Metabolic adaptation of a photosynthetic angiosperm to extreme CO2 levels in Yellowstone: living on the edge

doi:10.21203/rs.3.rs-3235087/v1

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Metabolic adaptation of a photosynthetic angiosperm to extreme CO₂ levels in Yellowstone: living on the edge

https://doi.org/10.21203/rs.3.rs-3235087/v1

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How do plants adapt to elevated CO₂ levels? This much-studied question has returned ambiguous answers. CO₂ is the carbon nutrient for all photosynthesis, underlying global biomass productivity. Small increases in CO₂ levels may boost photosynthetic efficiency in the short term, but at higher levels it inhibits biomass production and increases susceptibility to stress. Whether photosynthesis can favorably adapt to extreme CO₂ over prolonged periods of time remains yet unknown. Here, we investigate changes in photosynthetic metabolism in a C₃ plant growing along a natural CO₂ gradient in Yellowstone NP over many decades. We find that plants adapted to grow near high CO₂ sources (~ 6000 ppm) require similarly high CO₂ to open Linear Electron Flow (LEF) for NADPH reoxidation. Cyclic Electron Flow (CEF) for ATP generation is upregulated and photogenerated ATP is shorter-lived in high CO₂-Adapted plants where it is more rapidly lost in the dark. We attribute the shift in regulation away from LEF to higher CEF to the low chloroplast ATP levels caused by the stressful growth conditions, as revealed by stunted biomass and leaf chlorosis.

Biological sciences/Plant sciences/Photosynthesis

Biological sciences/Plant sciences/Light responses

environmental adaptation

carbon dioxide

carbon fixation

carbon metabolism

carboxylation

cyclic electron flow

plant photosynthesis

Predicting long-term consequences of elevated CO₂ on photosynthesis remains challenging owing to multiple interconnected processes in photosynthetic metabolism. For example, when atmospheric CO₂ levels rise, some plants can increase their photosynthetic productivity by suppression of the photorespiration process that competes with carboxylation by the enzyme RuBisCO (Sharkey et al., 1988). However, long term exposure to elevated CO₂ is associated with other adaptations that lower stomatal opening and lower chlorophyll concentration (Dusenge et al., 2019; Griffin & Seemann, 1996; Sims et al., 1998). At higher levels, CO₂ can inhibit both photosynthesis and respiration by acidification that causes accelerated plant mortality (Maschler et al., 2022).

Plants sense and respond to CO₂ levels at internal sites within the chloroplast, mitochondrion, and the cytoplasm. In the chloroplast, dissolved CO₂ and its hydrolysis products (H₂CO₃, HCO₃⁻, CO₃²⁻) regulate the flux of electrons/protons at sites along the photosynthetic electron transport chain (PETC) (Ananyev et al., 2018), terminating at the carboxylation reaction catalyzed by the enzyme RuBisCO in the Calvin-Benson-Bassham (CBB) Cycle (“CO₂ fixation”). A comprehensive understanding of the PETC and CCB cycle has emerged that describes the reactions of solar energy conversion (ATP and NADPH formation) that power the carboxylation reaction (Fig. 1). Three main rate-limiting processes have been identified in relation to CO₂ carboxylation: First, the availability and relative generation rates of ATP and NADPH can become rate-limiting as they provide the required energy and reductant (Kramer & Evans, 2010). This is also referred to as the light-limited electron transport rate (Farquhar et al., 2001). Second, RuBisCO has a catalytic turnover rate that depends on the concentration of both CO₂ and the receiver molecule (ribulose bisphosphate, RuBP), as well as direct competition between CO₂ and O₂ for reduction by RuBisCO (photorespiration). Consequently, the carboxylation rate changes with elevated CO₂ level and O₂ evolution in light (Dusenge et al., 2020; Qian et al., 2021; Wu et al., 2023). Third, the regeneration rate of RuBP (Schreiber et al., 1995) from phosphorylated intermediates in the CBB cycle requires ATP and NADPH to produce the triose phosphate products, which can limit the rate of CO₂ assimilation (Suzuki et al., 2020). In theory, the highest efficiency of light and CO₂ utilizations can be achieved when these three processes are kinetically in balance, i.e. synchronized to avoid reverse reactions and build-up of intermediates that are targets for competing reactions (Kramer & Evans, 2011; Sweetlove & Ratcliffe, 2011). Long-term adaptation to elevated CO₂ can be expected to bring about changes in the enzymes of the PETC and CCB cycle that control the rates of regeneration of ATP, NADPH and/or RuBP. One can predict that carboxylation could either increase or decrease, depending on up- or downregulation of ATP, NADPH and RuBP regeneration versus RuBisCO activity or content, respectively. The prediction is not easy to make with confidence owing to both known complexity and unknown interactions.

