Alternative Oxidase Enhances Metabolic Flexibility in Bioenergetic Metabolism of Physcomitrium patens

Alternative Oxidase Enhances Metabolic Flexibility in Bioenergetic Metabolism of Physcomitrium patens | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 July 2025 V1 Latest version Share on Alternative Oxidase Enhances Metabolic Flexibility in Bioenergetic Metabolism of Physcomitrium patens Authors : Shun-Ling Tan 0009-0006-0691-1229 , Antoni M. Vera-Vives 0000-0001-7761-5343 , Xing Huang 0000-0002-7087-6601 , Alessandro Alboresi 0000-0003-4818-7778 , and Tomas Morosinotto 0000-0002-0803-7591 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175152257.75470323/v1 428 views 224 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Mitochondrial respiration catalyses the electron transfer from NADH to oxygen through the activity of Complex I, II, III, and IV. Plant mitochondria possess additional alternative electron transport pathways, including one mediated by alternative oxidase (AOX), which transfers electrons from ubiquinone to O 2 , bypassing the cytochrome-dependent electron transport chain. This study investigates the functional role throughout plant evolution by analysing Physcomitrium patens plants missing or overexpressing AOX. In the moss P. patens, AOX has a remarkably high electron transport capacity and can fully compensate for the cytochrome pathway. AOX overexpression led to growth inhibition, possibly due to excessive energy dissipation. Despite the high potential activity, aox KO lines did not show significant impact in growth or photosynthetic activity under various conditions, due to the compensatory action of the cytochrome pathway. When the cytochrome pathway was inhibited, AOX activity impacted photosynthetic reactions, affecting in particular chloroplast ATPase. Simultaneous inactivation of AOX and complex III resulted in plant lethality, demonstrating the essential role of mitochondria respiration for plants cells, specifically in balancing reducing power and ATP availability. AOX thus enables mitochondria to sustain the oxidation of reducing equivalents even under conditions in which ATP consumption is saturated, providing flexibility to the bioenergetic metabolism. Alternative Oxidase Enhances Metabolic Flexibility in Bioenergetic Metabolism of Physcomitrium patens Shun-Ling Tan 1 , Antoni M Vera-Vives 1 , Xing Huang 1,2 , Alessandro Alboresi 1 , Tomas Morosinotto 1, Department of Biology, University of Padova, Padova, Italy Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China Abstract Mitochondrial respiration catalyses the electron transfer from NADH to oxygen through the activity of Complex I, II, III, and IV. Plant mitochondria possess additional alternative electron transport pathways, including one mediated by alternative oxidase (AOX), which transfers electrons from ubiquinone to O 2 , bypassing the cytochrome-dependent electron transport chain. This study investigates the functional role throughout plant evolution by analysing Physcomitrium patens plants missing or overexpressing AOX. In the moss P. patens, AOX has a remarkably high electron transport capacity and can fully compensate for the cytochrome pathway. AOX overexpression led to growth inhibition, possibly due to excessive energy dissipation. Despite the high potential activity, aox KO lines did not show significant impact in growth or photosynthetic activity under various conditions, due to the compensatory action of the cytochrome pathway. When the cytochrome pathway was inhibited, AOX activity impacted photosynthetic reactions, affecting in particular chloroplast ATPase. Simultaneous inactivation of AOX and complex III resulted in plant lethality, demonstrating the essential role of mitochondria respiration for plants cells, specifically in balancing reducing power and ATP availability. AOX thus enables mitochondria to sustain the oxidation of reducing equivalents even under conditions in which ATP consumption is saturated, providing flexibility to the bioenergetic metabolism. Introduction Plants rely on photosynthesis to convert sunlight into the chemical energy that supports their metabolism. The process involves a light-dependent chloroplast electron transport chain (cETC), located in the thylakoid membrane to oxidise water, generating NADPH and ATP. These energy carriers are then exploited by the carbon assimilation phase, specifically the Calvin-Benson cycle in the chloroplast stroma, fixing CO 2 into triose phosphates (Stitt et al. , 2010). In natural environments, the rate of carbon fixation and associated metabolic pathways are continuously changing (Foyer et al. , 2012; Ma et al. , 2014; Walker et al. , 2016), leading to highly dynamic demands for reducing power and ATP, in terms of quantity and balance. In this context, photosynthetic organis-ms have evolved multiple mechanisms to modulate cETC and energy production to prevent imbalances that could lead to over-reduction and photodamage. One key mechanism is cyclic electron transport around PSI (CEF), which redirects electrons from PSI back to cytochrome b6f complex , producing additional ATP without synthesis of NADPH and helps preventing PSI overreduction under light stress (Yamori & Shikanai, 2016; Tan et al. , 2020). Another route is pseudocyclic electron flow via flavodiiron proteins (FLV) or the Mehler reaction, where electrons are transferred to O 2 to H 2 O, supporting the generation of a proton motive force (Shimakawa et al. , 2017) while acting as a safety valve to dissipate excess electrons in cETC (Gerotto et al. , 2016). Additionally, nonphotochemical quenching (NPQ) is another important protective mechanism, which dissipates excess absorbed energy as heat (Demmig-Adams et al. , 2020). Even if all energy in plants ultimately originates from photosynthesis, respiration remains an essential metabolic process. Chloroplast and mitochondrial activities are tightly coordinated, and their interplay are essential for plant survival (Raghavendra & Padmasree, 2003; Noctor et al. , 2007). Key physiological processes rely on this interorganellar communication, such as photorespiration (Eisenhut et al. , 2019), carbon and nitrogen metabolism (Smith et al. , 2019; Medeiros et al. , 2021; Vera-Vives et al.,2025 ), and fatty acid synthesis (Guan et al. , 2020). Furthermore, mitochondrial respiration was also shown to be essential for the supply of ATP to the cytosol of plant cells (Gardeström & Igamberdiev, 2016; Voon et al. , 2018a; Vera‐Vives et al. , 2024). The mitochondrial electron transport chain (mETC) in plants consists of five complexes (Complex I-V) that mediate the transport of electrons from NADH and succinate to O 2 . CI and CII reduce ubiquinone (UQ) to ubiquinol (UQH 2 ). The cytochrome (cyt) pathway, which consists of CIII (cyt c ) and CIV (cyt c oxidase), transports electrons from the UQ pool to O 2 . In CI, CIII, and CIV, electron transport is coupled with proton translocation across the inner mitochondrial membrane, generating a proton motive force ( pmf ) that is exploited by mitochondrial ATP synthase (CV) to synthetize ATP. In addition to these five complexes, plant mitochondria possess another pathway, mediated by alternative oxidase (AOX), which transfers electrons from ubiquinol to O 2 bypassing two of the proton pumping sites, CIII and CIV (Vanlerberghe & McIntosh, 1997; Millar et al. , 2011). Since AOX does not contribute to proton translocation, this pathway partially uncouples electron transport from ATP biosynthesis, decreasing respiratory energy yield per reducing equivalent. AOX activity was shown to support mitochondrial electron transport when the cytochrome pathway is inhibited or saturated, thereby preventing UQH₂ pool overreduction and consequent reactive oxygen species (ROS) production, and thus constituting an electron security valve especially under stress conditions (Selinski et al. , 2018). AOX