# Manganese deficiency alters photosynthetic electron transport in Marchantia polymorpha

Umama Hani, Anja Krieger-Liszkay

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Umama Hani, Anja Krieger-Liszkay. Manganese deficiency alters photosynthetic electron transport in Marchantia polymorpha. Plant Physiology and Biochemistry, 2024, 215 (4), pp.109042. 10.1016/j.plaphy.2024.109042. hal-04791918

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# Manganese deficiency alters photosynthetic electron transport in *Marchantia polymorpha*

Umama Hani, Anja Krieger-Liszkay*

Université Paris-Saclay, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, 91198, Gif-sur-Yvette cedex, France

<figure><img src="image_2.png" /></figure>

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## ARTICLE INFO

Handling Editor: Dr. Mario De Tullio

**Keywords:**

Cyclic electron transport
In vivo absorption spectroscopy
Marchantia polymorpha
Photosystem I
Plastocyanin

## ABSTRACT

Manganese (Mn) is considered as an essential element for plant growth. Mn starvation has been shown to affect photosystem II, the site of the Mn₄CaO₅ cluster responsible for water oxidation. Less is known on the effect of Mn starvation on photosystem I. Here we studied the effects of Mn deficiency *in vivo* on redox changes of P700 and plastocyanin (Pc) in the liverwort *Marchantia polymorpha* using the KLAS-NIR spectrophotometer. Far-red illumination is used to excite preferentially photosystem I, thus facilitating cyclic electron transport. Under Mn starvation, we observed slower oxidation of P700 and a decrease in the Pc signal relative to P700. The lower Pc content under Mn deficiency was confirmed by western blots. Re-reduction kinetics of P700⁺ and Pc⁺ were faster in Mn deficient thalli than in the control. The above findings show that the kinetics studied under Mn deficiency not only depend on the number of available reductants but also on how quickly electrons are transferred from stromal donors via the intersystem chain to Pc⁺ and P700⁺. We suggest that under Mn deficiency a structural reorganization of the thylakoid membrane takes place favoring the formation of supercomplexes between ferredoxin, cytochrome *b*₆f complex, Pc and photosystem I, and thus an enhanced cyclic electron transport.

## 1. Introduction

Manganese (Mn) is one of the essential trace elements for plant growth and development. Mn is known as an indispensable constitutive element of the Mn₄O₅Ca cluster in photosystem II (PSII), where it oxidizes water molecules and thus provides the necessary electrons for oxygenic photosynthesis (Shen, 2015; Alejandro et al., 2020). Plants require an optimal supply of Mn to maintain homeostatic balance (Alejandro et al., 2020). Besides its role in PSII, Mn has an important role as co-factor of various enzymes including catalase (MnCAT), superoxide dismutase (MnSOD), decarboxylases of tricarboxylic acid (TCA) cycle (Marschner, 2011; Corpas et al., 2017) and in other metabolic processes (Schmidt et al., 2016; Bhat et al., 2020). Mn deficiency strongly impacts photosynthesis, mainly PSII, accompanied by subtle perturbation in chloroplast ultrastructure (Papadakis et al., 2007; Messant et al., 2023). Initial signs of Mn deficiency typically appear first in younger leaves, which includes paleness accompanied by interveinal chlorosis (Schmidt et al., 2016). However, symptoms of Mn deficiency vary depending on the species (Homann, 1967).

Contrary to the research done on effects of Mn deficiency on PSII, very limited information concerning its consequences on PSI is

