Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens

Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens View ORCID Profile Erika Csicsely , Norina Noor , View ORCID Profile Susanne Mühlbauer , View ORCID Profile Hans-Henning Kunz , View ORCID Profile Serena Schwenkert , Martin Lehmann , View ORCID Profile Andreas Klingl , View ORCID Profile Oguz Top , View ORCID Profile Wolfgang Frank doi: https://doi.org/10.1101/2025.04.14.648360 Erika Csicsely 1 Plant Molecular Cell Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Erika Csicsely Norina Noor 1 Plant Molecular Cell Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Susanne Mühlbauer 2 Department of Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Susanne Mühlbauer Hans-Henning Kunz 2 Department of Plant Biochemistry, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hans-Henning Kunz Serena Schwenkert 3 Mass Spectrometry of Biomolecules at LMU (MSBioLMU), Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Serena Schwenkert Martin Lehmann 3 Mass Spectrometry of Biomolecules at LMU (MSBioLMU), Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andreas Klingl 4 Plant Development, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andreas Klingl Oguz Top 1 Plant Molecular Cell Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Oguz Top For correspondence: wolfgang.frank{at}lmu.de oguz.top{at}lmu.de Wolfgang Frank 1 Plant Molecular Cell Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter , Großhaderner Str. 2-4, Planegg-Martinsried, 82152, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wolfgang Frank For correspondence: wolfgang.frank{at}lmu.de oguz.top{at}lmu.de Abstract Full Text Info/History Metrics Preview PDF Abstract The Band-7 proteins, known as FLOTILLINs (FLOT), are present at the plasma membranes of most land plants. They function in clathrin-independent endocytosis and contribute to nodule formation following symbiotic infections. This study reveals that the single FLOT variant in Physcomitrium patens is located at the thylakoid membranes in chloroplasts, serving an unanticipated function. Phenotypic analysis of knockout and overexpression lines demonstrates that PpFLOT overexpression significantly impairs the high salinity tolerance of P. patens . Additionally, liquid protonema cultures of PpFLOT- OEX lines exhibited a distinct color change due to necrotic events and developed brachycyte-like cells. These changes correlate with the strength of PpFLOT expression and do not occur when these lines are cultivated on solid medium. Our study found that PpFLOT -OEX lines display increased chlorophyll and H 2 O 2 production. We also discovered that PpFLOT is regulated by ABA and light, and its high expression can potentially affect retrograde signaling. Metabolomics and proteomics analyses revealed changes in the pigment and lipid composition as well as differentially accumulated proteins in PpFLOT mutant lines. We also observed changes in the expression of ion-transport related genes, accumulation of lipids crucial during pathogen defense, and differentially accumulated proteins taking part in multiple metabolomic pathways. Consequently, our study suggests a novel role for chloroplastic PpFLOT in plant terrestrialization, as it is putatively involved in Ca 2+ and reactive oxygen species (ROS) signaling in response to abiotic and biotic stress, along with the light-dependent regulation of chlorophyll biosynthesis. Introduction The Stomatin/Prohibitin/Flotillin/HflK/C (SPFH) protein family, also known as Band-7 proteins, is widespread across evolutionary lineages ( Rivera-Milla et al., 2006 ; Daněk et al., 2016 ; Martiniere and Zelazny, 2021 ). The SPFH-protein family includes FLOTILLINS (FLOT), STOMATINS (STOM), PROHIBITINS (PHB), ERLINS (ER), the bacterial membrane-specific HflK and HflC proteins, and the plant-specific HYPERSENSITIVE-INDUCED REACTION proteins (HIR). Despite differences in function, these proteins share the SPFH/Band-7 domain, ensuring cell membrane-associated localization, and a common accumulation in micro/nanodomains in cell membranes ( Daněk et al., 2016 ; Martiniere and Zelazny, 2021 ). Enriched in detergent-resistant membrane fractions (DRM), these SPFH-proteins are proposed to actively participate in microdomain formation ( Browman et al., 2007 ; Martiniere and Zelazny, 2021 ). Among these SPFH-proteins, FLOT seems to have evolved independently in multiple lineages since plant and fungi FLOT do not encode a Flotillin domain or a PDZ3-binding motif that are characteristic for metazoan flotillin/reggie proteins ( Rivera-Milla et al., 2006 ). A recent study suggests a horizontal gene transfer from fungi to plants, indicating FLOTś role in the infection of nitrogen-fixing bacteria, endocytosis, and seedling development evolved after this transfer ( Ma et al., 2022 ). In plants, FLOT is sterol-dependently recruited into nanodomains, as demonstrated by altered dynamics in response to sterol-depleting agents like methyl-β-cyclodextrin (mβCD) and disturbances in sterol biosynthesis like in cyclopropylsterol isomerase 1 ( cip-1 ) mutant lines of Arabidopsis thaliana which resulted in modified FLOT1 distribution in the root tissue ( Li et al., 2012 ; Cao et al., 2020 ; Martiniere and Zelazny, 2021 ). Studies on FLOT1 in A. thaliana revealed its crucial role in clathrin-independent endocytosis ( Li et al., 2012 ; Hao et al., 2014 ; Daněk et al., 2016 ). AtFLOT2 interaction partners in A. thaliana , including aquaporins and carbonic anhydrase 2, an early-responsive dehydration stress protein, have essential functions in biotic and abiotic stress response ( Junková et al., 2018 ). Additionally, both FLOT2 and FLOT4 of Medicago truncatula are critical in the early stages of symbiotic bacterial infection, with the loss of these genes impairing nodule formation induced by Sinorhizobium meliloti infection ( Haney and Long, 2010 ). Furthermore, Kroumanová et al. (2019) demonstrated increased expression of all three AtFLOT variants in response to Flagellin 22 (flg22) peptide treatment, while salt treatment suppressed AtFLOT1 and AtFLOT2 expression in A. thaliana . Notably, flg22 treatment increased trafficking of AtFLOT1 into late endosomes, suggesting its advantageous degradation during pathogen response ( Yu et al., 2017 ). Suppression of AtFLOT1 expression in A. thaliana leads to reduced seedling growth and root length ( Li et al., 2012 ), while silencing MtFLOT2 , MtFLOT3 , and MtFLOT4 in M. truncatula alters root development ( Haney and Long, 2010 ). Compared to A. thaliana , which encodes three FLOT variants, AtFLOT1 (AT5G25250), AtFLOT2 (AT5G25260), and AtFLOT3 (AT5G64870), Physcomitrium patens encodes only one (PpFLOT; Pp3c3_21910). This PpFLOT shares significant protein sequence similarity (73 – 75 %) with each of the three AtFLOT variants (Phytozome v.13; https://phytozome-next.jgi.doe.gov ). Since P. patens is a model organism for the land plant adaptation, the unique expression of a single FLOT compared to the multiple variants in seed plants, suggests an evolutionary conserved role potentially linked to plant terrestrialization. Notably, our recent study disrupting a DICER-LIKE1a ( PpDCL1a ) autoregulatory feedback loop based on intronic microRNA (miRNA) processing not only increased PpFLOT expression levels, but also led to salt sensitivity and an ABA hyposensitivity ( Arif et al., 2022 ). This indicates miRNA-mediated expression control of PpFLOT and its role in the abiotic stress tolerance in P. patens. Our analysis of Δ PpFLOT and PpFLOT -OEX lines reveals PpFLOT as a negative regulator of salt stress response, contrasting with its potentially positive role in infection events and the biotic stress response based on our proteomics analysis. In-depth investigations via ‘omics’ approaches revealed alterations in the accumulation of proteins, lipids, and pigments, impacting salt stress tolerance and further suggesting a potential role in pathogen infection. Localization studies indicated PpFLOT association with thylakoid membranes, which contrasts its subcellular localization prediction as well as the localization of FLOTs in other plant species that are localized to the plasma membrane. Changes in protein and lipid profiles further support the involvement of PpFLOT in multiple chloroplast-related metabolic pathways, linking it to both salt stress response and pathogen defense. Results Localization of single FLOT variant in P. patens Even though all SPFH-proteins are associated with cell membranes, their association is notably specific and determined by the N-terminal region of the protein. The N-terminus of SPFH-proteins, housing transmembrane domains or hydrophobic regions in conjunction with the Band-7 protein domain, facilitates interaction with cell membranes ( Rivera-Milla et al., 2006 ; Browman et al., 2007 ; Daněk et al., 2016 ). Previous studies in human cells have demonstrated that the membrane association of FLOT is determined by its N-terminal region ( Daněk et al., 2016 ). Localization studies in A. thaliana identified all three FLOT variants in the plasma membrane of root epidermal cells ( Li et al., 2012 ; Danek et al., 2020 ), and for AtFLOT1 and AtFLOT2 in epidermal cotyledon cells ( Junková et al., 2018 ; Cao et al., 2020 ). AtFLOT1 was also detected in the tonoplast of root epidermal cells ( Danek et al., 2020 ), most likely due to its primary role in clathrin-independent endocytosis ( Li et al., 2012 ). To determine the localization of FLOT in P. patens we constructed a PpFLOT CDS construct with an added citrine tag in-frame by cloning it into an empty Actin 5 (ACT5) vector containing the linked citrine sequence ( Top et al., 2021 ). P. patens WT protoplasts were transformed with this construct to generate transient PpFLOT::citrine lines. Three days after transformation the localization of PpFLOT::citrine was traced by confocal microscopy. In contrast to all three FLOT variants in A. thaliana, PpFLOT::citrine did not localize to the plasma membrane of the protoplast, but clearly colocalized with chlorophyll autofluorescence signals in the chloroplasts ( Figure 1A ). Close-up images further disclosed the accumulation of PpFLOT::citrine in thylakoids, suggesting the formation of nanodomains similar to the accumulation of AtFLOTs in plasma membranes. In silico prediction tools did not indicate chloroplast localization for PpFLOT, underscoring the limitations of computational approaches in predicting subcellular protein distribution. To determine whether PpFLOT exhibits a similar localization pattern in other plant species, we cloned the PpFLOT CDS into the pHKL0786 vector containing a venus tag and transiently expressed it in Nicotiana benthamiana leaves. Confocal microscopy analysis three days post-transformation revealed that PpFLOT::venus predominantly localized to the plasma membrane, consistent with FLOT localization in A. thaliana ( Li et al., 2012 ; Daněk et al., 2016 ). Notably, in contrast to P. patens , no detectable fluorescence signal was observed in the chloroplasts, indicating that PpFLOT does not associate with plastids in tobacco cells ( Supplementary Figure 1 ). Download figure Open in new tab Figure 1: Localization of PpFLOT::citrine and comparison of its peptide sequence to homologs in other plant species (A) Confocal microscopy images of a P. patens protoplast transiently transformed with the PpFLOT::citrine construct showing chlorophyll (chl) (red), PpFLOT::citrine (green), and a merged (orange) image. A close-up of a single chloroplast of the merged image is also presented. Scale bars are indicated in the respective images. (B) Phylogenetic tree generated by MEGA X using the maximum likelihood method, depicting relationships among PpFLOT homologs in various plant species: A. officinalis , A. thaliana , A. filiculoides , C. purpureus , C. braunii , C. subellipsoidea , K. nitens , M. polymorpha , M. truncatula , O. sativa , P. patens , S. cucullata , S. polyrhiza , Synechocystis sp. PCC 6803, Z. mays and Z. marina . The alignment was generated with the CLC workbench v20.0.4. Only branches with bootstrap values > 50 are shown. (C) Protein sequence alignment of all FLOT variants from A. thaliana , C. purpureus , P. patens, and M. polymorpha generated by CLC workbench v20.0.4. Sequence conservation is color-coded, from 0 % (red) to 100 % (black), and a sequence logo is provided below the alignment. Protein sequence hydropathicity is marked according to Kyte-Doolittle ( Kyte and Doolittle, 1982 ) from minimal (blue) to maximal (red). SPFH/Band-7 protein domain regions are highlighted in yellow and regions of potential coiled-coil structures are highlighted in orange. Analysis of the evolutionary relationship and the domain structure of PpFLOT Phylogenetic analysis of PpFLOT was conducted through reciprocal BLAST of full-length peptide sequences from various plant species, Asparagus officinalis , A. thaliana , Azolla filiculoides , Ceratodon purpureus , Chara braunii , Coccomyxa subellipsoidea , Klebsormidium nitens , Marchantia polymorpha , M. truncatula , Oryza sativa , P. patens , Salvinia cucullata , Spirodela polyrhiza , Synechocystis sp. PCC 6803, Zea mays , and Zostera marina . Results indicated a high similarity between bryophyte FLOT homologs, with PpFLOT showing a closer relationship to homologs in ferns, green algae and cyanobacteria than to seed plant homologs ( Figure 1B ). Notably, PpFLOT homologs in monocotyledons ( O. sativa , Z. mays , Z. marina and A. officinalis ) exhibit higher similarity compared to homologs in dicotyledonous species like A. thaliana and M. truncatula ( Figure 1B ). Interestingly, both O. sativa and Z. marina , which grow submerged or partially submerged in water, share higher similarity with PpFLOT. Given P. patens‵ ability to be cultivated in submerged conditions, this suggests that PpFLOT’s function may have evolved during the water-to-land transition and remains beneficial for plants regularly encountering anoxic environments. To examine the potential impact of amino acid (aa) sequence variations on the distinct locations of PpFLOT and its A. thaliana variants full-length homologs of PpFLOT were identified through reciprocal BLAST searches in A. thaliana , C. purpureus , and M. polymorpha . Alignment and analysis using CLC workbench v20.0.4 (Qiagen) revealed minimal changes in the aa sequence, with a generally high conservation among the FLOT variants, except for the last 100 aa at the C-terminus. Additionally, PpFLOT exhibited 22 aa from a repeated DAALY*K*KEA motif at positions 354 to 375 ( Figure 1C ). This conserved motif, also present in related bryophyte species, was absent in all three AtFLOT variants ( Figure 1C ). These minimal changes within an otherwise conserved sequence support a common origin of FLOT across all plant species. Evolutionary alterations in the FLOT peptide sequence might have occurred in the last common ancestor of bryophytes and tracheophytes, potentially resulting in an altered function of FLOT in bryophytes. The analyzed proteins encode an SPFH/Band-7 protein domain in the N-terminal region, beginning at position three, or position four in AtFLOT3, and ending at position 185/6 ( Figure 1C ). This region, crucial for membrane association, contains hydrophobic regions, displaying slight changes in hydrophobicity between bryophyte FLOT and the three A. thaliana FLOTs ( Figure 1C ). Another characteristic feature of FLOTs are coiled-coil domains that serve as recognition sites for interaction partners and facilitate oligomerization with themselves or other FLOT variants at the plasma membrane ( Rivera-Milla et al., 2006 ; Frick et al., 2007 ; Solis et al., 2007 ; Daněk et al., 2016 ). Interaction studies with truncated AtFLOT1/3 proteins in yeast demonstrated oligomerization between AtFLOT1 and AtFLOT3 ( Yu et al., 2017 ). These studies showed that AtFLOT1 aggregated at plasma membranes ( Yu et al., 2017 ). Further confirmation of the crucial role of coiled-coil regions in oligomerization was