activity was also shown to mitigate the risk of over-reduction of photosynthetic electron transport (Vishwakarma et al. , 2014; Florez-Sarasa et al. , 2020; Vanlerberghe et al. , 2020). The present literature suggests AOX modulates electron transport reactions, especially in response to dynamic environmental conditions (Vanlerberghe et al. , 2020). Its activity is dynamically regulated and induced by multiple stress conditions in plants (Arnholdt-Schmitt et al. , 2006; Clifton et al. , 2006). Due to its impact on ROS production and redox balance, AOX activity has also been associated with redox signaling networks (Van Aken et al. , 2009; Vanlerberghe et al. , 2020; Van Aken, 2021). AOX is a highly conserved protein in plant kingdom and is also present in fungi, protists, green algae, and some animal species (Moore et al. , 2013; Pennisi et al. , 2016). In Arabidopsis thaliana , AOX is encoded by five nuclear genes ( AtAOX1a-1d and AtAOX2 ), with AtAOX1a being the most abundant isoform in all tissues (Clifton et al. , 2006). AtAOX1a knockout plants were more sensitive than WT to low temperatures (Fiorani et al. , 2005), while its overexpression enhanced tolerance to salt stress (Smith et al. , 2009). Short-term high light treatment revealed that, when AOX was knocked out or inhibited by salicylhydroxamic acid (SHAM), Arabidopsis, tobacco and other C3 plants displayed increased UQ reduction levels and severe PSII photoinhibition (Yoshida et al. , 2011a,b; Zhang et al. , 2017). Various C4 plants, exposed to similar treatments, showed no detectable effect on PSII photoinhibition, suggesting species-specific differences in AOX-dependent photoprotection (Zhang et al. , 2017). Under longer term treatments, the growth of AtAOX1a knockout plants was not impaired compared with WT under both control and high light (Yoshida et al. , 2011a), while the antisense suppression of GmAOX2b in soybeans led to impaired growth and reproductive development linked to reduced CO 2 assimilation rate even under ambient condition (Chai et al. , 2010, 2012). The role of AOX in modulating photosynthetic metabolism has also been demonstrated in unicellular photosynthetic organisms. In the diatom Phaeodactylum tricornutum PtAOX contributed to optimize ATP/NADPH ratio in the chloroplasts and enhanced carbon fixation and growth (Bailleul et al. , 2015). In Chlamydomonas reinhardtii, CrAOX1 was found to be critical for photosynthetic performance and survival at high irradiance by limiting ROS accumulation and further preventing PSII damage (Mathy et al. , 2010; Kaye et al. , 2019). AOX thus is active in modulating bioenergetic metabolism but its physiological impact appears to be differentiated in various species and growing conditions. In this context, it is interesting to investigate the role of AOX in the moss Physcomitrium patens, a nonvascular plant that diverged from the vascular plant ancestors early after land colonisation. Due to its evolutionary position and the specific features that connect the aquatic and terrestrial lifestyples (Strotbek et al. , 2013), the investigation of P. patens enables us to assess evolutionary differences and conserved trends during evolution and land colonisation. As another reason of interest, in the moss Physcomitrium patens, AOX is encoded by a single nuclear gene, avoiding gene redundancy and facilitating the investigation of its biological role (Neimanis et al. , 2013). To this end, in this study, we generated PpAOX knockout ( aox KO) and overexpression lines (AOX OE) in Physcomitrium patens to explore the role of AOX and the interaction between mitochondria and chloroplast bioenergetic metabolism. Here, we show that AOX has a high electron transport capacity in P. patens and its overexpression reduces growth, possibly due to excessive energy waste. Depletion of AOX in KO plants has surprisingly a limited phenotype due to the strong complementarity with the cytochrome pathway. Inhibition of both pathways is lethal, showing that mitochondrial respiration plays an essential role in photosynthetic metabolism, with a direct effect on the regulation of chloroplast ATPase. In this context, AOX provides flexibility to mitochondrial ETC, enabling it to maintain the ability to consume reducing power produced from the chloroplast even under conditions where the ATP biosynthesis is saturated. Materials and Methods Plant materials and growth condition Protonemal tissue of P. patens WT (Gransden), aox KO plants and AOX OE plants was grown on minimum PPNO 3 medium in a growth chamber at 24 ℃, 50 μmol photons m -2 s -1 of light (control light in the text) and set in a long photoperiod (16h light /8h dark). Physiological and biochemical experiments were performed on 10-day-old protonema. Plant phenotyping Growth rate assays were started with fresh protonema, after 10 days of growth, 2 mm diameter protonema were transferred to new PPNO 3 medium plates or PPNO 3 medium contained with 8uM antimycin A or 50 uM Rotenone and grew at 50 μmol photons m -2 s -1 for 21 days, the plants for high light stress assessment were grew at 500 μmol photons m -2 s -1 . To access salt and osmotic stress, 2mm diameter 10-day-old protonema were grown on PPNO 3 medium with a filter for two weeks, then the filters with moss colonies were transferred to PPNO 3 medium contained 0.4M NaCl and 0.8M Sorbitol for 1 week respectively. The colony size was quantified as previous study. The plates contained with colonies were scan with Konica Minolta Bizhub C280 scanner in high resolution (600 ppi). Images were processed with FIJI (https://fiji.sc/) using the ‘threshold color’ plugin to remove the background. Then, integrated density (area x mean density) was measured for each colony and normalized by each initial area to characterize the 3D growth of gametophore. The maximum PSII efficiency ( Fv/Fm ) was detected using PAM imaging during growth. Moss transformation and line selection Selected upstream and downstream homologous recombination regions from PpAOX gene were amplified by PCR from WT gDNA and cloned respectively into BHRf and BNRf (Gerotto et al. , 2016) plasmids for aox knockout lines. Full-length coding sequence of PpAOX was amplified from WT cDNA with the PpAOX-attB1/2 primers and cloned to pT1OG (Aoyama et al. , 2012) plasmid with PpEF1-α promoter for AOX overexpression lines. Protoplast transformation and line screening were performed as previously described (Gerotto et al. , 2016). RNA was afterward purified with RNeasy Plant Mini Kit (Vazyme) and used as a template for cDNA synthesis with RevertAid Reverse Transcriptase (Thermo Scientific) to verify aox gene expression in WT, aox KO, and AOX OE plants. Immunoblot analysis Protonema and gametophore were ground in liquid nitrogen, and total proteins were extracted in sample buffer (50 mM TRIS pH 6.8, 100 mM DTT, 2% (w/v) SDS, and 10% (w/v) glycerol). Total protein extractions were quantified by chlorophyll content and then loaded to every well at different dilution factors. After SDS–PAGE, proteins were transferred to a PVDF membrane (Millipore). Membranes were blocked with 5% milk then probed with specific primary antibodies: anti-AOX1/2, Agrisera, catalogue number AS04054; custom-made anti-NAD9 (CI); custom-made anti-SDH1-1 (CII), anti-a-MPP (CIII), anti-COX2 (CIV), anti-b subunit (CV), and anti-AtUCP1. Oxygen consumption measurement Measurements of oxygen consumption (respirometry) were detected as in (Vera-Vives et al. , 2025), with 10-day-old intact 1 cm 2 protonema using the NextGen-O 2 k and the PhotoBiology (PB)-Module (Oroboros Instruments, Innsbruck). Briefly, protonema were placed on the sample holder in 2 ml chamber with sterile PPNO 3 medium containing 10 mM NaHCO3 after 40 minutes dark adaption. The oxygen concentration was monitored for 10 minutes at dark to assess the total respiration rate. KCN or SHAM (the final concentration of these inhibitors in chamber