Estimating leaf day respiration from conventional gas exchange measurements.

Leaf day respiration (Rd ) strongly influences carbon-use efficiencies of whole plants and the global terrestrial biosphere. It has long been thought that Rd is slower than respiration in the dark at a given temperature, but measuring Rd by gas exchange remains a challenge because leaves in the light are also photosynthesizing. The Kok method and the Laisk method are widely used to estimate Rd . We highlight theoretical limitations of these popular methods, and recent progress toward their improvement by using additional information from chlorophyll fluorescence and by accounting for the photosynthetic reassimilation of respired CO2 . The latest evidence for daytime CO2 and energy release from the oxidative pentose phosphate pathway in chloroplasts appears to be important to understanding Rd .

High photosynthesis rates in Brassiceae species are mediated by leaf anatomy enabling high biochemical capacity, rapid CO2 diffusion and efficient light use.

Certain species in the Brassicaceae family exhibit high photosynthesis rates, potentially providing a valuable route toward improving agricultural productivity. However, factors contributing to their high photosynthesis rates are still unknown. We compared Hirschfeldia incana, Brassica nigra, Brassica rapa and Arabidopsis thaliana, grown under two contrasting light intensities. Hirschfeldia incana matched B. nigra and B. rapa in achieving very high photosynthesis rates under high growth-light condition, outperforming A. thaliana. Photosynthesis was relatively more limited by maximum photosynthesis capacity in H. incana and B. rapa and by mesophyll conductance in A. thaliana and B. nigra. Leaf traits such as greater exposed mesophyll specific surface enabled by thicker leaf or high-density small palisade cells contributed to the variation in mesophyll conductance among the species. The species exhibited contrasting leaf construction strategies and acclimation responses to low light intensity. High-light plants distributed Chl deeper in leaf tissue, ensuring even distribution of photosynthesis capacity, unlike low-light plants. Leaf anatomy of H. incana, B. nigra and B. rapa facilitated effective CO2 diffusion, efficient light use and provided ample volume for their high maximum photosynthetic capacity, indicating that a combination of adaptations is required to increase CO2-assimilation rates in plants.

Roles for leakiness and O2 evolution in explaining lower-than-theoretical quantum yields of photosynthesis in the PEP-CK subtype of C4 plants.

Theoretically, the PEP-CK C4 subtype has a higher quantum yield of CO2 assimilation ( Φ CO 2 ) than NADP-ME or NAD-ME subtypes because ATP required for operating the CO2-concentrating mechanism is believed to mostly come from the mitochondrial electron transport chain (mETC). However, reported Φ CO 2 is not higher in PEP-CK than in the other subtypes. We hypothesise, more photorespiration, associated with higher leakiness and O2 evolution in bundle-sheath (BS) cells, cancels out energetic advantages in PEP-CK species. Nine species (two to four species per subtype) were evaluated by gas exchange, chlorophyll fluorescence, and two-photon microscopy to estimate the BS conductance (gbs) and leakiness using a biochemical model. Average gbs estimates were 2.9, 4.8, and 5.0 mmol m-2 s-1 bar-1, and leakiness values were 0.129, 0.179, and 0.180, in NADP-ME, NAD-ME, and PEP-CK species, respectively. The BS CO2 level was somewhat higher, O2 level was marginally lower, and thus, photorespiratory loss was slightly lower, in NADP-ME than in NAD-ME and PEP-CK species. Differences in these parameters existed among species within a subtype, and gbs was co-determined by biochemical decarboxylating sites and anatomical characteristics. Our hypothesis and results partially explain variations in observed Φ CO 2 , but suggest that PEP-CK species probably use less ATP from mETC than classically defined PEP-CK mechanisms.

Source-sink relationships during grain filling in wheat in response to various temperature, water deficit, and nitrogen deficit regimes.

