Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates inArabidopsis thaliana

Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana | 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 Downstream metabolites of (+)- cis -12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana Rina Saito , Yuho Nishizato , Tsumugi Kitajima , Misuzu Nakayama , Yousuke Takaoka , Nobuki Kato , View ORCID Profile Minoru Ueda doi: https://doi.org/10.1101/2024.05.01.592109 Rina Saito 1 Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yuho Nishizato 2 Department of Chemistry, Graduate School of Science, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tsumugi Kitajima 1 Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Misuzu Nakayama 2 Department of Chemistry, Graduate School of Science, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yousuke Takaoka 2 Department of Chemistry, Graduate School of Science, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nobuki Kato 2 Department of Chemistry, Graduate School of Science, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Minoru Ueda 1 Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University , Sendai 980-8578, Japan 2 Department of Chemistry, Graduate School of Science, Tohoku University , Sendai 980-8578, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Minoru Ueda For correspondence: minoru.ueda.d2{at}tohoku.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract (+)- cis -12-oxo-phytodienoic acid ( cis -OPDA) is a biosynthetic precursor of the plant hormone (+)-7- iso -jasmonoyl-L-isoleucine (JA-Ile). It functions as an endogenous chemical signal independent of the JA-Ile receptor COI1-JAZ in Arabidopsis thaliana . The bioactive form of cis -OPDA that induces COI1-JAZ-independent gene expression remains unknown. In this study, we hypothesized that the genuine bioactive forms of cis -OPDA are the downstream metabolites, which upregulate the expression of the OPDA marker genes such as ZAT10 / ERF5 in a JA-Ile-independent manner. These downstream metabolites function independently of the JA-Ile-COI1-JAZ-MYCs canonical jasmonate signaling module, and its electrophilic nature is essential for its bioactivity. Introduction Jasmonic acid and related fatty acid-derived oxylipins are collectively referred to as jasmonates. Jasmonate, (+)-7- iso -jasmonoyl-L-isoleucine (JA-Ile; Fig. 1 ), is a lipid-derived plant hormone that regulates growth, reproduction, and defense responses against pathogens and chewing insects 1 - 3 . In plant cells, JA-Ile is synthesized from α-linolenic acid via the biosynthetic precursor (+)- cis -12-oxo-phytodienoic acid ( cis -OPDA) ( Fig. 1A ), which is reduced to OPC-8 by OPDA reductase 3 (OPR3), followed by oxidation to naturally occurring (+)-7- iso -jasmonic acid (JA, a cis -form) through three rounds of β-oxidation and conjugation with L-isoleucine (Ile) by GH3 enzyme jasmonic acid-resistant 1 (JAR1) or At GH3.10 to produce JA-Ile 4 - 8 . Recently, an OPR3-independent route for JA-Ile biosynthesis from cis -OPDA was reported ( Fig. 1B ) 9 . In this bypass route, three rounds of β-oxidation occurred from cis -OPDA to dinor- cis -OPDA (dn- cis -OPDA), tetranor- cis -OPDA (tn- cis -OPDA), and (+)-7- iso -4,5-didehydro JA (4,5-ddh-JA), which is then reduced to JA by OPR2 ( Fig. 1B ). An increase in JA-Ile levels in plant cells leads to the triggering of protein-protein interactions between the F-box protein CORONATINE INSENSITIVE 1 (COI1) and jasmonate-ZIM domain (JAZ) repressors, causing proteasomal degradation of JAZ to derepress transcription factors, such as MYC2, and activating gene expression 10 - 13 . Download figure Open in new tab Fig. 1. Biosynthetic pathway of JA-Ile in planta . (A) Canonical OPR3-dependent synthesis of JA-Ile. In this route, cis -OPDA is reduced to OPC-8 by OPR3, followed by oxidization to JA through three β-oxidation and conjugation with Ile by JAR1. (B) OPR3-independent synthesis of JA-Ile. Through three β-oxidation, cis -OPDA is oxidized to 4,5-ddh-JA, which is then reduced to JA by OPR2. Several reports have demonstrated JA-Ile-independent biological activities of the biosynthetic intermediate cis -OPDA in Arabidopsis thaliana , tomato, and maize 14 - 23 . Taki et al . demonstrated cis- OPDA-specific expression of genes, including genes encoding a transcription factor involved in salt-tolerance zinc finger ( ZAT10 ), ethylene-responsive transcription factor ( ERF5) , dehydration-responsive element binding protein ( DREB2A ), cold-responsive zinc finger ( ZAT12 ), and iron deficiency-induced transcription factors ( FIT1 ) by comparing the gene expression mediated by cis -OPDA and JA 16 . ZAT10 encodes a transcription factor (TF) involved in salt tolerance 16 , and ERF5 encodes an ethylene-responsive TF involved in wounding and cold stress responses 24 . DREB2A encodes a TF involved in drought