The importance of species-specific and temperature-sensitive parameterisation of A/Ci models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO2 assimilation to variable intercellular CO2 concentrations (A/Ci response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, Vcmax) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; JPAR). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO2 (gm) derived from tobacco that may be inapplicable across species. To study the significance of using such parameters for other species, here we measured the temperature responses of key in vitro Rubisco catalytic properties and gm in cotton (Gossypium hirsutum cv. Sicot 71) and derived Vcmax and J2000 (JPAR at 2000 µmol m-2 s-1 PAR) from cotton A/Ci curves incrementally measured at 15°C-40°C using cotton and other species-specific sets of input parameters with our new automated fitting R package 'OptiFitACi'. Notably, parameterisation by a set of tobacco parameters produced unrealistic J2000:Vcmax ratio of <1 at 25°C, two- to three-fold higher estimates of Vcmax above 15°C, up to 2.3-fold higher estimates of J2000 and more variable estimates of Vcmax and J2000, for our cotton data compared to model parameterisation with cotton-derived values. We determined that errors arise when using a gm,25 of 2.3 mol m-2 s-1 MPa-1 or less and Rubisco CO2-affinities in 21% O2 (KC 21%O2) at 25°C outside the range of 46-63 Pa to model A/Ci responses in cotton. We show how the A/Ci modelling capabilities of 'OptiFitACi' serves as a robust, user-friendly, and flexible extension of 'plantecophys' by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to reduce variability when modelling Vcmax and J2000.

Gas exchange analysers exhibit large measurement error driven by internal thermal gradients.

Portable gas exchange analysers provide critical data for understanding plant-atmosphere carbon and water fluxes, and for parameterising Earth system models that forecast climate change effects and feedbacks. We characterised temperature measurement errors in the Li-Cor LI-6400XT and LI-6800, and estimated downstream errors in derived quantities, including stomatal conductance (gsw ) and leaf intercellular CO2 concentration (Ci ). The LI-6400XT exhibited air temperature errors (differences between reported air temperature and air temperature measured near the leaf) up to 7.2°C, leaf temperature errors up to 5.3°C, and relative errors in gsw and Ci that increased as temperatures departed from ambient. This caused errors in leaf-to-air temperature relationships, assimilation-temperature curves and CO2 response curves. Temperature dependencies of maximum Rubisco carboxylation rate (Vcmax ) and maximum RuBP regeneration rate (Jmax ) showed errors of 12% and 35%, respectively. These errors are likely to be idiosyncratic and may differ among machines and environmental conditions. The LI-6800 exhibited much smaller errors. Earth system model predictions may be erroneous, as much of their parametrisation data were measured on the LI-6400XT system, depending on the methods used. We make recommendations for minimising errors and correcting data in the LI-6400XT. We also recommend transitioning to the LI-6800 for future data collection.

A derivation error that affects carbon balance models exists in the current implementation of the modified Arrhenius function.

Understanding biological temperature responses is crucial to predicting global carbon fluxes. The current approach to modelling temperature responses of photosynthetic capacity in large scale modelling efforts uses a modified Arrhenius equation. We rederived the modified Arrhenius equation from the source publication from 1942 and uncovered a missing term that was dropped by 2002. We compare fitted temperature response parameters between the correct and incorrect derivation of the modified Arrhenius equation. We find that most parameters are minimally affected, though activation energy is impacted quite substantially. We then scaled the impact of these small errors to whole plant carbon balance and found that the impact of the rederivation of the modified Arrhenius equation on modelled daily carbon gain causes a meaningful deviation of c. 18% day-1 . This suggests that the error in the derivation of the modified Arrhenius equation has impacted the accuracy of predictions of carbon fluxes at larger scales since > 40% of Earth System Models contain the erroneous derivation. We recommend that the derivation error be corrected in modelling efforts moving forward.

Warming induces divergent stomatal dynamics in co-occurring boreal trees.

