Community recommendations on terminology and procedures used in flooding and low oxygen stress researchApart from playing a key role in important biochemical reactions, molecular oxygen (O2) and its by-products also have crucial signaling roles in shaping plant developmental programs and environmental responses. Even under normal conditions, sharp O2 gradients can occur within the plant when cellular O2 demand exceeds supply, especially in dense organs such as tubers, seeds and fruits. Spatial and temporal variations in O2 concentrations are important cues for plants to modulate development (van Dongen & Licausi, 2015; Considine et al., 2016). Environmental conditions can also expand the low O2 regions within the plant. For example, excessive rainfall can lead to partial or complete plant submergence resulting in O2 deficiency in the root or the entire plant (Voesenek & Bailey-Serres, 2015). Climate change-associated increases in precipitation events have made flooding a major abiotic stress threatening crop production and food sustainability. This increased flooding and associated crop losses highlight the urgency of understanding plant flooding responses and tolerance mechanisms. Timely manifestation of physiological and morphological changes triggering developmental adjustments or flooding survival strategies requires accurate sensing of O2 levels. Despite progress in understanding how plants sense and respond to changes in intracellular O2 concentrations (van Dongen & Licausi, 2015), several questions remain unanswered due to a lack of high resolution tools to accurately and noninvasively monitor (sub)cellular O2 concentrations. In the absence of such tools, it is therefore critical for researchers in the field to be aware of how experimental conditions can influence plant O2 levels, and thus on the importance of accurately reporting specific experimental details. This also requires a consensus on the definition of frequently used terms. At the 15th New Phytologist Workshop on Flooding stress (Voesenek et al., 2016), community members discussed and agreed on unified nomenclature and standard norms for low O2 and flooding stress research. This consensus on terminology and experimental guidelines is presented here. We expect that these norms will facilitate more effective interpretation, comparison and reproducibility of research in this field. We also highlight the current challenges in noninvasively monitoring and measuring O2 concentrations in plant cells, outlining the technologies currently available, their strengths and drawbacks, and their suitability for use in flooding and low O2 research. The inconsistent and sometimes inaccurate usage of flooding and low O2 stress-related terms together with incomplete details regarding experimental conditions have hindered the interpretation, reproducibility and comparison of independent studies in the field. Here, we define and clarify commonly used terms used in flooding and low-O2 related experimental conditions. A general term referring to excessively wet conditions, that is where excess water replaces gas-spaces surrounding roots and/or shoots. Flooding encompasses the following terms that describe natural events or experiments. Literally means ‘life without molecular O2’. Plants can only survive for a limited time without molecular O2. The term anaerobiosis frequently refers to the status of plants/organs exposed to a lack of O2 for a short time, during which acclimation occurs through altered gene expression and metabolism. Describes cellular energy production from carbohydrates without the benefit of oxidative phosphorylation and engagement of cytochrome c oxidase as the final electron acceptor. Anaerobic metabolism occurs when O2 is absent and is usually associated with (but not limited to) ethanolic and/or lactate fermentation. In plants, it is also associated with accumulation of alanine and gamma-aminobutyric acid due to altered metabolite fluxes involving the tricarboxylic acid cycle among others (Narsai et al., 2011; van Dongen & Licausi, 2015; Voesenek & Bailey-Serres, 2015). It can occur in cells within an ‘anoxic core’ in tissues/organs (e.g. vascular tissues of roots; Berry & Norris, 1949; Thomson & Greenway, 1991) even under externally aerobic conditions and in densely packed tissues or organs with a low surface to volume ratio (e.g. developing fruits, tubers, seeds, meristems; Geigenberger et al., 2000; Gibbs & Greenway, 2003; van Dongen & Licausi, 2015). The terms hypoxia and anoxia are often used interchangeably, which limits experimental reproducibility and can lead to misunderstanding of associated physiological, biochemical and molecular processes. When accurate quantification of the O2 status of biological samples or their environment is not possible, use of -oxic terms is valid, but care should be taken when inferring conclusions about O2 availability from these experiments. As a guideline, we describe common -oxic words, highlighting their limitations. Although it is advisable that O2 concentrations be described for each experimental system, authors may prefer to use -oxic conditions best suited to the study, as long as the description enables experimental replication. Detailed description of the O2 levels assessed externally or internally (within the plant), or physical parameters that affect its availability, will improve the reproducibility of observations and help the design of models and meta-analyses. Flooding is a compound stress imposing changes in O2 availability (and thus respiratory ATP production), CO2, light, ethylene, mineral nutrients and reactive oxygen species (ROS; Voesenek & Sasidharan, 2013; Voesenek & Bailey-Serres, 2015). The severity of the stress and the response elicited depends upon genotype, developmental age of the plant, organ, tissue, and other factors including flooding depth and duration, light availability, temperature, humidity and the amount of carbohydrate storage (such as sugars, starch, lipids, protein) in cells and tissues. It is not easy to predict what physiological changes occur in a spatial and dynamic fashion during flooding at the cellular level, especially with respect to O2 concentration. The way in which a flooding treatment is performed will strongly influence how fast plant tissues experience low O2 stress. Different factors, including light levels in the water, the temperature and the volume of the water used to submerge the plants, microbial activity in the submerged soil, and O2 concentration of the water at the beginning of the experiment, will all influence how the O2 availability to the plant changes during the treatment. Therefore, careful