and salt stress 25 , 26 . ZAT12 encodes a TF in cold stress and salt tolerance 27 , 28 . FIT1 encodes a TF in iron acquisition 29 . Biochemical, yeast two-hybrid (Y2H), and pull-down assays demonstrated that cis -OPDA is not recognized by Arabidopsis COI1-JAZ1/3/9 co-receptor pairs 12 , 30 , implying that cis -OPDA functions via a COI1-independent mode of action (MOA). However, in previous studies on cis -OPDA in A. thaliana , their biological activities have been distinguished from those of JA-Ile using the Arabidopsis mutants opr3-1 and jar1-1 , in which the biosynthetic genes of JA-Ile ( OPR3 or JAR1 ) are impaired 31 . This could be because cis -OPDA was not converted into JA-Ile in opr3-1 and jar1-1 . Nevertheless, recent studies have indicated that cis -OPDA can be converted into JA-Ile in opr3-1 or jar1-1 through OPR3-independent ( Fig. 1B ) 9 , 32 or JAR1-independent 33 bypass routes owing to the leaky nature of these mutant lines. As a result, the participation of cis -OPDA in JA-Ile-independent signaling pathways in A. thaliana remains debatable 34 , 35 . To date, the COI1-independent MOA of cis -OPDA has been reliably reported only in the thermotolerance of Marchantia polymorpha , in which the electrophilic α,β-unsaturated cyclopentenone moiety of cis -OPDA plays an essential role 36 . In this paper, we investigated the function of cis -OPDA in A. thaliana and the non-leaky mutant lines coi1-1 37 and opr2-1 opr3-3 9 ( Fig. 1 ) with impaired JA signaling and biosynthesis to identify the genuine bioactive form of cis -OPDA in A. thaliana . Results Analyses of cis -OPDA-induced gene expression in A. thaliana and the mutant lines on JA signaling cis -OPDA ( Fig. 1 ) was chemically synthesized using previously reported methods 38 , 39 . The subsequent experiments were performed using synthetic cis -OPDA. Gene expression analysis was performed based on the information from a previously reported comprehensive DNA microarray analysis 16 : the impact of (-)-JA (Fig. S1) or cis -OPDA was respectively evaluated on the expression of three JA marker genes, OPR3, JAZ8 , and MYC2 ( Fig. 2A-C ), and five cis -OPDA-specific marker genes, ZAT10, ERF5, DREB2A, ZAT12 , and FIT1 ( Fig. 2D-H ) 16 , 31 . Download figure Open in new tab Fig. 2. Validation of COI1-independence of cis -OPDA. ( A to H ) Gene expression analysis by RT-qPCR in 10-day-old WT (Col-0, white bar) and coi1-1 mutant (gray bar) treated with 30 μM (-)-JA or cis -OPDA for 30 min and with no treatments (mock). The data are presented as mean ± SD (n = 3–4). Samples were normalized to the UBQ10 level. Significant differences were evaluated by the ANOVA/Tukey Kramer test ( p < 0.05). The experiments were repeated three times with similar results. ( I ) Evaluation of the affinity between cis -OPDA and COI1-JAZs. Pull-down assay of GST- At COI1 with Fl- At JAZPs in the presence of JA-Ile (1 μM) or cis -OPDA (1/30 μM). Fl- At JAZ13 was used as a negative control because JAZ13 has no canonical JAZ degron sequence, which is necessary for JA-Ile perception. GST- At COI1 bound to Fl- At JAZPs was pulled down with anti-fluorescein antibody and Protein G magnetic beads and analyzed by immunoblotting (anti-GST-HRP conjugate for detection of GST- At COI1). Arrowheads indicate GST- At COI1 (95 kDa). Experiments were repeated three times with similar results (shown in Fig. S2 and S3 ). All compounds induced the expression of JA marker genes, OPR3, JAZ8 , and MYC2 in Col-0, and their expression was impaired in coi1-1 ( Fig. 2A-C ). cis -OPDA induced the expression of OPR3, JAZ8 , and MYC2 in a COI1-dependent manner ( Fig. 2A-C ). In contrast, complex effects were observed regarding ZAT10 expression, which was induced by all compounds in Col-0 ( Fig. 2D ). In the coi1-1 mutant, ZAT10 expression was not induced by (-)- JA, whereas moderate ZAT10 expression was observed for cis -OPDA treatment ( Fig. 2D ). These findings imply that the expression of ZAT10 by (-)-JA is COI1-dependent, whereas that of cis -OPDA depends on two distinct pathways, COI1-dependent and COI1-independent. Additionally, the expression of ERF5, DREB2A, ZAT12 , and FIT1 by cis -OPDA was not or slightly affected in coi1-1 , confirming COI1-independence ( Fig. 2E-H ). In all experiments, cis -OPDA affected the expression of JA and OPDA marker genes. In addition, cis -OPDA showed no affinity for the functional COI1-JAZ1-6/9-12 co-receptor pairs in the pull-down assay ( Fig. 2I and S2 and S3), confirming that cis -OPDA is not ligands for functional COI1-JAZ co-receptors. We also examined the effect of cis -OPDA on the expression of JA and OPDA marker genes in the myc2myc3myc4 triple mutant, in which the master TFs of JA signaling, MYC2/MYC3/MYC4, were impaired 40 (Fig. S4). The expression of the JA marker genes OPR3 and JAZ8 was suppressed in myc2myc3myc4 (Figs. S4A and B). Different effects were observed