Climate warming will alter photosynthesis and respiration not only via direct temperature effects on leaf biochemistry but also by increasing atmospheric dryness, thereby reducing stomatal conductance and suppressing photosynthesis. Our knowledge on how climate warming affects these processes is mainly derived from seedlings grown under highly controlled conditions. However, little is known regarding temperature responses of trees growing under field settings. We exposed mature tamarack and black spruce trees growing in a peatland ecosystem to whole-ecosystem warming of up to +9°C above ambient air temperatures in an ongoing long-term experiment (SPRUCE: Spruce and Peatland Responses Under Changing Environments). Here, we report the responses of leaf gas exchange after the first two years of warming. We show that the two species exhibit divergent stomatal responses to warming and vapor pressure deficit. Warming of up to 9°C increased leaf N in both spruce and tamarack. However, higher leaf N in the warmer plots translate into higher photosynthesis in tamarack but not in spruce, with photosynthesis being more constrained by stomatal limitations in spruce than in tamarack under warm conditions. Surprisingly, dark respiration did not acclimate to warming in spruce, and thermal acclimation of respiration was only seen in tamarack once changes in leaf N were considered. Our results highlight how warming can lead to differing stomatal responses to warming in co-occurring species, with consequent effects on both vegetation carbon and water dynamics.

Using multirate rapid A/Ci curves as a tool to explore new questions in the photosynthetic physiology of plants.

Steady-state photosynthetic CO2 responses (A/Ci curves) are used to assess environmental responses of photosynthetic traits and to predict future vegetative carbon uptake through modeling. The recent development of rapid A/Ci curves (RACiRs) permits faster assessment of these traits by continuously changing [CO2 ] around the leaf, and may reveal additional photosynthetic properties beyond what is practical or possible with steady-state methods. Gas exchange necessarily incorporates photosynthesis and (photo)respiration. Each process was expected to respond on different timescales due to differences in metabolite compartmentation, biochemistry and diffusive pathways. We hypothesized that metabolic lags in photorespiration relative to photosynthesis/respiration and CO2 diffusional limitations can be detected by varying the rate of change in [CO2 ] during RACiR assays. We tested these hypotheses through modeling and experiments at ambient and 2% oxygen. Our data show that photorespiratory delays cause offsets in predicted CO2 compensation points that are dependent on the rate of change in [CO2 ]. Diffusional limitations may reduce the rate of change in chloroplastic [CO2 ], causing a reduction in apparent RACiR slopes under high CO2 ramp rates. Multirate RACiRs may prove useful in assessing diffusional limitations to gas exchange and photorespiratory rates.

Using the rapid A-Ci response (RACiR) in the Li-Cor 6400 to measure developmental gradients of photosynthetic capacity in poplar.

The rapid A-Ci response (RACiR) technique alleviates limitations of measuring photosynthetic capacity by reducing the time needed to determine the maximum carboxylation rate (Vcmax ) and electron transport rate (Jmax ) in leaves. Photosynthetic capacity and its relationships with leaf development are important for understanding ecological and agricultural productivity; however, our current understanding is incomplete. Here, we show that RACiR can be used in previous generation gas exchange systems (i.e., the LI-6400) and apply this method to rapidly investigate developmental gradients of photosynthetic capacity in poplar. We compared RACiR-determined Vcmax and Jmax as well as respiration and stomatal conductance (gs ) across four stages of leaf expansion in Populus deltoides and the poplar hybrid 717-1B4 (Populus tremula × Populus alba). These physiological data were paired with leaf traits including nitrogen concentration, chlorophyll concentrations, and specific leaf area. Several traits displayed developmental trends that differed between the poplar species, demonstrating the utility of RACiR approaches to rapidly generate accurate measures of photosynthetic capacity. By using both new and old machines, we have shown how more investigators will be able to incorporate measurements of important photosynthetic traits in future studies and further our understanding of relationships between development and leaf-level physiology.

Modelled net carbon gain responses to climate change in boreal trees: Impacts of photosynthetic parameter selection and acclimation.

