Plant water status and genotype affect fruit respiration in grapevines.

An understanding of fruit gas exchange is necessary to determine the carbon balance in grapevines, but little attention has been paid to the relationships among fruit respiration, plant water status and genetic variability. The effect of plant water status and genotype on cluster respiration was studied over two seasons (2013 and 2014) under field conditions using a whole cluster respiration chamber. Whole cluster CO2 fluxes were measured in growing grapevines at hard-green, veraison and ripening stages under irrigated and non-irrigated conditions, and under light and dark conditions in two grapevine varieties, Tempranillo and Grenache. A direct relationship between cluster CO2 efflux and plant water status was found at hard-green stage. Genotype influenced the fruit CO2 efflux that resulted in higher carbon losses in Tempranillo than in Grenache. Fruit respiration rates decreased from the first berry developmental stages to ripening stage. The integration of fruit respiration rates under light and dark conditions showed the magnitude of fruit carbon losses and gains as well as interesting variety and environmental conditions effects on those processes.

Leaf growth rate and nitrogen content determine respiratory costs during leaf expansion in grapevines.

Respiration processes are well recognized as fundamental for the plant carbon balance, but little attention has been paid to the relationships among respiration rates, environment and genetic variability. This can be of particular interest to understand the differences in net carbon balances in crops as grapevines. Night respiration (Rn ) and its associated growth (Rg ) and maintenance (Rm ) components were evaluated during leaf expansion in two grapevine cultivars (Tempranillo cv. and Garnacha cv.) that differ in their plant growth pattern and carbon balance. Simultaneously, leaf traits as leaf mass area, nitrogen (N) and carbon (C) content were evaluated in order to relate to the respiratory processes and the leaf growth. The results showed the differences in respiration rates associated with the leaf expansion pattern. Tempranillo developed leaves with higher leaf area and lower dry weight per leaf unit than Garnacha. Although differences between cultivars were observed in terms of growth costs in expanding leaves, the maintenance costs were similar for both cultivars. Also, a significant linear regression was found between respiration rates and N content in expanding and mature leaves. The results indicate that differences in structure and nitrogen content of expanding leaves may lead to respiratory differences between cultivars. These results also demonstrate the importance of respiratory cost components in carbon balance calculations in grapevines.

Combined effect of virus infection and water stress on water flow and water economy in grapevines.

Water limitation is one of the major threats affecting grapevine production. Thus, improving water-use efficiency (WUE) is crucial for a sustainable viticulture industry in Mediterranean regions. Under field conditions, water stress (WS) is often combined with viral infections as those are present in major grape-growing areas worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting grapevines. Indeed, the optimization of water use in a real context of virus infection is an important topic that needs to be understood. In this work, we have focused our attention on determining the interaction of biotic and abiotic stresses on WUE and hydraulic conductance (Kh ) parameters in two white grapevine cultivars (Malvasia de Banyalbufar and Giró Ros). Under well-watered (WW) conditions, virus infection provokes a strong reduction (P < 0.001) in Kpetiole in both cultivars; however, Kleaf was only reduced in Malvasia de Banyalbufar. Moreover, the presence of virus also reduced whole-plant hydraulic conductance (Khplant ) in 2013 and 2014 for Malvasia de Banyalbufar and in 2014 for Giró Ros. Thus, the effect of virus infection on water flow might explain the imposed stomatal limitation. Under WS conditions, the virus effect on Kplant was negligible, because of the bigger effect of WS than virus infection. Whole-plant WUE (WUEWP ) was not affected by the presence of virus neither under WW nor under WS conditions, indicating that plants may adjust their physiology to counteract the virus infection by maintaining a tight stomatal control and by sustaining a balanced carbon change.

Exploring the genetic variability in water use efficiency: Evaluation of inter and intra cultivar genetic diversity in grapevines.

