The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation

The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation | 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 The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation View ORCID Profile Thalia Luden , Petra Amakorová , View ORCID Profile Ondřej Novák , View ORCID Profile Salma Balazadeh , View ORCID Profile Remko Offringa doi: https://doi.org/10.1101/2025.11.23.689984 Thalia Luden 1 Plant Developmental Genetics , Sylviusweg 72, 2333 BE Leiden, the Netherlands 4 Institute of Plant Sciences – Paris Saclay (IPS2), University of Paris-Saclay , Bat 630, Avenue des Sciences , 91190 Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thalia Luden Petra Amakorová 3 Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, The Czech Academy of Sciences , CZ-77900 Olomouc, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ondřej Novák 3 Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, The Czech Academy of Sciences , CZ-77900 Olomouc, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ondřej Novák Salma Balazadeh 2 Plant Stress Biology, Institute of Biology Leiden, Leiden University , Sylviusweg 72, 2333 BE Leiden, the Netherlands 5 University of Göttingen, Albrecht-von-Haller-Institute for Plant Sciences , Julia-Lermontowa-Weg 3, D-37077 Göttingen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Salma Balazadeh Remko Offringa 1 Plant Developmental Genetics , Sylviusweg 72, 2333 BE Leiden, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Remko Offringa For correspondence: r.offringa{at}biology.leidenuniv.nl Abstract Full Text Info/History Metrics Preview PDF Summary The Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) gene is associated with various longevity phenotypes and extends the life span of plants when overexpressed. In this study, we show that, in addition to previously described longevity phenotypes, constitutive overexpression of AHL15 in Arabidopsis delays leaf senescence, whereas ahl15 loss-of-function accelerates this process. Dexamethasone-induced nuclear localization of AHL15-GR during dark-triggered senescence results in a stay-green phenotype and represses the expression of several early senescence-associated genes. Among these, the ORESARA1 (ORE1) locus is directly bound by AHL15, suggesting a direct repressive effect of AHL15 on senescence. Furthermore, we demonstrate that AHL15 acts by directly repressing the expression of several CYTOKININ OXIDASE (CKX) genes involved in cytokinin inactivation, resulting in a delayed degradation of cytokinins during dark-induced senescence. Cytokinins are known to delay senescence, and together with the downregulation of ORE1 expression, this explains the repressive effect of AHL15 on senescence. Significance Here, we show that in addition to its previously reported effects on aging processes, the AT-HOOK MOTIF NUCLEAR LOCALIZED -family protein AHL15 also represses leaf senescence. Our results demonstrate that AHL15 delays the senescence program in two ways: by directly repressing the senescence master regulator ORESARA1, and by transcriptional repression of CYTOKININ OXIDASE genes, resulting in a delayed breakdown of the senescence-inhibiting hormone cytokinin, which together explain the strong stay-green phenotype of AHL15 -overexpressing plants. Introduction Leaf senescence is the process during which the photosynthetic machinery of leaves is catabolized and recycled for use in other parts of the plant ( Lim, Kim and Nam, 2007 ; Schippers et al ., 2015 ; Guo et al ., 2021 ). It is the final developmental stage of leaves, and its progression is tightly controlled by various transcription factors (TFs). Based on research primarily conducted in the model plant Arabidopsis thaliana (Arabidopsis), the TFs ORESARA1 (ORE1) and NAC-LIKE, ACTIVATED BY AP3/PI (AtNAP) have been identified as major regulators of senescence ( Guo and Gan, 2006 ; Kim et al ., 2009 ; Qiu et al ., 2015 ). Loss-of-function of ore1 , nap, or other positive regulators of senescence results in a stay-green phenotype ( Guo and Gan, 2006 ; Kim et al ., 2009 ), and ORE1 regulates the transcription of numerous genes in the senescence pathway ( Balazadeh et al ., 2010 ). Upon initiation of senescence, genes involved in chlorophyll degradation, also referred to as chlorophyll catabolic genes (CGGs) are upregulated by ORE1, NAP, and other TFs in the senescence pathway, and together the resulting enzymes degrade chlorophyll (Chl) into smaller molecules that can be exported to other tissues in the plant, resulting in the characteristic yellowing of senescent leaves ( Qiu et al ., 2015 ; Yang, Worley and Udvardi, 2015 ; Woo et al ., 2019 ). The remobilization of nutrients from older leaves to other parts of the plant helps regulate nutrient availability and distribution, thereby increasing the plant’s chances of survival during periods of nutrient scarcity ( Schippers et al ., 2015 ). However, when senescence negatively impacts the overall quality of a crop, such as in leafy vegetables or in ornamental plants, delayed senescence can be a desirable trait. Therefore, improving the control of leaf senescence is essential for enhancing crop quality. Senescence can be induced by various factors, including leaf age, dark incubation, nutrient shortage, and the application of hormones such as ethylene, jasmonic acid, abscisic acid, salicylic