available. We have previously studied the effects of Mn excess and deficiency in liverwort *Marchantia polymorpha*, for which we established the conditions for Mn concentration for optimal growth and a condition to induce Mn deficiency (Messant et al., 2023). With well-explored taxonomy and morphology, *M. polymorpha* stands as a complex thalloid liverwort (Bowman, 2016), residing mainly at the marginal area between aquatic and land environments. Today's use of *M. polymorpha* as a model plant is primarily based on its genomic simplicity (Bowman et al., 2016; Bowman, 2022) and ease of propagation (Naramoto et al., 2022). In our recent study, we investigated the effect of Mn deficiency on photosynthesis in *M. polymorpha*. Under Mn starvation, a change in the ratio of PSI/PSII capacities was observed. Moreover, we also showed changes in structural organization of thylakoid membrane. Most importantly for the present study, less limitation of electron donation to P700⁺ in actinic light was observed under Mn deficiency. We interpreted the difference in stoichiometry of PSI/PSII and the lower donor-side limitation of PSI as an increase in cyclic electron transport (CET) under Mn starvation (Messant et al., 2023). CET is one of the alternative electron pathways that comes into play under certain physiological conditions, especially upon stress (Sunil et al., 2019) with the main aim of balancing the ATP/NADPH ratio. CET involves cycling of

<footnote>* Corresponding author.
E-mail address: anja.liszkay@i2bc.paris-saclay.fr (A. Krieger-Liszkay).</footnote>
electrons around PSI, generating a proton gradient and thereby ATP without generating NADPH (for recent reviews see e.g., Nawrocki et al., 2019; Takahashi, 2022).

The current study aims to further explore the effects of Mn deficiency on PSI in *M. polymorpha*, with the main objective of investigating changes in oxido-reduction of P700 and plastocyanin (Pc) using KLAS-NIR spectrophotometer. KLAS-NIR is a pulse-amplitude modulation spectrophotometer that has been developed by Klughammer and Schreiber (2016) to study deconvoluted signals in the near infra-red of P700 and Pc *in vivo*, i.e. in thalli in our case. We particularly have chosen the approach of inducing CET by irradiating thalli with far-red (FR) light, thus facilitating the measurement of CET. FR light is preferentially absorbed by PSI and maximally oxidizes P700 to the extent of 80–90% (Chow et al., 2012; Pettai et al., 2005).

## 2. Materials and methods

### 2.1. Plant material

Gemmae from Takaragaike (Tak-1) male accession of *M. polymorpha* were cultured for two weeks under a light-dark cycle (16 h light, 22 °C, 100 µmol photons m⁻²s⁻¹, white fluorescent lamp; 8 h dark, 20 °C) on one-half-strength Gamborg's B5 media (GB5) with 1% Agar (Gamborg et al., 1968). To create Mn deficiency, thalli were transferred to ½ GB5 media containing 6% starch with or without addition of 33 µM MnCl₂ for a week prior to performing desired experiments (Messant et al., 2023).

### 2.2. Thylakoids isolation and immunoblots

Thalli from three-weeks old *M. polymorpha* were harvested and after careful washing with water to remove starch, were grounded in a buffer containing 0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl₂, 50 mM KCl, 25 mM MES-KOH (pH 6.1). The resulting slurry was filtered through two layers of cheese cloth and one layer of miracloth. After centrifugation, the pellet was re-suspended in 25 mM HEPES-KOH (pH 6.7), 0.33 M sorbitol, 1 mM EDTA, 1 mM MgCl₂, 50 mM KCl and washed twice. Finally, the isolated thylakoids were re-suspended and stored in 25 mM HEPES-KOH (pH 7.6), 0.33 M sorbitol, 1 mM MgCl₂, 50 mM KCl. All centrifugation steps were performed at 3,000×g for 3 min at 4 °C. Chlorophyll content was measured according to Arnon (1949).

For immunodetection, proteins from the thylakoids extract were separated using 12% (w/v) sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membrane (Amersham™ Protran, 0.45 µm pore size) and incubated with primary antibodies (Pc, PsaC, PetB and cytb₅₅₉) at room temperature in TBS-T (0.1 % Tween-20 (v/v), containing 5% non-fat milk powder). After washing with TBS-T, the membranes were decorated with anti-rabbit peroxidase-linked secondary antibodies. Bands were visualized with enhanced chemiluminescence (ChemiDoc™ Touch Imaging System, Bio-Rad) after 1–2 min incubation with ECL solution (Amersham ECL Prime Western Blotting Detection Reagent). Antibodies directed against Pc (AS06 141), PsaC (AS10 939), PetB (AS18 4169) were purchased from Agrisera (Vännäs, Sweden), antibodies directed against cytb₅₅₉ were kindly provided by P. Beyer, Freiburg University, Germany. Densitometric analysis of blots was performed with image J (https://imagej.nih.gov/ij/).

