Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H2O2homeostasis

Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H2O2 homeostasis | 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 Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H 2 O 2 homeostasis View ORCID Profile Yun Huang , View ORCID Profile Jiahui Gao , Guiming Ji , Wenjing Li , Jiaxue Wang , Qinghua Wang , View ORCID Profile Yuanyue Shen , View ORCID Profile Jiaxuan Guo , View ORCID Profile Fan Gao doi: https://doi.org/10.1101/2024.07.10.602942 Yun Huang 1 Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture , Beijing 102206, China ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yun Huang Jiahui Gao 1 Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture , Beijing 102206, China ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jiahui Gao Guiming Ji 2 Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Department of Resources and Environment, Beijing University of Agriculture , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wenjing Li 2 Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Department of Resources and Environment, Beijing University of Agriculture , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jiaxue Wang 1 Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture , Beijing 102206, China ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qinghua Wang 1 Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture , Beijing 102206, China ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yuanyue Shen 1 Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture , Beijing 102206, China ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuanyue Shen Jiaxuan Guo 2 Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Department of Resources and Environment, Beijing University of Agriculture , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jiaxuan Guo For correspondence: gaofan{at}bua.edu.cn guojiaxuangjx{at}163.com Fan Gao 2 Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Department of Resources and Environment, Beijing University of Agriculture , Beijing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fan Gao For correspondence: gaofan{at}bua.edu.cn guojiaxuangjx{at}163.com Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Polyamines (PAs), mainly including putrescine, spermidine, and spermine, are essential for plant growth and development. However, the post-translational regulation of PA metabolism remains unknown. Here, we report the COP9 signalosome subunit 5A (FvCSN5A)-mediated degradation of the PA oxidase FvPAO5, which catalyzes the conversion of spermidine/spermine to produce H 2 O 2 . FvCSN5A is localized in the cytoplasm and nucleus, ubiquitously expressed in strawberry plants, and rapidly increases during fruit ripening. FvCSN5A RNA interference (RNAi) transgenic strawberries exhibit pleiotropic effects on plant development, fertility, and fruit ripening by regulating PA and H 2 O 2 homeostasis, similar to FvPAO5 overexpression transgenic lines. FvCSN5A interacts with FvPAO5 in vitro and in vivo. The ubiquitination and degradation of FvPAO5 are impaired in FvCSN5A RNAi lines. Additionally, FvCSN5A interacts with cullin 1 (FvCUL1), a core component of the E3 ubiquitin-protein ligase complex. Transient genetic analysis in cultivated strawberry fruits showed that inhibiting FaPAO5 expression could partially rescue the ripening phenotype of FaCSN5A RNAi fruits. Collectively, the CSN5A-CUL1-PAO5 signaling pathway responsible for PA and H 2 O 2 homeostasis is crucial to strawberry vegetative and reproductive growth and, final to fruit ripening. Our findings may open a promising avenue for improving crop yield and quality by manipulating the CSN5-PAO5 pair. Introduction Polyamines (PAs) are small polycationic molecules, primarily consisting of putrescine (Put), spermidine (Spd), and spermine (Spm). These molecules have been demonstrated to participate in numerous molecular processes, such as serving as a nitrogen storage pool ( Siddappa and Marathe, 2020 ), protecting and stabilizing macromolecules ( Napieraj et al., 2023 ), regulating reactive oxygen species (ROS) homeostasis ( Pottosin et al., 2014 ; Jasso-Robles et al., 2020 ), and participating in signal transduction ( Gao et al., 2021 ). Consequently, PAs play important roles in various aspects of plant development and growth, including photosynthetic regulation, secondary metabolism, flowering, fruit ripening, and environmental responses ( Fortes et al., 2019 ; Wang et al., 2019 ; Aloisi et al., 2022 ; Nandy et al., 2022 ; Navakoudis and Kotzabasis, 2022 ; Blázquez, 2024 ). Although much progress has been made in understanding PA metabolism and function, the regulatory mechanisms governing PA homeostasis at the post-translational level remain elusive. In general, Put is involved in photosynthetic protection, while Spd/Spm, especially Spm, are involved in regulating ROS ( Zhao et al., 2021 ; Zhong et al., 2023 ; Blázquez, 2024 ; Furumoto et al., 2024 ). In response to developmental and environmental stimuli, PA equilibria are fine-tuned at biosynthetic and degradative levels ( Gupta et al., 2016 ; Wang et al., 2019 ; Zhan et al., 2023 ). That is, PA biosynthesis begins with Put, catalyzed by either arginine decarboxylase (ADC) or ornithine decarboxylase (ODC). Then, Put is utilized as a substrate for the successive biosynthesis of Spd and Spm, catalyzed separately by Spd synthase (SPDS) and Spm synthase (SPMS). This process requires the addition of an aminopropyl group derived from decarboxylated S-adenosyl-L-methionine (dcSAM). The dcSAM is generated from S-adenosyl-L-methionine (SAM), catalyzed by SAM decarboxylase (SAMDC) using SAM as a substrate ( Liu et al., 2006 ; Groppa and Benavides, 2008 ; Gupta et al., 2016 ). PA degradation involves copper-containing diamine oxidase (CuAO/DAO)-mediated Put degradation and flavin-containing polyamine oxidase (PAO)-mediated Spd/Spm degradation in a back-conversion (BC-type) or terminal-catabolism (TC-type) manner ( Paschalidis et al., 2005 ; Angelini et al., 2010 ; Yu et al., 2019 ). In particular, PA decomposition produces H 2 O 2 , which contributes to fine-tuning plant growth and development in response to environmental cues by PA and ROS homeostasis ( Navakoudis and Kotzabasis, 2022 ; Zhan et al., Notably, AtPAO5 has been demonstrated to participate in Arabidopsis xylem differentiation by auxin and cytokinin ( Alabdallah et al., 2017 ). Similarly, OsPAO5 plays an important role in rice mesocotyl and seedling growth, as well as grain weight, grain number, and yield potential through H 2 O 2 and ethylene ( Lv et al., 2021 ). Additionally, PAO5 acts as a negative regulator during strawberry fruit ripening by the TC-type degradation of Spm/Spd in concomitant with H 2 O 2 production ( Mo et al., 2020 ). However, the mechanism behind the continuously-decreased protein levels of FaPAO5 during fruit ripening ( Mo et al., 2020 ) remains unclear, this evokes us to speculate that ubiquitination-mediated degradation of PAO5 might be involved. The cullin-RING ubiquitin ligase (CRL) pathway, a common protein degradation mechanism, is prevalent in all eukaryotes and facilitates multiple cellular processes through the ubiquitin-mediated degradation of targeted proteins via an E1-E2-E3 cascade of the 26S proteasome system ( Barry and Früh, 2006 ; Hotton and Callis, 2008 ). Specifically, the 76-amino-acid ubiquitin (UB) is covalently attached to E1 (ubiquitin-activating enzyme) via ATP-derived adenylation, then the activated UB is successively transferred to E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) complex. Within the E3 ligase superfamily, CRL activity is regulated by RUB/NEDD8 (related to ubiquitin-like protein) and the COP9 signalosome (CSN; Hotton and Callis, 2008 ). As a component of the ubiquitin-proteasome system, the CSN regulates CRL assembly via neddylation and deneddylation processes ( Stratmann and Gusmaroli, 2012 ). Notably, the CSN complex was initially identified through genetic screening of light□induced photomorphogenic mutants in Arabidopsis, indicating that CSN plays a critical role in controlling CRL activity and various cellular processes ( Serino and Deng, 2003 ; Qin et al., 2020 ; Lu et al., 2024 ). In the COP9 signalosome complex, CSN1-4, CSN7, and CSN8 contain a PCI (proteasome, COP9, initiation factor 3) domain, while CSN5 and CSN6 contain a domain of MPN (Mov34 and Pad1p N□terminal), among which CSN5 acts as a zinc-ion-activating metalloprotease and has two paralogs, designated CSN5A and CSN5B ( Verma et al., 2002 ; Cope and Deshaies, 2003 ; Wei and Deng, 2003 ; Gusmaroli et al., 2004 ; Jin et al., 2014 ). Notably, CSN5A plays an important role in abscisic acid (ABA) signal transduction by regulating the stability of the bZIP transcription factor ABI5 ( Jin et al., 2014 ). The MPN domain is related to both CSN deneddylation and 26S-proteasome deubiquitination, while the PCI domain is responsible for subunit interaction ( Huang et al., 2005 ). The al., 2014; Reitsma et al., 2017 ; Liu et al., 2018 ; Mayor-Ruiz et al., 2019 ). For example, CSN regulates the NEDD8-modification of CUL-based E3 ligases in SCF TIR1 , SCF SLY , and SCF COI1 complexes, facilitating the deneddylation of these CRLs in vivo ( Qin et al., 2020 ). The E3 ubiquitin ligase SCF TIR1 directly interacts with the CSN, which is essential for the efficient degradation of its substrates in Arabidopsis ( Schwechheimer et al., 2001 ). A more recent report indicated that the CSN-mediated deneddylation of CUL1 is necessary for the proper assembly of SCF EBF1 in Arabidopsis ( Dong et al., 2024 ). CSN5 is localized in the nucleus and cytosol, and can cleave the NEDD8-CUL1 conjugate responsible for deneddylation and protein degradation ( Kwok et al., 1998 ; Lyapina et al., 2001 ; Schwechheimer et al., 2001 ). The CSN-mediated deneddylation not only prevents auto-ubiquitination of CRL components, but also facilitates the recycling of the cullin-RING heterodimer for numerous F-box/substrate interactions ( Fischer et al., 2011 ). Intriguingly, two more recent studies find that CSN5 inhibits autophagy by regulating the ubiquitination of Atg6 (autophagy-related) and TOR (target of rapamycin), thereby mediating the pathogenicity of Magnaporthe oryzae ( Shen et al., 2024 ). Under stress conditions, CSN5A regulates the stability of chloroplast proteins in tomato ( Solanum lycopersicum ) by degrading SlPsbS (S subunit of PSII) in the cytosol ( Lu et al., 2024 ). Collectively, the central role of CSN5 within the COP9 signalosome involves a wide range of cellular and biological processes, including light signaling, hormone signaling, and environmental responses in plants ( Qin et al., 2020 ). However, whether the COP9 signalosome participates in PA metabolism remains unknown. To explore the potential mechanism underlying the continued decline of PAO5 during strawberry fruit ripening ( Mo et al., 2020 ), we initially conducted a yeast two-hybrid library screening to identify the interaction protein of PAO5. Following confirmation of the interaction, FvCSN5A was identified through localization, spatiotemporal expression, and RNAi-stable transgenic analyses. Subsequently, the potential relationship between FvCSN5A and FvPAO5 in terms of ubiquitination degradation was explored. Finally, the results obtained from diploid strawberries during fruit ripening were confirmed in octaploid cultivated strawberries. Based on the data available and our current findings, we propose a post-translational regulatory model for PA and H 2 O 2 homeostasis, controlled by the FvCSN5A-FvCUL1-FvPAO5 pathway. This provides novel insights into the integration of the COP9 signalosome into PA metabolism, opening an attractive avenue for agricultural practices aimed at improving crop yield