In 25 years of surveys, more than 5000 confirmed exoplanets were found (https://exoplanets.nasa.gov/). Among them, small, rocky terrestrial-like exoplanets were discovered (Tinetti et al., 2013), mainly laying in the Habitable Zone (HZ, Kasting et al., 1993) of their host stars. Terrestrial-like exoplanets are rather common around M-dwarfs (M class Main Sequence stars, or also Red Dwarf stars) (Bonfils et al., 2013; Kopparapu et al., 2013; Hsu et al., 2020), very faint and cool stars (Pecaut and Mamajek, 2013) with most of their emission centred in the far-red (700 – 750 nm) and infrared (750 – 1000 nm), rather than in the visible (400 – 700 nm) like the Sun (G class Main Sequence star, or G2V). Despite high stellar activity during their early stages of life and stellar flares (Kiang et al., 2007; Segura et al., 2010; France et al., 2013; Cuntz and Guinan, 2016), orbiting exoplanets could remain habitable (O’Malley-James and Kaltenegger, 2017; Schwieterman et al., 2018). Moreover, because of the longevity of M-dwarfs (Adams and Laughlin, 1997), exoplanets in their HZ seem the best candidates to search for life beyond Earth. The closest exoplanet known to be orbiting an M-dwarf in its HZ, Proxima Centauri b (Anglada-Escudé et al., 2016) is 4.2 light-years away, meaning that the search for possible lifeforms will have to be made through detection of biosignatures: substances, molecules or patterns of biological origin (Seager et al., 2012; Schwieterman et al., 2018; Claudi and Alei, 2019). On Earth, the most known and referenced biosignature is oxygen (O₂) and its photoproduct ozone (O₃), derived from the oxygenic photosynthesis (OP) performed by cyanobacteria, algae and plants. This process is unique with respect to other photosynthetic processes (e.g., anoxygenic photosynthesis) due to the utilization of H₂O as a donor of electrons, an extremely abundant, yet very stable and very difficult molecule to oxidize. Thanks to OP, O₂ which was originally in traces reached 21% by volume of the Earth’s atmosphere, becoming a dominant gas. Abiotic processes on Earth account for less than 1 ppm of atmospheric O₂ and are considered negligible Harman et al. (2015). For exoplanets, O₂ could be considered a robust biosignature, even if care must be taken for possible false positives arising from photochemical reactions that have abiotic O₂ as a by-product (e.g., H₂O and CO₂ photoionization). A detailed discussion of several possible false positives is presented in Harman et al., 2015. On the other side, life on exoplanets could also not use oxygenic photosynthesis, and rely for example on anoxygenic photosynthesis which utilizes H₂, H₂S, Fe²⁺ as electron donors instead of H₂O. These molecules however are not as widely distributed as H₂O is, therefore anoxygenic photosynthesis would be less productive, leading to a harder detectability of possible by-product gasses (Kiang et al., 2007; Schwieterman et al., 2018). To evaluate the possibility, for an exoplanet orbiting the HZ of an M-dwarf, to undergo an oxygenation event as happened on Earth and reach through it detectable levels of O₂ in its atmosphere, one way is to select and study, through simulations, suitable organisms among terrestrial ones, keeping in mind some of the environmental characteristics expected to be present on those planets. Organisms need to be grown phototrophically under low light and/or far-red light conditions, and under continuous light irradiation, as exoplanets orbiting M-dwarfs can be tidally locked (Barnes, 2017). It’s also preferable to select among Earth’s extremophiles since very little information is available on the surface environmental conditions of those planets. Cyanobacteria fulfil all these requisites. These organisms drove important evolutionary events on Earth: i) being responsible for the emergence of oxygenic photosynthesis, which led to the accumulation of O₂ in the Earth’s atmosphere that caused the Great Oxidation Event and later promoted the evolution of more complex lifeforms (Lyons et al., 2014); ii) evolving the capability to withstand extreme environmental conditions, which allowed them to spread everywhere on Earth and become major primary producers from an ecological point of view (Gan and Bryant, 2015); iii) having high plasticity of their photosynthetic apparatus, which made them able to live in habitats characterized by deeply different light intensities and spectra (Gutu and Kehoe, 