Antonio Diaz-Espejo

Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis

Mesophyll diffusion conductance to CO(2) is a key photosynthetic trait that has been studied intensively in the past years. The intention of the present review is to update knowledge of g(m), and highlight the important unknown and controversial aspects that require future work. The photosynthetic limitation imposed by mesophyll conductance is large, and under certain conditions can be the most significant photosynthetic limitation. New evidence shows that anatomical traits, such as cell wall thickness and chloroplast distribution are amongst the stronger determinants of mesophyll conductance, although rapid variations in response to environmental changes might be regulated by other factors such as aquaporin conductance. Gaps in knowledge that should be research priorities for the near future include: how different is mesophyll conductance among phylogenetically distant groups and how has it evolved? Can mesophyll conductance be uncoupled from regulation of the water path? What are the main drivers of mesophyll conductance? The need for mechanistic and phenomenological models of mesophyll conductance and its incorporation in process-based photosynthesis models is also highlighted.

Role of mesophyll diffusion conductance in constraining potential photosynthetic productivity in the field

Limited mesophyll diffusion conductance to CO(2) (g(m)) can significantly constrain plant photosynthesis, but the extent of g(m)-limitation is still imperfectly known. As g(m) scales positively with foliage photosynthetic capacity (A), the CO(2) drawdown from substomatal cavities (C(i)) to chloroplasts (C(C), C(i)-C(C)=A/g(m)) rather than g(m) alone characterizes the mesophyll diffusion limitations of photosynthesis. The dependencies of g(m) on A, foliage structure (leaf dry mass per unit area, M(A)), and the resulting drawdowns across a dataset of 81 species of contrasting foliage structure and photosynthetic potentials measured under non-stressed conditions were analysed to describe the structure-driven potential photosynthetic limitations due to g(m). Further the effects of key environmental stress factors and leaf and plant developmental alterations on g(m) and CO(2) drawdown were evaluated and the implications of varying g(m) on foliage photosynthesis in the field were simulated. The meta-analysis demonstrated that g(m) of non-stressed leaves was negatively correlated with M(A), and despite the positive relationship between g(m) and A, the CO(2) drawdown was larger in leaves with more robust structure. The correlations were stronger with mass-based g(m) and A, probably reflecting the circumstance that mesophyll diffusion is a complex three-dimensional process that scales better with mesophyll volume-weighted than with leaf area-weighted traits. The analysis of key environmental stress effects on g(m) and CO(2) drawdowns demonstrated that the effect of individual stresses on CO(2) drawdowns varies depending on the stress effects on foliage structure and assimilation rates. Leaf diffusion limitations are larger in non-senescent older leaves and also in senescent leaves, again reflecting more robust leaf structure and/or non-co-ordinated alterations in leaf photosynthesis and g(m). According to simulation analyses, in plants with a larger part of the overall diffusion conductance from the ambient atmosphere to the chloroplasts in the mesophyll, photosynthesis is less sensitive to changes in stomatal conductance. Accordingly, in harsher environments that support vegetation with tougher long-living stress-tolerant leaves with lower g(m), reductions in stomatal conductance that are common during stress periods are expected to alter photosynthesis less than in species where a larger part of the total diffusion limitation is determined by stomata. While structural robustness improves plant performance under environmental stress, low g(m) and inherently large CO(2) drawdown in robust leaves limits the photosynthesis of these plants more severely under favourable conditions when stomatal conductance is high. The differences in overall responsiveness to environmental modifications of plants with varying g(m) need consideration in current large-scale ecosystem productivity models.

Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency

A key objective for sustainable agriculture and forestry is to breed plants with both high carbon gain and water-use efficiency (WUE). At the level of leaf physiology, this implies increasing net photosynthesis (AN) relative to stomatal conductance (gs). Here, we review evidence for CO2 diffusional constraints on photosynthesis and WUE. Analyzing past observations for an extensive pool of crop and wild plant species that vary widely in mesophyll conductance to CO2 (gm), gs, and foliage AN, it was shown that both gs and gm limit AN, although the relative importance of each of the two conductances depends on species and conditions. Based on Fick’s law of diffusion, intrinsic WUE (the ratio AN/gs) should correlate on the ratio gm/gs, and not gm itself. Such a correlation is indeed often observed in the data. However, since besides diffusion AN also depends on photosynthetic capacity (i.e., Vc,max), this relationship is not always sustained. It was shown that only in a very few cases, genotype selection has resulted in simultaneous increases of both AN and WUE. In fact, such a response has never been observed in genetically modified plants specifically engineered for either reduced gs or enhanced gm. Although increasing gm alone would result in increasing photosynthesis, and potentially increasing WUE, in practice, higher WUE seems to be only achieved when there are no parallel changes in gs. We conclude that for simultaneous improvement of AN and WUE, genetic manipulation of gm should avoid parallel changes in gs, and we suggest that the appropriate trait for selection for enhanced WUE is increased gm/gs.

