Implications of the mesophyll conductance to CO2 for photosynthesis and water-use efficiency during long-term water stress and recovery in two contrasting Eucalyptu species

Water stress (WS) slows growth and photosynthesis (An), but most knowledge comes from short-time studies that do not account for longer term acclimation processes that are especially relevant in tree species. Using two Eucalyptus species that contrast in drought tolerance, we induced moderate and severe water deficits by withholding water until stomatal conductance (gsw) decreased to two pre-defined values for 24 d, WS was maintained at the target gsw for 29 d and then plants were re-watered. Additionally, we developed new equations to simulate the effect on mesophyll conductance (gm) of accounting for the resistance to refixation of CO2. The diffusive limitations to CO2, dominated by the stomata, were the most important constraints to An. Full recovery of An was reached after re-watering, characterized by quick recovery of gm and even higher biochemical capacity, in contrast to the slower recovery of gsw. The acclimation to long-term WS led to decreased mesophyll and biochemical limitations, in contrast to studies in which stress was imposed more rapidly. Finally, we provide evidence that higher gm under WS contributes to higher intrinsic water-use efficiency (iWUE) and reduces the leaf oxidative stress, highlighting the importance of gm as a target for breeding/genetic engineering.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves1

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m−2. Mesophyll conductance (μ) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on Et saturated at Et = 30 μmol active sites m−2 and μ = 11 mm s−1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a μ of only 6 to 8 mm s−1. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm−1 s−1. Our data show that the saturation of the relationship between Et and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.

A Modeling Approach to Quantify the Effects of Stomatal Behavior and Mesophyll Conductance on Leaf Water Use Efficiency

Water use efficiency (WUE) is considered as a determinant of yield under stress and a component of crop drought resistance. Stomatal behavior regulates both transpiration rate and net assimilation and has been suggested to be crucial for improving crop WUE. In this work, a dynamic model was used to examine the impact of dynamic properties of stomata on WUE. The model includes sub-models of stomatal conductance dynamics, solute accumulation in the mesophyll, mesophyll water content, and water flow to the mesophyll. Using the instantaneous value of stomatal conductance, photosynthesis, and transpiration rate were simulated using a biochemical model and Penman-Monteith equation, respectively. The model was parameterized for a cucumber leaf and model outputs were evaluated using climatic data. Our simulations revealed that WUE was higher on a cloudy than a sunny day. Fast stomatal reaction to light decreased WUE during the period of increasing light (e.g., in the morning) by up to 10.2% and increased WUE during the period of decreasing light (afternoon) by up to 6.25%. Sensitivity of daily WUE to stomatal parameters and mesophyll conductance to CO2 was tested for sunny and cloudy days. Increasing mesophyll conductance to CO2 was more likely to increase WUE for all climatic conditions (up to 5.5% on the sunny day) than modifications of stomatal reaction speed to light and maximum stomatal conductance.

Towards an improved and more flexible representation of water stress in coupled photosynthesis–stomatal conductance models

Coupled photosynthesis–stomatal conductance (A–gs) models are commonly used in ecosystem models to represent the exchange rate of CO2 and H2O between vegetation and the atmosphere. The ways these models account for water stress differ greatly among modelling schemes. This study provides insight into the impact of contrasting model configurations of water stress on the simulated leaf-level values of net photosynthesis (A), stomatal conductance (gs), the functional relationship among them and their ratio, the intrinsic water use efficiency (A/gs), as soil dries. A simple, yet versatile, normalized soil moisture dependent function was used to account for the effects of water stress on gs, on mesophyll conductance (gm) and on the biochemical capacity. Model output was compared to leaf-level values obtained from the literature. The sensitivity analyses emphasized the necessity to combine both stomatal and non-stomatal limitations of A in coupled A–gs models to accurately capture the observed functional relationships A vs. gs and A/gsvs. gs in response to drought. Accounting for water stress in coupled A–gs models by imposing either stomatal or biochemical limitations of A, as commonly practiced in most ecosystem models, failed to reproduce the observed functional relationship between key leaf gas exchange attributes. A quantitative limitation analysis revealed that the general pattern of C3 photosynthetic response to water stress may be well represented in coupled A–gs models by imposing the highest limitation strength to gm, then to gs and finally to the biochemical capacity.