provided when truncated AtFLOT1/3 variants lacking these regions (position 201 - 470) ( Figure 1C ) failed to interact ( Yu et al., 2017 ). Using the Galaxy-based (ver. 5.0.0.1) ( Galaxy community, 2022 ) application pepcoil ( Rice et al., 2000 ; Blankenberg et al., 2007 ) and the web-based application CoCoNat ( Madeo et al., 2023 ), a search for coiled-coil regions in PpFLOT identified putative structures between positions 226 to 300 ( Figure 1C ). Given the presence of these structures, it is likely that PpFLOT undergoes homo-oligomerization, leading to the formation of PpFLOT scaffolds in thylakoid membranes, providing anchoring points for protein complex formation and interactions. Generation of Δ PpFLOT knockout and overexpression lines To probe the potential role of PpFLOT in P. patens development, we generated Δ PpFLOT lines by replacing exon 2 of the PpFLOT CDS with a nptII selection cassette via homologous recombination ( Figure 2A ) ( Schaefer, 2001 ; Frank et al., 2007 ). Following protoplast transformation with the linearized construct and subsequent selection via G418 sulfate (50 µg/ml), transformed plants were screened by amplifying the complete genomic PpFLOT sequence ( Figure 2B ). Examination of 3’ site and 5’ site integration of the construct ( Figure 2B ), along with confirming PpFLOT transcript loss ( Figure 2C ), identified three independent Δ PpFLOT lines, Δ PpFLOT-1, Δ PpFLOT-2 and Δ PpFLOT-3 , with a single integration confirmed by Southern blot analysis ( Figure 2D ). Given our previous observation of increased PpFLOT expression linked to salt sensitivity and ABA hyposensitivity in P. patens ( Arif et al., 2022 ), we also generated PpFLOT overexpression lines. To generate the PpFLOT- OEX lines we prepared a construct in which the PpFLOT CDS was under the control of an Act5 promoter for constitutive expression and the respective also harbored a hygromycin resistance cassette for plant selection. After protoplast transformation, screening and confirmation of construct insertion in the genome, an enhanced expression was confirmed by amplifying the PpFLOT transcript from cDNA for 25 and 35 cycles by PCR ( Figure 2E ). This approach identified three PpFLOT- OEX lines, PpFLOT -OEX1, PpFLOT -OEX2 and PpFLOT -OEX3 that showed a relative expression of 202.79 (SEM ± 62.01), 317.61 (SEM ± 102.18) and 674.49 (SEM ± 98.6), respectively, compared to the WT and normalized against the expression of ELONGATION FACTOR 1 ALPHA ( PpEF1α ) ( Figure 2F ). Download figure Open in new tab Figure 2: Generation of Δ PpFLOT and PpFLOT -OEX lines (A) Schematic representation of the transformation construct. The upper panel illustrates the WT PpFLOT coding sequence (CDS), while the lower panel displays the construct for Δ PpFLOT generation, involving exon 2 replacement with a nptII selection cassette. Arrows indicate primer positions during screening (B) Screening of Δ PpFLOT lines via PCR, amplifying the complete PpFLOT sequence and the 5’ and 3’ integration sites using genomic DNA (C) Transcript analysis of PpFLOT by PCR from cDNA, amplifying both the PpFLOT transcript sequence and exon 2 sequence. (D) Southern blot analysis for all three identified Δ PpFLOT lines, confirming single construct integration in the moss genome. In case of single integration, digestion of total genomic DNA with Xho I and detection of the nptII selection cassette with a complementary probe results in a band of 6523 bp since Xho I does not cut within the selection marker sequence. For better validation, the experiment was repeated with Nco I, an enzyme that cuts within the nptII selection cassette and produces two bands (1490 bp and 571 bp) upon single integration. (E) Confirmation of PpFLOT -OEX lines by PCR amplification of PpFLOT transcript from cDNA at 35 and 25 cycles, comparing band intensities to WT (F) qRT-PCR of relative PpFLOT expression compared to the WT, normalized to PpEF1α expression, following Schmittgen and Livak (2008) . Mean values with error bars indicating ± SEM (n = 3) are presented. Oligonucleotide sequences are listed in Supplementary Table 1. Δ PpFLOT lines display an enhanced salt tolerance Liquid cultures of P. patens WT and all three generated Δ PpFLOT lines were grown for 14 d under control conditions and in medium supplemented with 250 mM NaCl. To detect potential changes in the accumulation of biomass and their salt sensitivity every two to three days, the dry weight of the equalized cultures was determined, and images of the cultures were taken. Despite reported stunted growth in AtFLOT1 amiRNA lines ( Li et al., 2012 ), no changes in accumulated biomass over time were observed in the Δ PpFLOT lines compared to the WT, both under control conditions and in salt-treated cultures ( Figure 3A , 3B). Interestingly, unlike the WT, the Δ PpFLOT lines did not display bleaching in response to the salt treatment. Bleaching in plant cultures typically results from a decrease in chlorophyll content ( Marconi et al., 2001 ; Taïbi et al., 2016 ) to avoid reactive oxygen species (ROS) accumulation and oxidative damage ( Verma and Mishra, 2005 ). The apparent lack of chlorophyll suppression in response to salt treatment suggests a potential role for PpFLOT in detecting or regulating ROS, or in chlorophyll biogenesis regulation, a function that may be impaired in Δ PpFLOT lines. Download figure Open in new tab Figure 3: Growth phenotype of Δ PpFLOT and PpFLOT -OEX lines (A) Comparison of WT and Δ PpFLOT-1 liquid protonema cultures with an initial density of 0.1 mg/ml after 14 d of treatment with 250 mM NaCl (right) and an untreated control (left). (B) Growth curves of Δ PpFLOT lines and WT determined by dry weight measurement every three to four days for 14 d. Cultures were initially inoculated with an equal density of 0.1 mg/ml dry weight. Measurements were taken during treatment with 250 mM NaCl and under control conditions for Δ PpFLOT lines and a WT control. (C) Upper panel displays liquid protonema cultures starting with an equal density of 100 mg/L and grown for 16 weeks (left to right) of Δ PpFLOT-1 , WT, PpFLOT -OEX1 , PpFLOT -OEX2, and PpFLOT -OEX3 at a density of ∼ 1 mg/ml dry weight. The second panel presents close-ups of protonema cells from the respective lines when cultivated in standard liquid medium. Diaspore-like cells with brown coloration develop in PpFLOT -OEX2 and 3. Panels three to five show the development of the respective lines when cultivated for 0, 8 and 14 d on solid growth medium. Scale bars are indicated in the respective images. Increased PpFLOT overexpression strongly affects protonema growth Long-term cultivation of liquid protonema cultures from all generated PpFLOT mutant lines (including Δ PpFLOT-1 and PpFLOT -OEX1,2,3 lines) resulted in changes of the coloration. In Figure 3C depicted cultures started with an equal density of 100 mg/L and were cultivated for 16 weeks before dry weight was measured and images were taken. A comparison of the PpFLOT mutant lines with a WT control grown with the same density revealed subtle changes, with a discernible color gradient depending on the PpFLOT expression level ( Figure 3C ). This gradient ranged from light green in Δ PpFLOT-1 to dark brown-green in PpFLOT -OEX3 ( Figure 3C ), and close examination of single protonema cells indicated that increased PpFLOT expression led to the development of small round cells in addition to the characteristic cell filaments. These cells resemble vegetative diaspores or brachycytes observed in P. patens under exogenous ABA treatments or ABA-mediated stress responses ( Arif et al., 2019 ). These brachycytes are released from the protonema cell network by transforming the surrounding cells into empty tmema cells ( Arif et al., 2019 ), which could not be detected in the PpFLOT -OEX lines. These round cells, in contrast to these diaspores, turned reddish-brown with increasing PpFLOT levels, contributing to the observed change in culture coloration, especially in the strongest PpFLOT -OEX lines. Notably, this coloration expands to the surrounding cell filaments as well ( Figure 3C ). Interestingly, when PpFLOT -OEX protonema was plated on solid media, these structures receded, and after two weeks, they mostly recovered, developing WT-typical gametophores ( Figure 3C ). Additionally, mature gametophores submerged in liquid media without tissue disruption showed no changes in pigmentation ( Supplementary Figure 2 ). Salt and phytohormone sensitivity in P. patens are modulated by PpFLOT expression levels To evaluate the responses of the generated mutant lines to elevated salt concentrations, increased osmotic pressure, and exogenous phytohormone treatment, protonema cultures of an equal density (100 mg/L dry weight) were spotted on standard solid medium supplemented with 250 mM NaCl, 300 mM NaCl, 700 mM mannitol, 10 µM 2- cis ,4- trans -abscisic acid (ABA), 10 µM of the auxin analog 1-naphthylacetic acid (NAA) and 10 µM of the cytokinin derivate 6-y-y-(dimethylallylamino)-purine (2-ip). The spotted colonies were cultivated for eight weeks. Under control conditions and 250 mM NaCl treatment, Δ PpFLOT-1 and PpFLOT -OEX lines showed no significant changes in development compared to the WT ( Figure 4 ). However, at a salt concentration of 300 mM, the PpFLOT -OEX lines were unable to form colonies whereas both Δ PpFLOT-1 and the WT exhibited colony formation ( Figure 4 ). To assess the impact of increased osmotic pressure on the PpFLOT -OEX lines, which exhibit reduced salt tolerance, protonema cultures of all lines were placed on plates supplemented with 700 mM mannitol. This concentration was chosen to simulate the osmotic pressure equivalent to 300 mM NaCl for P. patens , following observations described by Saavedra et al. (2006) . All tested lines were able to develop colonies and exhibited growth similar to the WT. However, consistent with our observations in liquid culture, a gradient of pigmentation was observed in the colonies, shifting to a green-brownish hue with increased PpFLOT expression ( Figure 4 ). The findings suggest that increased expression of PpFLOT is detrimental to the salt tolerance of P. patens implicating that PpFLOT most likely acts as a negative regulator in abiotic stress response, either directly or indirectly. Notably, qRT-PCR measurements of the PpFLOT expression in WT protonema cultures treated with 250 mM NaCl over 24 h showed a decrease in the expression levels ( Figure 5A ) at 8 and 24 h. However, this decrease in expression was not statistically significant (ANOVA, p = 0.075) due to a high variance among the biological replicates. Conversely, the expression levels in the salt-treated PpFLOT -OEX1 line did not exhibit significant decrease (ANOVA, p = 0.26) ( Figure 5A ). Download figure Open in new tab Figure 4: Phenotypic analysis of Δ PpFLOT-1 and all PpFLOT -OEX lines Δ PpFLOT-1 , WT, PpFLOT -OEX1, PpFLOT -OEX2 and PpFLOT -OEX3 were inoculated with an equal density (100 mg/L dry weight) on standard solid medium (control) and media supplemented with 250 mM NaCl, 300 mM NaCl, 700 mM mannitol, 10 µM ABA, 10 µM NAA and 10 µM 2-ip and grown for at 8 weeks. The scale bar in all images represents 1 mm. The strength of PpFLOT expression is indicated on the right. Download figure Open in new tab Figure 5: PpFLOT expression is regulated by daytime, light, ABA and salt (A) Depiction of relative gene expression of PpFLOT and miR167 in P. patens WT protonema (upper panel) and expression of PpFLOT in PpFLOT -OEX1 (lower panel) measured over 24 h under control conditions and in response to 10 µM ABA and 250 mM NaCl in triplicates. Analysis performed according to Schmittgen and Livak (2008) . ANOVA analysis was conducted to identify statistically significant differences between time points, with p-values provided in the respective boxes. Asterisks denote time points with statistically significant changes in expression identified by Tukey’s HSD test compared to 0 h treatment. * p < 0.05, ** p < 0.01, **** p < 0.0001. (B) Relative gene expression of PpFLOT in P. patens WT protonema observed over 24 h under long day conditions (LD 16:8), complete darkness (D) and continuous light after adapting for three days to the respective light conditions. Mean values of biological triplicates are depicted with ± SEM (n = 3). Bars above the graphs indicate light conditions at the respective time points (black = light off, white = light on). Light intensity in all conditions was 85–100 µmol/m 2 s. P-values of the ANOVA analyses are given in the respective graphs. Cosinor curve calculated from acrophase, meseor, and amplitude is indicated by a red line. A continuous line indicates statistically significant rhythmicity (p 0,05). During ABA treatment of the respective lines, we further observed improved ABA sensitivity correlating with PpFLOT expression, as evidenced by larger PpFLOT -OEX3 colonies compared to the Δ PpFLOT-1 or the WT control ( Figure 4 ). Interestingly, 10 µM ABA treatment of WT protonema cultures led to a statistically significant decrease in PpFLOT transcript levels over time (ANOVA, p < 0.01) ( Figure 5A ). Starting from 1 h after ABA treatment, PpFLOT expression significantly decreased and continued to decrease steadily, reaching a relative expression of 0.01 after 24 h ( Figure 5A ). Treatment with NAA or 2-ip did not result in phenotypic changes for Δ PpFLOT-1 , PpFLOT -OEX1, or PpFLOT -OEX2 compared to the treated WT control ( Figure 4 ). In both cases the lines exhibited a similar growth pattern as the wild type, except line PpFLOT -OEX3. Upon NAA treatment this line failed to develop gametophores and formed reddish colonies. When treated with 2-ip PpFLOT -OEX3 exhibited slightly reduced growth compared to the other lines. ( Figure 4 ). Overall, the observed phenotypic alterations in response to all treatments were correlated with the levels of PpFLOT expression. The PpFLOT transcript is regulated by multiple pathways The phenotypic analysis of the generated PpFLOT mutant lines suggests a putative regulatory function for PpFLOT during abiotic stress responses in P. patens . Furthermore, the expression of WT PpFLOT is suppressed in response to ABA and salt treatment. Examination of the PpFLOT 5’UTR and 1.5 kb the genomic sequence upstream genomic sequence for cis -acting regulatory elements using PlantPAN 4.0 ( Chow et al., 2019 ) identified potential binding sites for various transcription factors, APETALA 2 (AP2), basic HELIX-LOOP-HELIX (bHLH), CG-1 DNA-binding domain containing transcription factors (CG-1), ETHYLENE INSENSITIVE 3 (EIN3), GATA transcription factors, DNA binding with ONE FINGER (DOF), LATERAL ORGAN BOUND (LOB) domain transcription factors, V-MYB MYELOBLASTOSIS VIRAL ONCOGENE HOMOLOG (MYB)/ SWITCH_DEFECTIVE PROTEIN 2 (SWI3), ADAPTOR 2 (ADA2), NUCLEAR RECEPTOR COREPRESSOR (N-CoR, TRANSCRIPTION FACTOR III B (TF) (SANT), NO APICAL MERISTEM/ARABIDOPSIS THALIANA ACTIVATING FACTOR/CUP-SHAPED COTYPEDON (NAC), WRKY, MINI CHROMOSOME MAINTENANCE1/AGAMOUS/DEFICIENCE/SERUM RESPONSE FACTOR (MADS) box, BASIC REGION/LEUCINE ZIPPER (bZIP), TATA BINDING PROTEIN (TBP) transcription factors as well as C2H2, TCR, homeodomain and AT-hook motifs. The diverse array of identified putative transcription factor binding sites suggests the involvement of PpFLOT in various biological processes. Interestingly bHLH, NAC, and WRKY, as well as, in some instances, MYB, AP2, DOF, and bZIP transcription factors, are recognized regulators of the abiotic stress response or are themselves regulated by ABA ( Golldack et al., 2011 ; Mizoi et al., 2012 ; Ambawat et al., 2013 ; Phukan et al., 2016 ; Das et al., 2019 ; Zou and Sun, 2023 ). Meanwhile, the presence of CG-1 and GATA binding sites suggests potential circadian or light-dependent regulation ( da Costa e Silva, 1994 ; Reyes et al., 2004 ). Previous studies in rice indicate that AT-hook motifs may interact with light-sensitive phytochromes ( Jorge Nieto-Sotelo, 1994 ). Subsequently, we investigated PpFLOT expression for light-dependent or circadian