Partitioning stomatal and non-stomatal limitations to photosynthesis

Abstract. Plant scientists concerned both with crop improvement and with understanding the control mechanisms of complex processes such as photosynthesis need to identify those processes that are most important in restricting the overall rate and to quantify the relative importance of different components. The techniques that have been used for quantifying the relative importance of component processes in limiting net assimilation rate are reviewed and related to a fundamental definition based on sensitivity analysis. It is concluded that many methods currently in use, including standard resistance analysis, frequently give very misleading answers.
In addition, possible methods for apportioning the contributions of different component processes to observed changes in net photosynthetic rate (for example after stress) are also reviewed and compared against a fundamental approach based on sensitivity analysis. Unfortunately, the detailed time course of changes in mesophyll and stomatal properties that is required for application of the basic sensitivity analysis is seldom likely to be available, so that it is usually necessary to adopt an approximate method. The standard approximation that is recommended for calculating the contributions of different component processes to a change in assimilation rate, involves measurements at the initial and final states only. The various methods discussed in this paper are compared using published photosynthetic data for a range of species.     

Division and growth of mesophyll cells isolated from Psoralea bituminosa leaves

Mesophyll cells have been isolated from Psoralea bituminosa plant by gentle homogenization in a liquid nutrient medium. Between 60 and 70% of the cells can be isolated from leaves using this method, of which 50 to 60% can be recovered morphologically intact. Under light the separated cells have rates of oxygen evolution under light of 3500 μl O₂ mg^?1 chlorophyll h^?1 (measured with a Clark-type electrode). During growth, this rate decreases rapidly, as does cell pigments (chlorophylls and carotenoids). As a first step in obtaining a photoautotrophic cell suspension, growth factors affecting cell division in free sugar medium were investigated. The starting culture contained between 1 and 2 × 10⁶ cells ml^?1. The best increase in cell number was obtained on a medium composed of Joshi and Ball's elements and vitamins and containing 1 mg l^?1 of naphthalene acetic acid, 0.1 mg l^?1 of benzylaminopurine and the grinding juice. The optimum culture pH was between 5 and 5.3.     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

Isolation of functional extensor and flexor protoplasts fromPhaseolus coccineus L. pulvini: potassium induced swelling

Methods are described for the isolation of functional protoplasts from secondary pulvinus tissue (flexor and extensor) and from leaf mesophyll tissue of primary leaves ofPhaseolus coccineus L. Integrity of the protoplasts was shown by vital staining and their ability to evolve oxygen in the light. Extensor-cell protoplasts increased their volume for up to 60% upon addition of 10 mM KCl. This K⁺-induced swelling was accompanied by increased rates of proton extrusion.     

Immunofluorescent Localization of a Connexin 26-like Protein at the Surface of Mesophyll Protoplasts from Vicia faba L. and Helianthus annuus L.

Mesophyll protoplasts from three week old leaves of Helianthus annuus L. and from four week old leaves of Vicia faba L. were incubated with polyclonal, monospecific antibodies, raised against either cx 32 or cx 26 mouse liver connexin. Crossreactions were visualized by FITC-labeled anti-rabbit antibodies. Incubations with the cx 26 antibody resulted in fluorescing spots on protoplast surfaces of both plant species, indicating the presence of a polypeptide, immunologically related to the animal cx 26. A plant protein, exhibiting similarities to cx 26, would present a new member of connexin-like plant proteins. Controls, performed with preimmune serum or with the FITC-conjugate alone, were negative. Immunofluorescing spots were not obtained after incubations with the cx 32 antibody. Since the existence of a cx 32-like plant protein, associated with ultrastructures of plasmodesmata and the plasma membrane, is meanwhile established, several explanations for the failed attempt to demonstrate a cx 32 antibody labeling at protoplast surfaces are discussed.     

Possible link between photosynthesis and leaf modulus of elasticity among vascular plants: a new player in leaf traits relationships?

A compromise between carbon assimilation and structure investment at the leaf level is broadly accepted, yet the relationship between net assimilation per area (A_n) and leaf mass per area has been elusive. We propose bulk modulus of elasticity (ε) as a suitable parameter to reflect both leaf structure and function, and an inverse relationship between ε and A_n and mesophyll conductance (g_m) is postulated. Using data for A_n, g_m and ε from previous studies and new measurements on a set of 20 species covering all major growth forms, a negative relationship between A_n or g_m and ε was observed. High ε was also related to low leaf capacitance and higher diffusive limitations to photosynthesis. In conclusion, ε emerges as a key trait linked with photosynthetic capacity across vascular plants, and its relationship with g_m suggests the existence of a common mechanistic basis, probably involving a key role of cell walls.     