Restrictions to Carbon Dioxide Conductance and Photosynthesis in Spinach Leaves Recovering from Salt Stress

Salt accumulation in spinach (Spinacia oleracea L.) leaves first inhibits photosynthesis by decreasing stomatal and mesophyll conductances to CO2 diffusion and then impairs ribulose-1,5-bisphosphate carboxylase/oxygenase (S. Delfine, A. Alvino, M. Zacchini, F. Loreto [1998] Aust J Plant Physiol 25: 395–402). We measured gas exchange and fluorescence in spinach recovering from salt accumulation. When a 21-d salt accumulation was reversed by 2 weeks of salt-free irrigation (rewatering), stomatal and mesophyll conductances and photosynthesis partially recovered. For the first time, to our knowledge, it is shown that a reduction of mesophyll conductance can be reversed and that this may influence photosynthesis. Photosynthesis and conductances did not recover when salt drainage was restricted and Na content in the leaves was greater than 3% of the dry matter. Incomplete recovery of photosynthesis in rewatered and control leaves may be attributed to an age-related reduction of conductances. Biochemical properties were not affected by the 21-d salt accumulation. However, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and content were reduced by a 36- to 50-d salt accumulation. Photochemical efficiency was reduced only in 50-d salt-stressed leaves because of a decrease in the fraction of open photosystem II centers. A reduction in chlorophyll content and an increase in the chlorophyll a/b ratio were observed in 43- and 50-d salt-stressed leaves. Low chlorophyll affects light absorptance but is unlikely to change light partitioning between photosystems.

Photosynthetic and anatomical responses of Eucalyptus grandis leaves to potassium and sodium supply in a field experiment

Although vast areas in tropical regions have weathered soils with low potassium (K) levels, little is known about the effects of K supply on the photosynthetic physiology of trees. This study assessed the effects of K and sodium (Na) supply on the diffusional and biochemical limitations to photosynthesis in Eucalyptus grandis leaves. A field experiment comparing treatments receiving K (+K) or Na (+Na) with a control treatment (C) was set up in a K-deficient soil. The net CO2 assimilation rates were twice as high in +K and 1.6 times higher in +Na than in the C as a result of lower stomatal and mesophyll resistance to CO2 diffusion and higher photosynthetic capacity. The starch content was higher and soluble sugar was lower in +K than in C and +Na, suggesting that K starvation disturbed carbon storage and transport. The specific leaf area, leaf thickness, parenchyma thickness, stomatal size and intercellular air spaces increased in +K and +Na compared to C. Nitrogen and chlorophyll concentrations were also higher in +K and +Na than in C. These results suggest a strong relationship between the K and Na supply to E. grandis trees and the functional and structural limitations to CO2 assimilation rates. © 2013 John Wiley & Sons Ltd.

Drought stress and carbon assimilation in a warming climate: Reversible and irreversible impacts

