TY - JOUR
AU1 - Giovagnetti,, Vasco
AU2 - Ruban, Alexander, V
AB - Abstract Photosystems possess distinct fluorescence emissions at low (77K) temperature. PSI emits in the long-wavelength region at ~710–740 nm. In diatoms, a successful clade of marine primary producers, the contribution of PSI-associated emission (710–717 nm) has been shown to be relatively small. However, in the pennate diatom Phaeodactylum tricornutum, the source of the long-wavelength emission at ~710 nm (F710) remains controversial. Here, we addressed the origin and modulation of F710 fluorescence in this alga grown under continuous and intermittent light. The latter condition led to a strong enhancement in F710. Biochemical and spectral properties of the photosynthetic complexes isolated from thylakoid membranes were investigated for both culture conditions. F710 emission appeared to be associated with PSI regardless of light acclimation. To further assess whether PSII could also contribute to this emission, we decreased the concentration of PSII reaction centres and core antenna by growing cells with lincomycin, a chloroplast protein synthesis inhibitor. The treatment did not diminish F710 fluorescence. Our data suggest that F710 emission originates from PSI under the conditions tested and is enhanced in intermittent light-grown cells due to increased energy flow from the FCP antenna to PSI. Chlorophyll fluorescence, chlorophyll red forms, diatoms, light harvesting, Phaeodactylum tricornutum, photosynthesis, PSI Introduction Oceans cover >70% of the Earth’s surface and are home to a hugely diverse multitude of microscopic planktonic organisms (Ibarbalz et al., 2019), the photosynthetic component of which contributes to roughly half of the net primary productivity on Earth (Field et al., 1998). Oceanic oxygenic photosynthesis is mainly carried out by single cell prokaryotes (cyanobacteria) or eukaryotes (microalgae; Field et al., 1998; Pierella Karlusich et al., 2020). Among the latter, diatoms (Bacillariophyta, photosynthetic Stramenopiles; see Keeling, 2013; Dorrell et al., 2017) are one of the most diverse, widespread, and successful lineages (Armbrust, 2009; Malviya et al., 2016; Tréguer et al., 2018). They are believed to contribute to as much as 20% of global primary productivity and sustain major biogeochemical cycles (Nelson et al., 1995; Field et al., 1998; Tréguer et al., 2018). Harbouring the so-called ‘secondary red plastids’ (Bowler et al., 2008; Armbrust, 2009; Keeling, 2013; Dorrell et al., 2017), they possess a unique photosynthetic apparatus (Büchel, 2020), and unusual physiological and metabolic features (e.g. Bowler et al., 2008; Armbrust, 2009; Dorrell et al., 2017; Falciatore et al., 2020), which enable them to withstand strong changes in light environment. Survival in aquatic and terrestrial habitats, which are characterized by unpredictable and variable light environments, depends on efficient regulation of light harvesting (Ruban, 2012). This ensures a continuous supply of excitation energy from a network of pigments embedded in light-harvesting antenna proteins to the reaction centres of PSII and PSI, where charge separation takes place (van Grondelle et al., 1994; Ruban et al., 2012; Croce and van Amerongen, 2014). By activating molecular mechanisms that regulate the flow of such excitation energy, fast and unexpected changes in the amount of absorbed solar energy can be safely controlled, thus preventing photodamage (Ruban et al., 2012). Light intensity strongly fluctuates, for example due to sunflecks, cloud cover, diurnal/seasonal solar cycles, as well as water turbulence and motion for microalgae (MacIntyre et al., 2000; Falkowski and Raven, 2007; Ruban, 2012). Additionally, photosynthetic organisms need to respond and acclimate to light quality, namely spectral changes (Falkowski and Raven, 2007; Kirk, 2011; Ruban, 2012). This is the case for the instance of higher plants growing under ‘shade light’ of the canopy, where the availability of the visible light is limited, while the amount of red/far-red light photons is high (see Anderson, 1986; Rivadossi et al., 1999). Likewise, light penetration through the water column causes variations in the light wavelengths available to microalgae, such as diatoms (MacIntyre et al., 2000; Kirk, 2011). Due to the optical properties of the water column and photosynthetic organisms inhabiting it, a strong attenuation of red and far-red (and UV) light occurs in the upper layer of the water column, while blue and blue-green light penetrates deeper (MacIntyre et al., 2000; Falkowski and Raven, 2007; Kirk, 2011). The ability to efficiently absorb light in the red/far-red region might thus have evolved to adapt to specific ecological niches (e.g. plant canopy for terrestrial habitats or dense cultures in shallow waters, microbial mats, or corals for aquatic habitats). In such types of light environment, the presence of long-wavelength spectral forms of chlorophyll—the so-called ‘red forms’ (Butler, 1960; Croce and van Amerongen, 2013)—have been proposed to play an important role in light harvesting (Anderson, 1986; Rivadossi et al., 1999). Chlorophyll red forms absorb at energies below those of the primary electron donors—P680 for PSII (absorbing at ~680 nm; Döring et al., 1967, 1968; Witt, 1979) and P700 for PSI (absorbing at ~700 nm; Kok, 1956). Hence, they transfer excitation energy to the reaction centre pigments by means of a thermally activated (‘energetically uphill’) process (Jennings et al., 2003). These low-energy forms of chlorophyll appeared to be a general feature of PSI light-harvesting systems or reaction centres. While in cyanobacteria they are associated with Chl a of the PSI core, in higher plants they are present in the peripheral light-harvesting antenna of PSI (LHCI, made of Lhca polypeptides) (e.g. Croce et al., 1998; Gobets and van Grondelle, 2001; Morosinotto et al., 2003; Qin et al., 2015). In the green alga Chlamydomonas reinhardtii, association of red forms with the PSI core complex (e.g. Wollman and Bennoun, 1982) or specific Lhcas (e.g. Mozzo et al., 2010) is still debated (see Santabarbara et al., 2020). Although present in a relatively low amount (~3–10% of the total chlorophylls), these low-energy chlorophylls strongly affect energy transfer and trapping in PSI (Gobets and van Grondelle, 2001). Different absorption and emission features have been observed in different organisms and species (see Croce and van Amerongen, 2013; Santabarbara et al., 2020). Typically, they are observed as a prominent absorption tail in the far-red region (up to ~740 nm; French, 1971; Gobets and van Grondelle, 2001), and as a rather broad, low-temperature (77K) steady-state fluorescence emission band (710–740 nm range; Butler, 1978; Gobets and van Grondelle, 2001). The 77K emission peaks associated with PSII are instead blue shifted and appear at 685 nm and 695 nm (Butler, 1978; Andrizhiyevskaya et al., 2005; Chen et al., 2015). While in plants the PSI-related low-temperature emission maximum is centred at ~735 nm (Butler, 1978; van Grondelle et al., 1994; Gobets and van Grondelle, 2001), variability has been reported for different photosynthetic organisms. This includes model green algae such as C. reinhardtii (~715 nm for in vivo and PSI–LHCI supercomplex measurements) or Chlorella spp. (715–720 nm in vivo; Germano et al., 2002; Gibasiewicz et al., 2005; Santabarbara et al., 2007, 2020), and cyanobacteria (720–760 nm, depending on the species studied and