HARVEST METHOD:

The methods for setting up each point frame plot are described below in both the section "POINT FRAME PIN-DROP METHODS" The methods here describe how each shoot was harvested from the plots which were used for the point frame method.

Six shoots were harvested from each of 19 1m x 1m point frame plots which were dominated either by Salix pulchra or Betula nana tall, shrub species. The "shoots" described here are branch clippings between eight to ten inches long. Whenever possible they were selected for appearing relatively healthy, intact leaves, and each shoot was taken from a different plant. As these shoots were used for shoot-level and leaf-level measurements, shoots with bi-furcated stems -- or two stems from the same branch/height were cut -- one for leaf-level and the second for shoot-level analyses.

Before being cut, we measured the distance from the point frame to the highest tip on the shoot as well as the distance from the point frame to the shoot five inches from the tip. This way, we could approximate the angle of the shoot relative to the ground. Using the same criteria as for the pin-drop measurements, we measured the distance from the top of the shoot to the soil. In addition, the row number and pin-hole number nearest to the shoot's location with respect to the point frame was also recorded .

We then measured the leaf area index (LAI) of the shoot by holding an LAI-2000 (Li-Cor Inc., Lincoln, Nebraska, USA) in the exact location where the shoot had been and taking the average of three readings (one above, three below). For these measurements we used the one-quarter cut out and took care to always hold the instrument level and with the technician's body casting a uniform shadow over the instrument's eye, with the technician standing between the instrument and the sun. On occasion when it was raining during the shoot harvest, the area near where the shoot had been was marked with flagging tape, and the the LAI measurement was taken at a later date, using the height from frame/distance from ground meausrements as well as the row number and pin hole measurements as a guide.

Once cut, the shoot was placed immediately into water and transported to the lab. Once in the lab, the end of each shoot was clipped under water to ensure that there were no air bubbles in the stem that would inhibit the flow of water. Shoots were then allowed to sit at ambient room conditions (~20-25 degrees Celsius) until the the photosynthetic rates could be measured.

LIGHT RESPONSE CURVE MEASUREMENT:

The light response curve measurements were taken using a Licor 6400 photosynthesis system (Li-Cor Inc., Lincoln, Nebraska, USA) with an opaque conifer chamber with red-blue-green (RGB) light source attachment (model number 6400-22L). The light response curve measurements were taken after each shoot completed an A-Ci curve (see "2012_GS_ITEX_PF_ShootACiData" for details). The methods for setting up the LI-6400 are detailed in the ITEX Manual as updated in 2011.

Each shoot was oriented in the chamber so that the leaves were facing the RGB light source. Care was taken to ensure that no leaves were caught between the foam gaskets. Aditionally, adhesive, mounting tack was used to ensure a seal around the end of the stem that protruded from the chamber and remained in water for the duration of measurements.

The light curve measurements were conducted with the reference CO2 set to a constant 400 umol CO2. Each shoot was allowed to acclimate to the 400 umol CO2 and to the highest light level for the light curve for a few minutes, or until the Ca:Ci ([CO2]amb to [CO2]internal to the cell) ratio was stable around ~0.7 +/- 0.1. The light curve measurements were taken at the following levels in the order listed (units are umol photosynthetically active radiation (PAR) m-2 s-1): 2200, 1800, 1200, 800, 600, 600, 400, 300, 200, 100, 50, 20, 0. The shoot was given a minimum of 60 seconds but not more than 85 seconds to adjust to each new light level before a measurement was taken, and the instrument matched the reference and sample chambers prior to every measurement. The block temperature of the infra-red gas analyzer (IRGA) was adjusted throughout the light curve measurement to maintain a leaf temperature as near as possible to 20 degrees Celsius.

Once the measurment was complete, each shoot was carefully cut from the stem that remained outside the chamber so that only the portion of the shoot inside the chamber remained. This whole shoot was then photographed for later silhouette-area analysis with Image-J software [See "2012_GS_ITEX_PF_ShootHarvestData" for details]. The leaves, stipules, petioles, green and brown stem, and other plant green organs were then clipped from the shoot, lain flat on a hinged, plexiglass folder, and scanned. These images were subsequently processed with the Image-J software to calculate the area of each tissue type; the sum of the area of the leaves, petioles, and stipules were used to correct the default area used when taking the light curve measurements.