acid, or strigolactones ( Woo et al ., 2019 ). In Arabidopsis, ethylene triggers a signaling cascade that activates the transcription factor ETHYLENE INSENSITIVE 3 (EIN3), which in turn promotes the transcription of ORE1 and NAP ( Li et al ., 2013 ; Kim et al ., 2014 ). Additionally, dark incubation induces transcription of both ORE1 and EIN3 via PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5 ( Song et al ., 2014 ). In monocarpic plants, flowering also triggers leaf senescence, allowing the relocation of nutrients from leaves to seeds and thereby enhancing the offspring’s chances of survival ( Thomas, 2013 ). For example, in Arabidopsis, the whole plant senesces within a few weeks after flowering. This process is controlled by several TFs, among which MYB2 and FRUITFULL (FUL), and coincides with reduced production of the hormone cytokinin (CK) ( Guo and Gan, 2011 ; Balanzà et al ., 2018 ). In contrast to most other plant hormones that induce senescence, CKs repress leaf senescence: exogenous CK application significantly delays leaf senescence in various plant species ( Richmond et al ., 1957 ; Gan and Amasino, 1995 ; Hallmark and Rashotte, 2020 ; Pokorná et al ., 2021 ). The senescence-delaying activity of CK is mediated by the CK receptor ARABIDOPSIS HISTIDINE KINASE 3 (AHK3), which phosphorylates ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2) upon CK perception ( Kim et al ., 2006 ). ARR2 then promotes expression of CYTOKININ RESPONSE FACTOR 6 ( CRF6 ), a TF that negatively regulates leaf senescence via an unknown mechanism ( Zwack et al ., 2013 ). Loss-of-function mutants of ahk3 show accelerated senescence phenotypes and the senescence-delaying effect of CK treatment is reduced in these plants as a result of their weakened ability to transduce CK signals ( Kim et al ., 2006 ). A similar phenotype can be seen in crf6 mutants ( Zwack et al ., 2013 ). CKs are synthesized predominantly by enzymes of the ISOPENTENYL TRANSFERASE (IPT) and LONELY GUY (LOG) families. IPT-family enzymes are responsible for adding a prenyl group to N 6 of ATP or ADP ( Kakimoto, 2001 ; Takei, Sakakibara and Sugiyama, 2001 ). Additional enzymes such as CYP735A are involved in making changes to this prenyl group, thereby producing precursors to different CK variants ( Argueso and Kieber, 2024 ). Biological activation of these CK precursors relies on the removal of the riboside group by LOG-family enzymes, which are highly redundant and of which LOG7 and LOG8 appear to be the most important in Arabidopsis ( Kuroha et al ., 2009 ; Tokunaga et al ., 2012 ). CK degradation is predominantly accomplished by enzymes of the CYTOKININ OXIDASE/DEHYDROGENASE (CKX) class. CKX enzymes irreversibly cleave off the N 6 side chains from active CKs, thereby inactivating them permanently ( Galuszka et al ., 2000 ; Schmülling et al ., 2003 ). Knockout of the rice ckx11 gene causes a delay in leaf senescence, indicating that reduced CK degradation negatively affects senescence ( W. Zhang et al ., 2021 ). On the other hand, overexpression of LOG4 delays leaf senescence in Arabidopsis ( Kuroha et al., 2009 ) and causes pleiotropic effects. In addition, ectopic expression of the Agrobacterium tumefaciens IPT gene under the senescence-specific SAG12 promoter delays leaf senescence in several crops ( Gan and Amasino, 1995 ; Jordi et al ., 2000 ; McCabe et al ., 2001 ). Thus, leaf senescence can be regulated by controlling the expression of the CK biosynthesis genes of the IPT and LOG families and by regulating the expression of the CK-degrading CKX genes. While CK plays an important role in senescence, the regulatory mechanisms that control CK-mediated inhibition of senescence remain elusive. So far, only two TFs that regulate CK levels during senescence have been identified: MYB2 and NAP ( Guo and Gan, 2011 ; Hu et al ., 2021 ; Li et al ., 2023 ). In wild type plants, IPT expression in the leaf is silenced by MYB2, whose expression increases with progressing age ( Guo and Gan, 2011 ). It was shown that MYB2 expression in Arabidopsis and the legume Caragana intermedia can be repressed by proteins of the S40 family, and overexpression of S40 -family genes results in enhanced CK levels and a stay-green phenotype ( Yang et al ., 2022 ). The expression of CRF6 also decreases in older leaves through an unknown mechanism ( Zwack et al ., 2013 ). At the same time, CKX3 expression is induced by the senescence-promoting TF NAP, resulting in enhanced CK degradation ( Hu et al ., 2021 ). The repression of CKX expression by NAP was observed in Arabidopsis and Chinese cabbage, indicating that this pathway is conserved ( Hu et al ., 2021 ; Li et al ., 2023 ). These results show a clear link between CK and age-induced senescence, but additional regulatory mechanisms that control the expression of CK biosynthesis- and degrading genes during the senescence process remain to be identified. Previously, we identified the Arabidopsis gene AT-HOOK MOTIF NUCLEAR LOCALIZED15 (AHL15) as a regulator of plant longevity: overexpression of AHL15 in Arabidopsis resulted in an increased life span and flowering time and enhanced vegetative development of axillary meristems, branching and secondary growth, whereas ahl15 loss-of-function or perturbed ahl function reduced all these aspects ( Karami et al ., 2020 ; Rahimi, Karami, Lestari, et al ., 2022 ). CK levels were increased in stems of AHL15 