A generalized stoichiometric model of C3, C2, C2+C4, and C4 photosynthetic metabolism.

The goal of suppressing photorespiration in crops to maximize assimilation and yield is stimulating considerable interest among researchers looking to bioengineer carbon-concentrating mechanisms into C3 plants. However, detailed quantification of the biochemical activities in the bundle sheath is lacking. This work presents a general stoichiometric model for C3, C2, C2+C4, and C4 assimilation (SMA) in which energetics, metabolite traffic, and the different decarboxylating enzymes (NAD-dependent malic enzyme, NADP-dependent malic enzyme, or phosphoenolpyruvate carboxykinase) are explicitly included. The SMA can be used to refine experimental data analysis or formulate hypothetical scenarios, and is coded in a freely available Microsoft Excel workbook. The theoretical underpinnings and general model behaviour are analysed with a range of simulations, including (i) an analysis of C3, C2, C2+C4, and C4 in operational conditions; (ii) manipulating photorespiration in a C3 plant; (iii) progressively upregulating a C2 shuttle in C3 photosynthesis; (iv) progressively upregulating a C4 cycle in C2 photosynthesis; and (v) manipulating processes that are hypothesized to respond to transient environmental inputs. Results quantify the functional trade-offs, such as the electron transport needed to meet ATP/NADPH demand, as well as metabolite traffic, inherent to different subtypes. The SMA refines our understanding of the stoichiometry of photosynthesis, which is of paramount importance for basic and applied research

Energetics of maize C4 physiology under light limiting conditions

In the present experimental programme I have eliminated two leading possibilities explaining why the C4 pathway is maladapted for deep shade: susceptibility to low light and changing spectral quality. Firstly I showed leakiness does not constrain assimilation under low light intensities and maize has efficient mechanisms to acclimate leakiness in response to irradiance. Secondly I showed that light quality does not constrain assimilation and maize has efficient biochemical mechanisms to take advantage of any light quality.C4 plants have a biochemical carbon concentrating mechanism (CCM) that increases CO2 concentration around Rubisco in the bundle sheath (BS). Maize CCM has two CO2 delivery pathways to the Bundle Sheath (BS) (respectively via malate, MAL or aspartate, ASP); rates of PGA reduction, carbohydrate synthesis and PEP regeneration vary between BS and Mesophyll (M) cells. For these anatomical and biochemical complexities, C4 plants are highly sensitive to light conditions. Under limiting light, the activity of the CCM generally decreases, causing an increase in leakiness, (Φ), the ratio of CO2 retrodiffusing from the BS relative to C4 carboxylation processes. This increase in Φ had been theoretically associated with a decrease in biochemical operating efficiency (expressed as ATP cost of gross assimilation, ATP / GA) under low light and, because a proportion of canopy photosynthesis is carried out by shaded leaves, to potential productivity losses at field scale. In C4 leaves, because of the concentric anatomy, light reaches M cells before the deeper BS (Evans et al., 2007), and could alter the energetic partitioning balance between BS and M and potentially cause efficiency losses. In this experimental programme I investigated strategies deployed by C4 plants to adjust operating efficiency under different illumination conditions. Firstly, maize plants were grown under high and low light regimes (respectively HL, 600 vs LL, 100 μE m-2 s-1). Short term acclimation of Φ was compared from isotopic discrimination (Δ), gas exchange and photochemistry using an improved modelling approach which does not suffer from elements of circularity. Long term acclimation to low light intensities brought about physiological changes which could potentially increase the operating efficiency under limiting ATP supplies. Secondly, profiles of light penetration across a leaf were used to derive the potential ATP supply for M and BS cells induced by changing light quality. Empirical measurements of net CO2 uptake, ATP production rate and carbon isotope discrimination were made on plants under a low light intensity. The overall conversion efficiency was not affected by light quality. A comprehensive metabolic model highlighted the importance of both CO2 delivery pathways in maize. Further, metabolic plasticity allowed the balancing of ATP and NADPH requirements between BS and M. Finally, I tested the hypothesis that plants can modify their physiology so as to reach a status of higher operating efficiency when exposed to high light and then to low light, so as to mimic the transition which leaves undergo when shaded by newly emerging leaves in a crop canopy. Plants were grown under high light and low light for three weeks, then, HL plants were transferred to low light for a further three weeks. Re-acclimation was very effective in reducing ATP cost of net assimilation under low light intensities. In addition, the hyperbolic leakiness increase observed under low light intensities was not associated to operating efficiency loss. Overall, in the three experimental Chapters I showed compelling theoretical and empirical evidence proving the hypothesis that C4 plants deal with low light conditions and with different light qualities without losing operating efficiency.EU FP7 Marie curie ITN, grant n° 23801