the oligomeric state of the PSI core; Gobets and van Grondelle, 2001; Santabarbara et al., 2020). Moreover, photosynthetic organisms belonging to the so-called ‘red lineage’ (including chromerids, cryptophytes, eustigmatophytes, phaeophytes, and diatoms; Keeling, 2013) show a lower content of red forms in PSI, which are blue shifted (~710–715 nm) relative to those observed in cyanobacteria and higher plants (e.g. Fujita and Ohki, 2004; Veith and Büchel, 2007; Ikeda et al., 2008; Yamagishi et al., 2010; Bína et al., 2014; Herbstová et al., 2015; Litvín et al., 2016, 2019; Belgio et al., 2017) . In diatoms, PSI emission is low at room temperature (e.g. Fujita and Ohki, 2004; Ikeda et al., 2008; Belgio et al., 2017). The diatom ‘red emission’ band is species dependent (710–717 nm; see Brown, 1967; French, 1967; Goedheer, 1973, 1981; Shimura and Fujita, 1973; Fujita and Ohki, 2004) and enhanced by acclimation to very low light intensities (Brown, 1967; Goedheer, 1973; Shimura and Fujita, 1973; Fujita and Ohki, 2004; Taddei et al., 2018) or red/far-red light (Fujita and Ohki, 2004; Herbstová et al., 2015, 2017; Ueno et al., 2019; Akimoto et al., 2020; Oka et al., 2020). However, in some photosynthetic organisms or specific species, moderately red-shifted forms (~710 nm) can also be present in the light-harvesting antenna complexes of PSII. Examples are the green alga Ostreobium sp. (Koehne et al., 1999; Wilhelm and Jakob, 2006), the moss Physcomitrella patens [via a unique, red-shifted antenna polypeptide, Lhcb9 (Alboresi et al., 2011), despite the fact that evidence for Lhcb9 association with PSI has also been shown (Iwai et al., 2015, 2018)], the chromerid alga Chromera velia (Bína et al., 2014; Kotabová et al., 2014), several eustigmatophyte algae (Wolf et al., 2018; Bína et al., 2019; Litvín et al., 2019; Niedzwiedzki et al., 2019), as well as the diatoms Phaeodactylum tricornutum (Fujita and Ohki, 2004; Herbstová et al., 2015, 2017; Oka et al., 2020) and Chaetoceros gracilis (Ueno et al., 2019; Akimoto et al., 2020). In the pennate diatom, P. tricornutum (De Martino et al., 2007; Bowler et al., 2008), a long-wavelength fluorescence band centred around 710 nm (F710) is detected at 77K (while being slightly blue shifted at room temperature and depending on the light conditions of growth; e.g. Brown, 1967; Goedheer, 1973, 1981). When P. tricornutum cells were grown under red light, F710 was shown to be functionally associated with PSII and attributed to an oligomeric complex made of the fucoxanthin Chl a/c-binding protein (FCP), Lhcf15 (Herbstová et al., 2015, 2017). This is consistent with previous studies showing that the Lhcf15 protein expression is controlled by light spectral quality and prolonged darkness (Nymark et al., 2013; Schellenberger Costa et al., 2013). The supramolecular oligomeric state of this Lhcf15 complex was shown to be labile, and the disruption of its connectivity to reaction centres induced a drastic and rapid reduction of the F710 band intensity (e.g. Brown, 1967; Sugahara et al., 1971; Goedheer, 1973; Berkaloff et al., 1990; Fujita and Ohki, 2004). Additionally, the appearance of F710 has been linked to diatom non-photochemical quenching capacity, among different species as well as different strains of the same species (e.g. P. tricornutum; Lavaud and Lepetit, 2013). The F710 band was indeed assigned to FCP complexes functionally uncoupled from PSII/PSI cores and aggregated in response to high light exposure. FCP aggregates were proposed to be involved in the formation of a second quenching site (Q1) in diatoms (Miloslavina et al., 2009). At the same time, no fast response to far-red light was found in the diatom F710 band (Lavaud and Lepetit, 2013), in line with the presumed absence of ‘state transitions’ (Owens, 1986a), which balance excitation energy between the two photosystems by phosphorylation-dependent migration of antenna complexes. How diatoms modulate the relative content, and energetic connectivity, of light-harvesting complexes and photosystem reaction centres in response to different light acclimations is poorly understood, especially when considering the lack of a key regulatory mechanism (i.e. state transitions) able to adjust the energy flow to photosystems. This prompted us to further address the functional energetic relationship of F710 emission with photosystems in P. tricornutum. Cells were acclimated to continuous (CL) and intermittent light (IL), with the latter condition causing chloroplast reorganization and possibly altering the energy transfer pathways from FCP antenna complexes to photosystem reaction centres (Giovagnetti and Ruban, 2017). By inhibiting the chloroplast protein synthesis with lincomycin (Belgio et al., 2012; Townsend et al., 2018), a preferential and strong decrease in PSII core content was induced for both acclimative states to further assess F710 association with the remaining complexes. Materials and methods Cell cultures and growth conditions Phaeodactylum tricornutum Bohlin CCAP 1052/1A (accession Pt2; De Martino et al., 2007) (Culture Collection of Algae and Protozoa, Scotland, UK) was grown in sterile artificial seawater F/2 medium, supplemented with silicate (Guillard and Ryther, 1962). Non-axenic, semi-continuous batch cultures were kept at 18 °C in glass bottles and continuously flushed with sterile air. Cells were grown under either continuous light (CL; 40 μmol photons m−2 s−1, in a photoperiod consisting of 14 h of light/10 h of darkness) or intermittent light (IL; 40 μmol photons m−2 s−1, with cycles of 5 min light/55 min dark) (Lavaud et al., 2002; Giovagnetti and Ruban, 2017). Light conditions and temperature were controlled using two Sanyo MLR-351-PE growth chambers. Cultures were grown using white light fluorescent bulbs. Cell concentration and growth rates were measured (3–4 biological replicates) to ensure experiments were performed with cells in exponential phase. An improved Neubauer haemocytometer (VWR International Ltd, UK) and a CH20 microscope (Olympus, UK) were used. All experiments were repeated at least three times on different biological samples, unless otherwise stated. Treatment with the chloroplast protein synthesis inhibitor, lincomycin (Sigma-Aldrich, Munich, Germany), was carried out on batch cultures kept in flasks and continuously shaken (Belgio et al., 2012; Townsend et al., 2018). Lincomycin at 0.55 mM was used for CL cultures to reach low values of PSII maximum quantum yield in the dark (Fv/Fm). Lincomycin treatment was repeated for IL cultures to decrease Fv/Fm (see the Results). Light conditions and temperature were kept as above, with lincomycin-treated and control cultures growing under the same conditions. Three to six biological replicates were tested. Determination of chlorophyll concentration Pulse amplitude modulation (PAM) fluorescence measurements, and low-temperature fluorescence emission and excitation spectra were carried out on cells harvested by centrifugation (1500 g for 3 min) and resuspended in F/2 medium to reach a final Chl a concentration of 10 μg ml−1. Chlorophyll content was quantified as previously described (Giovagnetti and Ruban, 2017). Chl a and c concentrations were determined spectrophotometrically (Ultrospec 2100 pro, GE Healthcare Ltd, UK), employing extinction coefficients and specific wavelengths according to Jeffrey and Humphrey (1975). Chlorophyll fluorescence analysis Chlorophyll fluorescence measurements were performed using a DUAL-PAM-100 measuring system (Walz Effeltrich, Germany) on 30 min dark-adapted cells of P. tricornutum acclimated to CL or IL. Measurements were performed in a quartz cuvette with cells gently stirred. Fo and Fm are the minimum and maximum fluorescence levels in the dark, used to calculate Fv/Fm, Fv=(Fm–Fo). Thylakoid isolation and sucrose gradient ultracentrifugation Thylakoid membranes were isolated from cells dark-adapted for 30 min. Thylakoid membranes were isolated as previously described (Veith and Büchel, 2007) with some modifications. All the following steps were carried out in dim light and at 4 °C. Cells were harvested by centrifugation (1500 g for 5 min) and resuspended in a homogenization buffer (10 mM HEPES pH 7.6, 10 mM KCl, 2 mM MnCl2, 5 mM MgCl2, 6 mM EDTA, 0.6 M sorbitol, 1 mM aminocaproic acid, 0.2 mM benzamidine, 1% BSA). Cells were broken by one passage through an LM20 Microfluidizer (Microfluidics, USA) at 18 000 psi. Cell debris and unbroken cells were removed by centrifugation at 1500 g for 3 min. The supernatant was collected and centrifuged for 20 min at 20 000 g, thus obtaining a final pellet that was resuspended in osmotic resuspension buffer (10 mM HEPES pH 7.6, 10 mM KCl, 2 mM MnCl2, 5 mM MgCl2, 6 mM EDTA, 1 mM aminocaproic acid, 0.2 mM benzamidine). Chl a concentration of thylakoid membranes was determined as described above. Separation of photosynthetic protein complexes was acheived by linear sucrose density gradient ultracentrifugation. Equal amounts of thylakoid membranes (~0.35 mg Chl a ml−1) isolated from CL and IL cells were solubilized to a final concentration of 2% n-dodecyl-α-d-maltoside (α-DDM, Generon, UK; w/v) at a final detergent/Chl a ratio of 100 for 30 min on ice. α-DDM (2%) was used to enhance separation between PSII and PSI core complexes, relative to previous attempts (Lepetit et al., 2007, 2010). During solubilization, samples were gently mixed every 5 min. The solubilized membranes were centrifuged at 14 000 rpm for 1 min to remove unsolubilized material. The supernatant, containing the solubilized pigment–protein complexes, was collected and immediately loaded onto linear sucrose gradients [0.1–0.7 M sucrose (w/v) in osmotic resuspension buffer supplemented with 0.03% α-DDM]. Sucrose density gradient separation was performed on biological triplicates, each replicate consisting of three tubes (technical replicates). A final Chl a concentration of 0.2 mg was loaded onto each tube. Photosynthetic protein complexes were fractionated by ultracentrifugation using a swinging bucket rotor (SW41 Ti, Beckman Coulter, UK) for 18 h at 273 620 g and at 4 °C (Optima L-80 XP Ultracentrifuge, Beckman Coulter, UK). After the separation, sucrose gradient bands and fractions were rapidly collected and characterized by UV–visible absorption and low-temperature fluorescence spectroscopy (see below). Immediately after collection, band/fraction aliquots were flash-frozen with liquid nitrogen and kept at –20 °C for further gel electrophoresis and immunoblotting. Absorption spectroscopy Absorption spectra were measured at room temperature. Measurements were performed on samples diluted to OD in the range 0.2–0.3 at the Qy maximum. Absorption spectra were measured between 350 nm and 750 nm with 1 nm increments, on a modernized Aminco DW-2000 UV/Vis spectrophotometer (Olis Inc., USA). Spectra were normalized at their absolute maximum in the Soret region, unless otherwise stated. Each spectrum shown is the average of four independent biological replicates. Absorption spectra were used to calculate the relative distribution of chlorophyll molecules in the bands and fractions harvested from sucrose density gradients as [chlorophyll]=(⁠ ∫600750A(λ)×d(λ)×V×D, where ∫600750A(λ)×d(λ) is the integral of the absorption as a function of wavelength (from 600 nm to 750 nm), V is the volume of the band harvested, and D is the dilution factor applied to samples prior to absorption spectrum acquisition. Low-temperature steady-state fluorescence spectroscopy Low-temperature (77K) fluorescence emission and excitation spectra were measured using a FluoroMax-3 spectrophotometer (HORIBA Jobin Yvon, Longjumeau, France) equipped with a cryostat cooled by liquid nitrogen. For cell measurements, a Chl a concentration of 10 μg ml−1 was used, which prevented re-absorption and re-emission artefacts during measurements (see Supplementarty Fig. S1 at JXB online). For samples harvested by sucrose gradient separation, sample dilutions equal to those used for absorption spectroscopy were applied. To assess fluorescence emission spectra, samples were excited at 436 nm and emission was acquired from 600 nm to 800 nm. Fluorescence excitation spectra were detected at 690 nm and 715 nm for PSII and PSI, respectively, and recorded between 380 nm and 580 nm (PSII) and between 380 nm and 706 nm (PSI). The emission spectral resolution was 1 nm, while excitation spectral resolution was 0.5 nm, with excitation and emission bandwidths of 1 nm and 5 nm, respectively. An integration time of 0.1 s was used to reduce the noise level. Five scans were taken for each spectrum and then averaged. Fluorescence excitation spectra were corrected for variations in the detector efficiency, and excitation lamp intensity and distribution, with files provided by the manufacturer. Measurements were performed on 3–4 biological replicates. SDS–PAGE and immunoblotting Thylakoid membranes isolated from dark-adapted CL and IL cells (5 μg of total protein amount), as well as samples of sucrose density gradient bands/fractions (loaded as 10 μl sample volume or 1.5 μg of total protein amount; details in figure legends) were loaded onto 16% Tricine–SDS–PAGE gels according to Schägger (2006). Protein content was quantified by Bradford assay (Bradford, 1976). Gels were either stained by InstantBlue protein stain (Expedeon Ltd, UK) or used for electroblotting onto nitrocellulose membranes (GE Healthcare, UK). Anti-PsaB (1:1000; PSI-B core subunit, AS10695, Agrisera, Sweden), the β-subunit of ATP synthase (1:4000; ATP-B, AS05085, Agrisera), PsbA (1:15 000; D1, AS05084, Agrisera), PsbB (1: 2000; CP47, AS04038, Agrisera), and PsbC (1:3000; CP43, AS111787, Agrisera) antibodies were used. Accurate blotting was verified by correct transfer of colour pre-stained protein standard (P7712 and P7719, New England BioLabs, UK) on the nitrocellulose membrane. InstantBlue-stained gels were scanned using a ChemiDoc Touch Imaging System (Bio-Rad, USA). Antibody signals were detected after incubation with a secondary goat anti-rabbit antibody (IRDye 800CW, LI-COR Biosciences Ltd, UK; 1:20 000) and visualized by near infrared fluorescence detection (Odyssey Imaging System, LI-COR Biosciences Ltd, UK). Quantitative densitometric analysis of protein signals was carried out on Image Studio Lite (LI-COR Biosciences Ltd, UK) or (Fiji) ImageJ. Quantification of protein signals shown in this study are averages of western blots or SDS–PAGE gels carried out on 3–6 independent biological replicates ±SE. Statistics and reproducibility Sample replicates and statistical tests for each figure are denoted in the text and figure legends. Experiments were performed on at least three biological replicates, often accompanied by technical replicates. All error bars represent the SE, and statistical significance between conditions is indicated in the text and figure legends (two-tailed t-test was applied). Results Acclimation to continuous and intermittent light affects chlorophyll fluorescence emission of