Boreal forests are crucial in regulating global vegetation-atmosphere feedbacks, but the impact of climate change on boreal tree carbon fluxes is still unclear. Given the sensitivity of global vegetation models to photosynthetic and respiration parameters, we determined how predictions of net carbon gain (C-gain) respond to variation in these parameters using a stand-level model (MAESTRA). We also modelled how thermal acclimation of photosynthetic and respiratory temperature sensitivity alters predicted net C-gain responses to climate change. We modelled net C-gain of seven common boreal tree species under eight climate scenarios across a latitudinal gradient to capture a range of seasonal temperature conditions. Physiological parameter values were taken from the literature together with different approaches for thermally acclimating photosynthesis and respiration. At high latitudes, net C-gain was stimulated up to 400% by elevated temperatures and CO2 in the autumn but suppressed at the lowest latitudes during midsummer under climate scenarios that included warming. Modelled net C-gain was more sensitive to photosynthetic capacity parameters (Vcmax , Jmax , Arrhenius temperature response parameters, and the ratio of Jmax to Vcmax ) than stomatal conductance or respiration parameters. The effect of photosynthetic thermal acclimation depended on the temperatures where it was applied: acclimation reduced net C-gain by 10%-15% within the temperature range where the equations were derived but decreased net C-gain by 175% at temperatures outside this range. Thermal acclimation of respiration had small, but positive, impacts on net C-gain. We show that model simulations are highly sensitive to variation in photosynthetic parameters and highlight the need to better understand the mechanisms and drivers underlying this variability (e.g., whether variability is environmentally and/or biologically driven) for further model improvement.

Improving models of photosynthetic thermal acclimation: Which parameters are most important and how many should be modified?

Photosynthetic temperature acclimation could strongly affect coupled vegetation-atmosphere feedbacks in the global carbon cycle, especially as the climate warms. Thermal acclimation of photosynthesis can be modelled as changes in the parameters describing the direct effect of temperature on photosynthetic capacity (i.e., activation energy, Ea ; deactivation energy, Hd ; entropy parameter, ΔS) or the basal value of photosynthetic capacity (i.e., photosynthetic capacity measured at 25°C). However, the impact of acclimating these parameters (individually or in combination) on vegetative carbon gain is relatively unexplored. Here we compare the ability of 66 photosynthetic temperature acclimation scenarios to improve the ability of a spatially explicit canopy carbon flux model, MAESTRA, to predict eddy covariance data from a loblolly pine forest. We show that: (1) incorporating seasonal temperature acclimation of basal photosynthetic capacity improves the model's ability to capture seasonal changes in carbon fluxes and outperforms acclimation of other single factors (i.e., Ea or ΔS alone); (2) multifactor scenarios of photosynthetic temperature acclimation provide minimal (if any) improvement in model performance over single factor acclimation scenarios; (3) acclimation of Ea should be restricted to the temperature ranges of the data from which the equations are derived; and (4) model performance is strongly affected by the Hd parameter. We suggest that a renewed effort be made into understanding whether basal photosynthetic capacity, Ea , Hd and ΔS co-acclimate across broad temperature ranges to determine whether and how multifactor thermal acclimation of photosynthesis occurs.

Autumn photosynthetic decline and growth cessation in seedlings of white spruce are decoupled under warming and photoperiod manipulations.

Climate warming is expected to increase the seasonal duration of photosynthetic carbon fixation and tree growth in high-latitude forests. However, photoperiod, a crucial cue for seasonality, will remain constant, which may constrain tree responses to warming. We investigated the effects of temperature and photoperiod on weekly changes in photosynthetic capacity, leaf biochemistry and growth in seedlings of a boreal evergreen conifer, white spruce [Picea glauca (Moench) Voss]. Warming delayed autumn declines in photosynthetic capacity, extending the period when seedlings had high carbon uptake. While photoperiod was correlated with photosynthetic capacity, short photoperiods did not constrain the maintenance of high photosynthetic capacity under warming. Rubisco concentration dynamics were affected by temperature but not photoperiod, while leaf pigment concentrations were unaffected by treatments. Respiration rates at 25 °C were stimulated by photoperiod, although respiration at the growth temperatures was increased in warming treatments. Seedling growth was stimulated by increased photoperiod and suppressed by warming. We demonstrate that temperature is a stronger control on the seasonal timing of photosynthetic down-regulation than is photoperiod. Thus, while warming can stimulate carbon uptake in boreal conifers, the extra carbon may be directed towards respiration rather than biomass, potentially limiting carbon sequestration under climate change.

The rapid A-Ci response: photosynthesis in the phenomic era.