Grain filling is a critical process for improving crop production under adverse conditions caused by climate change. Here, using a quantitative method, we quantified post-anthesis source-sink relationships of a large dataset to assess the contribution of remobilized pre-anthesis assimilates to grain growth for both biomass and nitrogen. The dataset came from 13 years of semi-controlled field experimentation, in which six bread wheat genotypes were grown at plot scale under contrasting temperature, water, and nitrogen regimes. On average, grain biomass was ~10% higher than post-anthesis above-ground biomass accumulation across regimes and genotypes. Overall, the estimated relative contribution (%) of remobilized assimilates to grain biomass became increasingly significant with increasing stress intensity, ranging from virtually nil to 100%. This percentage was altered more by water and nitrogen regimes than by temperature, indicating the greater impact of water or nitrogen regimes relative to high temperatures under our experimental conditions. Relationships between grain nitrogen demand and post-anthesis nitrogen uptake were generally insensitive to environmental conditions, as there was always significant remobilization of nitrogen from vegetative organs, which helped to stabilize the amount of grain nitrogen. Moreover, variations in the relative contribution of remobilized assimilates with environmental variables were genotype dependent. Our analysis provides an overall picture of post-anthesis source-sink relationships and pre-anthesis assimilate contributions to grain filling across (non-)environmental factors, and highlights that designing wheat adaptation to climate change should account for complex multifactor interactions.

Triose phosphate utilization in leaves is modulated by whole-plant sink-source ratios and nitrogen budgets in rice.

Triose phosphate utilization (TPU) is a biochemical process indicating carbon sink-source (im)balance within leaves. When TPU limits leaf photosynthesis, photorespiration-associated amino acid exports probably provide an additional carbon outlet and increase leaf CO2 uptake. However, whether TPU is modulated by whole-plant sink-source relations and nitrogen (N) budgets remains unclear. We address this question by model analyses of gas-exchange data measured on leaves at three growth stages of rice plants grown at two N levels. Sink-source ratio was manipulated by panicle pruning, by using yellower-leaf variant genotypes, and by measuring photosynthesis on adaxial and abaxial leaf sides. Across all these treatments, higher leaf N content resulted in the occurrence of TPU limitation at lower intercellular CO2 concentrations. Photorespiration-associated amino acid export was greater in high-N leaves, but was smaller in yellower-leaf genotypes, panicle-pruned plants, and for abaxial measurement. The feedback inhibition of panicle pruning on rates of TPU was not always observed, presumably because panicle pruning blocked N remobilization from leaves to grains and the increased leaf N content masked feedback inhibition. The leaf-level TPU limitation was thus modulated by whole-plant sink-source relations and N budgets during rice grain filling, suggesting a close link between within-leaf and whole-plant sink limitations.

The role of chloroplast movement in C4 photosynthesis: a theoretical analysis using a three-dimensional reaction-diffusion model for maize.

Chloroplasts movement within mesophyll cells in C4 plants is hypothesized to enhance the CO2 concentrating mechanism, but this is difficult to verify experimentally. A three-dimensional (3D) leaf model can help analyse how chloroplast movement influences the operation of the CO2 concentrating mechanism. The first volumetric reaction-diffusion model of C4 photosynthesis that incorporates detailed 3D leaf anatomy, light propagation, ATP and NADPH production, and CO2, O2 and bicarbonate concentration driven by diffusional and assimilation/emission processes was developed. It was implemented for maize leaves to simulate various chloroplast movement scenarios within mesophyll cells: the movement of all mesophyll chloroplasts towards bundle sheath cells (aggregative movement) and movement of only those of interveinal mesophyll cells towards bundle sheath cells (avoidance movement). Light absorbed by bundle sheath chloroplasts relative to mesophyll chloroplasts increased in both cases. Avoidance movement decreased light absorption by mesophyll chloroplasts considerably. Consequently, total ATP and NADPH production and net photosynthetic rate increased for aggregative movement and decreased for avoidance movement compared with the default case of no chloroplast movement at high light intensities. Leakiness increased in both chloroplast movement scenarios due to the imbalance in energy production and demand in mesophyll and bundle sheath cells. These results suggest the need to design strategies for coordinated increases in electron transport and Rubisco activities for an efficient CO2 concentrating mechanism at very high light intensities.

Neglecting acclimation of photosynthesis under drought can cause significant errors in predicting leaf photosynthesis in wheat.