### 2.3. RT-PCR

Plant Mini RNeasy kit (Qiagen, Venlo, Netherlands) was used for total RNA extraction, and samples were homogenized with RLT lysis buffer. RT reactions were done with the SuperScript IV Reverse Transcriptase kit (Invitrogen, Carlsbad, USA). cDNA was diluted 4 times with LightCycler 480 SYBR Green I Master (Roche, Penzberg, Germany) reaction medium (1X final concentration) containing 2.5 µM of each primer (Table S1). The mRNA levels of plastocyanin

(Mp4g02720.1_Sequence extracted from "MarpolBase"; genome database for Marchantia) were normalized to reference housekeeping gene ACTIN 1 (MpACT1: Mp6g10990).

### 2.4. Chlorophyll fluorescence and P700 measurements

Chlorophyll fluorescence and P700 absorbance were measured using a Dual-PAM-100 fluorometer (Walz, Effeltrich, Germany). Effective quantum yield of PSII: Y(II) = (Fm'-F)/Fm' and non-photochemical quenching: qN=(Fm'-Fm')/Fm, were measured as a function of light intensity. Each actinic light intensity was applied for 180 s. For P700 measurements, dark adapted thalli were exposed to three different actinic light intensities (64, 175 and 803 µmol quanta m⁻²s⁻¹) and far-red light followed by a saturating flash at the end of far-red light period.

### 2.5. KLAS-NIR measurements

All the measurements were made with kinetic-LED-array-spectrophotometer (KLAS) (Walz, Effeltrich, Germany) that utilizes pulse amplitude modulation within the NIR-Infrared (NIR) spectral range and operates in the dual-wavelength difference mode (785–840, 810–870, 870–970 and 795–970 nm) (Schreiber and Klughammer, 2016; Schreiber, 2017). Technical details of the KLAS-NIR spectrophotometer and the principle behind the deconvolution of P700, Pc and Fd are explained in detail by Klughammer and Schreiber (2016). All measurements were done under "Slow kinetics window". Transmittance changes were recorded with high-resolution trigger run file called "NIR max" along with data acquisition every ms (Fig. 2). Kinetics recording and illumination steps were fully automated. NIR max protocol includes three main components. After dark adaptation, the sample was illuminated with Actinic Light (AL10, 572 µmol quanta m⁻²s⁻¹) for 3 s and 30 ms pulse of light at saturating light intensity (6500 µmol quanta m⁻²s⁻¹) was applied during illumination with AL. The second component involves switching on the FR light (FR18) for 10 s that lead to almost maximal oxidation of P700 and Pc. At the end, a 300 ms saturating flash was given to fully induce oxidation of P700 and Pc (P700 and Pc MAX values). Kinetics of P700 oxidation (Fig. 4) and P700⁺ and Pc⁺ re-reduction (Figs. 5 and 6) were studied with FR-on (FR10) and FR-off respectively.

### 2.6. Transmission electron microscopy

About 1 mm strips of thalli were cut and fixed with glutaraldehyde/ paraformaldehyde fixative and later infused with 1% osmium (w/v) and 1.5% potassium hexacyanoferrate (w/v) en bloc staining protocol as described (Hawes et al., 1981; Juniper et al., 1982); EM UC6 ultramicrotome (Leica microsystems) was used for cutting 70 mm thick sections to be deposited on to copper grids. Ultrathin sections were stained with 2% uranyl acetate (w/v) (Merck) and Reynolds lead citrate according to standard procedures. Grids were examined under a JEOL 1400 TEM operating at 80 kV (JEOL, http://www.jeol.com). Images were acquired using a 9-megapixel high-speed camera (RIO9; Gatan, http://www.gatan.com) and processed using Digital Micrograph (Gatan).

