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Our primary measured response variables were leaf respiration measured in darkness (Rdark) and in the light (Rlight – made using the Kok method), estimated by CO2 release (using Licor 6400 gas exchange systems). To address Objective 3, we used the Licor 6400 to measure temperature responses of Rdark. In most cases, measurements were made on detached branches (sampled in the morning, with the xylem immediately re-cut under water). For leaves sampled at top and lower regions of the canopy, this required 2 hours canopy crane access per day [one hour in the morning (8-9 AM) and one in the afternoon (1-2 PM)]. Detached branches were transported to the laboratory where gas exchange rates would be measured. Immediately after measurements, leaf fresh mass and area were determined – thereafter, leaves were oven dried/weighed and then shipped to Canberra for chemical analysis. In addition to the above work on detached branches, we used an additional 2 hours of canopy crane access during the middle of the day (11 AM – 1 PM) to allow in situ measurements of light-saturated photosynthesis (both at ambient and elevated atmospheric CO2 concentration) and leaf respiration in darkness (after 30 mins of darkening to avoid post-illumination transients in respiratory CO2 release).

Measurements were made on species for which were sufficient replicates and for which removal of short branches containing several fully expanded leaves would not have an adverse effect on the growth/health of individual trees. Measurements were measured on 4 trees from each study species, using leaves sampled from two positions in the canopy (top and lower).

For two dominant species, we also determined whether rates of leaf respiration vary diurnally, via sampling branches at different time points in the day (e.g. 6 AM, NOON and 6 PM). The impact of cutting on leaf respiration and photosynthesis was accessed via first measuring rates of leaf R and photosynthesis on attached leaves, then cutting the branches and doing repeated measures of leaf respiration following cutting.

To address Objectives 1 and 2, leaf net CO2 exchange was measured at a set CO2 partial pressure and at several irradiance levels. Net CO2 exchange was first measured at saturating and growth irradiance, and then at several lower light levels (e.g. from 100 to 0?mol photons m-2 s-1); these measurements provided data on net assimilation (A) under ambient irradiance (Aamb) and light-saturation (Asat), as well as Rlight (calculated using the Kok method) and Rdark. Collectively, the data collected enabled us to calculate the % of A that is respired when assuming that R is, and is not, inhibited by light. Whilst cutting does not affect leaf respiration, we have found at other sites that sometimes (<20% of cases) stomatal conductance declines (affecting Asat) – thus, for upper canopy leaves, additional measurements of maximal photosynthesis (Amax, measured at elevated CO2 and under light-saturation) were made to allow assessment of the balance between respiratory and photosynthetic capacity.

We quantify the instantaneous temperature response of leaf respiration over a wide temperature range (using a programmable temperature controlled water bath to increase temperatures in a custom-made cuvette by 1 °C per min). Data were collected every 10 seconds over the 20-70 °C temperature range, enabling high precision temperature response curves to be generated. This will enable us to determine the extent to which the Q10 (i.e. proportional increase in respiration per 10 °C rise in temperature) of leaf respiration declined with increasing temperature.