Faster than expected Rubisco deactivation in shade reduces cowpea photosynthetic potential in variable light conditions

Cowpea is the major source of vegetable protein for rural populations in sub-Saharan Africa and average yields are not keeping pace with population growth. Each day, crop leaves experience many shade events and the speed of photosynthetic adjustment to this dynamic environment strongly affects daily carbon gain. Rubisco activity is particularly important because it depends on the speed and extent of deactivation in shade and recovers slowly on return to sun. Here, direct biochemical measurements showed a much faster rate of Rubisco deactivation in cowpea than prior estimates inferred from dynamics of leaf gas exchange in other species1–3. Shade-induced deactivation was driven by decarbamylation, and half-times for both deactivation in shade and activation in saturating light were shorter than estimates from gas exchange (≤53% and 79%, respectively). Incorporating these half-times into a model of diurnal canopy photosynthesis predicted a 21% diurnal loss of productivity and suggests slowing Rubisco deactivation during shade is an unexploited opportunity for improving crop productivity. The crucial enzyme for photosynthesis, Rubisco, is deactivated during periods of shade and slowly recovers when illuminated. In fluctuating light conditions crop productivity could be substantially increased by slowing Rubisco deactivation during shade.

The assumed mechanism of Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) activation is that Rca removes tightly bound inhibitory sugar-phosphates from catalytic sites, allowing carbamylation; that is, reversible binding of CO 2 and Mg 2+ , and in turn carboxylation or oxygenation of ribulose-1,5-bisphosphate (RuBP) 11 . Establishing the potential impact of Rubisco activation on photosynthetic productivity requires modelling the response of Rubisco activity to realistic within-crop canopy light regimes 1-3 .
Shade is an obvious limit on photosynthesis in forest and understorey plants 12 . Within dense short-stature crop canopies like soybean, wheat and cowpea, most leaves also experience many transitions between sun and shade 3,13,14 . Throughout a day, light reaching chloroplasts steps-up or steps-down by 90% within a second 3 . In shade, biochemical adjustments improve the efficiency with which chloroplasts use absorbed light 9 but the light-dependent supply of RuBP is insufficient to saturate Rubisco catalytic sites, allowing decarbamylation and/or sugar-phosphate inhibition to decrease Rubisco activity [15][16][17] . Following shade-sun transitions, Rubisco activation is among the slowest responding of the biochemical processes that tune photosynthetic capacity to match incoming light 18,19 .
Shade-sun transitions are initially followed by RuBP regeneration driven, fast increases in photosynthesis, quickly superseded by prolonged, slower recovery driven by Rubisco activation 19 . Rates of increase in CO 2 assimilation during induction have therefore been used to infer rates of Rubisco activation 16,20 and have shown diversity that could be exploited to improve crop productivity 2,21,22 . By contrast, the rate of Rubisco deactivation following sun-shade transitions has never been characterized in a grain crop using both in vitro assays and gas exchange. A foundational study using both methods with spinach 16 found long deactivation half-times of >1,440 s; however, subsequent gas exchange measurements estimated only 606 s for the same species 23 . Furthermore, basil and impatiens showed faster Rubisco deactivation on the basis of in vitro biochemistry than gas exchange 17 . Parameterization of Rubisco deactivation therefore remains a key uncertainty in addressing impacts of Rubisco activation on crop productivity 2 .
The match between in vivo (leaf gas exchange) and in vitro (Rubisco activity) measurements, and the potential gain in diurnal photosynthesis achievable by adjusting the response of Rubisco activity to shade, were evaluated in cowpea. Activation state during sun-shade-sun transitions was measured using an optimized in vitro leaf-disc approach 24,25 . A uniform light regime was imposed with balanced spectrum LED lighting and temperature control  Fig. 1) and light responses of Rubisco activation state were obtained under steady-state and with temporal resolution down to 15 s during sun-shade-sun sequences. Results were used to update a diurnal model that combines a light regime for a legume canopy 14 ; half-times (τ) for the Rubisco activation state (S) response to step changes in light 16,26 ; and net CO 2 assimilation (A) based on steady-state light-response curves 1 . In parallel, the model was parameterized using gas exchange-based τ for the maximum rate of carboxylation by Rubisco (V c,max ). To indicate potential for impacts of breeding on Rubisco activity, two V. unguiculata breeding lines (IT86D-1010 and IT82E-16), a sexually compatible wild relative Vigna sp. Savi (TVNu-1948) and a more distantly related perennial V. adenantha (L.) were compared.
For all accessions, S saturated at a photosynthetic photon flux density (PPFD) of ~600 μmol m −2 s −1 (Supplementary Fig. 2). Sunshade-sun sequences were simulated using 850 μmol m −2 s −1 (sun) and 150 μmol m −2 s −1 (shade) (Fig. 1a). In shade, S decreased with a half-time (τ d,S ) of 42-134 s, depending on the accession (F 3,374 = 13.2, P = 3.2 × 10 -8 ; Table 1). Deactivation of Rubisco in Vigna sp. Savi and IT86D-1010 was so rapid that τ d,S was not statistically resolvable from 0; by contrast, τ d,S for V. adenantha and IT82E-16 was ~120 s (Table 1). Thus, τ d,S was as different within cowpea as between Vigna species. In shade, S decreased by 18-28% and accessions with high S in sun also showed higher S in shade. S was greater in V. adenantha and IT86D-1010 than in the other two accessions (F 3,374 = 14.9, P < 3.4 × 10 -9 ; Table 1), so there was no clear association between S and τ d,S . For Rubisco activation, the half-time of induction (τ a,S ) did not differ among the accessions (F 3,371 = 1.56, P = 0.2) and was 144 s ( Table 1). Estimates of τ a for other crops derived from gas exchange range from ~100 to 350 s (refs. 1,2,17,20,21,27 ) and decrease at higher assay temperatures as used here 27 .
The behaviour of S and V c,max differed. Unlike S, V c,max of the four accessions was similar in high light (coefficient contrasts P ≥ 0.64). While confidence intervals (CIs, 95%) did indicate

