Variation in photosynthetic capacity of Salvia przewalskii along elevational gradients on the eastern Qinghai-Tibetan Plateau, China.

Elevational variation in plant growing environment drives diversification of photosynthetic capacity, however, the mechanism behind this reaction is poorly understood. We measured leaf gas exchange, chlorophyll fluorescence, anatomical characteristics, and biochemical traits of Salvia przewalskii at elevations ranging from 2400 m to 3400 m above sea level (a.s.l) on the eastern Qinghai-Tibetan Plateau, China. We found that photosynthetic capacity showed an initial increase and then a decrease with rising elevation, and the best state observed at 2800 m a.s.l. Environmental factors indirectly regulated photosynthetic capacity by affecting stomatal conductance (gs), mesophyll conductance (gm), maximum velocity of carboxylation (Vc max), and maximum capacity for photosynthetic electron transport (Jmax). The average temperature (T) and total precipitation (P) during the growing season had the highest contribution to the variation of photosynthetic capacity of S. przewalskii in subalpine areas, which were 25% and 24%, respectively. Photosynthetic capacity was mainly affected by diffusional limitations (71%-89%), and mesophyll limitation (lm) played a leading role. The variation of gm was attributed to the effects of environmental factors on the volume fraction of intercellular air space (fias), the thickness of cell wall (Tcw), the surface of mesophyll cells and chloroplasts exposed to intercellular airspace (Sm, Sc), and plasma membrane intrinsic protein (PIPs, PIP1, PIP2), independent of carbonic anhydrase (CA). Optimization of leaf tissue structure and adaptive physiological responses enabled plants to efficiently cope with variable climate conditions of high-elevation areas, and the while maintaining high levels of carbon assimilation.

Effect of light-induced changes in leaf anatomy on intercellular and cellular components of mesophyll resistance for CO2 in Fagus sylvatica.

Mesophyll resistance for CO2 diffusion (rm) is one of the main limitations for photosynthesis and plant growth. Breeding new varieties with lower rm requires knowledge of its distinct components. We tested new method for estimating the relative drawdowns of CO2 concentration (c) across hypostomatous leaves of Fagus sylvatica. This technique yields values of the ratio of the internal CO2 concentrations at the adaxial and abaxial leaf side, cd/cb, the drawdown in the intercellular air space (IAS), and intracellular drawdown between IAS and chloroplast stroma, cc/cbd. The method is based on carbon isotope composition of leaf dry matter and epicuticular wax isolated from upper and lower leaf sides. We investigated leaves from tree-canopy profile to analyse the effects of light and leaf anatomy on the drawdowns and partitioning of rm into its inter- (rIAS) and intracellular (rliq) components. Validity of the new method was tested by independent measurements of rm using conventional isotopic and gas exchange techniques. 73% of investigated leaves had adaxial epicuticular wax enriched in 13C compared to abaxial wax (by 0.50‰ on average), yielding 0.98 and 0.70 for average of cd/cb and cc/cbd, respectively. The rIAS to rliq proportion were 5.5:94.5% in sun-exposed and 14.8:85.2% in shaded leaves. cc dropped to less than half of the atmospheric value in the sunlit and to about two-thirds of it in shaded leaves. This method shows that rIAS is minor but not negligible part of rm and reflects leaf anatomy traits, i.e. leaf mass per area and thickness.

The long and tortuous path towards improving photosynthesis by engineering elevated mesophyll conductance.

The growing demand for global food production is likely to be a defining issue facing humanity over the next 50 years. To tackle this challenge, there is a desire to bioengineer crops with higher photosynthetic efficiencies, to increase yields. Recently, there has been a growing interest in engineering leaves with higher mesophyll conductance (gm), which would allow CO2 to move more efficiently from the substomatal cavities to the chloroplast stroma. However, if crop yield gains are to be realised through this approach, it is essential that the methodological limitations associated with estimating gm are fully appreciated. In this review, we summarise these limitations, and outline the uncertainties and assumptions that can affect the final estimation of gm. Furthermore, we critically assess the predicted quantitative effect that elevating gm will have on assimilation rates in crop species. We highlight the need for more theoretical modelling to determine whether altering gm is truly a viable route to improve crop performance. Finally, we offer suggestions to guide future research on gm, which will help mitigate the uncertainty inherently associated with estimating this parameter.

Harnessing photosynthetic C18O16O discrimination dynamics under leaf water nonsteady state to estimate mesophyll conductance: a new, regression-based method.