overexpressing plants, explaining the enhanced secondary growth and branching phenotypes in these plants ( Rahimi, Karami, Lestari, et al ., 2022 ). In Arabidopsis, the AHL gene family is comprised of 29 members that are subdivided into two clades (clades A and B) ( Zhao et al ., 2014 ). Previous research has demonstrated that members of the clade A AHL subfamily, including AHL15 , exhibit a high level of functional redundancy ( Yun et al ., 2012 ; Zhao et al ., 2013 ; Karami et al ., 2020 ). Lim et al. (2007) showed that overexpression of the clade A member AHL27 delays leaf senescence, whereas a recent study has identified the clade B member AHL9 as a promoter of leaf senescence, suggesting that clade A and B AHLs may function antagonistically ( Lim et al ., 2007 ; Zhou et al ., 2022 ). Here, we show that, similar to AHL27, overexpression of AHL15 delays leaf senescence, while loss-of-function ahl15 mutants exhibit early senescence. Our results show that the expression of several senescence marker genes, including ORESARA1 ( ORE1 ), is repressed when AHL15 activity is induced and that AHL15 directly binds to the ORE1 locus, indicating that AHL15 directly suppresses the senescence program. Additionally, we found that AHL15 binds upstream of the CKX2 , CKX3 , and CKX5 genes and that induction of AHL15 activity leads to nearly complete silencing of their expression. This silencing coincides with a slower decline in CK levels during dark-induced senescence. Together, our data reveal that AHL15 delays the senescence program through parallel pathways: by repressing the expression of ORE1 and by repressing CKX expression and subsequent CK degradation. Results AHL15 overexpression delays leaf senescence Given that overexpression of AHL15 extends overall plant longevity and because delayed senescence has previously been reported for AHL27 overexpressing lines ( Lim et al ., 2007 ), we tested whether a similar senescence phenotype could also be observed in p35S:3xFLAG-AHL15 plants (hereafter referred to as AHL15 OX). To this end, we compared senescence in the fifth leaf of Col-0 (wild type) and ahl15 knockout plants with two independent AHL15 OX lines in both dark-induced ( Figure 1 ) and age-dependent conditions ( Figure S1 ). To quantify senescence, we measured leaf Chlorophyll (Chl) using two methods: 1) Chl extraction using acetone followed by spectrophotometry ( Arnon, 1949 ), and 2) a recently developed colorimetric assay. This assay measures the pixel intensity value in Red, Green, and Blue channels of digital images, which can be used to calculate the normalized Red value (Rn; Red/(Red+Green+Blue)). Previously, we have demonstrated that the Rn value shows a significant negative correlation with the total Chl content of leaves, which makes it an appropriate non-invasive method for Chl quantification ( Luden et al ., 2025 ). Download figure Open in new tab Figure 1. Dark-induced leaf s enescence is delayed by AHL15 overexpression and accelerated by ahl15 loss-of-function. A-C: The fifth leaf of 23-day-old wild-type (Col-0), ahl15 mutant or p35S:3xFLAG-AHL15 ( AHL15 OX ) Arabidopsis plants was detached and floated on 5 ml (5 mM) MES buffer for five days in the dark to induce the senescence process. Leaves were imaged for chlorophyll quantification as the normalized Red value (Rn) ( A ). Chlorophyll was extracted and quantified by spectrophotometer ( B ). Representative images of leaves of each genotype ( C ). Differences between genotypes in A and B were compared by a Kruskal-Wallis test, and significance groups are indicated by letters (n=9-25; p < 0.05). White bars in C represent 1 cm. In dark-induced conditions, the senescence of the fifth leaf was significantly delayed in the AHL15 OX lines compared to the wild type, as evidenced by both the lower Rn value ( Figure 1A ) and the higher total Chl content ( Figure 1B ). In addition, ahl15 loss-of-function plants showed a mild but significant increase in Rn value and a decrease in total Chl content compared to Col-0. These findings demonstrate that AHL15 inhibits leaf senescence in dark-induced conditions. Similarly, at 42 days after germination, the older leaves of wild type and ahl15 knockout plants started to senesce, whereas leaves of the same age in AHL15 OX lines stayed green, indicating that age-dependent senescence could be delayed by AHL15 overexpression ( Figure S1A ). To monitor this in more detail, we harvested the fifth leaves of different plants at 21, 24, 27, 30, 34, and 36 days of emergence and used images of these leaves to measure the Rn value ( Figure S1B, C ). This showed that the progression of senescence in the fifth leaf was slightly delayed in AHL15 OX plants (most strongly in AHL15 OX line 2) compared to wild type and ahl15 loss-of-function plants, suggesting that AHL15 may play a role in age-dependent senescence as well. The latter is in line with the expression of AHL15 , which is on in young developing leaves and turned off when leaves are fully grown ( Figure S2 ) Overexpression of AHL15 and other clade-A AHLs generally delays flowering time ( Street et al., 2008 ; Xiao et al., 2009 ; Karami et al., 2020 ), and AHL15 has been shown to delay vegetative phase change ( Rahimi, Karami, Balazadeh, et al ., 2022 ). In addition, AHL15 is expressed primarily in juvenile, newly developing leaves and is absent in older and adult-stage leaves ( Rahimi, Karami, Balazadeh, et