Abstract Global change is characterized by increased {CO2} concentration in the atmosphere, increasing average temperature and more frequent extreme events including drought periods, heat waves and flooding. Especially the impacts of drought and of elevated temperature on carbon assimilation are considered in this review. Effects of extreme events on the subcellular level as well as on the whole plant level may be reversible, partially reversible or irreversible. The photosynthetically active biomass depends on the number and the size of mature leaves and the photosynthetic activity in this biomass during stress and subsequent recovery phases. The total area of active leaves is determined by leaf expansion and senescence, while net photosynthesis per leaf area is primarily influenced by stomatal opening (stomatal conductance), mesophyll conductance, activity of the photosynthetic apparatus (light absorption and electron transport, activity of the Calvin cycle) and {CO2} release by decarboxylation reactions (photorespiration, dark respiration). Water status, stomatal opening and leaf temperature represent a "magic triangle" of three strongly interacting parameters. The response of stomata to altered environmental conditions is important for stomatal limitations. Rubisco protein is quite thermotolerant, but the enzyme becomes at elevated temperature more rapidly inactivated (decarbamylation, reversible effect) and must be reactivated by Rubisco activase (carbamylation of a lysine residue). Rubisco activase is present under two forms (encoded by separate genes or products of alternative splicing of the pre-mRNA from one gene) and is very thermosensitive. Rubisco activase was identified as a key protein for photosynthesis at elevated temperature (non-stomatal limitation). During a moderate heat stress Rubisco activase is reversibly inactivated, but during a more severe stress (higher temperature and/or longer exposure) the protein is irreversibly inactivated, insolubilized and finally degraded. On the level of the leaf, this loss of photosynthetic activity may still be reversible when new Rubisco activase is produced by protein synthesis. Rubisco activase as well as enzymes involved in the detoxification of reactive oxygen species or in osmoregulation are considered as important targets for breeding crop plants which are still productive under drought and/or at elevated leaf temperature in a changing climate.

Conductancia del mesófilo en especies forestales: implicaciones en la respuesta fotosintética a la disponibilidad de agua y luz

