Build it, and they will shine: generating fluorescent sensors for H₂O₂ in a unicellular alga

Although sequencing of any kind is justly heralded as having revolutionized science, there have been—at least—two other seminal discoveries in my book: first, polymerase chain reaction; second, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. Fun fact: GFP was discovered while looking for the protein responsible for the blue fluorescence emitted by the jellyfish, which you may know as the calcium sensor aequorin (Prasher et al., 1992). Since the initial proof-of-concept publication demonstrating its fluorescence when accumulating in Escherichia coli and the nematode Caenorhabditis elegans (Chalfie et al., 1994), GFP has become a beacon illuminating our understanding of cell biology and signaling. Here, Justus Niemeyer and colleagues (Niemeyer et al., 2021) harness the power of mighty GFP to follow hydrogen peroxide (H₂O₂) levels and localization in a living plant cell.

GFP can be turned into a sensor for a number of ligands, either as an on–off switch, by using energy transfer between two fluorescent proteins flanking a protein that binds to a given molecule, or by following changes in the excitation spectrum of GFP. These latter sensors are dubbed ratiometric, since ligand binding alters the ratio between fluorescent states. While H₂O₂ is highly detrimental to cellular integrity, there are enzymes whose sole role is detoxification of H₂O₂: peroxidases, making them ideal H₂O₂-reactive proteins. The authors used here a modified version of yeast (Saccharomyces cerevisiae) 2-Cys peroxiredoxin thiol-specific antioxidant 2 (Tsa2), fused to the redox-sensitive GFP roGFP2. Wild-type Tsa2 normally forms an internal disulfide bond between two cysteines, but one was mutated to alanine to force the formation of a disulfide bond between Tsa2 and roGFP2, resulting in a quantifiable change in the roGFP2 excitation spectrum.

Niemeyer et al. (2021) wished to assess H₂O₂ levels in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii), which is uncannily good at gene silencing. They therefore used two tricks: first, they generated a series of expression constructs by combining the best promoters, regulatory elements, and subcellular targeting sequences available by capitalizing on the MoCLo toolbox (Crozet et al., 2018) for fast cloning. Second, they tested sensor fluorescence and accumulation in the UVM4 strain, which is less amenable to silencing transgenes (Neupert et al., 2020). After a lot of mix and matching, the authors came up with six constructs driving strong accumulation of the H₂O₂ sensor in the cytoplasm, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, or endoplasmic reticulum (ER) (see Figure). The addition of H₂O₂ to algal cells induced a shift to the oxidized form of the sensor in all compartments tested (with the exception of the ER, where the sensor was already fully oxidized), followed by a gradual return to a more reduced state reflecting the buffering capacity of each compartment against the oxidizing agent.

Figure.

A complement of optimized constructs drives high-level accumulation of a redox sensor in several compartments in the alga Chlamydomonas reinhardtii. All constructs and their individual components may be ordered from the Chlamydomonas Resource Center (crc.org). Adapted from Niemeyer et al. (2021)Figure 1.

And now the fun can finally begin! When exposed to high light intensities (1,000 µmol photons m⁻² s⁻¹), the chloroplast stroma experienced a burst of H₂O₂, as evidenced by the more oxidized state of the sensor. The rise in H₂O₂ was the result of photosynthetic linear electron transport, as it was abrogated by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Notably, the cytosolic sensor reported a similar, although weaker, accumulation of H₂O₂, while the sensors targeted to the nucleus and mitochondrial matrix did not, indicating only local diffusion of H₂O₂ from the chloroplast to the surrounding cytoplasm. Another condition that resulted in a burst of H₂O₂, this time in the cytoplasm and the nucleus, was exposure to high temperatures (40°C, 25°C being the typical growth temperature). What is causing this rise in H₂O₂ is unclear, but the tools are now available to dissect how, when, and where H₂O₂ is produced. Combined with chemical treatments and growth conditions, and perhaps even introducing these reporters into other strains or mutants, the exploration of H₂O₂ production and signaling has a bright future ahead in Chlamydomonas!