al ., 2022 ). To evaluate the effects of AHL15 overexpression on the senescence phenotype independently of other developmental processes, we used a dexamethasone (DEX)-inducible p35S:AHL15-GR line in which the AHL15-GR fusion protein translocates to the nucleus upon DEX treatment ( Karami et al ., 2020 ). During the dark incubation period, leaves were floated on 5 mL MES buffer, to which DEX was added to a final concentration of 10 μM to induce AHL15 activity simultaneously with the senescence process. DEX-treated p35S:AHL15-GR leaves showed a similar delayed senescence phenotype as AHL15 OX leaves, whereas mock-treated p35S:AHL15-GR leaves senesced like wild-type leaves ( Figure 2A-C ). Download figure Open in new tab Figure 2. DEX-induced nuclear localization of AHL15-GR inhibits leaf senescence. A-C: The fifth leaf was detached from 23-day-old wild-type (Col-0) or p35S:AHL15-GR plants and incubated in the dark on 5 mM MES buffer with or without 10 µM DEX for the indicated number of days. The leaf chlorophyll content was quantified over five days of dark incubation by image-based calculation of the normalized Red value ( A ) or by direct chlorophyll extraction ( B ) (n = 8 for days 1, 2, and 4, and n = 16 for days 0, 3, and 5). The quantification was repeated twice with similar results. Representative images of leaves from each group before and after three or five days of dark incubation ( C ). White bars in ( C ) represent 1 cm. D, E: Expression of the senescence-associated genes SAG13 ( D ) and SAG12 ( E ) in the DEX-or Mock-treated p35S:AHL15-GR leaves shown in C , as determined by RT-qPCR. RNA was extracted from three samples per time point and condition (n=3), with each sample consisting of the fifth leaf of three individual plants. Expression was normalized to ACTIN2 . SAG12 expression could not be detected (n.e. for not expressed) at day 0 and in DEX-treated leaves at day 3. Letters indicate significance groups calculated with the Kruskall-Wallis test (p < 0.05). We next investigated the effect of DEX-induced AHL15-GR nuclear localization on the expression of senescence-associated genes after increasing periods of dark incubation. Based on the Rn value and total Chl content of leaves incubated in the dark over five days, significant differences between DEX-induced and non-induced leaves (DEX-treated vs. mock-treated p35S:AHL15-GR, or DEX-treated Col-0) became evident after three days of dark incubation. This indicates that senescence had been initiated at this time point in non-induced leaves ( Figure 2A-C ). To determine whether AHL15 inhibits senescence at its onset or a later stage, we measured the expression of SENESCENCE-ASSOCIATED GENES (SAGs) SAG13 and SAG12, which are associated with early- and late senescence, respectively ( Lohman et al ., 1994 ). RNA was isolated from leaves right before dark incubation and DEX treatment (day 0), and after three or five days of dark incubation with and without DEX treatment. A clear and significant difference in the expression of the early senescence marker SAG13 as well as the late senescence marker SAG12 was visible between DEX-and mock-treated plants ( Figure 2D-E ), whereas the expression of SEN1 was slightly but significantly reduced in DEX-treated leaves after 5 days ( Figure S3 ). As the expression of SEN1 is induced by darkness ( Oh et al ., 1996 ; Chung et al ., 1997 ), these results indicate that overexpression of AHL15 predominantly affects the onset of senescence at an early stage and interferes with the response to darkness less strongly. AHL15 directly represses the expression of ORE1 and several CKX genes To understand how AHL15 suppresses leaf senescence, we investigated whether AHL15 binds any senescence genes directly by consulting AHL15 ChIP-seq data from AHL15 overexpressing plants (p35S:3xFLAG-AHL15) that we generated in a different study ( Luden, Chouaref and Offringa, 2025 ). ChIP-seq peaks of AHL15 were present up- and downstream of many genes, among which the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) gene ( Figure S4 ), whose expression was previously shown to be suppressed by AHL15 ( Rahimi, Karami, Balazadeh, et al ., 2022 ). Interestingly, significant peaks were present at 600 bp upstream and 1 kb downstream of the senescence regulator ORE1, which could explain the delayed senescence phenotype observed in AHL15 overexpressing plants ( Figure 3A ). We next measured the expression of ORE1 after senescence induction of DEX- or mock-treated leaves and saw that its expression was strongly repressed in DEX-treated leaves, implying a direct repression of this gene by AHL15 ( Figure 3B ). Download figure Open in new tab Figure 3. AHL15 binds to the ORE1 and CKX loci to repress gene expression. A: ChIP performed on the whole rosette of 31-day-old p35S:3xFLAG-AHL15 plants using an anti-FLAG antibody. ChIP seq detected peaks of AHL15 binding 1 kb downstream and 600bp upstream of ORE1 , in the second intron of CKX2 , 1.9 kb upstream and directly downstream of CKX3 , and 2.3 kb upstream and 2 kb downstream of CKX5 . Genes are shown in black (black blocks are exons, black lines are introns, grey blocks represent genes on the opposite strand), read alignments are shown in purple (bigwig track), and the location of significant peaks are indicated in blue, with a light blue bar indicating the peak summit (narrowpeak track). Similar results were obtained