Evolutionary CO₂ adaptation experiments have been conducted for species with relatively fast reproduction cycles, yet found no evidence for physiological or genomic changes by exposure to 200 to 800 ppm CO₂ in a C3 plant or green alga (Arabidopsis thaliana (Ward & Kelly, 2004); Chlamydomonas reinhardtii (Collins & Bell, 2004)). Long-term adaptation studies of three Japanese herbaceous species growing near natural sources of ~ 750ppm CO₂ were similarly inconclusive (Onoda et al., 2009), they found no changes in physiology or photosynthesis rates. Plants of a Mediterranean evergreen species exposed to ~ 2200 ppm CO₂ showed clear physiological changes in lower stomatal density and higher specific leaf area, but no significant differences in photosynthesis rates were detected (Jones et al., 1995).

Herein, we examine long-term adaptation of a native C3 wild plant (Prunella vulgaris) growing next to natural springs that release CO₂ to the atmosphere at two sites in Yellowstone National Park. The sites were selected owing to their long geological history: Mammoth Terraces date back to several hundred thousand years, the Gibbon Geyser Basin dates to 400,000 years ago (Bargar, 1978; Observatory, 2021). These areas have been generating travertine since the retreat of glaciers in the last ice age, circa 15,000 years ago (Bargar, 1978) and have been stable gaseous CO₂ sources according to chemical assays over the last two decades (Bergfeld et al., 2014; Hurwitz et al., 2020; Werner & Brantley, 2003). We used time-resolved chlorophyll fluorescence to study changes in photosynthetic electron transport in leaves over a range of fast (50 µsec) to slow (minutes) time-scales (using Fast Repetition Rate Fluorometry, FRRF). We monitored three kinetic Phases of the PETC reactions in leaves arising from bottlenecks where the supply of electrons temporarily exceeds forward transport by the downstream reactions (Fig. 1). These are Phase 1: water oxidation by Photosystem II (PSII) which fills the plastoquinone pool; Phase 2: electron transport from plastoquinol reoxidation to reduction of the Ferredoxin and NADP⁺ pools, and Phase 3: the terminal carboxylation reaction linked to removal of the triose sugar product in the CBB Cycle. We compare leaf size, pigment chlorosis, and Chl fluorescence response for plants growing in ~ 6,000 ppm near CO₂ vents to the same species growing nearby at ambient CO₂ (~ 400 ppm).

We show that following illumination and passing through Phases 1 and 2, the third bottleneck is overcome by supplementation of CO₂ and arises from the acceleration of the reoxidation of NADPH (accumulated in Phase 2) by the CBB cycle. This bottleneck is observed in both Control and Adapted plants. We observe two types of adaptation to long-term growth in high CO₂. The first is strong upregulation of CEF in the dark followed by ATP production in the light. We relate this upregulation to the faster decay of ATP or of the proton motive force (pmf) required for its production, by either faster leakage or faster utilization from the greater demand for cellular repair. The second is an increase in the CO₂ concentration required to overcome the flux bottleneck to carboxylation by inducing a switch from cyclic to linear electron flow (CEF ◊ LEF). Redirection of the PETC flux from CEF (under dark) to LEF (under illumination) in high CO₂ rebalances the ATP/NADPH production rates to achieve the necessary stoichiometries required for carboxylation in the CBB Cycle without wasteful accumulation of either.