Extreme climatic events, such as heat waves, cold snaps and drought spells, related to global climate change, have become more frequent and intense in recent years. Acclimation of plant physiological processes to changes in environmental conditions is a key component of plant adaptation to climate change. We assessed the temperature response of leaf photosynthetic parameters in wheat grown under contrasting water regimes and growth temperatures (Tgrowth ). Two independent experiments were conducted under controlled conditions. In Experiment 1, two wheat genotypes were subjected to well-watered or drought-stressed treatments; in Experiment 2, the two water regimes combined with high, medium and low Tgrowth were imposed on one genotype. Parameters of a biochemical C3 -photosynthesis model were estimated at six leaf temperatures for each factor combination. Photosynthesis acclimated more to drought than to Tgrowth . Drought affected photosynthesis by lowering its optimum temperature (Topt ) and the values at Topt of light-saturated net photosynthesis, stomatal conductance, mesophyll conductance, the maximum rate of electron transport (Jmax ) and the maximum rate of carboxylation by Rubisco (Vcmax ). Topt for Vcmax was up to 40°C under well-watered conditions but 24-34°C under drought. The decrease in photosynthesis under drought varied among Tgrowth but was similar between genotypes. The temperature response of photosynthetic quantum yield under drought was partly attributed to photorespiration but more to alternative electron transport. All these changes in biochemical parameters could not be fully explained by the changed leaf nitrogen content. Further model analysis showed that both diffusional and biochemical parameters of photosynthesis and their thermal sensitivity acclimate little to Tgrowth , but acclimate considerably to drought and the combination of drought and Tgrowth . The commonly used modelling approaches, which typically consider the response of diffusional parameters, but ignore acclimation responses of biochemical parameters to drought and Tgrowth , strongly overestimate leaf photosynthesis under variable temperature and drought.

Phenotyping and metabolome analysis reveal the role of AdoMetDC and Di19 genes in determining acquired tolerance to drought in rice.

Water-saving attempts for rice cultivation often reduce yields. Maintaining productivity under drought is possible when rice genotypes are bred with improved metabolism and spikelet fertility. Although attempts have been made to introgress water mining and water use efficiency traits, combining acquired tolerance traits (ATTs), that is, specific traits induced or upregulated to better tolerate severe stress, appears equally important. In our study, we screened 90 rice germplasm accessions that represented the molecular and phenotypic variations of 851 lines of the 3 K rice panel. Utilising phenomics, we identified markers linked to ATTs through association analysis of over 0.2 million SNPs derived from whole-genome sequences. Propensity to respond to 'induction' stress varied significantly among genotypes, reflecting differences in cellular protection against oxidative stress. Among the ATTs, the hydroxyl radical and proline contents exhibited the highest variability. Furthermore, these significant variations in ATTs were strongly correlated with spikelet fertility. The 43 significant markers associated with ATTs were further validated using a different subset of contrasting genotypes. Gene expression studies and metabolomic profiling of two well-known contrasting genotypes, APO (tolerant) and IR64 (sensitive), identified two ATT genes: AdoMetDC and Di19. Our study highlights the relevance of polyamine biosynthesis in modulating ATTs in rice. Genotypes with superior ATTs and the associated markers can be effectively employed in breeding rice varieties with sustained spikelet fertility and grain yield under drought.

Drought exerts a greater influence than growth temperature on the temperature response of leaf day respiration in wheat (Triticum aestivum).

We assessed how the temperature response of leaf day respiration (Rd ) in wheat responded to contrasting water regimes and growth temperatures. In Experiment 1, well-watered and drought-stressed conditions were imposed on two genotypes; in Experiment 2, the two water regimes combined with high (HT), medium (MT) and low (LT) growth temperatures were imposed on one of the genotypes. Rd was estimated from simultaneous gas exchange and chlorophyll fluorescence measurements at six leaf temperatures (Tleaf ) for each treatment, using the Yin method for nonphotorespiratory conditions and the nonrectangular hyperbolic fitting method for photorespiratory conditions. The two genotypes responded similarly to growth and measurement conditions. Estimates of Rd for nonphotorespiratory conditions were generally higher than those for photorespiratory conditions, but their responses to Tleaf were similar. Under well-watered conditions, Rd and its sensitivity to Tleaf slightly acclimated to LT, but did not acclimate to HT. Temperature sensitivities of Rd were considerably suppressed by drought, and the suppression varied among growth temperatures. Thus, it is necessary to quantify interactions between drought and growth temperature for reliably modelling Rd under climate change. Our study also demonstrated that the Kok method, one of the currently popular methods for estimating Rd , underestimated Rd significantly.

A model-guided holistic review of exploiting natural variation of photosynthesis traits in crop improvement.