La fotosíntesis es el proceso biológico que permite la producción primaria y, por tanto, la vida en nuestro planeta. La tasa fotosintética viene determinada por la ‘maquinaria’ bioquímica y las resistencias difusivas al paso del CO2 desde la atmósfera hasta su fijación en el interior de los cloroplastos. Históricamente la mayor resistencia difusiva se ha atribuido al cierre estomático, sin embargo ahora sabemos, debido a las mejoras en las técnicas experimentales, que existe también una resistencia grande que se opone a la difusión del CO2 desde los espacios intercelulares a los lugares de carboxilación. Esta resistencia, llamada normalmente por su inversa: la conductancia del mesófilo (gm), puede ser igual o incluso superior a la resistencia debida por el cierre estomático. En la presente tesis doctoral he caracterizado la limitación que ejerce la resistencia del mesófilo a la fijación de CO2 en diversas especies forestales y en distintos momentos de su ciclo biológico. En la fase de regenerado, hemos estudiado tres situaciones ambientales relevantes en el mayor éxito de su supervivencia, que son: el déficit hídrico, su interacción con la irradiancia y el paso del crecimiento en la sombra a mayor irradiancia, como puede suceder tras la apertura de un hueco en el dosel forestal. En la fase de arbolado adulto se ha caracterizado el estado hídrico y el intercambio gaseoso en hojas desarrolladas a distinta irradiancia dentro del dosel vegetal durante tres años contrastados en pluviometría. Para cada tipo de estudio se han empleado las técnicas ecofisiológicas más pertinentes para evaluar el estado hídrico y el intercambio gaseoso. Por su complejidad y la falta de un método que permita su cuantificación directa, la gm ha sido evaluada por los métodos más usados, que son: la discriminación isotópica del carbono 13, el método de la J variable, el método de la J constante y el método de la curvatura. Los resultados más significativos permiten concluir que la limitación relativa a la fotosíntesis por la conductancia estomática, del mesófilo y bioquímica es dependiente de la localización de la hoja en el dosel forestal. Por primera vez se ha documentado que bajo estrés hídrico las hojas desarrolladas a la sombra estuvieron más limitadas por una reducción en la gm, mientras que las hojas desarrolladas a pleno sol estuvieron más limitadas por reducción mayor de la conductancia estomática (gsw). Encontramos buena conexión entre el aparato fotosintético foliar y el sistema hídrico debido al alto grado de correlación entre la conductancia hidráulica foliar aparente y la concentración de CO2 en los cloroplastos en distintas especies forestales. Además, hemos mostrado diferentes pautas de regulación del intercambio gaseoso según las particularidades ecológicas de las especies estudiadas. Tanto en brinzales crecidos de forma natural y en el arbolado adulto como en plántulas cultivadas en el invernadero la ontogenia afectó a las limitaciones de la fotosíntesis producidas por estrés hídrico, resultando que las limitaciones estomáticas fueron dominantes en hojas más jóvenes mientras que las no estomáticas en hojas más maduras. La puesta en luz supuso un gran descenso en la gm durante los días siguientes a la transferencia, siendo este efecto mayor según el grado de sombreo previo en el que se han desarrollado las hojas. La aclimatación de las hojas a la alta irradiancia estuvo ligada a las modificaciones anatómicas foliares y al estado de desarrollo de la hoja. El ratio entre la gm/gsw determinó la mayor eficiencia en el uso del agua y un menor estado oxidativo durante la fase de estrés hídrico y su posterior rehidratación, lo cual sugiere el uso de este ratio en los programas de mejora genética frente al estrés hídrico. Debido a que la mayoría de modelos de estimación de la producción primaria bruta (GPP) de un ecosistema no incluye la gm, los mismos están incurriendo en una sobreestimación del GPP particularmente bajo condiciones de estrés hídrico, porque más de la mitad de la reducción en fotosíntesis en hojas desarrolladas a la sombra se debe a la reducción en gm. Finalmente se presenta un análisis de la importancia en las estimas de la gm bajo estrés hídrico de la refijación del CO2 emitido en la mitocondria a consecuencia de la fotorrespiración y la respiración mitocondrial en luz. ABSTRACT Photosynthesis is the biological process that supports primary production and, therefore, life on our planet. Rates of photosynthesis are determined by biochemical “machinery” and the diffusive resistance to the transfer of CO2 from the atmosphere to the place of fixation within the chloroplasts. Historically the largest diffusive resistance was attributed to the stomata, although we now know via improvements in experimental techniques that there is also a large resistance from sub-stomatal cavities to sites of carboxylation. This resistance, commonly quantified as mesophyll conductance (gm), can be as large or even larger than that due to stomatal resistance. In the present PhD I have characterized the limitation exerted by the mesophyll resistance to CO2 fixation in different forest species at different stages of their life cycle. In seedlings, we studied three environmental conditions that affect plant fitness, namely, water deficit, the interaction of water deficit with irradiance, and the transfer of plants grown in the shade to higher irradiance as can occur when a gap opens in the forest canopy. At the stage of mature trees we characterized water status and gas exchange in leaves developed at different irradiance within the canopy over the course of three years that had contrasting rainfall. For each study we used the most relevant ecophysiological techniques to quantify water relations and gas exchange. Due to its complexity and the lack of a method that allows direct quantification, gm was estimated by the most commonly used methods which are: carbon isotope discrimination, the J-variable, constant J and the curvature method The most significant results suggest that the relative limitation of photosynthesis by stomata, mesophyll and biochemistry depending on the position of the leaf within the canopy. For the first time it was documented that under water stress shaded leaves were more limited by a reduction in gm, while the sun-adapted leaves were more limited by stomatal conductance (gsw). The connection between leaf photosynthetic apparatus and the hydraulic system was shown by the good correlations found between the apparent leaf hydraulic conductance and the CO2 concentration in the chloroplasts in shade- and sun-adapted leaves of several tree species. In addition, we have revealed different patterns of gas exchange regulation according to the functional ecology of the species studied. In field grown trees and greenhouse-grown seedlings ontogeny affected limitations of photosynthesis due to water stress with stomatal limitations dominating in young leaves and nonstomatal limitations in older leaves. The transfer to high light resulted in major decrease of gm during the days following the transfer and this effect was greater as higher was the shade which leaves were developed. Acclimation to high light was linked to the leaf anatomical changes and the state of leaf development. The ratio between the gm/gsw determined the greater efficiency in water use and reduced the oxidative stress during the water stress and subsequent rehydration, suggesting the use of this ratio in breeding programs aiming to increase avoidance of water stress. Because most models to estimate gross primary production (GPP) of an ecosystem do not include gm, they are incurring an overestimation of GPP particularly under conditions of water stress because more than half of An decrease in shade-developed leaves may be due to reduction in gm. Finally, we present an analysis of the importance of how estimates of gm under water stress are affected by the refixation of CO2 that is emitted from mitochondria via photorespiration and mitochondrial respiration in light.