P. tricornutum cells The origin and energetic connectivity of the long-wavelength fluorescence emission was investigated in P. tricornutum, in response to cell acclimation to CL and IL. Given the low fluorescence yield of diatom PSI complexes at room temperature (e.g. Berkaloff et al., 1990; Fujita and Ohki, 2004; Ikeda et al., 2008; Belgio et al., 2017), we measured fluorescence emission on CL- and IL-acclimated cells at 77K. Low-temperature measurements revealed a long-wavelength emission band at 710 nm (F710) that accompanied a prominent band at 687 nm, typically associated with PSII (Fig. 1). The presence of re-absorption artefacts was excluded by testing different cellular concentrations of Chl a (Supplementary Fig. S1), and the position and relative extent of both bands agreed with previous reports on P. tricornutum (e.g. Fujita and Ohki, 2004; Lavaud and Lepetit, 2013; Nagao et al., 2019a; Oka et al., 2020) and other diatom species (e.g. Veith et al., 2009; Nagao et al., 2019b; Calvaruso et al., 2020). However, acclimation under IL strongly enhanced the F710 band (~3-fold) relative to CL acclimation (Fig. 1). While the PSII-associated 687 nm peak sits between emission bands previously assigned to PSII core antenna complexes (CP43 and CP47; Andrizhiyevskaya et al., 2005; Chen et al., 2015), F710 can originate from chlorophyll red forms associated either with PSI (core or FCP antenna) or with PSII (Herbstová et al., 2015). The latter scenario (i.e. ‘F710 association with PSII’) has been shown to occur in P. tricornutum cells grown under far-red light (Herbstová et al., 2015, 2017) and/or very low light intensities (e.g. Brown, 1967; Goedheer, 1973; Shimura and Fujita, 1973; Fujita and Ohki, 2004). Since our experiments were performed using white light, we hypothesized that at least part of those low-energy forms stemmed from the core and/or FCP antenna complexes of PSI (‘F710 association with PSI’). Fig. 1. Open in new tabDownload slide Low temperature (77K) fluorescence emission spectra of Phaeodactylum tricornutum cells acclimated to continuous (CL) and intermittent light (IL). A Chl a concentration of 10 μg ml–1 was used. The spectra shown are averages of 3–4 independent biological samples and normalized at 687 nm. The wavelengths used to detect fluorescence excitation from PSII (690 nm) and PSI (715 nm) are highlighted in grey. Fig. 1. Open in new tabDownload slide Low temperature (77K) fluorescence emission spectra of Phaeodactylum tricornutum cells acclimated to continuous (CL) and intermittent light (IL). A Chl a concentration of 10 μg ml–1 was used. The spectra shown are averages of 3–4 independent biological samples and normalized at 687 nm. The wavelengths used to detect fluorescence excitation from PSII (690 nm) and PSI (715 nm) are highlighted in grey. Biochemical and spectral properties of photosynthetic complexes isolated from continuous light- and intermittent light-grown cells: F710 emission is associated with PSI To test such a hypothesis and identify which photosystem contributed to F710 emission, we carried out linear sucrose density gradient ultracentrifugation of thylakoid membranes isolated from dark-adapted CL- and IL-grown P. tricornutum cells (Fig. 2A). Since P. tricornutum photosystem cores tend not to segregate as discrete bands along sucrose density gradients (Lepetit et al., 2007, 2010), thylakoid membranes were solubilized to a final concentration of 2% α-DDM (w/v) to improve separation of the photosynthetic membrane protein complexes. A similar pattern of bands was obtained regardless of the light acclimation of samples (Fig. 2A). For each band, spectroscopic and biochemical features were obtained. Below a yellow band indicative of some free pigment (B1), FCP antenna complexes (B2) appeared as a single, brown band, most probably isolated in the form of stable homodimers of Lhcf3/Lhcf4 protein (Wang et al., 2019). Denser, and spaced from FCPs by an unpigmented fraction (B3), green photosystem core bands were observed in the sucrose gradients. These bands were enriched in PSII cores (B4), PSII/PSI cores (B5), and PSI cores (B6), respectively (Fig. 2A). Because a clear separation between PSII and PSI was not fully achieved, B4–B6 bands were not pure and showed some contamination between photosystems (see below). Bands enriched in PSII (B4) and PSI (B6) were further characterized. Fig. 2. Open in new tabDownload slide Characterization of protein complexes isolated from thylakoid membranes of Phaeodactylum tricornutum cells. (A) Sucrose density gradient separation of thylakoid membranes isolated from continuous (CL) and intermittent light (IL)-acclimated cells (experiments were repeated using three independent biological samples). (B) Chlorophyll relative distribution (%) among bands and fractions harvested from CL and IL solubilized thylakoid membranes. Data are averages of three independent biological samples (total repeats measured, n=7) ±SE. (C) SDS–PAGE analysis of B2 (FCP), B4 (PSII-enriched), and B6 (PSI-enriched) band samples isolated from sucrose density gradient separation. Samples for SDS–PAGE were loaded as equal volumes (10 μl). SDS–PAGE of all samples was carried out in the same gel and repeated on three independent biological samples. For clarity, B2, B4, and B6 sample lanes only are shown. FP, free pigments; FCP, fucoxanthin Chl a/c binding protein antenna; Unpigm. F, unpigmented fraction. Fig. 2. Open in new tabDownload slide Characterization of protein complexes isolated from thylakoid membranes of Phaeodactylum tricornutum cells. (A) Sucrose density gradient separation of thylakoid membranes isolated from continuous (CL) and intermittent light (IL)-acclimated cells (experiments were repeated using three independent biological samples). (B) Chlorophyll relative distribution (%) among bands and fractions harvested from CL and IL solubilized thylakoid membranes. Data are averages of three independent biological samples (total repeats measured, n=7) ±SE. (C) SDS–PAGE analysis of B2 (FCP), B4 (PSII-enriched), and B6 (PSI-enriched) band samples isolated from sucrose density gradient separation. Samples for SDS–PAGE were loaded as equal volumes (10 μl). SDS–PAGE of all samples was carried out in the same gel and repeated on three independent biological samples. For clarity, B2, B4, and B6 sample lanes only are shown. FP, free pigments; FCP, fucoxanthin Chl a/c binding protein antenna; Unpigm. F, unpigmented fraction. The relative distribution of chlorophyll content among the isolated complexes was very similar between CL and IL samples (P>0.05), with minor differences only present for the FCP (B2) and PSI band (B6) (Fig. 2B). Protein composition and distribution along the sucrose density gradient were assessed by SDS–PAGE analysis (Fig. 2C). Together with a major protein band appearing at ~19 kDa for both light acclimations, a different composition of FCP antenna proteins (19–23 kDa) was found in CL and IL B2 samples (Fig. 2C), showing a minor accumulation of specific polypeptides in response to IL (Fig. 2C). It should be noted that the high molecular weight band (~66 kDa), visible in the CL B2 sample, is BSA present in the homogenization buffer used for thylakoid isolation from both CL and IL cells (protein identity was confirmed by LC-MS/MS analysis; data not shown). Analysis of PSII- and PSI-enriched bands (B4 and B6) showed (i) a similar core protein composition and content for both photosystems regardless of light acclimation; (ii) absence of FCP antenna