Breeding for improved leaf photosynthesis is considered as a viable approach to increase crop yield. Whether it should be improved in combination with other traits has not been assessed critically. Based on the quantitative crop model GECROS that interconnects various traits to crop productivity, we review natural variation in relevant traits, from biochemical aspects of leaf photosynthesis to morpho-physiological crop characteristics. While large phenotypic variations (sometimes >2-fold) for leaf photosynthesis and its underlying biochemical parameters were reported, few quantitative trait loci (QTL) were identified, accounting for a small percentage of phenotypic variation. More QTL were reported for sink size (that feeds back on photosynthesis) or morpho-physiological traits (that affect canopy productivity and duration), together explaining a much greater percentage of their phenotypic variation. Traits for both photosynthetic rate and sustaining it during grain filling were strongly related to nitrogen-related traits. Much of the molecular basis of known photosynthesis QTL thus resides in genes controlling photosynthesis indirectly. Simulation using GECROS demonstrated the overwhelming importance of electron transport parameters, compared with the maximum Rubisco activity that largely determines the commonly studied light-saturated photosynthetic rate. Exploiting photosynthetic natural variation might significantly improve crop yield if nitrogen uptake, sink capacity, and other morpho-physiological traits are co-selected synergistically.

Exploiting differences in the energy budget among C4 subtypes to improve crop productivity.

C4 crops of agricultural importance all belong to the NADP-malic enzyme (ME) subtype, and this subtype has been the template for C4 introductions into C3 crops, like rice, to improve their productivity. However, the ATP cost for the C4 cycle in both NADP-ME and NAD-ME subtypes accounts for > 40% of the total ATP requirement for CO2 assimilation. These high ATP costs, and the associated need for intense cyclic electron transport and low intrinsic quantum yield ΦCO2 , are major constraints in realizing strong improvements of canopy photosynthesis and crop productivity. Based on mathematical modelling, we propose a C4 ideotype that utilizes low chloroplastic ATP requirements present in the nondomesticated phosphoenolpyruvate carboxykinase (PEP-CK) subtype. The ideotype is a mixed form of NAD(P)-ME and PEP-CK types, requires no cyclic electron transport under low irradiances, and its theoretical ΦCO2 is c. 25% higher than that of a C4 crop type. Its cell-type-specific ATP and NADPH requirements can be fulfilled by local energy production. The ideotype is projected to have c. 10% yield advantage over NADP-ME-type crops and > 50% advantage over C3 counterparts. The ideotype provides a unique (theoretical) case where ΦCO2 could be improved, thereby paving a new avenue for improving photosynthesis in both C3 and C4 crops.

Role of bundle sheath conductance in sustaining photosynthesis competence in sugarcane plants under nitrogen deficiency.

The role of bundle sheath conductance (gbs) in sustaining sugarcane photosynthesis under nitrogen deficiency was investigated. Sugarcane was grown under different levels of nitrogen supply and gbs was estimated using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence at 21% or 2% [O2] and varying air [CO2] and light intensity. Maximum rates of PEPC carboxylation, Rubisco carboxylation, and ATP production increased with an increase in leaf nitrogen concentration (LNC) from 1 to 3 g m-2. Low nitrogen supply reduced Rubisco and PEPC abundancies, the quantum efficiency of CO2 assimilation and gbs. Because of reduced gbs, low photosynthetic rates were not associated with increased leakiness under nitrogen deficiency. In fact, low nitrogen supply increased bundle sheath cell wall thickness, probably accounting for low gbs and increased estimates of [CO2] at Rubisco sites. Effects of nitrogen on expression of ShPIP2;1 and ShPIP1;2 aquaporins did not explain changes in gbs. Our data revealed that reduced Rubisco carboxylation was the main factor causing low sugarcane photosynthesis at low nitrogen supply, in contrast to the previous report on the importance of an impaired CO2 concentration mechanism under N deficiency. Our findings suggest higher investment of nitrogen into Rubisco protein would favour photosynthesis and plant performance under low nitrogen availability.

Evolution of a biochemical model of steady-state photosynthesis.

On the occasion of the 40th anniversary of the publication of the landmark model by Farquhar, von Caemmerer & Berry on steady-state C3 photosynthesis (known as the "FvCB model"), we review three major further developments of the model. These include: (1) limitation by triose phosphate utilization, (2) alternative electron transport pathways, and (3) photorespiration-associated nitrogen and C1 metabolisms. We discussed the relation of the third extension with the two other extensions, and some equivalent extensions to model C4 photosynthesis. In addition, the FvCB model has been coupled with CO2 -diffusion models. We review how these extensions and integration have broadened the use of the FvCB model in understanding photosynthesis, especially with regard to bioenergetic stoichiometries associated with photosynthetic quantum yields. Based on the new insights, we present caveats in applying the FvCB model. Further research needs are highlighted.

The impact of global dimming on crop yields is determined by the source-sink imbalance of carbon during grain filling.