发根农杆菌附着植物细胞的离子效应

本文将光学显微镜、电镜技术和附着定量分析方法结合起来,研究了发根农杆菌各菌株附着烟草叶肉原生质体的离子效应。实验结果表明:发根农杆菌不同菌株对共培养基离子强度的敏感性有差异。菌株R1000附着植物细胞及其引起植物细胞的聚集对离子强度不敏感,而菌株A4和15834则非常敏感;农杆菌对植物细胞附着的多寡与其引起植物细胞的聚集程度有相关性;共培养基中Mg~(2 )、Mn~(2 )等二价刚离子(不包括Ca~(2 ))是农杆菌附着所必需的;植物凝集素ConA显著地促进了附着和植物细胞与细菌聚集团的形成。从菌株A4附着植物细胞的进程可以看出,附着至少是两步的过程:第一步主要是离子力的相互作用,是第二步附着发生的前提;第二步是位点专一的特异性附着,不可逆。发根农杆菌对植物细胞的附着机制不同于根癌农杆菌。     

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP+ and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C₄ plants are to provide ATP for the C₄ cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O₂, formation of H₂O₂ which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H₂O₂ to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (q_p) and the quantum yield of PS II. H₂O₂ also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (q_n), which was inhibited by addition of an uncoupler. These effects of H₂O₂ on q_p and q_n were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H₂O₂ is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C₄ photosynthesis. KCN decreased q_p and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_{_n}. KCN also decreased pH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished pH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C₄ pathway.     

Mesophyll conductance to CO2 and Rubisco as targets for improving intrinsic water use efficiency in C3 plants

Water limitation is a major global constraint for plant productivity that is likely to be exacerbated by climate change. Hence, improving plant water use efficiency (WUE) has become a major goal for the near future. At the leaf level, WUE is the ratio between photosynthesis and transpiration. Maintaining high photosynthesis under water stress, while improving WUE requires either increasing mesophyll conductance (g_m) and/or improving the biochemical capacity for CO₂ assimilation—in which Rubisco properties play a key role, especially in C₃ plants at current atmospheric CO₂. The goals of the present analysis are: (1) to summarize the evidence that improving g_m and/or Rubisco can result in increased WUE; (2) to review the degree of success of early attempts to genetically manipulate g_m or Rubisco; (3) to analyse how g_m, g_sw and the Rubisco's maximum velocity (V_cmax) co‐vary across different plant species in well‐watered and drought‐stressed conditions; (4) to examine how these variations cause differences in WUE and what is the overall extent of variation in individual determinants of WUE; and finally, (5) to use simulation analysis to provide a theoretical framework for the possible control of WUE by g_m and Rubisco catalytic constants vis‐à‐vis g_sw under water limitations.     

Separating the contribution of the upper and lower mesophyll to photosynthesis in Zea mays L. leaves

The appearance of transverse sections of maize leaves indicates the existence of two airspace systems serving the mesophyll, one connected to the stomata of the upper epidermis and the other to the stomata of the lower surface, with few or no connections between the two. This study tests the hypothesis that the air-space systems of the upper and lower mesophyll are separated by a defined barrier of measurable conductance. A mathematical procedure, based on this hypothesis, is developed for the quantitative separation of the contributions made by the upper and lower halves of the mesophyll to carbon assimilation using gasexchange data. Serial paradermal sections and three-dimensional scanning-electron-microscope images confirmed the hypothesis that there were few connections between the two air-systems. Simultaneous measurements of nitrous-oxide diffusion across the leaf and of transpiration from the two surfaces showed that the internal conductance was about 15% of the maximum observed stomatal conductance. This demonstrates that the poor air-space connections, indicated by microscopy, represent a substantial barrier to gas diffusion. By measuring the CO₂ and water-vapour fluxes from each surface independently, the intercellular CO₂ concentration (c _i) of each internal air-space system was determined and the flux between them calculated. This allowed correction of the apparent CO₂ uptake at each surface to derive the true CO₂ uptake by the mesophyll cells of the upper and lower halves of the leaf. This approach was used to analyse the contribution of the upper and lower mesophyll to CO₂ uptake by the leaf as a whole in response to varying light levels incident on the upper leaf surface. This showed that the upper mesophyll was light-saturated by a photon flux of approx. 1000 mol·m^-2·s^-1 (i.e. about one-half of full sunlight). The lower mesophyll was not fully saturated by photon fluxes of nearly double full sunlight. At low photon fluxes the c _i of the upper mesophyll was significantly less than that of the lower mesophyll, generating a significant upward flux of CO₂. At light levels equivalent to full sunlight, and above, c _i did not differ significantly between the two air space systems. The physiological importance of the separation of the air-space systems of the upper and lower mesophyll to gas exchange is discussed.Abbreviations and symbols A net leaf CO₂ uptake rate - A _upper ^app. and A _lower ^app. net rates of CO₂ uptake across the upper and lower surfaces - A _upper and A _lower derived net rates of CO₂ uptake by the upper and lower mesophyll - A _upward net flux of CO₂ from the lower to upper mesophyll - c _a, c _{a, upper} and c _{a, lower} the CO₂ concentrations in the air around the leaf above the upper surface and below the lower surface - c _N2O the concentration of N₂O in the air around the leaf - c _i, c _{i, upper} and c _{i, lower} the mesophyll intercellular CO₂ concentration of the whole leaf, the upper mesophyll and the lower mesophyll - g _i leaf internal conductance to CO₂ - g _s, g _{s, lower} and g _{s, upper} the stomatal conductance of the whole leaf, the lower surface and the upper surface - g the total conductance across the leaf - Q the photosynthetically active photon flux density