regulation. WT protonema cultures were analyzed for putative circadian regulation by measuring PpFLOT transcript levels every 4 h over 24 h. The experiment was conducted under standard growth conditions (16 h light: 8 h dark; LD), complete darkness (D), and continuous light (CL), with all samples pre-adapted to their respective photoperiods for 3 days. ANOVA analysis of the PpFLOT expression over 24 h revealed significant changes (p < 0.01) in LD, D and CL cultivated samples. LD samples showed a peak expression at time point (TP) 12, with the lowest PpFLOT expression at TP 16, while D samples peaked at TP 20, and TP 0 showed the lowest expression ( Figure 5B ). In contrast to the other two photoperiod conditions, CL did not display a single maximum or minimum of the expression ( Figure 5B ). While the overall expression levels of PpFLOT in LD and CL samples oscillated between a normalized relative expression of 0.5 – 3, D samples exhibited a higher overall expression level, fluctuating between a normalized relative expression of 10 – 50 ( Figure 5B ). Intriguingly, the highest PpFLOT expression in the LD samples coincided with the beginning of the dark period ( Figure 5B ), suggesting that PpFLOT expression is generally enhanced in the absence of irradiation. To assess if the observed oscillations follow circadian rhythmicity, we conducted a cosinor-based rhythometry analysis ( Cornelissen, 2014 ) for all three groups. Both LD and D expression levels could be fitted to a cosinor curve, meeting the criteria for circadian rhythmicity with p < 0.01. However, the CL group did not fulfill these conditions (p = 0.205). The detected arrhythmicity under CL is likely attributed to a dysfunctionality of the P. patens circadian clock at CL conditions, as proposed and demonstrated by Okada et al. (2009) for clock-related genes in P. patens ( Ichikawa et al., 2004 ; Ichikawa et al., 2008 ; Okada et al., 2009 ; Petersen et al., 2022 ). Due to the overall higher expression levels, we observed a shift in amplitude and acrophase between the LD and NL samples ( Figure 5B ). Additionally, a phase delay in D samples compared to LD samples was likely caused by the absence of irradiation impulses ( Fukuda et al., 2013 ). It is noteworthy that in mammals, stress and changes in hormone levels can induce a shift in the expression of genes regulated by the circadian clock ( Ota et al., 2021 ). Therefore, during stress, a delay in the circadian clock of P. patens could occur due to increased expression of PpFLOT or alterations in phytohormone profiles throughout the day. In our previous study, altered miRNA biogenesis led to an increase in PpFLOT transcript levels ( Arif et al., 2022 ). Subsequently, we searched the genomic PpFLOT sequence for a putative miRNA binding site with psRNATarget ( Dai et al., 2018 ), identifying miR167 as the best match with an expectation score of 3.5 and a putative cleavage site in the third exon of the PpFLOT sequence located at bp 1898-1918. Relative miR167 expression detected by stem-loop PCR revealed anticorrelating expression between miR167 and PpFLOT in WT protonema under control conditions and in response to salt treatment ( Figure 5A ). However, statistical analysis did not confirm the detected changes in miRNA expression as significant, likely due to high variance between replicates. While these changes in expression were not statistically significant, they suggest a potential regulation of PpFLOT on the transcript level. Overall, we identified four potential regulatory mechanisms controlling the PpFLOT transcript levels in the P. patens protonema cell. The evidence suggests that the expression of PpFLOT can be suppressed by ABA-responsive transcription factors. Additionally, PpFLOT is circadian regulated, with increased expression when cultivated without light impulses. Moreover, it cannot be dismissed that miR167 may regulate the PpFLOT transcript, although further experiments are necessary to confirm this type of regulation. We propose that PpFLOT, located in the chloroplasts of P. patens , serves as a scaffolding protein in various biological processes, particularly during the response to abiotic stress and in processes related to the day-night transition of chloroplasts. The diverse methods identified to regulate PpFLOT levels transcriptionally or posttranscriptionally in chloroplasts highlight its importance in multiple cellular functions. Detection of slight ROS accumulation in PpFLOT -OEX lines All generated PpFLOT -OEX lines, displaying lower salt tolerance than the WT or Δ PpFLOT-1 , potentially attributed to increased ROS accumulation ( Pandey et al., 2017 ) underwent 3,3-diamonobenzidine staining to detect H 2 O 2 overaccumulation. Protonema samples were incubated for 18 h in the staining solution under CL photoperiod conditions, and subsequent microscopic examination revealed higher H 2 O 2 levels in PpFLOT -OEX lines compared to the WT and Δ PpFLOT-1 ( Figure 6A ). Notably, PpFLOT -OEX2 and PpFLOT -OEX3 exhibited cells with persistent pigmentation, suggesting potential challenges in destaining by boiling in ethanol, making it challenging to ascertain the origin of H 2 O 2 overaccumulation in these structures. However, a general comparison of stained normal-grown protonema cells across all PpFLOT -OEX lines and the WT demonstrated a correlation between PpFLOT expression levels and H 2 O 2 accumulation ( Figure 6A ). Download figure Open in new tab Figure 6: Changes in H 2 O 2 levels and gene expression corresponding to changes in PpFLOT expression (A) H 2 O 2 levels were visualized in Δ PpFLOT-1 , WT, and all three PpFLOT -OEX lines through staining with 3, 3-diamonobenzidine. The upper panel depicts images of treated protonema cells, while the lower panel exhibits images of untreated mock controls. The scale bar in all images represents 10 µm. (B) Box plots present qRT-PCR results of relative gene expression for (top to bottom) AP2/ERF domain transcription factor Pp3c8_7340 , copper transport HMA domain protein Pp3c3_6890 , PpGLK2 and PpCRY1b , comparing expression levels to WT at 0 h of treatment and normalized against PpEF1α following the method by Schmittgen and Livak (2008) . The depicted relative gene expression of the respective genes in Δ PpFLOT-1 , WT and PpFLOT -OEX1 protonema is shown under control conditions (left), upon ABA (middle) and salt-(right) treatment measured over 24 h in triplicates. ANOVA was used to determine statistically significant changes in expression between the three genotypes, with p-values indicated in the respective graphs (black asterisk). Tukey’s HSD results for time-dependent expression in one line are marked by red asterisk when significant compared to 0 h of treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Elevated H 2 O 2 levels, indicative of stress in P. patens , prompted an examination of transcript levels of abiotic stress-induced genes ( Boursiac et al., 2005 ; Saavedra et al., 2006 ; Frank et al., 2007 ; Hauser et al., 2011 ; Li et al., 2011 ; Li et al., 2012 ). These measurements were performed under control conditions and when treated with either 250 mM NaCl or 10µM ABA in WT, Δ PpFLOT-1 all PpFLOT -OEX1. Interestingly, the expression of AQUAPORINE TIP ( Pp1s44_31/ Pp3c20_15350 ), 9′- cis EPOXYCAROTENOID DIOXIGENASE ( NCED ) ( Pp1s412_7/ Pp3c25_4810 ), TRANSLOCATOR PROTEIN 1 ( TSPO1 ) ( Pp1s281_123/ Pp3c2_17540 ) and DEHYDRIN B ( DHNB ) ( Pp1s442_22/Pp3c5_11880 ) barely showed any significant changes in the gene expression among the tested genotypes ( Supplementary Figure 3 ). Specifically, only PpTSPO1 displayed slightly lower expression in PpFLOT -OEX1 compared to the WT and Δ PpFLOT-1 (ANOVA p = 0.04) in response to salt treatment ( Supplementary Figure 3 ). Conversely, the expression of a mRNA encoding an AP2/ERF domain transcription factor Pp1s8_127/ Pp3c8_7340 changed significantly between the genotypes under control conditions (ANOVA, p = 0.02) since Pp3c8_7340 (AP2/ERF) exhibited significantly (Tukey’s HSD, p = 0.0235) higher expression in PpFLOT -OEX1 compared to Δ PpFLOT-1 . Significant upregulation of Pp3c8_7340 (AP2/ERF) was observed at 0 h of salt treatment (Tukey’s HSD, p = 0.0429) and at 8 h of ABA treatment (Tukey’s HSD, p = 0.0229) in PpFLOT -OEX1 compared to the WT. Notably, both the WT and Δ PpFLOT-1 samples treated with ABA for 1, 2, and 4 h exhibited a significant increase in Pp3c8_7340 (AP2/ERF) expression compared to untreated samples (ANOVA, p < 0.01). Statistically significant differences in expression levels between the genotypes were observed at 2 h (Tukey’s HSD Δ PpFLOT-1 vs WT, p < 0.01) and 8 h (Tukey’s HSD PpFLOT -OEX1 vs WT, p = 0.0295). However, the overall changes in expression between the tested genotypes in response to ABA treatment were not statistically significant (ANOVA, p = 0.09) ( Figure 6B ). Similarly, upon salt treatment, the overall change in expression between the tested genotypes was not significant (ANOVA, p = 0.187). At 0 h, a significantly higher expression of Pp3c8_7340 (AP2/ERF) was detected in PpFLOT -OEX1 (Tukey’s HSD PpFLOT -OEX1 vs WT, p = 0.0429) ( Figure 6B ). This upregulation in untreated samples of PpFLOT -OEX1 is most likely an attempt to suppress the ectopic PpFLOT expression. Heavy metal-associated domain (HMA) containing proteins are vital for heavy metal transport, detoxification in plants, and respond to various stresses ( Li et al., 2020 ; Barr et al., 2023 ). Proteins in this family are upregulated in response to drought, cold, hypoxia, and bacterial infection ( Barr et al., 2023 ). The gene expression of the copper transport protein Pp1s296_27/Pp3c3_6890, encoding such an HMA domain, exhibited significant differences in expression between the three genotypes upon ABA and salt treatment (ANOVA, p < 0.01). For instance, while all genotypes showed increasing Pp3c3_6890 levels in response to ABA, this upregulation was extreme in PpFLOT -OEX1 1 h after ABA treatment compared to the other two lines (Tukey’s HSD, p < 0.05) ( Figure 6B ). On the other hand, in response to salt Δ PpFLOT-1 displayed an extreme upregulation of Pp3c3_6890 (Cu-HMA) compared to both WT (Tukey’s HSD, p < 0.01) and PpFLOT- OEX1 (Tukey’s HSD, p < 0.01). Comparatively Pp3c3_6890 (Cu-HMA) expression is subdued in PpFLOT -OEX1 in response to salt ( Figure 6B ). Despite the salt-sensitive phenotype of PpFLOT -OEX lines, only minor changes in the expression levels of salt and ABA-induced genes were detected upon induction. For instance, Aquaporine TIP exhibited slight repression in PpFLOT -OEX1 at all TP compared to 0 h under control conditions. Comparatively, PpFLOT -OEX1 showed a peak in Aquaporine TIP expression at 2 h instead of 24 h ( Supplementary Figure 3 ). In the WT, NCED expression significantly increased in response to ABA (ANOVA, p = 0.015) and salt treatment (ANOVA, p < 0.01). While this change in expression was also detected in PpFLOT -OEX1 and Δ PpFLOT-1 , it was not statistically significant ( PpFLOT -OEX1: ANOVA salt p = 0.585, ABA p = 0.427 and Δ PpFLOT- 1: ANOVA salt p = 0.629; ABA p = 0.078) ( Supplementary Figure 3 ). Similarly, no changes in the expression of PpDHNB were detected among the three genotypes (ANOVA, p = 0.865) over 24 h. However, at 24 h, PpDHNB expression was significantly upregulated in the WT compared to Δ PpFLOT-1 and PpFLOT -OEX1 (Tukey’s HSD, p < 0.01). Additionally, the peak expression of PpDHNB upon salt treatment for both WT and PpFLOT -OEX1 occurred at 2 h, while in Δ PpFLOT-1 , it peaked at 24 h ( Supplementary Figure 3 ). Most abiotic stress markers behaved similarly to the WT, suggesting no upregulation of ABA biosynthesis or other known abiotic stress-sensing pathways in in Δ PpFLOT-1 and PpFLOT -OEX lines. Abnormal expression of stress-induced genes and increased H 2 O 2 levels may impact retrograde signaling pathways, involving chloroplast-to-nucleus communication using ROS as signaling molecules ( Locato et al., 2018 ; Li and Kim, 2022 ). The gene GOLDEN 2-LIKE PROTEIN 2 ( Pp3c11_21140 ) ( GLK2 ), associated with retrograde signaling ( Sun et al., 2022 ; Zeng et al., 2023 ), exhibited significant expression changes under control conditions and salt treatment in WT, Δ PpFLOT-1 and all PpFLOT -OEX lines (ANOVA, salt p = 0.014, cont p < 0.01). Δ PpFLOT-1 displayed consistently higher PpGLK2 expression than WT and PpFLOT -OEX1 under control conditions (Tukey’s HSD, p <0.01) ( Figure 6B ). In response to salt treatment, PpFLOT -OEX1 showed significantly lower PpGLK2 expression (Tukey’s HSD, p < 0.0119) compared to Δ PpFLOT-1 but not compared to the WT (Tukey’s HSD, p = 0.581) ( Figure 6B ). Another retrograde signaling gene, CRYPTOCHROME 1b ( Pp3c7_20480V3 ) ( PpCRY1b ), displayed a statistically significant expression change (ANOVA, p = 0.041) between the genotypes upon ABA treatment. PpCRY1b was generally expressed at higher levels in WT than Δ PpFLOT-1 (Tukey’s HSD, p < 0.0356) except at 1 h. However, under control conditions, PpCRY1b showed higher transcript levels at 8 and 24 h after start of the measurements compared to Δ PpFLOT-1 and PpFLOT -OEX1, with no statistically significant differences under control conditions ( Figure 6B ). Increased chlorophyll a/b content correlates with strength of PpFLOT expression The total chlorophyll content of WT, Δ PpFLOT-1 , PpFLOT -OEX1, PpFLOT -OEX2, and PpFLOT -OEX3 was assessed to determine if the observed PpFLOT -dependent change in pigmentation resulted in altered chlorophyll content in the PpFLOT mutant lines. Chlorophyll a and b were extracted using 80 % acetone from equal amounts of protonema harvested per line. After separating cell debris, chlorophyll absorption was measured, and total chlorophyll content was calculated following the method outlined by Frank et al. (2005) . Significant changes (ANOVA, p < 0.01) in the chlorophyll content were observed between PpFLOT mutant lines and the WT control. Δ PpFLOT-1 exhibited only half as much chlorophyll as the WT, with 0.4 mg chl/g dry weight ( Figure 7A ). Surprisingly, the PpFLOT -OEX1 line exhibited a similar amount of chlorophyll (0.49 mg chl/g dry weight). In contrast, both PpFLOT -OEX2 and PpFLOT -OEX3 had significantly higher chlorophyll content, with 2.6 and 2.5 mg chl/g dry weight, respectively ( Figure 7A ). This suggests an enhanced chlorophyll biosynthesis, potentially leading to increased photosynthetic activity. To assess this, we performed Pulse-Amplitude-Modulation (PAM) measurements to quantify the chlorophyll fluorescence and calculate the quantum yield of the photosystem II (PSII) activity and non-photochemical quenching during photosynthesis activation by light. As previously mentioned, we observed massive differences in the pigmentation between PpFLOT mutant lines grown in liquid cultures and those cultivated on solid media. Therefore, we conducted separate measurements for mature gametophores grown on solid medium and protonema liquid cultures with the same number of biological replicates (n = 9) using identical parameters. Interestingly, while statistical analysis of PSII activity among all tested genotypes in the gametophores revealed significant differences (ANOVA, p = 0.023), only PpFLOT -OEX1 displayed a lower PSII activity compared to WT and Δ PpFLOT-1 , while PpFLOT -OEX2 and 3 showed no differences in activity compared to the WT. During non-photochemical quenching, we found no changes between the WT, Δ PpFLOT-1 , and all PpFLOT -OEX lines in the gametophores (ANOVA, p =0.34) ( Figure 7B ). To ensure comparability between the gametophore and the protonema samples, we measured fluorescence at 450 nm and an actinic light intensity of 55 µmol/m 2 s photosynthetically active radiation (PAR). However, we were unable to detect the fluorescence of PpFLOT -OEX3 protonema under these conditions, and the measuring light parameter had to be adjusted to an intensity of 60 µmol/m 2 s PAR to detect a fluorescence signal. Therefore, PAM measurements of PpFLOT -OEX3 protonema were excluded from the analysis to maintain