Phenotyping for photosynthetic gas exchange parameters is limiting our ability to select plants for enhanced photosynthetic carbon gain and to assess plant function in current and future natural environments. This is due, in part, to the time required to generate estimates of the maximum rate of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) carboxylation (Vc,max ) and the maximal rate of electron transport (Jmax ) from the response of photosynthesis (A) to the CO2 concentration inside leaf air spaces (Ci ). To relieve this bottleneck, we developed a method for rapid photosynthetic carbon assimilation CO2 responses [rapid A-Ci response (RACiR)] utilizing non-steady-state measurements of gas exchange. Using high temporal resolution measurements under rapidly changing CO2 concentrations, we show that RACiR techniques can obtain measures of Vc,max and Jmax in ~5 min, and possibly even faster. This is a small fraction of the time required for even the most advanced gas exchange instrumentation. The RACiR technique, owing to its increased throughput, will allow for more rapid screening of crops, mutants and populations of plants in natural environments, bringing gas exchange into the phenomic era.

Rapid desiccation hardening changes the cuticular hydrocarbon profile of Drosophila melanogaster.

The success of insects in terrestrial environments is due in large part to their ability to resist desiccation stress. Since the majority of water is lost across the cuticle, a relatively water-impermeable cuticle is a major component of insect desiccation resistance. Cuticular permeability is affected by the properties and mixing effects of component hydrocarbons, and changes in cuticular hydrocarbons can affect desiccation tolerance. A pre-exposure to a mild desiccation stress increases duration of desiccation survival in adult female Drosophila melanogaster, via a decrease in cuticular permeability. To test whether this acute response to desiccation stress is due to a change in cuticular hydrocarbons, we treated male and female D. melanogaster to a rapid desiccation hardening (RDH) treatment and used gas chromatography to examine the effects on cuticular hydrocarbon composition. RDH led to reduced proportions of unsaturated and methylated hydrocarbons compared to controls in females, but although RDH modified the cuticular hydrocarbon profile in males, there was no coordinated pattern. These data suggest that the phenomenon of RDH leading to reduced cuticular water loss occurs via an acute change in cuticular hydrocarbons that enhances desiccation tolerance in female, but not male, D. melanogaster.

Real-time measurement of metabolic rate during freezing and thawing of the wood frog, Rana sylvatica: implications for overwinter energy use.

Ectotherms overwintering in temperate ecosystems must survive low temperatures while conserving energy to fuel post-winter reproduction. Freeze-tolerant wood frogs, Rana sylvatica, have an active response to the initiation of ice formation that includes mobilising glucose from glycogen and circulating it around the body to act as a cryoprotectant. We used flow-through respirometry to measure CO(2) production ( ) in real time during cooling, freezing and thawing. CO(2) production increases sharply at three points during freeze-thaw: at +1°C during cooling prior to ice formation (total of 104±17 µl CO(2) frog(-1) event(-1)), at the initiation of freezing (565±85 µl CO(2) frog(-1) freezing event(-1)) and after the frog has thawed (564±75 µ l CO(2) frog(-1) freezing event(-1)). We interpret these increases in metabolic rate to represent the energetic costs of preparation for freezing, the response to freezing and the re-establishment of homeostasis and repair of damage after thawing, respectively. We assumed that frogs metabolise lipid when unfrozen and that carbohydrate fuels metabolism during cooling, freezing and thawing, and when frozen. We then used microclimate temperature data to predict overwinter energetics of wood frogs. Based on the freezing and melting points we measured, frogs in the field were predicted to experience as many as 23 freeze-thaw cycles in the winter of our microclimate recordings. Overwinter carbohydrate consumption appears to be driven by the frequency of freeze-thaw events, and changes in overwinter climate that affect the frequency of freeze-thaw will influence carbohydrate consumption, but changes that affect mean temperatures and the frequency of winter warm spells will modify lipid consumption.

Effects of cold acclimation on rectal macromorphology, ultrastructure, and cytoskeletal stability in Gryllus pennsylvanicus crickets.