Experimental design

A detailed description of our sampling method and the measurement technique is presented in Methods. We used the Fast Repetition Rate Fluorometry (FRRF) technique which differs in important ways from the more common Pulse Amplitude Modulation (PAM) fluorometry, as described in the Supporting Information (SI.I). Specifically, it samples photochemical quenching and suppresses non-photochemical quenching by applying light saturating flashes (50μs) to drive photochemical turnover and to monitor chlorophyll variable fluorescence at variable frequency (100 Hz in this study). We collected 14 whole plants (Prunella vulgaris) at Terrace Spring (TP) in the Gibbon Geyser Basin and at Poison Spring (PS) at Mammoth Terrace. At both sites gas composition at the vent source is very high in CO₂ (96.1-99.8% normalized dry gas composition), water vapor (10.3-37.7%), and low levels in CH₄ (0.0002% and 0.002% TP and PS), He (0.003%) and H₂S (<0.01% TP, 0.03% PS) (Bergfeld et al., 2014; Hurwitz et al., 2020; Werner & Brantley, 2003). Plants were taken from field sites close to the source (~6000 ppm CO₂) and at about 10 meters distance (~400 ppm CO₂, denoted ambient). More details on sampling conditions are provided in Methods. For each specimen we measured leaf size, leaf pigmentation, leaf stomatal conductance and CO₂ concentration in the atmosphere surrounding the plants (Table 1).

Adaptation to high environmental CO₂ levels results in highly stunted growth and chlorosis

Leaves of Prunella v. plants growing near high CO₂ sources (~6000 ppm) are more than 50% smaller in size compared to control plants growing in ambient CO₂and show yellow-brown discoloration indicative of chlorosis which increases in severity at closer distance to source. Stressful growth conditions imposed by the extreme CO₂ sources are proportional to the environmental CO₂ level as suggested by observations at 3 distances (CO₂ levels) from the source (Table 1).

Exposure to high CO₂ accelerates regeneration of NADP⁺ by LEF, but does not affect electron flux from water oxidation into PSII

Three Phases of Q_A reduction/Q_A^- reoxidation are observed in the Fv/Fm kinetics obtained by the FRR method, denoted as Phases 1, 2 and 3 opening and closing (Figure 2a, all flashes and 2b, average of each flash train; 50 flashes/train). The amplitude of oscillations on the first train, associated with Period-4 cycling through the S-States in the Water Oxidation Complex (WOC), is unchanged in high compared to ambient CO₂ (Figure 2c, orange and black trace). This implies that the rate of delivery of electrons from water oxidation into Q_A via PSII (Phase 1 opening) does not change in elevated CO₂ , nor does the rate of filling of the PQ pool. Following damping of the Period-4 oscillations, Fv/Fm decreases from above 0.8 (PSII fully open) to below 0.1 (PSII closed) before the end of the first train (Figure 2c, summary statistics for all leaf samples are available in Extended Data). This represents the functional size of the PQ pool, by plastoquinone reduction to plastoquinol (PQH₂), as previously assigned in many plants and microalgae (Ananyev et al., 2017).

Phase 2, the rise in Fv/Fm followed by decrease to a local minimum, ends after about 1250 flashes (25 seconds) in both elevated and ambient CO₂. In this Phase, PSII first opens by Q_A^- reoxidation induced by downstream flux into the FDX^-/FDX pool, then partly closes as reduced Q_A^-accumulates in response to slowing of the downstream electron flux when the FDX^- and NADPH pools get reduced. The assignment of the Phase 2 minimum as the turning point for the onset of FDX^- reoxidation by FNR/NADP⁺ for use in the CCB cycle is consistent with what is seen by O₂ evolution (see SI.II) and the standard model of the PETC (Figure 1). Further evidence for assigning the Phase 2 flux comes from laboratory inhibitor studies targeting Cytb6/f (results not shown). The time required to reach the minimum of Phase 2 does not change systematically in high compared to ambient CO₂, hence the kinetic bottleneck controlling reduction of FDX^- and NADPH remains constant. While the Phase 2 minimum is slightly higher in this plant in high CO₂ (Figure 2a, 2b) it does not change systematically across all leaf samples (see Extended data).

A strong enhancement occurs in the rise of Fv/Fm in Phase 3 upon high CO₂ exposure, both in the time-scale (earlier and steeper rise) and the maximum Fv/Fm level reached. Regeneration of NADP+ in this phase is strongly accelerated in high CO₂. Fv/Fm eventually reaches 75% of the original dark-adapted value versus 62% in ambient CO₂ (Figure 2a). The time to reach maximum Fv/Fm in Phase 3 (Phase 3 opening) accelerates by a factor of at least 1.5 (from >150 s in 400 ppm to 100 s in 6000 ppm CO₂). The Fv/Fm variations between Q_A (oxidized) and Q_A^- (reduced) states in Phase 3, referred to as “comb width” represent the closing and reopening of PSIIs during the short flashes and following the 0.5 s dark interval. High CO₂ exposure accelerates Q_A^- oxidation at this time-scale by more than 2-fold compared to ambient CO₂ (0.08 versus 0.18, Figure 2d) caused by accelerating the downstream reactions in the CBB Cycle (Figure 2d). Results for Phase 3 are consistent for all leaf samples (Extended Data). A shallow negative slope (decreasing Fv/Fm) appears in high CO₂ at the end of Phase 3 that we designate Phase 3 closing (Figure 2a, 2b). We assign this decrease in Fv/Fm to the depletion of the carboxylation receiver molecule RuBP which is required stoichiometrically for carboxylation, also referred to as Triose Phosphate Utilization (TPU) limitation (Figure 1).