comparability between all measurements. In contrast to the fluorescence measurements in gametophores, we detected statistically significant differences in the Y(II) and NPQ/4 (ANOVA, p < 0.01) between Δ PpFLOT-1 and PpFLOT -OEX1 and 2 protonema cultures. Compared to the WT samples, neither the Δ PpFLOT-1 nor the two PpFLOT -OEX lines showed any significant differences in the calculated photosynthetic activity (Tukey’s HSD, Δ PpFLOT-1 vs WT p = 0.898, PpFLOT -OEX1 vs WT p = 0.0719, PpFLOT -OEX1 vs WT p = 0.165). However, a comparison between the Δ PpFLOT-1 and the PpFLOT -OEX lines revealed a significantly higher quantum yield of PSII in the PpFLOT -OEX lines (Tukey’s HSD, PpFLOT -OEX1 vs Δ PpFLOT-1 p < 0.01, PpFLOT -OEX2 vs Δ PpFLOT-1 p = 0.0298) compared to the knockout line ( Figure 7B ). This change in activity was also detected during non-photochemical quenching. For instance, both PpFLOT -OEX lines process less energy by non-photochemical quenching than Δ PpFLOT-1 (ANOVA, p < 0.01; Tukey’s HSD, PpFLOT -OEX1/2 vs Δ PpFLOT-1 p < 0.01) ( Figure 7B ). We found that the level of photosynthetic activity in protonema cultures slightly increases in correlation with the increased expression of PpFLOT . However, a statistical comparison between the WT and all PpFLOT mutant lines did not yield any significant results. Interestingly, we detected a significantly higher (ANOVA, p < 0,01) NPQ/4 signal in Δ PpFLOT-1 compared to PpFLOT -OEX1 and 2 (Tukey’s HSD, p < 0.01). The absence of PpFLOT in the knockout line results in lower photosynthetic activity and increased non-photochemical quenching, potentially due to reduced chlorophyll levels. We suggest that adjusting the amount of PpFLOT in chloroplasts of protonema cells can fine-tune the photosynthetic activity to accommodate the specific needs of the respective cells. This seems to be relevant only during the protonema life stage of P. patens as PpFLOT overexpression, with the exception of PpFLOT -OEX1, has barely an effect on the photosynthetic activity of the gametophores. Download figure Open in new tab Figure 7: Effects of changes in PpFLOT expression on chlorophyll content and photosynthetic activity (A) Chlorophyll content of 0.4 g protonema from WT, Δ PpFLOT-1 and all PpFLOT-OEX lines. Mean values ± SEM (n =3) are shown. ANOVA results are provided, and statistically significant (p < 0.05) differences in the PpFLOT mutants compared to the WT were determined by Tukey’s HSD test and are indicated by asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (B) Effect of PpFLOT expression on Y(II) and NPQ/4 in the gametophore and protonema life stage of P. patens . Mean values ± SD (n = 9) are shown. Measurements were performed after 3 h of dark adaptations and actinic light of 55 µmol/m 2 s at 450 nm for 315 s after saturation pulse stimulation. (C) qRT-PCR results shown as box blots of relative gene expression of PpLHCB1 (Pp3c2_35930), PpLHCA1.1 (Pp3c13_14980), PpPsaD1 (Pp3c16_23780) over 24 h relative to WT expression at 0 h, normalized against PpEF1α as described by Schmittgen and Livak (2008) . Depicted is the relative gene expression of the respective genes in Δ PpFLOT-1 , WT and PpFLOT-OEX1 protonema under control conditions measured in biological triplicates. Statistically significant changes in expression between the three genotypes were determined by ANOVA, with p-values provided in the respective graphs. Significant differential gene expression at a specific time point is marked by black asterisks. Results of the Tukey’s HSD for the time-dependent expression within the same genotype are marked by red asterisks when significant compared to 0 h of treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 Since photosynthetic activity is slightly altered in the PpFLOT mutant protonema liquid cultures and these lines display changes in the chlorophyll biosynthesis rate, we measured the transcript levels of photosystem one (PSI) and two (PSII) related components. Under control conditions, the expression of the LIGHT-HARVESTING COMPLEX B1 ( LHCB1 ) ( Pp3c2_35930 ) displayed significant (ANOVA, p < 0.01) differential expression when compared between WT, Δ PpFLOT-1 and PpFLOT -OEX1 over 24 h. Δ PpFLOT-1 showed significantly higher PpLHCB1 expression compared to the WT (Tukey’s HSD, p = 0.0347) and PpFLOT -OEX1 (Tukey’s HSD, p < 0.0347). Although not statistically significant, PpLHCB1 expression appears subdued in the PpFLOT -OEX1 line in general ( Figure 7C ). Meanwhile, the expression of the LIGHT-HARVESTING COMPLEX A1.1 ( LHCA1.1 ) ( Pp3c13_14980 ) not only changes under control conditions (ANOVA, p < 0.01) ( Figure 7C ), but also in response to salt (ANOVA, p = 0.031) and ABA treatment (ANOVA, p < 0.01) ( Supplementary Figure 3 ). Under all conditions, PpLHCA1.1 showed a lower expression in the PpFLOT -OEX1 line compared to both the WT and Δ PpFLOT-1 ( Figure 7C , Supplementary Figure 3 ). A similar trend was observed for the expression of the PSI SUBUNIT D1 ( PsaD1 ) ( Pp3c16_23780 ). Comparison among the three tested lines revealed a statistically significant change in expression under control conditions (ANOVA, p < 0.01) ( Figure 7C ), ABA (ANOVA, p = 0.013) and salt treatment (ANOVA, p = 0.019) ( Supplementary Figure 3 ). Under control conditions, PpPsaD1 expression was significantly higher in the Δ PpFLOT-1 line compared to the WT (Tukey’s, p < 0.01) and PpFLOT -OEX1 (Tukey’s, p < 0.01). Overall, the alterations in chlorophyll content, photosynthetic activity of PSII, and the expression of PS antenna-complex components in correlation with PpFLOT expression indicate an involvement of PpFLOT in the photosynthesis of P. patens . Zeaxanthin level is depleted in correlation to PpFLOT expression The observed color change in the PpFLOT -OEX liquid cultures may result from specific alterations in the pigment profile compared to the WT. Thus, the pigment composition of Δ PpFLOT-1, all PpFLOT -OEX lines, and a WT control was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS) in four replicates. Among 24 detected pigments, 19 could not be identified. To identify differentially accumulated pigments, statistical analysis was conducted using Perseus v 2.0.11 (MaxQuant) ( Tyanova et al., 2016 ). Pigments were classified as DE when both the p-value and the permutation-based false discovery rate (FDR; q-value) of the Student’s t -test of Δ PpFLOT-1 and all PpFLOT -OEX lines using the WT as a control group were less than 0.05. The analysis employed the log 2 transformed fold change (FC) of the relative pigment abundance. It was found that two detected pigments, lutein and unknown pigment 16, were differentially accumulated in all groups. Additionally, zeaxanthin and unknown pigment 10, displayed an altered abundance in the PpFLOT -OEX lines ( Figure 8A , Supplementary Table 2) that correlates with PpFLOT expression. Notably, all shared differentially accumulated pigments of the PpFLOT mutant lines were downregulated compared to the WT ( Figure 8A , Supplementary Table 2). However, while the detected amount of these pigments decreased in the PpFLOT mutant lines, only zeaxanthin and unknown pigment 10 decreased in correlation with the PpFLOT expression ( Figure 8A ). As zeaxanthin is part of the xanthophyll cycle, we further analyzed the accumulation of its other main components alongside lutein in the PpFLOT mutant lines ( Figure 8B ). Download figure Open in new tab Figure 8: Pigment composition of all PpFLOT mutant lines (A) The clustered heatmap displays log 2 (FC) values of identified pigments, which were deemed differentially accumulated by Student’s t -test (p < 0.05) and the permutation-based false discovery rate (FDR) (p < 0.05) in at least one PpFLOT mutant line in comparison to the WT. (B) The log 2 (FC) of key xanthophyll cycle components, including lycopene, lutein, violaxanthin, β-carotene, and zeaxanthin is shown for Δ PpFLOT-1 and all PpFLOT -OEX lines compared to the WT. Differentially accumulated pigments identified by Student’s t -test and FDR are denoted with an asterisk. It is noteworthy that the lycopene pool was significantly depleted in Δ PpFLOT-1 , PpFLOT -OEX1 and 3 when compared to the WT ( Figure 8A , Supplementary Table 2). β-carotene also showed a slight depletion in PpFLOT -OEX1 and 2 but recovered in PpFLOT -OEX3. The abundance of violaxanthin exhibited only minor changes in the PpFLOT mutant lines compared to the WT ( Figure 8B ). No other differentially accumulated components of the xanthophyll cycle were detected or identified. Interestingly unknown pigments 6, 12, 19, and 25 all showed increased abundance in at least three PpFLOT mutant lines compared to the WT. Identification of these unknown pigments may elucidate the molecular processes responsible for the observed change in culture coloration. It is also interesting to note that with the exception of zeaxanthin, no gradual change in the analyzed pigment levels can be detected since PpFLOT -OEX2 seems to behave differently from PpFLOT -OEX1 and 3. These differences most likely occurred due to a higher variability between the technical replicates and extreme outliers in this group compared to the other PpFLOT mutant lines. Changes in the proteome of PpFLOT -OEX lines convey influence on metabolic pathways in chloroplasts To identify potential proteins that could explain the observed phenotype and altered salt tolerance, a proteomics analysis was carried out. Since the change in protonema cell shape and coloration, as well as the altered salt tolerance, was limited to PpFLOT-OEX lines, the proteomic analysis was carried out for the three PpFLOT -OEX and a WT control. For this analysis 50 mg fresh weight protonema liquid culture of WT control and all PpFLOT -OEX lines were harvested in four replicates and LC-MS was conducted. First analysis of the generated proteomic data confirmed that the increase of PpFLOT transcript in all three PpFLOT -OEX lines translated into correspondingly higher protein levels. The detected log 2 (FC) of PpFLOT abundance in PpFLOT -OEX 1, 2 and 3 lines compared to the WT amounted to 8.12, 8.88 and 9.79, respectively. The statistical analysis of the generated proteomics data was performed with Perseus ( Tyanova et al., 2016 ). To identify statistically significant differentially expressed protein groups (DEP), we performed ANOVA analysis and used the permutation-based FDR of 0.05. A total of 2163 detected protein groups were identified, with 1484 showing significant DE between at least two of the analyzed genotypes (Supplementary Table 3). By visualizing the z-scores of the DEP using a clustered heatmap, a shift in the protein expression correlated with the amount of synthesized PpFLOT ( Figure 9A ) was shown. Cluster one contained proteins whose expression increased with enhanced PpFLOT expression, while the expression of protein groups in cluster three was suppressed ( Figure 9A , Supplementary Table 3). Interestingly, clusters two and four both showed changes in protein abundance in P pFLOT -OEX1 and 2 compared to the WT, while the protein expression profile of PpFLOT -OEX3 was WT like ( Figure 9A , Supplementary Table 3). To identify the molecular functions, cellular components, and biological processes influenced by PpFLOT overexpression, a gene ontology (GO) enrichment analysis was performed using shinyGO v 0.8 ( Ge et al., 2020 ). The majority of affected cellular components are part of the thylakoids and the chloroplasts, supporting the localization of PpFLOT associated to thylakoid membranes. This implies a function during photosynthetic or photosynthesis-related processes, most likely by assisting protein complex assembly ( Figure 9B ). Interestingly among the affected GO-terms in the category molecular function were copper ion binding, structural molecular activity, and oxidoreductase activity, particularly focusing on oxidoreductase activity when acting on CH-OH groups ( Figure 9B ). The detected enriched biological processes also suggest an involvement of PpFLOT in photosynthesis and biosynthesis of carbohydrates, amides, organic acids, and peptides ( Figure 9B , Supplementary Tables 3 and 4). Download figure Open in new tab Figure 9: Proteomics and GO term analysis of all PpFLOT -OEX lines compared to P. patens WT (A) Clustered heatmap of z-score transformed log 2 (LFQ intensities) values of all differentially expressed protein groups (DEP) identified by ANOVA (p < 0.05) and a significant permutation-based false discovery rate (FDR) (q < 0.05). The depicted protein groups are DEP between at least two of the analyzed genotypes. Clusters are numbered and proteins contained in each cluster are listed in Supplementary Table 3. (B - D) Results of the GO term analysis performed by shinyGO ( Ge et al., 2020 ) and sorted after (B) biological processes, (C) cellular components, and (D) molecular functions. Shown are the significant (p < 0.05) GO terms also displaying a significant enrichment (FDR) q < 0.05. The number of detected proteins and the ratio between total genes of a pathway and the detected genes/proteins of that pathways are given. To identify DEP in each PpFLOT -OEX line compared to the WT specifically, we performed a Student’s t -test for each overexpression line. Protein groups with a p-value and a permutation-based FDR of 0.05 and a log 2 (FC) ≤ −1 and ≥ + 1 were identified as DEP (only main IDs shown). This way, we identified 89 DEPs (76 up and 13 down) in PpFLOT -OEX1, 92 in PpFLOT -OEX2 (62 up and 30 down), and 216 in PpFLOT -OEX3 (135 up and 81 down) ( Figure 10A , 10B, Supplementary Table 5). The majority of these DEPs are upregulated in all three overexpression lines, and only a fraction of them are downregulated ( Figure 10C ). Download figure Open in new tab Figure 10: Identification of differentially expressed protein groups in all PpFLOT -OEX lines (A) Volcano plots of all differentially expressed protein groups (DEP) identified by Student’s t -test with p < 0.05 and permutation-based false discovery rate (FDR) q < 0.05 using WT expression as the control group. All DEP exhibited a log 2 (FC) of 1. UniProt protein ID was added to the DEP common in all PpFLOT -OEX lines and either correlated with the PpFLOT expression or showed a log 2 (FC) of 2. Downregulated DEPs are denoted in blue and upregulated DEPs are denoted in red. (B) UpSet plot of all identified DEP in the three PpFLOT -OEX lines. (C) Bar chart showing the amount of up- or downregulated DEP in all three PpFLOT -OEX lines. (D) Bar chart shows 14 common DEP between the three PpFLOT -OEX lines either showing a PpFLOT correlating expression or a log 2 (FC) of 2. DEP: PpTRX-Y2 (A0A2K1ICI4); PpNDUFA2/B8 (A0A2K1IUJ9); PpPME53 (A0A2K1KJF2); PpLHCB2 (A0A2K1KKR9); PpFLOT (A0A2K1KVH5); PpMEE51 (A0A2K1L177); unknown protein (A0A7I4CH40); EF1B-γ GST (A9RWY6); aminoacyl t-RNA ligase (A9RZT5); PpSEN1 (A9SIC2); PpNDUFS8 (A9SIS3); 60S ribosomal protein L36 (A9SJ72); PpHSP70-1 (A9TRK2); PpEXPA9 (Q84V44). However, the number of downregulated DEP increases with the strength of PpFLOT expression ( Figure 10C , Supplementary Table 5). Our analysis shows that PpFLOT -OEX3 displays the largest modification in its proteomics profile with 160 DEP, compared to PpFLOT -OEX1 and PpFLOT -OEX2, which display 36 and 31 DEP, respectively, that are specific to these lines and do not overlap with any of the other two overexpression lines. However, 20 DEP were found to be differentially accumulated in all three lines ( Figure 10 B, Supplementary Table 5). Notably, among these, 14 proteins (including PpFLOT) displayed either a log 2 (FC) ≤ −2 and ≥ + 2 or a correlated DE to the strength of PpFLOT expression ( Figure 10D , Supplementary Table 5). THIOREDOXIN Y2 (PpTRX-Y2) (Pp3c26_10440/A0A2K1ICI4), HEAT SHOCK COGNATE PROTEIN 70-1 (PpHSP70-1) (Pp3c4_21500/ A9TRK2) and one unknown protein (A0A7I4CH40) showed decreased protein levels. TRX-Y2 plays a role in oxidative stress response signaling pathways in chloroplasts ( Geigenberger et al., 2017 ; Wittmann et al., 2021 ), while PpHSP70-1 is a cytosolic