2012); iv) acquiring the ability to fix N₂ beside CO_2, thus causing significant changes both in the Earth’s atmosphere and in the N₂ and CO₂ geochemical cycles (Schirrmeister et al., 2016). Oxygenic photosynthesis employs two types of pigment-protein complexes, photosystems I and II (PSI, PSII), both composed of a reaction centre, which converts light into chemical energy, and an antenna, which harvests and funnels the light energy into the reaction centre (Bryant and Canniffe, 2018). The reaction centres are highly conserved in all oxygenic photosynthetic organisms and link chlorophyll a and carotenoids, which have major absorption bands in the blue (400-500 nm) and red (600-700 nm) wavebands. Antenna systems, instead, are diversified in the different taxa depending on the specific light-harvesting pigments associated with proteins, conferring them a distinctive capability to absorb photons in the visible portion of the solar spectrum, generally defined as Photosynthetically Active Radiation or PAR (400 – 700 nm). Most oxygenic photosynthetic organisms absorb blue and red light, while cyanobacteria are able to harvest a larger spectrum of wavelengths thanks to antenna complexes called phycobilisomes (PBS), made by different species-specific combinations of phycobiliproteins (PBPs) such as phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (AP), which have peak absorptions at roughly 565, 615 and 655 nm (Bryant and Canniffe, 2018). Moreover, even if most cyanobacteria use visible light-absorbing pigments to perform oxygenic photosynthesis, it has been shown that some species are able to harvest photons in the far-red region (700 – 800 nm), thanks to the synthesis of alternative types of chlorophylls beside chlorophyll a. They live indeed in habitats depleted of visible light and enriched in far-red, due to physical light scattering and attenuation (sands, caves, water columns) or by absorption of other photosynthetic organisms living on top of them (plant canopies, microbial mats, stromatolites) (Gan and Bryant, 2015). They evolved to utilize this light through the so-called Far-Red Light Photoacclimation (FaRLiP). This process was first discovered in Leptolyngbya sp. JSC-1 and then identified in a dozen species of terrestrial cyanobacteria (Gan et al., 2014a; Gan et al., 2014b; Li et al., 2016) and seems to be widely distributed in natural environments (Zhang et al., 2019; Antonaru et al., 2020; Kühl et al., 2020). These cyanobacteria deeply modify their photosynthetic machinery to absorb far-red light: they synthesize chlorophylls d and f (Chl d, Chl f) (Chen, 2019), but also far-red-absorbing forms of AP. They substitute core subunits of the PSI, PSII and PBS with far-red paralogs and modify the morphology of the PBS antennae to better absorb light above 700 nm, as can be observed in their in vivo absorption spectra (Gan et al., 2014a; Gan and Bryant, 2015; Li et al., 2016). With respect to other oxygenic photosynthetic organisms, cyanobacteria are thus characterized by high photosynthetic plasticity and can integrate the absorption of chlorophylls and carotenoids expanding the wavelengths of light they can harvest for photosynthesis. Recently (Claudi et al., 2021) we obtained preliminary important data exposing few cyanobacterial species to a simulated M-dwarf light spectrum, demonstrating their survival and growth under this exotic light condition. Based on chlorophyll fluorescence measurements, we also showed their capability to perform photosynthesis. We here exposed two species of cyanobacteria, one, Chlorogloeopsis fritschii PCC6912, able to perform FaRLiP and another, Synechocystis sp. PCC6803, unable to perform it, to different light spectra. Their growth, photosynthetic apparatus organization and changes in pigment composition, as well as their oxygenic photosynthetic evolution rate, were investigated under the simulated M-dwarf light spectrum along with two control conditions: a simulated solar light spectrum, to assess the behaviour of the cyanobacteria in Earth-like conditions, and a far-red light spectrum, to check the activation of the FaRLiP response. Results demonstrate that both cyanobacteria grown under a simulated M-dwarf spectrum acclimate in different ways, growing and evolving oxygen as efficiently as in a solar simulated spectrum, despite the lesser amount of visible light. They could therefore produce atmospheric biosignatures detectable from remote.