and quality through manipulation of the CSN5-PAO5 pair. Results Identification of FvCSN5A as FvPAO5 interacting protein Given the continual decrease in FaPAO5 protein levels during strawberry fruit ripening ( Mo et al., 2020 ), it is potential to FaPAO5 involved in protein degradation. To investigate this hypothesis, we used diploid strawberries ( Fragaria vesca , Ruegen) and performed a yeast two-hybrid (Y2H) screen using FvPAO5 as a bait. As a result, we identified an interacting protein of FvPAO5, designated FvCSN5A, which is homologous to the Arabidopsis CSN5A protein ( Fig. 1A ). We subsequently validated the physical interaction between FvPAO5 and FvCSN5A using a variety of complementary experimental approaches. In the GST pull-down assay conducted in vitro, the FvPAO5-GST fusion protein was precipitated by FvCSN5A-His ( Fig. 1B ). Furthermore, the firefly luciferase complementation (FLC) assay revealed a strong fluorescent signal upon co-transformation of the FvPAO5-NLUC and FvCSN5A-CLUC constructs in tobacco leaves, while no such signal was observed in the control ( Fig. 1C ). To corroborate the interaction in vivo, we performed co-immunoprecipitation (CoIP) experiments by co-expressing MYC-tagged FvCSN5A with either FvPAO5-GFP or GFP alone in Nicotiana benthamiana epidermal cells via Agroinfiltration. The results demonstrated that MYC-FvCSN5A could specifically associate with FvPAO5-GFP, but not with GFP alone, indicating that FvPAO5 interacts with FvCSN5A ( Fig. 1D ). Download figure Open in new tab Figure 1. FvCSN5A interacted with FvPAO5 in vitro and in vivo . (A) FvCSN5A interacted with FvPAO5 in yeast two-hybrid assays. SD-LW: synthetic dropout medium without Leu and Trp; SD-LWHA: synthetic dropout medium without Leu, Trp, His, and Ade. ( B) FvCSN5A interacted with FvPAO5 in vitro pull-down assays. Recombinant GST or FvPAO5-GST bounded to glutathione sepharose beads was incubated with recombinant FvCSN5A-His protein. Input and elution proteins immunoblotted with anti-His and anti-GST antibodies, respectively. ( C) FvCSN5A interacted with FvPAO5 in firefly luciferase complementation (FLC) assays. The CDS of FvCSN5A was constructed into the pCAMBIA1300-CLUC vector, and the CDS of FvPAO5 was constructed into the pCAMBIA1300-NLUC vector. ( D) FvCSN5A interacted with FvPAO5 in vivo Co-Immunoprecipitation (Co-IP) assays. FvCSN5A-MYC protein bounded to MYC beads was incubated with GFP or FvPAO5-GFP and immunoblotted with anti-GFP antibody and anti-MYC antibody, respectively. ( E) FvCSN5A interacted with FvPAO5 in bimolecular fluorescence complementation (BiFC) assays. FvCSN5A was fused to the N terminus of YFP and FvPAO5 to the C terminus of YFP. FvPAO5 250-450aa is a truncated form of FvPAO5 protein that acts as a negative control. The encoding constructs were co-infiltrated into N. benthamiana leaves. Bars, 20 μm. Finally, we employed a bimolecular fluorescence complementation (BiFC) assay to further validate the FvCSN5A-FvPAO5 interaction in plant cells. The FvCSN5A-YNE (N-terminal of YFP) and FvPAO5-YCE (C-terminal of YFP) constructs were delivered into N. benthamiana leaf cells, and distinct fluorescence was observed in the cytoplasm and nucleus, providing additional evidence for the interaction between FvPAO5 and FvCSN5A ( Fig. 1E ). Collectively, these complementary experimental results provide consolidated evidence that FvPAO5 interacts with FvCSN5A both in vitro and in vivo. Expression pattern and subcellular localization of FvCSN5A To explore the expression pattern of FvCSN5A , we tracked its transcriptional level in different organs of strawberry plants and across five developmental stages of strawberry fruit using RT-qPCR. The expression level of FvCSN5A was highest in roots, followed by red seeds, stems, and leaves, while it was lowest in flowers and fruits ( Fig. 2A ). The FvCSN5A transcript level increased gradually from the white (Wt) to partial red (PR) stage and reached a peak during the full red (FR) stage ( Fig. 2B ). In contrast, the expression level of FvPAO5 decreased rapidly from the small green (SG) to FR stage ( Mo et al., 2020 ). These results suggest that FvCSN5A may exert an effect on the fruit ripening process, potentially related to the decreased FvPAO5 levels. Download figure Open in new tab Figure 2. FvCSN5A expression pattern and subcellular localization. ( A) FvCSN5A expression levels in different tissues of diploid strawberries ( F. vesca , Ruegen). Different letters indicate statistically significant differences at P<0.05 as determined by one-way ANOVA. ( B) FvCSN5A expression levels in fruit receptacles at different developmental stages of diploid strawberries ( F. vesca , Ruegen). SG, small green fruit stage; Wt, white fruit stage; IR, initial red fruit stage; PR, partial red fruit stage; FR, full ripening fruit stage. Relative expression values were relative to receptacles at the SG stage, which was assigned an arbitrary value equal to one. Different letters indicate statistically significant differences at P<0.05 as determined by one-way ANOVA. ( C) Subcellular localization of FvCSN5A-GFP fusions in transiently transformed N. benthamiana leaves. All experiments were performed 48 h post-infiltration. Bars, 20 μm. For the locations of the nuclei, N. benthamiana leaves were stained with DAPI. Px-rk/CD3-983 was a marker for the locations of the peroxisome. To determine the subcellular localization of FvCSN5A, we expressed the Super1300:FvCSN5A-GFP construct or the empty vector (EV, expressing GFP alone) in tobacco leaves and performed fluorescence imaging observations. The results showed that the FvCSN5A-GFP signals were detectable in the cytoplasm and nucleus, the latter of which overlapped with the 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, a dye used to label nuclei ( Fig. 2C ). In comparison, FvPAO5 was localized in the nucleus and peroxisomes, as evidenced by its co-localization with the peroxisome marker px-RK/CD3-983 ( Fig. 2C ). Overall, the common localization of the two proteins in the nucleus suggests their potential roles important to strawberry plant development. Inhibition of FvCSN5A expression led to developmental defects in leaves and flowers CSN5 has been found to participate in crucial biological functions, such as development and stress responses in Arabidopsis and tomato ( Shang et al., 2019 ; Qin et al., 2020 ; Lu et al., 2024 ); however, the function of strawberry homolog FvCSN5A remains unknown. To elucidate the role of FvCSN5A, we generated RNAi-based FvCSN5A-knockdown F. vesca plants. Two independent transgenic lines exhibiting different expression levels of FvCSN5A were obtained and verified by red fluorescence (Fig. S1). The expression levels of FvCSN5A in the RNAi-1 and RNAi-2 lines were decreased by approximately 8-fold and 46-fold, respectively ( Fig. 3A ). In addition, the chromosome ploidy of transgenic strawberry was analyzed by flow cytometry, and it was found that FvCSN5A RNAi-1 and RNAi-2 were both diploid strawberries (Fig. S2). Download figure Open in new tab Figure 3. Phenotypes of the FvCSN5A RNAi transgenic plants. ( A) Relative expression levels of FvCSN5A were determined by RT-qPCR. ( B) The fresh weight of wild-type (WT, F. vesca , Ruegen) and FvCSN5A RNAi transgenic plants. The seedlings grown 45 days after germination were collected for the experiments. ( C) The photos of WT and FvCSN5A RNAi transgenic plants in soil. Bars, 1 cm. ( D) One compound leaf of WT and FvCSN5A RNAi transgenic plants. Bars, 1 cm. ( E) Depth of the serrations of WT and FvCSN5A RNAi transgenic plants. ( F) The flowers of WT and FvCSN5A RNAi transgenic plants. Bars, 1 cm. ( G) The sepals of WT and FvCSN5A RNAi transgenic plants. Bars, 1 cm. ( H) The percentages of different types of sepals in WT and FvCSN5A RNAi transgenic plants. n = 36. ( I) The percentages of different types of petals in WT and FvCSN5A RNAi transgenic plants. n = 36. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. The fresh weight of RNAi seedlings was smaller than that of the wild-type ( Fig. 3B ). The RNAi plants maintained a similar leaf shape index and number of serrations compared to the wild-type leaves (Fig. S3A-B). However, the leaves of FvCSN5A RNAi-1 and FvCSN5A RNAi-2 plants exhibited deeper serrations with sharper serrated blade angles ( Fig. 3C-E ). Moreover, the petal and sepal numbers of the RNAi plants exhibited significant variation. In the wild-type, each flower typically has 10 to 14 sepals and 5 petals ( Fig. 3F-I ). In contrast, some flowers of the FvCSN5A RNAi plants had 6 to 9 sepals (6% in RNAi-1 and 44% in RNAi-2) and 4 petals (11% in RNAi-1 and 28% in RNAi-2), while the wild-type plant did not show such variations ( Fig. 3F-I ). Collectively, the reduction in FvCSN5A expression resulted in substantial developmental defects in both leaves and flowers. FvCSN5A affected the fertility of strawberries, especially pollen viability Since plant flowering and seed formation are fundamental to strawberry fruit development ( Liao et al., 2018 ), we examined the influence of FvCSN5A on seed development. The FvCSN5A RNAi-1 and FvCSN5A RNAi-2 transgenic plants exhibited no observable defects in the appearance of the stigmas, carpels, or stamens (Fig. S3C-D). However, the number of carpels and anthers was reduced in FvCSN5A RNAi flowers (Fig. S3E-F). Subsequently, we performed Alexander’s staining and in vitro pollen germination assays to assess pollen viability ( Fig. 4A-B ). Based on Alexander’s staining, we determined that there was near-complete pollen activity abortion in the lower expression line ( FvCSN5A RNAi-2; 10.29±0.98) and a subtle reduction in the moderate expression line ( FvCSN5A RNAi-1; 91.35±1.22; Fig. 4C ). A significant decrease in pollen germination in vitro was observed in FvCSN5A RNAi-1 (65.93±7.07) and FvCSN5A RNAi-2 (8.49±2.42) compared to the wild-type (96.97±3.03; Fig. 4D ). Furthermore, the germinated pollen tube length of FvCSN5A RNAi plants was shorter than that of the wild-type ( Fig. 4E ). These results demonstrate that the inhibition of FvCSN5A expression leads to reduced pollen viability in strawberry. Download figure Open in new tab Figure 4. Pollen grain fertility in FvCSN5A RNAi transgenic plants. ( A) The images of Alexander’s staining of pollen grains from WT and FvCSN5A RNAi transgenic plants. The red-stained pollen grains are viable and fertile. Bars, 20 μm. ( B) The images of pollen tubes from WT and FvCSN5A RNAi transgenic plants germinating in vitro . Bars, 50 μm. ( C) The percentage of Alexander’s staining of pollen grains from WT and FvCSN5A RNAi transgenic plants. ( D) The percentage of pollen tubes from WT and FvCSN5A RNAi transgenic plants germinating in vitro . ( E) The length of pollen tubes from WT and FvCSN5A RNAi transgenic plants germinating in vitro . Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. Non-significant: ns To further investigate the relative influence of male and female gametophytes on seed formation and fruit expansion, we performed reciprocal crosses between wild-type and FvCSN5A RNAi-2 plants. As shown in Fig. S4A, more seeds were produced, and fruit expanded to a greater extent when the wild-type pollen was applied to FvCSN5A RNAi-2 stigmas, compared to using FvCSN5A RNAi-2 as the paternal plant. Collectively, these data indicate that FvCSN5A is essential for strawberry reproductive success, with particular importance for pollen development. FvCSN5A positively regulated fruit ripening To gain insight into the function of FvCSN5A on fruit ripening, we observed the fruit coloring phenotype of FvCSN5A RNAi. The results demonstrated that strawberry fruit coloration was retarded in FvCSN5A RNAi-1 plants ( Fig. 5A ), while FvCSN5A RNAi-2 fruits were impeded from completing the normal development and maturation process (Fig. S4B). The content of anthocyanin was lower in FvCSN5A RNAi-1 fruits compared to the control, consistent with the observed phenotype ( Fig. 5B ). The firmness analysis also revealed that the softening process was inhibited in FvCSN5A RNAi fruits ( Fig. 5C ). The fresh weight was further determined, indicating that the fresh weight of FvCSN5A RNAi fruits was smaller than that of the control ( Fig. 5D ). The contents of sucrose, glucose, fructose, and total soluble sugar were significantly lower in the fruit of FvCSN5A RNAi ( Fig. 5E ). Download figure Open in new tab Figure 5. Fruit ripening changes in FvCSN5A transgenic fruits. ( A) Photos were taken at 15, 17, 19, 21, 23, and 25 days after pollination (DAP) respectively to record the fruit phenotype. Bars, 0.5 cm. ( B-E) The anthocyanin content (B) , firmness (C) , fresh weight (D) , and soluble sugar content (E) of WT and FvCSN5A RNAi transgenic fruits. Fruits were collected for experiments 21 days after pollination (DAP). Bars are means SEs of three independent experiments. ( F) RT-qPCR was used to analyze the genes’ expression in WT and FvCSN5A RNAi transgenic fruits, and three biological replicates were tested for all samples using the Actin gene from strawberries as an internal control. The relative expression levels were calculated using the 2 -△△Ct method. FvCSN5A: COP9 Signalosome5; FvPG: polygalacturonase; FvCEL: cellulose; FvCHS: chalcone synthase; FvANS: anthocyanidin synthase; FvSUT1: sucrose transporter1; FvSS: sucrose synthase. FvADC1: arginine decarboxylase1; FvSAMDC1: S-adenosyl-methionine decarboxylase1; FvSPDS: spermidine synthase; FvSPMS: spermine synthase; FvPAO5: polyamine oxidase5. Statistical significance was determined by one-way ANOVA: *, P < 0.05; **, P < 0.01. The expression levels of genes related to sugar and anthocyanin accumulation, fruit softening, and PA homeostasis in transgenic strawberry fruits were also examined using RT-qPCR. The expression levels of sucrose transporter FvSUT1 , chalcone synthase FvCHS and anthocyanidin synthase FvANS , polygalacturonase FvPG and cellulose FvCEL were significantly inhibited in the RNAi fruit, while sucrose synthase FvSS , which negatively regulates sugar accumulation, was elevated ( Fig. 5F ). Based on the expression of these marker genes, the physiological results, and phenotypic observations, these findings indicate that FvCSN5A regulated fruit ripening and fruit quality. To explore the effect of FvCSN5A on PA metabolism, the relative gene expression levels in the WT and FvCSN5A RNAi fruits were detected. The results showed that the expression levels of polyamine synthetase ( FvADC1 , FvSAMDC1 , FvSPDS , and FvSPMS ) and polyamine oxidase ( FvPAO5 ) in FvCSN5A RNAi fruits were significantly lower than that in WT. At the same time, we found that in the FvCSN5A RNAi-1 fruit, the expression level of all COP9 signalosome subunits, except FvCSN1 , was significantly reduced (Fig. S3G). To further verify the role of strawberry CSN5A, we manipulated FaCSN5A expression in octoploid cultivated strawberry fruits by overexpression or RNAi construction through transient transformation. The phenotype and RT-qPCR analysis demonstrated that the fruit with lower FaCSN5A mRNA levels ( FaCSN5A RNAi) colored slowly compared to the control fruit, while the fruit with higher FaCSN5A mRNA levels ( FaCSN5A OE) colored rapidly (Fig. S5A-B). The anthocyanin, soluble sugar, firmness and their relative gene expression were detected and significantly changed in FaCSN5A OE and RNAi fruits (Fig. S5C-D). We also examined the expression of PA metabolism-related genes. The results showed that the expression levels of FaADC1 , FaSAMDC1 , FaSPDS , FaSPMS , and FaPAO5 in the FaCSN5A RNAi fruit were significantly lower than those in the WT. The expression levels of these genes in the FaCSN5A OE fruit were significantly higher than those in the WT (Fig. S5D). Altogether, these results demonstrate that strawberry CSN5A controls fruit ripening as a positive regulator at both physiological and molecular levels. FvCSN5A promoted FvPAO5 ubiquitination degradation The CSN (COP9 signalosome) complex typically regulates plant development and stress response by modulating the E3 ubiquitin ligase complex. Therefore, we propose that the FvCSN5A-FvPAO5 interaction might affect FvPAO5 protein stability and regulate strawberry development and fruit ripening. Firstly, we examined the protein levels of FvPAO5 and FvCSN5A at different stages of fruit development. The results showed a decrease in the levels of FvPAO5 during fruit ripening, while the levels of FvCSN5A increased gradually ( Fig. 6A ). Additionally, FvPAO5 exhibited relatively higher accumulation in the FvCSN5A RNAi plants compared to the WT plant ( Fig. 6B ). Secondly, we investigated the degradation pathway of FvPAO5 through the 26S proteasome in a cell-free system. The results showed that the protein level of FvPAO5 decreased when incubated with strawberry total protein, and this degradation was inhibited by the proteasome inhibitor MG132 ( Fig. 6C ). These data indicate that FvCSN5A may promote the degradation of FvPAO5. Download figure Open in new tab Figure 6. The ubiquitination degradation of FvPAO5 is dependent on FvCSN5A. ( A) FvPAO5 and FvCSN5A protein levels in fruit receptacles at different developmental stages of diploid strawberry ( F. vesca , Ruegen). SG, small green fruit stage; Wt, white fruit stage; FR, full ripening fruit stage. ( B) FvPAO5 and FvCSN5A protein abundance detection in the leaves from FvCSN5A RNAi and WT plants. FvPAO5 and FvCSN5A were detected by anti-FvPAO5 and anti-FvCSN5A, respectively. ( C) Cell-free FvPAO5 protein degradation. Total protein was extracted from strawberry leaves and incubated with FvPAO5-GST protein for 0.5, and 1 h with MG132 treatment or DMSO treatment as a control. FvPAO5-GST protein and total plant protein were detected by anti-GST and anti-Actin, respectively. ( D) In vivo FvPAO5 polyubiquitination in FvCSN5A RNAi and WT plants. Three-month-old then harvested for protein extraction. Each total protein was incubated with P62-agarose to obtain ubiquitinated proteins. Polyubiquitinated FvPAO5 was detected with an anti-FvPAO5 antibody. The accumulation of Actin in the total protein was tested as a control. The polyubiquitination of FvPAO5 was assessed in the FvCSN5A RNAi and WT plants. Tissue culture seedlings of the FvCSN5A RNAi and WT plants were treated with or without 50 μM MG132 for 5 h and then harvested for protein extraction. The ubiquitination-modified proteins were enriched with P62-agarose, and ubiquitinated FvPAO5 was detected by an anti-FvPAO5 antibody. Without MG132 treatment, polyubiquitinated FvPAO5 was weak in the WT plants, while it appeared significantly stronger under MG132 treatment. However, the FvPAO5 polyubiquitination signal was hardly visible in the FvCSN5A RNAi plants, regardless of whether MG132 was used or not ( Fig. 6D ). These data suggest that FvCSN5A may promote the proteasome-dependent degradation of FvPAO5 in a ubiquitin-mediated manner. FvCSN5A interacted with FvCUL1 in vitro and in vivo Many studies have reported that CSN interacts with CUL within the E3 ubiquitin ligase complex, and the deneddylation of AtCUL1 was blocked in the atcsn5 mutant ( Schwechheimer et al., 2001 ; Cope and Deshaies, 2003 ; Gusmaroli et al., 2004 ). To determine whether FvCSN5A, FvCULs, and FvPAO5 function within the same complex, we performed a CoIP mass spectrometry assay with an anti-FvPAO5 antibody. The results showed that, in addition to FvCSN5A, FvCSN1 and FvCUL1 were also detected in the anti-FvPAO5 antibody-enriched proteins from the WT (Supplemental Data Set 1). However, FvPAO5 and FvCUL1 did not interact directly in the yeast two-hybrid experiment (data not shown). This implies that FvPAO5 might associate with FvCUL1 via FvCSN5A. To examine the relationship between FvCSN5A and FvCUL1, we investigated the interaction between FvCUL1 and FvCSN5A. The results showed that FvCUL1 interacts with FvCSN5A in Y2H and FLC assays ( Fig. 7A-B ). Therefore, we hypothesize that FvCSN5A may collaborate with FvCUL1 to regulate FvPAO5 protein stability. Download figure Open in new tab Figure 7. FvCUL1 interacted with FvCSN5A in vitro and in vivo . ( A) FvCUL1 interacted with FvCSN5A in FLC assay. The CDS of FvCSN5A was constructed into pCAMBIA1300-CLUC vector, and the CDS of FvCUL1 was constructed into pCAMBIA1300-NLUC vector. ( B) FvCUL1 interacted with FvCSN5A in yeast two-hybrid assay. SD-LW: synthetic dropout medium without Leu and Trp; SD-LWHA: synthetic dropout medium without Leu, Trp, His, and Ade. ( C) The transcript level of FvCUL1 in different tissues of strawberry. Different letters indicate statistically significant differences at P<0.05 as determined by one-way ANOVA. ( D) FvCUL1 transcript levels in fruit receptacles at different developmental stages of diploid strawberry. Different letters indicate statistically significant differences at P<0.05 as determined by one-way ANOVA. ( E) FvCUL1 protein level in fruit receptacles at different developmental stages of diploid strawberries. ( F) Detection of FvCUL1 protein level in FvCSN5A RNAi and WT plants using antibody anti-CUL1. FvCUL1 Nedd8 : FvCUL1 with neddylation; FvCUL1: FvCUL1 without neddylation. In strawberries, the FvCUL1 transcript level was highest in the root, and lowest in flowers ( Fig. 7C ). During fruit ripening, the expression pattern of FvCUL1 was similar to FvCSN5A , rising rapidly and reaching its peak at the FR stage in both mRNA and protein levels ( Fig. 7D-E ). Subcellular localization analysis was performed and found that FvCUL1 was localized in the cytoplasm (Fig. S6). We also tested the influence of FvCSN5A on the FvCUL1 protein level using an anti-CUL1 antibody. We found the deneddylated and neddylated forms of FvCUL1 in the wild-type, and the FvCUL1 protein level in the FvCSN5A RNAi line markedly decreased ( Fig. 7F ). These results suggested that FvCSN5A, the subunit of CSN, interacts with FvCUL1, which may affect the protein stability of FvCUL1. Polyamine, phytohormone, H 2 O 2 and Dap levels in the FvCSN5A RNAi plants Previous studies have reported that CSN5A is involved in numerous phytohormones, including indoleacetic acid (IAA), jasmonic acid (JA), and gibberellic acid (GA; Jin et al., 2014 ; Qin et al., 2020 ). In the current investigation, we have determined that FvCSN5A promotes the ubiquitin-mediated degradation of FvPAO5, a key enzyme that catalyzes the conversion of Spd/Spm to H 2 O 2 and 1,3-diaminopropane (Dap; Mo et al., 2020 ). To elucidate the regulatory roles of FvCSN5A, we assessed the alterations in endogenous PAs, phytohormones, H 2 O 2 , and Dap in the FvCSN5A RNAi plant. Firstly, we quantified PA levels in strawberries. The contents of Spd and Spm were significantly lower in the FvCSN5A RNAi plant compared to the WT plant, with Spd being almost undetectable in the FvCSN5A RNAi-2 plant ( Fig. 8A-B ). However, Put accumulation varied, showing a reduction in in the FvCSN5A RNAi-1 line but not significantly different from WT in the FvCSN5A RNAi-2 line ( Fig. 8C ). These results indicate that the inhibition of strawberry FvCSN5A expression leads to a substantial decrease in Spd and Spm levels, which may be attributable to the over-accumulation of the FvPAO5 protein. Download figure Open in new tab Figure 8. Polyamine and phytohormone contents in FvCSN5A RNAi transgenic plants ( A-C) Spm (A) , Spd (B) , and Put (C) content in WT and FvCSN5A RNAi transgenic leaves. ( D-L) Phytohormone IAA, ABA, JA, GA3, SA, tZR, DHZR, HZ-2 and tZ content in WT and FvCSN5A RNAi transgenic leaves. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. Non-significant: ns Secondly, we investigated the contents of phytohormones related to plant development and environmental responses, including IAA, ABA, JA, GA, cytokinin (CK), and salicylic acid (SA). The synthesis of IAA, ABA, and JA was suppressed in FvCSN5A RNAi plants, with IAA displaying an 83% reduction ( Fig. 8D-F ). In strawberries, the most abundant GA compound was GA3, and the predominant CK compounds were TZR and DHZR, especially, the levels of GA3, TZR, DHZR, and SA were elevated in FvCSN5A RNAi plants ( Fig. 8G-I ). At last, we assessed the amount of H 2 O 2 and Dap in the FvCSN5A RNAi plants. The DAB staining results showed that the pollen grains, leaves and roots of FvCSN5A RNAi plants, particularly FvCSN5A RNAi-2, were stained more intensely, indicating higher H 2 O 2 levels compared to the lighter staining observed in the WT ( Fig. 9A-B , Fig. S7). Furthermore, the ROS generation, as detected by 2,7□dichlorodihydrofluorescein diacetate (DCFH□DA) fluorescence, was markedly higher in the pollen grains and leaves of FvCSN5A RNAi plants ( Fig. 9C-D ). The content analysis also confirmed that the FvCSN5A RNAi plant had significantly elevated H 2 O 2 production compared to the WT ( Fig. 9E ). The content of Dap, another product of the FvPAO5-catalyzed reaction, was significantly higher in the FvCSN5A RNAi plant compared to the WT ( Fig. 9F ). Moreover, when strawberries were treated with exogenous Spd, the Dap content in FvCSN5A RNAi plants decreased, while it increased in the WT ( Fig. 9F ), reducing the gap between the two. Download figure Open in new tab Figure 9. Detection of ROS and Dap accumulation in FvCSN5A RNAi transgenic plants ( A) DAB dye of pollen grains from WT and FvCSN5A RNAi transgenic plants. Bars, 20 μm. ( C) ROS signal determination of pollen grains by DCFH-DA. Bars, 20 μm. ( D) ROS signal determination of leaves by DCFH-DA. Bars, 50 μm. ( E) H 2 O 2 content in WT and FvCSN5A RNAi transgenic plants. ( F) Dap content in WT and FvCSN5A RNAi transgenic plants after Spd treatment. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. Overall, these results indicate that the reduction of FvCSN5A expression leads to a significant decrease in the contents of PAs and IAA, accompanied by an excessive accumulation of H 2 O 2 and Dap. The disruption of this homeostatic balance in FvCSN5A RNAi plants may contribute to the Overexpression of FvPAO5 affected plant development and pollen viability We previously found that FaPAO5 negatively regulated fruit ripening ( Mo et al., 2020 ). The above results suggest that FvCSN5A plays a role in plant development and fruit ripening by affecting FvPAO5 stability. To determine which functions of FvCSN5A are mediated by FvPAO5, we produced and characterized FvPAO5 OE lines using Super1300:GFP (empty vector, EV) and Super1300:FvPAO5-GFP ( FvPAO5 OE) transgenic strawberry plants via the leaf disk transformation method with GFP as a selection tag (Fig. S8A-C). Two overexpression transgenic lines with different expression levels were used for subsequent experiments ( Fig. 10A ). Subcellular localization experiments suggested that FvPAO5 is expressed in both the cytoplasm and nucleus of FvPAO5 OE plants, which was consistent with previous results ( Fig. 10B ). Download figure Open in new tab Figure 10. Phenotypes of the FvPAO5 overexpression transgenic plants. ( A) Relative expression levels of FvPAO5 were determined in FvPAO5 overexpression (OE) transgenic plants by RT-qPCR. EV: Super1300:GFP; FvPAO5 OE: Super1300:FvPAO5-GFP. ( B) Subcellular localization of FvPAO5 in FvPAO5 OE transgenic plant leaves mesophyll cells. Bars, 20 μm. ( C) Compound leaves in WT and FvPAO5 OE transgenic plants. Bars, 1 cm. ( D) Fruit development and ripening process in WT and FvPAO5 OE transgenic plants. Bars, 0.5 cm. Photos were taken at 5 and 21 days after pollination respectively to record the fruit phenotype. ( E) The images showing Alexander’s staining of pollen grains in WT and FvPAO5 OE transgenic plants. The red-stained pollen grains are viable and fertile. ( F) The percentages of Alexander’s staining of pollen grains in WT and FvPAO5 OE transgenic plants. ( G-I) : Spm (G) , Spd (H) , and Put (I) content in WT and FvPAO5 OE transgenic leaves. ( J) DAB dye of pollen grains in WT and FvPAO5 OE transgenic plants. Bars, 20 μm. ( K) DAB dye of one compound leaf in WT and FvPAO5 OE transgenic plants. Bars, 1 cm. ( L) H 2 O 2 content in WT and FvPAO5 OE transgenic plants. ( M) Dap content in WT and FvPAO5 OE transgenic plants. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. The leaves of FvPAO5 OE-1 and FvPAO5 OE-2 plants exhibited different leaf shapes, numbers, and depths of serrations ( Fig. 10C ), with no significant differences in flower organ development between FvPAO5 OE and EV plants (Fig. S7C). Observations of the fruit developmental process in FvPAO5 OE plants revealed that the fruits of FvPAO5 OE were deformed and fruit ripening was inhibited ( Fig. 10D ). Alexander’s staining showed a significant reduction in pollen activity in the FvPAO5 OE lines ( Fig. 10E-F ). PAs and products of the FvPAO5-catalyzed reaction, including H 2 O 2 and Dap, were determined in the FvPAO5 OE plants. The results showed significantly lower PA contents in the FvPAO5 OE plant compared to the EV plant ( Fig. 10G-I ), while the accumulation of H 2 O 2 and Dap was greater in FvPAO5 OE plants than in EV plants ( Fig. 10J-M ). These experiments indicate that the overexpression of FvPAO5 also affect plant development and pollen viability, similar to the phenotype observed in FvCSN5A RNAi plants, suggesting a functional association between FvCSN5A and FvPAO5. FaCSN5A regulates fruit ripening and H 2 O 2 homeostasis partly depending on FaPAO5 To explore the genetic interactions between strawberry CSN5A and PAO5, we conducted a transient transgenic fruit experiment to obtain FaCSN5A and FaPAO5 co-RNAi strawberry fruits ( FaCSN5A RNAi/ FaPAO5 RNAi). Agrobacterium containing FaCSN5A RNAi or FaPAO5 RNAi vectors were injected into strawberry fruit individually or in combination, and photos were taken at 0, 3, 6, and 7 days after injection. The results showed that fruit coloring was inhibited in the FaCSN5A RNAi fruit, while it was promoted in the FaPAO5 RNAi fruit ( Fig. 11A-B ). The FaCSN5A RNAi/ FaPAO5 RNAi fruits colored more rapidly than the FaCSN5A RNAi fruits ( Fig. 11A-B ). The content of anthocyanin was similar to the observed phenotype ( Fig. 11C ). Additionally, the H 2 O 2 accumulation was also measured, revealing that the content of H 2 O 2 in FaCSN5A RNAi/ FaPAO5 RNAi fruits was lower than FaCSN5A RNAi fruits but higher than that in FaPAO5 RNAi fruits ( Fig. 11D ). These findings suggest that FaCSN5A regulates fruit ripening and H 2 O 2 homeostasis, partly depending on FaPAO5. Download figure Open in new tab Figure 11. FaCSN5A control fruit ripening and H 2 O 2 homeostasis partly depending on FaPAO5. ( A) Phenotypes of FaCSN5A RNAi, FaPAO5 RNAi, and FaCSN5A RNAi/ FaPAO5 RNAi strawberry fruits. Agrobacterium GV3101 strains containing recombinant plasmids were injected into DG fruits attached to the plant. The fruit phenotype was recorded at 0, 3, 6, and 7 days after injection. Bars, 0.5 cm. ( B) Analysis of FaCSN5A and FaPAO5 transcript levels in FaCSN5A RNAi, FaPAO5 RNAi, and FaCSN5A RNAi/ FaPAO5 RNAi strawberry fruits. Fruits were collected for experiments 7 days after injection. ( C-D) The anthocyanin content (C) and H 2 O 2 content (D) of FaCSN5A RNAi, FaPAO5 RNAi, and FaCSN5A RNAi/ FaPAO5 RNAi strawberry fruits. Fruits were collected for experiments 7 days after injection. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01; different letters indicate statistically significant differences at P<0.05. DISCUSSION Strawberries, as a perennial herbaceous plant, has emerged as a model organism for investigating the non-climacteric fruit ripening process, regulated by the intricate crosstalk of PAs with ABA, IAA, and ethylene ( Guo et al., 2018 ; Bai et al., 2021; Gao et al., 2021 ). Consistent with the earlier notion of "no growth, no PA production" ( Russell, 1973 ), the present data not only further corroborate the pivotal role of PAs in fruit ripening, but also provide novel insights into the efficacy of PAs in modulating plant growth, flowering, as well as fruit yield and quality in strawberries, especially by the CSN5-PAO5 module in strawberries. This underscores the significance of the COP9 signalosome-ubiquitination system in PA metabolism, underlying plant vegetative growth, reproductive growth, and ultimately fruit ripening and quality. Spd/Spm plays essential roles in strawberry vegetative and reproductive growth Our previous investigations have demonstrated that during strawberry fruit development, Put content gradually declined, reaching its nadir in the ripe fruit, while spermidine (Spd) content remained at low levels, albeit exhibiting a slight rise during fruit ripening ( Guo et al., 2018 ). Remarkably, spermine (Spm) content rapidly increased at the onset of ripening, becoming a dominant component of PAs in the ripe fruit. This proves an important role of Spd/Spm, particularly Spm, confirmed by the functional analysis of FaSAMDC1, a key enzyme for Spd/Spm biosynthesis ( Guo et al., 2018 ). Moreover, the protein expression levels of FaPAO5 gradually decreased during fruit development, indicating its function as a negative regulator of ripening, in line with observed the function of FaPAO5 and H 2 O 2 levels ( Mo et al., 2020 ). These studies uncover that elevated FaSAMDC1 and depressed FaPAO5 levels during fruit ripening synergistically promote Spd/Spm accumulation and regulate H 2 O 2 homeostasis, suggesting a cooperative interplay between PAs and ROS. Indeed, it was found that the expression of OsPAO5 in rice seedlings is induced by light and suppressed by darkness, with ospao5 mutants exhibiting lower H 2 O 2 levels, resulting in a longer mesocotyl, faster seedling growth, and improved crop yield ( Lv et al., 2021 ). However, the post-translational regulation of PAO5 remains elusive. Fortunately, an interactor of FvPAO5, FvCNS5A, has now been identified through comprehensive physiological, biochemical, and molecular evidence. Notably, FvCNS5A RNAi plants exhibited elevated levels of both FvPAO5 and H 2 O 2 ( Fig. 6 and 9 ) and reduced amounts of IAA, ABA, and JA ( Fig. 8 ), ultimately impairing plant growth and flowering, as well as fruit yield and ripening ( Fig. 3 - 5 ). Intriguingly, the contents of Spd and Spm were significantly lower in the RNAi plants ( Fig. 8 ), while Put levels remained unchanged ( Fig. 8 ). Given that strawberry PAO5 acts as a negative regulator of ripening by catalyzing the degradation of Spm/Spd in the TC-type ( Mo et al., 2020 ), thus the stable Put accumulation is not derived from Spd/Spm degradation. The downregulation of both ADC1 and SAMDC1 expression in the RNAi plants (Fig. S5) suggests that the stable Put level might result from the inhibited Spd biosynthesis, while the PAO5-mediated degradation may function as a feedback control mechanism, potentially via H 2 O 2 . To a large extent, the homeostasis and balance among Put, Spd, and Spm as well as H 2 O 2 are essential for strawberry vegetative and reproductive growth. When this balance is disrupted by abundant FvPAO5 in the FvCSN5A RNAi plants, the conversion of Put to Spm is blocked as a sign of the undetectable Spd, ultimately leading to abnormal plant development, particularly in the reproductive phase. Based on our current data and a recent report that the regulation of PA composition, especially the Put/Spm ratio, may be an effective strategy to enhance plant survival and fitness ( Navakoudis and Kotzabasis, 2022 ), we conclude that PA homeostasis is essential for strawberry growth and development in response to environmental cues through the CSN5A-PAO5 model-mediated PA and H 2 O 2 regulation. FvCSN5-mediated FvPAO5 degradation coordinates PA and ROS homeostasis to fine-tune strawberry