HSP70 involved in protein trafficking and maintenance ( Cazalé et al., 2009 ; Shi and Theg, 2010 ; Leng et al., 2017 ). Double mutants of functionally redundant HSP70-1 and −4 in A. thaliana exhibited salt hypersensitivity and ABA hyposensitivity ( Leng et al., 2017 ). Conversely, HSP70-1 overexpression lines showed increased tolerance to abiotic stress, including salinity stress ( Cazalé et al., 2009 ). Therefore, the downregulation of both PpTRX-Y2 and PpHSP70-1 might contribute to the altered salt sensitivity and response to enhanced osmotic pressure in PpFLOT -OEX lines. Additionally, a homolog to A. thaliana PECTIN METHYLESTERASE 53 (PpPME53) (Pp3c5_12660/A0A2K1KJF2) and EXPANSIN A9 (PpEXPA9) (Pp3c12_4560/Q84V44) are both upregulated in all PpFLOT -OEX lines. Since both proteins are involved in restructuring the cell wall ( Li et al., 2002 ; Cosgrove, 2015 ; Gigli-Bisceglia et al., 2022 ), and cell wall integrity is an important factor during salt sensing ( Shin et al., 2021 ; Gigli-Bisceglia et al., 2022 ), these differences in expression might influence the salt sensing capability of the PpFLOT -OEX lines. Besides these cell wall-related proteins, two proteins related to electron transport in mitochondria, NADH DEHYDROGENASE UBIQUINONE IRON-SULFUR PROTEIN 8 (PpNDUFS8) (Pp3c15_4330/A9SIS3) and NADH-ubiquinone oxidoreductase B8 subunit (PpNDUFA2/B8) (Pp3c20_8510/A0A2K1IUJ9) were upregulated, indicating increased respiration due to PpFLOT overexpression ( Klodmann and Braun, 2011 ; Domergue et al., 2022 ). Also, the A. thaliana MATERNAL EFFECT EMBRYO ARREST 51 (MEE51) homolog (Pp3c2_11980/A0A2K1L177) is upregulated. PpMEE51 belongs to the phosphofructokinase family, and its upregulation might indicate an increasingly anoxic environment in the PpFLOT -OEX lines since both ATP-dependent phosphofructokinases (PFK) and pyrophosphate-fructose-6-phosphotransferases (PFP) are upregulated in O. sativa upon anoxia ( Mustroph et al., 2013 ). Furthermore, knockout of PpNDUFS8 homolog in A. thaliana led to increased lipid content, suggesting that its higher expression in P. patens might also lead to changes in the lipid profile ( Domergue et al., 2022 ). An EF1B-γ glutathione S-transferase (GST) (Pp3c18_20060/A9RWY6) is also upregulated. In plants, GSTs are not only involved in the abiotic but also in the biotic stress response ( Gullner et al., 2018 ; Hernández Estévez and Rodríguez Hernández, 2020 ). Interestingly, while our transcript expression analysis in PpFLOT -OEX1 revealed suppression of PpLHCB1 and PpLHCA1.1 , we detected increased levels of PpLHCB2 (Pp3c5_22920/A0A2K1KKR9) in all three PpFLOT -OEX lines. It is noteworthy that PpLHCB2 was the sole PS antenna component that displayed a change in protein level. Additionally, a Rhodanese-containing protein, homologous to A. thaliana SENESCENCE 1 (AtSEN1) also known as DARK INDUCIBLE 1 (AtDIN1) (Pp3c16_18500/A9SIC2), exhibited elevated protein levels. Similar to PpFLOT , AtSEN1 expression is induced in darkness and exhibits increased transcript levels in response to bacterial infection ( Schenk et al., 2005 ; Fernandez-Calvino et al., 2016 ). Furthermore, AtSEN1 participates in senescence ( Fernandez-Calvino et al., 2016 ). Thus, the dependence of PpSEN1 accumulation on PpFLOT suggests a role for PpFLOT in the pathogen response. Overexpression of PpFLOT leads to changes in the fatty acid profile an increases monogalactosyldiacylglycerol (MGDG) accumulation Chloroplasts do not only serve as the site of photosynthesis, but also for multiple metabolic pathways, including fatty acid (FA) biosynthesis ( Block et al., 2007 ; Eberhard et al., 2008 ; Johnson, 2016 ; He et al., 2020 ). Fatty acids are essential building blocks for many metabolic compounds, including the cuticle layer that covers the outer cell wall of P. patens and plasma membrane components ( Renault et al., 2017 ; Resemann et al., 2019 ; Batsale et al., 2021 ). Previous studies have shown that plasma membrane fluidity and cell wall structure are crucial factors during the abiotic stress tolerance, particularly during drought and cold stress of P. patens and A. thaliana ( Bhyan et al., 2012 ; Barrero-Sicilia et al., 2017 ; Batsale et al., 2021 ). Moreover, increased salt concentrations lead to changes in the cell wall composition, which, in turn, activate the salt stress response ( Gigli-Bisceglia et al., 2022 ). Since the salt tolerance of the PpFLOT -OEX lines is severely impaired, the relative conductivity for all PpFLOT -OEX lines and a WT control was measured during cold stress. Observations of the relative conductivity in a temperature range of 0 °C to −7 °C for all tested genotypes revealed that the electrolyte leakage, and in turn, relative conductivity, increased in correlation with increased PpFLOT expression ( Figure 11A ). However, the variances between the measured replicates were too high to make a statistically significant statement on the data distribution. LC-MS analysis of the lipid profile of Δ PpFLOT-1 and all PpFLOT -OEX lines revealed significant changes in their respective lipid levels with 27 differentially accumulated lipid classes in Δ PpFLOT-1 , 13 in PpFLOT -OEX1, 17 in PpFLOT -OEX2 and 26 in PpFLOT -OEX3 (Supplementary Table 6). Interestingly, there was little overlap of the differentially abundant lipid classes among the four PpFLOT mutant lines. Only one lipid class, FA 20:4 (arachidonic acid), displayed significantly altered accumulation in all tested lines ( Figure 11B , Supplementary Table 6). When comparing the PpFLOT -OEX lines, only digalactosyldiglyceride (DGDG) 34:6 and arachidonic acid were found to be differentially abundant in all lines ( Figure 11B , 11C, Supplementary Table 6), with DGDG 34:6 decreasing and arachidonic acid increasing in the three overexpression lines correlating with the level of PpFLOT expression ( Figure 11C ). Similar to the results of the pigment and protein profile analyses, PpFLOT -OEX3 showed the greatest alterations in its lipid profile with 10 additional differentially accumulated lipid classes compared to the other PpFLOT mutant lines. Interestingly, when we looked for lipid classes that display altered levels in at least one PpFLOT -OEX line and show a change in the abundance correlating with the PpFLOT expression, we found one linolenic acid (FA 18:2) derivate displaying an increase correlated with the amount of PpFLOT. Similarly, the FA heptadecane acid (FA 17:0), ethyl linoleate (FA 20:2), heneicosanoic acid (FA 21:0), and triacontanoic acid (FA 30:0) showed their lowest abundance in the PpFLOT -OEX1 and their highest abundance in PpFLOT -OEX3 ( Figure 11C ). In addition to the FA, the glycerophosphoglycerol (PG) PG 34:2, glycerophosphocholines (PC), and phosphatidylethanolamine (PE) displayed similar behavior with the exception of PC 34:6 and PE 36:2, whose levels decreased with increasing PpFLOT expression ( Figure 11C ). Besides decreased DGDG 36:4 levels, MGDG 34:6 and 36:4 also displayed a low abundance. However, MGDG 36:4 shows higher levels in PpFLOT -OEX3 compared to PpFLOT -OEX1 and 2. This, combined with the general increase in the arachidonic acid pool, demonstrates the strongest effect of increasing PpFLOT expression on the lipid profile. These changes in the lipid profiles in the PpFLOT -OEX lines indicate alterations in composition of thylakoid membranes, attributed to variations in the abundance of DGDG and MGDG classes, which are primary constituents of thylakoid membranes. Additionally, the levels of FAs, crucial for cell wall component biosynthesis, potentially impacting the cell wall composition and subsequently, the sensitivity of these lines to abiotic stress, appear to be influenced by PpFLOT expression. Furthermore, the accumulation of arachidonic acid in PpFLOT -OEX plants suggests a potential role in plant defense mechanisms, as low concentrations can induce systemic resistance against pathogens, while high concentrations may lead to necrosis and phytoalexin accumulation ( Dedyukhina et al., 2014 ). Arachidonic acid serves as precursor for oxylipins, oxygenated derivatives involved in plant defense, implying potential alterations in oxylipin biosynthesis and pathogen response, thus highlighting the involvement of PpFLOT not only in abiotic stress responses but also in pathogen defense mechanisms ( Blée, 2002 ). Download figure Open in new tab Figure 11: Analysis of changes in the lipid profile of PpFLOT mutant lines (A) Conductivity measurements of 5 mg WT and PpFLOT -OEX gametophores cultivated for 11 weeks on solid medium at decreasing temperatures. Mean values ± SEM (n =3) of the relative conductivity are shown. (B) UpSet plot depicting the overlap of all identified differentially expressed (DE) lipids between Δ PpFLOT-1 and all PpFLOT -OEX lines compared to the WT. Lipid levels are labeled as differentially accumulated when Student’s t -test using WT expression as control yielded p < 0.05 and the permutation-based false discovery rate (FDR) q < 0.05. (C) Clustered heatmap showing the log 2 (FC) of DE lipids of all PpFLOT -OEX lines. Lipids that show altered abundance in at least one line correlating to the amount of PpFLOT are displayed. PpFLOT -OEX affects grana stack assembly and enlarges the thylakoid lumen Since PpFLOT localizes to the thylakoid membrane and alterations in thylakoid-membrane related proteins were observed in response to changes in the PpFLOT expression, we hypothesized that chloroplast structure might be impacted by PpFLOT overexpression. Transmission electron microscopy (TEM) analysis of chloroplast structures revealed significant deviations in the PpFLOT -OEX lines compared to WT and Δ PpFLOT-1 , characterized by disordered thylakoid membranes, disrupted grana stacks, and enlarged thylakoid lumens ( Figure 12 ). Interestingly, similar thylakoid alterations have been reported under high-light stress in Chlorophyta species and in seed plants during cold stress adaptation and salt treatment ( Gorelova et al., 2019 ; Venzhik et al., 2019 ). Furthermore, examination of cell wall structure revealed a potential increase in cuticle layer density with increasing PpFLOT levels ( Figure 12 ), possibly indicating changes induced by PpFLOT expression, supported by observed alteration in relative conductivity and fatty acid abundance in PpFLOT -OEX lines. Download figure Open in new tab Figure 12: Altered PpFLOT expression leads to changes in the chloroplast thylakoid structure Transmission electron microscopy (TEM) images of complete chloroplasts (upper panel) in protonema cells of Δ PpFLOT-1 , WT and all three PpFLOT -OEX lines, along with close-up images of the cell wall in the respective lines (lower panel). Structural abbreviations include: chloroplast (CP), cytosol (CS), mitochondria (MT), cell wall (CW), plasma membrane (PM), vacuole (V), starch granula (S), plastoglobuli (PG), cuticle layer (CL) and multi vesicular bodies (MVB). Scale bars represent 1 µm (upper panel) and 0.1 µm (lower panel), respectively. Discussion As mosses were among the first plants to adapt to life on land, the segregation of bryophyte FLOT in terms of function and localization from other plant FLOT is intriguing in the context of adapting to the new environment. Our observations suggest that both loss and overexpression of PpFLOT are less detrimental in the leafy gametophore than in the protonema life stage of P. patens , indicating the involvement of PpFLOT in the water-to-land transition. Our findings demonstrate that PpFLOT localizes to the plasma membrane in tobacco leaves, with no detectable presence in chloroplasts. This contrasts with its localization in P. patens and suggests an evolutionary divergence in FLOT targeting between bryophytes and seed plants. The absence of PpFLOT in tobacco chloroplasts raises questions about the molecular mechanisms governing its subcellular distribution, potentially influenced by species-specific regulatory elements such as post-translational modifications or protein-protein interactions. Whether at least one FLOT variant is positioned in the chloroplasts of other bryophyte, fern, or green algae species and what might have caused a shift in localization remains to be seen. To determine the most probable position of FLOT in the last common ancestor of land plants, further investigations in streptophyte algae, bryophytes, and seed plants are required. However, it is suggested that FLOT was acquired through horizontal gene transfer from fungi into ancient plant lineages ( Ma et al., 2022 ), supported by the localization of the fungus FLOT homolog, FloA of Aspergillus nidulans , to plasma membranes ( Takeshita et al., 2012 ). Thus, it is highly likely that a change in FLOT localization was driven by exaptation of FLOT function in P. patens and potentially in other bryophytes. At the protonema stage, we detected altered pigmentation in PpFLOT -OEX lines when grown in liquid culture under normal growth conditions. No such changes were detected in the leafy gametophores grown on solid medium, implying greater importance of FLOT under either submerged conditions or at the developmental stage of protonema cells. Indeed, changes in the cell shape were observed in response to PpFLOT overexpression, reminiscent of ABA-induced brachycytes that develop in P. patens as a survival mechanism in response to abiotic stress conditions ( Arif et al., 2019 ). However, no significant changes were detected in the transcript expression of the ABA biosynthesis rate-limiting enzyme PpNCED ( Hauser et al., 2011 ). The accumulation of ABA levels to counter the increased PpFLOT levels is unlikely to be the only source of these changes, especially since 100 µm ABA is necessary to induce brachycytes ( Arif et al., 2019 ). However, accumulation of PpLHCB2 correlated with increased PpFLOT levels. A study in 2012 showed that altered LHCB expression can influence ABA sensing and signaling ( Xu et al., 2012 ), potentially affecting the ABA response in PpFLOT -OEX lines without impacting the ABA biosynthesis rate. Meanwhile, in M. truncatula , increased expression of MtFLOT2 and 4 is crucial during nodule initiation upon infection with nitrogen-fixing bacteria ( Haney and Long, 2010 ), and artificially activating CCaMK-IPD3 module in P. patens results in constitutively developed brood cells ( Kleist et al., 2022 ). The CCaMK-IPD3 module is activated by symbiotic infection and oscillating calcium signals ( Lévy et al., 2004 ; Miller et al., 2013 ; Kleist et al., 2022 ), suggesting that the formation of round, brachycyte-like cells in response to PpFLOT overexpression may involve slightly increased ABA levels and changes in Ca 2+ signaling. Additionally, slight changes in PpCRY1b expression were detected, and since Ca 2+ waves in P. patens can be induced by light and altered by changes in cryptochrome expression ( Tucker et al., 2005 ), the circadian-regulated putative scaffolding protein PpFLOT might be involved in light-dependent Ca 2+ signaling. External signals such as light-dark cycle, salt, mannitol treatment, and oxidative stress can influence Ca 2+ dynamics between plastids and the cytosol ( Sello et al., 2016 ; Martí Ruiz et al., 2020 ; Navazio et al., 2020 ). It has been proposed that FLOT regulates the positioning of A. thaliana AQUAPORINE PIP1;2 (AtAPIP1;2) by initiating lipid rafts since AtAPIP1;2 colocalizes with AtFLOT1 ( Browman et al., 2007 ; Li et al., 2011 ; Martiniere and Zelazny, 2021 ). Thus, by altering the composition of the thylakoid membrane, FLOT may putatively regulate the positioning of ion channels. For instance, PpFLOT might be involved in altering membrane fluidity or colocalize with Ca 2+ channels, determining their position in thylakoid membranes. Moreover, Ca 2+ transporters might not be the only ion channels affected by this. Our transcript expression analysis of the copper transport and HMA protein Pp3c3_6890 showed a different response to ABA and salt treatment in PpFLOT -OEX1 than in both the WT and Δ PpFLOT-1 . The GO-term analysis of all identified