in three biological replicates. B: The expression of ORE1 , CKX2, CKX3 and CKX5 is repressed by DEX induced AHL15-GR activation in dark-incubated leaves. Leaves of 23-day-old p35S:AHL15-GR plants were floated on 5 mM MES buffer with or without 10 µM DEX for the indicated number of days in the dark. RNA used for RT-qPCR was extracted from three samples per time point and condition (n=3), with each sample consisting of the fifth leaf of three individual plants. Expression was normalized to ACTIN2 . Letters indicate significance groups as calculated by the Kruskall-Wallis test (p < 0.05). In addition, we identified the CKX genes 2 , 3 , and 5 ( Figure 3A ) and several CK biosynthesis genes ( IPT7, LOG1, LOG7, LOG8; Figure S5A ) among the significant ChIP-seq peaks. Because of the previous finding that AHL15 promotes wood formation by enhancing CK levels ( Rahimi, Karami, Lestari, et al ., 2022 ), and because CKs are known to have an delaying effect on leaf senescence ( Kim et al ., 2006 ; Guo and Gan, 2011 ; Hu et al ., 2021 ; Yang et al ., 2022 ), we hypothesized that the delayed senescence phenotype of AHL15 overexpressing plants could in part be a result of increased CK levels. To test whether AHL15 directly regulates the expression of these genes, we measured the expression of each of these genes at 0, 3, or 5 days after senescence induction in DEX- or mock-treated p35S:AHL15-GR leaves. The expression of LOG8 and the three CKX genes in mock-treated leaves followed a similar pattern as the senescence markers SAG13 and SAG12 , and were all increased after 3 and 5 days of dark incubation ( Figure 3B , Figure S5B ). In contrast, the expression of IPT7 and LOG1 in mock-treated leaves was lower than at day 0, and the expression of LOG7 was not affected ( Figure S5B ). Interestingly, the expression of the CKX genes was significantly lower in DEX-treated leaves compared to the mock treatment, implying that these genes are directly repressed by AHL15 ( Figure 3B ). The repression of IPT7 expression upon dark incubation was delayed in DEX-treated leaves, whereas the expression of LOG1 and LOG8 was repressed in comparison to mock-treated leaves and LOG7 expression was unaffected ( Figure S5B ). These data suggest that unlike in stem tissue, where AHL15 promotes CK biosynthesis ( Rahimi, Karami, Lestari, et al ., 2022 ), it delays leaf senescence rather by repressing the expression of cytokinin-degrading CKX proteins. Cytokinin levels are increased in AHL15 overexpressing plants The repression of CKX genes by AHL15 suggests that the inactivation of CKs is delayed in AHL15- overexpressing leaves, resulting in increased CK levels that repress the onset of senescence. To test whether this is the case, we measured the levels of a wide range of CKs in dark-induced AHL15-GR leaves with and without DEX treatment before and during senescence ( Figure 4 , Figures S6 - S11 ). These measurements showed that total CK levels drop upon senescence induction in both DEX- and mock-treated leaves, but that this decrease in CK levels is less pronounced in DEX-treated leaves ( Figure 4A ). This trend was especially clear for the isopentenyladenine (iP)-glucosides ( Figure 4B , Figure S6 ), implying that they may be especially sensitive to degradation by CKX enzymes during the senescence process. Previous research has shown that both iP and iP-9-glucoside (IP9G), but not iP-7-glucoside (iP7G), can inhibit senescence in Arabidopsis cotyledons ( Hallmark and Rashotte, 2020 ). Although iP in its base form was not present at detectable levels in any of the samples, the elevated levels of iP9G in DEX-treated samples may partially explain the delayed senescence phenotype. In addition, the levels of trans-zeatin base and trans-zeatin Riboside (tZ and tZR) were reduced in all dark-incubated samples, but their concentration remained significantly higher in DEX-treated leaves ( Figure 4C-D , Figure S7 ). Of all CK types, tZ-base and tZR are among the most potent inhibitors of senescence ( Hönig et al ., 2018 ; Pokorná et al ., 2021 ), and their elevated concentration in DEX-versus mock-treated leaves could therefore play an important role in senescence inhibition in DEX-treated leaves. Download figure Open in new tab Figure 4. DEX-induced AHL15-GR activation delays the reduction in cytokinin (CK) levels in dark-incubated leaves. A-D: Quantification of the total CK ( A ), isopentenyladenine-9-glucoside ( B ), trans-zeatin base ( C ) or trans-zeatin riboside ( D ) level in the fifth leaf of 23-day old p35S:AHL15-GR plants floated on 5 mM MES with or without 10 µM DEX in the dark for the indicated number of days. For each sample three leaves were pooled and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). In addition to tZ and iP, we profiled the levels of cis-zeatine (cZ; Figure S8 ) and dihydrozeatin (DHZ; Figure S9 ) during the senescence process. Interestingly, cZ-base and cZG levels increased upon dark induction and were especially high after five days of DEX treatment ( Figure S8 ). cZ has a weak effect on leaf senescence, and its function in the plant remains largely unknown ( Gajdošová et al ., 2011 ). Previous research has shown that DHZ levels drop during the senescence process in tobacco leaves ( Hönig et al ., 2018 ), and we observed a similar trend in our experiments ( Figure S9 ). While DHZ-base levels were undetectable, we found that DHZ derivatives were