Leaf water potential measurements using the pressure chamber: Synthetic testing of assumptions towards best practices for precision and accuracy.

Leaf water potential (ψleaf ), typically measured using the pressure chamber, is the most important metric of plant water status, providing high theoretical value and information content for multiple applications in quantifying critical physiological processes including drought responses. Pressure chamber measurements of ψleaf (ψleafPC ) are most typical, yet, the practical complexity of the technique and of the underlying theory has led to ambiguous understanding of the conditions to optimize measurements. Consequently, specific techniques and precautions diversified across the global research community, raising questions of reliability and repeatability. Here, we surveyed specific methods of ψleafPC from multiple laboratories, and synthesized experiments testing common assumptions and practices in ψleafPC for diverse species: (i) the need for equilibration of previously transpiring leaves; (ii) leaf storage before measurement; (iii) the equilibration of ψleaf for leaves on bagged branches of a range of dehydration; (iv) the equilibration of ψleaf across the lamina for bagged leaves, and the accuracy of measuring leaves with artificially 'elongated petioles'; (v) the need in ψleaf measurements for bagging leaves and high humidity within the chamber; (vi) the need to avoid liquid water on leaf surfaces; (vii) the use of 'pulse' pressurization versus gradual pressurization; and (viii) variation among experimenters in ψleafPC determination. Based on our findings we provide a best practice protocol to maximise accuracy, and provide recommendations for ongoing species-specific tests of important assumptions in future studies.

The interplay of hydraulic failure and cell vitality explains tree capacity to recover from drought.

Global climatic models predict an increment in the frequency and intensity of drought events, which have important consequences on forest dieback. However, the mechanisms leading to tree mortality under drought conditions and the physiological thresholds for recovery are not totally understood yet. This study aimed to identify what are the key physiological traits that determine the tree capacity to recover from drought. Individuals of a conifer (Pseudotsuga menziesii M.) and an angiosperm (Prunus lusitanica L.) species were exposed to drought and their ability to recover after rehydration monitored. Results showed that the actual thresholds used for recovery from drought based on percentage loss of conductance (PLC) (i.e., 50% for conifers, 88% for angiosperms) do not provide accurate insights about the tree capacity for surviving extreme drought events. On the contrary, differences in stem relative water content (RWCStem ) and the level of electrolytes leakage (EL) were directly related to the capacity of the trees to recover from drought. This was the case for the conifer species, P. menziesii, for which higher RWCStem and lower EL values were related to the recovery capacity. Even if results showed a similar trend for the angiosperm P. lusitanica as for the conifers, differences between the two traits were much more subtle and did not allow an accurate differentiation between trees able to recover and those that were not. RWCStem and EL could work as indicators of tree capacity to recover from drought for conifers but more studies are required to confirm this observation for angiosperms.

Water contamination with atrazine: is nitric oxide able to improve Pistia stratiotes phytoremediation capacity?

Atrazine is an herbicide commonly used in several countries. Due to its long half-life, associated with its use in large scales, atrazine residues remain as environmental pollutants in water bodies. Phytoremediation is often pointed out as an interesting approach to remove atrazine from the aquatic environment, but its practical application is limited by the high toxicity of this herbicide. Here, we characterize the damages triggered by atrazine in Pistia stratiotes, evaluating the role of nitric oxide (NO), a cell-signaling molecule, in increasing the tolerance to the pollutant and the phytoremediation potential of this species. Pistia stratiotes plants were exposed to four treatments: Control; Sodium nitroprusside (SNP) (0.05 mg L-1); Atrazine (ATZ) (150 µg L-1) and ATZ + SNP. The plants remained under those conditions for 24 h for biochemical and physiological analysis and 3 days for the evaluation of relative growth rate. The presence of atrazine in plant cells triggered a series of biochemical and physiological damages, such as the increase in the generation of reactive oxygen species, damages to cell membranes, photosynthesis impairment, and negative carbon balance. Despite this, the plants maintained greater growth rates than other aquatic macrophytes exposed to atrazine and showed high bioconcentration and translocation factors. The addition of SNP, a NO donor, decreased the herbicide toxicity, with an increase of over 60% in the IC50 value (Inhibitor Concentration). Indeed, the NO signaling action was able to increase the tolerance of plants to atrazine, which resulted in increments in pollutant uptake and translocation, with the maintenance of overall cell (e.g. membranes) and organs (root system) structure, and the functioning of central physiological processes (e.g. photosynthesis). These factors allowed for more quickly and efficient removal of the pollutant from the environment, reducing costs, and increasing the viability of the phytoremediation process.