significant protein groups between WT and all PpFLOT -OEX lines also detected an effect of PpFLOT abundance on copper ion binding molecular function. Maintaining Cu homeostasis in chloroplasts is crucial, as Cu is a cofactor for the electron transporter plastocyanin (PCY), polyphenol oxidases (PPO), and Cu/Zinc superoxide dismutase (Cu/ZnSOD). PPO and Cu/ZnSOD participate in biotic and oxidative stress protection, respectively ( Aguirre and Pilon, 2015 ; Printz et al., 2016 ; Schmidt et al., 2020 ). Interestingly, no significant change in the abundance of these Cu-dependent proteins was detected. Nonetheless, the expression levels of other copper transporters similar to Pp3c3_6890 , might be affected, leading to changes in the copper homeostasis in PpFLOT -OEX lines. Further examination is necessary to determine if PpFLOT influences both Ca 2+ signaling and copper transport. As a scaffolding protein, PpFLOT is likely involved in multiple pathways and can oligomerize with multiple proteins ( Garbett and Bretscher, 2014 ; Daněk et al., 2016 ). However, overexpression of PpFLOT impairs the high tolerance of P. patens against salinity and osmolarity stress ( Frank et al., 2005 ), suggesting an additional function as a negative regulator during salt stress response. In response to long-term salt treatment, PpFLOT expression in WT is suppressed, likely due to elevated ABA levels, since ABA is a signaling molecule initiating the abiotic stress response ( Hauser et al., 2011 ) and suppressing PpFLOT expression. Interestingly, PpTRX-Y2 and PpHSP70-1 are both suppressed in correlation to elevated PpFLOT levels. AtHSP70-1 participates in abiotic stress response regulation in A. thaliana ( Cazalé et al., 2009 ; Leng et al., 2017 ), while AtTRX-Y2 is a known regulator of the light-dependent ( Valerio et al., 2011 ; Seung et al., 2013 ; Geigenberger et al., 2017 ) and osmotic stress-induced starch degradation via amylases ( Valerio et al., 2011 ). AtTRX-Y2 is also a crucial component of the antioxidative defense system in chloroplasts ( Geigenberger et al., 2017 ). Hence, suppression of both proteins likely contributes to the observed altered salt tolerance. Although causality between PpFLOT expression and subsequent suppression of these two proteins is currently unknown, we can speculate about their relationship to PpFLOT. For example, HSP-70 proteins in plants drive protein translocation into organelles, including mitochondria and plastids ( Shi and Theg, 2010 ; Berka et al., 2022 ). Downregulation of PpHSP70-1 might be a way for the cells to impede PpFLOT transportation in the chloroplasts of PpFLOT -OEX lines. Furthermore, AtTRX-Y2 reduces antioxidant enzymes, including peroxiredoxins ( Collin et al., 2004 ; Jurado-Flores et al., 2020 ), glutathione peroxidases, and methionine sulfoxide reductases ( Vieira Dos Santos et al., 2007 ; Laugier et al., 2013 ; Vanacker et al., 2018 ). Thus, changes in PpTRX-Y2 levels likely lead to the elevated ROS levels in response to elevated PpFLOT protein expression. Previous studies also showed that the LON DOMAIN-CONTAINING PROTEIN 1 suppresses TRX-Y2 activity and regulates ROS levels by controlling TRX-Y2 activity in A. thaliana ( Shin et al., 2020 ). PpFLOT might putatively activate a similar protein in P. patens, and thus its overexpression leads to suppression of PpTRX-Y2 and increased ROS levels. Upon detection of pathogens, ROS production within the chloroplast increases that activates stress signaling pathways to induce the plant defense against pathogens ( Bleau and Spoel, 2021 ). Overexpression of PpFLOT leads to the accumulation of ROS, which is linked to the pathogen defense response ( Hernández et al., 2016 ; Bleau and Spoel, 2021 ). Therefore, PpFLOT may have a putative role in the pathogen defense. In A. thaliana , treatment with flg22 alters the mobility of AtFLOT1, and AtFLOT1 overexpression increases callose deposition. Flg22 also induces AtFLOT aggregation ( Yu et al., 2017 ; Junková et al., 2018 ). Our findings suggest that the cuticle layer and cell wall composition in the PpFLOT -OEX lines are altered due to an altered fatty acid profile. These changes of the cell wall components may be due to increased PpEXPA9 and PpPME53 accumulation in response to PpFLOT activated or guided pathogen defense mechanisms in P. patens . For example, an increase in Ca 2+ -dependent PME activity can lead to cell wall remodeling during abiotic stress response, and pectin fragments can be used as damage-associated signals ( Shin et al., 2021 ). Moreover, PME activity increases after pathogen treatment in A. thaliana ( Bethke et al., 2014 ). Increased PpFLOT expression not only leads to changes in the cell wall composition, but also to a higher abundance of linoleic acid derivatives and the accumulation of arachidonic acid. Pathogen defense signaling pathways depend on unsaturated FA (UFA), such as linoleic acid derivates ( He and Ding, 2020 ). These UFA function not only as constituents for components of the cuticle layer, such as cutin and suberin, but also as intermediates in the biosynthesis of jasmonates and other active biomolecules of pathogen defense ( Resemann et al., 2019 ; He and Ding, 2020 ). During pathogen defense, one way to counter the rising ROS is the oxidation of C18 UFAs into oxylipins, which themselves are building blocks of jasmonate biosynthesis ( Blée, 2002 ; Resemann et al., 2019 ; He and Ding, 2020 ). The increase in the arachidonic acid pool further supports this hypothesis. Arachidonic acid is an elicitor of plant pathogen defense and depending on its abundance, its accumulation can also lead to either systemic resistance or to accumulation of phytoalexins and necrosis of plant tissues ( Dedyukhina et al., 2014 ). There is a distinct possibility that the detected changes in coloration of PpFLOT -OEX cultures are the result of increased necrotic events. Interestingly, we also detected increased levels of PpSEN1 in all three PpFLOT -OEX lines. SEN1 not only shows increased expression in A. thaliana in response to infection events but also triggers the senescence response resulting in necrosis upon pathogen infection ( Schenk et al., 2005 ; Fernandez-Calvino et al., 2016). A senescent phenotype in P. patens can also lead to changes in the FA composition, including arachidonic acid ( Chen et al., 2020 ). Changes in culture coloration of PpFLOT -OEX lines might thus be attributed to necrosis rather than changes in pigments of the xanthophyll cycle. Even though these pigments displayed an overall low abundance in PpFLOT -OEX lines, changes in coloration due to altered expression of the unidentified pigments are still a distinct possibility. Curiously, no changes in coloration were detected in the gametophores of PpFLOT -OEX lines. We suggest that the increased production of ROS triggers SEN1 accumulation ( Schenk et al., 2005 ) and may cause enhanced hypoxia in chloroplasts ( Pucciariello and Perata, 2021 ), which cannot be compensated by P. patens protonema cells in an already anoxic environment. The molecular changes that enable 3D growth of P. patens may help the plant to counteract this effect. For example, PpFLOT expression in the WT gametophore is lower compared to its expression in rhizoids, caulonema cells and protoplasts (eFP browser, https://bar.utoronto.ca/efp_physcomitrella/cgi-bin/efpWeb.cgi ) ( Winter et al., 2007 ). Other regulators of systemic acquired resistance (SAR) in response to pathogen infection include DGDG and MGDG ( Gao et al., 2014 ). DGDG promotes SAR by regulating NO and salicylic acid synthesis, while MGDG regulates signals downstream of NO-guided SAR, such as azelaic acid and glycerol-3-phosphate ( Gao et al., 2014 ). Although proteomics and metabolomics analysis did not detect any DE of these signals, overexpression of PpFLOT severely altered MGDG and DGDG levels. Therefore, overexpressed MGDG species may be beneficial during the pathogen response of PpFLOT -OEX lines. In addition to its proposed contribution to pathogen defense, PpFLOT seems to be involved in chlorophyll biosynthesis. An increase in PpLHCB2 protein levels was detected in correlation with PpFLOT levels, and the two PpFLOT -OEX lines with the highest PpFLOT and PpLHCB2 expression also showed elevated chlorophyll levels. PpLHCB2 could potentially be a direct interaction partner of PpFLOT, but it is also a chlorophyll-binding molecule, and its overexpression could be due to a need for binding excessive chlorophyll ( Eberhard et al., 2008 ; Johnson, 2016 ). Even slight changes in the expression of one LHCB influence the expression and overall composition of the light-harvesting antenna of PSII ( Xu et al., 2012 ). Furthermore, LHCB expression takes part in regulating both ABA and ROS homeostasis ( Xu et al., 2012 ), and high ABA levels can induce LHCB expression ( Liu et al., 2013 ). Interestingly, ROS is one of the signaling molecules used for plastid to nucleus communication the retrograde signaling ( Eberhard et al., 2008 ; Li and Kim, 2022 ). GLK2 , a gene involved in retrograde signaling and chlorophyll biosynthesis regulation ( Yasumura et al., 2005 ; Kim et al., 2023 ; Lee et al., 2023 ), is upregulated in response to PpFLOT knockout, but downregulated in PpFLOT -OEX lines. This gene acts as a transcription factor for photosynthetic genes, and its knockout in A. thaliana leads to diminished chlorophyll content, while AtGLK2 overexpression results in higher chlorophyll levels ( Yasumura et al., 2005 ; Kim et al., 2023 ). Both knockout and overexpression of PpFLOT lead to changes in the expression of PpGLK2 , suggesting a role of PpFLOT in chlorophyll biosynthesis. However, no changes in the protein levels of known factors participating in chlorophyll biosynthesis could be detected, suggesting a regulating function rather than a direct involvement. Since the chlorophyll content is decreased in Δ PpFLOT-1 and increases with the PpFLOT expression, the anticorrelating expression of PpGLK2 to the respective chlorophyll content suggests an attempt to regulate PpFLOT-dependent changes in the chlorophyll biosynthesis via PpGLK2 . We propose that PpFLOT most likely participates in the recruitment of protein complexes involved in chlorophyll biosynthesis to the thylakoid membrane. Due to the circadian nature of PpFLOT and its inducibility by darkness, one of its functions might contribute to the light-dependent regulation of chlorophyll biosynthesis. For instance, FLUORESCENT IN BLUE LIGHT (FLU) suppresses the chlorophyll biosynthesis in the dark by inactivating the GLUTAMYL-tRNA-REDUCTASE (GluTR) and repressing the 5-aminolevulinic acid (ALA) synthesis ( Meskauskiene and Apel, 2002 ; Kauss et al., 2012 ; Fang et al., 2016 ; Hou et al., 2019 ; Wittmann et al., 2021 ). ALA is an intermediate compound during chlorophyll biosynthesis, and its decline during the night prevents the accumulation of phototoxic products ( Wittmann et al., 2021 ). How GluTR is recruited into a complex together with the membrane-bound FLU and other components of the Mg 2+ branch of the chlorophyll biosynthesis is unclear ( Wittmann et al., 2021 ). Our findings suggest that at least in P. patens , PpFLOT might fulfill such a role. Since, in this case, the activity of FLU would be dependent on PpFLOT abundance, chlorophyll biosynthesis might be suppressed in the PpFLOT -OEX1, leading to the detected lower chlorophyll levels. In this case, both PpFLOT -OEX2 and 3, as well as the ΔPpFLOT-1 lines, might depend on other ways to regulate the chlorophyll synthesis under changing light conditions, leading to higher and lower chlorophyll levels, respectively. However, concrete evidence supporting this hypothesis is still lacking. It is possible that PpFLOT influences chlorophyll biosynthesis in another way. However, altered chlorophyll levels are the most likely explanation for the detected changes in the Y(II) of PpFLOT -OEX1 and 2 and the NPQ/4 of Δ PpFLOT-1 . In Δ PpFLOT-1 , excess energy that cannot be processed due to inefficient chlorophyll levels is redistributed and redirected into non-photochemical quenching ( Eberhard et al., 2008 ). On the other hand, an excess of chlorophyll leads to a higher photorespiration in PpFLOT -OEX lines. This increase in photorespiration most likely also leads to an increase in respiration, supported by the measured increase in PpNDUFS8 and PpNDUFA2/B8 expression. Previous studies showed that the thylakoid lumen enlarges in response to abiotic stresses ( Gorelova et al., 2019 ; Venzhik et al., 2019 ) to increase the travel distance and diffusion rate of electron carriers to the photosystems, thus slowing down the photosynthesis rate ( Mullineaux, 2008 ; Kirchhoff et al., 2011 ; Jarvi et al., 2013 ; Gorelova et al., 2019 ). Such a reaction would also explain the observed lumen enlargement in PpFLOT -OEX chloroplasts. Increased PpFLOT expression most likely contributes to this effect. Detected changes in the lipid composition of thylakoid membranes could also explain the increase in thylakoid lumen and structural changes in these membranes. Thylakoid membranes mainly consist of the glycolipids MGDG, DGDG, and sulfoquinosyl-diacylglycerol (SQDG), with MGDG making up more than 50 % of the total lipid content, while DGDG makes up about 30 % ( Rast et al., 2015 ; Garab et al., 2016 ). Unlike DGDG, MGDG is a non-bilayer lipid that is forced into a bilayer by interaction with LHCII proteins ( Garab et al., 2016 ). PpFLOT may be involved in the localization or transport of PpLHCB2 to the PS II antenna complexes along the thylakoid membranes, which could affect the structure of the thylakoid membrane. Meanwhile, DGDG is the main driver behind the bilayer formation, and changes in the MGDG/DGDG ratio affect both membrane organization and protein complex stability ( Pribil et al., 2014 ; Rast et al., 2015 ; Garab et al., 2016 ). Although the exact mechanism behind increased PpFLOT levels and subsequent changes in thylakoid membrane composition are unknown, it can be assumed that this change in lipid composition contributes to the detected alterations in the thylakoid structure of the PpFLOT -OEX lines. Interestingly, increased levels of MGDG can also lead to a higher photosynthetic activity ( Zhou et al., 2009 ; Pribil et al., 2014 ). One way that PpFLOT could adjust the lipid composition of thylakoid membranes directly is by participating in the formation of stromal vesicles that transport lipids generated in the chloroplast envelope to the thylakoid membranes. Vesicle formation has been observed in proplastids and developing chloroplasts ( Pribil et al., 2014 ; Mechela et al., 2019 ), potentially playing a crucial role in early thylakoid membrane development ( Mechela et al., 2019 ). While AtFLOT1 has been implicated in clathrin-independent endocytosis ( Li et al., 2012 ; Cao et al., 2020 ) and vesicle formation, it is challenging to extrapolate the molecular function of PpFLOT from AtFLOT due to their differing locations. Besides, the absence of evidence for chloroplast-related FLOT in other plant species, coupled with the lack of observable changes in thylakoid structure in Δ PpFLOT- 1, suggests that FLOT may not significantly contribute to chloroplastic vesicle formation outside of P. patens . It is difficult to distinguish between direct and indirect effects of PpFLOT overexpression since no claims can be made about its direct interaction partners. However, this study has revealed a few important points. PpFLOT is more versatile than previously assumed, and while its association to thylakoid membranes may be unique to bryophytes, the differences in the gametophore and protonema phenotype suggest a change in function during the transition of plant terrestrialization. Although the exact function of PpFLOT is still a mystery, this study has shed light on a few pathways in chloroplast metabolism where PpFLOT appears to be crucial. High concentrations of PpFLOT are detrimental during salt adaptations and to cope with elevated osmotic pressure. However, evidence