is 1mM) is added to the chamber using Hamilton syringes. The oxygen concentration was monitored for another 10 minutes at dark to assess alternative or cytochrome respiration rate. All the data were normalized by the chlorophyll content in each sample. Spectroscopic analysis A Dual PAM-100 (Heinz Walz, Effeltrich, Germany) was used to measure PSI and PSII parameters at room temperature imultaneously. In photosynthetic phenotype measurement, after 40 min dark adaptation, the maximum fluorescence and the maximum change in P700 was measured using a saturating pulse. Afterward, 10-day-old protonema or 2-month-old gametophore without rhizoid were illuminated at 63 or 330 μmol photons m −2 s −1 for 8 min followed by 5 min darkness. In short-term antimycin A measurement, 10-day-old protonema grown with PPNO 3 medium were submerged in H 2 O or 16uM antimycin A during 40 min dark adaptation. Then protonema were exposed to 330 μmol photons m −2 s −1 for 8 min followed by 10 min darkness. PSI and PSII parameters were calculated as following: Y(I) as (Pm’ − P)/ Pm, Y(ND) as P/Pm, Y(NA) as (Pm−Pm’)/Pm, Y(II) as (Fm’ − Fs)/Fm’, NPQ as (Fm − Fm’)/Fm’, qL as (Fm’ − F)/(Fm’-F0) X F0’/F. Y(I), quantum yield of PSI photochemistry; Y(ND), quantum yield of non-photochemical quenching due to PSI donor side imitation; Y(NA), quantum yield of non-photochemical quenching due to PSI acceptor side imitation; Y(II), effective quantum yield of PSII photochemistry; NPQ, non-photochemical quenching in PSII. Electro Chromic Shift (ECS) spectra were recorded with a JTS-10 system (Biologic) as described in (Gerotto et al. , 2016). Before test, protonema were imbibed with HEPES buffer (20 mM HEPES, pH 7.5 and 10 mM KCl) or 16uM antimycin A during 40 min dark acclimation. For each measure, the background signal at 546 nm was subtracted from the 520 nm signal to eliminate the contribution of scattering and cytochromes. Single turnover flash spectroscopy was performed to obtain the total number of active reaction centres in the chloroplasts. Protonema were exposed to 350 μmol photons m −2 s −1 for 6 min, the relaxation kinetics were recorded after light was switched off. The pmf was calculated as the difference between the maximum signal at the light steady state and the minimum level of ECS in the dark. ATPase activity in chloroplast was accessed by fitting the first 300 ms of the ECS decay curve with a first order exponential decay kinetic and indicated as the inverse of the decay time constant (Avenson et al. , 2005). Gas exchange measurement The CO 2 assimilation rate was measured with the LI-6800 portable photosynthetic system Li-cor Biosciences, Lincoln, NE, USA) on 8-week-old gametophytes of P. patens . Gametophytes were cultivated on the PPNO 3 medium with a cellophane filter to separate them easier from the medium after measurement to obtain accurate dry weight. Gametophytes with medium block were placed in the bryophyte chamber (part no.: 6800-24) with the large light source (part no.: 6800-03). All measurements were made at an air humidity of 80-85%, CO 2 concentration of 400 ppm, and chamber temperature of 25 °C. After gas exchange measurements, the CO 2 assimilation rate was recalculated by dry weight. The light response curve was performed after 10 min light induction at 300 μmol photons m −2 s −1 . For each light intensity (600, 400, 250, 150, 100, 50, 20, 0 μmol photons m −2 s −1 ), the CO 2 assimilation rate was logged upon reaching a steady condition after 2 min. Subcellular protein localization The PpAOX coding region was cloned to the PT1OGY plasmid. 5 or 6-day-old protonemal tissue grown on PPNH 4 medium was collected for protoplast generation. PEG-mediated transformation was performed as pervious described (Alboresi et al. , 2010). The transfected protoplasts were incubated in dark at 23 ℃ for 24-72h before taking images. YFP signals and chlorophyll auto-fluorescent signals were visualized under the ZEISS LSM900 confocal microscope. The YFP signals were excited using 488 nm and detected at 505-576 nm for detection of YFP. The chloroplasts auto-fluorescence were taken with excitation at 640 nm and emission at 600-700 nm. AOX has high electron transport capacity in the moss Physcomitrium patens The subcellular localisation of AOX in P. patens was first confirmed by transient expression of AOX-YFP fusion protein in WT protoplasts. Microscopy analysis showed that AOX-YFP did not colocalize with chlorophyll fluorescence and exhibited the distribution characteristic of a mitochondrial protein (Figure S1), confirming previous studies (Wu et al. , 2019). To investigate the functional role of AOX, multiple independent aox KO plants ( aox ) and overexpressing plants (AOX OE) were generated by homologous recombination-mediated gene targeting. Rescue lines were also generated by overexpressing AOX in an aox KO background. RT-PCR confirmed the expected AOX expression in all the transformed lines (Figure S2). Protein accumulation was assessed using a specific anti-AOX antibody (Figure 1A). In the KO lines, the accumulation of AOX was undetectable, whereas protein levels in AOX OE1 and AOX OE2 were more than tenfold higher as compared to the WT plants, confirming also the recovery of the protein accumulation in the rescue line (Figure 1A). Immunoblot analysis using specific antibodies against different subunits of mitochondrial respiratory complexes revealed no major changes in the accumulation of other respiratory complexes except AOX. (Figure 1B). To quantify the impact of AOX on respiratory activity, the O 2 consumption rate in the dark was assessed using inhibitors of CIV (KCN) and AOX (SHAM) to estimate the capacity of cytochrome and alternative pathways. Both inhibitors showed no effect on O 2 consumption of WT plants, whereas respiration was almost completely inhibited when both were applied together (Figure 2) as observed previously (Vera-Vives et al. , 2025). Figure 1. Immunoblot analysis of AOX and mitochondrial respiratory complex proteins in Physcomitrium patens . A. AOX protein accumulation in WT, two independent aox KO lines, two independent AOX OE lines and one rescue line ( aox KO complemented with AOX-OE). Three dilutions of total protein extracts were loaded for WT (0.5 µg, 1 µg and 2 µg of chlorophyll) while 1 µg of chlorophyll equivalent was loaded for aox KO lines, AOX OE lines and aox rescue line. RuBisCO band is shown as loading control. B. Impact of AOX levels on the accumulation of other mitochondrial respiratory complexes. Total protein extracts equivalent to 0.5 µg, 1 µg and 2 µg of chlorophyll were loaded for WT, while 1 µg of chlorophyll equivalent was loaded for aox KO lines and AOX OE lines. RuBisCO band is shown as representative loading control. Similar measurements in aox KO plants showed no difference in respiratory activity compared to WT, and also SHAM treatment had no effect on aox KO plants (Figure 2). However, in the presence of KCN alone respiratory activity was inhibited to the same extent as in WT and aox KO plants treated with both KCN and SHAM together (Figure 2). These results confirmed that AOX pathway was inactive in aox KO but also suggested that both the cytochrome and alternative pathway had a high capacity in WT plants and each one of them was able, alone, to sustain full respiratory activity, even if the other was inhibited (Vera-Vives et al. , 2025). Figure 2 Effect of AOX mutation on respiration in Physcomitrium patens protonema. After 40 minutes of dark adaptation, O 2 consumption rate was measured in darkness for 10 minutes. Respiration was monitored under three conditions: in the absence of inhibitors (total respiration rate; grey), in the presence of 1 mM SHAM (yellow), KCN (blue) and both to estimate capacity of cytochrome and alternative pathways. Bars represent the average ± SD (n≧4). The different letters indicate statistically significant differences between