Each tissue type was separated, placed in a coin envelope, labeled, and dried at 60 degrees Celsius at least three days before being weighed on a four-point balance with glass enclosure. The mass of each sample can be found in the "2012_GS_ITEX_PF_ShootHarvestData" file.

CHN ANALYSIS:
Grinding: All leaf samples were dried in an oven at 60°C before grinding. Samples were small enough to grind the entire sample without subsampling. Leaves were ground using the Retsch MM 200 for 3 minutes or until a talcum powder consistency was achieved.

Weighing: After grinding, samples were stored in glass scintillation vials and dried again at 60°C for at least 36 hours. Once samples were removed, vials were tightly re-capped. When not in use, vials were stored in dessicators. 3.5-4.5 mg of each sample was weighed into a 10x12 mm tin capsule. A standard calibration curve was created using increasing amounts of aspartic acid (from about 0.2 mg to 5.0 mg). A chemical standard, acetanilide, and an organic sample, apple leaf, were run after the standard curve. Every ten samples, an aspartic acid check standard and a duplicate of an already-packed sample were run.

CN analysis: CN analysis was run between 10/4/2012 and 11/15/2012 by Rachel Rubin using the ThermoScientific 2000 at the Ecosystems Center, MBL, Woods Hole, MA. Duplicate sample values were averaged (mass, %N and %C) before inclusion into final results. The CHN data is available in the file "PF_CHN_Data".

CURVE MODELS:

Once corrected for the actual leaf area, the light curve for each shoot was modelled with the five photosynthetic models listed below. Although the formulas for each model are different, they were each performed using the Excel Solver function set to minimize the sum of squared difference between the measured photosynthetic rates and the modelled rates. The Solver function was used again to find the light level (umol PAR m-2 s-1) in each model equation that was closest to zero.

The parameters listed in the "PF_ShootLightCurve_Data" are those from the best-fit model (refer to the Best-fit Model (1-5) column).

Model 1: Rectangular Hyperbola (Cosby and Hornberger 1984)

Where:
P = rate of photosynthesis (umol CO2 m-2 ground s-1)
Jmax = light saturated rate of photosynthesis (umol CO2 m-2 ground s-1)
Phi (φ) = parameter for quantum yield (Eo) (umol CO2 umol-1 PAR)
I = irradiance (umol PAR m-2 ground s-1)
R = modeled respiration rate (umol CO2 m-2 ground s-1)

Model 2: Incorporating Elevated CO2 - Mechanistic Photosynthesis Model (McMurtie et al. 1992)

Where:
Theta (θ) = describes curvature of quantum response
P = rate of photosynthesis (umol CO2 m-2 ground s-1)
R = modeled respiration rate (umol CO2 m-2 ground s-1)
Jmax = light saturated rate of photosynthesis (umol CO2 m-2 ground s-1)
Phi (φ) = parameter for quantum yield (Eo) (umol CO2 m-2 ground s-1)
I = irradiance (umol PAR m-2 ground s-1)

Model 3: Leaf Nitrogen Distribution (Chen et al. 1993)

Where:
P = rate of photosynthesis (umol CO2 m-2 ground s-1)
Jmax = light saturated rate of photosynthesis (umol CO2 m-2 ground s-1)
Phi (φ) = parameter for quantum yield (Eo) (umol CO2 m-2 ground s-1)
I = irradiance (umol PAR m-2 ground s-1)
R = modeled respiration rate (umol CO2 m-2 ground s-1)
Gamma (γ) = describes curvature of quantum response

Model 4: Photoinhibition Model (Platt et al. 1980)

Where:
P = rate of photosynthesis (umol CO2 m-2 ground s-1)
Jmax = light saturated rate of photosynthesis (umol CO2 m-2 ground s-1)
Alpha (α) = parameter for quantum yield (Eo) (umol CO2 m-2 ground s-1)
I = irradiace (umol PAR m-2 ground s-1)
Beta (β) = photoinhibition parameter
R = modeled respiration rate (umol CO2 m-2 ground s-1)

