Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes fromEuglena gracilisstrain Z

Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes from Euglena gracilis strain Z | 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 Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes from Euglena gracilis strain Z Runa Sakamoto , Koji Kato , Yoshiki Nakajima , Jian-Ren Shen , View ORCID Profile Ryo Nagao doi: https://doi.org/10.1101/2025.06.29.662240 Runa Sakamoto 1 Research Institute for Interdisciplinary Science, Advanced Research Field, and Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , Okayama 700-8530, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Koji Kato 1 Research Institute for Interdisciplinary Science, Advanced Research Field, and Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , Okayama 700-8530, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yoshiki Nakajima 1 Research Institute for Interdisciplinary Science, Advanced Research Field, and Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , Okayama 700-8530, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian-Ren Shen 1 Research Institute for Interdisciplinary Science, Advanced Research Field, and Graduate School of Environmental, Life, Natural Science and Technology, Okayama University , Okayama 700-8530, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ryo Nagao 2 Faculty of Agriculture, Shizuoka University , Shizuoka 422-8529, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ryo Nagao For correspondence: nagry{at}shizuoka.ac.jp Abstract Full Text Info/History Metrics Preview PDF Abstract Euglena gracilis , a phototrophic flagellate, possesses light-harvesting complexes (LHCs) with a pigment composition distinct from that of land plants and green algae, despite notable similarities in LHC polypeptide sequences to green algae. Here, we purified photosystem I-LHCI (PSI-LHCI) supercomplexes from E. gracilis strain Z and characterized their biochemical and spectroscopic properties. The purified complex exhibited a unique pigment profile, notably including diadinoxanthin that is typically found in red-lineage organisms, setting E. gracilis apart from green-lineage organisms. The absorption spectrum displayed the Qy band of chlorophyll a at 675□nm, while the 77-K fluorescence-emission spectrum revealed a prominent peak at 732□nm, closely resembling those in land plants. These features suggest that long-wavelength chlorophylls bound to LHCI may be evolutionarily conserved. Nevertheless, the absence of neoxanthin, lutein, and violaxanthin further differentiates the Euglena LHCIs from other oxyphototrophs. Together, these results illuminate the evolutionary diversification of PSI-LHCI supercomplexes and offer insights into the unique pigment-binding features of the Euglena light-harvesting system. Introduction Photosystem I (PSI) catalyzes the light-driven electron transfer from plastocyanin or cytochrome c 6 at the lumenal side of the thylakoid membrane to ferredoxin at the stromal side in oxygenic photosynthetic organisms ( Brettel and Leibl 2001 ; Fromme et al. 2001 ; Golbeck 1992 ; Nelson and Junge 2015 ). In most species, PSI is associated with light-harvesting complexes (LHCs), forming PSI-LHCI supercomplexes that facilitate efficient excitation energy transfer. These pigment-protein assemblies vary considerably in both protein composition and pigment content across photosynthetic lineages ( Croce and van Amerongen 2013 , 2020 ; Hippler and Nelson 2021 ; Shen 2022 ). Euglena gracilis , a phototrophic flagellate derived from secondary endosymbiosis ( Novák Vanclová et al. 2020 ; Turmel et al. 2009 ), possesses an unusual carotenoid (Car) profile. Its LHCs contain diadinoxanthin and diatoxanthin—Cars typically found in red-lineage organisms such as diatoms and haptophytes ( Falkowski et al. 2004 )—while lacking lutein, violaxanthin, and zeaxanthin, which are common in green-lineage phototrophs such as land plants and green algae ( Casper-Lindley and Björkman 1998 ; Cunningham Jr. and Schiff 1986; Kato et al. 2017 ; Nagao et al. 2021 ). Despite these differences, the LHC polypeptides of E. gracilis exhibited notable similarity to those of green algae ( Houlné and Schantz 1988 ; Muchhal and Schwartzbach 1992 ), suggesting a conserved evolutionary origin. However, the