different treatments within each genotype (WT and aox KO, one-way ANOVA, P< 0.05). Overexpression of AOX negatively impacts Physcomitrium patens growth. Considering the high electron transport capacity of AOX, its physiological impact was assessed by growing WT, aox KO and AOX OE plants under control light conditions (50 μmol photons m -2 s -1 , Figure 3A). Growth quantification showed no observable differences in aox KO mutant, while AOX OE plants exhibited a significant growth penalty as compared to WT plants (Figure 3A, B). Plants were exposed to various abiotic stresses to evaluate the potential impact of AOX in those conditions. Under strong illumination (500 μmol photons m -2 s -1 ), growth of WT and aox KO plants remained comparable, with no significant differences (Figure 3A). Both genotypes also displayed a similar decrease in Fv/Fm under excess light treatment (Figure 3C) . AOX OE grew better under HL than CL but remained smaller than WT (Figure 3A-B). Based on previous results with Arabidopsis thaliana , wheat and rice (Bartoli et al. , 2005; Giraud et al. , 2008; Challabathula et al. , 2022), we further examined the response to salt (0.4M NaCl) and osmotic (0.8M sorbitol) stress. Experimental conditions were selected to be severe enough to cause a reduction of Fv/Fm and growth inhibition, but again no differences were visible between WT and aox KO plants (Figure S3). Although it is possible that harsher or moderate conditions could yield different results, under tested conditions of excess light, sufficient induce measurable photoinhibition, the absence of AOX had no measurable impact, while AOX OE plants showed no rescue nor exacerbation of the phenotype (Figure S3). Figure 3 Growth phenotype of Physcomitrium patens plants under control light and high light. 10-day-old protonema with a 2mm diameter were transferred on fresh PPNO 3 medium and grown under control and high light (50 and 500 μmol photons m -2 s -1 respectively) for 21 days. Representative images (A), growth quantification (B) and PSII maximum efficiency (C) of 21-day-old colonies. Data are shown as average ± SD (n≧4). Bar = 2 mm. “n.s.” indicates no statistically significant differences, the asterisk indicates statistically significant differences (one-way ANOVA, P<0.05). AOX absence has a limited impact on Physcomitrium patens photosynthetic properties, but its overexpression affects carbon fixation. To determine whether AOX accumulation affects photosynthetic electron transport, photosynthetic properties were analysed using PAM fluorometry under growth light (63 μmol photons m -2 s -1 ) and saturating light intensity (330 μmol photons m -2 s -1 ). Both aox KO mutants and AOX OE lines were indistinguishable from WT plants in all light intensities and considering all measured parameters, including PSI and PSII efficiency, plastoquinone pool (PQ) and PSI redox state and the activation of photoprotective mechanisms such as NPQ (Figure S4 and S5). Public RNA-seq data from P. patens plants (https://peatmoss.plantcode.cup.uni-freiburg.de/) show that AOX expression is higher in gametophores than in protonema (Figure S6), the tissue analysed in above results. Immunoblotting confirmed that, while Complex I, Complex V and uncoupling protein (UCP1) were similarly accumulated in both developmental stages, Complex II was less abundant in gametophore whereas AOX and Complex III showed higher accumulation (Figure S6). To assess if higher protein accumulation corresponded to a more prominent functional role, fluorescence measurements were repeated on WT and aox KO mutant gametophores. Similar to protonema, however, no major differences were observed but for a small reduction in NPQ activation and higher PSII efficiency in aox KO plants under increasing light intensity (Figure S7). Figure 4 Carbon fixation capacity of Physcomitrium patens . A) Light response curve of CO 2 assimilation rate. B) Steady-state CO 2 assimilation rate at saturated light intensity (300 μmol photons m -2 s -1 ). Gametophores were grown under control light (50 μmol photons m -2 s -1 ) for 30-day-old and then were measured with Li-cor 6800. WT, aox KO, and AOX OE lines are shown in black, red, and blue, respectively. Average ± SD (n=4) are reported. The different letters indicate statistically significant differences in WT, aox KO plants and AOX OE plants, respectively (one-way ANOVA, P< 0.05). To further investigate the impact of AOX alteration on photosynthesis beyond the light phase, we measured the CO 2 assimilation rate. WT and aox KO plants showed similar CO 2 assimilation rates, whereas AOX OE plants showed a significant reduction (Figure 4A). In these plants the maximum CO 2 assimilation rate, recorded after 10 minutes of light adaptation at saturating light (300 μmol photons m -2 s -1 ), was approximately 35% lower than that WT (Figure 4B). Similar results were obtained in plants grown under high light conditions, with aox KO plants indistinguishable from WT, while AOX OE plants showed reduced CO 2 fixation capacity (Figure S8). These results indicated that the overexpression of AOX impacted negatively photosynthetic carbon fixation capacity, aligning with the observed growth reduction. AOX plays an essential role when the cytochrome pathway is inhibited. Considering the high complementarity in O 2 consumption capacity between the cytochrome and AOX pathways, the phenotype of aox KO/OE plants was further investigated by treating plants with the Complex III inhibitor antimycin A. Both WT and AOX OE plants exhibited approximately a 50% reduction in growth following the treatment (Figure 5). aox KO plants were instead much more severely affected, being unable to survive in the presence of the inhibitor (Figure 5). To confirm that this growth impairment was specifically due to the absence of AOX and not to any secondary effect, aox rescue plants (Figure 1) were also tested. These plants recovered their ability to grow in the presence of antimycin A, confirming that AOX activity was indeed essential when the cytochrome pathway was inhibited. The same experiment was repeated using Rotenone, a respiratory inhibitor targeting Complex I (Figure 5). Figure 5 Impact of antimycin A and Rotenone on Physcomitrium patens growth. 10 days old protonema of WT, aox KO, AOX-OE and a rescue lines ( aox KO complemented with AOX-OE) with a diameter of 2 mm were transferred to fresh PPNO 3 medium and cultivated for 21 days without inhibitors (Control) or in the presence of 8 µM antimycin A or 50 µM Rotenone to inhibit the activity of Complex III and I respectively. Rotenone also inhibited growth in all genotypes, demonstrating that the lethality of antimycin A in aox KO plants was specifically associated with the inhibition of Complex III and not a general effect due to disruption of respiration. Impairment of mitochondrial electron transport impacts photosynthetic electron transport. Analysis above confirms that AOX and the cytochrome pathway are highly complementary and, given their high electron transport capacity, the AOX impact might be underestimated by the analysis of aox KO plants alone. To address this limitation, the photosynthetic properties of aox KO and OE plants were re-assessed following short-term treatment with antimycin A. Fv/Fm of aox KO plants, unlike other genotypes, decreased to 0.5 after just one day of treatment, suggesting that the simultaneous inhibition of both the cytochrome pathway impacted chloroplast activity (Figure S9). To investigate this in more detail, photosynthetic properties were analysed using samples incubated with the inhibitor for 40 min during dark adaptation before measurements, focusing on a short treatment to assess the impact on photosynthesis functionality while minimizing other longer-term effects. In antimycin A-treated WT plants, Y(I) and Y(II) showed only minor reductions under light, but their recovery in darkness was significantly