The possibility of Oxygenic Photosynthesis (OP) on terrestrial exoplanets has been discussed for a long time now. OP is the most important bioprocess known on Earth: it shaped the atmosphere we breathe and allowed complex forms of life to evolve and spread on the planet (Gale and Wandel, 2016), and nowadays it produces nearly 100% of Earth’s biomass (Covone et al., 2021). Of course, the only OP we are aware of is that arose on Earth, therefore, we don’t know how a similar process could have evolved on other planets of the Solar System (if any) or on planets orbiting different kinds of stars. As M-dwarfs are the most abundant stars present in our galaxy and recent surveys showed they can host terrestrial exoplanets in their Habitable Zone (HZ) (Hsu et al., 2020), the question shifted to whether or not OP could be performed in exoplanets orbiting those stars. This is not trivial, since water splitting, a crucial step in OP, requires photons energetic enough to trigger the reaction. These photons in the visible range are abundant for terrestrial-like planets orbiting Sun-like stars but are limited in the same conditions for an M-dwarf spectrum. Photons in the IR instead, abundant in the M-dwarf spectrum, could not be energetic enough to trigger the process if terrestrial OP took place (Kiang et al., 2007). Some researchers believe however OP could work similarly as on Earth (Ritchie et al., 2018; Wandel and Gale, 2020; Covone et al., 2021). It was calculated indeed that on tidally locked exoplanets orbiting the HZ of M-dwarfs, there would be enough photon flux in the visible to support Earth-like photosynthesis and that productivity could range from 13 to 22% to that on Earth (Ritchie et al., 2018; Wandel and Gale, 2020). Also in Kiang et al. (2007) a similar conclusion was made, even if they also speculated on the possibility of performing OP utilizing multiple chained photosystems (e.g. 3 or 4) and photons of longer wavelengths (up to 1400 nm) or more photons per molecule of O₂, both popular hypotheses in the scientific community (Wolstencroft and Raven, 2002; Tinetti et al., 2006; Chen and Blankenship, 2011; Takizawa et al., 2017; Lehmer et al., 2018). Lingam et al. (2021) went even further, proposing OP performed through far-red absorbing pigments not belonging to the family of chlorophylls. Wandel and Gale (2020) however disagree on the evolution of far-red absorbing pigments, as they conclude there would be no evolutionary pressure for them to appear since there could be a surplus of light in the visible for M-dwarf planets. As an example, the exoplanet Proxima Centauri b (Anglada-Escudé et al., 2016) orbiting the M-dwarf Proxima Centauri, the closest star to our Solar System (4.2 light-years), experiences an irradiance of about 64 µmol m^-2 s^-1 in the visible, approximately the 3% of Earth’s irradiance by the Sun. This is more than 20 and 180 times the amount light of required by some land plants and cyanobacteria to survive and grow, respectively (Raven et al., 2000; Nobel, 2009). Moreover, a model based on that stellar environment shows that organisms would be light-limited, but still have gross productivities higher than those obtained for most terrestrial grasslands and the open ocean on Earth (Ritchie et al., 2018). Despite the debate, OP hypotheses for M-dwarf exoplanets are indeed mainly based on theoretical models or argued from studies focusing on ecological niches enriched in far-red light: under forest canopies, in water columns, caves, microbial mats, stromatolites, endolithic environments, or under the sands (Gilbert et al., 2001; Chen et al., 2012; Behrendt et al., 2015; Gan and Bryant, 2015; Wolf and Blankenship, 2019; Zhang et al., 2019; Behrendt et al., 2020; Kühl et al., 2020). This field remains largely unexplored from an experimental point of view, with only one experiment to date (Claudi et al., 2021) carried out by acclimating different cyanobacteria strains growing in solid media to a simulated spectrum of an M-dwarf and assessing both their growth and their photosynthetic efficiency using a PAM fluorometer, thus with indirect measurements of chlorophyll fluorescence parameters. Here we physiologically characterized liquid cultures acclimated to an M7 simulated light spectrum, comparing the responses to FR and SOL light sources, measuring their growth through OD and dry weight, and assessing their photosynthetic apparatus features by recording in vivo absorption and 77K fluorescence spectra. Moreover, we measured the O₂ evolution of acclimated cultures when directly growing under the simulated M-dwarf, solar or far-red light spectra, thanks to the newly developed setup including the ASC. This was particularly important to evaluate the contribution of complex light spectra (as