## 3. Results

In the current study, we aimed to investigate the effect of Mn deficiency on photosynthetic electron transport in *M. polymorpha* in more detail compared with the study by Messant et al. (2023). Fig. 1 shows that Y(II) was lower and qN was higher in Mn-deficient thalli than in thalli grown in control conditions. In addition to PSII, P700 oxidation is also affected under Mn deficiency. At light intensities higher than growth light, P700 oxidation was slower and a lower steady state oxidation level was reached at an actinic red light intensity of 175 µmol quanta m⁻²s⁻¹. We have reported previously that limitation of electron donation to PSI was lower in Mn-deficient thalli than in thalli grown in
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Chlorophyll fluorescence and P700 measurements in response to different light intensities in *Marchantia thalli* grown in control and Mn-deficient conditions. Effective quantum yield of PSII (YII) and non-photochemical quenching (qN) measured as function of light intensity. Data are represented as the mean ± SD of 5 biological replicates (A). For P700 oxidation measurements, red actinic light (803, 175 and 64 µmol quanta m⁻²s⁻¹) and far-red light were given as indicated in the coloured bars. To get the maximum oxidation amplitude of P700, a saturation flash was given at the end of the far-red illumination. Measurements with three independent biological replicates were performed and representative traces are shown (B).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Redox changes in P700 and Plastocyanin in *Marchantia thalli* grown in control and Mn-deficient conditions. Changes in the redox states of P700 (red) and plastocyanin (Pc; blue) in thalli grown in control conditions (+Mn; A) and in Mn-deficiency (-Mn; B) upon illumination with red actinic light (570 µmol quanta m⁻²s⁻¹) and far-red light. Saturation flashes were applied at 4 s and 20 s as indicated by black arrows. Horizontal marks indicate the maximum intensity levels (under far-red light) and the zero levels (in the dark) for P700 and Pc. Top of the figure indicates the illumination protocol (red = Actinic light, dark red = Far-red light). Figures A, B show representative traces. The mean absorption values of the calculated ratio (Pc/P700) for + Mn and -Mn are given in C, 3 independently grown biological replicates, n = 9. Statistically significance (*P* < 0.05) was assessed by Student's t-test.</figcaption></figure>

control conditions while the capacity of PSII electron transport was lowered (Messant et al., 2023). To study oxidation of P700 and Pc in parallel, we used the KLAS NIR spectrophotometer. The kinetics shown in Fig. 2 were obtained using the “NIR-Max” protocol of KLAS NIR spectrophotometer. Changes in the redox states of P700 and Pc in

response to red-actinic and far-red light are shown for 3-weeks old thalli of *Marchantia polymorpha*. The maximum oxidation levels for P700 and Pc were determined through a saturation flash following the far-red light. Calculated “relative” values indicate the amount of photo-oxidized (and reduced) P700 and Pc. Compared with P700⁺
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Western blots and RT-PCR analysis of plastocyanin in *Marchantia* grown in control and Mn-deficient conditions. Representative immunoblots showing the content of Pc (Plastocyanin), PsaC (PSI subunit), *cytb*<sub>559</sub> (PSII subunit) and PetB (cyt *b*<sub>6f</sub> subunit) in thylakoids extracted from *Marchantia polymorpha*, grown in the presence of Mn (+Mn) and under Mn deficiency (-Mn). Gels were loaded based on chlorophyll content and ponceau red staining is shown as loading control (A). Densitometric analysis (B), mean value ± SE of three to six biological replicates are shown. Statistically significance (*P* < 0.05) was assessed by Student's t-test. RT-PCR data for Pc (mean value ± SE) of two biological replicates are shown (C).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Far-red light (FR) illumination shows differences in the kinetics of P700 oxidation in Mn-deficient samples. Thalli grown in control (A) and Mn-deficient (B) conditions were illuminated with FR light for 15 s. Top of the figure indicates the illumination protocol. The representative curves shown are an average of 10 traces. In total, 3 biological replicates were measured, each with 2 technical replicates.</figcaption></figure>