Genetic improvement of crop Water Use Efficiency (WUE) is a general goal because the increasing water scarcity and the trend to a more sustainable agriculture. For grapevines, this subject is relevant and need an urgent response because their wide distribution in semi-arid areas. New cultivars are difficult to introduce in viticulture due to the narrow dependency of consumer appreciation often linked to a certain particular wine taste. Clones of reputed cultivars would presumably be more accepted but little is known on the intra-cultivar genetic variability of the WUE. The present work compares, on the basis of two field assays, the variability of intrinsic water use efficiency (WUEi) in a large collection of cultivars in contrast with a collection of clones of Tempranillo cultivar. The results show that clonal variability of WUEi was around 80% of the inter-cultivar, thus providing a first assessment on the opportunity for clonal selection by WUE. Plotting the WUEi data against stem water potential or stomatal conductance it was possible to identify cultivars and clones out of the confidence intervals of this linear regression thus with significantly higher and lower WUEi values. The present results contribute to open the expectative for a genetic improvement of grapevine WUE.

The photosynthetic capacity in 35 ferns and fern allies: mesophyll CO2 diffusion as a key trait.

Ferns and fern allies have low photosynthetic rates compared with seed plants. Their photosynthesis is thought to be limited principally by physical CO2 diffusion from the atmosphere to chloroplasts. The aim of this study was to understand the reasons for low photosynthesis in species of ferns and fern allies (Lycopodiopsida and Polypodiopsida). We performed a comprehensive assessment of the foliar gas-exchange and mesophyll structural traits involved in photosynthetic function for 35 species of ferns and fern allies. Additionally, the leaf economics spectrum (the interrelationships between photosynthetic capacity and leaf/frond traits such as leaf dry mass per unit area or nitrogen content) was tested. Low mesophyll conductance to CO2 was the main cause for low photosynthesis in ferns and fern allies, which, in turn, was associated with thick cell walls and reduced chloroplast distribution towards intercellular mesophyll air spaces. Generally, the leaf economics spectrum in ferns follows a trend similar to that in seed plants. Nevertheless, ferns and allies had less nitrogen per unit DW than seed plants (i.e. the same slope but a different intercept) and lower photosynthesis rates per leaf mass area and per unit of nitrogen.

Plasticity of vulnerability to leaf hydraulic dysfunction during acclimation to drought in grapevines: an osmotic-mediated process.

Previous studies have reported correlation of leaf hydraulic vulnerability with pressure-volume parameters related to cell turgor. This link has been explained on the basis of the effects of turgor on connectivity among cells and tissue structural integrity, which affect leaf water transport. In this study, we tested the hypothesis that osmotic adjustment to water stress would shift the leaf vulnerability curve toward more negative water potential (Ψ leaf ) by increasing turgor at low Ψ leaf . We measured leaf hydraulic conductance (K leaf ), K leaf vulnerability [50 and 80% loss of K leaf (P50 and P80 ); |Ψ leaf | at 50 and 80% loss of K leaf , respectively), bulk leaf water relations, leaf gas exchange and sap flow in two Vitis vinifera cultivars (Tempranillo and Grenache), under two water treatments. We found that P50 , P80 and maximum K leaf decreased seasonally by more than 20% in both cultivars and watering treatments. However, K leaf at 2 MPa increased threefold, while osmotic potential at full turgor and turgor loss point decreased. Our results indicate that leaf resistance to hydraulic dysfunction is seasonally plastic, and this plasticity may be mediated by osmotic adjustment.

Is stomatal conductance optimized over both time and space in plant crowns? A field test in grapevine (Vitis vinifera).

Crown carbon gain is maximized for a given total water loss if stomatal conductance (gs ) varies such that the marginal carbon product of water (∂A/∂E) remains invariant both over time and among leaves in a plant crown, provided the curvature of assimilation rate (A) versus transpiration rate (E) is negative. We tested this prediction across distinct crown positions in situ for the first time by parameterizing a biophysical model across 14 positions in four grapevine crowns (Vitis vinifera), computing optimal patterns of gs and E over a day and comparing these to the observed patterns. Observed water use was higher than optimal for leaves in the crown interior, but lower than optimal in most other positions. Crown carbon gain was 18% lower under measured gs than under optimal gs . Positive curvature occurred in 39.6% of cases due to low boundary layer conductance (gbw ), and optimal gs was zero in 11% of cases because ∂A/∂E was below the target value at all gs . Some conclusions changed if we assumed infinite gbw , but optimal and measured E still diverged systematically in time and space. We conclude that the theory's spatial dimension and assumption of positive curvature require further experimental testing.

