Brown algae are among the major primary producers in coastal ecosystems. Similar to land plants, alternating generation and morphology in response to physical conditions, such as light, temperature, and dehydration, are important for the environmental adaptation of immobile brown algae. Seasonal changes are among the most influential environmental factors for brown algae. They sense the seasonal transitions based on day length, regulate physiological activities, and alternate generations. Many studies have investigated photoperiodism in land plants, with most focusing on morphological formation, such as flower bud formation and fruit ripening (Thomas and Vince-Prue, 1997). The response of an organism to day length is known as photoperiodism. Many studies have reported photoperiodism in animals, fungi and terrestrial plants (Nelson et al., 2010). In angiosperms, night-break (NB) treatment, which involves a short period of light irradiation in the middle of the dark period, is among the most useful methods for photoperiodism analysis (Thomas and Vince-Prue, 1997). As the day length affects the total amount of photosynthetic products involved in growth and physiological functions, NB treatment helps in determining whether certain morphogenesis is induced by a photoperiodic response (photoperiodism) (Parker et al., 1950). Moreover, NB treatment with monochromatic light helps to identify the photoreceptors controlling photoperiodic responses (Parker et al., 1945). In land plants, photoperiodically controlled responses are dependent on photoreceptors rather than photosynthesis. Analysis of the NB action spectrum revealed that photoperiodicity in short-day (SD) plants is regulated by the perception of phytochromes to red light (RL) and far-red light (FR) (Thomas and Vince-Prue, 1997).

To date, only physiological experiments have been conducted on brown algae, with only a few studies investigating photoreceptors using molecular biology techniques. Photomorphogenesis controlled by the photoperiod has been reported in several genera of brown algae, including erect thallus formation in Scytosiphon lomentaria (Dring and Lüning, 1975a), gamete formation in Sphacelaria rigidula (Ten Hoopen et al., 1983), sorus formation in Saccharina and Undaria (Lüning, 1986, Lüning, 1988; Pang and Lüning, 2004), stem elongation in Sargassum muticum (Hwang and Dring, 2002), and receptacle initiation in Ascophyllum nodosum and Sargassum horneri (Terry and Moss, 1980; Yoshikawa et al., 2014). These reports suggest that the photoperiod is an essential environmental factor affecting the seasonality of brown algae and that brown algae possess photoreceptors to perceive day length. However, the specific receptor molecules involved in photoperiodism in brown algae remain elusive. This is due to the scarcity of studies analyzing the wavelengths controlling photoperiodism in brown algae (Dring and Lüning, 1975a; Terry and Moss, 1980; Yoshikawa et al., 2014) despite the important effect of light quality on photoperiodic responses and photoreceptor molecules.

Analysis of the effect of NB treatment on erect thallus formation in S. lomentaria revealed that the photoperiodic responses are regulated only by blue light (BL) (Dring and Lüning, 1975a). In contrast, NB treatment with RL and BL induces receptacle formation in A. nodosum (Terry and Moss, 1980). We previously showed that NB treatment with green light (GL) and BL effectively initiates receptacle formation in Sargassum horneri (Yoshikawa et al., 2014). However, whether brown algae have common photoreceptors associated with photoperiodism remains unclear. Moreover, differences in light quality related to photoperiodism hinder the comprehensive understanding of photoperiodism in brown algae. To determine the photoreceptors controlling photoperiodism in brown algae, we focused on the photoperiodic responses affecting erect thallus formation in Petalonia fascia in this study. A previous study reported that P. fascia forms an erect thallus from a discoid thallus under SD conditions (Roeleveld et al., 1974), suggesting that the erect thallus formation is a photoperiodic response in P. fascia. However, detailed physiological analysis of the photoperiodism of P. fascia has not yet been performed. Here, we analyzed the effects of various light intensities and qualities on erect thallus formation and photoperiodic responses in P. fascia.

Previous studies on the life cycle of P. fascia have reported erect thallus formation under SD conditions (Roeleveld et al., 1974). However, to date, no study has conducted physiological analysis to determine whether this phenomenon is controlled by a genuine photoperiodic response or the total light period. In this study, we demonstrated that erect thallus formation in P. fascia is regulated by photoperiodism.

Previous studies reported that the light quality affects photoperiodism in three brown algal species: Scytosiphon lomentaria, Ascophyllum nodosum, and Sargassum horneri (Dring and Lüning, 1975a; Terry and Moss, 1980; Yoshikawa et al., 2014). These reports are consistent with our findings on BL-controlled photoperiodism. Therefore, BL is possibly a common mechanism for day-length perception in brown algae. Here, 10 s of dim light NB treatment with BL (total quantum: 30 µmol photons m^-2) inhibited erect thallus formation, indicating the high sensitivity of P. fascia to BL. This effect is comparable to that observed in S. lomentaria after NB treatment with BL (total quantum 20 µmol photons m^-2) (Dring and Lüning, 1975a).