Model 5: Hyperbolic Tangent Model (Cosby and Hornberger 1984)

Where:
P = rate of photosynthesis (umol CO2 m-2 ground s-1)
Jmax = light saturated rate of photosynthesis (umol CO2 m-2 ground s-1)
Alpha (α) - parameter for quantum yield (Eo) (umol CO2 m-2 ground s-1)
I = irradiance (umol PAR m-2 ground s-1)
R = modeled respiration rate (umol CO2 m-2 ground s-1)

POINT FRAME PIN-DROP METHODS:
We preferentially selected tall shrub canopies dominated either by Betula nana or Salix pulchra, that is canopies that were greater than 75 cm height. Care was taken to select fairly uniform canopies, that is avoiding the edge of a shrub stand or areas where the canopy had a large gaps, suggesting the area may have been disturbed.

We used point frames constructed from a 1.1 m x 1.1 m aluminum square with holes in each corner to accomodate steel rod posts used as the legs of the point frame. In this way, the frame could rest upon the four leg posts that had been hammered into the ground and remain adjustable in each corner. The frame had a level on each side, and great care was taken to ensure that the frame was (a) unable to be pushed deeper into the ground and, (b) level on all four sides prior to taking measurements. These factors were important to the measurement to have accurate data regarding the distance from the frame and the overall height of each point sampled in the canopy.

The aluminum frame had numbered, regularly spaced holes on two opposite sides in order to accomodate a metal bar that could be placed across the frame and locked into place. [These holes on the frame are the row numbers.] The bar that was placed across the frame similarly had numbered, evenly spaced holes in order to accomodate a pin--a long (100-200cm) metal rod with a diameter of ~3.175 mm. [The holes on this bar are the pin hole numbers.] Measurements were only ever taken from odd row numbers, and alternated even/odd pin hole numbers with each row; in this way, for every plot 25 evenly spaced locations were sampled covering an area of one square meter.

The length of the pin was marked every half-centimeter so that the distance could be read easily. Measurements were made by lowering the pin through a pin hole and, once encountering a leaf or stem, recording the following: row#, pin hole#, hit#, and the species hit. If the object hit was not a leaf, the plant tissue was noted; the diameter of each stem hit was estimated in millimeters, and the length of every graminoid blade hit was recorded from the point at which it was hit to the tip. As the primary species of interest for this project were for a select number of species (B. nana, S. pulchra, S. glauca, S. reticulata, V. uliginosum, V. vitis, L. palustre), species that were not the target of interest were classified as functional groups--e.g. graminoid spp., forb, moss.

The last pin-hit recorded for each pin hole was always at the "soil" which was considered to be the transition between the green and brown plant material, often in a mossy layer.

REFERENCES:
Chen J-L., Reynolds J.F., Harley P.C., Tenhunen J.D., 1993. Coordination theory of leaf nitrogen distribution in a canopy. Oecologia. 93: 63-9.

Cosby B.J. and Hornberger, G.M., 1984. Identification of photosynthesis-light models for aquatic systems. I. Theory and simulations. Ecol. Modelling. 23: 1 - 24.
ITEX Manual Updated in 2011
McMurtie R.E., Comins H.N., Kirschbaum M.U.F., and Wang Y-P., 1992. Modifying existing forest growth models to take account of effects of elevated CO2. Aust. J. Bot. 40: 657-77.
Platt, T, CL Gallegos, and WG Harrison. 1980. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res. 38:687-701.

Street L. E. Shaver G. R. Williams M. and van Wijk M. T. 2007. What is the relationship between changes in canopy leaf area and changes in
photosynthetic CO2 flux in arctic ecosystems? Journal of Ecology 95: 139-150.

van Wijk M. T. Williams M. and. Shaver G. R. 2005. Tight coupling between leaf area index and foliage N content in arctic plant communities. Oecologia 142: 421–427.