present at reduced levels in all samples and did not differ significantly between treatments, indicating that this form of CK is not affected by AHL15 or its downstream targets ( Figure S9 ). Finally, we observed an overall increase in CK bases in DEX-treated samples compared to untreated and mock-treated samples ( Figure S10 ), suggesting that other CK bases may also contribute to the AHL15-mediated delay of senescence. Taken together, our results indicate that AHL15 delays senescence through multiple mechanisms: by directly repressing ORE1 expression, and by downregulating CKX genes, thereby delaying the inactivation of CKs in leaves. The latter process results in elevated CK levels in AHL15 -overexpressing leaves, ultimately contributing to the delay in leaf senescence ( Figure 5 ). Download figure Open in new tab Figure 5. Model of AHL15-mediated repression of senescence. AHL15 represses ORESARA1 (ORE1), which is a key positive regulator of the senescence transcriptional network. ORE1 can be induced by dark incubation via the PHYTOCHROME INTERACTING FACTORS (PIFs), by ethylene via ETHYLENE INSENSITIVE 3 (EIN3), or by age. ORE1 activates several downstream targets among which chlorophyll catabolic genes ( CCG s), ultimately leading to chlorophyll catabolism and causing the characteristic yellowing of senescent leaves. The NAC-LIKE, ACTIVATED BY AP3/PI (NAP) transcription factor acts in parallel to ORE1 and activates the expression of CCG s as well as the CYTOKININ OXIDASE/DEHYDROGENASE ( CKX ) genes, leading to the irreversible inactivation of cytokinins (CKs). CKs activate ARABIDOPSIS HISTIDINE KINASE 3 (AHK3), which represses leaf senescence via CYTOKININ RESPONSE FACTOR 6 (CRF6). CKs are produced by ISOPENTENYL TRANSFERASE (IPT) enzymes, which are transcriptionally repressed by MYB2 in ageing plants. AHL15 represses CKX expression and promotes the expression of IPT7 . Lower CKX levels result in slower breakdown of CKs, resulting in higher CK levels and delayed senescence. TFs are indicated in bold letters, enzymes are indicated in red, and hormones and external abiotic factors are in italics. Discussion AHL15 has previously been shown to delay several developmental processes, including vegetative phase change, flowering time, and axillary meristem maturation ( Karami et al ., 2020 ; Rahimi, Karami, Balazadeh, et al ., 2022 ). Our data show that in addition to these processes, AHL15 delays leaf senescence by directly repressing the expression of ORE1, which encodes a key senescence regulator, and by limiting CK degradation through repression of CKX gene expression. These two processes are likely to act in parallel and together explain the strong stay-green phenotype observed in AHL15 overexpressing plants ( Figure 5 ). Moreover, our data suggest that AHL15 acts predominantly by repressing gene expression, as observed for ORE1 and three CKX genes. ORE1 acts as a key transcriptional regulator of senescence, and its expression is induced by several senescence-promoting environmental cues. Ethylene promotes the expression of ORE1 and NAP via EIN3 ( Li et al., 2013 ; Kim et al., 2014 ), ABA promotes ORE1 expression via the TF ACTIVATING FACTOR1 (ATAF1) ( Garapati et al ., 2015 ), and dark incubation leads to enhanced ORE1 transcription via the PIF TFs ( Song et al ., 2014 ). It was also shown that ORE1 expression is post-transcriptionally silenced in young leaves by miR164 and that this silencing is relieved with progressing age ( Kim et al ., 2009 ; Li et al ., 2013 ). Here, we have identified AHL15 as a novel repressor of ORE1 expression, suggesting that AHL15 can antagonize the ethylene- and dark-induced senescence processes mediated by ORE1. In addition, AHL15 is strongly expressed in juvenile plants and developing leaves and is gradually silenced over developmental age ( Rahimi, Karami, Balazadeh, et al ., 2022 ). It is therefore possible that AHL15 can act as an additional age-dependent repressor of ORE1 expression in parallel to miR164 ( Figure 5 ). CK has long been recognized as an inhibitor of leaf senescence, yet its precise role and position within the senescence regulatory network remain relatively poorly understood. Application or overproduction of CK can block the onset of leaf senescence ( Richmond et al ., 1957 ; Gan and Amasino, 1995 ; Kim et al ., 2006 ; Argueso and Kieber, 2024 ), and it has been shown that CK degradation is promoted during the senescence process by NAP-mediated upregulation of CKX expression ( Hu et al ., 2021 ; Li et al ., 2023 ). MYB2 was shown to lower CK production in an age-dependent manner by repressing the expression of several IPT genes, which are the rate-limiting enzymes in the CK biosynthesis pathway ( Guo and Gan, 2011 ). Our data show that AHL15 acts antagonistically to NAP in CKX transcriptional regulation and represses senescence via inhibition of CK degradation. In addition, we found that IPT7 expression decreases in response to dark-induced senescence and that this process is delayed after activation of AHL15-GR by DEX treatment. Taken together, our results indicate that AHL15 acts as a positive regulator of CK in the senescence pathway, and thereby acts antagonistically to NAP and MYB2 in this process ( Figure 5 ). Our experiments show that the expression of the CK biosynthesis genes LOG1 and IPT7 is downregulated upon senescence induction by dark treatment. On the other hand, we