Global dimming reduces incident global radiation but increases the fraction of diffuse radiation, and thus affects crop yields; however, the underlying mechanisms of such an effect have not been revealed. We hypothesized that crop source-sink imbalance of either carbon (C) or nitrogen (N) during grain filling is a key factor underlying the effect of global dimming on yields. We presented a practical framework to assess both C and N source-sink relationships, using data of biomass and N accumulation from periodical sampling conducted in field experiments for wheat and rice from 2013 to 2016. We found a fertilization effect of the increased diffuse radiation fraction under global dimming, which alleviated the negative impact of decreased global radiation on source supply and sink growth, but the source supply and sink growth were still decreased by dimming, for both C and N. In wheat, the C source supply decreased more than the C sink demand, and as a result, crops remobilized more pre-heading C reserves, in response to dimming. However, these responses were converse in rice, which presumably stemmed from the more increment in radiation use efficiency and the more limited sink size in rice than wheat. The global dimming affected source supply and sink growth of C more significantly than that of N. Therefore, yields in both crops were dependent more on the source-sink imbalance of C than that of N during grain filling. Our revealed source-sink relationships, and their differences and similarities between wheat and rice, provide a basis for designing strategies to alleviate the impact of global dimming on crop productivity.

The Kok effect revisited.

The Kok effect refers to the abrupt decrease around the light compensation point in the slope of net photosynthetic rate vs irradiance. Arguably, this switch arises from light inhibition of respiration, allowing the Kok method to estimate day respiration (Rd ). Recent analysis suggests that increasing proportions of photorespiration (quantified as Γ*/Cc , the ratio of CO2 compensation point Γ* to chloroplast CO2 concentration, Cc ) with irradiance explain much of the Kok effect. Also, the Kok method has been modified to account for the decrease in PSII photochemical efficiency (Φ2 ) with irradiance. Using a model that illustrates how varying Rd , Γ*/Cc , Φ2 and proportions of alternative electron transport could engender the Kok effect, we quantified the contribution of these parameters to the Kok effect measured in sunflower across various O2 and CO2 concentrations and various temperatures. Overall, the decreasing Φ2 with irradiance explained c. 12%, and the varying Γ*/Cc explained c. 25%, of the Kok effect. Maximum real light inhibition of Rd was much lower than the inhibition derived from the Kok method, but still increased with photorespiration. Photorespiration had a dual contribution to the Kok effect, one via the varying Γ*/Cc and the other via its participation in light inhibition of Rd .

Using photorespiratory oxygen response to analyse leaf mesophyll resistance.

Classical approaches to estimate mesophyll conductance ignore differences in resistance components for CO2 from intercellular air spaces (IAS) and CO2 from photorespiration (F) and respiration (Rd). Consequently, mesophyll conductance apparently becomes sensitive to (photo)respiration relative to net photosynthesis, (F + Rd)/A. This sensitivity depends on several hard-to-measure anatomical properties of mesophyll cells. We developed a method to estimate the parameter m (0 ≤ m ≤ 1) that lumps these anatomical properties, using gas exchange and chlorophyll fluorescence measurements where (F + Rd)/A ratios vary. This method was applied to tomato and rice leaves measured at five O2 levels. The estimated m was 0.3 for tomato but 0.0 for rice, suggesting that classical approaches implying m = 0 work well for rice. The mesophyll conductance taking the m factor into account still responded to irradiance, CO2, and O2 levels, similar to response patterns of stomatal conductance to these variables. Largely due to different m values, the fraction of (photo)respired CO2 being refixed within mesophyll cells was lower in tomato than in rice. But that was compensated for by the higher fraction via IAS, making the total re-fixation similar for both species. These results, agreeing with CO2 compensation point estimates, support our method of effectively analysing mesophyll resistance.

Genotypic variation in source and sink traits affects the response of photosynthesis and growth to elevated atmospheric CO2.

This study aimed to understand the response of photosynthesis and growth to e-CO2 conditions (800 vs. 400 µmol mol-1 ) of rice genotypes differing in source-sink relationships. A proxy trait called local C source-sink ratio was defined as the ratio of flag leaf area to the number of spikelets on the corresponding panicle, and five genotypes differing in this ratio were grown in a controlled greenhouse. Differential CO2 resources were applied either during the 2 weeks following heading (EXP1) or during the whole growth cycle (EXP2). Under e-CO2 , low source-sink ratio cultivars (LSS) had greater gains in photosynthesis, and they accumulated less nonstructural carbohydrate in the flag leaf than high source-sink ratio cultivars (HSS). In EXP2, grain yield and biomass gain was also greater in LSS probably caused by their strong sink. Photosynthetic capacity response to e-CO2 was negatively correlated across genotypes with local C source-sink ratio, a trait highly conserved across environments. HSS were sink-limited under e-CO2 , probably associated with low triose phosphate utilization (TPU) capacity. We suggest that the local C source-sink ratio is a potential target for selecting more CO2 -responsive cultivars, pending validation for a broader genotypic spectrum and for field conditions.