Cold-acclimated insects maintain ion and water balance in the cold, potentially by reducing permeability or increasing diffusion distance across ionoregulatory epithelia such as the rectum. We explored whether cold acclimation induces structural modifications that minimize water and ion diffusion across the rectum and maintain rectal cell integrity. We investigated rectal structure and cytoskeletal stability in chill-susceptible adult Gryllus pennsylvanicus crickets acclimated for one week to either warm (25 °C) or cold (12 °C) conditions. After acclimation, we used light and transmission electron microscopy to examine rectal macromorphology and rectal pad paracellular ultrastructure. We also used fluorescence microscopy and a filamentous-actin (F-actin) specific phalloidin stain to compare the polymerization state of the actin cytoskeleton for each of the acclimation groups before and after a cold shock (1 h at -4 °C). Cold acclimation did not alter rectal pad cell density, or the thickness of the rectal pads, muscle, or cuticle. The tortuosity and width of the rectal pad paracellular channels also did not differ between warm- and cold-acclimated crickets. Rectal pad cells had clear basal and apical regions with differing densities of F-actin. Cold shock reduced the density of F-actin in warm-acclimated crickets, whereas cold-acclimated crickets appeared to have unchanged (basal) or enhanced (apical) F-actin density after cold shock. This suggests that while cold acclimation does not modify rectal permeability through structural modifications to increase diffusion distance for water and ions, cold-acclimated crickets have a modified cytoskeleton that resists the depolymerising effects of cold shock.

How well do growing season dynamics of photosynthetic capacity correlate with leaf biochemistry and climate fluctuations?

Accurate values of photosynthetic capacity are needed in Earth System Models to predict gross primary productivity. Seasonal changes in photosynthetic capacity in these models are primarily driven by temperature, but recent work has suggested that photoperiod may be a better predictor of seasonal photosynthetic capacity. Using field-grown kudzu (Pueraria lobata (Willd.) Ohwi), a nitrogen-fixing vine species, we took weekly measurements of photosynthetic capacity, leaf nitrogen, and pigment and photosynthetic protein concentrations and correlated these with temperature, irradiance and photoperiod over the growing season. Photosynthetic capacity was more strongly correlated with photoperiod than with temperature or daily irradiance, while the growing season pattern in photosynthetic capacity was uncoupled from changes in leaf nitrogen, chlorophyll and Rubisco. Daily estimates of the maximum carboxylation rate of Rubisco (Vcmax) based on either photoperiod or temperature were correlated in a non-linear manner, but Vcmax estimates from both approaches that also accounted for diurnal temperature fluctuations were similar, indicating that differences between these models depend on the relevant time step. We advocate for considering photoperiod, and not just temperature, when estimating photosynthetic capacity across the year, particularly as climate change alters temperatures but not photoperiod. We also caution that the use of leaf biochemical traits as proxies for estimating photosynthetic capacity may be unreliable when the underlying relationships between proxy leaf traits and photosynthetic capacity are established outside of a seasonal framework.

Warming delays autumn declines in photosynthetic capacity in a boreal conifer, Norway spruce (Picea abies).

Climate change, via warmer springs and autumns, may lengthen the carbon uptake period of boreal tree species, increasing the potential for carbon sequestration in boreal forests, which could help slow climate change. However, if other seasonal cues such as photoperiod dictate when photosynthetic capacity declines, warmer autumn temperatures may have little effect on when carbon uptake capacity decreases in these species. We investigated whether autumn warming would delay photosynthetic decline in Norway spruce (Picea abies (L.) H. Karst.) by growing seedlings under declining weekly photoperiods and weekly temperatures either at ambient temperature or a warming treatment 4 °C above ambient. Photosynthetic capacity was relatively constant in both treatments when weekly temperatures were >8 °C, but declined rapidly at lower temperatures, leading to a delay in the autumn decline in photosynthetic capacity in the warming treatment. The decline in photosynthetic capacity was not related to changes in leaf nitrogen or chlorophyll concentrations, but was correlated with a decrease in the apparent fraction of leaf nitrogen invested in Rubisco, implicating a shift in nitrogen allocation away from the Calvin cycle at low autumn growing temperatures. Our data suggest that as the climate warms, the period of net carbon uptake will be extended in the autumn for boreal forests dominated by Norway spruce, which could increase total carbon uptake in these forests.