A novel approach for detecting molecular O₂ at the subcellular level in plants

Short abstract

This article is a Commentary on Perri et al. (2025), 247: 2998–3009.

What is the typical oxygen status of plant tissues? The answer, perhaps unsurprisingly, is ‘it depends’. A recent meta‐analysis revealed that plant tissues may experience oxygen levels ranging from anoxia to hyperoxia exceeding 60 kPa oxygen partial pressure (pO₂), depending on tissue type and the environmental conditions under which oxygen status is assessed (Herzog et al., ²⁰²³). Most studies included in the analysis employed physical O₂ sensors inserted into cells or tissues across various plant organs. Arguably, the insertion of such sensors induces injury, leading some researchers to question whether the resulting measurements accurately reflect the in vivo oxygen status of the investigated cells, tissues, or organs. Excitingly, in an article published in this issue of New Phytologist, Perri, Kahn & Wallabregue led a study (Perri et al., ²⁰²⁵; 247: 2998–3009) in which they introduced a novel fluorescent probe for detecting subcellular oxygen levels, employing two distinct chemical resorufins. Although the technique is still in its early stages, it holds considerable promise for advancing our understanding of subcellular oxygen dynamics in plants.

The authors elegantly demonstrate that their resorufin‐based chemical probes can penetrate plant cells and even intact tissues.

In plant tissues, the primary role of O₂ is to sustain respiration (Plaxton & Podestá, 2006), although molecular O₂ can also function as a signaling molecule (Gibbs et al., ²⁰¹¹). Whether the focus is on respiration or signaling, the adage ‘to measure is to know’ (Thomson, 1889) holds true, underscoring the need for reliable tools to quantify molecular O₂. The toolbox of O₂ microsensors has recently been reviewed (Pedersen et al., ²⁰²⁰; Panicucci et al., ²⁰²⁴), and so the main challenge is to select the correct sensor in a given situation (Box 1).

Box 1. O₂ partial pressure vs concentration.

All O₂ sensors that incorporate a membrane respond to oxygen partial pressure, not concentration. This is due to the phase transition that occurs as O₂ diffuses from the external environment (gas or liquid phase), through the membrane, and into the internal electrolyte or sensing matrix. Consequently, all sensors based on the Clark‐type or galvanic principles respond to O₂ partial pressure. Similarly, O₂ optodes also measure partial pressure, as the fluorophore is embedded in a plastic polymer matrix fused to the tip of a glass fiber.

It is a significant advantage that these sensors measure O₂ partial pressure, as the researcher may not always know whether the sensing tip is located within a cell (liquid phase) or in intracellular gas‐filled spaces, such as aerenchyma. If the sensor responded to O₂ concentration rather than partial pressure, transitions between liquid and gas phases would produce substantial signal artifacts due to the c. 30‐fold higher solubility of O₂ in the gas phase than in the liquid phase. Therefore, a Clark‐type O₂ microsensor or an O₂ microoptode can be calibrated in the gas phase and used in the liquid phase, or vice versa, providing valuable flexibility in laboratory applications.

However, both Clark‐type O₂ microsensors and O₂ microoptodes are highly sensitive to temperature. Considering the typical Q₁₀ value of c. 2 for inorganic reactions, one can appreciate the primary reason for this strong temperature dependence. Additionally, the molecular diffusion rate of O₂ is also markedly temperature‐dependent. For this reason, temperature must be carefully controlled – or at the very least measured near the sensing tip – so that appropriate corrections can be applied to ensure accurate determination of O₂ status.

In many cases, the researcher aims to assess the O₂ status of plant tissues, a task that typically requires the insertion of a sensor directly into the tissue. If internal O₂ levels are expected to fluctuate over time, a dynamic sensor with a sufficiently rapid response time is essential to capture these variations accurately. Furthermore, such measurements are often conducted ‘blind’, meaning the precise position of the sensor within the tissue cannot be visually confirmed during the experiment. Under these conditions, a sensor that responds to pO₂ is necessary. By contrast, a sensor that measures oxygen concentration would produce artefactual peaks when transitioning between the gas phase and aqueous cell sap, due to the markedly lower solubility of O₂ in the latter compared with the intercellular gas spaces (Box 1). Consequently, a miniaturized Clark‐type O₂ sensor or an optode is generally the preferred choice for such applications (Fig. 1).

Fig. 1.

Conceptual illustration depicting a cross section of a rice root with three different O₂ sensors. A Clark‐type O₂ microsensor is shown positioned within either the cortical aerenchyma (a) or inserted into a cortical cell (b). A slightly larger optical O₂ sensor is inserted such that it spans both the aerenchyma and a radial file of dead cortical tissue (c). The inset illustrates the tip of the Clark‐type microsensor located inside a cortical cell (d), alongside green fluorescent resorufin probes distributed within individual cortical cells (e). Note that the size of the resorufin probes is not drawn to scale. Photo courtesy: photographs of microsensors and microoptodes by Unisense A/S and photograph of rice root cross section by Dr Zhiwei Song.