Evidence of drought memory in Dipteryx alata indicates differential acclimation of plants to savanna conditions.

The remarkable phytogeographic characteristics of the Brazilian savanna (Cerrado) resulted in a vegetation domain composed of plants with high structural and functional diversity to tolerate climate extremes. Here we used a key Cerrado species (Dipteryx alata) to evaluate if species of this domain present a mechanism of stress memory, responding more quickly and efficiently when exposed to recurrent drought episodes. The exposure of D. alata seedlings to drought resulted in several changes, mainly in physiological and biochemical traits, and these changes differed substantially when the water deficit was imposed as an isolated event or when the plants were subjected to drought cycles, suggesting the existence of a drought memory mechanism. Plants submitted to recurrent drought events were able to maintain essential processes for plant survival when compared to those submitted to drought for the first time. This differential acclimation to drought was the result of orchestrated changes in several metabolic pathways, involving differential carbon allocation for defense responses and the reprogramming and coordination of primary, secondary and antioxidant metabolism. The stress memory in D. alata is probably linked the evolutionary history of the species and reflects the environment in which it evolved.

Improving water use efficiency by changing hydraulic and stomatal characteristics in soybean exposed to drought: the involvement of nitric oxide.

A variety of cellular responses is needed to ensure the plants survival during drought, but little is known about the signaling mechanisms involved in this process. Soybean cultivars (EMBRAPA 48 and BR 16, tolerant and sensitive to drought, respectively) were exposed to the following treatments: control conditions (plants in field capacity), drought (20% of available water in the soil), sodium nitroprusside (SNP) treatment (plants irrigated and treated with 100-µM SNP [SNP-nitric oxide (NO) donor molecule], and Drought + SNP (plants subjected to drought and SNP treatment). Plants remained in these conditions until the reproductive stage and were evaluated for physiological (photosynthetic pigments, chlorophyll a fluorescence and gas exchange rates), hydraulic (water potential, osmotic potential and leaf hydraulic conductivity) and morpho-anatomical traits (biomass, venation density and stomatal characterization). Exposure to water deficit considerably reduced water potential in both cultivars and resulted in decrease in photosynthesis and biomass accumulation. The addition of the NO donor attenuated these damaging effects of water deficit and increased the tolerance index of both cultivars. The results showed that NO was able to reduce plant's water loss, while maintaining their biomass production through alteration in stomatal characteristics, hydraulic conductivity and the biomass distribution pattern. These hydraulic and morpho-anatomical alterations allowed the plants to obtain, transport and lose less water to the atmosphere, even in water deficit conditions.

Photosynthetic and metabolic acclimation to repeated drought events play key roles in drought tolerance in coffee.

Over the last decades, most information on the mechanisms underlying tolerance to drought has been gained by considering this stress as a single event that happens just once in the life of a plant, in contrast to what occurs under natural conditions where recurrent drought episodes are the rule. Here we explored mechanisms of drought tolerance in coffee (Coffea canephora) plants from a broader perspective, integrating key aspects of plant physiology and biochemistry. We show that plants exposed to multiple drought events displayed higher photosynthetic rates, which were largely accounted for by biochemical rather than diffusive or hydraulic factors, than those submitted to drought for the first time. Indeed, these plants displayed higher activities of RuBisCO and other enzymes associated with carbon and antioxidant metabolism. Acclimation to multiple drought events involved the expression of trainable genes related to drought tolerance and was also associated with a deep metabolite reprogramming with concordant alterations in central metabolic processes such as respiration and photorespiration. Our results demonstrate that plants exposed to multiple drought cycles can develop a differential acclimation that potentiates their defence mechanisms, allowing them to be kept in an 'alert state' to successfully cope with further drought events.

Corrigendum: The Involvement of Nitric Oxide in Integration of Plant Physiological and Ultrastructural Adjustments in Response to Arsenic.

[This corrects the article on p. 516 in vol. 8, PMID: 28469622.].

The Involvement of Nitric Oxide in Integration of Plant Physiological and Ultrastructural Adjustments in Response to Arsenic.