impaired (Figure 6). The dark recovery was particularly impacted for NPQ, indicating that antimycin A interfered with thylakoid ∆pH relaxation in the dark (Figure 6). The PQ (1-qL) and PSI redox state (Y(NA) and Y(ND)) were not impacted under light, while PSI acceptor side limitation (Y(NA)) was higher after antimycin A treatment (Figure S10). The effects of antimycin A on Y(I) and Y(II) were more pronounced in aox KO plants, that showed larger differences with respect to untreated plants and a complete lack of NPQ relaxation after light exposure (Figure 6). In aox KO plants, PSI donor-side limitation was also higher in the light (Figure S10), suggesting that the impact on electron transport by antimycin A treatment was generally amplified by the absence of AOX. Even more interestingly, in AOX OE plants photosystem efficiency was able to recover in darkness to levels similar to the untreated control (Figure 6). Additionally, PQ and PSI redox states in AOX OE plants were also comparable to control conditions (Figure S10), showing that the higher AOX activity was able to rescue the effect of the Complex III inhibition. These results indicated that the inhibition of Complex III led to increased NPQ activation and impaired NPQ relaxation, effects that were amplified when both cytochrome and alternative pathways were affected. However, the overexpression of AOX partially rescued the effect of the inhibitor and maintained the electron transport in chloroplast. The combined activity of CIII and AOX thus has an impact on relaxation of ΔpH across thylakoid membrane in the dark. Figure 6 Photosynthetic properties of WT, aox KO, AOX OE in P.patens protonema treated with antimycin A. Photosynthetic parameters of 10-day -old protonema grown in control condition were measured with Dual PAM 100 incubating the tissues with HEPES buffer (control) or 16 μM antimycin A during the 40 min dark adaptation. During measurements plants were exposed to 330 μmol photons m -2 s -1 actinic light for 8 min (indicated in yellow, top bar) followed by 10 min darkness (indicated in black, top bar). WT, aox KO , and AOX OE plants are shown as black squares, red cycles, and blue triangles, respectively, full or empty for control and samples treated with the inhibitor. Data are shown as average ± SD (n=4). In specific time points (after 4 and 8 minutes of illumination and 5 and 10 min in the dark) the statistically significant differences are reported (one-way ANOVA, asterisk: P0.05). As the observed specific effect on NPQ kinetics suggests a possible alteration in the ΔpH across thylakoid membranes, proton motive force ( pmf ) was assessed in the same plants treated with antimycin A using ECS relaxation kinetics (Figure 7A). Under normal conditions, AOX alterations had no effects on total pmf . However, when cytochrome pathway was inhibited by antimycin A, pmf increased in WT and aox KO plants, whereas it was maintained in AOX OE plants (Figure 7B). Proton conductivity (gH + ) through the ATPase complex, quantified from ECS signal decay, showed no difference between control and antimycin A treated WT (Figure 7C). In contrast, the gH + was strongly reduced in aox KO plants when cytochrome pathway was inhibited. AOX overexpression instead enhanced the gH + with respect to WT even in the presence of antimycin A, suggesting that AOX stimulated proton translocation rate (gH + ) in chloroplast (Figure 7C). These results indicated how a functional mitochondrial electron transport chain impacts photosynthetic electron transport, particularly affecting chloroplast ATPase activity. While this effect was not evident in the aox KO plant because of the complementary activity of complex III, it became apparent when both AOX and CIII were inhibited. Figure 7. Effect of AOX alteration on pmf and proton conductivity (gH + ) in chloroplast. Proton motive force was estimated by monitoring the carotenoid electrochromic shift. A) Relaxation electrochromic shift signal in the dark after 400s exposition to saturating light (300 μmol photons m -2 s -1 ). Before measurements samples were incubated in the dark for 40 minutes with a HEPES buffer (control, left) or with 16µM antimycin A (Right). B) Proton motive force ( pmf ) size at the end of the illumination. C. Proton conductivity of protons (gH + ) across the thylakoid membrane. WT, aox KO, and AOX OE are shown in black, red, and blue respectively. Average ± SD (n≥6) are reported. Different letters indicate statistically significant differences in WT, aox KO plants and AOX OE plants, respectively (one-way ANOVA, P< 0.05). Discussion AOX activity is highly complementary with Complex III/IV in P. patens AOX in vascular plants has been shown to play an important role in response to environmental stress (Vanlerberghe, 2013) and developmental processes such as seed germination and flowering (Wagner et al. , 2008; Fang et al. , 2022; Rodrigues et al. , 2023). AOX was suggested to have a protective effect by avoiding over-reduction in stress conditions when excess electrons overflowing from electron transport chain (ETC) in chloroplast and mitochondria might react with O 2 to produce reactive oxygen species (ROS) (Mittova et al. , 2003; Vanlerberghe, 2013). AOX has been shown to play a role in modulation of metabolism also in eukaryotic algae: various stresses increased the AOX gene expression in Phaeodactylum tricornutum and photosynthetic rates were reduced in AOX silencing lines (Murik et al. , 2019). In Chlamydomonas reinhardtii, the AOX pathway was proposed to supply additional energy to complement LEF for CO 2 fixation, the lack of CrAOX1 increased ROS accumulation and sensitivity to high light (Kaye et al. , 2019; Peltier et al. , 2023). Studies investigating AOX, however, have shown variable results in different species and tissues, suggesting that its role may also be differentiated depending on the environmental adaptation (Gonzàlez-Meler et al. , 1999; Clifton et al. , 2006; Florez‐Sarasa et al. , 2011; Selinski et al. , 2018). Considering this diversity, it is interesting to assess the biological role of AOX in the moss P. patens , to highlight its role in non-seed plants and to identify eventual evolutionary differences with vascular plants. The respiratory O 2 consumption rate in P. patens WT plants showed a limited impact of both CIV (KCN) or AOX (SHAM) inhibitors (Figure 2), while activity was drastically reduced when both inhibitors were applied simultaneously. Thus, both the cytochrome and AOX pathway alone can support the full respiratory activity in P.patens even if the other is inhibited, at least in the conditions tested here (Figure 2). These results suggest the presence of a strong overcapacity for respiration that is not fully exploited in WT plants, likely because of other limiting factors. This hypothesis is confirmed by the observation that aox KO plants show the same respiratory activity as WT, but treatment with KCN caused a complete inhibition. This result is consistent with previous studies in P. patens showing that AOX was able to support a large fraction of O 2 consumption in complex I or IV knockout mutants to complement inhibition of cytochrome pathway (Mellon et al. , 2021; Vera-Vives et al., 2025 ). Based on these data, AOX capacity in P. patens can be estimated to be 73 % of total respiratory O 2 consumption, a higher value that most vascular plants where values around 40% - 50% have been reported, as measured in Arabidopsis, tomato and pea (Kühn et al. , 2014; Dinakar et al. , 2016; Zhu et al. , 2018). The high value observed here, however, is not a unique feature of P. patens, since 74 % capacity of the AOX pathway was also reported for Nicotiana benthamiana (Lee et al. , 2011). In the green alga Chlamydomonas reinhardtii, the AOX capacity was estimated to be 50 % of the total respiration rate (Mathy et al. , 2010), thus a value closer to most plants. These data thus