amplitude, in far-red light, the amplitude of Pc oxidation was signifi-
cantly smaller (≤50 %) under Mn deficiency (Fig. 2B) than in the
presence of Mn (Fig. 2A). Fig. 2C shows significant changes for the
calculated Pc to P700 ratio. The decrease in Pc content was confirmed by
immunoblotting using specific antibodies. A clear decrease in the Pc
content was visible under Mn deficiency (Fig. 3A), while PsaC (PSI
subunit), *cytb*<sub>559</sub> (PSII subunit) and PetB (cyt *b*<sub>6f</sub> subunit) remained
stable under both conditions. RT-PCR analysis of Pc (Fig. 3C) showed no
significant differences between the two growth condition, indicating
that it is not the expression levels but rather the protein stability that is
responsible for the low Pc content under Mn deficiency.

The question arises how a low content of Pc affects electron donation to P700<sup>+</sup>. We reported earlier lower donor-side limitation in Mn-deficient *Marchantia* when illuminated with red actinic light. Fig. 4 shows P700 oxidation kinetics induced by far-red light of dark-adapted *Marchantia* thalli. A considerable difference in P700 oxidation was noticed between control (+Mn) and Mn-deficient (-Mn) samples. In the presence of Mn (Fig. 4A), far red light triggered P700 oxidation within 8s, whereas under Mn deficiency (Fig. 4B) P700 is oxidized more slowly,

indicating that more electrons are available to be donated to P700<sup>+</sup>,
thereby delaying the time until maximum P700<sup>+</sup> accumulation is
observed. This slow rise of P700 oxidation may indicate a higher cyclic
electron transport.

To further see the effect of Mn deficiency on PSI, reduction of P700<sup>+</sup> was studied after turning off the far-red light. Fig. 5 shows a significantly slower re-reduction of P700<sup>+</sup> under control conditions than under Mn deficiency. Half times (t<sub>1/2</sub>) of P700<sup>+</sup> were determined as 3.7 s ± 0.01 and 1.9 s ± 0.007 for + Mn and -Mn, respectively. The faster re-reduction of P700<sup>+</sup> indicates again that more electrons are available to be donated to P700<sup>+</sup>, suggesting a higher CET or less strong photo-synthetic control under Mn deficiency. Moreover, we also reported faster re-reduction of Pc<sup>+</sup> in Mn-deficient thalli than in control thalli (Fig. 6). Pc<sup>+</sup> reduction was about 2.5-fold slower in comparison with P700<sup>+</sup>. In the presence of the uncoupler nigericin the opposite effect was observed (Fig. S1). Both, the t<sub>1/2</sub> for P700<sup>+</sup> reduction and for Pc<sup>+</sup> reduction were slower under Mn deficiency than in control conditions. This shows that in the presence of nigericin under Mn deficiency less electron donors are available to reduce P700<sup>+</sup> and Pc<sup>+</sup>.
<figure><img src="image_7.png" /></figure>

**Fig. 5.** Re-reduction kinetics of P700⁺ after FR illumination. The kinetics of P700⁺ re-reduction in Marchantia thalli indicated in black and grey for + Mn and -Mn, respectively, were monitored in the dark, after 5s exposure to FR light. Decay signals for P700⁺ were fitted with single exponential function (red traces). The curves represent an average of 6 measurements for each sample. The inset show the half times (t₁/₂) calculated for P700⁺ decay (samples from three independent cultures, n = 6). Statistical significance (*P* < 0.05) was assessed by Student's t-test.

<figure><img src="image_8.png" /></figure>

**Fig. 6.** Re-reduction kinetics of Pc⁺ after FR illumination. The kinetics of Pc⁺ re-reduction in Marchantia thalli indicated as black and grey for + Mn and -Mn respectively, were monitored in the dark, after 5s exposure to FR light. Decay signals for P700⁺ were fitted with single exponential function (red traces). The curves represent an average of 6 measurements for each sample. The inset show the half times (t₁/₂) calculated for Pc⁺ decay (samples from three independent cultures, n = 6). Statistical significance (*P* < 0.05) was assessed by Student's t-test.