PSI-LHCI supercomplex from E. gracilis remained unpurified, and its molecular and functional characteristics remained unexplored. In this study, we successfully purified PSI-LHCI supercomplexes from Euglena gracilis strain Z and characterized their biochemical and spectroscopic properties. The complex was found to contain diadinoxanthin as a major Car component, clearly distinguishing it from PSI-LHCI supercomplexes in land plants and green algae. The absorption and 77 K fluorescence spectra revealed unique features, including a long-wavelength emission peak at 732 nm, suggesting the presence of conserved LHCI-bound chlorophyll (Chl) species. These results shed light on the evolutionary adaptations of light-harvesting systems in E. gracilis and expand our understanding of PSI-LHCI diversity across photosynthetic eukaryotes. Materials and methods Cell culture and thylakoid preparation Euglena gracilis strain Z (hereafter referred to as Euglena ) was cultured in a Cramer-Myers medium ( Cramer and Myers 1952 ) supplemented with 1/1000 volume of KW21 (Daiichi Seimo) at 30 °C under continuous aeration and a photosynthetic photon flux density of 30 µmol photons m −2 s −1 ( Nagao et al. 2021 ). Harvested cells were pelleted by centrifugation and resuspended in buffer A (20 mM Mes-NaOH (pH 6.5), 0.2 M trehalose, 5 mM CaCl 2 , and 10 mM MgCl 2 ). Cell disruption was performed using glass beads under dark and cold conditions on ice, employing 19 cycles of 10□s agitation and 3□min rest ( Nagao et al. 2017 ). Unbroken cells were removed by centrifugation at 3,000□×□g for 10□min at 4 °C. The resultant pellet was subjected to a second round of disruption under the same conditions, followed by centrifugation again at 3,000□×□g for 10□min at 4 °C. The supernatant was then centrifuged at 125,000□×□g for 20□min at 4 °C to isolate thylakoid membranes, which were resuspended in buffer A. Chl concentrations were determined in 100% methanol ( Porra et al. 1989 ). Purification of PSI-LHCI supercomplexes All purification steps were performed at 4 °C unless otherwise stated. Thylakoid membranes were solubilized in the dark with 1% (w/v) sucrose monolaurate (SM; Carbosynth) at a Chl concentration of 0.25□mg□mL −1 by gentle stirring for 20□min on ice. After centrifugation at 100,000□×□g for 20□min, the supernatant was applied to a HiTrap Q HP column (5□mL; Cytiva) equilibrated with buffer B (20□mM Mes-NaOH (pH 6.5), 0.2□M trehalose, and 0.03% SM). The column was washed with buffer B at a flow rate of 1.0□mL□min −1 until the eluate became colorless. Proteins were eluted using the following gradient: 0–5□min, 10% buffer C (buffer B with 500□mM NaCl); 5–35□min, 10–60% buffer C; 35–55□min, 60–100% buffer C; and 55–75□min, 100% buffer C ( Figure 1A ). The peak labeled as 1 ( Figure 1 ) was collected and loaded onto a 10–40% linear trehalose gradient prepared in 20□mM Mes-NaOH (pH 6.5), 10□mM NaCl, and 0.03% SM. After centrifugation at 154,000□×□g for 18□h (P40ST rotor; Hitachi), the green band (indicated by a red arrow in Figure 1B ) was harvested, concentrated using a 150□kDa cut-off filter (Apollo; Orbital Biosciences), and stored in liquid nitrogen. Download figure Open in new tab Figure 1. Purification of the Euglena PSI-LHCI. A , Elution profile by anion exchange chromatography. A peak labeled as 1 was collected. B , Trehalose density gradient centrifugation profile of the peak 1. A green fraction marked as a red arrow was collected as the Euglena PSI-LHCI supercomplex. SDS-PAGE Protein composition was analyzed by SDS-PAGE as described by Ikeuchi and Inoue (1988) . PSI-LHCI samples were solubilized in 3% lithium lauryl sulfate and 75□mM dithiothreitol at 60 °C for 10□min, and then applied to a 16% polyacrylamide gel containing 7.5□M urea. A molecular weight marker (SP-0110; APRO Science) was used. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250. Pigment analysis Pigments associated with PSI-LHCI were analyzed by HPLC as described previously ( Nagao et al. 2020 ). Pigments were extracted in 100% methanol and separated using a flow rate of 0.9□mL□min −1 . The mobile phases consisted of solvent A (methanol:acetonitrile:0.25 M pyridine = 50:25:25 (v:v:v)) and solvent B (methanol:acetonitrile:acetone = 20:60:20 (v:v:v)) ( Zapata et al. 2000 ). Pigments were identified by their absorption spectra and retention times ( Taniguchi and Lindsey 2021 ; Zapata et al. 2000 ). Spectroscopic measurements Absorption spectra were measured at room temperature using a spectrophotometer (UV-2450; Shimadzu). Fluorescence-emission spectra were recorded at 77 K using a spectrofluorometer (RF-5300PC; Shimadzu). Each of the spectra was averaged and normalized. Results The PSI-LHCI supercomplexes from E. gracilis were purified by anion-exchange chromatography followed by trehalose density gradient centrifugation ( Figure 1 ). The polypeptide composition of the purified complexes was examined by SDS-PAGE ( Figure□2 ). Multiple bands were observed, including a prominent band (marked with an asterisk) corresponding to PsaA/B, as inferred from its apparent molecular weight between 45.0 and 66.4 kDa. Additional bands within the 14.3–29.0 kDa range likely represent PSI and LHCI subunits, and the overall band pattern resembled those reported for PSI-LHCI from land plants ( Croce et al. 1996 ; Dunahay and Staehelin 1985 ) and green algae ( Germano et al. 2002 ; Qin et al. 2015 ). Download figure Open in new tab Figure 2. Polypeptide composition of the Euglena PSI-LHCI. The Euglena PSI-LHCI was subjected to SDS-PAGE and its protein profile showed a typical PSI protein band of PsaA/B marked as an asterisk. The pigment composition of the Euglena PSI-LHCI was analyzed by HPLC ( Figure□3 ). Four pigments—diadinoxanthin, Chl b , Chl a , and β-carotene—were detected. Diadinoxanthin appeared to be the major Car, consistent with earlier analyses of isolated Euglena LHCs ( Cunningham Jr. and Schiff 1986 ). The Car composition differed markedly from that of PSI-LHCI in land plants and green algae, whose LHCs lack diadinoxanthin ( Klimmek et al. 2005 ; Qin et al. 2015 ; Schmid et al. 2002 ; Shen 2022 ). Download figure Open in new tab Figure 3. HPLC profile of pigments extracted from the Euglena PSI-LHCI. The HPLC chromatogram of the Euglena PSI-LHCI was monitored at 440 nm. Ddx, diadinoxanthin; b , Chl b ; a , Chl a ; β-Car, β-carotene. The absorption spectrum of Euglena PSI-LHCI measured at room temperature showed the Qy band of Chl a at 675□nm and additional peaks attributable to Chls and Cars in the 400–500□nm region ( Figure□4 ). Compared with PSI-LHCI from Spinacia oleracea ( Qin et al. 2006 ) and Chlamydomonas reinhardtii ( Drop et al. 2011 ), the Euglena complex exhibited relatively lower absorbance in the 450–500 nm range, likely reflecting differences in Car composition, particularly the absence of lutein and neoxanthin. Furthermore, the Qy peak of Chl a in the Euglena PSI-LHCI appeared at a shorter wavelength (675□nm) than those in S. oleracea and C. reinhardtii (679□nm) ( Drop et al. 2011 ; Qin et al. 2006 ), implying differences in Chl composition and binding properties. Download figure Open in new tab Figure 4. Absorption spectrum of the Euglena PSI-LHCI. The absorption spectra of the Euglena PSI-LHCI were measured at room temperature, and then averaged and normalized to the intensity of the Qy band of Chl a . The 77-K fluorescence-emission spectrum of Euglena PSI-LHCI displayed a maximum peak at 732 nm and a broad shoulder around 677 nm ( Figure□5 ). The 732-nm peak closely matches that observed in PSI-LHCI from land plants ( Drop et al. 2011 ; Qin et al. 2006 ), but differs from the 714-nm peak characteristic of C. reinhardtii ( Le Quiniou et al. 2015 ; Turmel et al. 2009 ). As the long-wavelength fluorescence near 730□nm originates from LHCI-bound Chls in land plants ( Croce et al. 1998 ; Lam et al. 1984 ; Pålsson et al. 1995 ; Qin et al. 2006 ), it is plausible that the 732-nm emission in Euglena also arises from LHCI-associated Chls. In contrast, the 677-nm emission may originate from higher-energy Chls within LHCIs and/or dissociated LHCIs, consistent with the fluorescence maximum of isolated LHCIs from S. oleracea , which peaked at 675–680 ( Lam et al. 1984 ; Qin et al. 2006 ). Download figure Open in new tab Figure 5. Fluorescence emission spectrum of the Euglena PSI-LHCI. The fluorescence emission spectra of the Euglena PSI-LHCI were measured at 77 K upon excitation at 430 nm, and then averaged and normalized to the maximum-peak intensity. Discussion In this study, we encountered considerable difficulty in isolating thylakoid membranes