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 (A N) relative to stomatal conductance (g s). 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 (g m), g s, and foliage A N, it was shown that both g s and g m limit A N, 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 A N/g s) should correlate on the ratio g m/g s, and not g m itself. Such a correlation is indeed often observed in the data. However, since besides diffusion A N also depends on photosynthetic capacity (i.e., V c,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 A N and WUE. In fact, such a response has never been observed in genetically modified plants specifically engineered for either reduced g s or enhanced g m. Although increasing g m 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 g s. We conclude that for simultaneous improvement of A N and WUE, genetic manipulation of g m should avoid parallel changes in g s, and we suggest that the appropriate trait for selection for enhanced WUE is increased g m/g s.

Importance of leaf anatomy in determining mesophyll diffusion conductance to CO2 across species: quantitative limitations and scaling up by models.

Foliage photosynthetic and structural traits were studied in 15 species with a wide range of foliage anatomies to gain insight into the importance of key anatomical traits in the limitation of diffusion of CO2 from substomatal cavities to chloroplasts. The relative importance of different anatomical traits in constraining CO2 diffusion was evaluated using a quantitative model. Mesophyll conductance (g m) was most strongly correlated with chloroplast exposed surface to leaf area ratio (S c/S) and cell wall thickness (T cw), but, depending on foliage structure, the overall importance of g m in constraining photosynthesis and the importance of different anatomical traits in the restriction of CO2 diffusion varied. In species with mesophytic leaves, membrane permeabilities and cytosol and stromal conductance dominated the variation in g m. However, in species with sclerophytic leaves, g m was mostly limited by T cw. These results demonstrate the major role of anatomy in constraining mesophyll diffusion conductance and, consequently, in determining the variability in photosynthetic capacity among species.

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.

Photosynthesis limitations during water stress acclimation and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandierixV. rupestris).

The hybrid Richter-110 (Vitis berlandierixVitis rupestris) has the reputation of being a genotype strongly adapted to drought. A study was performed with plants of R-110 subjected to sustained water-withholding to induce acclimation to two different levels of water stress, followed by rewatering to induce recovery. The goal was to analyse how photosynthesis is regulated during acclimation to water stress and recovery. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence and thermoluminescence), and biochemistry (V(c,max)) were assessed. During water stress, g(s) declined to 0.1 and less than 0.05 mol CO(2) m(-2) s(-1) in moderately and severely water-stressed plants, respectively, and was kept quite constant during an acclimation period of 1-week. Leaf photochemistry proved to be very resistant to the applied water-stress conditions. By contrast, g(m) and V(c,max) were affected by water stress, but they were not kept constant during the acclimation period. g(m) was initially unaffected by water stress, and V(c,max) even increased above control values. However, after several days of acclimation to water stress, both parameters declined below (g(m)) or at (V(c,max)) control values. For the latter two parameters there seemed to be an interaction between water stress and cumulative irradiance, since both recovered to control values after several cloudy days despite water stress. A photosynthesis limitation analysis revealed that diffusional limitations and not biochemical limitations accounted for the observed decline in photosynthesis during water stress and slow recovery after rewatering, both in moderately and severely stressed plants. However, the relative contribution of stomatal (SL) and mesophyll conductance (MCL) limitations changes during acclimation to water stress, from predominant SL early during water stress to similar SL and MCL after acclimation. Finally, photosynthesis recovery after rewatering was mostly limited by SL, since stomatal closure recovered much more slowly than g(m).

The role of mesophyll conductance during water stress and recovery in tobacco (Nicotiana sylvestris): acclimation or limitation?