The use of sap flow measurements for scheduling irrigation in olive, apple and Asian pear trees and in grapevines

We evaluated three approaches for scheduling irrigation in wine grape vineyards and in olive, apple and Asian pear tree orchards, based on sap flow measurements and models of plant transpiration. In the first approach, we analysed how the shape of the sap-flow profile changed in response to root-zone soil water conditions and potential evaporative demand. The second approach was based on a transpiration ratio, as defined from the actual daily water use of a target plant divided by the potential daily water use of similar-sized plants under non-limiting soil water conditions (“well-irrigated” plants). Values of the actual plant water use were always determined from measured sap flow. Two independent methods were assessed for the calculation of potential plant water use; either sap flow was measured in well-irrigated plants or we used a leaf-area based model of plant transpiration. On some occasions water stress was found to modify the shape of the sap velocity profile. However, most of the time the velocity profile was found to be an insensitive indicator for triggering irrigation. The transpiration ratio method, using measured sap flow in well-irrigated plants, was more useful for irrigation scheduling, at least for the two species (i.e. olive and grape) that were investigated here. Nonetheless, realization of such an approach in a commercial orchard may not be practical due to problems associated with irrigation management e.g. excessive vegetative growth may occur on the reference plants over time. Besides, irrigating the orchard to maintain non-limiting soil water conditions is not always the best option for water and nutrient management. The alternative transpiration ratio method based on a leaf-area based model of plant water use, yielded the best results. Modelled transpiration rates always provided reliable information not only for well-irrigated plants, but also for deficit-irrigated plants. This result lends support to the use of the method for irrigation scheduling of vineyard and orchard trees. However, the use of models does require detailed microclimate data as well as a user-friendly technique to quantify plant leaf area. From a practical viewpoint the method should encompass the spatial variability of the soil and plants within the orchard. Accurate quantification of these factors is a cornerstone of precision horticulture and such information would help to minimise risks associated with insufficient as well as excessive irrigation applications.

Importance of mesophyll diffusion conductance in estimation of plant photosynthesis in the field

Mesophyll diffusion conductance to CO(2) (g(m)) is an important leaf characteristic determining the drawdown of CO(2) from substomatal cavities (C(i)) to chloroplasts (C(C)). Finite g(m) results in modifications in the shape of the net assimilation (A) versus C(i) response curves, with the final outcome of reduced maximal carboxylase activity of Rubisco (V(cmax)), and a greater ratio of the capacity for photosynthetic electron transport to V(cmax) (J(max)/V(cmax)) and alterations in mitochondrial respiration rate (R(d)) when estimated from A/C(i) responses without considering g(m). The influence of different Farquhar et al. model parameterizations on daily photosynthesis under non-stressed (C(i) kept constant throughout the day) and stressed conditions (mid-day reduction in C(i)) was compared. The model was parameterized on the basis of A/C(C) curves and A/C(i) curves using both the conventional fitting procedure (V(cmax) and R(d) fitted separately to the linear part of the response curve and J(max) to the saturating part) and a procedure that fitted all parameters simultaneously. The analyses demonstrated that A/C(i) parameterizations overestimated daily assimilation by 6-8% for high g(m) values, while they underestimated if by up to 70% for low g(m) values. Qualitative differences between the A/C(i) and A/C(C) parameterizations were observed under stressed conditions, when underestimated V(cmax) and overestimated R(d) of A/C(i) parameterizations resulted in excessive mid-day depression of photosynthesis. Comparison with measured diurnal assimilation rates in the Mediterranean sclerophyll species Quercus ilex under drought further supported this bias of A/C(i) parameterizations. While A/C(i) parameterization predicted negative carbon balance at mid-day, actual measurements and simulations with the A/C(C) approach yielded positive carbon gain under these conditions. In addition, overall variation captured by the best A/C(i) parameterization was poor compared with the A/C(C) approach. This analysis strongly suggests that for correct parameterization of daily time-courses of photosynthesis under realistic field conditions, g(m) must be included in photosynthesis models.

