Data set and R code supporting the New Phytologist manuscript "Influence of vapour pressure deficit and CO₂ on the thermal sensitivity of stomatal coupling in tropical trees"

Data set consists of biomass, leaf-level functional traits, and leaf-level gas exchange data from saplings of three species of tropical tree grown under controlled conditions and either ambient (420ppm) or elevated (820ppm) [CO₂].

R Code attached includes that used to analyze data in the manuscript and to explore the potential temperature sensitivity of the modelling term g1 in the Medlyn et al (2011) stomatal conductance model.

Study species and experimental material

We examined the behaviour of leaf-level gas exchange at elevated temperatures in three broadly distributed tropical tree species found within the Australian Wet Tropics: Mallotus philippensis (Lam.) Mull.Arg. (Euphorbiaceae), Ficus congesta Roxb. var. congesta (Moraceae) and Elaeocarpus grandis F.Muell. (Elaeocarpaceae). All plant material was obtained from a local nursery, and for each species originated from a single seed source in the lowlands. All three species show a broad geographical distribution and are typically found within the Australian Wet Tropics between sea level and 1100m.

Seedlings were potted into 145mm square pots (4L) containing a high organic matter potting mix with ¼ (by volume) of a local volcanic stone, Quincan (Northside Raw Materials & Timber Supplies, Cairns, Australia), to improve drainage. Before treatments, seedlings were established in a shade house (75% solar transmission) for 1 month. At the start of the experiment, six plants per species were placed into one of two glasshouse chambers (each 2.8 x 5 m) at James Cook University’s Environmental Research Complex (https://www.jcu.edu.au/environmental-research-complex). The chambers are conditioned by individual air handling units and controlled by a building management system able to provide independent control of temperature, CO₂ concentration, and humidity (Middleby et al., 2024).

Both chambers tracked external air temperatures, and had ultrasonic humidifiers set to turn on if VPD of the air (VPD_a) rose above 1.5 kPa. The two chambers differed in the target CO₂ concentration, set to be either ambient (400ppm) or elevated (i.e. 800 ppm). Each chamber was fitted with a combined temperature/ relative humidity probe (HMP60, Vaisala) and data logger (CR300, Campbell Scientific) to monitor conditions independently of the building management system. To avoid unintended bias between and within chambers, pots were periodically rotated within chambers and treatments rotated between chambers every ~2 months.

Gas Exchange Equipment

Leaf-level gas exchange measurements were conducted in a third climate-controlled chamber using one of three LI6400/XT portable photosynthesis systems (Li-Cor Inc., Lincoln, NE, USA) equipped with a 2 × 3 cm leaf cuvette with a red-blue LED light source (6400-02B, Li-Cor). Given the inability of standard LI6400/XT environmental controls to provide continuous and stable humidity and CO₂ control at the same time we pretreated inlet air to the LI6400XTs and bypassed the local sodalime and drierite scrubbers. Specifically, inlet air to the LI6400/XT machines was first scrubbed of CO₂ with an external soda lime cartridge (2L) and pump, and then passed through one of three dew point generators (LI-610, Li-Cor Inc) before being delivered at excess to T-junctions from which the LI6400/XTs could sample CO₂ free air of a pre-determined dew point. The CO₂ cylinder in each individual LI6400/XT was then used to deliver an inlet air stream with stable, user-defined CO₂ and H₂O concentrations.

The LI6400/XTs were networked to a computer external to the growth chamber running LI6400XTerm v3.4.2 whilst the dewpoint generators were controlled by independent 0-5V control signals supplied by an Arbitrary Waveform Generator (OWON, AG1022). To avoid issues of condensation, air lines delivering conditioned air to the LI6400/XTs were made of PTFE and the growth chamber temperature was generally set 2 °C above the Li6400XT cuvette set point. Nonetheless care was still needed to stabilize the temperature profile of all components and so they were shielded from direct solar radiation with reflective panels.

Temperature Ramping Protocol

On days of data collection, three well-watered plants grown under low VPD conditions and either near-ambient or elevated CO₂ were moved to the third chamber where CO₂ concentration was matched to growth conditions, and the Li6400xt cuvette affixed to leaves with a standard block temperature of 30 °C, PPFD of 1000 µmol m⁻² s⁻¹, and CO₂ concentration of incoming reference air at either 420 or 820 ppm depending on growth condition.