from Euglena gracilis . Conventionally, thylakoid membranes are obtained by pelleting the supernatant following low-speed centrifugation after glass bead disruption (see Materials and Methods). However, in Euglena , although thylakoid membranes were successfully solubilized, the extracted protein complexes consistently aggregated during anion-exchange chromatography and trehalose density gradient centrifugation. In contrast, reproducible isolation of PSI-LHCI was achieved when the initial low-speed pellet was subjected to an additional round of glass bead disruption. Although the mechanistic basis remains unclear, Euglena cultured under our conditions required this modified approach for successful thylakoid membrane preparation. The Car composition of the Euglena PSI-LHCI was markedly distinct from that of land plants and green algae ( Figure 2 ). In particular, the presence of diadinoxanthin is notable, as this Car is typically found in red-lineage organisms. Conversely, neoxanthin, lutein, and violaxanthin—dominant in the LHCs of green-lineage organisms—were absent. Given that the LHC proteins of Euglena share sequence similarity with those of green algae ( Houlné and Schantz 1988 ; Muchhal and Schwartzbach 1992 ) and are classified within the LHC protein superfamily ( Engelken et al. 2010 ; Sturm et al. 2013 ), a degree of structural conservation is expected. Nevertheless, the divergent Car profile raises important questions about the mechanisms underlying Car binding specificity in Euglena LHCs. The 77-K fluorescence-emission spectrum of Euglena PSI-LHCI exhibited a maximum at 732 nm ( Figure□5 ), closely resembling that of S. oleracea ( Qin et al. 2006 ) yet distinct from the 714-nm peak characteristic of C. reinhardtii ( Le Quiniou et al. 2015 ; Turmel et al. 2009 ). In other plant species, major long-wavelength fluorescence peaks near 750 nm have also been reported ( Li et al. 2024 ). Structural analyses have attributed such long-wavelength emission to Chls bound within LHCI ( Li et al. 2024 ), raising the possibility that the 732-nm-emitting Chls in Euglena are likewise associated with LHCIs. In summary, the presence of diadinoxanthin and the absence of canonical green-lineage Cars underscores the distinct pigment composition of the Euglena PSI-LHCI, highlighting its divergence from typical land plant and green algae. Nevertheless, the 732-nm fluorescence emission implies a degree of functional conservation with LHCI in land plants. Despite the similarity in LHC protein sequences to green algae, the unique Car profile in Euglena suggests independent adaptations in pigment-binding properties. These findings broaden our understanding of PSI-LHCI diversity and offer new perspectives on the structural and pigment-binding variations in photosynthetic light-harvesting systems. Author Contributions R.N. conceived the project; R.S. performed isolation and characterization of the PSI-LHCI supercomplexes; K.K, Y.N., J.-R.S., and R.N. provided laboratory resources and supervised experimental work; R.N. drafted the original manuscript; J.-R.S. modified the manuscript; and R.N. wrote the final manuscript, and all authors joined the discussion of the results. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We thank Kumiyo Kato and Satoko Kakiuchi for helpful assistance in this study. 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Share Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes from Euglena gracilis strain Z Runa Sakamoto , Koji Kato , Yoshiki Nakajima , Jian-Ren Shen , Ryo Nagao bioRxiv 2025.06.29.662240; doi: https://doi.org/10.1101/2025.06.29.662240 Share This Article: Copy Citation Tools Purification and pigment analysis of diadinoxanthin-binding PSI-LHCI supercomplexes from Euglena gracilis strain Z Runa Sakamoto , Koji Kato , Yoshiki Nakajima , Jian-Ren Shen , Ryo Nagao bioRxiv 2025.06.29.662240; doi: https://doi.org/10.1101/2025.06.29.662240 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 (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41910) Biophysics (21436) Cancer Biology (18576) Cell Biology (25480) Clinical Trials (138) Developmental Biology (13368) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40360) Molecular Biology (17163) Neuroscience (88534) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)