the simulated M-dwarf one) to oxygenic photosynthesis. The acclimation experiments have been carried out at low light intensity to further reduce the amount of photons in the visible portion of an M-dwarf spectrum (roughly one-sixth the light that would be present on Proxima Centauri b), to assess even unfavorable conditions. We could confirm our preliminary results in solid media cultures (Claudi et al., 2021) and further investigate the acclimation features of selected strains from that study. Strains capable and incapable of FaRLiP alike could acclimate and grow similarly under the simulated M7 and SOL lights, despite the different availability of visible light in the two spectra (about 10 and 26 µmol m^-2 s^-1, respectively). As for PCC6912, capable of FaRLiP response, it could be expected a slower growth in M7 with respect to SOL: FaRLiP response would indeed redirect energy from growth to the biosynthetic pathways involved in the synthesis of chlorophylls d and f, and all the alternative subunits of the major components of the PSI, PSII and PBS, necessary for the reorganization of the photosynthetic apparatus in far-red light (Gan et al., 2014a; Gan et al., 2014b). This was not the case, as optical density and dry weight measurements in M7 at 21 days were comparable to those in SOL ( Figures 4A, C ). As for PCC6803, incapable of FaRLiP response, much lower growth in M7 than in SOL was expected, as the strain could only rely on the modulation of pigments able to absorb red light but poorly capable of absorbing FR light. It is interesting to note that both strains acclimated to M7 by increasing the concentration of pigments per dry weight absorbing the few photons in the visible ( Table 1 ). This is an acclimation process which has been observed previously under red monochromatic light or low light (Mullineaux, 2001; Bernát et al., 2021). Moreover, PCC6912 activated FaRLiP to grow in M7, even if its activation was slower compared to FR, as seen after 3 days from the in vivo spectra ( Figure S3 ). Evidence of FaRLiP activation in M7 could be found eventually through the detection of chlorophylls d and f at the HPLC at 3 days ( Figure S4 ) and the detection of the rearranging photosynthetic apparatus through 77 K fluorescent measurements ( Figure 8 ). The progression of the acclimation process was confirmed by the same measurements after 21 days ( Figures 6A , 7A , 9A ). In cyanobacteria, under normal light conditions (e.g. white light, WL) photosystems have 3 peaks of fluorescence when excited at 440 nm, of which two come from PSII antenna pigments (WL-PSI, 685, 695 nm) and one comes from PSI antenna pigments and is variable in wavelength (WL-PSII, 710-735 nm), depending on the species (Murakami, 1997). Conversely, under far-red light conditions (far-red light, FRL), strains capable of activating FaRLiP show a variation in the emission spectra that can be detected. PSI shifts its peak emission from 710-735 nm to roughly 740 nm (FRL-PSI), due to the reorganization of the photosynthetic apparatus and the presence of Chl f as an antenna pigment in the PSI (Gan et al., 2014a). Under M7, PCC6912 activated FaRLiP extending the absorption of light in the far-red by synthesizing chlorophylls d and f and the subunits of the PSI necessary to host them. After 3 days, only a small shift in the PSI emission at 440 nm of cells acclimated to M7 compared to FR could be seen, which meant only a small portion of WL-PSI was substituted with FR-PSI. Conversely, after 21 days, the shift in M7 acclimated samples was identical to that in 3 days FR acclimated samples, meaning that in M7 a similar reorganization of the photosynthetic apparatus to that in FR was taking place at a slower pace. The reorganization of the photosynthetic apparatus allowed PCC6912 to absorb light in M7 and evolve O₂ efficiently ( Figure 10A ). Data from 77K spectroscopy highlighted an acclimation response to FR also in PCC6803. This strain, incapable of FaRLiP, responded to FR by deeply changing the ratio of PSII to PSI ( Figure 9B ), similarly to what had been seen in the past under long-wavelength red light (Murakami, 1997). This reorganization however didn’t lead to any net O₂ production in FR light ( Figure 10B ). On the contrary, though, PCC6803 could exploit efficiently the light in M7 by only adjusting its light-harvesting capabilities, evolving afterwards comparable amounts of O₂ in M7 and SOL. Recent studies on plants show that far-red light is used efficiently for oxygenic photosynthesis when in combination with shorter wavelength, while instead is poorly utilized when provided alone (Zhen, 2020; Zhen and Bugbee, 2020; Zhen et al., 2022), even in the absence of exotic acclimations like those performed by cyanobacteria. This has led the