High arsenic (As) concentrations are toxic to all the living organisms and the cellular response to this metalloid requires the involvement of cell signaling agents, such as nitric oxide (NO). The As toxicity and NO signaling were analyzed in Pistia stratiotes leaves. Plants were exposed to four treatments, for 24 h: control; SNP [sodium nitroprusside (NO donor); 0.1 mg L-1]; As (1.5 mg L-1) and As + SNP (1.5 and 0.1 mg L-1, respectively). The absorption of As increased the concentration of reactive oxygen species and triggered changes in the primary metabolism of the plants. While photosynthesis and photorespiration showed sharp decrease, the respiration process increased, probably due to chemical similarity between arsenate and phosphate, which compromised the energy status of the cell. These harmful effects were reflected in the cellular structure of P. stratiotes, leading to the disruption of the cells and a possible programmed cell death. The damages were attenuated by NO, which was able to integrate central plant physiological processes, with increases in non-photochemical quenching and respiration rates, while the photorespiration level decreased. The increase in respiratory rates was essential to achieve cellular homeostasis by the generation of carbon skeletons and metabolic energy to support processes involved in responses to stress, as well to maintaining the structure of organelles and prevent cell death. Overall, our results provide an integrated view of plant metabolism in response to As, focusing on the central role of NO as a signaling agent able to change the whole plant physiology.

When Bad Guys Become Good Ones: The Key Role of Reactive Oxygen Species and Nitric Oxide in the Plant Responses to Abiotic Stress.

The natural environment of plants is composed of a complex set of abiotic stresses and their ability to respond to these stresses is highly flexible and finely balanced through the interaction between signaling molecules. In this review, we highlight the integrated action between reactive oxygen species (ROS) and reactive nitrogen species (RNS), particularly nitric oxide (NO), involved in the acclimation to different abiotic stresses. Under stressful conditions, the biosynthesis transport and the metabolism of ROS and NO influence plant response mechanisms. The enzymes involved in ROS and NO synthesis and scavenging can be found in different cells compartments and their temporal and spatial locations are determinant for signaling mechanisms. Both ROS and NO are involved in long distances signaling (ROS wave and GSNO transport), promoting an acquired systemic acclimation to abiotic stresses. The mechanisms of abiotic stresses response triggered by ROS and NO involve some general steps, as the enhancement of antioxidant systems, but also stress-specific mechanisms, according to the stress type (drought, hypoxia, heavy metals, etc.), and demand the interaction with other signaling molecules, such as MAPK, plant hormones, and calcium. The transduction of ROS and NO bioactivity involves post-translational modifications of proteins, particularly S-glutathionylation for ROS, and S-nitrosylation for NO. These changes may alter the activity, stability, and interaction with other molecules or subcellular location of proteins, changing the entire cell dynamics and contributing to the maintenance of homeostasis. However, despite the recent advances about the roles of ROS and NO in signaling cascades, many challenges remain, and future studies focusing on the signaling of these molecules in planta are still necessary.

Sustained enhancement of photosynthesis in coffee trees grown under free-air CO2 enrichment conditions: disentangling the contributions of stomatal, mesophyll, and biochemical limitations.

Coffee (Coffea spp.), a globally traded commodity, is a slow-growing tropical tree species that displays an improved photosynthetic performance when grown under elevated atmospheric CO2 concentrations ([CO2]). To investigate the mechanisms underlying this response, two commercial coffee cultivars (Catuaí and Obatã) were grown using the first free-air CO2 enrichment (FACE) facility in Latin America. Measurements were conducted in two contrasting growth seasons, which were characterized by the high (February) and low (August) sink demand. Elevated [CO2] led to increases in net photosynthetic rates (A) in parallel with decreased photorespiration rates, with no photochemical limitations to A. The stimulation of A by elevated CO2 supply was more prominent in August (56% on average) than in February (40% on average). Overall, the stomatal and mesophyll conductances, as well as the leaf nitrogen and phosphorus concentrations, were unresponsive to the treatments. Photosynthesis was strongly limited by diffusional constraints, particularly at the stomata level, and this pattern was little, if at all, affected by elevated [CO2]. Relative to February, starch pools (but not soluble sugars) increased remarkably (>500%) in August, with no detectable alteration in the maximum carboxylation capacity estimated on a chloroplast [CO2] basis. Upregulation of A by elevated [CO2] took place with no signs of photosynthetic downregulation, even during the period of low sink demand, when acclimation would be expected to be greatest.