Plants adapted to growth in elevated CO₂ require high CO₂ to open LEF for NADPH reoxidation

Prunella v. plants adapted to growth in high CO₂ (~6000 ppm) reach appreciably lower Fv/Fm levels in Phase 3 when exposed to ambient CO₂(Figure 3b compared to 3a). They recover only 47% of the dark-adapted Fv/Fm level compared to 66% for Control plants growing in ambient CO₂. The lower Q_A^- reoxidation rate in Phase 3 reflects slower downstream carboxylation flux through the CBB Cycle. By contrast, upon exposure to 6000 ppm CO₂, Adapted plants recover carboxylation flux to the same level as the Control, reaching similar Q_A^- reoxidation rate (Fv/Fm values at the end of Phase 3). The Fv/Fm “comb width” during Phase 3 reduces by about 2-fold in both Control and Adapted plants when measured in high compared to ambient CO₂ (0.18 to 0.08 and 0.18 to 0.10 respectively, Figure 3d, 3f), reflecting greater and faster flux from PSII to the terminal carboxylation reaction at 0.5 s intervals. The time to open Phase 3 (slope of the Fv/Fm trace) also strongly accelerates in high CO₂, for both Adapted and Control plants (from >180 s to 100 s, and from > 180 s to 120 s, in 400 ppm vs 6000ppm CO₂, respectively). A shallow negative slope appears upon high CO₂ exposure, postulated as due to TPU limitation in both plants (Figure 3a, 3b).

Lastly, the flux of electrons and protons from water oxidation by PSII does not change between Adapted and Control plants, as reflected by the identical amplitude of Period-4 oscillations; nor does the functional size of the PQ-pool, as measured by the change in Fv/Fm level during the first flash train (Figure 3c and 3e).

Photogenerated ATP is shorter-lived in high CO₂-Adapted plants

During alternating light-dark conditions, 3 minutes flashing (180 trains of 50 flashes) followed by 4 minutes dark, PSII remains more open upon successive flash train sequences in the Control plants, both in ambient and high CO₂. Fv/Fm levels for Phases 1 and 2 are considerably higher in Sequences 2, 3 and 4 compared to Sequence 1, while maximum levels reached in Phase 3 are the same albeit with smaller comb width when exposed to high CO₂ (Figure 4a). This shows that PSII transiently remains more open in alternating light-dark conditions as ATP remains available for reoxidation of NADPH by carboxylation by the CBB cycle. By contrast, in the Adapted plants PSII remains much more closed in ambient CO₂, in all sequences. In high CO₂ PSII opens to nearly the same Fv/Fm level in Phase 3 as in the Control. However, in Phase 1 the PQ pool remains more closed (55% filled compared to 23% in the Control plant, Figure 4d) and the Phase 2 minimum associated with the FDX^-/NADPH pool bottleneck, is systematically lower in the Adapted plants than the Control (0.2-0.25 compared to 0.3-0.45, Figure 4a, 4b). FDX^-/NADPH cannot be reoxidized at the same rate in the Adapted plants as in the Control plants, indicating that ATP is lost during the intermediate 4 minute dark time and is no longer available for carboxylation in the CBB cycle. The kinetics of Phase 3 do accelerate in both Control and Adapted in high CO₂, as seen by the 50% reduction of the “comb width” compared to the first Sequence (Figure 3a, 3b). That the maximum level reached in Phase 3 does not change compared to the first Sequence confirms that the acceleration seen in the Control plants is a transient phenomenon associated with conservation of resources (ATP) during the intermediate dark time that does not occur in the Adapted plants. The decrease in first Fv/Fm value on successive flash train sequences, as seen in Figure 4, is completely reversible if sufficient dark time between sequences is allowed. The recovery rate of the initial Fv/Fm relative to the dark time between trains is approximately exponential and has a half-time of 7-8 min (see Figure SI.3). Note that the order of sequencing of measurements (air 1^st, CO₂ 2^nd, or vice versa) does not change the outcome provided sufficient time to remove excess CO₂ by streaming of air is used (Extended data, Figure E1, E2).