While the responses of photosynthesis to water stress have been widely studied, acclimation to sustained water stress and recovery after re-watering is poorly understood. In particular, the factors limiting photosynthesis under these conditions, and their possible interactions with other environmental conditions, are unknown. To assess these issues, changes of photosynthetic CO(2) assimilation (A(N)) and its underlying limitations were followed during prolonged water stress and subsequent re-watering in tobacco (Nicotiana sylvestris) plants growing under three different climatic conditions: outdoors in summer, outdoors in spring, and indoors in a growth chamber. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence), and biochemistry (V(c,max)) were assessed. Leaf gas exchange and chlorophyll fluorescence data revealed that water stress induced a similar degree of stomatal closure and decreased A(N) under all three conditions, while V(c,max) was unaffected. However, the behaviour of g(m) differed depending on the climatic conditions. In outdoor plants, g(m) strongly declined with water stress, but it recovered rapidly (1-2 d) after re-watering in spring while it remained low many days after re-watering in summer. In indoor plants, g(m) initially declined with water stress, but then recovered to control values during the acclimation period. These differences were reflected in different velocities of recovery of A(N) after re-watering, being the slowest in outdoor summer plants and the fastest in indoor plants. It is suggested that these differences among the experiments are related to the prevailing climatic conditions, i.e. to the fact that stress factors other than water stress have been superimposed (e.g. excessive light and elevated temperature). In conclusion, besides g(s), g(m) contributes greatly to the limitation of photosynthesis during water stress and during recovery from water stress, but its role is strongly dependent on the impact of additional environmental factors.

Adjustments of water use efficiency by stomatal regulation during drought and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandieri x V. rupestris).

The hybrid Richter-110 (Vitis berlandieri x Vitis rupestris) (R-110) has the reputation of being a genotype strongly adapted to drought. A study was performed with plants of R-110 subjected to water withholding followed by re-watering. The goal was to analyze how stomatal conductance (g(s)) is regulated with respect to different physiological variables under water stress and recovery, as well as how water stress affects adjustments of water use efficiency (WUE) at the leaf level. Water stress induced a substantial stomatal closure and an increase in WUE, which persisted many days after re-watering. The g(s) during water stress was mainly related to the content of ABA in the xylem and partly related to plant hydraulic conductivity but not to leaf water potential. By contrast, low g(s) during re-watering did not correlate with ABA contents and was only related to a sustained decreased hydraulic conductivity. In addition to a complex physiological regulation of stomatal closure, g(s) and rate of transpiration (E) were strongly affected by leaf-to-air vapor pressure deficit (VPD) in a way dependent of the treatment. Interestingly, E increased with increasing VPD in control plants, but decreased with increasing VPD in severely stressed plants. All together, the fine stomatal regulation in R-110 resulted in very high WUE at the leaf level. This genotype is revealed to be very interesting for further studies on the physiological mechanisms leading to regulation of stomatal responsiveness and WUE in response to drought.

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA.

The reactions of glyoxal with 2'-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, (1)H and (13)C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx(2)-dG) and N(2)-(carboxymethyl)-9-(2'-deoxy-beta-D-erythro-pentofuranosyl)-purin-6(9H)-one (Gx(1)-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx(2)-dG were found to be unstable and partly transformed to Gx(1)-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2'-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2'-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx(1)-dG was the only stable adduct. The transformation of Gx-dG to Gx(1)-dG seemed to take place in single-stranded DNA and therefore, Gx(1)-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.

Aquaporin expression in response to different water stress intensities and recovery in Richter-110 (Vitis sp.): relationship with ecophysiological status.

Aquaporins seem essential for the regulation of plant water status and expenses. Richter-110 is a Vitis hybrid (Vitis berlandieri x rupestris) reputed to be strongly drought-tolerant. Three irrigation treatments were established in Richter-110 plants growing outdoors defined by the resulting maximum stomatal conductance (g (s)), and ensuring water stress situations not severe enough as to stop photosynthesis and growth: well-watered plants (g (s) about 250 mmol H(2)O m(-2) s(-1)), moderate water stress (g (s) about 150 mmol H(2)O m(-2) s(-1)) and severe water stress (g (s) about 50 mmol H(2)O m(-2) s(-1)). Plants under water stress were kept at constant water availability for 7 days to check for possible acclimation. Finally, plants were re-watered, and allowed to recover, for 3 days. Stomatal conductance, leaf water potential, xylem abscisic acid (ABA) content and root and stem hydraulic conductivity were determined. The relative amounts of expression of mRNA encoding seven putative aquaporins were determined in roots and leaves by RT-PCR. The decrease in stomatal conductance with moderate and severe water stress was associated with increasing ABA contents, but not with the leaf water potential and hydraulic conductivities, which remained unchanged during the entire experiment. Aquaporin gene expression varied depending on which aquaporin, water stress level and the plant organ. We suggest that aquaporin expression was responsive to water stress as part of the homeostasis, which resulted in constant leaf water potential and hydraulic conductivity.