gathered suggests a potential role of PpFLOT in pathogen defense response, chlorophyll biosynthesis, and involvement in Ca 2+ signaling ( Figure 13 ). Download figure Open in new tab Figure 13: Effect of PpFLOT overexpression in protonema cells The expression of PpFLOT is regulated by the circadian clock, ABA, light stimuli, and potentially by miR167. Elevated PpFLOT levels in chloroplasts alter fatty acid biosynthesis, increase chlorophyll biosynthesis, decrease PpHSP70-1 levels, and likely impact ion channel activity or localization. Moreover, high PpFLOT levels increase PpLHCB2 while reducing zeaxanthin and PpTRX-Y2 levels. Changes in fatty acid biosynthesis affect thylakoid membrane composition due to an increase in MGDG and a decrease in DGDG levels. These changes enlarge the lumen and impair grana formation in the thylakoid membrane, enhance photosynthesis, H2O2 production, and possibly Ca 2+ signaling. Elevated photosynthesis affects metabolic processes including respiration, as evidenced by upregulated PpNDUFA2/B8 and NDUFAS8. H 2 O 2 accumulation triggers retrograde signaling increasing the expression of PpEXPA9 and PpPME53 and suppressing the expression of PpCRY1b and PpGLK2 . Increased PpEXPA1 and PpPME53 levels alter cell wall, plasma membrane, and cuticle composition. Together with low levels of PpHSP70-1, PpTRX-Y2, and the overall increased metabolic activity changes in these components are reducing the salt tolerance. Altered fatty acid biosynthesis leads to linoleic acid and arachidonic acid accumulation, potentially contributing to pathogen-associated molecular patterns (PAMP), including the accumulation of PpSEN1, leading to necrotic events. The arrows in the graph denote experiments validating changes in expression levels or protein abundance. Blue: qRT-PCR measurements, red: Proteomics analysis, yellow: Metabolomics analysis, black: PAM and DAB staining. This graphical representation was generated using BioRender. Material and Methods PpFLOT::citrine localization in P. patens protoplasts The PpFLOT transcript (Pp3c3_21910) was tagged with citrine by cloning its CDS into a vector containing the Actin 5 promotor (ACT5_P), citrine sequence with required linkers for the tag (citrine) ( Tian et al., 2004 ; Top et al., 2021 ), and a nopaline synthase terminator (NOS_T). The complete PpFLOT CDS was amplified using the oligonucleotides 5’-AGCTCTCGAGATGGCGTTCCATACCGC-3’ and 5’-TCTAGATCTGGCTTGGGGAAGCTTGG-3’, which generated a PpFLOT sequence flanked by two restriction enzyme sites for Bgl II and Xho I. This sequence was then ligated into a linearized Act5-citrineL vector using T4 ligase (Invitrogen) following manufacturer’s instructions. The generated PpFLOT::citrine construct was transformed into P. patens protoplasts to generate lines that transiently express PpFLOT::citrine . Images of PpFLOT::citrine protoplasts were captured three days after transformation with a Stellaris 5 Point Scanning Confocal Microscope (Leica Microsystems) equipped with a turnable white light laser (485 - 685 nm) and five power hybrid HyD S detectors. The imaging was performed using the immersion oil objective HC PL APO CS2 63x/1.40. Chlorophyll autofluorescence (chl) served as a localization marker, excited at 405 nm and recorded at 623 nm–750 nm while the fluorescent citrine protein (citrine) was excited at 512 nm and recorded at 524 nm–560 nm. Image processing was conducted using the LAS X Office software. PpFLOT::venus localization in N. benthamiana The PpFLOT CDS was cloned into the pHKL0786 vector using Gibson Assembly (NEB), transformed into Escherichia coli , and confirmed by sequencing. For Agrobacterium tumefaciens -mediated expression, Agrobacterium tumefaciens GV3101 containing the pSOUP helper plasmid ( Hellens et al., 2000 ) was co-injected with the 19k vector ( Voinnet et al., 2003 ) and infiltrated as described by Waadt et al. (2014) with minor modifications. The HygR selection marker was replaced with a fast-red selection cassette, which expresses RFP under the control of the Olesin promoter in the seed coat. A. tumefaciens cultures transformed with pHKL0786- PpFLOT::venus and pSoup were electroporated at 1.66–1.9 kV, recovered in LB medium, and plated on selective LB agar for incubation at 28°C overnight. Transformed A. tumefaciens cultures were then used for transient transformation of N. benthamiana leaves. Three days post-transformation, leaf samples were collected for fluorescence imaging. Confocal imaging of leaf tissue was performed using a Stellaris 8 Point Scanning Confocal Microscope equipped with a tunable white light laser (485–685 nm) and an HC PL FLUOTAR L 20x/0.40 DRY objective. Additionally, imaging was also conducted using a Stellaris 5 Confocal Laser Scanning Microscope (Leica, Wetzlar), equipped with a 405 nm diode and a supercontinuum White Light Laser (WLL), with fluorescence detection using a Power Hybrid Detector HyD S. For visualization, the recombinant protein fused to mvenus (YFP) was excited at 514 nm, and emission was recorded between 520–580 nm. Chlorophyll autofluorescence was excited at 405 nm, with emission detected in the 623–813 nm range. In the Stellaris 8 system, chlorophyll autofluorescence was excited at 440 nm and recorded at 670–685 nm, while venus fluorescence was excited at 510 nm and recorded at 539–550 nm. Image processing was conducted using LAS X Office software. Phylogenetic analysis To analyze the evolutionary relationship of PpFLOT to FLOT homologs in other species we performed query searches with the full-length amino acid sequence (aa) of PpFLOT in multiple plant species. Reciprocal searches with the BLASTp function of Phytozome v13 ( phytozome-next.jgi.doe.gov ), UniProt (re_2024_01; uniprot.org ) and MarpolBase (marchantia.info) databases revealed PpFLOT homologs in A. thaliana , A.us officinalis , A. filiculoides , C. purpureus , C. braunii , C. subellipsoidea , K. nitens , M. polymorpha , M. truncatula , O. sativa , S. cucullata , S. polyrhiza , Synechocystis sp. PCC 6803, Z. mays , and Z. marina . The full-length aa sequences of the detected PpFLOT homologs were aligned using CLC Genomics Workbench v20.0.4 (Quiagen), followed by phylogenetic analysis employing the Maximum Likelihood method with 1000 bootstrap replicates and the Jones-Taylor-Thornton method as the substitution model. The phylogenetic tree was generated using the Nearest-Neighbor-Interchange method in MEGA X ( Kumar et al., 2018 ). Generation of PpFLOT knockout and overexpression lines To create Δ PpFLOT lines, exon 2 of the PpFLOT was replaced with a neomycin phosphotransferase II ( nptII ) selection cassette through homologous recombination. A PpFLOT CDS construct with the embedded nptII cassette was generated using Gibson assembly (NEB) (oligonucleotides listed in Supplementary Table 1) and then cloned into pJET1.2/blunt (Thermo Fisher Scientific) vector. After amplification, the construct was cut out of the plasmid via a BglII restriction site, separated from the pJET1.2 backbone by agarose gel electrophoresis and purified. The construct was transformed into P. patens protoplasts, and transformed plants were selected by growing them on medium supplemented with 50 µg/ml G418 sulfate. The correct integration of the nptII cassette was confirmed by amplifying the full-length PpFLOT genomic sequence containing the nptII cassette and the predicted integration sites by PCR (oligonucleotides listed in Supplementary Table 1). Loss of the PpFLOT transcript was confirmed by qRT-PCR using primers located within the PpFLOT CDS (oligonucleotides listed in Supplementary Table 1). To create PpFLOT overexpression lines, the entire PpFLOT CDS was ligated into a vector carrying an ACT5_P sequence, a NOS_T sequence and a hygromycin selection cassette. ACT5_P-controlled PpFLOT CDS fragments encoding additional hygromycin resistance were transformed into P. patens protoplasts. The transformed plants were selected by cultivating them on hygromycin-containing medium and pre-screened by detecting the hygromycin resistance cassette by PCR (oligonucleotides listed in Supplementary Table 1) and by amplifying ACT5_P controlled PpFLOT CDS with primers spanning from the ACT5_P region to the NOS_T region of the newly inserted PpFLOT sequence. The overexpression of PpFLOT was confirmed by amplifying of the PpFLOT transcript from cDNA using Pp_Flot_fwd and Pp_Flot_rev primers (Supplementary Table 1). The strength of the overexpression was determined by qRT-PCR with the primers Flot_qRT_fwd and Flot_qRT_rev (Supplementary Table 1). Confirmation of single integration lines by Southern blot analysis Genomic DNA was extracted following the CTAB DNA extraction protocol as described by Inglis et al. (2018) . From each Δ PpFLOT line, 10 µg DNA was digested overnight with X hoI and NcoI . After digestion the genomic DNA was separated by agarose gel electrophoresis and then transferred to an Hydrobond-N+ membrane (GE Healthcare) using an adapted alkalic desaturating blotting following the capillary method ( Brown, 2001 ). After washing and pre-hybridization of the membrane, the hybridization probe was radioactive labeled with the Prime-a-Gene® Labeling System (Promega). A complementary probe against the nptII selection cassette was generated by PCR with the primers 5’-TCCATCATGGCTGATGCAAT-3’ and 5’-GGCGATACCGTAAAGCACGA −3’ using the nptII containing pUC- nptII vector ( Top et al., 2021 ) as template. After overnight hybridization and subsequent washing of the blot, radiation was detected with phosphor image screen for at least 4 h before scanning the screen with the Typhoon Trio Varible Mode Imager (Amersham Biosciences). The brightness of the image was adjusted with the open-source program ImageJ ( Schneider et al., 2012 ). Plant material cultivation and phenotypic analysis Physcomitrium patens ssp. patens (Hedwig) ecotype “Gransden” 2004 and all generated mutant lines were grown under long-day conditions (16 h light/ 8 h dark) at 23 °C and a light intensity of 85–100 µmol/m 2 s. Axenic cultivation was performed in liquid or on solid medium as previously described ( Frank et al., 2005 ). Liquid cultures were homogenized and transferred to fresh media every 14 d. Plants were cultivated on solid medium supplemented with 250 mM NaCl, 300 mM NaCl, 700 mM mannitol, 10 µM 2- cis ,4- trans -abscisic acid (ABA)(Sigma-Aldrich), 10 µM 1-naphthylacetic acid (NAA) (1mg/ml, Sigma-Aldrich), 10 µM 6-y-y-(dimethylallylamino)-purine (2-ip) (Duchefa), respectively. For the phenotypic analysis, liquid cultures of all lines were adjusted to an equal density of 100 mg dry weight/L, and 5 µl of the generated knockout, overexpression, and WT control lines were spotted on solid medium. The phenotype assay was performed in triplicates and culture growth was observed and documented for 8 weeks. Pictures were taken with a SMZ 1500 stereomicroscope and a DS-U3 camera (Nikon). Growth assay To assess changes in the growth rate between the WT and the generated Δ PpFLOT lines under control conditions and salt treatment, protonema cultures were adjusted to an equal density of 100 mg dry weight/L in liquid medium, with and without supplementation of 250 mM NaCl. Cultures were monitored for 14 d, with dry weight measurements taken every two to three days. Culture pigmentation was documented using a DS-U3 camera (Nikon). These measurements were conducted in three independent experiments. Analysis of circadian expression control of PpFLOT To investigate whether PpFLOT expression is influenced by light or daytime, WT subcultures of 30 mg dry weight protonema were cultivated for three days under different light conditions: long-day 16 h light/8 h dark photoperiod (LD 16h:8h), continuous light (CL), and complete darkness (D). After three days, protonema cultures were harvested at 4 h intervals over a 24 h period (0, 4, 8, 12, 16, 20 and 24 h). Light intensity and temperature conditions were kept to standard cultivation conditions. RNA extraction, sample preparation, and qRT-PCR were performed using EVAGREEN®DYE (Biotium) containing reaction mixes as previously described by Arif et al. (2022) . To detect time-dependent changes in expression and differences between light treatments, normal distribution and equality of variances of the data sets were confirmed using Shapiro-Wilk and Levene tests, respectively, before ANOVA analysis. Statistically significant differences in PpFLOT expression among light treatments and time points were determined using Tukey’s HSD test. Cosinor analysis was additionally performed to determine potential rhythmicity in PpFLOT expression. All statistical analyses were performed in R (v. 4.2.2) ( R Core Team, 2023 ) using the packaged “rstatix” ( Kassambara, 2023b ) and “cosinor2” ( Mutak, 2018 ). Results were considered significant when p < 0.05. Gene expression analysis after ABA and salt treatments Liquid cultures of WT, Δ PpFLOT-1 and PpFLOT -OEX1 were adjusted to a density of 0.4 mg dry weight/ml. The cultures were then divided into three groups per line, untreated control cultures and cultures treated with 10 µM ABA and 250 mM NaCl, respectively. Samples were harvested from each line and treatment in triplicates at 0, 1, 2, 4, 8 and 24 h after treatment. RNA extraction and qRT-PCR were performed as described above and statistical ANOVA analysis was performed to determine statistically significant differences between the analyzed lines under control conditions and after the treatment. When p < 0.05 a Tukey’s HSD test was performed to identify the significantly different lines and the time points of treatment where these differences could be identified. The statistical analysis was conducted with R (v4.2.2) as previously described. MiRNA expression was analyzed using the stem loop PCR method according to Kramer (2011) following the protocol described by Tiwari et al. (2021) adapted for P. patens and using PpEF1α as cDNA synthesis control. Analysis of photosynthetic activity Pulse-Amplitude-Modulation (PAM) fluorometry was used to measure the photosynthetic activity of WT, Δ PpFLOT-1 , PpFLOT -OEX1, PpFLOT -OEX2, and PpFLOT -OEX3 during their gametophore and protonema life stages. For the analysis of photosynthetic activity in the gametophores, liquid cultures of all lines were standardized to a density of 100 mg/L and spotted in triplicate 5 µl spots on solid medium before cultivating them for three months to ensure densely grown moss colonies. Three independent experiments were prepared for the gametophores (n = 9 per analyzed line). To measure the fluorescence in protonema, liquid cultures of all lines were adjusted to a concentration of 1 mg dry weight/ml. Five ml of the liquid cultures were filtered through Miracloth (Merck) tissue and placed into six well plates (n = 9 per analyzed line). All plates were dark adapted for 3 h before PAM measurements were performed with the IMAGING PAM M-Series chlorophyll fluorescence imaging system (Walz GmbH) and the ImagingWinGigE V2.56zn software program. Actinic light of 450 nm (IMAG-Max/L) was used at intensity of 55 µmol/m²s photosynthetically active radiation (PAR) to mimic normal growth conditions and the saturation pulse was given for 240 ms to determine maximal fluorescence after dark-adaptation. After 40 s, the steady-state fluorescence (F) levels were determined. The steady-state F levels were measured at actinic light intensities of 55 µmol/m²s PAR in 20 intervals for 315 s. Since protonema cultures of PpFLOT -OEX3 displayed low fluorescence signals and fluorescence could not be detected with these parameters the measuring light intensity was adjusted, resulting in an actinic light intensity of 60 µmol/m²s PAR. Due to these adjustments, PAM measurements of PpFLOT -OEX3 protonema cultures were not considered during the statistical evaluation. PSII quantum yield [Y(II)] was calculated as (Fm’ -F)/Fm’ ( Genty et al., 1989 ) and the non-photochemical quenching (NPQ/4) as ((Fm-Fm’)/Fm’)/4 ( Kramer et al., 2004 ; Gao et al., 2022 ). To detect statistically significant differences