We collected physiological observations and studied kinetics of the PETC to examine three current hypotheses for long-term adaptation to high environmental CO₂ on photosynthetic metabolism: 1) Does higher CO₂ enhance the PET carboxylation rate by the RuBisCO enzyme? 2) Is RuBisCO content lowered or its affinity shifted to the higher growth CO₂ concentration? 3) Can plants adapt to indiscriminate transport of CO₂ across cell walls and membranes, ie., harm due to acidification and other harmful non-enzymatic carboxylation reactions?

Our results show that decades-long adaptation of a photosynthetic angiosperm to ten-fold elevated CO₂ in the atmosphere does not realize the predicted benefit of enhanced PET carboxylation efficiency over photorespiration (Hypothesis 1). Instead, the carboxylation efficiency is lowered due to insufficient ATP and NADPH availability for operation of the CBB cycle. Our fluorescence observations establish that plants adapt to growth in extreme CO₂ by shifting their CO₂ requirement for carboxylation to higher CO₂ concentrations equal to their growth environment (6000 ppm in this study). When measured in ambient CO₂, their PSIIs remain mostly closed and the carboxylation rate remains low (Fig. 3b, 4b, Extended Data Figure E3). This arises from a shift in regulation to CEF status and downregulating LEF, which results in a bottleneck in NADPH reoxidation by the CBB Cycle. However, when exposed to high CO₂, Adapted plants recover the same Fv/Fm kinetics as Control plants growing in ambient CO₂. They reach the same time to the onset of carboxylation, same maximum carboxylation flux (Fig. 3b, 4b) and identical RuBP consumption rate (Phase 3 negative slope, Extended Data Figure E3e). Hence adaptation does not occur by making less RuBisCO, or less RuBP or lowering its maximum turnover rate at saturation; all are excluded by our data (Hypothesis 2).

We conclude from our observations that a C3 plant adapts to growth in a high CO₂ natural environment by strongly upregulating CEF for ATP production. This presumably occurs to cope with the higher energy demand for homeostasis in the high CO₂ environment (Hypothesis 3). When stromal ATP levels are low, CEF has been shown to dominate over LEF flux levels in the PETC (Fischer et al., 2019). The result of upregulating CEF relative to LEF is more reduced FDX⁻ and NADPH pools, hence closing PSII, as seen in Adapted plants at ambient CO₂. Only once the ATP/NADPH ratio becomes sufficiently high, as occurs at the high CO₂ level by accelerated ATP production and NADPH reoxidation, CEF becomes downregulated to allow more light-induced generation of NADPH by LEF. In ambient CO₂, with inherently slow CBB Cycle kinetics resulting in low NADPH reoxidation, CEF remains strongly upregulated in the Adapted plants. The faster decay of photo-generated ATP observed in adapted plants in the light-adapted state (Hypothesis 3), demonstrates that they do not fully switch from dark (predominantly CEF) to light (higher LEF) states under illumination. Rather, they decay back more quickly in the dark than do Control plants. We conclude that the diminished yield and shorter lifetime of CEF-generated energy (pmf and ATP) and the higher CO₂ threshold for carboxylation in high-CO₂-adapted plants are evidence of strong CEF upregulation as the main adaptive strategy for growth in extreme CO₂.

Environmental conditions that induce the state of maximum demand for cellular energy do not necessarily mean that CEF can occur. Several mechanisms can be involved in pmf and ATP generation (Allen, 2003; Kramer & Evans, 2010). Energy generation by CEF requires a mechanism to properly balance the PQ/PQH₂ ratio, as both molecules are needed to operate the Q-cycle. Hence, LEF or some other mechanism is needed to regenerate sufficient oxidized PQ, especially under high light and reducing conditions (Nicholas Fisher et al., 2019). Several mechanisms can potentially contribute to regeneration of oxidized PQ: 1) LEF to either CO₂ or O₂ (Sunil et al., 2019), 2) Ferredoxin-mediated O₂ reduction by PSI, 3) PSII-mediated reverse electron flow (PSII-cyclic electron flow, (Ananyev et al., 2017), and 4) upregulation of the PSI/PSII ratio. Which mechanism occurs likely depends on the species and on balancing the other cellular needs under the stress condition, for example photoprotection, nutrient and water uptake and thermal management (Shikanai, 2014).