proteins in the band enriched in PSII complexes (B4); and (iii) presence of a specific FCP antenna bound to PSI complexes (19–23 kDa range in B6; Fig. 2C). The latter feature is consistent with previous reports on diatoms (Veith and Büchel, 2007; Veith et al., 2009) and suggests that band B6 contains PSI–FCP complexes. In band B6, a slight up-regulation of PSI–FCP antenna polypeptides was found in CL relative to IL samples (Fig. 2C), in line with the larger relative chlorophyll content observed in CL B6 relative to IL B6 (Fig. 2B), as well as PSI core content assessed by western blotting (Supplementary Fig. S2). Absorption spectra of bands B2, B4, and B5 are compared in Fig. 3A, showing almost no difference between CL and IL samples. In FCP complexes (B2), the Soret and Qy absorption bands of Chl a were located at ~440 nm and 672 nm, respectively, with those for Chl c instead visible as a shoulder at ~460 nm and a band at ~635 nm, respectively. A broad absorption due to carotenoid pigments (480–550 nm; mainly fucoxanthin and, in part, diadinoxanthin) was also present (Fig. 3A). In contrast, PSII core (B4) and PSI–FCP-enriched complexes (B6) were largely deprived of carotenoid absorption and Chl c (no shoulder at ~460 nm, despite retaining the 635 nm band; Fig. 3A). A peak centred at ~489 nm, and assigned to lipid-dissolved diadinoxanthin (Lepetit et al., 2010), was observed in the carotenoid region of both photosystems (Fig. 3A). Most importantly, the Soret and Qy bands of both photosystems were blue and red shifted, respectively, relative to those of the FCP antenna (Fig. 3A). This was coupled to a strong broadening of the Qy band (Fig. 3A; Supplementary Fig. S3). Both features were more prominent for the PSI–FCP complexes than for the PSII cores, suggesting accumulation of (low-energy) chlorophyll red forms in the former and possibly different interactions among these chlorophylls with respect to the FCP antenna pigments. Fig. 3. Open in new tabDownload slide Spectroscopic properties of protein complexes isolated from thylakoid membranes of Phaeodactylum tricornutum cells. (A) Absorption spectra of bands B2 (FCP), B4 (PSII-enriched), and B6 (PSI-enriched), harvested via sucrose density gradient separation of solubilized thylakoid membranes isolated from continuous (CL) and intermittent light (IL)-grown P. tricornutum cells. The spectra shown are averages of four biological samples. (B–D) Low temperature (77K) fluorescence emission spectra of bands B2, B4, and B6 (averages of three biological samples). The spectra were normalized at 675 nm (B) or 686 nm (C, D). Fig. 3. Open in new tabDownload slide Spectroscopic properties of protein complexes isolated from thylakoid membranes of Phaeodactylum tricornutum cells. (A) Absorption spectra of bands B2 (FCP), B4 (PSII-enriched), and B6 (PSI-enriched), harvested via sucrose density gradient separation of solubilized thylakoid membranes isolated from continuous (CL) and intermittent light (IL)-grown P. tricornutum cells. The spectra shown are averages of four biological samples. (B–D) Low temperature (77K) fluorescence emission spectra of bands B2, B4, and B6 (averages of three biological samples). The spectra were normalized at 675 nm (B) or 686 nm (C, D). The identity and functional state of the different complexes separated by sucrose density gradient were further addressed by 77K emission spectroscopy (Fig. 3B–D). the 77K fluorescence spectra of FCP complexes (B2) isolated from CL- and IL-grown cells were identical and showed a peak positioned at 675 nm with a minor band at ~740 nm assigned to its vibronic satellite (Fig. 3B) (Johnson and Ruban, 2009; Ruban, 2012). This emission agrees with previous studies on isolated FCP complexes of diatoms (e.g. Lavaud et al., 2003; Guglielmi et al., 2005; Ikeda et al., 2008; Yokono et al., 2015). The fluorescence emission spectra of isolated PSII complexes (B4) displayed maxima at 686 nm and 692 nm—with a slightly more pronounced 692 nm peak in IL samples—and a vibronic sub-band at ~750 nm (Fig. 3C). These emission fingerprints are typical for the 77K fluorescence spectrum of PSII and previously reported for purified PSII complexes from higher plants and diatoms (Johnson and Ruban, 2009; Nagao et al., 2010; Ruban, 2012). A small shoulder at ~675 nm was visible, underlining only a minor content of uncoupled FCP antennae (Fig. 3C). Notably, no long-wavelength emission was found in PSII complexes isolated from both culture conditions. Instead, a broad F710 emission band appeared in band B6, which is enriched in PSI–FCP complexes (Fig. 3D). PSII-associated maxima (686 nm and 692 nm) were also retained (Fig. 3D), consistent with contamination by PSII cores (Fig. 2C; Supplementary Fig. S2). Relative to PSII emission peaks, F710 emission was greater in CL than in IL samples, and accompanied by a shoulder at ~675 nm that revealed the presence of some FCP antenna energetically uncoupled from the PSI core (Fig. 3D). Both aspects are consistent with the greater relative chlorophyll and FCP protein content, as well as PSI core content, observed in CL PSI–FCP complexes relative to that of IL samples (sample B6 in Fig. 2 and Supplementary Fig. S2). These data show that F710 emission originates from PSI–FCP complexes in both culture conditions, while being absent in PSII core-enriched complexes. FCP antenna transfers more excitation energy to PSI under intermittent light relative to continuous light acclimation Fluorescence excitation spectra of CL- and IL-acclimated cells were measured at 77K (Fig. 4; see Fig. 1 for emissions detected). Upon normalization of spectra at 436–438 nm, no major difference was observed between PSII excitation spectra (detected at 690 nm) from CL and IL cells (Fig. 4A), while differences were visible for PSI excitation spectra (detected at 715 nm) (see 450–550 nm and 668–700 nm regions; Fig. 4B). A shoulder was indeed visible in the far-red region of the CL spectrum, appearing as a clear peak at ~686 nm in the CL–IL difference spectrum (Fig. 4B). Following normalization of the spectra at 686 nm, the IL–CL difference spectrum was obtained (Fig. 4C), revealing the presence of an additional FCP antenna that is intact and energetically connected to PSI in IL cells, relative to CL cells (Fig. 4D). The excitation spectrum (detected at 715 nm) from isolated FCP antenna complexes and the IL–CL difference spectrum were plotted together to show some of the similarities observed (Fig. 4D). It should be noted that while the red excitation peaks are very similar, the Soret and carotenoid regions differ probably because of cell scattering. Nonetheless, carotenoid- and Chl c-specific peaks were observed, further indicating that it represents excitation from FCP complexes. These data suggest that IL acclimation induces either a greater accumulation or more efficient energetic coupling of FCP antenna complexes (see also Fig. 2) that feeds energy into the PSI cores, in comparison with CL acclimation. Fig. 4. Open in new tabDownload slide Assessing excitation energy transfer from antenna complexes to photosystem cores. (A, B) Low temperature (77K) fluorescence excitation spectra of PSII (PSII emission detected at 690 nm) and PSI core (PSI emission detected at 715 nm) measured on Phaeodactylum tricornutum cells grown under continuous (CL; black line) and intermittent light conditions (IL; red line). The spectra shown are averages of 3–4 biological samples. Spectra were normalized at 436–438 nm. Difference spectra (CL–IL) are