May 14, 2018

Public workspaceMeasuring specific leaf area and water content V.1

DOI
dx.doi.org/10.17504/protocols.io.p3tdqnn
  • 1Université de Montréal
  • Plant Functional Ecology Lab
  • Canadian Airborne Biodiversity Observatory
Etienne Laliberté
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DOI: dx.doi.org/10.17504/protocols.io.p3tdqnn
External link: www.caboscience.org
Protocol Citation: Etienne Laliberté 2018. Measuring specific leaf area and water content. protocols.io https://dx.doi.org/10.17504/protocols.io.p3tdqnn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol.
Created: May 11, 2018
Last Modified: April 21, 2023
Protocol Integer ID: 12115
Abstract
Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf water content and specific leaf area, using the WinFOLIA™ software (Régent Instruments). These leaf area and water measurements are done on a subset of leaves from the same bulk leaf sample used to measure leaf spectral reflectance and transmittance. Briefly, after removing their petioles, fresh leaves are weighed, rehydrated for 6 h, scanned for total leaf area and weighed again; they are then oven-dried at 65 ºC for 72 h, and weighed one last time. This allows us to measure leaf dry matter content and its complement, leaf water content, as well as leaf relative water content. Leaf area measurements are used to estimate specific leaf area, a key functional trait central to the leaf economics spectrum. Specific leaf area allows us to estimate equivalent water thickness and to convert concentrations of foliar biochemical constitutents from a leaf mass to a leaf area basis.
Guidelines
Equipment
  • Sartorius Secura 213-1S 1 mg (3 decimal places) balance or other similar balance
  • Sartorius Secura 1102-1S 0.01 g (2 decimal places) balance or other similar balance
  • Canon LiDE 220 portable scanner or other suitable flatbed scanner
  • WinFOLIA™ leaf area software (Régent Instruments Inc.)
  • Forced air drying oven
  • Dessicator
  • RezChecker color/resolution target (optional)
Consumables
  • Paper towels
  • Paper envelopes and/or bags
  • Stapler and staples (for paper bags)
  • Sealed plastic bags
  • Weighing trays
Leaf Sample Selection And Preparation
Leaf Sample Selection And Preparation
Select a sub-sample of leaves from the same bulk fresh leaf sample on which spectral reflectance and transmittance was measured.
Note
The selected leaves do not have to be the same ones used for spectral measurements, but should be as similar as possible to those.
Note
Select enough leaves to entirely fill the scanner bed. Leaves larger than the scanner bed should be cut and scanned in multiple files.
Cut the petiole of each leaf that will be scanned.
Note
For a compound leaf, the petiole is the extension of the rachis beyond which there are no leaflets. The rachis should remain on the leaf, since it is the functional analogue of the midrib vein for a simple leaf.
Record Leaf Fresh Mass
Record Leaf Fresh Mass
Immediately weigh the selected leaves (with petioles removed) and record the leaf fresh mass (g).
Note
This fresh leaf mass should be done as close as possible to the spectral measurements; keep the fresh leaves in a sealed bag in which you have breathed into to prevent them from losing water.
Note
Use a 3-decimal place balance if possible. A 2-decimal place may be sufficient for very large leaves.
Leaf Rehydration
Leaf Rehydration
Store the selected leaves in a sealed plastic bag in which you have breathed into. Add a piece of damp paper towel (use deionised water).
Note
Label the sealed plastic bag with the sample ID and/or a barcode label.
Store the sealed sample in the dark, in the fridge (never a freezer) or a chilled cooler for 12 h.
Record Leaf Rehydrated Mass
Record Leaf Rehydrated Mass
Gently pat dry the rehydrated leaves to remove surface water.
Weigh the rehydrated leaves as a whole and record rehydrated leaf mass (g).
Note
Use a 3-decimal place balance. A 2-decimal place balance can be sufficient for very large leaves.
Create Working Folder
Create Working Folder
Go the the shared 'leafscans' CABO Google Drive Folder for your project.
Note
If you do not yet have a shared Google Drive folder for your project, contact the CABO data manager to create one (etienne.laliberte@umontreal.ca or jeremy.goimard@umontreal.ca).
Create a new folder named YYYY-MM-DD-SiteID (without spaces) within that 'leafscans' folder.


Note
The 'Site ID' should be the same as the Site ID defined in the field for that site.
Note
This working folder is where the data file and all acquired images for that site on that day will be stored.
WinFOLIA Set-Up
WinFOLIA Set-Up
Open WinFOLIA.


Choose the same settings as the last time WinFOLIA was run.


Select the scanner to use; it should start with WIA...


Note
The scanner drivers must be installed first. Choose the scanned starting with WIA-...
Ensure that the scanner calibration file is loaded.


