disc are exposed to air for less than 40 seconds for all the steps.
Record value C0 when the difference between consecutive 2-min measurements fell below strictly 5 mmol.kg-1 after at least three runs.
If error! or Nr_Run > 10 :
try a 2nd cycle with same leaf
try a 3rd cycle with another leaf
otherwise record NA
Beware of the stuck leaf inside the vapro! If so cf Vapro_cheatsheet
4.1.2.3.3 End measurements
Clean Vapro
For more information on the vapro machine, please refer to the cf Vapro_cheatsheet.
4.3 Relative water content RWC (%)
4.3.1 Material
Ziplock bag Paper towel Distilled water Fridge Analytical balance Sharpie cooler envelop
4.3.2 Method
Before going on the field: * write individual code on ziplock bag * preweight ziplock bag
On the field: * collect leaf * clean leaf with clean paper towel * place leaf in preweighted corresponding ziplock bag * place bag in cooler for transport to the lab
In the lab: * weight the closed ziplock with the leaf (fresh weight) * delicately take the leaf out of the bag, wrap it in moist paper towel and place the wrap back in the ziplock bag * place ziplock bag in the fridge during 24 hours * 24 hours later, take out the leaf and wipe off water excess * weight re-hydrated leaf (saturated weight) * place leaf in envelop for oven-drying during at least 72h * weight dry leaf (dry weight)
Calculate RWC (%):
RWC = \(\frac{(fresh\ weight-dry\ weight)}{(saturated\ weight-dry\ weight)}\) X 100
From Sapes et al 2020 and Barrs & Weatherley 1962.