Zero-bias conductance and energy gap measurements in LaNiO3-YBa2Cu3O7-x junctions

Conductance measurements of junctions between a high- superconductor and a metallic oxide have been carried out along the a-b plane to examine the tunnel-junction spectra. For these measurements, in situ films have been grown on c-axis oriented thin films using the pulsed laser deposition technique. Two distinctive energy gaps have been observed along with conductance peaks around zero bias. The analysis of zero-bias conductance and energy gap data suggests the presence of midgap states located at the centre of a finite energy gap. The results obtained are also in accordance with the d-wave nature of high- superconductors.

Ion conductance in electrolyte solutions

We develop a new theoretical formulation to study ion conductance in electrolyte solutions, based on a mode coupling theory treatment of the electrolyte friction. The new theory provides expressions for both the ion atmosphere relaxation and electrophoretic contributions to the total electrolyte friction that acts on a moving ion. While the ion atmosphere relaxation term arises from the time-dependent microscopic interaction of the moving ion with the surrounding ions in the solution, the electrophoretic term originates from the coupling of the ion's velocity to the collective current mode of the ion atmosphere. Mode coupling theory, combined with time-dependent density functional theory of ion atmosphere fluctuations, leads to self-consistent expressions for these two terms which also include the effects of self-motion of the ion under consideration. These expressions have been solved for the concentration dependence of electrolyte friction and ion conductance. It is shown that in the limit of very low ion concentration, the present theory correctly reduces to the well-known Debye-Huckel-Onsager limiting law which predicts a linear dependence of conductance on the square root of ion concentration (c). At moderate and high concentrations, the present theory predicts a significant nonlinear and weaker dependence on root c which is in very good agreement with experimental results. The present theory is self-contained and does not involve any adjustable parameter.

The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.

Constant conductance fluctuations in disordered metals

We discuss the effect of fluctuations of the random potential in directions transverse to the current flow in a modified Migdal-Kadanoff approach to probabilistic scaling of conductance with size L, in d-dimensional metallic systems. The conductance cumulants are finite and vary as Ld−1−n for n greater-or-equal, slanted 2 i.e. conductance fluctuations are constant for d = 3. The mean conductance has a non-classical correction with Image Full-size image (<1K) for d greater-or-equal, slanted 2. The form of the higher cumulants is strongly influenced by the transverse potential fluctuations and may be compared with the results of perturbative diagrammatic approaches.

Quantum Interference in Coherent Molecular Conductance

Coherent electronic transport through individual molecules is crucially sensitive to quantum interference. We investigate the zero-bias and zero-temperature conductance through pi-conjugated annulene molecules weakly coupled to two leads for different source-drain configurations, finding an important reduction for certain transmission channels and for particular geometries as a consequence of destructive quantum interference between states with definite momenta. When translational symmetry is broken by an external perturbation we find an abrupt increase of the conductance through those channels. Previous studies concentrated on the effect at the Fermi energy, where this effect is very small. By analyzing the effect of symmetry breaking on the main transmission channels we find a much larger response thus leading to the possibility of a larger switching of the conductance through single molecules.

Tuning the Conductance of Dirac Fermions on the Surface of a Topological Insulator

We study the transport properties of the Dirac fermions with a Fermi velocity v(F) on the surface of a topological insulator across a ferromagnetic strip providing an exchange field J over a region of width d. We show that the conductance of such a junction, in the clean limit and at low temperature, changes from oscillatory to a monotonically decreasing function of d beyond a critical J. This leads to the possible realization of a magnetic switch using these junctions. We also study the conductance of these Dirac fermions across a potential barrier of width d and potential V-0 in the presence of such a ferromagnetic strip and show that beyond a critical J, the criteria of conductance maxima changes from chi = eV(0)d/(h) over barv(F) = n pi to chi = (n + 1/2)pi for integer n. We point out that these novel phenomena have no analogs in graphene and suggest experiments which can probe them.