for the five OPDA marker genes, ZAT10, ERF5, DREB2A, ZAT12 , and FIT1 ; expressions of ZAT10 and DREB2A were moderately suppressed, and that of ERF5, ZAT12 , and FIT1 were enhanced or not affected (Fig. S4C-G), respectively. The current results demonstrated that cis -OPDA mediated the expression of all OPDA marker genes independently of the canonical COI1-JAZ-MYC signaling pathway. cis -OPDA induces the expression of OPDA-marker genes independent of the conversion into JA-Ile Given that cis -OPDA is a major biosynthetic precursor of JA-Ile, we examined whether cis -OPDA-induced gene expression depends on the in planta conversion into JA-Ile. This was investigated using the synthesized cis -OPDA- d 5 ( Fig. 3A and Scheme S1). The conversion of cis -OPDA into JA-Ile was examined using Arabidopsis opr2-1opr3-3 double mutant with impaired conversion of cis -OPDA into JA-Ile 9 . UPLC-MS/MS analysis demonstrated that cis -OPDA- d 5 was converted into JA- d 5 -Ile within 30 min in Col-0, whereas this conversion was suppressed in opr2-1opr3-3 ( Fig. 3A ). For the JA marker gene, cis -OPDA-induced expression of JAZ8 in Col-0 was significantly suppressed in opr2-1opr3-3 ( Fig. 3B ). In contrast, (-)-JA-mediated JAZ8 expression in Col-0 cells was not affected by opr2-1opr3-3 ( Fig. 3B ). Additionally, cis -OPDA-induced expression of ZAT10 in Col-0 was not suppressed in opr2-1opr3-3 ( Fig. 3C ). The expression of ERF5, DREB2A, ZAT12 , and FIT1 was not affected or suppressed in opr2-1opr3-3 ( Fig. 3D-3G ). The undetectable level of JA-Ile in opr2-1opr3-3 in Fig. 3A 9 implies that cis -OPDA induces the expression of OPDA maker genes independently of their conversion to JA-Ile. Download figure Open in new tab Fig. 3. Gene expression analyses induced by cis -OPDA in opr2-1opr3-3 . ( A ) Schematic diagram of the experimental design assessing the conversion of cis -OPDA to JA-Ile in Col-0 or opr2-1opr3-3 by using deuterium-labeled precursors ( cis -OPDA- d 5 ); accumulation (pmol/fresh weight (g)) of JA- d 5 -Ile in 10-day-old WT (Col-0) and opr2-1opr3-3 mutant treated with 30 μM cis -OPDA- d 5 for 30 min. The data are presented as mean ± SD (n = 4). The experiments were repeated three times with similar results. ( B to G ) Gene expression analysis by RT-qPCR in 10-day-old WT (Col-0, white bar) and opr2-1opr3-3 mutants (blue bar) treated with 30 μM (-)-JA or cis -OPDA for 30 min and with no treatments (mock). The data are presented as mean ± SD (n = 3–4). Samples were normalized to the UBQ10 level. Significant differences were evaluated by the ANOVA/Tukey Kramer test ( p < 0.05). The experiments were repeated three times with similar results. Fig3D should be reexamined! The Arabidopsis cis -OPDA transporter mutant and metabolism showed that cis -OPDA is not a genuine bioactive form cis -OPDA is converted to JA in the peroxisomes, and the peroxisomal ATP-binding cassette (ABC) transporter COMATOSE (CTS) is involved in the import of cis -OPDA into the peroxisomes in A. thaliana ( Fig. 4A , left) 35 . Then, we examined whether cis -OPDA itself is a genuine bioactive form by using the Arabidopsis cis -OPDA transporter mutant. In the cts1 mutant line, in which CTS is impaired, wound-induced accumulation of JA-Ile was significantly suppressed but not completely abolished 41 - 44 . In the cts1 mutant line, the level of cis -OPDA was significantly increased ( Fig. 4A , right), however, to our surprise, cis -OPDA-induced expressions of ZAT10, ERF5, DREB2A, ZAT12 , and FIT1 were suppressed in cts1 ( Fig. 4B ). Peroxisomal conversion of cis- OPDA into JA-Ile occurs through the canonical OPR3-dependent or OPR3-independent route via several downstream metabolites in A. thaliana ( Fig. 1 and 3A ). This finding indicated that the downstream metabolites, not cis -OPDA itself, are genuine bioactive forms. Download figure Open in new tab Fig. 4. Downstream metabolites of cis -OPDA are the genuine bioactive form. ( A ) Left: Conversion of cis -OPDA into JA-Ile. The CTS transporter is localized on the peroxisomal membrane and involved in the import of cis -OPDA into the peroxisome. cis -OPDA is oxidized to dn- cis -OPDA, tn- cis -OPDA, and 4,5-ddh-JA in the peroxisome and finally converted to JA-Ile. Right: Accumulation (pmol/fresh weight (g)) of deuterium-labeled cis -OPDA- d 5 . 