growth In plants, ROS functions as a double-edged sword, highlighting the importance of ROS homeostasis to accommodate diverse cellular roles and environmental responses ( Farooq et al., 2019 ). Based on the available data and our present findings related to PAs, we summarize several key points, including (1) the dual roles of PAs as both ROS scavengers and triggers; (2) the multiple PA components with common and specialized cellular and biological processes, such as Put in photosynthesis, Spm in antioxidants, and Spd in both photosynthetic protection and oxidation resistance; (3) the essential role of Spd/Spm in reproductive growth and fitness; (4) the relationship between increased PAO5 activity and higher H 2 O 2 levels; (5) the CSN5-mediated FvPAO5 degradation participates is plant vegetative and reproductive growth, suggesting that PAO5 may serve as a hub regulatory checkpoint for the balance between PAs and ROS, which are crucial for plant development and adaptation. Earlier reports indicate that CSN5 is a key component of the COP9 signalosome and participates in various plant processes, including light signaling, circadian rhythm control, cell recycling, and IAA signaling ( Chamovitz and Segal, 2001 ; Serino and Deng, 2003 ). CSN5 functions by the deneddylation of NEDD8 from the cullin subunit of CRL E3 ligases ( Schwechheimer and Isono, 2010 ). This NEDD8-deneddylation is a post-translational modification that affects the activity of cullin-based and non-cullin substrates, while the CSN-mediated deneddylation is required but not essential for CRL-mediated processes ( Mergner and Schwechheimer, 2014 ). Consistent with previous reports, the complete loss of CSN function results in seedling lethality ( Stratmann and Gusmaroli, 2012 ). We observed that the inhibition of FvCSN5A expression led to developmental defects in strawberry leaves, flowers, and fruits ( Fig. 3 - 5 and Fig. S4), and also affected PA levels and H 2 O 2 production ( Fig. 8 - 9 ). Interestingly, the overexpression of FvPAO5 similarly affected plant development and pollen viability, akin to the phenotype of FvCSN5A RNAi plants ( Fig. 10 ), suggesting a functional link between FvCSN5A with FvPAO5. This association is confirmed by a transient fruit transgenic experiment, showing that inhibiting FaPAO5 expression could partially rescue the ripening phenotype of FaCSN5A RNAi fruits ( Fig. 11 ). These data highlight that the post-translational modification of FvPAO5 by FvCSN5A contributes to the regulation of PA and ROS homeostasis, fine-tuning plant vegetative and reproductive growth. The COP9 signalosome (CSN) controls the RUB/NEDD8 modification of several cullin-based E3 ligases, such as CUL□CDD□RBX1, and CUL□DDB1□COP1□SPA ( Fischer et al., 2011 ; Qin et al., 2020 ; Dong et al., 2024 ). In the present study, we found that the expression pattern of FvCUL1 was similar to that of FvCSN5A during fruit ripening, and the two proteins co-localized in the cytoplasm. Specially, FvCSN5A interacted in vitro and in vivo with FvCUL1 ( Fig. 2 and 7 ), a core component of E3 ubiquitin-protein ligase complexes. We further found the deneddylated and neddylated forms of FvCUL1 in the wild-type, with the protein levels of FvCUL1 markedly decreased in the FvCSN5A RNAi plants ( Fig. 7 ). Additionally, FvCSN5A promoted the proteasome-dependent degradation of FvPAO5 in a ubiquitin-mediated manner ( Fig. 6 ). Altogether, the CSN5A-CUL1-PAO5 pathway responsible for PA and H 2 O 2 homeostasis, is crucial for strawberry plant growth and fruit ripening. An integrative model for FvCSN5A and FvPAO5 synergistically regulates the balance among Put, Spd and Spm essential to strawberry vegetative and reproductive growth by phytohormones and H 2 O 2 To date, we have gained a more comprehensive understanding of the roles of PAs in embryo development, seed germination, seedling growth, reproductive development, and fruit ripening, as well as the relationship between PAs, hydrogen peroxide (H 2 O 2 ), and phytohormone signaling. There is a close relationship between PAs and phytohormones in plant stomatal movement, survival fitness, and fruit ripening ( Mo et al., 2020 ; Liu et al., 2023 ; Song et al., 2023 ; Gao et al., 2021 , 2024a ), with a particular emphasis on the interaction of PAs with ethylene in climacteric tomato fruit ripening and with ABA in non-climacteric strawberry fruit ripening ( Gao et al., 2021 ). During the initiation of strawberry fruit ripening, increased ABA suppresses the expression of FaPAO5 and reduces H 2 O 2 production, ultimately facilitating the accumulation of Spm/ Spd and fruit ripening ( Mo et al., 2020 ). Largely, PAs, especially Spm, regulate strawberry fruit ripening in an ABA-dominated, IAA-participating, and ethylene-coordinated manner ( Guo et al., 2018 ). Similarly, in response to drought stress, the production of ABA controls PA homeostasis through both reducing Put biosynthesis and accelerating PA metabolism, in the process of which IAA is suggested to link to not only Put-regulating photosynthesis and oxidative phosphorylation, but also ABA-regulating sugar and flavonoid metabolism ( Gao et al., 2024b ). Thus, we consider that PAs, ABA, and IAA weave a core signaling network essential for plant growth and adaptation, focusing on the role of Put in photosynthesis and primary metabolism related to vegetative growth and plant fitness, and of Spd/Spm in secondary metabolism and survival fitness related to reproductive growth and fruit ripening. To a large extent, this may highlight PA metabolism acting as a feedback control mechanism in response to developmental and environmental stimuli. In the present study, we found that the levels of IAA, ABA, and JA were suppressed in FvCSN5A RNAi plants, especially IAA with an 83% reduction; while the levels of both H 2 O 2 production and FvPAO5 expression were significantly elevated compared to the WT ( Fig. 8 and 9 ). These data suggest that the inhibition of FvCSN5A expression significantly suppresses PA and IAA accumulation, accompanied by H 2 O 2 burst, ultimately resulting in growth defects, retarded fruit ripening, and poor fertility phenotype. This is consistent with a previous report that OsPAO5 serves as a negative regulator for the mesocotyl elongation of rice seedlings, thus its knockout mutants facilitate faster seedling growth, ultimately increasing grain weight and yield by the higher accumulation of Spm and lower accumulation of H 2 O 2 and JA ( Lv et al., 2021 ). Also, in Arabidopsis, the loss of AtPAO5 function inhibits the CK-induced shoot meristem formation from lateral root primordia ( Kaszler et al., 2023 ), potentially as a result of the interference of CK and IAA signaling in xylem differentiation ( Alabdallah et al., 2017 ). Overall, the synergistic interaction between PAs and phytohormones lays a basis for the CSN5-PAO5 module responsible for the trade-off vegetative and reproductive growth mediated by H 2 O 2 . Based on the available data and the present study, we propose a comprehensive model for the FvCSN5A-FvPAO5 coordinate regulation of PA metabolism and H 2 O 2 homeostasis, which aligns with plant growth in response to environmental cues in strawberries ( Fig. 12 ). The FvCSN5A-FvCUL1-FvPAO5 model provides novel insights into the integration of the COP9 signalosome into PA metabolism linked to IAA/ABA/JA by ROS, at least in model plant strawberries. Anyway, this may open a promising door for improvement of crop yield and quality through manipulating CSN5-PAO5 pair. Next, how the proposed model integrates early environmental cues by H 2 O 2 underlying plant development and adaptation, is an intriguing question for further investigation. Download figure Open in new tab Figure 12. Proposed working model An equilibrium regulation for the composition and content of Put, Spd and Spm, is programmed by PA biosynthesis and degradation: Put-related key enzymes is ADC1; Spd/Spm-related key enzymes include SAMDC1, SPDS, and SPDM for biosynthesis and PAO5 for degradation. In response to environments, the two equilibria from Put/Spd/Spm metabolism to vegetative/reproductive growth are fine-tuned at the transcriptional, translational, and post-translational levels to optimize plant growth and breeding. The interaction of CSN5A with CUL1 mediates the degradation of PAO5, thereby rapidly controlling PA balance and H 2 O 2 production, which can be integrated with various phytohormones. Finally, PAs, ROS, and phytohormones synergistically regulate strawberry development and adaptation. CSN5A is essential for the deneddylation of CUL1, potentially affecting the protein stability of CUL1 and the activity of E3 ubiquitin ligase. In FvCSN5A RNAi plants, CUL1 protein levels markedly decrease, while PAO5 levels increase. These changes lead to significant decreases in PA, Auxin, ABA, JA content, and excessive accumulation of H 2 O 2. The homeostasis disruption in FvCSN5A RNAi plants may be responsible for the growth defects, slow fruit ripening and poor fertility phenotype. Materials and methods Plant materials and growth conditions Octoploid strawberries ( Fragaria × ananassa Duch. cv Benihoppe) grown in a greenhouse under natural sunlight conditions were used for transient transgenic experiments. Diploid strawberries ( Fragaria vesca , Ruegen) and Nicotiana benthamiana were cultured in an artificial climate chamber at 25°C with a 16-h light/8-h dark photoperiod, 55% relative humidity, and a light intensity of 100 μmol m -2 s -1 . RNA isolation and RT-qPCR Total RNA was extracted using the Plant Total RNA Mini-Extraction Kit (Magen, China) and qPCR Kit (YEASEN, China). RT-qPCR was performed on a Bio-Rad CFX96 system using the Trans Start Top Green qPCR Super Mix Kit (YEASEN, China) with Actin as an internal control. Yeast two-hybrid assay For the yeast two-hybrid cDNA library screening, the coding sequence (CDS) of FvPAO5 was cloned into a pGBKT7 vector. FvPAO5-BD was transformed into a yeast gold strain. The transformed yeast strain expressing FvPAO5 was subsequently mated with a strawberry fruit cDNA library. After 22 to 24 h, the culture was plated onto SD/-Ade-His-Leu-Trp medium and incubated at 30°C for 3 to 5 days. Yeast colonies that grew on the medium were subsequently identified by PCR amplification and sequencing. For the one-to-one yeast two-hybrid assay, the CDS of FvPAO5 and FvCUL1 were individually cloned into the pGBKT7 vector, while the CDS of FvCSN5A was cloned into the pGADT7 vector. BD-FvPAO5 and AD-FvCSN5A, as well as BD-FvCUL1 and AD-FvCSN5A, were transformed into yeast cells, respectively, as previously reported ( Huang et al., 2024 ). The transformed yeast cells were cultured on SD/-Leu-Trp medium and incubated at 28°C for 3 days. Subsequently, the yeast cells were transferred to both SD/-Leu-Trp and SD/-Ade-His-Leu-Trp media, and incubated at 28°C for an additional 3 days before photos were taken. Autoactivation assays were performed for each bait and prey construct with the corresponding empty vectors to exclude potential false positives. Subcellular localization The CDS regions of FvCSN5A , FvCUL1 , and FvPAO5 were constructed into the pCAMBIA1300-GFP vector and transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. Nuclei were stained with DAPI in N. benthamiana leaves, and px-RK/CD3-983/CD3-983 was used as a marker for the peroxisome ( Nelson et al., 2007 ). Visualization and imaging were performed using a Leica Stellaris 5 laser confocal microscope. Firefly luciferase complementation (FLC) assay The CDS of FvPAO5 and FvCUL1 were cloned into pCAMBIA1300-NLUC, while the CDS of FvCSN5A was introduced into pCAMBIA1300-CLUC. These constructs were co-transfected into Nicotiana benthamiana leaves, and luminescence signals were recorded with a cold CCD camera 3 days after post-transfection. Bimolecular fluorescence