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significantly lower shade values in wild Vigna compared with IT82E-16, between-accessions patterns of difference between S and V c,max did not correspond (Table 1 and Fig. 1b). Such correspondence is not expected because, in addition to S, V c,max depends on Rubisco amount and catalytic properties. The apparently larger decrease in Rubisco activity in shade based on V c,max (48-60%; Table 1 and Fig. 1b) compared to S was linked with longer τ V than τ S . Similarly, the half-time for increasing V c,max (τ a,V ) was 26% longer than τ a,S (P ≤ 0.05 on the basis of 95% CIs; Table 1). Half-times for decreasing V c,max (τ d,V ) were calculated dependent on V c,max,H and V c,max,L (equation (7)) so CIs were not estimated for τ d,V but at 241-253 s they were 1.9-5.8 × τ d,S , depending on accession and were longer than the upper 95% CIs for τ d,S (Table 1). Because estimates of τ d,V assumed that after 20 min shade V c,max was within 1 μmol m −2 s −1 of the asymptote (V c,max,L ) (equations (6), (7)) and S stabilized faster than this, τ d,V overestimated τ d (Fig. 1a). Initial activity of Rubisco in shaded leaf discs stabilized at ~70% of the value at high light (Fig. 1c). Assays of total activity, following carbamylation of catalytic sites free of sugar-phosphates, showed no response to PPFD (Fig. 1d). Carbamylation relies on stromal pH, [CO 2 ] and [Mg 2+ ] and the availability of inhibitor-free Rubisco catalytic sites depends on [RuBP] and Rca activity 28 . In shade, A diminishes and stomata will open at low [CO 2 ], so CO 2 seems unlikely to be limiting. The relative importance of stromal pH and [Mg 2+ ] as companions to Rca activity controlling Rubisco carbamylation in shade remain to be established but in model plant species expressing varying amounts 20 and isoforms 25 of Rca, slowing deactivation and speeding induction by Rca-mediated maintenance of Rubisco activity shows promise as a strategy to enhance productivity. Important diurnal impacts of Rubisco activation previously reported for wheat 1 were based on in vivo estimates of Rubisco activity (τ d,V and τ a,V ). Here, Rubisco deactivation and activation half-times determined both in vivo and in vitro (τ d,S and τ a,S ), were used to model photosynthetic adjustment to diurnal light fluctuations within the second layer of a canopy (Fig. 2). Both in vivo and in vitro approaches predicted foregone assimilation linked with Rubisco activation (A f ) matching the 21% of diurnal photosynthetic potential (A Q ; Table 2 and Fig. 2c) predicted for wheat 1 . Significant differences in light-response characteristics between the four Vigna accessions (Table 1) had little impact on diurnal photosynthesis (A Q : coefficient of variation, 3.9%; Table 2) relative to the ~21% reduction linked with Rubisco regulation (A f ; Table 2). Noting that τ d,V represents an upper limit for reasons given above, and that τ V were longer than τ S , we used these values reciprocally to establish the potential impact of modifying τ d and τ a . Both slowing-down deactivation (τ d,V + τ a,S versus τ d,S + τ a,S ) and speeding-up activation (τ d,V + τ a,S versus τ d,V + τ a,V ) significantly decreased A f to 17% (on the basis of 95% CIs; Table 2). Similarly, slowing activation following shade (τ d,S + τ a,V versus τ d,S + τ a,S ) significantly increased A f to 24%. Therefore, small but significant differences in τ are sufficient to drive improvements in diurnal carbon gain.
New, high-frequency sampling during sun-shade transitions for biochemical analysis of Rubisco activation in cowpea, revealed far more rapid deactivation than previously appreciated on the basis of gas exchange measurements 2 . Modelling of these results augments predictions of 2-20% impacts of shade-induced changes in Rubisco activity on diurnal photosynthesis 2,3 . Prior estimates have relied on gas exchange in wheat 1 , where estimated τ d was slightly longer than τ a , consistent with measurements using S in spinach, basil and impatiens 16,17 . Longer deactivation times, important for exploitation of sunflecks, have also been reported in the tropical understorey species Alocasia macrorrhiza 15 . By contrast, the fast decline in S measured in cowpea suggests that shade-induced Rubisco limitation may have been underestimated for some crops. An answer to the question of why cowpea does not exhibit longer deactivation times may be that its wild ancestors exploited warm, dry climates 29 where shading was less important than in forest or contemporary cropping environments.
Using S to establish Rubisco activity in shade required a carefully constructed, laboratory-based set-up and more work is needed    (7)): not modelled using mixed effects.