## 4. Discussion

Here, we studied transient absorption changes in P700 and Pc using the KLAS-NIR spectrophotometer in the liverwort *M. polymorpha* grown

in control and Mn-deficient conditions. According to the maximum signals for Pc⁺ (Fig. 2) and immunoblot analysis (Fig. 3), the Pc signal relative to the P700 signal is drastically decreased upon Mn deficiency (Fig. 2). In *Arabidopsis*, it has been shown that the Pc content can be significantly decreased without pronounced changes in the overall photosynthetic activity, suggesting that Pc is present in large excess under control growth conditions (Pesaresi et al., 2009). However, in tobacco *Schöttler et al.* (2004) observed a reduction of electron transport and assimilation capacity when the amount of Pc was decreased. A decrease in Pc content has previously also been reported in early stages of senescence in tobacco and barley (*Schöttler et al.*, 2004; *Shimakawa et al.*, 2020). In Marchantia, lower Pc content induced by Mn deprivation, affects electron donation to PSI. The slower rate of P700 oxidation upon illumination of dark-adapted sample with red and FR light under Mn deficiency (Fig. 2B and 4B, see also Fig. 7 in *Messant et al.*, 2023) and the faster re-reduction of P700⁺ (Fig. 5) and Pc⁺ (Fig. 6), show that either more electron donors are available for the reduction of these compounds, or that the electron transport becomes faster. Extra electron donation could originate from enhanced chlororespiration; lower photosynthetic control or from enhanced cyclic electron transport under Mn deficiency. Since lower donor-side limitation is also observed upon illuminations with actinic red light, a condition that outcompetes the chlororespiration pathway, we suggest that either less strong photosynthetic control or more cyclic electron transport is taking place under Mn deficiency than under control conditions. In Figs. 4–6, we have studied the effect of FR illumination. FR light excites preferentially PSI and cyclic electron transport is favored compared with red light illumination (Heber et al., 1978). In KLAS-NIR, using FR light at 720 nm, in a leaf the quantum yield of PSII of absorbed light is about 15% compared with illumination at 620 nm (Pettai et al., 2005). This low PSII activity is sufficient to supply electrons at a low rate via linear electron flow to sustain cyclic flow. After illumination with FR light, the re-reduction kinetics of P700⁺ takes place with a t₁/₂ of 3.7 s ± 0.01 in control and 1.9 s ± 0.007 in Mn-deficient samples (Fig. 5). These kinetics are rather slow compared to the kinetics observed after illumination with red actinic light in higher plants (see for example *Shimakawa et al.*, 2020). However, comparable half-time of 1.7 s for PSI CET has been previously reported for *Arabidopsis thaliana* (Jensen et al., 2000). The faster reduction of P700⁺ and Pc⁺ could also be explained by a lower photosynthetic control under Mn deficiency. Donor-side limitation to PSI is lower under Mn deficiency as we have reported earlier (Messant et al., 2023). This could be either caused by increased cyclic flow or lower photosynthetic control. qN was higher under Mn deficiency (Fig. 1), speaking for a larger proton gradient and, presumably, a higher photosynthetic control. When the thalli had been infiltrated with nigericin the reduction of P700⁺ and Pc⁺ was slower under Mn deficiency (Fig. S1), opposite to what has been observed in the absence of an uncoupler (Figs. 5 and 6). This observation could be explained by lower photosynthetic electron transport and lower NADPH production since the PSII activity is decreased under Mn deficiency (Fig. 1A, see also data in Messant et al., 2023).