The slope of assimilation rate against stomatal conductance should not be used as a measure of water use efficiency or stomatal control over assimilation

Quantifying water use efficiency, and the impact of stomata on CO2 uptake are pivotal in physiology and efforts to improve crop yields. Although tempting, relying on regression slopes from assimilation-stomatal conductance plots to estimate water use efficiency or stomatal control over assimilation is erroneous. Through numerical simulations, I substantiate this assertion. I propose the term ‘instantaneous transpiration efficiency’ for the assimilation-to-transpiration ratio to avoid confusion with ‘intrinsic water use efficiency’ which refers to the assimilation-to-stomatal conductance ratio, and recommend to compute both metrics for each gas exchange data point

Stomatal and non-stomatal limitations in savanna trees and C₄ grasses grown at low, ambient and high atmospheric CO₂

By the end of the century, atmospheric CO₂ concentration ([CO₂]a) could reach 800 ppm, having risen from ~200 ppm ~24 Myr ago. Carbon dioxide enters plant leaves through stomata that limit CO₂ diffusion and assimilation, imposing stomatal limitation (LS). Other factors limiting assimilation are collectively called non-stomatal limitations (LNS). C₄ photosynthesis concentrates CO₂ around Rubisco, typically reducing LS. C₄-dominated savanna grasslands expanded under low [CO₂]a and are metastable ecosystems where the response of trees and C₄ grasses to rising [CO2]a will determine shifting vegetation patterns. How LS and LNS differ between savanna trees and C₄ grasses under different [CO₂]a will govern the responses of CO₂ fixation and plant cover to [CO₂]a – but quantitative comparisons are lacking. We measured assimilation, within soil wetting–drying cycles, of three C₃ trees and three C₄ grasses grown at 200, 400 or 800 ppm [CO₂]a. Using assimilation–response curves, we resolved LS and LNS and show that rising [CO₂]a alleviated LS, particularly for the C₃ trees, but LNS was unaffected and remained substantially higher for the grasses across all [CO₂]a treatments. Because LNS incurs higher metabolic costs and recovery compared with LS, our findings indicate that C₄ grasses will be comparatively disadvantaged as [CO₂]a rises.We acknowledge funding through an ERC advanced grant (CDREG, 322998) awarded to DJB. CB acknowledges funding through a H2020 MSCA individual fellowship (DILIPHO, ID: 702755)

Anatomical constraints to C4 evolution: light harvesting capacity in the bundle sheath.

In C4 photosynthesis CO2 assimilation and reduction are typically coordinated across mesophyll (M) and bundle sheath (BS) cells, respectively. This system consequently requires sufficient light to reach BS to generate enough ATP to allow ribulose-1,5-bisphosphate (RuBP) regeneration in BS. Leaf anatomy influences BS light penetration and therefore constrains C4 cycle functionality. Using an absorption scattering model (coded in Excel, and freely downloadable) we simulate light penetration profiles and rates of ATP production in BS across the C3 , C3 -C4 and C4 anatomical continua. We present a trade-off for light absorption between BS pigment concentration and space allocation. C3 BS anatomy limits light absorption and benefits little from high pigment concentrations. Unpigmented BS extensions increase BS light penetration. C4 and C3 -C4 anatomies have the potential to generate sufficient ATP in the BS, whereas typical C3 anatomy does not, except some C3 taxa closely related to C4 groups. Insufficient volume of BS, relative to M, will hamper a C4 cycle via insufficient BS light absorption. Thus, BS ATP production and RuBP regeneration, coupled with increased BS investments, allow greater operational plasticity. We propose that larger BS in C3 lineages may be co-opted for C3 -C4 and C4 biochemistry requirements

Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity

The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach

C4 maize and sorghum are more sensitive to rapid dehydration than C3 wheat and sunflower

The high productive potential, heat resilience, and greater water use efficiency of C4 over C3 plants attract considerable interest in the face of global warming and increasing population, but C4 plants are often sensitive to dehydration, questioning the feasibility of their wider adoption. To resolve the primary effect of dehydration from slower from secondary leaf responses originating within leaves to combat stress, we conducted an innovative dehydration experiment. Four crops grown in hydroponics were forced to a rapid yet controlled decrease in leaf water potential by progressively raising roots of out of the solution while measuring leaf gas exchange. We show that, under rapid dehydration, assimilation decreased more steeply in C4 maize and sorghum than in C3 wheat and sunflower. This reduction was due to a rise of nonstomatal limitation at triple the rate in maize and sorghum than in wheat and sunflower. Rapid reductions in assimilation were previously measured in numerous C4 species across both laboratory and natural conditions. Hence, we deduce that high sensitivity to rapid dehydration might stem from the disturbance of an intrinsic aspect of C4 bicellular photosynthesis. We posit that an obstruction to metabolite transport between mesophyll and bundle sheath cells could be the cause.European Commission Horizon 2020Science Foundation IrelandAustralian National Universit

The corewood of 25-year-old Hevea brasiliensis from two rubber plantations has high starch content

In Brazil after 25 to 30 years of rubber production, when yield starts to drop, rubber trees are felled and destined for firewood and charcoal, despite the good mechanical properties and workability of the wood, and relatively low production costs. Wood with low starch content could be destined for the production of higher added-value products with potential to spare deforestation of many native forest species, but in rubberwood, starch increases palatability by wood borers and accelerates fungal degradation, thus compromising wood durability and the quality of timber. The aim of this study is to determine whether removal of the outer part of wood or varying the season of logging would result in wood with lower starch content. We measured the content of starch using enzymatic hydrolysis, the radial distribution of starch grains by light microscopy, and the corresponding seasonal variation of starch in 25-year-old felled trees. Rubberwood had large amount of starch in its entire trunk, increasing from the inner to the outer region, before decreasing in the outermost sapwood. Starch content was lower in summer, although higher than in other timber species. After relating the data to a comprehensive bibliographic survey of starch quantification in rubberwood, it was concluded that there are no technological arguments to destine the inner part of rubber tree trunks to the production of higher value products.Science Foundation IrelandEuropean Commission Horizon 2020European Research CouncilMinistry of Education of BrazilFundação de Amparo à Pesquisa do Estado de São Paulo - BrasilNPq Research Productivity Fellowshi

Computer Reconstruction of Plant Growth and Chlorophyll Fluorescence Emission in Three Spatial Dimensions

Plant leaves grow and change their orientation as well their emission of chlorophyll fluorescence in time. All these dynamic plant properties can be semi-automatically monitored by a 3D imaging system that generates plant models by the method of coded light illumination, fluorescence imaging and computer 3D reconstruction. Here, we describe the essentials of the method, as well as the system hardware. We show that the technique can reconstruct, with a high fidelity, the leaf size, the leaf angle and the plant height. The method fails with wilted plants when leaves overlap obscuring their true area. This effect, naturally, also interferes when the method is applied to measure plant growth under water stress. The method is, however, very potent in capturing the plant dynamics under mild stress and without stress. The 3D reconstruction is also highly effective in correcting geometrical factors that distort measurements of chlorophyll fluorescence emission of naturally positioned plant leaves