between the analyzed lines, ANOVA analysis was performed, and when p < 0.05 than the statistical analysis was followed up with a Tukey’s HSD test to determine which lines show significant differences. Statistical analysis for all parameters was performed with R (v. 4.2.2) using the “rstatix” ( Kassambara, 2023b ) package, and graphs were generated with R using the “ggpubr” ( Kassambara, 2023a ) package. H 2 O 2 detection by 3,3-Diamonobenzidine staining Accumulation of H 2 O 2 in all mutant lines was analyzed by harvesting 15 ml of WT, Δ PpFLOT-1 , PpFLOT -OEX1, PpFLOT -OEX2, and PpFLOT -OEX3 protonema cultures grown under standard conditions. The harvested protonema was equally divided into samples treated with sterile MilliQ H 2 O (mock), 1 mg/ml 3,3-Diamonobenzidine (DAB) pH 3.8 ( Rea et al., 2004 ), and medium (control) for each line before incubating the samples for 18 h at CL. Subsequently, the solutions of the mock and DAB samples were replaced with 99.9 % ethanol, boiled for 10 min, and stored in fresh ethanol, while the control remained untreated. Microscopy images of mock, DAB and untreated control of all lines were taken with an Axiophot light-microscope (Carl Zeiss Microscopy GmbH). Reddish-brown colorations are indicating the sites of H 2 O 2 accumulation. Evaluation of chlorophyll content To evaluate the chlorophyll content of WT, Δ PpFLOT-1 , and all PpFLOT overexpression lines, 0.4 g protonema fresh-weight was harvested in triplicates for each line and immediately frozen in liquid nitrogen. The harvested plant material was homogenized with metal beads in a TissueLyser™ (Quiagen) to obtain a fine powder, and chlorophyll was extracted by adding 1.5 ml of 80% (v/v) acetone to all samples. The samples were then mixed at 300 rpm for 5 min before centrifugation at 14,000 g for 5 min to obtain cell free chlorophyll solution. Chlorophyll content was determined by measuring the absorbance of the supernatant at 645 and 663 nm. Subsequently, the supernatant was transferred back into the reaction tube containing the cell debris of the respective lines, and the samples were dried in a speed-vac centrifuge at room temperature overnight. Finally, the samples were dried for 2 h at 105°C to obtain the dry weight for all samples. The chlorophyll content was then calculated after the following method: Electrolyte leakage assay To assess electrolyte leakage in WT and all PpFLOT overexpression lines, 5 mg of gametophores grown on solid medium for 8 weeks were collected without damaging the tissue and placed into clean round glass tubes. A clean, adjusted cell sieve was placed into each glass tube without damaging the harvested plant material to serve as physical barrier between the plant material and the electrode of the conductivity meter during measurements. Glass tubes were filled with 5 ml ddH 2 O and incubated in a cryostat for 1 h at 0 °C. Ice formation was induced by introducing a metal wire pre-cooled in liquid nitrogen to the water surface without disturbing the plant material. Samples were returned to the cryostat, and every 30 min, the temperature was decreased by 1 °C. Incubation was stopped for all lines in triplicates at 0, −1, −2, −3, −5, and −7 °C. Samples were left to thaw while gently shaking overnight at 4 °C until all ice crystals were completely dissolved. After adding 5 ml ddH 2 O samples were gently shaken additional 30 min before measuring their conductivity with an inoLab® Cond 7110 (Xylem Inc.) conductivity meter. Samples were autoclaved for 20 min, cooled for 30 min at 4 °C, and then allowed to adjust to room temperature by gently shaking at room temperature for 45 min before measuring total conductivity. The relative conductivity was calculated as follows: Proteomics analysis DE protein expression in PpFLOT -OEX1, PpFLOT -OEX2, and PpFLOT -OEX3 lines was analyzed by generating total protein extracts from 50 mg protonema (fresh weight) for these lines and the WT control in four replicates. Protein extraction and trypsin digestion were performed as described in ( Marino et al., 2019 ), Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed following the protocol described by Espinoza-Corral et al. (2023) with the exception that peptides were separated over a 90 min linear gradient of 5 – 80% (v/v) acetonitrile (ACN). Raw files were processed using the MaxQuant software version 2.1.0.0 ( Cox and Mann, 2008 ), annotating detected peaks against the P. patens reference proteome (UniProt, www.Uniport.org ) using the “match-between-runs” setting. Proteins were quantified via label-free quantification method (LFQ) previously described by Cox et al. (2014) , and subsequent analysis utilized Perseus version 2.0.1.1. ( Tyanova et al., 2016 ; Tyanova and Cox, 2018 ). Contaminants, reverse hits, and proteins identified solely by site modifications were excluded from further analysis. Only protein groups quantifiable by LFQ algorithm in at least three out of four technical replicates of one of the analyzed lines were used. LFQ intensities were log 2 -transformed, and missing values were imputed from a normal distribution using default settings. ANOVA tests in Perseus identified statistically significant protein groups between all tested lines (p < 0.05, permutation-based false discovery rate (FDR) q < 0.05). Z-scores of significant log 2 -transformed LFQ intensities were used to generate a clustered heatmap for easy comparison. GO-term enrichment analysis of differentially expressed protein groups (DEP) was performed using web based shinyGO v0.8 application ( Ge et al., 2020 ). Additional Student’s t -tests between all PpFLOT -OEX lines and the WT control identified significantly differentially expressed protein groups (all p < 0.05, permutation-based FDR q < 0.05 and log 2 (LFQ intensities) + 1). Heatmaps, UpSet plots, and Volcano plots were generated with R v4.3.1 using the “pheatmap” ( Kolde, 2019 ), “UpSetR” ( Gehlenborg, 2019 ) and “ggplot2” ( Wickham, 2016 ) packages, respectively. Metabolite analysis Approximately 50 mg (fresh weight) of liquid nitrogen frozen and pulverized protonema tissue of WT, Δ PpFLOT-1, and all PpFLOT -OEX lines was mixed with 700 µL of basic acetone (acetone: 0.2 M NH 4 OH, 9:1 v/v) containing 2 µl corticosterone (2 mg/mL) per sample as an internal standard, in a 2 ml Eppendorf tube. Samples were stored at −20°C for 20 min with gentle mixing every 5 min, then centrifuged for 10 min at 4 °C at maximum speed. The supernatant was transferred to a new tube, and the pellet was extracted a second time using 300 µl of basic acetone. Both supernatants were combined, mixed, and 750 µL of the mixture was vacuum dried (Concentrator 5301; Eppendorf). Sample preparation was performed under low light, and the dried supernatant was stored at −80°C until further analysis, with argon added to prevent oxidation. The dry pellet was resuspended in 100 µl methanol, and subsequent LC-MS/MS was performed using a Dionex Ultimate 3000 UHPLC with a diode array detector (DAD) (Thermo Fisher Scientific). For pigment analysis, a 5 µl injection volume was separated at a flow rate of 500 µL min-1 on a C30 reversed-phase column (Acclaim C30, 3 µm, 2.1 x 150 mm, Thermo Fisher Scientific) at 15°C. ACN (solution A) and a mixture of methanol and ethyl acetate (50/50; v/v) (solution B) both containing 0.1% formic acid were used to form a solvents gradient. The gradient started with 14.5 % solution B followed by a ramp to 34.5 % solution B within 15 min that was then maintained for 10 min, before returning to 14.5 % solution B with additional 5 min of re-equilibration. Pigments were quantified by DAD. Analysis of the lipid composition was performed by separating 5 µl injection volume at a flow rate of 500 µL min -1 on a C30 reversed-phase column (Acclaim C30, 3 µm, 2.1 x 150 mm, Thermo Fisher Scientific) at 15°C. The solvent used for lipid separation was water (solution C) and an ACN:isopropanol mixture (7:3; v/v) (solution D), both including 1 % ammonium acetate and 0.1 % (v/v) acetic acid. The 26 min gradient started at 55% solution D, followed by a ramp to 99% solution D within 15 min. After a 5 min washing step at 99% solution D, the gradient was returned to 55% solution D and kept constant for 5 min equilibration. For lipid detection, an electrospray ionization (ESI) source was used in positive mode and negative mode to detect fatty acids. Nitrogen was used as the dry gas, at 8 L min −1 , 8 bar, and 200°C. Mass spectra were recorded in MS mode from 50 m/z to 1300 m/z with 40.000 resolution, 1 Hz scan speed, and 0.3 ppm mass accuracy using the timsTOF (Bruker) mass spectra were recorded in MS mode. Compounds were compared to reference standards or annotated in a targeted approach using DAD data as well as the specific mass (m/z) at retention time and the isotopic pattern. Data were acquired by OTOF Control 4.0 and evaluated using DataAnalysis 5.0 and MetaboScape 4.0. All software tools were provided by Bruker. All analyses were performed in four replicates, and extreme outliers were removed while ensuring that at least three replicates per line remained for statistical analysis. To identify significantly enriched or depleted pigments and lipids, log 2 transformation of the DAD data was performed, and the fold change compared to the WT was calculated. Subsequently, statistical analysis was conducted using Perseus v 2.0.1.1. ( Tyanova et al., 2016 ; Tyanova and Cox, 2018 ). A two-sample Student’s t -test with was performed for all generated mutant lines using the WT as the control group. Metabolites were considered statistically significant when p < 0.05, and permutation-based FDR q < 0.05. All presented heatmaps and UpSet plots were generated using R v4.3.1 with the “pheatmap” ( Kolde, 2019 ) and “UpSetR” ( Gehlenborg, 2019 ) packages, respectively. TEM microscopy Protonema cultures of WT, Δ PpFLOT-1 , and all PpFLOT -OEX lines were harvested and fixed for three days in fixation buffer made of 75 mM cacodylate, 2mM MgCl 2 (pH = 7.0) and 2.5 % glutaraldehyde. Subsequently, the samples underwent sequential washing steps in fresh fixation buffer for 5, 20, 40, and 60 min. Following this, samples were incubated for 90 min in fixation buffer supplemented with 1% OsO 4 , followed by washing in fresh buffer for 25 min. After overnight incubation in fresh buffer, the samples underwent additional washing steps in ddH 2 O for 5, 15 and 30 min. The samples were then gradually dehydrated by incubating them for 20 min in 10 % acetone, 20 min in a 20 % acetone/ 1% UrAc mixture, 110 min in 40 % acetone and for 15 min in 60 % acetone, 80 % acetone. Prior to embedding in 100 % resin, the samples were subjected to a final incubation in 100 % acetone in three steps. Ultrathin sections were prepared, and the slides were analyzed using a Zeiss EM912 (Carl Zeiss Microscopy GmbH) equipped with a 2k x 2k Tröndle slow-scan CCD camera (TRS, Tröndle Restlichtverstärkersysteme, Moorenweis, Germany) operated at 80 kV. Author contributions EC, OT and WF designed the research. EC and NN performed the research. SM and H-HK performed confocal microscopy and SS and ML performed LC-MS experiments. Transmission electron microscopy was performed by AK. EC, SS and ML performed bioinformatical analysis of proteomics and metabolomics data. Statistical analysis was performed by EC. All authors analyzed the data. EC, OT and WT wrote the manuscript. All authors reviewed the manuscript. Conflict of interest No conflict of interest declared. Data availability statement All data generated or analyzed during this study are included in this article and its supplementary information. The raw proteomics and metabolomics data will be open to the public at the proteomics identification database (PRIDE) after the acceptance of the manuscript. LIST OF SUPPLEMENTARY TABELS AND FIGURES SUPPLEMENTARY FIGURES Download figure Open in new tab Supplementary Figure 1: Confocal microscopy images of PpFLOT::venus in tobacco leaf cells showing PpFLOT::venus (A, yellow) and chlorophyll (B, red). N. benthamiana leaves. Scale bars are indicated in the respective images. Download figure Open in new tab Supplementary Figure 2: Growth of gametophores from PpFLOT -OEX lines submerged in liquid medium. P. patens WT and all three generated PpFLOT -OEX lines previously grown on solid medium were transferred to 50 ml liquid medium and observed for three months. Cultures were grown with continuous shaking at long day conditions (16 h light, 8 h dark; LD 16:8). Liquid media levels were maintained as needed to keep growing gametophore colonies submerged in the media. Images were captured at 0 d, 21 d, 42 d, and three months after transfer. Scale bar represents 1 cm. Download figure Open in new tab Supplementary Figure 3: Relative gene expression analyses of salt-induced genes and genes encoding components of photosystem I and II under control conditions and after ABA and salt treatment. qRT-PCR results are shown as box blots illustrating the relative gene expression of PpAquaporine TIP (Pp1s44_31V6.1), PpNCED9 (Pp1s412_7V6.1), PpTSPO1 (Pp3c2_17540), PpDHNB (Pp3c5_11880), PpLHCB1 (Pp3c2_35930), PpLHCA1.1 (Pp3c13_14980) and PpPsaD1 (Pp3c16_23780) compared to WT expression at 0 h and normalized against PpEF1α as described by Schmittgen and Livak (2008) . Measurements were conducted over 24 h under control conditions (left) and treated with ABA (middle), and 250 mM NaCl treatment (right). The relative gene expression of the respective genes was measured for Δ PpFLOT-1 , WT and PpFLOT -OEX1 protonema under control conditions in biological triplicates. Statistically significant changes in expression between the three genotypes was determined by ANOVA, with p-values provided in the respective graphs. Significant differential gene expression at a specific time point is indicated by black asterisks. Results of the Tukey’s HSD of the time-dependent expression within the same genotype are marked by red asterisks when significant compared to 0 h of treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Supplementary Table 1: Oligonucleotides used in this study. Supplementary Table 2: Significantly differentially accumulated pigments in all PpFLOT mutant lines compared to the WT. Supplementary Table 3: Results of ANOVA analysis comparing z-scores between WT and all PpFLOT -OEX lines. Supplementary Table 4: Results of the GO-term analysis of all differentially expressed protein groups between at least one tested genotype. Supplementary Table 5: Results of Student’s t -test comparing protein expression of all PpFLOT -OEX lines with the WT. Supplementary Table 6: Significantly differentially accumulated lipids in all PpFLOT mutant lines compared to the WT. Acknowledgments This project was carried out in the framework of MAdLand ( https://madland.science/ , DFG Priority Program 2237). WF and EC are grateful for funding by the Deutsche Forschungsgemeinschaft (DFG; FR 1677/5-1). We also acknowledge help by Peter Geigenberger and David González-Gampo introducing the measurement of electrolyte leakage and technical help by Ioana Miruna Raulea. Funding Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64 , FR 1677/5-1 References ↵ Aguirre , G. , and Pilon , M . 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Share Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens Erika Csicsely , Norina Noor , Susanne Mühlbauer , Hans-Henning Kunz , Serena Schwenkert , Martin Lehmann , Andreas Klingl , Oguz Top , Wolfgang Frank bioRxiv 2025.04.14.648360; doi: https://doi.org/10.1101/2025.04.14.648360 Share This Article: Copy Citation Tools Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens Erika Csicsely , Norina Noor , Susanne Mühlbauer , Hans-Henning Kunz , Serena Schwenkert , Martin Lehmann , Andreas Klingl , Oguz Top , Wolfgang Frank bioRxiv 2025.04.14.648360; doi: https://doi.org/10.1101/2025.04.14.648360 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41936) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15153) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)