The observed CEF upregulation in high-CO₂ adapted plants is not successful in meeting the higher energy demand for homeostasis, and growth. It could not prevent severe stunted growth and chlorosis, even at the intermediate environmental CO₂ levels we examined (Table 1). The unregulated (physico-chemical) nature of CO₂ transport means that CO₂ poisoning will occur in all phototrophs to some degree and would not be restricted to C3 plants. This points at a low threshold below which elevated CO₂ might possibly be beneficial for photosynthetic metabolism. Enhancing biomass production by growth at elevated CO₂ as applied for instance in the greenhouse industry has been shown to be beneficial at levels of up to about 1000 ppm (Dieleman et al., 2005; Kaiser et al., 2015). However, long term adaptation has not been investigated. What the limits are for photosynthetic adaptive capacity to elevated CO₂ levels remains an open question for future investigation.

Acknowledgements:

Travel for this research was funded by a Waksman-Busch seed grant and the 4TU Federation (Plantenna). Instrumentation construction was supported by the US Department of Energy Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, grant DE-FG02-10ER16195.

Sampling sites and sample collection

We collected whole Prunella vulgaris plants from two sites in Yellowstone National Park between 13 and 18 July 2022, under supervision of the NPS (Permit#: YELL-2021-SCI-8098). Prunella vulgaris is a perennial low-growing, flowering C3 plant. It is native to north America and is found throughout the park in diverse environments and one of the few species that is found within one meter of elevated CO₂ sources. A second reason to choose Prunella v. for study is its stomatal regulation. Stomatal conductance values measured in the field varied between 0.5 to 1,0 mol.m^-2.s^-1, with no apparent variation between ambient conditions and close to CO₂ sources (Table 2). This ensures rapid and reversible gas exchange for our experiments. Soil and air temperatures varied between 22 and 26°C on the days of sampling. Sampling sites were well-drained (above the spring). Our sampling sites were sufficiently close (~10 m distance) to expect soil nutrient conditions are the same for Control and high CO₂-adapted samples. For each specimen we measured leaf size, pigmentation of the leaves, mean CO₂ concentration in the atmosphere surrounding the plants, ground temperature at sampling sites (all in the range 20-25⁰C) and leaf stomatal conductance (SC-1 leaf porometer; Meter Group) directly in the field and after transfer to the lab. In total 14 whole plants were collected from the field, 10 from TS and 4 from PS. Samples were wrapped in moist tissue and kept in plastic bags in a non-transparent box for transfer to the lab for fluorescence measurements. As no biological samples were allowed to be removed from the park under conditions of the NPS permit, all measurements were done on-site, directly on the day of the sampling.

Table 2 Sampling site conditions and leaf stomatal conductance

Sampling day and site	CO₂ levels (ppm) Air	*Stomatal conductance Prunella* leaves at growth site (mol.m².s^-1)**	*Stomatal conductance Prunella* leaves after transfer to lab (mol.m².s^-1)**
13 July 2022, TS. Air temperature: 23 C, RH: 30-32%
At source	> 6000	-	-
At 0.5m	> 6000	0.9 - 1.1	0.2 - 0.6
At 0.75 m	> 6000	0.9 - 1.0	-
At > 10 m	360 - 720	0.8 – 0.9	0.2 - 0.6
16 July 2022, PS. Air temperature: 25 C, RH: 48-50%
At source	> 6000	-	-
At 0.5m	> 6000	0.2 - 0.5	0.2 - 0.5
At > 7 m	500 - 600	0.5 - 0.6	0.4 - 0.5
18 July 2022, TS. Air temperature: 25.9 C, RH: 42%
At source	> 6000
At 0.5m	> 6000	0.8	0.4 - 0.8
At 0.75 m	1500-3000	0.7-0.8	-
At > 10 m	420 - 930	0.8	0.1 - 0.2

Sampling. Intact leaves were cut from collected Prunella vulgaris plants and transferred directly to the FIRe sample chamber. Fluorescence measurements were conducted at room temperature (in a temperature controlled room). Leaves were measured in humidified air, either at ambient CO₂ or in air with added CO₂to a level of 6000ppm. We collected fluorescence data from 24 Prunella v. plant leaves, 13 from Control plants growing in ambient, and 11 from Adapted plants growing at high CO₂. We had to discard data from 8 plant leaves due to weak fluorescence signals from some of the leaves and instrument problems due to several power cuts we experienced in the park.