suggest that AOX capacity is a variable feature among different species, and that this may represent a species-specific feature rather than any evolutionary trend. The high electron transport capacity of AOX can explain the growth penalty exhibited by AOX OE plants with respect to WT, which is also correlated with a reduced CO 2 fixation rate (Figure 3 and 4). Since AOX accepts electrons from ubiquinol to reduce oxygen bypassing two of three proton pumping sites, it reduces the mitochondrial electron transport energy efficiency in ATP production, and in non-stress conditions an excess AOX protein accumulation could thus drive to a wasteful energy dissipation. However, this is likely not the sole possible explanation for the observed phenotype. AOX is also involved in mitochondrial and chloroplast retrograde signalling pathways, responding to multiple regulators, such as ANAC017-dependent and ABI4-dependent pathways (León et al. , 2013; Ng et al. , 2013; Merendino et al. , 2020). The phenotype in AOX OE plants could be associated with a possible misregulation of these signalling pathways that could easily drive a growth penalty. In vascular plants, AOX has been reported to be important in response to various abiotic stresses such as excess light, salt, and osmotic stress (Smith et al. , 2009; Vishwakarma et al. , 2014; Vanlerberghe et al. , 2016; Zhu, 2016; Wanniarachchi et al. , 2018). However, reports are not always consistent and, as example, an increased AOX capacity increases salt tolerance in Arabidopsis, but P. patens gametophore with overexpressing AOX are more sensitive to salt treatment (Smith et al. , 2009; Wu et al. , 2019). Even in vascular plants, plants with reduced AOX levels do not always show increased stress sensitivity as observed in Arabidopsis, tomato, and tobacco (Lee et al. , 2011; Zhu et al. , 2018; Del-Saz et al. , 2022). Despite the high electron transport capacity of the alternative pathway, P. patens aox KO plants did not show any significant differences in growth and photosynthetic parameters compared to WT under growth light (Figure 3 and 4). This was the case in testing different tissues and in particular gametophores where AOX is more abundant (Figure S7). Even after exposing plants to abiotic stress, we could not observe any effect on growth and maximum PSII efficiency associated with the absence of AOX (Figure S3), despite all conditions tested being impactful for WT plants. Although it is clearly possible that different stress levels could eventually enable to detect an impact these data suggest that P. patens could cope well with moderate stress even in the absence of AOX. These observations can be explained by considering that in WT plants CIII alone is fully capable of supporting the full respiratory activity, and thus in aox KO the cytochrome pathway is most likely compensating thus minimising the negative repercussions. This strong complementarity between AOX and the cytochrome pathway emerges clearly from analyses of plants treated with the CIII inhibitor antimycin A, where AOX becomes essential, leading to a lethal phenotype for aox KO plants (Figure 5). We can conclude that while mitochondrial ETR is essential for P. patens metabolism, AOX biological function is largely underestimated in P.patens aox KO plants due to the high complementarity of mitochondrial electron transport pathways. Flexibility in mitochondrial respiration supports regulation of photosynthetic metabolism. P. patens therefore appears to have a mETR capacity in excess of metabolic necessities, at least in the conditions tested here. The impact of AOX on photosynthetic activity was thus assessed by a short-term inhibition of cytochrome III using antimycin as a strategy to bypass the strong complementarity of the two pathways. In the chloroplast, PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron transport is also an antimycin A sensitive pathway (Munekage et al. , 2002; DalCorso et al. , 2008), but this is not the case in P. patens where the protein lacks amino acids responsible of the inhibitor binding (Sugimoto et al. , 2013). This is confirmed also here since the addition of antimycin A did not impact the Y(II) and NPQ activation in WT plants (Figure 6). While long-term treatment with antimycin A is lethal for aox KO, a short-term treatment enables highlighting the impact of its depletion on photosynthetic electron transport avoiding the compensation from CIII activity. In these conditions, the presence of AOX indeed impacted photosynthetic activity, with the most evident effect being the slower deactivation of NPQ in the dark. This phenotype is the consequence of a decrease in proton conductivity (gH + ) of chloroplast ATPase (Figure 7) that maintains a ΔpH across the membrane of thylakoids. The association of this phenotype with AOX is confirmed by the fact that plants overexpressing AOX showed the opposite trend with higher chloroplast ATPase activity. This effect is not specific to AOX but is rather associated with mETC activity, as suggested by the fact that antimycin A treatment alone showed a similar effect, which was amplified by AOX depletion and mitigated by overexpression of AOX. These results suggest an overall picture where respiratory activity has a strong impact in modulating the ratio of reducing power/ATP. Even in autotrophic organisms such as plants, where photosynthesis is the ultimate source of energy, in fact, the chloroplasts have a limited capacity to export ATP in the cytosol whose supply is instead strongly dependent on mETC (Shameer et al. , 2019) and mitochondria has been shown to be essential for cytosol ATP supply (Vera‐Vives et al. , 2024) (Figure 8). The results shown here, when both cytochrome and alternative pathways were inhibited, confirm that active mETC is indeed essential for plant survival (Figure 5), even in fully autotrophic conditions where the energetic contribution of respiration could in principle be dispensable. When cells are actively growing under steady-state photosynthesis with high CO 2 fixation rate and high demand for ATP from metabolism, the stromal reductant exported from the chloroplast using redox shuttles such as the malate valve or the TP/3-phosphoglycerate shuttle (Taniguchi & Miyake, 2012; Voon et al. , 2018b; Shameer et al. , 2019) is exploited mainly by cytochrome pathway meets to produce ATP to be supplied to the cytosol and the rest of the cell (Figure 8)(Vanlerberghe et al. , 2020; Vera‐Vives et al. , 2024). Results obtained with AOX enables to expand even further the understanding of the role of mitochondrial respiration beyond the synthesis of ATP. When the demand for ATP is low, like in stress conditions when external factors limit growth, the activity of Complex V and therefore the dissipation of proton motive force across the inner mitochondrial membrane (p) will also be slowed down. Under these conditions, electron flow through proton pumping complexes III and IV becomes restricted by increase in ∆p, in a mechanism called “adenylate control” that is a major regulator of electron flow through the cytochrome pathway (Igamberdiev & Kleczkowski, 2006; O’Leary et al. , 2019). In these conditions however, also the mitochondria ability to consume reducing power would be limiting, possibly driving to excess reducing power and over saturation of the photosynthetic ETC. AOX, however, is not constrained by the availability of ADP, which enables maintaining high rates of electron flow to oxygen and continuous consumption of reducing equivalents, even under energy-limiting conditions (Vanlerberghe & McIntosh, 1997; Chadee et al. , 2021). Thus, AOX activity allows to maintain the mitochondrial electron transport capacity and thus its ability to consume reducing power even when metabolic demand is limited by external factors such as stresses (Figure 8). AOX thus enables mitochondria to act as electron sink, oxidizing reductants