observed an increased expression of LOG8 after senescence induction, and that LOG7 expression is stable throughout the senescence process. In addition, the levels of cZ in its base form and most of its derivatives were increased in senescent leaves. Together, these data show that CK biosynthesis is not completely repressed but partially maintained during the senescence developmental program. Our data also confirm the previously reported induction of CKX expression during senescence ( Hu et al ., 2021 ; Li et al ., 2023 ). This indicates that CK levels are regulated both by biosynthesis and by degradation during the senescence process and suggests a role for some CK variants in the senescence process. Finally, our data show that inhibition of CK breakdown by direct silencing of CKX genes partially explains the delayed senescence phenotype of AHL15 overexpressing plants. Previously, it was shown that expression of LOG4 , IPT3 and IPT7 was decreased in ahl15 knockout plants, resulting in lower CK levels and a decrease in radial xylem width ( Rahimi, Karami, Lestari, et al ., 2022 ). Here, we observed delayed downregulation of IPT7 in dark-incubated leaves following DEX-induced nuclear localization of AHL15-GR, indicating that AHL15 targets are partially shared between stem and leaf tissue. The leaf senescence phenotype of ahl15 loss-of-function mutants is relatively mild ( Figure S1 ), suggesting that either AHL15 itself plays a minor role in leaf senescence, or that other AHL genes take over its role in the mutant plants. AHL genes have been shown to be highly functionally redundant, and developmental phenotypes are observed only in higher-order ahl mutants ( Xiao et al ., 2009 ; Zhao et al ., 2013 ; Karami et al ., 2020 ; Rahimi, Karami, Balazadeh, et al ., 2022 ). In addition, AHL15 is expressed primarily in young juvenile leaves as its expression in adult leaves is repressed by SPL TFs ( Rahimi, Karami, Balazadeh, et al ., 2022 ). In fact, with the pAHL15:GUS reporter, no expression is observed in fully expanded juvenile leaves or in adult leaves ( Figure S2 ). It is therefore likely that higher-order ahl mutants, especially of the clade-A AHL genes that are expressed in adult leaves, show a stronger leaf senescence phenotype compared to the ahl15 single mutant. The functional redundancy among AHL proteins suggests that overexpression of other clade-A AHLs could have similar effects as AHL15 on the expression of ORE1 and CKX genes and the overall CK levels in the plant, and that the repression of CK degradation is a general effect of high AHL dosage. Overexpression of at least one other clade-A AHL, AHL27, has been shown to inhibit senescence ( Lim et al ., 2007 ), indicating that, like flowering time, the senescence-inhibiting phenotype could be induced by multiple AHLs in this clade. It would therefore be interesting to see whether overexpression of other clade-A AHLs has a similar senescence phenotype as AHL15 and AHL27 , and whether this coincides with increased CK levels in the leaves. Arabidopsis contains 15 clade A AHLs , and the genomes of most crop species contain a similar or higher number of AHL genes ( Zhao et al ., 2014 , 2020 ; Machaj and Grzebelus, 2021 ; W. M. Zhang et al ., 2021 ; Wang et al ., 2023 ). Breeding efforts to improve plant longevity and shelf life could focus on enhancing the expression of clade-A AHLs, which, due to their functional overlap and large number, represent promising targets for crop improvement. Materials and Methods Plant material and growth conditions Seeds were sown on damp soil (90% turf soil with 10% sand) and stratified for three days at 4 °C before transfer to a climate chamber set at 21°C with a long day (16h photoperiod) and 65% relative humidity. Seedlings were transferred to individual pots one week after sowing and plants were watered weekly. The p35S:AHL15-GR overexpression and ahl15 loss-of-function lines have been described previously ( Karami et al ., 2020 ). p35S:3xFLAG-AHL15 overexpression lines were generated by replacing the AHL15-GR construct in the p35S:AHL15-GR vector made by Karami et al. (2020) with a synthetic 3xFLAG-AHL15 construct, which was transformed to Agrobacterium tumefaciens strain AGL1 by electroporation ( Dulk-Ras and Hooykaas, 1995 ). Transgenic p35S:AHL15 Arabidopsis Col-0 lines were obtained by the floral dip method ( Clough and Bent, 1998 ) and by subsequently selecting T1 and T2 plants on ½ MS medium supplemented with 15 mg/L phosphinotricin. Single locus transgenic lines, showing a 3:1 segregation ratio of resistant: susceptible plants in the T2 generation, were selected and T3 seeds from homozygous T2 plants were subsequently used for senescence experiments. For dark-induced senescence assays, fifth leaves were detached from the plants at 23 days after the start of germination and floated on ca 5mL 5mM MES buffer (pH 5.6) in 5 cm ø Petri dishes, with a maximum of 10 leaves per dish. Petri dishes were sealed with parafilm, wrapped in aluminum foil, and stored in a cardboard box which was placed in the growth chamber to ensure a constant temperature of 20 °C. For the hormone treatments, a 10 mM dexamethasone (Thermo Fisher, Vilnius, Lithuania) stock solution dissolved in DMSO was diluted 1000-fold in 5 mM MES buffer to a final concentration of 10 μM. For mock treatment, DMSO was diluted 1000-fold in 5 mM MES buffer. Senescence measurements For