Note
If the scanner calibration file is not loaded, refer to the WinFOLIA manual to load it.
Ensure that the Image > Acquisition Parameters... are set as shown in the image below.


Note
The parameters shown are for the Canon LiDE 220 portable scanner used in the field, and may differ on another scanner.
Note
Although the leaf area analysis is done on greyscale images, scan in color at 150 dpi.
For very small leaves, a higher resolution (e.g. 300 dpi) might be required.
Ensure that the Analysis > Parameters... are set as shown in the image below.


Ensure that the Analysis > Measurements... are set to Total Are Only.


Create New Data File
Create New Data File
Create a new data file using Data > New File...


Save the data file as YYYY-MM-DD-SiteID.txt in the 'leafscans' CABO shared Google Drive folder for your project.


Leaf Scan
Leaf Scan
Position the leaves to fill the scanner bed:
  • position leaf apex at the top of the image
  • leave margins of scanned bed free
  • ensure leaves are not folder
  • ensure leaves do not overlap each other
Add the RezChecker target in the top left section of the image (optional; if target is available).


Note
This target can be used to calibrate the color and/or resolution of the image later on.
Acquire a scan.


Enter the Sample ID, and name of operator.


Note
The 'Sample ID' should be exactly the same sample ID of the bulk fresh leaf sample, as defined in the field.
Click anywhere on the scanned image to start the analysis on the whole image, or select a particular region for analysis, and record the total leaf area (cm2).


Note
The total leaf area (cm2) for that image gets automatically saved in the data file.
Note
If the leaf sample requires multiple scans, repeat steps 15-17 as many times as required. In that case, add an identifier at the end of the Sample ID (e.g. 4663504-A, 4663504-B).
Save the analysed image(s) in the Google Drive working folder.


Name the image file using the sample ID.


Record Leaf Dry Mass
Record Leaf Dry Mass
Transfer the scanned leaves in a labelled paper envelope or small paper bag.
Note
Stape the envelope or paper bag to prevent leaves from falling out.
Dehydrate the leaves in forced air drying oven at 65 ºC for 72 h.
Ideally, cool the samples down to room temperature in a dessicator prior to weighing.
Note
If no dessicator is available, lower the drying oven temperature to room temperature but with forced air still on while samples are being weighed.
Weigh the dried leaves and record leaf dry mass (g).
Note
Use a 3 or 4-decimal-place balance, depending on sample size. A 2-decimal place balance might be sufficient for very large leaves.
Close Data File
Close Data File
Once all leaf samples for that site/day are scanned and analysed for area using WinFOLIA™, close the data file using Data > Close File.
Calculating Specific Leaf Area (SLA) and Leaf Mass per Area (LMA)
Calculating Specific Leaf Area (SLA) and Leaf Mass per Area (LMA)
Specific leaf area (SLA; m2 kg-1) is calculated as:
SLA = ( LA ÷ LDM ) ÷ 10
where
LA is the total area of the leaf sample (cm2)
LDM is the total leaf dry mass (g).
Leaf mass per area (LMA; g m-2), the inverse of SLA, is calculated as:
LMA = LDM ÷ ( LA ÷ 10 000)
Calculating Leaf Dry Matter Content (LDMC) and Leaf Water Content (LWC)
Calculating Leaf Dry Matter Content (LDMC) and Leaf Water Content (LWC)
Leaf dy matter content (LDMC; mg g-1) is calculated as:
LDMC = ( LDM × 1000 ) ÷ RLM
where
LDM is the total leaf dry mass (g)
RLM is the rehydrated leaf mass (g)
Leaf water content (LWC; mg g-1), the complement of LDMC, is calculated as:
LWC = 1000 - LDMC
Calculating Leaf Relative Water Content (RWC) and Equivalent Water Thickness (EWT)
Calculating Leaf Relative Water Content (RWC) and Equivalent Water Thickness (EWT)
The leaf relative water content (RWC; %) is expressed as:
RWC = [ ( LFM - LDM) ÷ ( RLM - LDM) ] × 100
where
LFM is the total leaf fresh mass (g),
LDM is the total leaf dry mass (g),
RLM is the rehydrated leaf mass (g).
Equivalent water thickness (EWT, g cm-2, or cm3 cm-2 = cm) is calculated as:
EWT = ( LFM - LDM ) ÷ LA
where
LA is the total leaf area (cm2).
Note
The relative water content expresses the actual water content of a given amount of leaf relative to the amount of water it contains in its fully hydrated state. It is one measure of leaf water stress at the time when spectral measurements were made.
Equivalent water content expresses the amount of water per leaf area, also at the time when spectral measurements were made.