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to understand the offset in τ a and evidence that (de-)carbamylation rather than RuBP/inhibitor-binding drove Rubisco activity under our assay conditions. Gas exchange-based methods therefore remain the best option for evaluating Rubisco regulation in, for example, breeders plots 2,3,10,18,21,22 . Here, the use of equation (6) with a constrained point-estimate of V c,max,L overestimated τ d,V . This will be improved by experiments that establish how V c,max responds to shade periods of different durations. Our finding that S in cowpea stabilized within 10 min of shade also suggests use of <20 min of shade, with the benefit that stomatal closure would be less and so less complicating to gas exchange assays. Significant variation in Rubisco deactivation half-times (τ d,S ) among Vigna accessions suggests that τ d would be amenable to selection for improvement in breeding programmes. Variation between two cowpea lines from the same geographical origin (IT86D-1010 and IT82E-16) also suggests that greater variation is probably available from more diverse germplasm. Induction is relatively easy to study using field portable gas exchange equipment, so has been a focus in recent studies highlighting Rubisco regulation in crop plants 2,3,10,21,22,27 ; however, measurements of S suggest that, at least in cowpea, the speed of response to shade differs more than speed of induction. Slowing Rubisco deactivation during shade is a new target for crop improvement, with potential to improve productivity in food crops like cowpea.   Mean ± 95% CI 433 ± 6.1 130 ± 5 23.9 ± 0.91 Perfect tracking of changes in PPFD by net CO 2 assimilation based on steady-state light-response curves (A Q ), is compared with models in which the rate of change in Rubisco activity during deactivation (τ d ) and activation (τ a ) in response to fluctuating PPFD are alternatively parameterized using one-point V c,max from leaf gas exchange (τ -,V ) or Rubisco activation state (S; τ -,S ). Foregone assimilation (A f ) is the difference between A Q and the respective alternative models. Sampling under changing irradiance for Rubisco activation. The leaf-disc method used is a variant of previously described light assays conducted in vitro 24,25 .