produced in the chloroplast, even in conditions of low ATP demand (Vanlerberghe et al. , 2020) (Figure 8). In the absence of AOX, the rate of reducing power consumption would be strictly linked to ATP production and mitochondria thus could consume reducing power effectively only in conditions of high demand. The cells would thus need other mechanisms to drive dissipation of excess reducing power in conditions of low ATP demand. The presence of AOX instead enables mitochondrial electron transport with the flexibility necessary for to respond to environmental conditions and thus expand its role of modulator of reducing power produced by photosynthesis. Figure 8. AOX provides flexibility to mitochondrial respiration activity. Mitochondrial respiration converts reducing power exported for the chloroplasts into ATP for metabolic demand. in case of high ATP demand cytochrome (Cyt) pathway is main responsible of this activity. In conditions of stress or low ATP consumption cytochrome activity is restricted. AOX because of its lower ATP yield is able to maintain capacity of reducing power consumption even in these conditions, providing flexibility to the mETC. Evolutionary it is interesting to observe that unicellular algae like Chlamydomonas have active mitochondrial respiration but can survive photo-autotrophically even when this is completely inactivated by mutations or anoxia (Salinas et al. , 2014; Larosa et al. , 2018). In plants, instead, there is an even stronger interdependence between respiratory and photosynthetic activity, and respiration has become essential for survival even in fully photosynthetically active tissues, as the ones analysed here. Acknowledgements SLT is grateful for support by the Chinese Scholarship Council. XH is grateful for support by the Chinese Academy Science Scholarship. Authors thank Anna Segalla (University of Padova) for the technical support in transformations. Competing interests None declared. Author contributions S-L T contributed to all data generation and analysis. AMV-V contributed to the O 2 consumption measurement and lines generation. XH contributed to CO 2 assimilation and JTS measurement. TM and AA contributed to the data analysis and supervision. S-L T and TM contributed to the writing - original draft. AA, XH and AMV-V contributed to the review and editing. Data availability The data supporting the findings of this study are available within the article and its supporting information. ORCID Shun-Ling Tan https://orcid.org/0009-0006-0691-1229 Antoni M. Vera-Vives https://orcid.org/0000-0001-7761-5343 Xing Huang https://orcid.org/0000-0002-7087-6601 Alessandro Alboresi https://orcid.org/0000-0003-4818-7778 Tomas Morosinotto https://orcid.org/0000-0002-0803-7591 References. Alboresi A, Gerotto C, Giacometti GM, Bassi R, Morosinotto T . 2010 . Physcomitrella patens mutants affected on heat dissipation clarify the evolution of photoprotection mechanisms upon land colonization. Proceedings of the National Academy of Sciences 107 : 11128–11133. Aoyama T, Hiwatashi Y, Shigyo M, Kofuji R, Kubo M, Ito M, Hasebe M . 2012 . AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens . Development 139 : 3120–3129. Arnholdt-Schmitt B, Costa JH, De Melo DF . 2006 . 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Immunoblot analysis of AOX and mitochondrial respiratory complex proteins in Physcomitrium patens . A. AOX protein accumulation in WT, two independent aox KO lines, two independent AOX OE lines and one rescue line ( aox KO complemented with AOX-OE). Three dilutions of total protein extracts were loaded for WT (0.5 µg, 1 µg and 2 µg of chlorophyll) while 1 µg of chlorophyll equivalent was loaded for aox KO lines, AOX OE lines and aox rescue line. RuBisCO band is shown as loading control. B. Impact of AOX levels on the accumulation of other mitochondrial respiratory complexes. Total protein extracts equivalent to 0.5 µg, 1 µg and 2 µg of chlorophyll were loaded for WT, while 1 µg of chlorophyll equivalent was loaded for aox KO lines and AOX OE lines. RuBisCO band is shown as representative loading control. Figure 2 Effect of AOX mutation on respiration in Physcomitrium patens protonema. After 40 minutes of dark adaptation, O 2 consumption rate was measured in darkness for 10 minutes. Respiration was monitored under three conditions: in the absence of inhibitors (total respiration rate; grey), in the presence of 1 mM SHAM (yellow), KCN (blue) and both to estimate capacity of cytochrome and alternative pathways. Bars represent the average ± SD (n≧4). The different letters indicate statistically significant differences between different treatments within each genotype (WT and aox KO, one-way ANOVA, P< 0.05). Figure 3 Growth phenotype of Physcomitrium patens plants under control light and high light. 10-day-old protonema with a 2mm diameter were transferred on fresh PPNO 3 medium and grown under control and high light (50 and 500 μmol photons m -2 s -1 respectively) for 21 days. Representative images (A), growth quantification (B) and PSII maximum efficiency (C) of 21-day-old colonies. Data are shown as average ± SD (n≧4). Bar = 2 mm. “n.s.” indicates no statistically significant differences, the asterisk indicates statistically significant differences (one-way ANOVA, P<0.05). Figure 4 Carbon fixation capacity of Physcomitrium patens. A) Light response curve of CO 2 assimilation rate. B) Steady-state CO 2 assimilation rate at saturated light intensity (300 μmol photons m -2 s -1 ). Gametophores were grown under control light (50 μmol photons m -2 s -1 ) for 30-day-old and then were measured with Li-cor 6800. WT, aox KO, and AOX OE lines are shown in black, red, and blue, respectively. Average ± SD (n=4) are reported. The different letters indicate statistically significant differences in WT, aox KO plants and AOX OE plants, respectively (one-way ANOVA, P< 0.05). Figure 5 Impact of antimycin A and Rotenone on Physcomitrium patens growth. 10 days old protonema of WT, aox KO, AOX-OE and a rescue lines ( aox KO complemented with AOX-OE) with a diameter of 2 mm were transferred to fresh PPNO 3 medium and cultivated for 21 days without inhibitors (Control) or in the presence of 8 µM antimycin A or 50 µM Rotenone to inhibit the activity of Complex III and I respectively. Figure 6 Photosynthetic properties of WT, aox KO, AOX OE in P.patens protonema treated with antimycin A. Photosynthetic parameters of 10-day -old protonema grown in control condition were measured with Dual PAM 100 incubating the tissues with HEPES buffer (control) or 16 μM antimycin A during the 40 min dark adaptation. During measurements plants were exposed to 330 μmol photons m -2 s -1 actinic light for 8 min (indicated in yellow, top bar) followed by 10 min darkness (indicated in black, top bar). WT, aox KO , and AOX OE plants are shown as black squares, red cycles, and blue triangles, respectively, full or empty for control and samples treated with the inhibitor. Data are shown as average ± SD (n=4). In specific time points (after 4 and 8 minutes of illumination and 5 and 10 min in the dark) the statistically significant differences are reported (one-way ANOVA, asterisk: P0.05). Figure 7. Effect of AOX alteration on pmf and proton conductivity (gH + ) in chloroplast. Proton motive force was estimated by monitoring the carotenoid electrochromic shift . A) Relaxation electrochromic shift signal in the dark after 400s exposition to saturating light (300 μmol photons m -2 s -1 ). Before measurements samples were incubated in the dark for 40 minutes with a HEPES buffer (control, left) or with 16µM antimycin A (Right) B) Proton motive force ( pmf ) size at the end of the illumination. C. Proton conductivity of protons (gH + ) across the thylakoid membrane. WT, aox KO, and AOX OE are shown in black, red, and blue respectively. Average ± SD (n≥6) are reported. Different letters indicate statistically