The acclimation of leaf photosynthesis of wheat and rice to seasonal temperature changes in T-FACE environments.

Crops show considerable capacity to adjust their photosynthetic characteristics to seasonal changes in temperature. However, how photosynthesis acclimates to changes in seasonal temperature under future climate conditions has not been revealed. We measured leaf photosynthesis (An ) of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) grown under four combinations of two levels of CO2 (ambient and enriched up to 500 µmol/mol) and two levels of canopy temperature (ambient and increased by 1.5-2.0°C) in temperature by free-air CO2 enrichment (T-FACE) systems. Parameters of a biochemical C3 -photosynthesis model and of a stomatal conductance (gs ) model were estimated for the four conditions and for several crop stages. Some biochemical parameters related to electron transport and most gs parameters showed acclimation to seasonal growth temperature in both crops. The acclimation response did not differ much between wheat and rice, nor among the four treatments of the T-FACE systems, when the difference in the seasonal growth temperature was accounted for. The relationships between biochemical parameters and leaf nitrogen content were consistent across leaf ranks, developmental stages, and treatment conditions. The acclimation had a strong impact on gs model parameters: when parameter values of a particular stage were used, the model failed to correctly estimate gs values of other stages. Further analysis using the coupled gs -biochemical photosynthesis model showed that ignoring the acclimation effect did not result in critical errors in estimating leaf photosynthesis under future climate, as long as parameter values were measured or derived from data obtained before flowering.

The fertilization effect of global dimming on crop yields is not attributed to an improved light interception.

Global dimming, a decadal decrease in incident global radiation, is often accompanied with an increase in the diffuse radiation fraction, and, therefore, the impact of global dimming on crop production is hard to predict. A popular approach to quantify this impact is the statistical analysis of historical climate and crop data, or use of dynamic crop simulation modelling approach. Here, we show that statistical analysis of historical data did not provide plausible values for the effect of diffuse radiation versus direct radiation on rice or wheat yield. In contrast, our field experimental study of 3 years demonstrated a fertilization effect of increased diffuse radiation fraction, which partly offset yield losses caused by decreased global radiation, in both crops. The fertilization effect was not attributed to any improved canopy light interception but mainly to the increased radiation use efficiency (RUE). The increased RUE was explained not only by the saturating shape of photosynthetic light response curves but also by plant acclimation to dimming that gradually increased leaf nitrogen concentration. Crop harvest index slightly decreased under dimming, thereby discounting the fertilization effect on crop yields. These results challenge existing modelling paradigms, which assume that the fertilization effect on crop yields is mainly attributed to an improved light interception. Further studies on the physiological mechanism of plant acclimation are required to better quantify the global dimming impact on agroecosystem productivity under future climate change.

Using a reaction-diffusion model to estimate day respiration and reassimilation of (photo)respired CO2 in leaves.

Methods using gas exchange measurements to estimate respiration in the light (day respiration Rd ) make implicit assumptions about reassimilation of (photo)respired CO2 ; however, this reassimilation depends on the positions of mitochondria. We used a reaction-diffusion model without making these assumptions to analyse datasets on gas exchange, chlorophyll fluorescence and anatomy for tomato leaves. We investigated how Rd values obtained by the Kok and the Yin methods are affected by these assumptions and how those by the Laisk method are affected by the positions of mitochondria. The Kok method always underestimated Rd . Estimates of Rd by the Yin method and by the reaction-diffusion model agreed only for nonphotorespiratory conditions. Both the Yin and Kok methods ignore reassimilation of (photo)respired CO2 , and thus underestimated Rd for photorespiratory conditions, but this was less so in the Yin than in the Kok method. Estimates by the Laisk method were affected by assumed positions of mitochondria. It did not work if mitochondria were in the cytosol between the plasmamembrane and the chloroplast envelope. However, mitochondria were found to be most likely between the tonoplast and chloroplasts. Our reaction-diffusion model effectively estimates Rd , enlightens the dependence of Rd estimates on reassimilation and clarifies (dis)advantages of existing methods.