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The artificial sunlight simulation rig (light-rig; Supplementary Fig. 1) consisted of two high-intensity dimmable LED grow lights (Specialty Lighting Holland BV), jointly capable of supplying a PPFD of >1,200 µmol m -2 s -1 with a spectrum designed to closely match clear-sky solar irradiance. A steel frame allowed precise positioning of the lights and was enclosed on three sides using white reflective shielding to improve uniformity of lighting (MCPET M4, Furukawa Electric Europe). Light treatments were implemented using a SLESA-UE7 lighting controller incorporated into a custom control interface (Specialty Lighting Holland BV), programmed using the Easy Stand Alone 2 software (Nicolaudie Architectural Lighting). Leaf discs (0.55 cm 2 ) were excised from intact plants in the glasshouse and immediately placed with the abaxial surfaces in contact with 25 mM MES-NaOH pH 5.5, in 50 ml beakers filled to within 5 mm of the rim, maintaining the usual orientation with respect to irradiance. A circulating water bath containing a 37 × 25 cm 2 metal rack coated with non-reflective primer was used to hold the beakers in the light-rig. PPFD was measured at leaf-disc-level for each position within the rack to control uniformity of treatment levels and the water bath maintained the buffer at a constant temperature of 30 ± 0.1 °C (Omega Thermocouple Thermometer RDXL4SD, equipped with a type-K thermocouple; Omega). The rack had a 6 × 4 array to meet our randomized sampling design. Leaf discs were sampled for Rubisco assays by snap-freezing into liquid nitrogen after blotting on Whatman filter paper. Samples were stored at −80 °C until biochemical analysis. The sampling method by incubation of leaf discs at specific light and temperature conditions in the light-rig enabled accurate determination of Rubisco activity and activation state, representative of that found in intact leaves. Comparable results were obtained using leaf-disc samples collected from intact leaves and after incubation of leaf discs by floating in 25 mM MES-NaOH pH 5.5 or H 2 O for 60 min under the same light and temperature conditions in the glasshouse ( Supplementary Fig. 3). The incubation time was also tested, with 20, 40 and 60 min producing comparable results ( Supplementary Fig. 4). The source of CO 2 to the leaf discs during incubation in the light-rig is the ambient air in contact with the adaxial leaf surface. Ambient air was circulated using two fans positioned at the top of the partially enclosed light-rig. In addition to the comparison with intact leaves, comparable Rubisco activation states in leaf discs floated in 25 mM MES-NaOH pH 5.5 with and without 10 mM NaHCO 3 showed that leaf discs were not CO 2 limited ( Supplementary Fig. 5).
To establish the light response of Rubisco activation ( Supplementary Fig. 2), one leaf disc per plant from four to six replicates of every genotype, was illuminated for 40 min at PPFD of 0, 80, 160, 240, 320, 400, 500, 850 and 1,200 µmol m -2 s -1 . PPFD at the level of the leaf discs was measured before each assay (Q203 Quantum Radiometer with PFD filter, Irradian). Using the same system, time series were sampled to establish changes in Rubisco activation following sun-shade (deactivation) and shade-sun (activation) transitions. Each time series consisted of 32 discs collected from the youngest fully expanded trifoliate leaf on an individual plant. Treatments during time series consisted of high light for sun (850 µmol m -2 s -1 PPFD) for 40 min; low light for shade (150 µmol m -2 s -1 PPFD) for 20 min and a return to high light for sun ('postshade'). Leaf discs were first sampled 1 and 3 min before the transition to shade. Then, during both the shade and postshade periods, discs were sampled every 15 s for 2 min, then every 2 min until 20 min after the change in irradiance.