significant differences in WT, aox KO plants and AOX OE plants, respectively (one-way ANOVA, P< 0.05). Figure 8. AOX provides flexibility to mitochondrial respiration activity. Mitochondrial respiration converts reducing power exported for the chloroplasts into ATP for metabolic demand. in case of high ATP demand cytochrome (Cyt) pathway is main responsible of this activity. In conditions of stress or low ATP consumption cytochrome activity is restricted. AOX because of its lower ATP yield is able to maintain capacity of reducing power consumption even in these conditions, providing flexibility to the mETC. Figure S1 Subcellular location of PpAOX-YFP in moss P.patens protoplast . PpAOX-YFP was transformed in P. patens protoplasts and YFP signal was detected under ZEISS LSM900 confocal microscope. Bars = 5 um. Figure S2. A, Scheme for representation of aox KO event. Blue boxes represent 5’ and 3’UTR, green boxes represent exons, black continued lines introns and black dashed lines adjacent non-coding regions, grey box represents hygromycin resistance cassette. Homology regions were indicated by brackets. B. AOX expression in WT, aox KO and AOX OE lines. PpAOX gene expression was assessed by RT-PCR in WT, two independent aox KO, two independent AOX OE lines and one aox rescue line protonema grown under control light. Expression of Actin 2 was also reported as reference. Figure S3 Salt and osmotic stress growth phenotypes of P. patens WT, aox KO lines and AOX OE lines. 10-day-old protonema colonies with a diameter of about 2 mm were transferred and grown under control light (50 μmol photons m -2 s -1 ) on PPNO 3 medium supplemented 0.4M NaCl or 0.8M Sorbitol, respectively. Photographs were taken and PSII maximum efficiency ( Fv/Fm ) was measured after 21-day growth. Bar = 2 mm. Data are shown as average ± SD (n=6). The asterisk indicates statistically significant differences (Independent sample test, P<0.05). Figure S4 PSI photosynthetic characteristics of WT, aox KO, AOX OE protonema at different light intensities. 10-day-old protonema grown under control light (50 μmol photons m -2 s -1 ) were measured with Dual PAM 100. After 40 min dark adaptation, plants were exposed to 63 μmol photons m -2 s -1 or 330 μmol photons m -2 s -1 actinic light for 8 min (indicated in yellow, top bar) followed by 5 min darkness ( indicated in black, top bar). WT, aox KO and AOX OE are shown in black square, red cycle, and blue triangle, respectively. Data are shown as average ± SD (n=4). The n.s. indicate statistically no significant differences of three plants (one-way ANOVA, P>0.05) after 4, and 8 min of illumination and after 2 and 5 min in the dark. Figure S5 PSII photosynthetic characteristics of WT, aox KO, AOX OE protonema at different light intensities. 10-day-old protonema grown under control light (50 μmol photons m -2 s -1 ) were measured with Dual PAM 100. After 40 min dark adaptation, plants were exposed to 63 μmol photons m -2 s -1 or 330 μmol photons m -2 s -1 actinic light for 8 min (indicated in yellow, top bar) followed by 5 min darkness ( indicated in black, top bar). WT, aox KO, AOX OE are shown in black square, red cycle, and blue triangle, respectively. Data are shown as average ± SD (n=4). The n.s. indicate statistically no significant differences of three plants (one-way ANOVA, P>0.05) after 4, and 8 min of illumination and after 2 and 5 min in the dark. Figure S6 AOX expression in different developmental stages of P.patens . A. AOX gene expression map describing the transcript expression patterns of AOX during development in P.patens Gransden and Reute ecotypes. The graph was recomposed from PEAT moss https://peatmoss.plantcode.cup.uni-freiburg.de/. B. Transcript expression of the AOX in protonema and gametophore in P.patens Gransden. Data was downloaded from PEAT moss. The asterisk indicates statistically significant differences (Independent sample test, P<0.01) C. The amounts of protein on respiratory electron transport chain in protonema and gametophore. 10-day-old protonema and 2-month-old gametophore were grown on PPNO 3 medium under control light. Total protein extraction were loaded, 1x loading corresponded to 2 ug of total chlorophyll. Figure S7 Photosynthetic fluorescence parameters in P.patens gametophores. Gametophores were cultivated on PPNO 3 medium at control light (50 μmol photons m -2 s -1 ). 30-day-old gametophores were cut and measured at 63 μmol photons m -2 s -1 or 330 μmol photons m -2 s -1 . WT and aox KO lines are shown in dark green squares and light green cycles, respectively. Average ± SD (n=4) are reported. Figure S8 CO 2 fixation capacity of Physcomitrium patens grown under high light. A, Light response curve of CO 2 assimilation rate. B, Steady-state CO 2 assimilation rate at saturated light intensity (300 μmol photons m -2 s -1 ). Gametophores were cultivated on PPNO 3 medium at high light (500 μmol photons m -2 s -1 ). CO 2 assimilation rate was measured after 30 days of growth. WT, aox KO, and AOX OE lines are shown in black, red, and blue, respectively. Average ± SD (n=4) are reported. The different letters indicate statistically significant differences in WT, aox KO plants and AOX OE plants, respectively (one-way ANOVA, P< 0.05). Figure S9 PSII maximum photosynthetic efficiency of WT, aox KO, AOX OE in P.patens treated with antimycin A. 10-day-old protonema colonies with a diameter of about 2 mm were transferred and grown under control light (50 μmol photons m -2 s -1 ) on PPNO 3 medium supplemented with 8 μM antimycin A. PSII maximum efficiency ( Fv/Fm ) were measured after one day of growth. Data are shown as average ± SD (n=4). The n.s. and asterisk indicate statistically no or significant differences between control and treatment, respectively (Independent sample test, P>0.05). Figure S10 PQ and PSI redox state of WT, aox KO, AOX OE lines in P.patens protonema treated with antimycin A. 10-day-old protonema grown at control light (50 μmol photons m -2 s -1 ) and were measured with Dual PAM 100. Plants were submerged with HEPES buffer (control) or 16 μM antimycin A during the 40 min dark adaptation, then plants were exposed to 330 μmol photons m -2 s -1 actinic light for 8 min (indicated in yellow, top bar) followed by 10 min darkness ( indicated in black, top bar). WT, aox KO, and AOX OE plants are shown in black square, red cycle, and blue triangle respectively. Data are shown as average ± SD (n=4). The different symbols indicate statistically significant differences of the three plants (one-way ANOVA, asterisk: P0.05) after 4, and 8 min of illumination and after 5 and 10 min in the dark. Supplementary Material File (tan et al. supporting information.pdf) Download 923.80 KB Information & Authors Information Version history V1 Version 1 03 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords bioenergetics moss photosynthesis photosynthesis: electron transport respiration Authors Affiliations Shun-Ling Tan 0009-0006-0691-1229 Universita degli Studi di Padova View all articles by this author Antoni M. Vera-Vives 0000-0001-7761-5343 Universita degli Studi di Padova View all articles by this author Xing Huang 0000-0002-7087-6601 Universita degli Studi di Padova View all articles by this author Alessandro Alboresi 0000-0003-4818-7778 Universita degli Studi di Padova View all articles by this author Tomas Morosinotto 0000-0002-0803-7591 [email protected] Universita degli Studi di Padova View all articles by this author Metrics & Citations Metrics Article Usage 428 views 224 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shun-Ling Tan, Antoni M. Vera-Vives, Xing Huang, et al. Alternative Oxidase Enhances Metabolic Flexibility in Bioenergetic Metabolism of Physcomitrium patens. Authorea . 03 July 2025. DOI: https://doi.org/10.22541/au.175152257.75470323/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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