complementation (BiFC) assay The CDS of FvCSN5A was cloned into pSPYNE173, and the CDS of FvPAO5 was cloned into pSPYCE. FvPAO5-YCE and FvCSN5A-YNE were co-transfected into Nicotiana benthamiana leaves. GFP signals were detected using laser confocal microscopy (Leica Stellaris 5) after 3 days. Co-Immunoprecipitation (CoIP) assay Super1300:FvCSN5A-MYC and Super1300:FvPAO5-GFP constructs were cotransfected into Nicotiana benthamiana leaves. Leaves were collected after 2 days, and total proteins were extracted with a protein lysis buffer (100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% TritonX-100, 1% Tween-20 and 1% NP-40). The extracted proteins were incubated with anti-MYC beads (AlpalifeBio, Cat. No.:KTSM1306) for 3 h. The target proteins were detected with an anti-MYC antibody (TransGen Biotech, Cat. No.:HT101-01; dilution 1:3000) and an anti-GFP antibody (TransGen Biotech, Cat. No.: HT801-01; dilution 1:3000). Co-immunoprecipitation mass spectrometry (MS) This experiment was conducted according to the instructions of the ProteinA/G Immunoprecipitation Kit (Solarbio, Cat. No.: M2400). Total proteins were extracted from WT and FvCSN5A RNAi-1 leaves (10 g) using a protein extraction solution (100 mM HEPES, 1 mM EDTA, 10% glycerol, 1% TritonX-100, 1 mM DTT, 1 mM PMSF, 1% protease inhibitor cocktail, pH 7.8). Anti-FvPAO5 antibody was added to the pre-treated magnetic beads, and then total proteins were incubated with the beads for 1 h. The magnetic beads with bound proteins were washed 3 times using the extraction buffer. Finally, the magnetic beads with proteins were frozen in liquid nitrogen and used for mass spectrometry detection. GST pull-down assay The CDS of FvPAO5 was cloned into pGEX4T-1 (GST-tagged) and the CDS of FvCSN5A was cloned into pET-30a (His-tagged). Proteins were expressed in BL21 Escherichia coli . Recombinant FvPAO5-GST was purified and incubated with 20 µL glutathione Sepharose 4B in pull-down buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM β-mercaptoethanol, 3 mM EDTA, pH 8.0, and 1% NP-40) for 1 h, and then FvCSN5A-His was added and incubated for 1 h. The proteins were then detected by immunoblotting using anti-His (TransGen Biotech, Cat. No.: HT501-01; dilution 1:3000) and anti-GST (TransGen Biotech, Cat. No.: HT601-01; dilution 1:3000) antibodies. Cell-free protein degradation assay Cell-free degradation assays were carried out as described previously with little modification ( Kong et al., 2015 ). The leaves of three-month-old ‘Ruegen’ strawberry seedlings were harvested and ground in liquid nitrogen. Total plant proteins were extracted using 0.5 g powder with 1.5 mL protein lysis buffer (100 mM HEPES, 1 mM EDTA, 10% glycerol, 1% TritonX-100, 1 mM DTT, 1 mM PMSF, 1% protease inhibitor cocktail, pH 7.8). The purified FvPAO5-GST protein (2.5 µg) and total strawberry protein were mixed with either DMSO or 50 µM MG132, respectively, and the samples were incubated at 20°C for various durations. Anti-GST (TransGen Biotech, Cat. No.: HT601-01; dilution 1:3000) and anti-Actin (BBI, Cat. No.: D110007-0100; dilution 1:3000) antibodies were used for immunoblotting detection. In vivo ubiquitination assay In vivo ubiquitination assays were carried out as described previously ( Ye et al., 2018 ). Three-month-old WT and FvCSN5A RNAi transgenic seedlings were cultured in liquid MS medium at 25°C for 5 h, with or without DMSO or 50 μM MG132 (stock solution prepared with 0.5% DMSO) and 100 μM CHX (cycloheximide, an inhibitor of protein synthesis). The total proteins were extracted as described above, and the total protein was incubated with ubiquitin-trap bead p62-agarose at 4°C for 4 h. Centrifuge at 1000 g for 1 min at 4 □, followed by discarding the supernatant, washing with 1 mL of PBS, and repeating this wash step twice. The ubiquitination signal was detected by an anti-PAO5 antibody (Beijing Protein Innovation; dilution 1:3000), while the input sample was detected by an anti-Actin antibody (BBI, Cat. No.: D110007-0100; dilution 1:3000). Protein changes in strawberry fruit development The small green, white, and full red fruits of diploid strawberries were selected, achenes were removed, and samples were ground in liquid nitrogen. Total plant proteins were then extracted using 0.4 g powder with 200 µL protein lysis buffer (100 mM HEPES, 1 mM EDTA, 10% glycerol, 1% TritonX-100, 1 mM DTT, 1 mM PMSF, 1% protease inhibitor cocktail, pH 7.8). The target proteins were detected using the following antibodies: anti-FvPAO5 (Beijing Protein Innovation; dilution 1:3000), anti-FvCSN5A (Beijing Protein Innovation; dilution 1:3000), anti-CUL1 (PHYTO, Cat. No.: PHY1861S; dilution 1:3000), and anti-Actin (BBI, Cat. No.: D110007-0100; dilution 1:3000) antibody. Transient transgene expression in strawberry fruits was performed as previously described ( Chen et al., 2024 ). The CDS of FaCSN5A and FaPAO5 was constructed into pCAMBIA1300-GFP vector to obtain the overexpression vector, while a 300 bp FaCSN5A and FaPAO5 fragment was constructed into pK7GWIWG2 (II) vector for RNAi. Agrobacterium transformed FaCSN5A OE, control and FaCSN5A RNAi were incubated at 28°C with a final OD 600 value of 0.5, and de-greening stage fruits were selected to inject. Ten fruits were used in each treatment. Photos were taken at 0, 2, 3, 4, and 5 days to record the phenotypes. Samples were harvested and achenes were removed 5 days after injection. The injection site was cut, and the injected parts were frozen in liquid nitrogen and stored at −80°C. To explore the genetic interactions of strawberry CSN5A and PAO5, Agrobacterium transformed FaCSN5A RNAi, control, and FaPAO5 RNAi were incubated at 28°C until reaching an OD 600 value of 0.5, and de-greening stage fruits were selected injection according to control, FaCSN5A RNAi, FaPAO5 RNAi, FaCSN5A RNAi and FaPAO5 RNAi combinations. Each treatment included ten fruits. The fruit phenotype was recorded through photography at 0, 3, 6, and 7 days after injection. Samples were harvested and achenes were removed 7 days post-injection. The injection sites were excised, frozen in liquid nitrogen, and stored at −80°C. Stable transformation in diploid strawberry The FvCSN5A RNAi (pK7GWIWG2 II: FvCSN5A 300 bp -DsRed) and FvPAO5 OE (Super1300:FvPAO5-GFP) constructs were transformed into the Agrobacterium tumefaciens strain GV3101, respectively. Agrobacterium tumefaciens cultures were incubated at 28°C, with a final OD 600 value of 0.5, and then infiltrated into the diploid strawberry "Ruegen" using diploid strawberry leaf disks as described ( Mao et al., 2022 ; Li et al., 2023 ; Lu et al., 2024 ). In this process, positive transgenic calli and regenerated plants were selected using DsRed or GFP fluorescence examined under a fluorescent protein observation lamp (LUYOR-3415RG). Fourteen FvCSN5A RNAi and ten FvPAO5 OE transgenic seedlings were obtained. The experiment was carried out with T1 generation materials. In vitro germination of pollen grains The anthers from freshly opened WT and FvCSN5A RNAi transgenic flowers were harvested. Pollen grains were released and incubated with pollen grain germination medium (10% sucrose, 0.002% boric acid, and 0.5% agar) at 22°C for 5 h. The pollen grains were taken to slides and photographed using a Leica DM750 microscope. Alexander’s staining The anthers from freshly opened WT, FvCSN5A RNAi and FvPAO5 OE transgenic flowers were harvested and Alexander’s staining solution (Coolabor, China) was applied to the anthers. The anthers were gently peeled off with tweezers, and the pollen grains were released into the staining solution for 6 h. Subsequently, the pollen grains were observed and photographed using a Leica DM750 microscope. Determination of anthocyanin contents Samples (n=3) for determination of anthocyanin content was carried out according to the procedure described in the plant anthocyanin content detection kit (Solarbio, China). 0.1 g of strawberry sample powder was used in this experiment. Determination of soluble sugar contents Soluble sugar content was determined by the previous method ( Huang et al., 2019 ). Samples (n=3) of 0.5 g strawberry fruit powder was dissolved in 80% ethanol at 80°C for 30 min and repeated three times. After centrifugation, the supernatant was evaporated to dryness and dissolved in ultrapure water. The solution was filtered using LC-18 SPE and determined by HPLC. D-(+) glucose, D-(-) fructose and sucrose were used as standards. Determination of polyamines, 1, 3-diaminopropane (Dap), and phytohormones PAs and Dap were determined according to the previous method ( Guo et al., 2018 ) with little modification. Samples (n=3) of fresh strawberry leaves (0.5 g) were homogenized in 4 mL of 5% (v/v) cold perchloric acid and incubated at 4 °C for 1 h. Centrifugation was carried out at 4□ for 30 min at 12,000 g. The extracted supernatant was mixed with benzoyl chloride and subsequently analyzed for PAs and Dap using an HPLC system. To determine the accumulation of Dap after exogenous Spd treatment, leaves were sampled 24 h post-treatment, and Dap content was carried out using the aforementioned methods. Each treatment was replicated three times. Phytohormone contents were determined out according to the method described by Du et al. (2012) with minor modifications. Strawberry leaf samples (0.5 g FW) were ground into a fine powder in phytohormones and extracted with a modified Bieleski’s solvent (methanol/formic acid/water 15:1:4) overnight at 4°C. The extract was centrifuged at 12,000 rpm for 15 min at 4°C, followed by concentration and purification using primary secondary amine (PSA) and C 18 tandem dual SPE cartridges. Finally, the extract was passed through a 0.45 µm organic filter membrane and analyzed by HPLC-MS/MS. DAB staining For DAB (3,3’-Diaminobenzidine) staining, strawberry leaves, and anthers were incubated in DAB working staining solution (DAB color development kit, Solarbio, China) at room temperature for 4 h. Subsequently, the samples were decolorized using 95% ethanol at 80°C for 20 min. The DAB samples were then immersed in the preservation solution for 30 min. Pollen grains and leaves were photographed using a Leica DM750 microscope. Measurement of ROS production For fluorescent detection of ROS using DCFH-DA, strawberry leaves and anthers were incubated with 5 µM DCFH-DA working staining solution (Solarbio, China) at room temperature for 10 min, and washed twice in PBS. Pollen grains and leaves were observed and photographed with a Leica Stellaris 5 laser confocal microscope. Samples (n=3) for measurement of H 2 O 2 following the method reported by Gay and Gebicki (2003) , 0.2 g of strawberry leaf powder was incubated with pre-cooled acetone extraction buffer, and then extractant [(CCL4: CHCL3=3:1 (V:V)] and distilled water were added. The solution was centrifuged at 5000 r/min for 10 min, and the upper aqueous phase was taken to add the working reagent. After 30 min 30 □ water bath, the content of H 2 O 2 was determined at A560 nm. Ploidy analysis Cell nuclei were extracted and stained according to the guidelines of the CyStainR UV Precise P kit. The chromosome ploidy of young strawberry leaves from WT (diploid and tetraploid), FvCSN5A RNAi-1, FvCSN5A RNAi-2, Super1300-GFP (EV), Super1300:FvPAO5-GFP ( FvPAO5 OE-1 and FvPAO5 OE-2) plants was analyzed using a CyFlowR space flow cytometer. Statistical analysis All data are presented as means of at least three independent biological replicates. Statistical analyses were performed using SigmaPlot version 12.0 (Systat Software, USA). Data are presented as the analysis of variance (ANOVA) was performed. Asterisks indicate significant differences between treatments, assessed by Student’s t-test: *, P < 0.05; **, P < 0.01. Different letters indicate statistically significant differences at P < 0.05 as determined