Igor Florez-Sarasa

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.

The Mediterranean evergreen Quercus ilex and the semi-deciduous Cistus albidus differ in their leaf gas exchange regulation and acclimation to repeated drought and re-watering cycles

Plants may exhibit some degree of acclimation after experiencing drought, but physiological adjustments to consecutive cycles of drought and re-watering (recovery) have scarcely been studied. The Mediterranean evergreen holm oak (Q. ilex) and the semi-deciduous rockrose (C. albidus) showed some degree of acclimation after the first of three drought cycles (S1, S2, and S3). For instance, during S2 and S3 both species retained higher relative leaf water contents than during S1, despite reaching similar leaf water potentials. However, both species showed remarkable differences in their photosynthetic acclimation to repeated drought cycles. Both species decreased photosynthesis to a similar extent during the three cycles (20–40% of control values). However, after S1 and S2, photosynthesis recovered only to 80% of control values in holm oak, due to persistently low stomatal (gs) and mesophyll (gm) conductances to CO2. Moreover, leaf intrinsic water use efficiency (WUE) was kept almost constant in this species during the entire experiment. By contrast, photosynthesis of rockrose recovered almost completely after each drought cycle (90–100% of control values), while the WUE was largely and permanently increased (by 50–150%, depending on the day) after S1. This was due to a regulation which consisted in keeping gs low (recovering to 50–60% of control values after re-watering) while maintaining a high gm (even exceeding control values during re-watering). While the mechanisms to achieve such particular regulation of water and CO2 diffusion in leaves are unknown, it clearly represents a unique acclimation feature of this species after a drought cycle, which allows it a much better performance during successive drought events. Thus, differences in the photosynthetic acclimation to repeated drought cycles can have important consequences on the relative fitness of different Mediterranean species or growth forms within the frame of climate change scenarios.

Stomatal and mesophyll conductances to CO2 are the main limitations to photosynthesis in sugar beet (Beta vulgaris) plants grown with excess zinc

*The effects of zinc (Zn) toxicity on photosynthesis and respiration were investigated in sugar beet (Beta vulgaris) plants grown hydroponically with 1.2, 100 and 300 microM Zn. *A photosynthesis limitation analysis was used to assess the stomatal, mesophyll, photochemical and biochemical contributions to the reduced photosynthesis observed under Zn toxicity. *The main limitation to photosynthesis was attributable to stomata, with stomatal conductances decreasing by 76% under Zn excess and stomata being unable to respond to physiological and chemical stimuli. The effects of excess Zn on photochemistry were minor. Scanning electron microscopy showed morphological changes in stomata and mesophyll tissue. Stomatal size and density were smaller, and stomatal slits were sealed in plants grown under high Zn. Moreover, the mesophyll conductance to CO(2) decreased by 48% under Zn excess, despite a marked increase in carbonic anhydrase activity. Respiration, including that through both cytochrome and alternative pathways, was doubled by high Zn. *It can be concluded that, in sugar beet plants grown in the presence of excess Zn, photosynthesis is impaired due to a depletion of CO(2) at the Rubisco carboxylation site, as a consequence of major decreases in stomatal and mesophyll conductances to CO(2).

Metabolite Profiles of Maize Leaves in Drought, Heat, and Combined Stress Field Trials Reveal the Relationship between Metabolism and Grain Yield

Foliar metabolite levels, including myoinositol, show correlation with grain yield in tropical maize field trials during drought, heat, and simultaneous drought/heat stresses. The development of abiotic stress-resistant cultivars is of premium importance for the agriculture of developing countries. Further progress in maize (Zea mays) performance under stresses is expected by combining marker-assisted breeding with metabolite markers. In order to dissect metabolic responses and to identify promising metabolite marker candidates, metabolite profiles of maize leaves were analyzed and compared with grain yield in field trials. Plants were grown under well-watered conditions (control) or exposed to drought, heat, and both stresses simultaneously. Trials were conducted in 2010 and 2011 using 10 tropical hybrids selected to exhibit diverse abiotic stress tolerance. Drought stress evoked the accumulation of many amino acids, including isoleucine, valine, threonine, and 4-aminobutanoate, which has been commonly reported in both field and greenhouse experiments in many plant species. Two photorespiratory amino acids, glycine and serine, and myoinositol also accumulated under drought. The combination of drought and heat evoked relatively few specific responses, and most of the metabolic changes were predictable from the sum of the responses to individual stresses. Statistical analysis revealed significant correlation between levels of glycine and myoinositol and grain yield under drought. Levels of myoinositol in control conditions were also related to grain yield under drought. Furthermore, multiple linear regression models very well explained the variation of grain yield via the combination of several metabolites. These results indicate the importance of photorespiration and raffinose family oligosaccharide metabolism in grain yield under drought and suggest single or multiple metabolites as potential metabolic markers for the breeding of abiotic stress-tolerant maize.

The Peclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO2

Leaf water gets isotopically enriched through transpiration, and diffusion of enriched water through the leaf depends on transpiration flow and the effective path length (L). The aim of this work was to relate L with physiological variables likely to respond to similar processes. We studied the response to drought and vein severing of leaf lamina hydraulic conductance (K(lamina) ), mesophyll conductance for CO(2) (g(m) ) and leaf water isotope enrichment in Vitis vinifera L cv. Grenache. We hypothesized that restrictions in water pathways would reduce K(lamina) and increase L. As a secondary hypothesis, we proposed that, given the common pathways for water and CO(2) involved, a similar response should be found in g(m) . Our results showed that L was strongly related to mesophyll variables, such as K(lamina) or g(m) across experimental drought and vein-cutting treatments, showing stronger relationships than with variables included as input parameters for the models, such as transpiration. Our findings were further supported by a literature survey showing a close link between L and leaf hydraulic conductance (K(leaf) = 31.5 × L(-0.43) , r(2) = 0.60, n = 24). The strong correlation found between L, K(lamina) and g(m) supports the idea that water and CO(2) share an important part of their diffusion pathways through the mesophyll.

In vivo cytochrome and alternative pathway respiration in leaves of Arabidopsis thaliana plants with altered alternative oxidase under different light conditions

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Effects of drought stress and subsequent rewatering on photosynthetic and respiratory pathways in Nicotiana sylvestris wild type and the mitochondrial complex I-deficient CMSII mutant

The interaction of photosynthesis and respiration has been studied in vivo under conditions of limited water supply and after consecutive rewatering. The role of the alternative (valt) and cytochrome (vcyt) pathways on drought stress-induced suppression of photosynthesis and during photosynthetic recovery was examined in the Nicotiana sylvestris wild type (WT) and the complex I-deficient CMSII mutant. Although photosynthetic traits, including net photosynthesis (AN), stomatal (gs) and mesophyll conductances (gm), as well as respiration (vcyt and valt) differed between well-watered CMSII and WT, similar reductions of AN, gs, and gm were observed during severe drought stress. However, total respiration (Vt) remained slightly higher in CMSII due to the still increased vcyt (to match ATP demand). valt and maximum carboxylation rates remained almost unaltered in both genotypes, while in CMSII, changes in photosynthetic light harvesting (i.e. Chl a/b ratio) were detected. In both genotypes, photosynthesis and respiration were restored after 2 d of rewatering, predominantly limited by a delayed stomatal response. Despite complex I dysfunction and hence altered redox balance, the CMSII mutant seems to be able to adjust its photosynthetic machinery during and after drought stress to reduce photo-oxidation and to maintain the cell redox state and the ATP level.

Changes of alternative oxidase activity, capacity and protein content in leaves of Cucumis sativus wild-type and MSC16 mutant grown under different light intensities

In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results.

The alternative respiratory pathway mediates carboxylate synthesis in white lupin cluster roots under phosphorus deprivation

Plant adaptations associated with a high efficiency of phosphorus (P) acquisition can be used to increase productivity and sustainability in a world with a growing population and decreasing rock phosphate reserves. White lupin (Lupinus albus) produces cluster roots that release carboxylates to efficiently mobilize P from P-sorbing soils. It has been hypothesized that an increase in the activity of the alternative oxidase (AOX) would allow for the mitochondrial oxidation of NAD(P)H produced during citrate synthesis in cluster roots at a developmental stage when there is a low demand for ATP. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in different root sections of white lupins grown hydroponically with and without P. In parallel, AOX protein levels and internal carboxylate concentrations were determined in cluster and non-cluster roots. Higher in vivo AOP activity was measured in cluster roots when malate and citrate concentrations were also high, thus confirming our hypothesis. AOX protein levels were not always correlated with in vivo AOP activity, suggesting post-translational regulation of AOX.