When the research question demands exceptionally high spatial resolution, the choice of sensor becomes less straightforward. The miniaturized Clark‐type O₂ microsensor remains an excellent option for many applications, as it can be constructed with a tip diameter of just 3–5 μm (Weits et al., ²⁰¹⁹), in which the O₂ sensitivity is localized. As a general rule, the spatial resolution of a microsensor – whether a Clark‐type or an optode – is approximately twice the sensor's tip diameter. Thus, the smallest available Clark‐type microsensors offer outstanding spatial resolution, enabling assessment of intracellular O₂ status. In addition, these sensors respond extremely rapidly, with a 90% response time of less than 1 s, making them suitable for capturing rapid temporal fluctuations in O₂ associated with processes such as photosynthesis and respiration (Lassen et al., ¹⁹⁹⁸). Finally, the conical shape of the glass capillary effectively seals the insertion point, preventing the escape of gas bubbles from the tissue – an important feature when tissues become pressurized due to photosynthetic O₂ production. By contrast, needle‐embedded sensors (whether Clark‐type or optode‐based) lack this sealing mechanism due to the parallel wall of the needles, which can result in unstable or ‘zigzagging’ signals as internal pressure builds and is then abruptly released (Pedersen et al., ²⁰¹⁶). In summary, the Clark‐type O₂ microsensor provides excellent spatial and temporal resolution, but only at the precise location of the sensor tip. Consequently, alternative techniques are needed when two‐dimensional imaging of O₂ is required.

Planar O₂ optodes provide a valuable technique for accurately mapping molecular O₂ in two dimensions (Tschiersch et al., ²⁰¹²). Numerous examples exist of their application in plant science, particularly in visualizing O₂ dynamics at root surfaces. However, since current planar optodes cannot be inserted into plant tissues, they are limited to detecting O₂ in the external medium or at tissue interfaces. Nevertheless, they have been effectively used to demonstrate O₂ leakage or consumption at root surfaces, especially in studies involving plants growing in waterlogged soils (Larsen et al., ²⁰¹⁵). Notably, recent technological advances have enabled the integration of multiple fluorophores into a single planar film, allowing simultaneous imaging of variables such as O₂, CO₂, and pH (Lenzewski et al., ²⁰¹⁸). Despite their current limitations in mapping intratissue O₂ distribution, planar optodes remain powerful tools for investigating spatial patterns of gas exchange and plant–environment interactions at tissue interfaces.

However, the novel approach by Perri et al. exemplifies a technique that enables intratissue O₂ mapping. The authors elegantly demonstrate that their resorufin‐based chemical probes can penetrate plant cells and even intact tissues. The probe is enzymatically cleaved from a carrier molecule (either 4‐nitrobenzyl or methyl‐indolequinone) when intracellular O₂ levels fall below specific thresholds. The two carriers differ in their sensitivity to O₂, allowing detection across contrasting oxygen concentrations. Based on fluorescence intensity, the authors propose that the methyl‐indolequinone‐resorufin probe is cleaved at moderately low O₂ levels, peaking c. 0.5–1 kPa pO₂, whereas the 4‐nitrobenzyl‐resorufin probe responds to more severe hypoxia, with cleavage occurring at ≤ 0.2 kPa pO₂. Using the methyl‐indolequinone‐resorufin probe, the authors skillfully reveal low O₂ tensions in the root meristem (a hypoxic niche) even under normoxic conditions.

These are still early days for chemical O₂ probes, and even traditional O₂ microsensors and microoptodes are likely to benefit from continued refinement. In the case of the chemical O₂ probes reported by Perri et al., the two most notable limitations are their relatively low resolution in terms of O₂ concentration and the irreversible nature of their chemical response. While these features may seem like distant goals, it would be highly desirable to achieve a chemical resolution of 1 kPa pO₂ in the range from normoxia down to c. 5 kPa, and 0.1 kPa resolution below that threshold. Achieving this would require specific emission wavelengths for each probe to enable accurate discrimination between concentration ranges – that is, a chemical cocktail of c. 65 unique fluorophores, each dissolved in a nontoxic solvent and capable of penetrating plant cell walls. By contrast, overcoming the issue of irreversibility may be more readily achievable, for example by replacing enzymatic cleavage of the fluorophore with a redox‐based reaction. While these are ambitious targets, even modest, incremental improvements to the current probes will be timely and could substantially advance our understanding of intracellular O₂ dynamics in plant cells.

Together, established microsensing techniques and emerging chemical probes form a complementary toolkit that, with continued refinement, promises to significantly progress our understanding of O₂ dynamics in plant tissues at unprecedented spatial and temporal scales.

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