shown (dashed line). (C) PSI excitation spectra normalized at 686 nm. The difference spectrum (IL–CL; dashed line) is shown. (D) IL–CL difference spectrum (from C) and FCP excitation spectrum (grey line) are compared. Spectra were normalized at 668 nm. Fig. 4. Open in new tabDownload slide Assessing excitation energy transfer from antenna complexes to photosystem cores. (A, B) Low temperature (77K) fluorescence excitation spectra of PSII (PSII emission detected at 690 nm) and PSI core (PSI emission detected at 715 nm) measured on Phaeodactylum tricornutum cells grown under continuous (CL; black line) and intermittent light conditions (IL; red line). The spectra shown are averages of 3–4 biological samples. Spectra were normalized at 436–438 nm. Difference spectra (CL–IL) are shown (dashed line). (C) PSI excitation spectra normalized at 686 nm. The difference spectrum (IL–CL; dashed line) is shown. (D) IL–CL difference spectrum (from C) and FCP excitation spectrum (grey line) are compared. Spectra were normalized at 668 nm. To test the energetic connectivity of the F710 band to the PSII core in vivo, we decreased the concentration of PSII reaction centres within the thylakoid membrane by growing cultures with the chloroplast protein synthesis inhibitor, lincomycin (Belgio et al., 2012; Townsend et al., 2018). Phaeodactylum tricornutum cells grown under both light conditions underwent lincomycin treatment, during which growth capacity and maximum quantum yield of PSII in the dark (Fv/Fm) were assessed and compared with control (untreated) cultures (Supplementary Fig. S4). Lincomycin treatment (0.55 mM) started during the early exponential phase of CL-grown cells and, after 2 d, cells grew less and Fv/Fm was significantly reduced (0.15±0.008, P<0.001; see Supplementary Table S1 and Supplementary Fig. S4 to compare control and lincomycin-treated cultures). Because IL acclimation strongly reduces cell growth rates (Lavaud et al., 2002; Giovagnetti and Ruban, 2017), 0.55 mM lincomycin was added three times to IL-grown cells over 44 d until the Fv/Fm was 0.28±0.009 (P<0.001; Supplementary Table S1; SupplementaryFig. S4). Reduction in growth capacity of IL cells was indeed barely visible despite the prolonged lincomycin treatment (Supplementary Fig. S4). Western blotting analysis was performed to quantify the decrease induced in the content of PSII and PSI cores (assessed as D1 and PsaB protein content changes, respectively; Fig. 5A). Loading the same total protein content accumulated in thylakoid membranes for each condition (Supplementary Fig. S5 for loading control), we found a significant reduction in PSII core content for both acclimations (P<0.001, Fig. 5B; in line with the low Fv/Fm values measured, Supplementary Fig. S4). The higher level of D1 found in IL relative to CL control thylakoids might be due to the presence of a greater content of photosystems embedded in IL thylakoid membranes, as well as the lower excitation pressure experienced under IL (Giovagnetti and Ruban, 2017). A minor decrease in PSI cores was instead obtained (P>0.05; Fig. 5B). As an additional control, a decrease in the chloroplast-encoded ATP-B was also appreciated (Fig. 5A). Similar reduction in photosystem cores was previously observed in higher plants in response to lincomycin treatment (Belgio et al., 2012; Townsend et al., 2018). Fig. 5. Open in new tabDownload slide Effect of lincomycin treatment on PSI and PSII content of Phaeodactylum tricornutum cells. (A) Western blot analysis of PsaB, ATPase β subunit (ATP-B), and PsbA (D1) proteins accumulated in thylakoid membranes isolated from dark-adapted continuous (CL) and intermittent light (IL)-acclimated cells of P. tricornutum. Instant blue staining is shown for the high molecular weight bands of PSI reaction centres. Lincomycin-treated cultures (+L) are compared with control cultures (–L; 3–6 independent biological replicates were tested). A 5 μg aliquot of total protein content was loaded for CL and IL conditions. (B) Quantification of PsaB and D1 protein content was performed from western blot analysis using three (PsaB, P>0.05) and six biological replicates (D1; an asterisk indicates P<0.001, t-test). Fig. 5. Open in new tabDownload slide Effect of lincomycin treatment on PSI and PSII content of Phaeodactylum tricornutum cells. (A) Western blot analysis of PsaB, ATPase β subunit (ATP-B), and PsbA (D1) proteins accumulated in thylakoid membranes isolated from dark-adapted continuous (CL) and intermittent light (IL)-acclimated cells of P. tricornutum. Instant blue staining is shown for the high molecular weight bands of PSI reaction centres. Lincomycin-treated cultures (+L) are compared with control cultures (–L; 3–6 independent biological replicates were tested). A 5 μg aliquot of total protein content was loaded for CL and IL conditions. (B) Quantification of PsaB and D1 protein content was performed from western blot analysis using three (PsaB, P>0.05) and six biological replicates (D1; an asterisk indicates P<0.001, t-test). To probe if and how F710 emission was altered by the significant decrease in PSII cores, emission spectra were measured either at room temperature or at 77K (Fig. 6). Room temperature emission spectra showed a single peak (~681 nm and ~683 nm for CL and IL cells, respectively), with a shoulder positioned around 710 nm more prominent in IL than CL cultures (Fig. 6A, B). At room temperature, a slight broadening of the peak and enhancement of the F710 shoulder was observed in lincomycin-treated relative to control cultures (Fig. 6A, B). When measured at 77K and normalized at the PSII-associated 687 nm band, we found that, for both CL and IL acclimations, the loss of functional PSII cores increased F710 emission relative to the emission measured in control cells (Fig. 6C, D). Moreover, the lincomycin-induced F710 band became broader than that emitted from untreated cells, especially for the CL acclimation (Fig. 6C, D). This reveals that the removal of PSII cores brings about the accumulation of a poorly coupled FCP antenna that serves PSI, resulting in broadening of F710 emission for both light acclimations. Fig. 6. Open in new tabDownload slide Effect of the loss of functional PSII cores on F710 emission band. Room temperature (A, B) and low temperature (77K) fluorescence emission spectra (C, D) of Phaeodactylum tricornutum cells acclimated to continuous (CL) and intermittent light (IL). Lincomycin-treated cultures (+L) are compared with control cultures (–L). A similar amount of cells was used during experiments (Supplementary Table S1). The spectra shown are averages of 3–4 biological samples. The spectra were normalized at the PSII maximum fluorescence emission peaks (681–683 nm and 687 nm). Fig. 6. Open in new tabDownload slide Effect of the loss of functional PSII cores on F710 emission band. Room temperature (A, B) and low temperature (77K) fluorescence emission spectra (C, D) of Phaeodactylum tricornutum cells acclimated to continuous (CL) and intermittent light (IL). Lincomycin-treated cultures (+L) are compared with control cultures (–L). A similar amount of cells was used during experiments (Supplementary Table S1). The spectra shown are averages of 3–4 biological samples. The spectra were normalized at the PSII maximum fluorescence emission peaks (681–683 nm and 687 nm). To visualize the absolute change in F687 and F710 emission bands in response to lincomycin treatment, normalization to (5 μM) fluorescein emission was applied (Mullineaux