A comparison between P700⁺ and Pc⁺ reduction kinetics shows that Pc⁺ reduction is 2.5-fold slower than that of P700⁺, both in control and under Mn deficiency. This can be explained by an amount of Pc larger than that of PSI reaction centers. Upon Mn deficiency, the ratio Pc/P700 decreased by about 50% while the reduction of P700⁺ and Pc⁺ was accelerated. The faster reduction of P700⁺ and of Pc⁺ under Mn deficiency shows clearly that the kinetics not only depend on the number of available reductants in stroma and Pc electron carriers in the lumen but also on how quickly electrons can transfer from the stromal donors via the intersystem chain to Pc⁺ and from there to P700⁺. It has been reported previously that a decrease in the amount of Pc limits electron transport on the donor side of PSI probably caused by the limitation of lateral diffusion of Pc between the cyt b6f complex and PSI (Drepper et al., 1996; Haehnel, 1982; Cruz et al., 2001). In the present case, however, the electron transport is accelerated when the Pc pool is small.
Using the data shown in Fig. 2 and the extinction coefficient of P700 and Pc determined *in vitro* (Sétif et al., 2019), the molar ratio of Pc to P700 is about 1.6 in control and 0.8 under Mn starvation. Even if this determination is an underestimation of the Pc content, under Mn deficiency too few Pc would be available for maintaining efficient electron transport between cyt b<sub>6f</sub> complex, Pc and PSI. This speaks for a structural reorganization of the complexes and the formation of supercomplexes formed between ferredoxin, cyt<sub>b6f</sub> complex, Pc and PSI. According to the study conducted by Joliot and Joliot (2002), PSI, cyt<sub>b6f</sub> complex, Fd and Ferredoxin NADP<sup>+</sup>-reductase (FNR) are physically associated and serve as a platform for Fd-dependent CET, leading to slow leakage of electrons and ultimately limited photo-oxidation of P700. In the green alga *Chlamydomonas reinhardtii* supercomplexes composed of PSI, Pc, cyt<sub>b6f</sub> complex, Fd have been biochemically isolated (Iwai et al., 2010), and functional supercomplexes have been biophysically characterized (Joliot et al., 2022). Moreover, a low-resolution structure of a PSI-LHCI-cytb<sub>6f</sub> complex has been also revealed in *C. reinhardtii* (Steinbeck et al., 2018). Alterations of the organization of the thylakoid membrane may favor the formation of such supercomplexes. According to transmission electron microscopy images (Fig. S2), the organization of the thylakoid membrane is affected by Mn deprivation. In *in vivo* super-resolution chlorophyll fluorescence emission microscopy strongly fluorescent regions were localized more distinctly in chloroplasts of Mn-deficient thalli, indicating a change in the mesoscopic organization of the thylakoid membrane (Messant et al., 2023). This indicates smaller grana stacks with a more pronounced segregation between them and the stroma lamellae.

Enhancement of cyclic electron transport upon abiotic stress conditions allows to generate a higher pmf and a higher ATP/NADPH ratio. CET is known to increase under certain stress conditions like drought, low CO₂, low N, low temperature, and heavy metal stress (for review, see Sunil et al., 2019). CET is crucial for protecting PSI by alleviating the over-reduction of the acceptor side of PSI. *Marchantia* possess flagoviiron proteins and over-reduction of the acceptor side of PSI is less critical than in angiosperms (*Shimakawa et al., 2017*), however, upon Mn deficiency the capacity of linear electron flow is diminished and maintaining a high ATP level may be physiologically important to survive photoautrophically. Future work is needed to show if CET is increased under deprivation of other micronutrients than Mn. In addition, the biochemical composition of the putative supercomplex involved in CET under Mn deficiency in *Marchantia* should be analyzed.

## Author contribution

U. H., and A.K.-L. designed the project. U.H. performed the experiments; U.H. and A.K.-L. analyzed the data. U. H., and A.K.-L. wrote the manuscript.

## Funding

This work was supported by the Labex Saclay Plant Sciences-SPS (ANR-17-EUR-0007), the platform of Biophysics of the I2BC supported by the French Infrastructure for Integrated Structural Biology (FRISBI; grant number ANR-10-INSB-05), BIISA and France-BioImaging infrastructure (ANR-10-INBS-04, call "investissements d'Avenir"). U.H. is supported by a CNRS PhD contract.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anja Krieger-Liszky reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

## Acknowledgements

We would like to thank Sébastien Thomine (I2BC) for hosting U.H. in his laboratory, Claire Boulogne (I2BC) for her help with electron microscopy and Ginga Shimakawa (Kobe university, Japan) for scientific discussions.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.plaphy.2024.109042.

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