monitoring and reporting of the O2 concentration around submerged plant tissue is required. Polarographic electrodes (such as the Clark-type electrode) are still most widely used for this. However, fiber-optic based sensor methods have become more popular during recent years (Rolletschek et al., 2009; Ast et al., 2012), since these are fast and selective. Moreover, optical sensors have the advantage that the same sensor can be used to measure molecular O2 concentrations in solution as well as in air, and the baseline of the measurement is more stable as compared with polarographic methods, which makes optical sensing more suited for long-term (days to weeks) measurements. To avoid technical difficulties in controlling the O2 concentration around a plant by submergence, many studies use a chamber filled with O2-free or O2-poor air. This has the advantage that O2 concentrations can be changed much faster as compared to a submergence treatment, and that the actual external concentration can be controlled precisely. One should be aware, however, that a treatment with air containing little O2 does not mimic submergence, but only changes one out of many parameters that are affected by submergence. Apart from the importance of controlling the environmental O2 concentration during experimental treatments, there is a strong need to obtain precise information about the plant internal-O2 concentration as well. To date, measurements of plant internal O2 have only been accomplished via invasive means (Ast et al., 2012; Ast & Draaijer, 2014; van Dongen & Licausi, 2015). Most commonly, a small sensor needle is inserted into a plant organ and O2 concentrations are measured at the tip of the needle. The smallest needle-type sensors that currently exist are based on the Clark-type sensor system (Revsbech, 1989) and commercially available sensors have a diameter of c. 4 μm. These sensors are extremely fragile. More robust glass-fiber-based optical sensors typically have a diameter of c. 50 μm. A disadvantage of needle-type sensors is that the tissue will be damaged upon insertion, which can lead to local changes in the rate of respiratory O2 consumption. Moreover, external O2 is likely to diffuse through the insertion wound into the interior of the tissue, which could lead to an overestimation of the actual in planta O2 concentration. An alternative invasive method to determine local differences or changes in O2 concentration is by using O2-sensitive reporter foil (Tschiersch et al., 2012). Here, a special camera is used to determine O2 concentration-dependent light emission from a special coated sensor foil that is placed on the surface of the plant tissue (Jensen et al., 2005). This method has been used to describe local differences in O2 concentration of plant organs, such as stems and seeds, that were cut in order to access the interior tissues with the sensor foil. By doing so, these measurements allowed detection of differential O2-consumption patterns within the plant organ (Tschiersch et al., 2012). More recently, nano particles coated with a fluorescent dye have been successfully used in rhizosphere studies (Koren et al., 2015). These nano particles are possible future candidates for O2 studies at the cell level when working with large, transparent model cells such as cells of Chara. To date, no method exists that enables noninvasive analysis of plant internal-O2 concentrations. The best alternative that is currently being applied makes use of reporter proteins (such as GUS, GFP or Luciferase) that are expressed under the control of low O2-induced promotor sequences (Gasch et al., 2016). Interpretation of the expression pattern of the reporter protein allows conclusions about relative variation in the O2 concentration between regions, or through time. It will not provide, however, an exact value for the actual local concentration of O2. Moreover, the reaction time of such reporter systems is relatively long, making it difficult to investigate rapid changes. Several other suggestions are being discussed to design alternative noninvasive molecular O2-reporter systems. Fluorescent resonance energy transfer (FRET)-based methods, in which the FRET efficiency is affected by O2-dependent protein maturation, have already been applied successfully in bacterial cell cultures (Potzkei et al., 2012), but there are no reports yet of the successful application of such O2 sensors in plants. In medical research, various noninvasive O2 monitoring techniques are being used, including positron emission tomography (PET) and nuclear magnetic resonance (NMR) technology (Roussakis et al., 2015). In plants, such methods have not yet been reported to determine O2 gradients, because of the poor resolution, and because homogenous application of the required radioisotopes or contrast agents (such as Fluorine-19 (19F)-based probes) appears difficult in plants. Further research to develop methods to determine plant internal-O2 concentrations will remain of utmost importance for the research field to advance further. It is extremely important that researchers carefully detail the experimental imposition of flooding or low O2 stress. We suggest that, in addition to details essential to any methods description, the following details specific to low O2 and flooding studies are necessary: A recovery period following the removal of flooding/hypoxia/anoxia stress is essential for scoring survival (Striker, 2012). Plants should be photographed immediately before and after the treatment and at the end of the recovery period. When scoring damage, quantitative rather than qualitative data are more reproducible and can be analyzed statistically (e.g. chlorophyll levels, biomass, green leaf area). Careful descriptions of growth and treatment conditions, especially factors that can influence both plant external- and internal-O2 concentrations are essential for clarity, reproducibility and progress in research on plant responses to flooding and low O2. Reporting on O2 concentrations, whenever possible, using the most suitable, currently available methods is recommended. Ultimately, the challenge is also to achieve an understanding of the spatial and temporal dynamics of the major flooding signals, O2, ethylene, nitric oxide, ROS and low-energy, their interactions, and how signaling modulates response from the subcellular to the whole-plant level. Furthermore, the focus of many studies has been on short-term molecular signatures, often under severe conditions, whereas responses associated with long-term, less severe and more chronic O2 limitations that influence developmental plasticity, deserve greater attention.