and Allen, 1987; Supplementary Fig. S6), showing not only that F710 emission was increased at 77K, but also that F687 was enhanced (CL, Supplementary Fig. S6C) or similar (IL, Supplementary Fig. S6D) in comparison with control cells. This is likely to be due to possessing a large bed of poorly coupled FCP antenna complexes that surrounds the few functional PSII reaction centres and emits strongly (Belgio et al., 2012). Thus, despite the strong reduction of PSII cores observed in lincomycin-treated cultures, the enhancement of the low-temperature F710 emission relative to that of control cultures shows that F710 is exclusively associated with the PSI core in response to our light acclimations. Discussion Thylakoid function and structure are modified by changes in the relative amount of light-harvesting complexes, PSII and PSI reaction centres, as well as electron carriers, occurring while photosynthetic organisms adapt to a specific light environment. Physical separation of photosystems is essential for optimal light absorption and efficient regulation of electron transport chain(s) across the different protein complexes embedded in the thylakoid membrane. In contrast to the lateral heterogeneity observed in plant thylakoids (Anderson, 1986), the chloroplast ultrastructure of diatoms consists of loosely appressed stacks of three thylakoid membranes, where functional segregation of PSII and PSI is nonetheless achieved (Flori et al., 2017). Overall, photons are absorbed by light-harvesting antenna complexes at a shorter wavelength than the absorption of the so-called ‘special pair’ of chlorophylls responsible for charge separation in the reaction centres of PSII and PSI. This allows for an energetically favoured (‘downhill’) energy transfer from light-harvesting antenna pigments to those of photosystem cores (van Grondelle et al., 1994). Even so, long-wavelength spectral forms of chlorophyll, capable of capturing light at lower energies than those of photosystem primary electron donors, have been found in a number of photosynthetic organisms (Gobets and van Grondelle, 2001; Croce and van Amerongen, 2013; Santabarbara et al., 2020). Often associated with PSI (Gobets and van Grondelle, 2001; Croce and van Amerongen, 2013; Santabarbara et al., 2020), their spectral characteristics have been reported to originate from mixing of excitonic interactions and charge transfer states (e.g. Croce et al., 2007a; Romero et al., 2009). Although it is widely accepted that these low-energy red forms enhance the absorption capacity of light-harvesting systems in the near infrared region and provide adaptive advantages (Rivadossi et al., 1999), their biological role has not yet been completely elucidated. They have been reported to be involved in focusing the excitation energy close to the reaction centres (Trissl, 1993), as well as contributing to photoprotection by enhancing triplet quenching formation in antenna protein domains (Carbonera et al., 2005; Santabarbara et al., 2005; Croce et al., 2007b), while not being in quenched conformations (Passarini et al., 2010; Wientjes et al., 2011; Jennings et al., 2013). Transfer of excess energy to the low-energy Chl a found in PSI and subsequent quenching by P700+ have also been postulated (Yokono et al., 2019). In diatoms, far-red-absorbing and emitting chlorophylls have been reported to be associated either with PSII (e.g. Brown, 1967; Shimura and Fujita, 1973; Owens, 1986b) or with PSI (e.g. Sugahara et al., 1971; Goedheer, 1973, 1981). Establishing their origin and assignment has proven challenging. The relative contribution of PSI emission (~710–717 nm at 77K) is indeed low in diatoms, and chlorophyll red forms arising from PSII, both at room temperature and at 77K, have been observed (e.g. Brown, 1967; Goedheer, 1973; Shimura and Fujita, 1973; Fujita and Ohki, 2004). The ability of ‘chromatic adaptation’ under far-red light has been documented in different photoautotrophs, among which different centric and pennate diatom species (e.g. Fujita and Ohki, 2004; Herbstová et al., 2015, 2017; Ueno et al., 2019; Akimoto et al., 2020; Oka et al., 2020). In P. tricornutum, growth under red light induces the formation of a red-shifted oligomeric antenna complex (consisting of the protein Lhcf15) and appearance of F710 emission, already visible at room temperature (Herbstová et al., 2015, 2017). Recently, two forms of low-energy Chl a (F713 and F718), which were energetically bound to the PSII core, were found in P. tricornutum red-shifted FCP antenna when cells were grown under red light (Oka et al., 2020). Spectroscopic and biochemical evidence supporting PSII association of such red forms in diatoms was obtained: (i) red-shifted emission bands at room temperature were found to respond to fluorescence induction and closure of PSII [by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCMU] in a comparable fashion to those bands associated with PSII (Brown et al., 1967; Goedheer, 1973, 1981; Fujita and Ohki, 2004; Herbstová et al., 2015); (ii) their origin from PSII was revealed via time-resolved spectroscopy at room and low temperature using DCMU or preferential excitation of fucoxanthin (Miloslavina et al., 2009; Chukhutsina et al., 2013); and (iii) complexes harbouring these red forms were purified and characterized (Herbstová et al., 2015, 2017). At the same time, a number of studies demonstrated the energetic coupling of F710–F717 bands to PSI (core or FCP antenna) in several diatom species (e.g. Sugahara et al., 1971; Goedheer, 1973; Berkaloff et al., 1990; Veith and Büchel, 2007; Ikeda et al., 2008; Yamagishi et al., 2010; Oka et al., 2020; Tanabe et al., 2020). Moreover, despite having a minor contribution to the overall emission at 77K, a PSI-located F710 component was also found in P. tricornutum cells grown under red light (Herbstová et al., 2015; Oka et al., 2020). Here, we combined spectroscopic and biochemical measurements in vivo and in vitro to investigate the origin and energetic connectivity of F710 emission in P. tricornutum acclimated to CL and IL. IL acclimation was shown to cause modifications in the chloroplast ultrastructure, likely to affect the excitation energy transfer from FCP antenna complexes to photosystem reaction centres (Giovagnetti and Ruban, 2017). Using a white light source results in the absence of a clear F710 peak at room temperature, which was only visible as a minor and more pronounced shoulder in IL relative to CL cells. This indicates that no red-shifted antenna associated with PSII was present in vivo, differently from studies on cells acclimated to far-red light (Fujita and Ohki, 2004; Herbstová et al., 2015). A clear F710 emission band only appears in both conditions upon performing measurements at 77K, with IL-grown cells capable of much larger F710 emission relative to CL-grown cells (Fig. 1). By characterizing the photosynthetic protein complexes isolated from thylakoids, we show that F710 emission only originates when PSI complexes are coupled to a specific FCP antenna (PSI–FCP complexes; Veith and Büchel, 2007; Veith et al., 2009) (Figs 2C, 3D). This is found regardless of the light acclimation imposed, and importantly despite the marked low-light acclimation brought by the IL regime (Giovagnetti and Ruban, 2017). Notably, the differences observed in protein composition of complexes isolated from CL and IL thylakoid membranes are minor (Fig. 2C), and in line with a similar relative content of Chl a and absorption between