authors to question the real capabilities of oxygenic photosynthetic organisms to absorb far-red light and to extend the definition of PAR to a new broader definition, extended PAR (or ePAR, 400 – 750 nm) (Zhen et al., 2021). In this frame then we could hypothesize that PCC6803 is able to grow in M7 similarly to SOL but not in FR, because it can utilise far-red light efficiently only when in combination with visible light, as in the case of M7, and not alone, as in the case of FR. The overall picture that we obtained then, seems to be that cyanobacteria could utilize an M-dwarf light to photosynthesize, without synthesizing new forms of far-red absorbing pigments. For those who are capable of FaRLiP instead, the response is active and seems to be slower in M7 with respect to FR. So, even if in theory OP shouldn’t be as efficient under an M-dwarf spectrum as it is under a G-type one like the Sun, in practice Earth-like OP may be a possibility on the surface of those planets and could produce O₂ biosignatures detectable remotely if other boundaries conditions are met (Rugheimer and Kaltenegger, 2018; Schwieterman et al., 2018). However, it cannot be excluded that organisms evolved under an M-dwarf spectrum could rely on other types of oxygenic or anoxygenic photosynthesis, which can use a variety of organic and inorganic molecules as donors of electrons (George et al., 2020). Those molecules, in the case of anoxygenic photosynthesis for example, could be hardly available as H₂O is on Earth, thus leading to low global productivities of anaerobic organisms (Kiang et al., 2007). Such life would be thus difficult to detect remotely and biosignatures would be not robust enough (e.g., keen to false positives). Finally, it is important to highlight that the activation of FaRLiP in M7 and its detectability in vivo is of interest for the search of other biosignatures, in particular the red-edge (Seager et al., 2005). This is a so-called surface biosignature and is a spectral feature that arises from the reflectance properties of photosynthetic pigments, which absorb light in the visible and reflect it in the infrared, and is a feature displayed at roughly 700 nm only by oxygenic photosynthetic organisms (Seager et al., 2005; Tinetti et al., 2006; Kiang et al., 2007; Takizawa et al., 2017; Cavalazzi and Westall, 2019; Lingam and Loeb, 2021). Given that the in vivo absorption and reflectance spectra of oxygenic photosynthetic organisms are related (in fact one is almost the complementary to the other), results obtained for PCC6912 ( Figure 6A ) imply that, if life utilizing a far-red photoacclimation took place in a distant exoplanet orbiting an M-dwarf star, we could possibly see a shift or a distortion in the Red-Edge feature of the planet, making it easier to distinguish a positive detection from false ones. In conclusion, the objective of this study was to assess the growth, acclimation strategies and photosynthetic rate of selected cyanobacterial strains under a simulated M-dwarf light spectrum. Both cyanobacteria tested (Chlorogloeopsis fritschii PCC6912, able to activate the FaRLiP response, and Synechocystis sp PCC6803, unable to perform such acclimation) could exploit the simulated light conditions to grow and photosynthesize efficiently. C. fritschii PCC6912 by activating FaRLiP and expanding its capabilities for oxygenic photosynthesis beyond 700 nm, Synechocystis sp. PCC6803 possibly by utilizing far-red light efficiently only in combination with visible light, even if present in a very low amount as in the simulated M7 spectrum. Both acclimations led to efficient O₂ evolution, showing the potentiality of cyanobacteria to utilize light regimes that could arise on tidally locked planets orbiting the HZ of M-dwarf stars and to produce potentially detectable O₂ biosignatures detectable from remote. Finally, FaRLiP activation in C. fritschii PCC6912 under M-dwarf simulated light conditions opens up to the detectability of peculiar surface biosignatures produced by far-red acclimated oxygenic photosynthetic organisms on the surface of these exoplanets, increasing our chances to detect life beyond our Solar System.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

NL, RC and LP made the conceptualisation and supervision of the whole work. MB, NL, and LC conceived the Solar Simulator and the Far-red Simulator. NL, RC, LP, LC and MB conceived the Atmosphere Simulator Chamber (ASC), LC realized it. LC and MB assembled and tested the ASC setup. NL, MB and TM conceived the biological experiments, MB, AP, AS, DS performed and analysed them, NL, TM and LC helped in the analysis. MB and NL wrote the manuscript, all authors critically read it. All authors contributed to the article and approved the submitted version.