The dew point of incoming air was then modified to result in an initial VPD_L of between 1.5 and 1.8 kPa. After stabilization, initial gas-exchange data were collected (three rounds of logging every 20 seconds for 3min) and ramping begun. Ramping consisted of increasing the glasshouse and block temperature of the Li6400xts by intervals of 1°C and either a) raising associated inlet dew point to maintain a constant VPD_L (mean curve-average VPD_L of 1.6 kPa and maximum curve VPD_L of 2.2kPa) or b) maintain a constant dew point allowing VPD_L to increase which resulted in a VPD_L of 5 kPa at a leaf temperatures (T_L) ~ of 42.5°C. In both cases gas exchange was allowed to stabilize before the next incremental increase with data collected as per initial conditions every 2°C. Temperature ramping typically took 3.5 h and ran from a block temperature of 30 to 40 °C resulting in T_L ranging, on average, from between 32.4 to 42.5 °C.

After ramping experiments, plants were returned to their growth chambers and were not worked on again for at least 3 days. Response curves were collected on all three species in plants grown under both control and elevated CO_2, with a constant VPD. In the control CO₂ plants only, a second set of curves was also collected with the increasing VPD protocol. For each species, approximately six (five to seven) high-quality curves were obtained per treatment-scenario tested.

Leaf-level functional Traits

To provide species-level context for observed leaf-level gas exchange, at the conclusion of temperature ramping experiments leaf-level functional traits were determined on at least one leaf per replicate plant (i.e. minimum n = 6 per species, per growth CO₂). This included performing photosynthetic CO₂ response curves (A–Ci) at a cuvette temperature of 30 °C to obtain estimates of the maximum rates of RuBisCO carboxylation (V_cmax) and the rates of electron transport at 1000 µmol PPFD m⁻² s⁻¹ (J₁₀₀₀) (Buckley & Diaz-Espejo, 2015). The A-Ci curves were analysed using the ‘fitacis’ function in the plantecophys package in R (Duursma, 2015). To account for the control over block temperature instead of T_L  (which differed from block temperature by up to 2°C), the fitted parameters V_cmax and J₁₀₀₀ were normalised to the typically reported 25°C using temperature response parameters from Kelly (2014). Leaf mass per unit area (LMA) was determined by scanning leaves for area calculations, then drying at 70°C until constant mass.  

Minimal processing of gas-exchange data

After removal on non-sensical data (i.e. negative Ci) due to condensation in lines or a lack of stability data were analyzed using R Statistical Software, version 4.2.2 (R Core Team, 2022). Logged data points for each curve and set-point were used to determine Water Use Efficiency (WUE) calculated as A/E. The g₁ parameter was calculated using the inversion-approach set out by Potkay et al (2025). Given the controlled nature of the data collection protocol and stability of observed gas-exchange, this approach was found to be equivalent to g₁ determined at each curve’s individual setpoints via the typical regression method using the R plantecophys package (Duursma, 2015). All gas-exchnage data parameters (i.e. A, g_s, E, WUE, g₁) were then averaged for each leaf at each cuvette setpoint prior to data being archived.

References

Buckley TN, Diaz-Espejo A. 2015. Reporting estimates of maximum potential electron transport rate. New Phytologist 205(1): 14-17.

Duursma RA. 2015. Plantecophys - An R Package for Analysing and Modelling Leaf Gas Exchange Data. Plos One 10(11): 13.

Kelly J. 2014. Productivity and water use of Australian tree species under climate change. Macquarie University

Middleby KB, Cheesman AW, Cernusak LA. 2024. Impacts of elevated temperature and vapour pressure deficit on leaf gas exchange and plant growth across six tropical rainforest tree species. New Phytologist 243(2): 648-661.

Potkay A, Sloan B, Feng X. 2025. Stomatal Parameters in a Changing Environment. Plant, Cell & Environment 48: 2986-2997.

R Core Team 2022. R: A language and environmental for statistical computing. Vienna, Austria: R Foundation for Statistical Computing.