10-day-old Arabidopsis WT (Ler-0, white bar) and cts1 mutant (orange bar) were treated with cis -OPDA- d 5 (30 μM). The data are presented as mean ± SD (n = 4). Significant differences were evaluated by the Student’s t-test (* p < 0.05). ( B ) Gene expression analysis by RT-qPCR in 10-day-old WT (Ler-0, white bar) and cts1 mutant (orange bar) with no treatments (mock) and 30 μM cis -OPDA treatment for 30 min. The data are presented as mean ± SD (n = 3–4). Samples were normalized to the UBQ10 level. Significant differences were evaluated by the ANOVA/Tukey Kramer test ( p < 0.05). The experiments were repeated three times with similar results. The downstream metabolites of cis -OPDA, dn- cis -OPDA, tn- cis -OPDA, and (+)-7- iso -4,5-didehydrojasmonic acid, are bioactive forms of cis -OPDA cis -OPDA significantly upregulated the expressions of ERF5, DREB2A, ZAT12 , and FIT1 in opr2-1opr3-3 compared to Col-0 ( Fig. 3D-G ). This finding implies that metabolites located downstream of cis -OPDA and upstream of JA in the OPR3-independent bypass route ( Fig. 1B ) are potential candidates for the bioactive form of cis -OPDA. As a result, we performed UPLC-MS/MS analyses of the downstream metabolites of cis -OPDA in the canonical OPR3-dependent (OPC-4, Fig. 1A ) and OPR3-independent routes (dn- cis -OPDA, tn- cis -OPDA, and 4,5-ddh-JA; Fig. 1B ) using Col-0 and opr2-1opr3-3 ( Fig. 5 ). For UPLC-MS/MS analysis, Col-0 and opr2-1opr3-3 were treated with cis -OPDA- d 5 . Compared to Col-0, cis -OPDA- d 5 -treatment enhanced the accumulation of 4,5-ddh-JA- d 5 and tn- cis -OPDA -d 5 in the opr2-1opr3-3 mutant but did not affect the level of dn- cis -OPDA -d 5 ( Fig. 5 ). Among these metabolites, the accumulation of tn- cis -OPDA -d 5 (ca.4200 pmol/g FW in opr2-1opr3-3 and ca. 2400 pmol/g FW in Col-0) and 4,5-ddh-JA -d 5 (ca.3700 pmol/g FW in opr2-1opr3-3 and ca. 1400 pmol/g FW in Col-0) was significantly higher than dn- cis -OPDA -d 5 (ca.400 pmol/g FW). Notably, the accumulation of tn- ci s-OPDA -d 5 and 4,5-ddh-JA -d 5 was enhanced in opr2-1opr3-3 . In contrast, the accumulation of OPC-4 -d 5 , a downstream metabolite of the OPR3-dependent route, was below the detection limit in opr2-1opr3-3 ( Fig. 5 ). Download figure Open in new tab Fig. 5. Conversion of cis -OPDA- d 5 into jasmonate derivatives in planta . 10-day-old WT (Col-0) and opr2-1opr3-3 were treated with cis -OPDA- d 5 (30 μM) for 30 min. The data are presented as mean ± SD (n = 4). Significant differences were evaluated by Student’s t-test (* p < 0.05). The experiments were repeated three times with similar results. The downstream metabolites of cis -OPDA were similarly effective as cis -OPDA on the expression of OPDA-marker genes We examined the effects of dn- cis -OPDA, tn- cis -OPDA, and 4,5-ddh-MeJA, the methyl ester of 4,5-ddh-JA ( Fig. 6A and Scheme S2 and S3), on the expression of OPDA marker genes in A. thaliana . Tn -cis -OPDA and 4,5-ddh-MeJA were equally effective in upregulating expression of ZAT10, ERF5, DREB2A, ZAT12 , and FIT1 in the opr2-1opr3-3 mutant ( Fig. 6B, C , and S5A). Tn -cis -OPDA and 4,5-ddh-MeJA upregulated the expression of OPDA marker genes in a concentration-dependent manner ( Fig. 6D and E ). Download figure Open in new tab Fig. 6. Gene expression mediated by cis -OPDA, tn- cis -OPDA and 4,5-ddh-MeJA in the mutant lines. ( A ) OPR3-independent bypassing route and chemical structure of 4,5-ddh-JA and 4,5-ddh-MeJA. ( B and C ) Gene expression analysis by RT-qPCR in 10-day-old WT (Col-0, white bar) and opr2-1opr3-3 mutants (blue bar) treated with 30 μM cis -OPDA, tn- cis -OPDA, and 4,5-ddh-MeJA for 30 min. Mock indicates findings from WT and opr2-1opr3-3 mutant without any treatments. ( D and E ) Gene expression analysis by RT-qPCR in 10-day-old opr2-1opr3-3 mutants treated with 1, 10, 30, or 100 μM cis -OPDA (left), tn- cis -OPDA (middle), and 4,5-ddh-MeJA (right) for 30 min. Mock indicates findings from WT and opr2-1opr3-3 mutants without any treatments. The data are presented as mean ± SD (n = 3–4). Samples were normalized to the UBQ10 level. Significant differences were evaluated by ANOVA/Tukey Kramer test ( p < 0.05). The experiments were repeated three times with similar results. The downstream metabolites of cis -OPDA caused gene expression through their electrophilic property Monte et al . reported that cis -OPDA and dn- cis -OPDA upregulate the expression of HSP genes in A. thaliana and M. polymorpha in a COI1-independent manner 36 . In addition, the upregulated gene expression depends on the electrophilic properties of cis -OPDA and dn- cis -OPDA because dn- iso -OPDA, which is an isomer of dn- cis -OPDA and less reactive as an electrophile, is significantly less effective than dn- cis -OPDA. Therefore, we compared the effects of tn- cis -OPDA and tn- iso -OPDA ( Fig. 7A ), 4,5-ddh-MeJA and 3,7-ddh-MeJA ( Fig. 7A ), corresponding to an iso -isomer of 4,5-ddh-MeJA, on the expression of ZAT10, ERF5 , and HSP genes. The effect on the expression of DREB2A, ZAT12 , and FIT1 genes was also investigated (Fig. S6). As shown in Fig. 7 and S6, tn- cis -OPDA and 4,5-ddh-MeJA upregulated the expression of ZAT10 ( Fig. 7B ), ERF5 ( Fig. 7C ), DREB2A (Fig. S5A), ZAT12 (Fig. S5B), and FIT1 (Fig. S5C) whereas tn- iso -OPDA and 3,7-ddh-MeJA did not. Similarly, cis -OPDA, tn- cis -OPDA, and 4,5-ddh-MeJA upregulated the expression of HSP , whereas tn- iso -OPDA and 3,7-ddh-MeJA did not ( Fig. 7D-F ). This suggests that the electrophilic nature of tn- cis -OPDA and 4,5-ddh-JA might be responsible for upregulating ZAT10, ERF5, DREB2A, ZAT12, FIT1 , and HSP s. Download figure Open in new tab Fig. 7. cis -OPDA, tn- cis -OPDA, and 4,5-ddh-MeJA mediated gene expression through their electrophilic properties. ( A ) Chemical structures of tn- iso -OPDA, 3,7-ddh-JA, and 3,7-ddh-MeJA. ( B to F ) Gene expression analysis by RT-qPCR in 10-day-old WT (Col-0, white bar) and opr2-1opr3-3 mutant (blue bar) or without any treatments (mock) or treated with 30 μM tn- cis -OPDA, tn- iso -OPDA, 4,5-ddh-MeJA, and 3,7-ddh-MeJA for 30 min ( B, C ) or 30 μM cis -OPDA, tn- cis -OPDA, tn- iso -OPDA, 4,5-ddh-MeJA, and 3,7-ddh-MeJA, for 180 min ( D, E, F ). The data are presented as mean ± SD (n = 3–4). Samples were normalized to the UBQ10 level. Significant differences were evaluated by the ANOVA/Tukey Kramer test ( p < 0.05). The experiments were repeated three times with similar results. Discussion Previous studies reported that cis -OPDA is a bioactive jasmonate in A. thaliana 14 - 21 . Here, we reexamined its bioactivities by combining bioassays using the complete loss-of-function Arabidopsis mutant lines, coi1-1 , and opr2-1opr3-3 , and in vitro biochemical assays to assess the affinity between cis -OPDA and COI1-JAZs, and concluded that cis -OPDA is not the genuine bioactive form responsible for the expression of OPDA-marker genes. Our findings revealed that the downstream metabolites of cis -OPDA in the OPR3-independent biosynthetic route of JA-Ile are responsible for cis -OPDA-induced expression of ZAT10 and ERF5 ( Fig. 8 ). Download figure Open in new tab Fig. 8. A model demonstrating that downstream metabolites of cis -OPDA function as bioactive jasmonates independent of canonical JA signaling. According to the hypothesis, downstream metabolites of cis -OPDA are responsible for the expression of OPDA marker genes such as ZAT10 / ERF5 through the unknown target. The finding that the expression of OPDA marker genes was suppressed in the cts1 mutant lines 43 , in which cis -OPDA transport to peroxisomes was inhibited, strongly supports our conclusion ( Fig. 4 ). This finding demonstrated that the downstream metabolites of cis -OPDA, and not cis -OPDA itself, were genuine bioactive forms. Next, we focused on the fact that cis -OPDA caused higher expression of ERF5, DREB2A, ZAT12 , and FIT1 in opr2-1opr3-3 mutant compared to Col-0 ( Fig. 3D-G ). This result indicates that common metabolites of cis -OPDA are responsible for the upregulation of these genes in opr2-1opr3-3 . Chini et al . reported that the OPR3-independent JA biosynthetic pathway operates weakly in Col-0 and is predominant in opr2-1opr3-3 / opr3-3 mutants 9 . In opr2-1opr3-3 , the downstream metabolites of the OPR3-independent route, tn- cis -OPDA and 4,5-ddh-JA, accumulated more than in Col-0, whereas little accumulation was observed for the downstream metabolites of cis -OPDA in the canonical OPR3-dependent route ( Fig. 5 ). The accumulation of metabolites in opr3-3 was previously reported using deuterium-labeled α-linolenic acid 9 ; however, we used deuterium-labeled cis -OPDA- d 5 to exclude the effect of metabolites from the branched metabolic routes of α-linolenic acid, such as hydroperoxide lyase/isomerase, epoxyalcohol synthase, divinyl ether synthase, and 9-LOX routes 4 , 45 . As a result, downstream metabolites of cis -OPDA in the OPR3-independent route are expected to be bioactive forms of cis -OPDA. Unfortunately, we could not identify one of them or all of them function as endogenous chemical signals. Identification of the genuine endogenous signal must await the target identification and subsequent evaluation of ligand-receptor binding affinity. Considering that UPLC-MS/MS analysis revealed that the accumulation of tn- cis -OPDA and 4,5-ddh-JA was increased in opr2-1opr3-3 compared to Col-0, while the accumulation of dn- cis -OPDA was unchanged ( Fig. 5 ), suggesting that tn- cis -OPDA and 4,5-ddh-JA are plausible endogenous signaling molecule ( Fig. 8 ). However, our current data cannot exclude the possible function of dn- cis -OPDA as an endogenous chemical signal. Dn- cis -OPDA is converted into tn- cis -OPDA and then 4,5-ddh-JA through β-oxidation in the peroxisome and this conversion cannot be impaired because of the lack of a mutant line ( Fig. 1 ). But, 4,5-ddh-JA is the most downstream metabolite produced in peroxisome, and previous report have shown that 4,5-ddh-JA is reduced to JA by cytosol-localized OPR2 ( Figure 8 ). 