Mesophyll conductance (gm) is a crucial plant trait that can significantly limit photosynthesis. Measurement of photosynthetic C18O16O discrimination (Δ18O) has proved to be the only viable means of resolving gm in both C3 and C4 plants. However, the currently available methods to exploit Δ18O for gm estimation are error prone due to their inadequacy in constraining the degree of oxygen isotope exchange (θ) during mesophyll CO2 hydration. Here, we capitalized on experimental manipulation of leaf water isotopic dynamics to establish a novel, nonsteady state, regression-based approach for simultaneous determination of gm and θ from online Δ18O measurements. We demonstrated the methodological and theoretical robustness of this new Δ18O-gm estimation approach and showed through measurements on several C3 and C4 species that this approach can serve as a benchmark method against which to identify previously-unrecognized biases of the existing Δ18O-gm methods. Our results highlight the unique value of this nonsteady state-based approach for contributing to ongoing efforts toward quantitative understanding of mesophyll conductance for crop yield improvement and carbon cycle modeling.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

In Situ Accumulation of CaOx Crystals in C. quitensis Leaves and Its Relationship with Anatomy and Gas Exchange.

The accumulation of crystal calcium oxalate (CaOx) in plants is linked to a type of stress-induced photosynthesis termed 'alarm photosynthesis', serving as a carbon reservoir when carbon dioxide (CO2) exchange is constrained. Colobanthus quitensis is an extremophyte found from southern Mexico to Antarctica, which thrives in high-altitude Andean regions. Growing under common garden conditions, C. quitensis from different latitudinal provenances display significant variations in CaOx crystal accumulation. This raises the following questions: are these differences maintained under natural conditions? And is the CaOx accumulation related to mesophyll conductance (gm) and net photosynthesis (AN) performed in situ? It is hypothesized that in provenances with lower gm, C. quitensis will exhibit an increase in the use of CaOx crystals, resulting in reduced crystal leaf abundance. Plants from Central Chile (33°), Patagonia (51°), and Antarctica (62°) were measured in situ and sampled to determine gas exchange and CaOx crystal accumulation, respectively. Both AN and gm decrease towards higher latitudes, correlating with increases in leaf mass area and leaf density. The crystal accumulation decreases at higher latitudes, correlating positively with AN and gm. Thus, in provenances where environmental conditions induce more xeric traits, the CO2 availability for photosynthesis decreases, making the activation of alarm photosynthesis feasible as an internal source of CO2.

Greater mesophyll conductance and leaf photosynthesis in the field through modified cell wall porosity and thickness via AtCGR3 expression in tobacco.

Mesophyll conductance (gm) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing gm is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase gm. Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and gm measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing gm in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

Leaf diffusional capacity largely contributes to the reduced photosynthesis in rice plants under magnesium deficiency.

Numerous studies have clarified the impacts of magnesium (Mg) on leaf photosynthesis from the perspectives of protein synthesis, enzymes activation and carbohydrate partitioning. However, it still remains largely unknown how stomatal and mesophyll conductances (gs and gm, respectively) are regulated by Mg. In the present study, leaf gas exchanges, leaf hydraulic parameters, leaf structural traits and cell wall composition were examined in rice plants grown under high and low Mg treatments to elucidate the impacts of Mg on gs and gm. Our results showed that reduction of leaf photosynthesis under Mg deficiency was mainly caused by the decreased gm, followed by reduced leaf biochemical capacity and gs, and leaf outside-xylem hydraulic conductance (Kox) was the major factor restricting gs under Mg deficiency. Moreover, increased leaf hemicellulose, lignin and pectin contents and decreased cell wall effective porosity were observed in low Mg plants relative to high Mg plants. These results suggest that Kox and cell wall composition play important roles in regulating gs and gm, respectively, in rice plants under Mg shortages.

The interplay of short-term mesophyll and stomatal conductance responses under variable environmental conditions.

Understanding the short-term responses of mesophyll conductance (gm) and stomatal conductance (gsc) to environmental changes remains a challenging yet central aspect of plant physiology. This review synthesises our current knowledge of these short-term responses, which underpin CO2 diffusion within leaves. Recent methodological advances in measuring gm using online isotopic discrimination and chlorophyll fluorescence have improved our confidence in detecting short-term gm responses, but results need to be carefully evaluated. Environmental factors like vapour pressure deficit and CO2 concentration indirectly impact gm through gsc changes, highlighting some of the complex interactions between the two parameters. Evidence suggests that short-term responses of gm are not, or at least not fully, mechanistically linked to changes in gsc, cautioning against using gsc as a reliable proxy for gm. The overarching challenge lies in unravelling the mechanistic basis of short-term gm responses, which will contribute to the development of accurate models bridging laboratory insights with broader ecological implications. Addressing these gaps in understanding is crucial for refining predictions of gm behaviour under changing environmental conditions.

Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques.

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO2 and H2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (gs ) and net photosynthesis rate (An ) decoupled across all tested species, with gs increasing but An decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency.

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.