by ANOVA. A summary of the statistical analyses is provided in Supplemental Data Set 3. Author contributions GF and GJX conceived the study and managed the projects. HY, GF, GJX, and SYY designed and guided the experiments. GJH, HY, and JGM performed most experiments. LWJ performed the determination of polyamines, Dap and H 2 O 2 . WQH performed yeast two-hybrid cDNA library screening assays. WJX assisted with the ploidy analysis. All authors analyzed the data. HY, GF, GJX, and GJH wrote and revised the article. Accession Numbers The strawberry gene sequences from the F. vesca genome ver4.0 can be downloaded from the Genome Database for Rosaceae ( http://ww.rosaceae.org ) with accession numbers: FvPAO5, FvH4_7g01430; FvCSN5A, FvH4_2g36890; FvCUL1, FvH4_2g25090; FvCSN1, FvH4_7g1333; FvCSN2, FvH4_4g27570; FvCSN3, FvH4_6g52990; FvCSN4, FvH4_2g40000; FvCSN6A, FvH4_3g41360; FvCSN7, FvH4_7g10110; FvCSN7L, FvH4_5g19860; FvCSN8, FvH4_5g29770; FvSUT1, FvH4_5g33660; FvCHS, FvH4_7g01160; FvANS, FvH4_5g01170; FvPG, FvH4_7g01140; FvCEL, FvH4_4g33940; FvSS, FvH4_1g09360; FvADC1, FvH4_2g10570; FvSAMDC1, FvH4_6g45760; FvSPDS, FvH4_4g34680; FvSPMS, FvH4_7g31820. Data availability The data underlying this article are available in the article and its online supplementary material. Competing interests The authors declare that no competing interests exist. Supplemental files Supplemental Figure 1. F v CSN5A RNAi transgenic plants screening. ( A) The red calluses were FvCSN5A RNAi successful transgenic calluses, which were named FvCSN5A RNAi-1 and FvCSN5A RNAi-2. ( B) Visualization of DsRed accumulation in FvCSN5A RNAi transgenic plants (right). Bars, 1 cm. Supplemental Figure 2. Peak plot of chromosome ploidy of FvCSN5A RNAi strawberries. The young leaves of WT and FvCSN5A RNAi transgenic plants were used for chromosome ploidy analysis. The x-axes show the DNA contents, and the y-axes indicate cell numbers. Supplemental Figure 3. Phenotype of FvCSN5A RNAi transgenic plants ( A) Leaf shape index of WT and FvCSN5A RNAi transgenic plants. ( B) Numbers of the serrations in WT and FvCSN5A RNAi transgenic plants. ( C-D) The images of pistil (C) and anther (D) in WT and FvCSN5A RNAi transgenic plants. Bars, 0.5 mm. ( E-F) Numbers of pistil (E) and anther (F) in WT and FvCSN5A RNAi transgenic plants. ( G) The expression of COP9 signalosome subunits in WT and FvCSN5A RNAi-1 fruits. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. Non-significant: ns Supplemental Figure 4. Fruit Phenotype of FvCSN5A RNAi-2 transgenic fruits ( A) Reciprocal crosses between WT and FvCSN5A RNAi-2 transgenic fruits. ( B) Observation of fruit development process of two representative FvCSN5A RNAi-2 transgenic fruits. Photos were taken at 15, 17, 19, 21, 23, and 25 days after pollination (DAP) respectively to record the fruit phenotype. Bars, 0.5 cm. Supplemental Figure 5. Manipulation of FaCSN5A expression affected physiological parameters and expression levels of ripening□related genes ( A) Phenotypes of FaCSN5A OE and FaCSN5A RNAi strawberry fruits. Agrobacterium GV3101 strains containing RNAi or overexpression FaCSN5A recombinant plasmids were injected into DG fruits attached to the plant. The fruit phenotype was recorded at 0, 2, 3, 4, and 5 days after injection. Bars, 1 cm. ( B) FaCSN5A expression levels in FaCSN5A OE and FaCSN5A RNAi fruits compared with the control. ( C-E) The anthocyanin content (C) , soluble sugar content (D) , and firmness (E) in FaCSN5A OE and FaCSN5A RNAi strawberry fruits. ( F) RT-qPCR was used to analyze the genes’ expression in FaCSN5A OE and FaCSN5A RNAi fruits. Relative expression levels were calculated using the 2 -△△Ct method. FaPG: polygalacturonase; FaCEL: cellulose; FaCHS: chalcone synthase; FaANS: anthocyanidin synthase; FaSUT1: sucrose transporter1; FaSS: sucrose synthase. FaADC1: arginine decarboxylase1; FaSAMDC1: S-adenosyl-methionine decarboxylase1; FaSPDS1: spermidine synthase1; FaSPMS: spermine synthase; FaPAO5: polyamine oxidase5. Statistical significance of one-way ANOVA: *, P < 0.05; **, P < 0.01. Supplemental Figure 6. Subcellular localization of FvCUL1 Subcellular localization of FvCUL1-GFP fusions in transiently transformed N. benthamiana leaves. All experiments were performed 48 h post-infiltration. Bars, 20 μm Supplemental Figure 7. DAB dye of roots from WT and FvCSN5A RNAi transgenic plants. Seeds of WT and FvCSN5A RNAi transgenic plants were grown on MS medium for 3 months, and then the roots were harvested and stained with DAB. Supplemental Figure 8. F v PAO5 OE transgenic plants screening and flower phenotype. ( A) Detection of FvPAO5 OE (Super1300: FvPAO5-GFP) successful transgenic callus. ( B) Visualization of GFP fluorescence in FvPAO5 OE plants. Bars, 1 cm. ( C) Peak plot of chromosome ploidy of FvPAO5 OE strawberries. ( D) The flower phenotype of FvPAO5 OE transgenic plants. Bars, 1cm. Supplemental Data Set 1 Identification of FvPAO5 associated proteins by CoIP/MS. Supplemental Data Set 2 The list of primers used in the experiments. Supplemental Data Set 3 Statistical analyses. Acknowledgments This work was supported by the National Natural Science Foundation of China (32030100; 32272648; 32072516; 32476225; 32372672) and R&D Program of Beijing Municipal Education Commission (KM202310020013). Partly supported by the open funds of the State Key Laboratory of Plant Environmental Resilience (SKLPERKF2407). Footnotes Authors’ email address: Yun Huang: yunhuang{at}bua.edu.cn , Jiahui Gao: gjh696969{at}126.com , Guiming Ji: 1074166581{at}qq.com , Wenjing Li: 17316112071{at}163.com , Jiaxue Wang: 1024963127{at}qq.com , Qinghua Wang: qinghuasummer{at}163.com , Yuanyue Shen: shenyuanyuesyy{at}163.com Section on Results updated to clarify FvCSN5A interacted with FvCUL1 in vitro and in vivo, and FvCSN5-mediated FvPAO5 degradation coordinates PA and ROS homeostasis; Figure 1 (E), Figure 3 (B-C), Figure 7 (E), and Figure 9 (F) revised; Figure 10 and 11 added; author affFliations updated; Supplemental files updated (Figure S1 (B) and Figure S4 (B) revised; Figure S2, S3, S6, S7 and S8 added; Data set 1 CoIP/MS and Data Set 3 Statistical analyses added). References ↵ Alabdallah O , Ahou A , Mancuso N , Pompili V , Macone A , Pashkoulov D , Stano P , Cona A , Angelini R , Tavladoraki P . The Arabidopsis polyamine oxidase/dehydrogenase 5 interferes with cytokinin and auxin signaling pathways to control xylem differentiation . Journal of Experimental Botany , 2017 , 68 ( 5 ): 997 – 1012 . OpenUrl CrossRef PubMed ↵ Aloisi I , Piccini C , Cai G , Del Duca S . Male Fertility under Environmental Stress: Do Polyamines Act as Pollen Tube Growth Protectants? . International Journal of Molecular Sciences , 2022 , 23 ( 3 ): 1874 . OpenUrl CrossRef PubMed ↵ Angelini R , Cona A , Federico R , Fincato P , Tavladoraki P , Tisi A . Plant amine oxidases "on the move": an update . Plant Physiology and Biochemistry , 2010 , 48 ( 7 ): 560 – 564 . OpenUrl CrossRef PubMed Web of Science ↵ Barry M , Früh K . Viral modulators of cullin RING ubiquitin ligases: culling the host defense . Science’s STKE , 2006 , 2006 ( 335 ): p e21 . OpenUrl ↵ Blázquez MA. Polyamines: Their Role in Plant Development and Stress . Annual Review of Plant Biology , 2024 , 75 . Available from : doi: 10.1146/annurev-arplant-070623-110056 OpenUrl CrossRef ↵ Chamovitz DA , Segal D . JAB1/CSN5 and the COP9 signalosome. A complex situation . EMBO Reports , 2001 , 2 ( 2 ): 96 – 101 . OpenUrl Abstract / FREE Full Text ↵ Chen X , Gao J , Shen Y . Abscisic acid controls sugar accumulation essential to strawberry fruit ripening via the FaRIPK1-FaTCP7-FaSTP13/FaSPT module . The Plant Journal , 2024 , 119 ( 3 ): 1400 – 1417 . OpenUrl CrossRef PubMed ↵ Cope GA , Deshaies RJ . COP9 signalosome: A multifunctional regulator of SCF and other cullinDbased ubiquitin ligases . Cell , 2003 , 114 : 663 – 671 . OpenUrl CrossRef PubMed Web of Science ↵ Dong J , Li Y , Cheng S , Li X , Wei N . COP9 signalosome-mediated deneddylation of CULLIN1 is necessary for SCF EBF1 assembly in Arabidopsis thaliana . Cell reports , 2024 , 43 ( 1 ): 113638 . OpenUrl CrossRef PubMed ↵ Du F , Ruan G , Liu H . Analytical methods for tracing plant hormones . Analytical and Bioanalytical Chemistry , 2012 , 403 ( 1 ): 55 – 74 . OpenUrl CrossRef PubMed ↵ Farooq MA , Niazi AK , Akhtar J , Saifullah , Farooq M , Souri Z , Karimi N , Rengel Z . Acquiring control: The evolution of ROS-Induced oxidative stress and redox signaling pathways in plant stress responses . Plant Physiology and Biochemistry . 2019 , 141 : 353 – 369 . OpenUrl CrossRef PubMed ↵ Fischer ES , Scrima A , Böhm K , Matsumoto S , Lingaraju GM , Faty M , Yasuda T , Cavadini S , Wakasugi M , Hanaoka F , Iwai S , Gut H , Sugasawa K , Thomä NH . The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation . Cell , 2011 , 147 : 1030 – 1039 . OpenUrl ↵ Fortes AM , Agudelo-Romero P , Pimentel D , Alkan N . Transcriptional modulation of polyamine metabolism in fruit species under abiotic and biotic stress . Frontiers in Plant Science , 2019 , 10 : 816 . OpenUrl CrossRef PubMed ↵ Furumoto T , Yamaoka S , Kohchi T , Motose H , Takahashi T . Thermospermine is an evolutionarily ancestral phytohormone required for organ development and stress responses in Marchantia polymorpha . Plant & Cell Physiology , 2024 , 65 ( 3 ): 460 – 471 . OpenUrl CrossRef PubMed ↵ Gao F , Mei XR , Li YZ , Guo JX , Shen YY . Update on the roles of polyamines in fleshy fruit ripening, senescence, and quality . Frontiers in Plant Science , 2021 , 12 : 610313 . OpenUrl CrossRef PubMed ↵ Gao F , Li JY , Li WJ , Shi S , Song SH , Shen YY , Guo JX . Abscisic acid and polyamines coordinately regulate strawberry drought responses . Plant Stress , 2024a , 11 : 100387 . OpenUrl CrossRef ↵ Gao F , Guo J , Shen Y . Advances from chlorophyll biosynthesis to photosynthetic adaptation, evolution and signaling . Plant Stress , 2024b , 12 : 100470 . OpenUrl CrossRef ↵ Gay CA , Gebicki JM . Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method . Anal Biochemistry , 2003 , 315 ( 1 ): 29 – 35 . OpenUrl CrossRef PubMed ↵ Groppa MD , Benavides MP . Polyamines and abiotic stress: recent advances . Amino Acids , 2008 , 34 ( 1 ): 35 – 45 . OpenUrl CrossRef PubMed Web of Science ↵ Guo J , Wang S , Yu X , Dong R , Li Y , Mei X , Shen Y . Polyamines regulate strawberry fruit ripening by abscisic Acid, auxin, and ethylene . Plant Physiology . 2018 , 177 ( 1 ): 339 – 351 . OpenUrl Abstract / FREE Full Text ↵ Gupta K , Sengupta A , Chakraborty M , Gupta B . Hydrogen peroxide and polyamines act as double edged swords in plant abiotic stress responses . Frontiers in Plant Science , 2016 , 7 : 1343 . OpenUrl PubMed ↵ Gusmaroli G , Feng S , Deng XW . The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome complexes, and mutations in their JAMM domains exhibit differential dominant negative effects on development . The Plant Cell , 2004 , 16 : 2984 – 3001 . OpenUrl Abstract / FREE Full Text ↵ Hotton SK , Callis J . Regulation of cullin RING ligases . Annual Review of Plant Biology , 2008 , 59 : 467 – 489 . OpenUrl CrossRef PubMed Web of Science ↵ Huang F , Sun M , Yao Z , Zhou J , Bai Q , Chen X , Huang Y , Shen Y . Protein kinase FaSnRK2.6 phosphorylates transcription factor FabHLH3 to regulate anthocyanin homeostasis during strawberry fruit ripening . Journal of Experimental Botany , 2024 , 29 : erae250 . OpenUrl ↵ Huang X , Hetfeld BK , Seifert U , Kahne T , Kloetzel PM , Naumann M , BechDOtschir D , Dubiel W . Consequences of COP9 signalosome and 26S proteasome interaction . The FEBS Journal , 2005 , 272 : 3909 – 3917 . OpenUrl CrossRef PubMed ↵ Huang Y , Xu PH , Hou BZ , Shen YY . Strawberry tonoplast transporter, FaVPT1, controls phosphate accumulation and fruit quality . Plant Cell & Environment , 2019 , 42 ( 9 ): 2715 – 2729 . OpenUrl CrossRef ↵ Jasso-Robles FI , Gonzalez ME , Pieckenstain FL , Ramírez-García JM , Guerrero-González ML , Jiménez-Bremont JF , Rodríguez-Kessler M . Decrease of Arabidopsis PAO activity entails increased RBOH activity, ROS content and altered responses to Pseudomonas . Plant Science , 2020 , 292 : 110372 . ↵ Jin D , Li B , Deng XW , Wei N . Plant COP9 signalosome subunit 5, CSN5 . Plant Science , 2014 , 224 : 54 – 61 . OpenUrl CrossRef PubMed ↵ Kaszler N , Benkő P , Molnár Á , Zámbori A , Fehér A , Gémes K . Absence of Arabidopsis polyamine oxidase 5 influences the cytokinin-induced shoot meristem formation from lateral root primordia . Plants , 2023 , 12 ( 3 ): 454 . OpenUrl CrossRef PubMed ↵ Kong L , Cheng J , Zhu Y , Ding Y , Meng J , Chen Z , Xie Q , Guo Y , Li J , Yang S , Gong Z . Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases . Nature Communications , 2015 , 6 : 8630 . OpenUrl CrossRef PubMed ↵ Kwok SF , Solano R , Tsuge T , Chamovitz DA , Ecker JR , Matsui M , Deng XW . Arabidopsis homologs of a cDJun coactivator are present both in monomeric form and in the COP9 complex, and their abundance is differentially affected by the pleiotropic cop/det/fus mutations . The Plant Cell , 1998 , 10 : 1779 – 1790 . OpenUrl Abstract / FREE Full Text Li W , Zhang J , Sun H , Wang S , Chen K , Liu Y , Li H , Ma Y , Zhang Z . FveRGA1, encoding a DELLA protein, negatively regulates runner production in Fragaria vesca . Planta , 2018 , 247 ( 4 ): 941 – 951 . OpenUrl CrossRef PubMed ↵ Li X , Martín-Pizarro C , Zhou L , Hou B , Wang Y , Shen Y , Li B , Posé D , Qin G . Deciphering the regulatory network of the NAC transcription factor FvRIF, a key regulator of strawberry ( Fragaria vesca ) fruit ripening . The Plant Cell , 2023 , 35 ( 11 ): 4020 – 4045 . OpenUrl CrossRef PubMed ↵ Liao X , Li M , Liu B , Yan M , Yu X , Zi H , Liu R , Yamamuro C . Interlinked regulatory loops of ABA catabolism and biosynthesis coordinate fruit growth and ripening in woodland strawberry . Proceedings of the National Academy of Sciences of the United States of America , 2018 , 115 ( 49 ): E11542 – E11550 . OpenUrl Abstract / FREE Full Text Lingaraju GM , Bunker RD , Cavadini S , Hess D , Hassiepen U , Renatus M , Fischer ES , Thoma NH . Crystal structure of the human COP9 signalosome . Nature , 2014 , 512 : 161 – 165 . OpenUrl CrossRef PubMed Web of Science ↵ Liu J , Nada K , Pang X , Honda C , Kitashiba H , Moriguchi T . Role of polyamines in peach fruit development and storage . Tree Physiology , 2006 , 26 ( 6 ): 791 – 798 . OpenUrl CrossRef PubMed Web of Science ↵ Liu X , Reitsma JM , Mamrosh JL , Zhang Y , Straube R , Deshaies RJ . Cand1Dmediated adaptive exchange mechanism enables variation in FDbox protein expression . Molecular Cell , 2018 , 69 ( 5 ): 773 – 786 . OpenUrl CrossRef PubMed ↵ Liu XD , Zeng YY , Zhang XY , Tian XQ , Hasan MM , Yao GQ , Fang XW . Polyamines inhibit abscisic acid-induced stomatal closure by scavenging hydrogen peroxide . Physiologia Plantarum , 2023 , 175 ( 2 ): e13903 . OpenUrl CrossRef ↵ Lu J , Yu J , Liu P , Gu J , Chen Y , Zhang T , Li J , Wang T , Yang W , Lin R , Wang F , Qi M , Li T , Liu Y . Ubiquitin-mediated degradation of SlPsbS regulates low night temperature tolerance in tomatoes . Cell Reports , 2024 , 43 ( 10 ): 114757 . OpenUrl CrossRef PubMed Lu R , Hu S , Feng J , Liu Z , Kang C . The AP2 transcription factor BARE RECEPTACLE regulates floral organogenesis via auxin pathways in woodland strawberry . The Plant Cell , 2024 , koae270 , doi: 10.1093/plcell/koae270 OpenUrl CrossRef ↵ Lv Y , Shao G , Jiao G , Sheng Z , Xie L , Hu S , Tang S , Wei X , Hu P . Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield . Molecular plant , 2021 , 14 ( 2 ): 344 – 351 . OpenUrl CrossRef PubMed ↵ Lyapina S , Cope G , Shevchenko A , Serino G , Tsuge T , Zhou C , Wolf DA , Wei N , Shevchenko A , Deshaies RJ . Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome . Science , 2001 , 292 : 1382 – 1385 . OpenUrl Abstract / FREE Full Text ↵ Mao W , Han Y , Chen Y , Sun M , Feng Q , Li L , Liu L , Zhang K , Wei L , Han Z , Li B . Low temperature inhibits anthocyanin accumulation in strawberry fruit by activating FvMAPK3-induced phosphorylation of FvMYB10 and degradation of chalcone synthase 1 . The Plant Cell , 2022 , 34 ( 4 ): 1226 – 1249 . OpenUrl CrossRef PubMed ↵ Mayor-Ruiz C , Jaeger MG , Bauer S , Brand M , Sin C , Hanzl A , Mueller AC , Menche J , Winter GE . Plasticity of the cullin-RING ligase repertoire shapes sensitivity to ligand-induced protein degradation . Molecular Cell , 2019 , 75 : 849 – 858 . OpenUrl CrossRef PubMed ↵ Mergner J , Schwechheimer C . The NEDD8 modification pathway in plants . Frontiers in Plant Science . 2014 , 5 : 103 . ↵ Mo AW , Xu T , Bai Q , Shen YY , Gao F , Guo JX . FaPAO5 regulates Spm/Spd levels as a signaling during strawberry fruit ripening . Plant Direct , 2020 , 4 ( 5 ): e00217 . OpenUrl ↵ Nandy S , Das T , Tudu CK , Mishra T , Ghorai M , Gadekar VS , Anand U , Kumar M , Behl T , Shaikh NK , Jha NK , Shekhawat MS , Pandey DK , Dwivedi P , Radha , Dey A . Unravelling the multi-faceted regulatory role of polyamines in plant biotechnology, transgenics and secondary metabolomics . Applied Microbiology and Biotechnology , 2022 , 106 ( 3 ): 905 – 929 . OpenUrl CrossRef ↵ Napieraj N , Janicka M , Reda M . Interactions of polyamines and phytohormones in plant response to abiotic stress . Plants , 2023 , 12 ( 5 ): 1159 . OpenUrl CrossRef PubMed ↵ Navakoudis E , Kotzabasis K . Polyamines: Α bioenergetic smart switch for plant protection and development . Journal of Plant Physiology , 2022 , 270 : 153618 . OpenUrl CrossRef PubMed ↵ Nelson BK , Cai X , Nebenführ A . A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants . The Plant Journal , 2007 , 51 ( 6 ): 1126 – 1136 . OpenUrl CrossRef PubMed Web of Science ↵ Paschalidis KA , Roubelakis-Angelakis KA . Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development . Plant Physiology , 2005 , 138 ( 4 ): 2174 – 2184 . OpenUrl Abstract / FREE Full Text ↵ Pottosin I , Velarde-Buendía AM , Bose J , Zepeda-Jazo I , Shabala S , Dobrovinskaya O . Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses . Journal of Experimental Botany , 2014 , 65 ( 5 ): 1271 – 1283 . OpenUrl CrossRef PubMed Web of Science ↵ Qin N , Xu D , Li J , Deng XW . COP9 signalosome: Discovery, conservation, activity, and function . Journal of integrative plant biology , 2020 , 62 ( 1 ): 90 – 103 . OpenUrl CrossRef PubMed ↵ Reitsma JM , Liu X , Reichermeier KM , Moradian A , Sweredoski MJ , Hess S , Deshaies RJ . Composition and regulation of the cellular repertoire of SCF ubiquitin ligases . Cell , 2017 , 171 ( 6 ): 1326 – 1339 . OpenUrl CrossRef PubMed ↵ Russell DH . The roles of polyamines, putrescine, spermidine and spermine in normal and malignant tissues . Life Science , 1973 , 13 : 1635 – 1647 . OpenUrl CrossRef PubMed Web of Science ↵ Schwechheimer C , Serino G , Deng XW . Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response . Science , 2001 , 292 : 1379 – 1382 . OpenUrl Abstract / FREE Full Text ↵ Schwechheimer C , Isono E . The COP9 signalosome and its role in plant development . European Journal of Cell Biology . 2010 , 89 ( 2 ): 157 – 162 . OpenUrl CrossRef PubMed Web of Science ↵ Serino G , Deng XW . The COP9 signalosome: regulating plant development through the control of proteolysis . Annual Review of Plant Biology , 2003 , 54 ( 1 ): 165 – 182 . OpenUrl CrossRef PubMed Web of Science ↵ Shang Y , Wang K , Sun S , Zhou J , Yu JQ . COP9 Signalosome CSN4 and CSN5 subunits are involved in jasmonate-dependent defense against root-knot nematode in tomato . Frontier in Plant Science , 2019 , 10 : 1223 . OpenUrl CrossRef ↵ Shen ZF , Li L , Wang JY , Liao J , Zhang YR , Zhu XM , Wang ZH , Lu JP , Liu XH , Lin FC . CSN5 inhibits autophagy by regulating the ubiquitination of Atg6 and Tor to mediate the pathogenicity of Magnaporthe oryzae . Cell Communication and Signaling , 2024 , 22 ( 1 ): 222 . OpenUrl CrossRef ↵ Siddappa S , Marathe GK . What we know about plant arginases? Plant Physiology and Biochemistry , 2020 , 156 : 600 – 610 . OpenUrl CrossRef PubMed ↵ Song J , Sun P , Kong W , Xie Z , Li C , Liu JH . SnRK2.4-mediated phosphorylation of ABF2 regulates ARGININE DECARBOXYLASE expression and putrescine accumulation under drought stress . New Phytologist , 2023 , 238 ( 1 ): 216 – 236 . OpenUrl CrossRef PubMed ↵ Stratmann JW , Gusmaroli G . Many jobs for one good cop - the COP9 signalosome guard development and defense . Plant Science , 2012 , 185-186 : 50 – 64 . OpenUrl CrossRef ↵ Verma R , Aravind L , Oania R , McDonald WH , Yates JR 3rd . , Koonin EV , Deshaies RJ. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome . Science . 2002 , 298 ( 5593 ): 611 – 615 . OpenUrl Abstract / FREE Full Text ↵ Wang W , Paschalidis K , Feng JC , Song J , Liu JH . Polyamine catabolism in plants: A universal process with diverse functions . Frontiers in Plant Science , 2019 , 10 : 561 . OpenUrl CrossRef PubMed ↵ Wei N , Deng XW . The COP9 signalosome . Annual Review of Cell and Developmental Biology , 2003 , 19 ( 1 ): 261 – 286 . OpenUrl CrossRef PubMed Web of Science ↵ Ye Q , Wang H , Su T , Wu WH , Chen YF . The Ubiquitin E3 Ligase PRU1 Regulates WRKY6 Degradation to Modulate Phosphate Homeostasis in Response to Low-Pi Stress in Arabidopsis . The Plant Cell , 2018 , 30 ( 5 ): 1062 – 1076 . OpenUrl Abstract / FREE Full Text ↵ Yu Z , Jia DY , Liu TB . Polyamine oxidases play various roles in plant development and abiotic stress tolerance . Plants , 2019 , 8 ( 6 ): 184 . OpenUrl CrossRef PubMed ↵ Zhan ZN , Wang N , Chen ZM , Zhang YX , Geng KQ , Li DM , Wang ZP . Effects of water stress on endogenous hormones and free polyamines in different tissues of grapevines ( Vitis vinifera L. cv. ‘Merlot’ ) . Functional Plant Biology , 2023 , 50 ( 12 ): 993 – 1009 . OpenUrl CrossRef PubMed ↵ Zhao JQ , Wang XF , Pan XB , Jiang QQ , Xi ZM . Exogenous putrescine alleviates drought stress by altering reactive oxygen species scavenging and biosynthesis of polyamines in the seedlings of Cabernet Sauvignon . Frontiers in Plant Science , 2021 , 12 : 767992 . ↵ Zhong M , Yue L , Liu W , Qin H , Lei B , Huang R , Yang X , Kang Y . Genome-wide identification and characterization of the polyamine uptake transporter (Put) gene family in tomatoes and the role of Put2 in response to salt stress . Antioxidants , 2023 , 12 ( 2 ): 228 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 14, 2024. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H2O2 homeostasis Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H 2 O 2 homeostasis Yun Huang , Jiahui Gao , Guiming Ji , Wenjing Li , Jiaxue Wang , Qinghua Wang , Yuanyue Shen , Jiaxuan Guo , Fan Gao bioRxiv 2024.07.10.602942; doi: https://doi.org/10.1101/2024.07.10.602942 Share This Article: Copy Citation Tools Strawberry COP9 signalosome FvCSN5A regulates plant development and fruit ripening by facilitating polyamine oxidase FvPAO5 degradation to control polyamine and H 2 O 2 homeostasis Yun Huang , Jiahui Gao , Guiming Ji , Wenjing Li , Jiaxue Wang , Qinghua Wang , Yuanyue Shen , Jiaxuan Guo , Fan Gao bioRxiv 2024.07.10.602942; doi: https://doi.org/10.1101/2024.07.10.602942 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 (7652) Biochemistry (17752) Bioengineering (13936) Bioinformatics (42084) Biophysics (21501) Cancer Biology (18655) Cell Biology (25586) Clinical Trials (138) Developmental Biology (13410) Ecology (19949) Epidemiology (2067) Evolutionary Biology (24378) Genetics (15639) Genomics (22562) Immunology (17779) Microbiology (40505) Molecular Biology (17219) Neuroscience (88825) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4840) Physiology (7666) Plant Biology (15182) Scientific Communication and Education (2048) Synthetic Biology (4305) Systems Biology (9840) Zoology (2274)