9 Thus, 4,5-ddh-JA is a promising candidate as an endogenous signaling molecule. Our findings also indicated that the electrophilic reactivity of 4,5-ddh-JA and tn- cis -OPDA may be responsible for the expression of OPDA marker genes ZAT10 and ERF5 . Monte et al . demonstrated that cis -OPDA and dn- cis -OPDA upregulate Mp COI1-independent expression of HSP genes in M. polymorpha through electrophilic reactivities 36 . They concluded that electrophilic reactivity is essential for HSP expression because it is not induced by dn- iso -OPDA, an isomer of dn- cis -OPDA with significantly lower electrophilic reactivity, due to the tetra-substituted α,β-unsaturated ketone moiety 36 , 46 . Similarly, tn- iso -OPDA and 3,7-ddh-JA, iso -isomers of tn- cis -OPDA and 4,5-ddh-JA with little electrophilic reactivity, did not induce the expression of OPDA maker genes ( Fig. 7 and S6). Downstream metabolites of cis -OPDA function as chemical signals that cause the upregulation of OPDA marker genes independently of COI1 and MYCs ( Fig. 2 and S4). COI1-independent gene upregulation may be attributed to non-COI1-targets of cis -OPDA, such as cyclophilin 20-3 and glutathione S-transferase 19, which are proposed putative targets of cis -OPDA ( Fig. 8 ) 18 , 47 . Further studies identifying the non-COI1-target of cis- OPDA will enable detailed genetic studies of the unknown MOA of cis- OPDA. In this study, we examined the effects of cis -OPDA on coi1-1 and opr2-1opr3-3 mutants in which JA signaling and biosynthesis were impaired. In addition, we demonstrated that cis -OPDA had no affinity for any of the functional COI1-JAZ co-receptor pairs. Combining the results of gene expression and UPLC-MS/MS analyses, downstream metabolites of cis -OPDA are responsible for the expression of OPDA marker genes through a non-COI1 target. Materials and Methods Plant Materials A. thaliana ecotype Col-0 and Ler-0 seeds were surface-sterilized in 5% sodium hypochlorite with 0.3% Tween 20 and vernalized for 2–3 d at 4 °C. All seedlings were grown (118 μmol m -2 s -1 ) under a 16 h light/8 h dark cycle at 22 °C in a CL-301 growth chamber (Tomy Seiko Co., Ltd., Japan). Seedlings were grown in 1/2 Murashige and Skoog (MS) liquid medium. The mutant lines used in this study were coi1-1 37 , opr2-1opr3-3 9 , myc2myc3myc4 40 , and cts1 41 . The seeds of coi1-1 and opr2-1opr3-3 mutants were kindly gifted from Professor Roberto Solano and Dr. Andrea Chini (CNB-CSIC, Spain). The cts1 mutant was purchased from the Nottingham Arabidopsis Stock Centre (NASC). To select homozygous coi1-1 , heterozygous coi1-1 seeds were geminated on a 1/2 MS plate containing 10 μM JA, and well-grown 4 d seedlings (homozygous coi1-1 ) were transferred to a 1/2 MS liquid medium. After 6 d of culture, the seedling was treated with each compound ((-)-JA, cis -OPDA, cis -OPDA- d 5 , dn- cis -OPDA, tn- cis -OPDA, 4,5-ddh-MeJA, tn- iso -OPDA, 3,7-ddh-MeJA). For cts1 , surface-sterilized seeds were pipetted onto 1/2 MS plate medium, and seed coats were disrupted using sterile tweezers to stimulate germination 42 . Chemical synthesis Synthesis of (+)- cis -OPDA- d 5 (+)-12-( R )-Hydroxy-phytodienoic alcohol- d 5 was synthesized as described in the Supplementary Text. To a solution of the diol (+)-12-( R )-hydroxyphytodienoic alcohol- d 5 (32.7 mg, 0.11 mmol) in acetone (9.8 mL), Jones reagent (4.0 M solution) at −20 °C was added until the orange color of the reagent persisted (8 drops). After 10 min of stirring at −20 °C, i -PrOH was added to quench the remaining reagent. AcOEt/ n -hexane (1:1) and H 2 O were then added, and the water layer was extracted with AcOEt. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4, and concentrated under reduced pressures. The residue was purified by medium-pressure chromatography (Isolera, eluent: 0.1:88:12 AcOH/ n -hexane/EtOAc to 0.1:99.9 AcOH/EtOAc) to obtain cis -OPDA- d 5 (14.4 mg, 42%) as a colorless oil. [α] d 23 +127.2 ( c 0.34, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ) δ H : 7.74 (dd, J = 6.0, 2.8 Hz, 1H), 6.19 (dd, J = 6.0, 2.0 Hz, 1H), 5.32–5.45 (m, 2H), 2.93–3.02 (m, 1H), 2.40–2.56 (m, 2H), 2.36 (t, J =7.8 Hz, 2H), 2.08–2.19 (m, 1H), 1.09–1.82 (m, 12H); 13 C NMR (100 MHz, CDCl 3 ) δ C : 211.1, 179.7, 167.3, 132.9, 132.5, 127.0, 49.8, 44.3, 34.0, 30.7, 29.6, 29.1, 28.9, 27.6, 24.6, 23.8, 19.3–20.3 (m), 12.4–13.3 (m); IR (neat) cm −1 : 2929, 2222, 1707, 1213; HRMS (ESI, negative) m/z [M-H] - calcd for C 18 H 22 D 5 O 3 : 296.2279, found: 296.2268. Synthesis of tn- cis -OPDA (+)-8-( R )-hydroxyphytodienoyl alcohol was synthesized as described in the Supplementary Text. To a solution of (+)-8-( R )-hydroxyphytodienoyl alcohol (18.0 mg, 81.1 μmol) in acetone (7 mL), Jones reagent (4.0 M solution) was added at −20 °C dropwise until the color of the reagent persisted (7 drops). i -PrOH and AcOEt/ n -hexane (1:1) were then added to quench the remaining reagents. The mixture was extracted using EtOAc. The organic layer was washed with saturated aqueous NaCl, dried over Na 2 SO 4 , and then filtered. The reaction mixture was purified using medium-pressure chromatography (Isolera, eluent: 88:12:0.1 n-hexane/EtOAc/AcOH to 100:0.1 EtOAc/AcOH) to obtain