the complexes isolated from the two culture conditions (Figs 2B, 3A). Given the absence of F710 emission in PSII-enriched complexes (band B4), the contamination of PSII core and inner antenna complexes found in the PSI–FCP complex-enriched band (B6) is unlikely to be responsible for F710 emission. To understand what causes the enhancement of F710 emission observed in IL-acclimated cells, we measured fluorescence excitation spectra for both photosystems at 77K. The obtained excitation spectra of PSII are comparable between CL and IL cells, in agreement with the same PSII antenna cross-section found in dark-adapted CL and IL cells (Giovagnetti and Ruban, 2017). On the contrary, when excitation was probed for PSI, we found that cells possess an additional cross-section associated with PSI, with an excitation spectrum that resembles that of an intact FCP antenna (Fig. 4). This is consistent with the difference in the PSI-assigned lifetime component (at Fm state) measured in P. tricornutum cells grown under CL (86 ps) or IL (110 ps; Giovagnetti and Ruban, 2017). Finding an ~28% increase in PSI lifetime under IL suggests indeed the presence of a larger antenna serving PSI in this acclimation, as well as no contribution of FCP aggregation to F710 emission in the conditions tested (i.e. FCP aggregation would have induced shortening of PSI-assigned lifetime). This indicates that PSI receives more excitation energy under IL, relative to CL, by means of FCP antenna complexes that are either more abundant or more efficiently connected to PSI cores. It is possible that a part of these PSI-associated FCP antenna complexes was detached by detergent during thylakoid solubilization, thus partially accumulating as FCP antenna proteins in IL samples (compare B2 samples in Fig. 2B and C). Yet, no strong changes in polypeptide composition are present between CL- and IL-isolated (FCP or core) complexes, suggesting that F710 emission originates from FCP antenna-sensitized PSI cores, without compositional changes of specific antenna proteins. The detected enhancement of IL-induced F710 emission appears to involve direct connectivity of FCP antenna complexes to PSI cores, and no major changes in PSII/PSI stoichiometry (see Fig. 2; Supplementary Fig. S2). However, we cannot rule out the possibility for some spillover to occur in vivo under IL (i.e. a PSII–FCP that transfers energy to PSI). If present at all, spillover was shown to be negligible in P. tricornutum cells acclimated to low light (Miloslavina et al., 2009; Flori et al., 2017), while other reports measured different contributions of spillover in regulating energy transfer processes in the same species subjected to different growth conditions (e.g. Yokono et al., 2015; Nagao et al., 2019a; Ueno et al., 2019; Tanabe et al., 2020). To the best of our knowledge, we report here for the first time on growing diatoms with lincomycin to decrease PSII cores. The loss of functional PSII reaction centres in both culture conditions does not diminish the F710 emission in vivo, but rather enhances and broadens it (Fig. 6). This further indicates that F710 emission originates from PSI in the conditions tested. Increased amplitude and broadening of F710 emission might be caused by the accumulation of a poorly coupled PSI–FCP antenna with modified pigment interactions. Overall, our data suggest that the presence of a low-temperature F710 emission band is uniquely associated with the PSI complex in P. tricornutum, regardless of CL or IL acclimation. The latter requires a larger or more effective FCP antenna channelling excitation energy towards the cores of PSI. Because only PSI–FCP complexes were isolated (Fig. 2), we cannot discern if the chlorophyll red forms contributing to F710 emission belong exclusively to PSI–FCP antenna complexes or PSI cores, or both. The presence of an enhanced F710 emission correlates with the ability of forming very large levels of non-photochemical quenching in IL-acclimated cells (Giovagnetti and Ruban, 2017). Previous studies proposed connection linking the F710 extent to diatom capacity for excess energy dissipation via FCP antenna detachment (Miloslavina et al., 2009; Lavaud and Lepetit, 2013). However, since we showed that a comparable FCP antenna detachment (~50%) from reaction centre II occurred upon non-photochemical quenching formation in CL and IL cells (Giovagnetti and Ruban, 2017), this suggests that the quenching and F710 emission are not mechanistically connected. Instead, our data show that enhanced F710 in IL cells is an adaptive strategy of P. tricornutum to enhance PSI cross-section under very low light. This redistribution of excitation energy between photosystems, without alteration of the PSII/PSI ratio, could facilitate the switch between linear and cyclic electron flow, as well as their regulation, under IL. Beyond the diversity of experimental (light) conditions tested, much of the variability described regarding the origin and connectivity of diatom chlorophyll red forms seems to reflect intrinsic differences of diatom species/groups (i.e. pennates versus centrics). Clear differences in (i) fluorescence emission of FCP antenna complexes (e.g. Berkaloff et al., 1990; Lavaud et al., 2003; Ikeda et al., 2008; Veith et al., 2009) and chlorophyll red forms of PSI (e.g. Ikeda et al., 2008; Veith et al., 2009; Yokono et al., 2015); (ii) concentrations of low-energy chlorophylls in the SI core and/or PSI–FCP antenna (e.g. Nagao et al., 2019a, 2019c); (iii) functions of the PSII-associated red-shifted forms (i.e. light-harvesting versus quenching roles; Ueno et al., 2019; Oka et al., 2020); and (iv) the states of oligomerization of FCP complexes (and possibly the structural organization of PSII and PSI supercomplexes; Nagao et al., 2019d; Pi et al., 2019; Wang et al., 2019) have been observed between pennate (e.g. P. tricornutum) and centric diatoms (e.g. C. gracilis and Cyclotella meneghiniana). We believe that such modularity and flexibility in the photosynthetic machinery of diatoms evolved as a crucial feature ensuring their ecological success. Supplementary data The following supplementary data are available at JXB online. Table S1. Growth and physiological state of P. tricornutum lincomycin-treated and control cultures. Fig. S1. Testing for re-absorption artefacts during low temperature (77K) fluorescence emission measurements. Fig. S2. Western blot analysis of PsaB (PSI), PsbB (CP47), and PsbC (CP43) proteins. Fig. S3. Comparison of Qy absorption bands. Fig. S4. Growth and PSII quantum yield in the dark (Fv/Fm) of Phaeodactylum tricornutum lincomycin-treated and control cultures. Fig. S5. Protein composition of thylakoid membranes isolated from Phaeodactylum tricornutum lincomycin-treated and control cultures. Fig. S6. Room temperature and low temperature (77K) fluorescence emission from Phaeodactylum tricornutum lincomycin-treated and control cultures. Acknowledgements We thank F. Saccon and S. Wilson for discussions during preliminary stages of this work. S. 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Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The mechanism of regulation of photosystem I cross-section in the pennate diatom Phaeodactylum tricornutum
JF - Journal of Experimental Botany
DO - 10.1093/jxb/eraa478
DA - 2021-02-02
UR - https://www.deepdyve.com/lp/oxford-university-press/the-mechanism-of-regulation-of-photosystem-i-cross-section-in-the-pv0vrNGE3Q
SP - 561
EP - 575
VL - 72
IS - 2
DP - DeepDyve
ER -