Stomatal conductance. Previous studies on photosynthesis in elevated CO₂ environments reported effects on stomatal conductance, where stomata remained more closed in a high CO₂ environment in some of the species investigated (Ainsworth & Rogers, 2007; Ware et al., 2015) We measured conductance on all samples and did not observe this effect in Prunella vulgaris growing near CO₂ sources in Yellowstone. The stomatal conductance values measured in the field ranged from 0.5 (in a shady growth site) to 1.0 mol.m^-2.s^-1 (sunny growth site), for plants growing both close and far from the CO₂ sources (Table 2). Indeed, this is one of the reasons we chose Prunella over other plants that were growing both near and at distance from CO₂ sources and showed much lower stomatal conductance values (eg., panic grass, Dichanthelium lanuginosum).

FRRF measurement protocol and instrumentation

The Fast Repetition Rate Fluorometry (FRRF) technique was used here to enable monitoring of chlorophyll variable fluorescence yield for both fast and slow PETC kinetics, from 50 μsec (flash duration) to several minutes. The FRRF single turnover flashes (STFs) provide both the photochemical turnover and the measurement excitation. This flash duration and intensity produced visible period-4 oscillations of Fv which indicates light saturation on the time scale of water oxidation by PSII (Ananyev & Dismukes, 2005). The FRRF technique records Fo and Fm for every flash to compute variable fluorescence, ensuring that Fv is strictly related to photochemical charge separation (see SI.I for a more detailed explanation of fluorescence monitoring by FRR). We used the FRRF technique because it provides time-resolved observations of photochemical yield and is not sensitive to NPQ effects. The average light intensity over the flash + dark periods using our flash protocol (see below) is only 5 μMol/m²/s. There is no background actinic light used at all for any experiments described herein. This average light intensity produced an imperceptible yield of non-photochemical quenching (NPQ < 0.03) in the model plant Arabidopsis using standard PAM fluorometry method and actinic light intensities of 1500 μMol/m²/s (Ware et al., 2014). The FRRF method rejects background fluorescence changes that occur on slower time scales than the sampling window (50 μsec flash duration). For illustration, we show the Fo, Fm and Fv/Fm responses from a model algal sample that was exposed to continuous white light at 180 μMol/m²/s for 150 minutes to intentionally induce light adaptation. The data shows that a visible change in Fm that occurs on the 1 second time scale, arising from a non-photochemical process, does not affect Fv/Fm observations, as it is appears in both Fo and Fm that are both continuously monitored.

Instrumentation. We used a miniaturized Fluorescence Induction and Relaxation Systems instrument (Satlantic, Mini-FIRe) suitable for transport to and application in field environments (Gorbunov & Falkowski, 2021). The mini-FiRE fluorometer uses a 450nm LED for excitation, a non-reflective sample holder and detects fluorescence from Chl > 680nm. We used single turnover flashes of 50 μs duration to excite both PSI and PSII simultaneously, while monitoring Chl emission. The peak light intensity of a single flash is 2000 μMol/m2/s. The average light intensity received by the samples is 5 μMol/m2 /s using our flash protocol. The flash intensity and duration achieved > 90% saturation of Fm.

Chlorophyll variable fluorescence measurement protocol

We used two timescales for excitation as illustrated in Figure 7. This flash protocol enables us to monitor fluorescence changes in relation to kinetics of the PETC from 100 Hz time-scale, capturing Period-4 oscillations associated with S-States in the WOC (Ananyev & Dismukes, 2005) to 0.5 second (time between trains) and minutes time-scale, capturing the slower downstream reactions of the PETC. To investigate dark recovery of the PETC kinetics, we ran sequences of up to 6 fluorescence experiments, applying 180 trains of 50 flashes followed by a dark time of 4 to 15 minutes, followed by another 180 flash trains etc.

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Metabolic adaptation of a photosynthetic angiosperm to extreme CO₂ levels in Yellowstone: living on the edge

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