Rubisco activation state (S) measurements.
Leaf samples (0.55 cm 2 ) were ground in a mortar and pestle for up to 1 min in 250 µl of ice-cold buffer containing 50 mM Bicine-NaOH, pH 8.2, 20 mM MgCl 2 , 1 mM EDTA, 2 mM benzamidine, 5 mM ε-aminocaproic acid, 50 mM 2-mercaptoethanol, 10 mM DTT, 1 mM phenylmethylsulphonyl fluoride and 1% (v/v) protease inhibitors 30 . The leaf lysate was cleared by centrifugation (14,000g for 1 min) at 4 °C. The supernatant was collected into a new tube, quickly mixed by pipetting and immediately used to initiate the Rubisco reactions. Rubisco initial and total activities at 30 °C were measured by the incorporation of 14 CO 2 into 3-phosphoglycerate, following the carboxylation reaction by Rubisco 31 . Initial activities were obtained by adding 25 µl of supernatant to the assay mix containing 100 mM Bicine-NaOH, pH 8.2, 20 mM MgCl 2 , 10 mM [ 14 C]-NaHCO 3 (18.5 kBq µmol -1 ), 2 mM KH 2 PO 4 and 0.6 mM RuBP. Total activities were obtained by incubating 25 µl of supernatant in the assay mix for 3 min, in the absence of RuBP. A test using IT68D-1010 showed the 3 min of incubation in the total activity assay was sufficient to allow available Rubisco catalytic sites to be carbamylated, resulting in the same S as 5 min of incubation (3 min, 79.7 ± 1.7%; 5 min, 79.6 ± 1.8%; n = 5). Reactions containing activated Rubisco were initiated by the addition of 0.6 mM RuBP. Both initial and total reactions were quenched after 30 s with 100 µl of 20% formic acid. Reaction vials were dried at 100 °C, rehydrated with 400 µl of ultrapure H 2 O, then mixed with 3.6 ml of scintillation cocktail (Gold Star Quanta, Meridian). Radioactive content of acid-stable 14 C products was determined using a Liquid Scintillation Analyzer (Packard Tri-Carb, PerkinElmer). Rubisco activation state (S) is the ratio of initial to total Rubisco activity [32][33][34] .
Leaf gas exchange. Photosynthesis in terminal leaflets of recently expanded first or second trifoliate leaves ( Supplementary Fig. 6), consistent with material used for Rubisco activity assays, was characterized in the glasshouse using two portable gas exchange systems (LI-6800F Photosynthesis Systems LI-COR; with Bluestem v.1.  (20 min) to that used in Rubisco activity assays; and following return to the steady-state PPFD of 1,500 μmol m −2 s −1 . Control of cuvette conditions during sun-shade-sun assays was achieved using set-points for air temperature (30 °C), relative humidity (60-70%, fixed at steady-state value) and CO 2 supply (430 μmol mol −1 ).

One-point estimates of Rubisco maximum carboxylation rates (V c,max ).
The recovery of V c,max following shade was predicted point-by-point from gas exchange measurements of A and c i by rearranging the Farquhar et al. 35

equation:
where The parameters R d (respiration in the light) and g m (mesophyll conductance) were determined from steady-state A/c i curves fit to the [CO 2 ] assay data (measured from the same leaf and during the same diurnal period as induction measurements; Supplementary Fig. 7 and Supplementary Methods). For simplicity, g m was assumed constant during induction, on the basis of recent measurements that show limited changes in g m responding to similar sun-shade sequences that used 200 μmol m −2 s −1 as the shade irradiance in tobacco 36 . Parameters K C , K O (Rubisco Michaelis-Menten coefficients for CO 2 and O 2 , respectively) and Γ* (CO 2 compensation point in the absence of R d ) were predicted at the mean leaf temperature measured by the LI-6800F leaf thermocouple, using published equation sets for tobacco 37 . The concentration of O 2 (O) was assumed to be the current atmospheric level of 209.5 mmol mol −1 and gas concentrations were converted to partial pressures before fitting the model.

Statistical models of S and V c,max time series.
To obtain estimates of half-times for S and V c,max in response to changes in light, the piecewise model of activation state was where a, b and c are set to 1 in timesteps where the submodel is relevant: a, t ≤ −t L ; b, −t L < t ≤ 0; c, t > 0; and are otherwise set to 0. Time (t, s) is relative to the beginning of induction and t L is the duration of low light (shade). Transitions between the steady-state Rubisco activity in high light (S H ) and low light (S L ) follow exponential trajectories. The coefficient determining the rate of decline in S after a high-to low-light transition (deactivation) is the half-time τ d,S ; conversely, the half-time τ a,S determines the rate of increase in S following transition from low to high light (activation).