tn- cis -OPDA as a colorless oil (18.5 mg, 96%). [α] d 23 +207.2 ( c 0.14, CHCl 3 ); 1 H NMR (400 MHz, CDCl 3 ) δ H : 7.75 (dd, J = 5.8, 2.8 Hz, 1H), 6.21 (dd, J = 5.8, 1.8 Hz, 1H), 5.52-5.27 (m, 2H), 3.09–2.92 (m, 1H), 2.65–2.30 (m, 4H), 2.27–1.93 (m, 3H), 1.89–1.59 (m, 3H), 1.34–1.11 (m, 1H), 0.97 (t, J = 7.6 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ C : 210.56, 178.52, 166.28, 133.21, 132.91, 126.71, 49.67, 44.04, 33.96, 30.19, 23.79, 22.76, 20.80, 14.01; IR (neat) cm −1 : 3050, 1732, 1702, 1109; HRMS (ESI negative) m/z [M-H] - calcd for C 14 H 19 O 3 : 235.1340, found: 235.1328. Synthesis of 4,5-ddh-MeJA (+)-Methyl 4,5-ddh-6- epi -cucurbate was synthesized as described in the Supplementary Text. To a solution of (+)-methyl 4,5-ddh-6- epi -cucurbate (9.8 mg, 43.7 μmol) in acetone (4.5 mL), Jones reagent (4.0 M solution) was added at −20 °C until the orange color of the reagent persisted (16 drops). After 10 min of stirring at −20 °C, i -PrOH was added to quench the remaining reagent. Subsequently, n -hexane and H 2 O were added, and the water layer was extracted with n -hexane. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4, and concentrated under reduced pressures. The residue was purified by medium-pressure chromatography (Isolera, eluent: 98:2 n -hexane/EtOAc to 80:20 n -hexane/EtOAc) to obtain 4,5-ddh- cis -MeJA (5.3 mg, 55%) as a colorless oil. Diastereomeric purity of 4,5-ddh- cis -MeJA was > 99% by 1 H NMR spectroscopy (δ H =7.71 (dd, J = 5.7, 2.7 Hz, 1H) for 4,5-ddh- cis -MeJA; 7.63 (dd, J = 5.7, 2.4 Hz, 1 H) for the trans isomer). [α] d 24 +127.4 ( c 0.26, CHCl 3 ). 1 H NMR (400 MHz, CDCl 3 ) δ H : 7.71 (dd, J = 5.7, 2.7 Hz, 1H), 6.22 (dd, J = 5.7, 2.0 Hz, 1H), 5.45 (dtt, J = 10.9, 7.4, 1.8 Hz, 1H), 5.32 (dddt, J = 10.9, 6.8, 5.0, 1.4 Hz, 1H), 3.72 (s, 3H), 3.55-3.45 (m, 1H), 2.76 (dd, J = 16.4, 5.8 Hz, 1H), 2.57 (dt, J = 6.8, 5.0 Hz, 1H), 2.53 (brdt, J = 15.7, 5.0 Hz, 1H), 2.21 (dd, J = 16.4, 10.1 Hz, 1H), 2.13 (brdt, J = 15.7, 6.8 Hz, 1H), 2.05 (quintet, J = 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ C : 210.03, 172.34, 165.87, 133.66, 133.19, 126.17, 51.87, 48.32, 40.41, 34.44, 24.39, 20.79, 13.85; IR (film) cm −1 :2961, 1733, 1717, 1198; HRMS (ESI, positive) m/z [M+Na] + calcd for C 13 H 18 NaO 3 : 245.1154, found: 245.1148. Gene expression analyses Surface sterilized A. thaliana seeds were germinated (118 μmol m -2 s -1 ) under a 16 h light/8 h dark cycle at 22 °C in a CL-301 growth chamber (TOMY SEIKO Co., Ltd., Japan) after vernalization in the dark at 4 °C for 2 d. 10-day-old seedlings (5–6 seedlings/sample) grown in 1/2 MS liquid medium were treated with each compound ((-)-JA, cis -OPDA, dn- cis -OPDA, tn- cis -OPDA, 4,5-ddh-MeJA, tn- iso -OPDA, 3,7-ddh-MeJA). Based on the results of previously reported comprehensive DNA microarray analyses 16 , we performed gene expression analyses by adding cis -OPDA to Arabidopsis WT (Col-0) and the coi1-1 mutant. Arabidopsis WT and coi1-1 plants were treated with (-)-JA (a mixture of cis and trans = 5/95; fig. S1) or cis -OPDA, to examine their effects on the expression of three JA marker genes, OPR3, JAZ8 , and MYC2 , as well as cis -OPDA-specific marker genes, ZAT10, ERF5, DREB2A, ZAT12 , and FIT1 16 , 31 . Each sample was frozen after treatment for 30 min, and total RNA was extracted using ISOGEN (NIPPON GENE, Japan). First-strand cDNA was obtained using ReverTra Ace ® reverse transcriptase (TOYOBO, Japan) with oligo-dT primers. A StepOnePlus Real-Time PCR System (Life Technologies, USA) was used for quantitative PCR (qPCR). PCR conditions were followed: an initial hold at 95 °C for 30 s, followed by a two-step PCR program of 95 °C for 5 s and 60 °C for 30 s for 40 cycles. Primer sequences used in this study are listed in Supplementary Table S1. Polyubiquitin 10 was used as the reference gene. UPLC-MS/MS analysis of cis -OPDA metabolites Surface sterilized A. thaliana seeds were germinated (118 μmol m -2 s -1 ) under a 16 h light/8 h dark cycle at 22 °C in a CL-301 growth chamber (TOMY SEIKO Co., Ltd., Japan) after vernalization in the dark at 4 °C for 2 d. 10-day-old seedlings (5 seedlings/sample) grown on 1/2 MS liquid medium were treated with cis -OPDA- d 5 (30 μM). The frozen plant materials were homogenized and extracted using 600 μL of ice-cold (pre-cooled at −20 °C) 25% MeOH/H 2 O. The samples were sonicated for 10 min and extracted for 30 min at 4 °C using a rotator. After centrifugation at 15,000 g for 15 min, supernatants were collected. The pooled supernatants were purified by one-step reversed-phase polymer-based solid-phase extraction using Oasis ® HLB cartridges (Waters, USA). Before sample loading, the SPE sorbent was conditioned with 1 mL of 100% MeOH and equilibrated with 1 mL of 0.1% HCOOH/H 2 O (v/v). After the samples had been loaded onto the Oasis ® HLB column, interfering compounds were removed by