Interactive effects of soil water deficit and air vapour pressure deficit on mesophyll conductance to CO2 in Vitis vinifera and Olea europaea

The present work aims to study the interactive effect of drought stress and high vapour pressure deficit (VPD) on leaf gas exchange, and especially on mesophyll conductance to CO(2) (g(m)), in two woody species of great agronomical importance in the Mediterranean basin: Vitis vinifera L. cv. Tempranillo and Olea europaea L. cv. Manzanilla. Plants were grown in specially designed outdoor chambers with ambient and below ambient VPD, under both well-irrigated and drought conditions. g(m) was estimated by the variable J method from simultaneous measurements of gas exchange and fluorescence. In both species, the response to soil water deficit was larger in g(s) than in g(m), and more important than the response to VPD. Olea europaea was apparently more sensitive to VPD, so that plants growing in more humid chambers showed higher g(s) and g(m). In V. vinifera, in contrast, soil water deficit dominated the response of g(s) and g(m). Consequently, changes in g(m)/g(s) were more related to VPD in O. europaea and to soil water deficit in V. vinifera. Most of the limitations of photosynthesis were diffusional and especially due to stomatal closure. No biochemical limitation was detected. The results showed that structural parameters played an important role in determining g(m) during the acclimation process. Although the relationship between leaf mass per unit area (M(A)) with g(m) was scattered, it imposed a limitation to the maximum g(m) achievable, with higher values of M(A) in O. europaea at lower g(m) values. M(A) decreased under water stress in O. europaea but it increased in V. vinifera. This resulted in a negative relationship between M(A) and the CO(2) draw-down between substomatal cavities and chloroplasts in O. europaea, while being positive in V. vinifera.

Regulation of photosynthesis and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins

Summary In plants with sclerophyll leaves, the response of stomatal and mesophyll conductance to CO2 to water stress and recovery is correlated with the expression of aquaporins and carbonic anhydrase.

Rapid hydraulic recovery in Eucalyptus pauciflora after drought: linkages between stem hydraulics and leaf gas exchange.

In woody plants, photosynthetic capacity is closely linked to rates at which the plant hydraulic system can supply water to the leaf surface. Drought-induced embolism can cause sharp declines in xylem hydraulic conductivity that coincide with stomatal closure and reduced photosynthesis. Recovery of photosynthetic capacity after drought is dependent on restored xylem function, although few data exist to elucidate this coordination. We examined the dynamics of leaf gas exchange and xylem function in Eucalyptus pauciflora seedlings exposed to a cycle of severe water stress and recovery after re-watering. Stomatal closure and leaf turgor loss occurred at water potentials that delayed the extensive spread of embolism through the stem xylem. Stem hydraulic conductance recovered to control levels within 6 h after re-watering despite a severe drought treatment, suggesting an active mechanism embolism repair. However, stomatal conductance did not recover after 10 d of re-watering, effecting tighter control of transpiration post drought. The dynamics of recovery suggest that a combination of hydraulic and non-hydraulic factors influenced stomatal behaviour post drought.

Vulnerability to cavitation in Olea europaea current-year shoots: further evidence of an open-vessel artifact associated with centrifuge and air-injection techniques

Different methods have been devised to analyze vulnerability to cavitation of plants. Although a good agreement between them is usually found, some discrepancies have been reported when measuring samples from long-vesseled species. The aim of this study was to evaluate possible artifacts derived from different methods and sample sizes. Current-year shoot segments of mature olive trees (Olea europaea), a long-vesseled species, were used to generate vulnerability curves (VCs) by bench dehydration, pressure collar and both static- and flow-centrifuge methods. For the latter, two different rotors were used to test possible effects of the rotor design on the curves. Indeed, high-resolution computed tomography (HRCT) images were used to evaluate the functional status of xylem at different water potentials. Measurements of native embolism were used to validate the methods used. The pressure collar and the two centrifugal methods showed greater vulnerability to cavitation than the dehydration method. The shift in vulnerability thresholds in centrifuge methods was more pronounced in shorter samples, supporting the open-vessel artifact hypothesis as a higher proportion of vessels were open in short samples. The two different rotor designs used for the flow-centrifuge method revealed similar vulnerability to cavitation. Only the bench dehydration or HRCT methods produced VCs that agreed with native levels of embolism and water potential values measured in the field.

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 (gm ) 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 gm and/or Rubisco can result in increased WUE; (2) to review the degree of success of early attempts to genetically manipulate gm or Rubisco; (3) to analyse how gm , gsw and the Rubiscos maximum velocity (Vcmax ) 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 gm and Rubisco catalytic constants vis-à-vis gsw under water limitations.