Notably, effects of NB treatment with GL on P. fascia in this study were clearly different from those in previous studies on S. lomentaria. Dring and Lüning reported that the action spectrum for erect thallus formation in S. lomentaria had no effect in the wavelength range of 501–724 nm (Dring and Lüning, 1975a cf.; Figure 5 ). Differences between our results and previous findings on the effects of GL may be due to differences in the total quantum rather than differences in the experimental organisms. Here, NB treatment with 1 µmol photons m^-2 s^-1 (60 min; total quantum 3600 µmol photons m^-2) GL inhibited erect thallus formation in P. fascia. In contrast, Dring and Lüning reported that NB treatment with 1 µmol photons m^-2 s^-1 (1 min; total quantum 60 µmol photons m^-2) GL has no effect on erect thallus formation. As the total quantum of GL irradiation in this study was 60-times higher than that in the study by Dring and Lüning, GL effectively suppressed erect thallus formation in P. fascia.

The light intensity and duration of GL exposure used in this study were comparable to those observed in the natural growth environment of P. fascia. GL intensity of 1 µmol photons m^-2 s^-1 may cause disc thallus formation in P. fascia. As GL is more prominent than BL in phytoplankton-rich environments (Jaubert et al., 2017), the mechanism to measure day length using GL is helpful for the environmental adaptation of P. fascia. BL-and GL mediating photoperiodic responses have been reported in S. horneri (Yoshikawa et al., 2014). However, only a few studies have investigated the spectral quality related to photoperiodism in brown algae. Moreover, whether most brown algae use GL in the same manner as BL to measure day length remains unclear. Future studies should investigate the photoperiodicity and light quality in other brown algal groups to determine whether photoperiodicity control via GL is a shared characteristic among brown algae.

The BL photoreceptors, aureochromes and cryptochromes, are possible photoreceptors controlling photoperiodism in P. fascia based on the identified photoreceptor genes in the brown alga Ectocarpus siliculosus (Cock et al., 2010).

Aureochromes are BL receptors unique to stramenopiles. Although their functions in brown algae remain unknown, they are involved in branch formation in the xanthophyte Vaucheria frigida (Takahashi et al., 2007) and cell division in diatoms (Huysman et al., 2013). Recent studies in diatoms have reported that aureochromes are involved in light-dependent responses, including photoprotection (Zhang et al., 2024) and the regulation of the circadian clock (Madhuri et al., 2024). As aureochromes are composed of a BL-sensing LOV domain and b-zip domain that binds to DNA, they perform dual functions: photoreception and regulation of gene expression (Takahashi et al., 2007; Takahashi, 2016). Therefore, aureochromes may be the key photoreceptors involved in photoperiodism and gene expression control in brown algae. However, as aureochromes exhibit an absorption spectrum similar to that of typical flavin proteins and the absorption does not include the GL region (> 500 nm) (Hisatomi et al., 2013), involvement of aureochromes in the regulation of photoperiodism in P. fascia suggesting the existence of another GL photoreceptor for photoperiodism in P. fascia. Cryptochromes are BL receptors that function as day-length sensors in Arabidopsis (Mockler et al., 2003). Although cryptochrome is a receptor that primarily absorbs BL, reduced cryptochrome has an absorbance spectrum that extends into the GL range (Lin et al., 1995a, b), suggesting that cryptochromes may perceive both BL and GL in P. fascia. Although homologous cryptochrome genes have been identified in brown algae, their physiological functions remain unknown.

In this study, we demonstrated that NB treatment with RL enhanced erect thallus formation. Similarly, Lüning also reported that continuous irradiation with RL enhances erect thallus formation in P. fascia (1973). Some studies have reported BL-induced photomorphogenesis in brown algae (Dring and Lüning, 1975a, b, Lüning, 1981; Lüning and Dring, 1975; Wang et al., 2010; Yoshikawa et al., 2014); however, studies on RL-induced photomorphogenesis are rare (Duncan and Foreman, 1980; Lüning, 1973; Müller and Clauss, 1976). RL photoreceptor phytochrome-mediated photoresponse, which is also observed in terrestrial plants, is cancelled by FR irradiation shortly after RL irradiation (Thomas and Vince-Prue, 1997). In brown algae, only one response, potentially involving phytochromes, has been reported. FR induces stipe elongation in Nereocystis luetkeana, which is cancelled by RL irradiation (Duncan and Foreman, 1980). In this study, FR did not counteract the RL response in P. fascia ( Figure 5 ). The response of P. fascia to RL may be a different type of photoreceptor-mediated response from that of N. luetkeana.

The phytochrome gene has been identified in E. siliculosus genome (Cock et al., 2010; Rockwell et al., 2014). As the spectrum of the recombinant protein of E. siliculosus phytochrome shows RL and GL photoconversion (Rockwell et al., 2014), we investigated whether the effects of GL are counteracted by RL irradiation immediately after GL irradiation and vice versa in this study. Notably, only the effects of GL were observed in experiments with GL irradiation, regardless of whether it was performed before or after RL irradiation. This result suggests that photoreceptors undergoing photoconversion with RL and GL are not involved in erect thallus formation in P. fascia. When GL irradiation preceded RL irradiation, RL did not promote erect thallus formation. This suggests that RL-induced erect thallus formation is not involved in day length perception but rather acts as a promoter under SD. Therefore, RL photoreceptors may function downstream of BL and GL receptors for day-length perception.

In conclusion, this study revealed that P. fascia uses both BL and GL for photoperiodic regulation, showing higher sensitivity to BL for erect thallus formation under SD conditions. RL further promotes erect thallus formation in NB treatment under SD conditions. Overall, this study provides insights into the photoperiodic strategies of brown algae from a physiological perspective and establishes a basis for future molecular biological studies on photoreceptor mechanisms.