washing with 1 mL of 25% MeOH/H 2 O, following which the pre-concentrated analytes on the sorbent were eluted with 2 mL of 50% MeOH/H 2 O and 2 mL 100% MeOH. The eluent solution was evaporated to dryness at 30 °C under reduced pressure through freeze-drying. The samples were then resuspended in 100 μL of 100% MeOH. The samples were examined by UPLC-MS/MS using a TripleTOF 5600 system (AB SCIEX, USA) operating in the negative mode. Liquid chromatography separation was performed with an Eclipse Plus C18 RRHD 1.8 μm (Φ1.05 × 50 mm, Waters, USA) at 35 °C and a flow rate of 0.3 mL/min. The elution was performed using a gradient of water (solvent A) and MeOH (solvent B), both containing 0.1% formic acid (v/v). The proportion of solvent B in the eluent was increased linearly from 10% to 60% for 2 min and from 60% to 90% for 8 min of the elution phase, followed by maintaining a flow of 90% B for 1 min. The column was re-equilibrated with 10% solvent B for 3 min. Pull-down assay For the pull-down experiments using fluorescein-tagged JAZ peptides (Fl- At JAZPs), purified GST- At COI1 (5 nM), Fl- At JAZP (10 nM), and each compound (JA-Ile or cis -OPDA) in 350 μL of incubation buffer (50 mM Tris-HCl buffer, pH 7.8, 100 mM NaCl, 10% glycerol, 0.1% Tween20, 100 nM inositol-1,2,4,5,6-pentakisphosphate (IP5)) were combined with anti-fluorescein antibody (0.2 μL, GeneTex, GTX26644, USA), and incubated for 10–15 h at 4 °C with rotation. After incubation, the samples were combined with SureBeads™ Protein G (10 μL in 50% incubation buffer slurry; Bio-Rad, Hercules, CA, USA). After 3 h of incubation at 4 °C with rotation, the samples were washed three times with 350 μL of wash buffer (phosphate-buffered saline containing 0.1% Tween 20). The washed beads were resuspended in 35 μL of SDS-PAGE loading buffer containing dithiothreitol (DTT, 100 mM). After heating for 10 min at 60 °C, the samples were subjected to SDS-PAGE and analyzed by western blotting. The bound GST-COI1 protein was detected using an anti-GST HRP conjugate (RPN1236, GE Healthcare, USA, 5,000-fold dilution in blocking buffer (Nakalai Tesque, Inc., Japan)). Statistical analyses Samples were analyzed in triplicate, and the data are presented as the mean ± standard deviation (SD). An analysis of variance (ANOVA) was used for data analysis. Different letters within a column indicate statistically significant differences by the Tukey-Kramer multiple range test ( p < 0.05, CoStat version 6.400). A Student’s t-test was performed to examine differences between the two groups. Funding This work was financially supported by Grants-in-Aid for Scientific Research from JSPS, Japan (Nos. 23H00316, 23H04883, 23K17967, 22KK0076, 21K19037, 20H00402, JPJSBP120229905, and JPJSBP120239903 to MU). Author contributions Conceptualization, M.U.; methodology, M.U., R.S., N.K., and Y.T.; validation, M.U., Y.T., R.S., and N.K..; formal analysis, R.S., Y.N., N.K., M.N., T.K., H.Y.; investigation, R.S., Y.N., N.K., M.N., T.K., H.Y.; resources, R.S., Y.N., N.K., M.N., T.K., H.Y.; data curation, M.U., Y.T., R.S., and N. K.; writing—original draft preparation, M.U.; writing—review and editing, M.U., Y.T., N.K., R.S.; visualization, Y.T., R.S., and M.N.; supervision, M.U.; project administration, M.U.; funding acquisition, M.U. All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare that they have no competing interests. Data availability statement All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. Acknowledgments Seeds of the opr2-1opr3-3 double mutant and coi1-1 mutant were provided by Professor Roberto Solano and Dr. Andrea Chini (CNB-CSIC, Spain). 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Share Downstream metabolites of (+)- cis -12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana Rina Saito , Yuho Nishizato , Tsumugi Kitajima , Misuzu Nakayama , Yousuke Takaoka , Nobuki Kato , Minoru Ueda bioRxiv 2024.05.01.592109; doi: https://doi.org/10.1101/2024.05.01.592109 Share This Article: Copy Citation Tools Downstream metabolites of (+)- cis -12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana Rina Saito , Yuho Nishizato , Tsumugi Kitajima , Misuzu Nakayama , Yousuke Takaoka , Nobuki Kato , Minoru Ueda bioRxiv 2024.05.01.592109; doi: https://doi.org/10.1101/2024.05.01.592109 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 (7649) Biochemistry (17738) Bioengineering (13925) Bioinformatics (42059) Biophysics (21496) Cancer Biology (18643) Cell Biology (25577) Clinical Trials (138) Developmental Biology (13406) Ecology (19946) Epidemiology (2067) Evolutionary Biology (24370) Genetics (15627) Genomics (22551) Immunology (17772) Microbiology (40497) Molecular Biology (17212) Neuroscience (88786) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4835) Physiology (7663) Plant Biology (15177) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9838) Zoology (2272)