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The response of V c,max reflecting Rubisco activation during induction was modelled as Vc,max = Vc,max,H − (Vc,max,H − Vc,max,L) e −t τa,V (4) where V c,max,H and V c,max,L are high-light and low-light steady-state values, respectively; and t is time from the start of shade (s). The rate of increase in V c,max declines exponentially with half-time τ a,V . This model was fit to data collected between 1 and 5 min after shade, a period that followed the initial inflection in A associated with the end of the RuBP-regeneration phase and during which photosynthesis was determined to be consistently limited by V c,max (Supplementary Fig. 8).
To establish the half-time for decrease in V c,max on transfer to shade (τ d,V ), we used the equation for decreasing V c,max Further assuming that V c,max at the end of 20 min shade is ~V c,max,L + 1 (for context, 95% CI of V c,max,L are ±17-22 μmol m −2 s −1 ; Table 1), so that V c,max,L − V c,max = −1, simplifies to This is an upper limit on τ d,V , the value of which decreases as V c,max,L − V c,max → 0. Nonlinear-least-squares models were initially fit to individual replicates, providing starting models (S, Supplementary Table 2; V c,max , Supplementary Table 3) from which we aimed to identify significant differences at the level of accessions, the level relevant to crop improvement. Differences between the starting models were used to inform construction and simplification of nonlinear mixed-effects models (S, Supplementary Fig. 9; V c,max , Supplementary Fig. 10). Maximal models, that is complete parameterization at the level of individual replicates, with individuals treated as random effects, were progressively simplified. Using evidence from likelihood ratio testing, Wald tests and plots of residuals and model coefficients, fixed effects were introduced, their importance established and unnecessary fixed or random terms removed 38 .
Diurnal assimilation models. The diurnal impact of shade-responsive changes in Rubisco activity on potential A, was predicted on the basis of fitted net CO 2 assimilation-light-responses (A/PPFD) ( Table 1, Supplementary Fig. 11 and Supplementary Methods) and an irradiance regime relevant to chloroplasts in second-layer leaves of a legume crop (Fig. 2a): irradiance values had been derived at ~60 s intervals by reverse ray tracing, with shade-generating structures in the canopy distributed at random within each layer and assuming a clear-sky day in June at latitude 44° N (ref. 16 ).
When PPFD was increasing, Rubisco limited A (A R ) was modelled as 16 The rate of change in A R decreases exponentially over the duration of each timestep (t) in proportion to the Rubisco activation half-time (τ a ). The net CO 2 assimilation rate at the final PPFD (A F ) is approximated using the PPFD response where ϕ is an initial slope, Q is PPFD, A sat is the light-saturated rate and θ a curvature parameter. In each timestep, the initial net CO 2 assimilation rate (A I ) is the A R achieved at the end of the previous timestep (taken to be 0 at first light).
Assuming that [RuBP] is saturating, integrated, Rubisco activity-limited CO 2 assimilation ( ∫ t 0 A, annotated as A τ ) is Setting τ a = 0 integrates potential assimilation rate with instantaneous response to PPFD/quantum input (A Q = A F t). An estimate of foregone assimilation, A f , is A Q − A τ (refs. 16,26 ), which is expressed as a percentage of potential assimilation: When PPFD was decreasing, CO 2 assimilation was modelled as responding immediately to PPFD: A τ = A Q . However, to provide an appropriate A I on return to non-light-limiting conditions, we predicted A R as declining at a rate determined by τ d : (12) Outcomes of diurnal modelling (A Q and A f ) were compared using linear mixed effects, treating models using alternative (estimated from S or V c,max ) τ a and τ d as fixed effects, while accounting for variation among accessions as a random effect.

Code availability
Code used for analysis and figure preparation are available through GitHub (https://github.com/smuel-tylor/Fast-Deactivation-of-Rubisco); data can also be obtained from this location.

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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the findings of this study are available in the Lancaster University Research Directory: https://doi.org/10.17635/lancaster/researchdata/493. They are also included, for ease of use, with the aforementioned GitHub submission (https://github.com/smuel-tylor/Fast-Deactivation-of-Rubisco) nature research | reporting summary April 2020 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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Sample size
The minimum sample size of 3 independent biological replicates was limited by the effort required to obtain each set of measurements. More samples were collected where possible, up to a maximum of n = 6.
Data exclusions Data that failed to match physiological expectations, were noisily imprecise, or failed to produce adequate fits when carrying out non-linear modelling were excluded. These exclusions and their rationale are documented for the complete gas exchange dataset in data analysis scripts made available on GitHub. Rubisco activity data was quality checked for errors in experimental procedure.

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Biological replicates were analysed independently and all genotypes were measured at the same time. A minimum of 3 and maximum of 6 independent biological replicates were collected in each experiment.
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There was no blinding. Due to the experiment design, researchers were aware of the identity of each plant because, for example, visual checking for leaf age prior to sampling was a necessary component of the protocols and the genotypes have different leaf shapes. For Rubisco activity experiments, leaf disc samples undergo processing subsequent to sampling; processing was done in batches that ensured all treatments and accessions were treated similarly.
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