colorimetric measurements, the leaves were imaged on a dark background with a white reference card under uniform lighting with a Nikon DC3000 camera, with the following settings: ISO 160, exposure time 1/160 s. Image analysis was done in ImageJ with a custom plugin that measures Chl using RGB images ( Luden et al ., 2025 ). The analysis consisted of the following steps: white balancing of images based on a white reference sheet included in each picture; removal of background, conversion to an RGB image stack, automatic selection of individual leaves, and measurement of pixel intensity for each leaf in each of the red, green, and blue channels. The mean red, green, and blue values for each leaf were used to calculate the normalized red value (Rn; Red/(Red+Green+Blue)), which we previously showed correlates best with absolute Chl content ( Luden et al ., 2025 ). After imaging for colorimetric measurements, two 5 mm ø leaf disks were taken from each leaf and stored at −80 °C. Acetone-based Chl extraction was done following the method described by Arnon (1949) . Briefly, frozen leaf material was pulverized with a metal bead and dissolved in 200 μL 25 mM sodium phosphate buffer (pH 7) and 800 μL 80% acetone. Samples were then incubated at room temperature for 1 hour with gentle shaking and centrifuged for 10 minutes at 2500x g . 200 μL supernatant was then transferred to a clear-bottomed 96-well plate and the absorption (D) at 645 and 663 nm was measured with a TECAN Spark plate reader (Männedorf, Switzerland). The total Chl content in mg/L was then calculated with the following formula: Chl Total = 20,2*D 645 + 8,02*D 663 , as described by ( Arnon, 1949 ), and then corrected for the total area of leaf tissue used for Chl extraction. Spectrophotometry results and colorimetric results from ImageJ were processed in R ( R Core Team, 2023 ). Plots were generated using ggplot2 ( Wickham, 2016 ) and ggpubr ( Kassambara, 2023 ), and genotypes or treatments were compared with the Kruskal-Wallis test by using the package Agricolae ( de Mendiburu, 2023 ). RNA isolation and RT-qPCR After imaging for colorimetric measurements, leaves were cut in half, of which one half was used for RNA extraction, while the other half of each leaf was used for Chl extraction. For each RNA extraction, material from three plants was pooled and snap-frozen in liquid nitrogen, and tissue was pulverized with a metal bead using the Qiagen Tissuelyser. RNA was isolated with Trizol© reagent (Thermo Fisher Scientific, Vilnius, Lithuania) by following the standard protocol for Trizol-based RNA extraction, with an additional ethanol wash step at the RNA precipitation stage. Next, 1 μg of RNA was aliquoted to a new tube and treated with DNase I (Thermo Fisher Scientific, Vilnius, Lithuania) at 37 °C for 30 minutes and used for cDNA synthesis with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, Lithuania) according to the kit’s instructions. RT-qPCR was performed with TB green ex-Taq II (Takara Bio Europe, Saint-Germain-en-Laye, France) on the QuantStudio 5 Real-Time PCR machine (Applied Biosystems, Foster City, USA) in a reaction volume of 5 μL. Primers used for RT-qPCR ( Supplementary Table 1 ) were synthesized by Sigma Aldrich (Haverhill, UK). Gene expression was normalized to ACTIN2 expression by using the 2^-ΔΔCT method ( Livak and Schmittgen, 2001 ), and groups were compared with the Kruskall-Wallis test in R as described above. Cytokinin measurements Quantification of cytokinin metabolites was performed on biological replicates according to the method described by Svačinová et al. (2012) , including modifications described by Antoniadi et al. (2015) . Samples (10 mg fresh weight) were homogenized and extracted in 1 ml of modified Bieleski buffer (60% methanol, 10% formic acid and 30% water) together with a cocktail of stable isotope-labeled internal standards (0.2 pmol of CK bases, ribosides, N-glucosides, and 0.5 pmol of CK O-glucosides, nucleotides per sample added). The extracts were applied onto an Oasis MCX column (30 mg/1 ml, Waters) conditioned with 1 ml each of 100% methanol and water, equilibrated sequentially with 1ml of 50% (v/v) nitric acid, 1 ml of water, and 1 ml of 1M formic acid, and washed with 1 ml of 1M formic acid and 1 ml 100% methanol. Analytes were then eluted by two-step elution using 1 ml of 0.35M ammonium hydroxide aqueous solution and 2 ml of 0.35M NH4OH in 60% (v/v) methanol solution. The eluates were then evaporated to dryness in vacuo and stored at −20°C. Cytokinin levels were determined by an ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS) using stable isotope-labelled internal standards as a reference ( Rittenberg and Foster, 1940 ). Separation was performed on an Acquity UPLC® i-Class System (Waters, Milford, MA, USA) equipped with an Acquity UPLC BEH Shield RP18 column (150×2.1 mm, 1.7 μm; Waters), and the effluent was introduced into the electrospray ion source of a triple quadrupole mass spectrometer Xevo™ TQ-S MS (Waters, Milford, MA, USA). Five independent biological replicates, each consisting of material from the fifth leaf of three plants, were performed, including two technical replicates of each. Data statement Raw data and materials are available upon request to R.O. Author Contributions The project was conceived by T. L. and R. O. with input from S. B.. All experiments were performed by T. L., except for the CK measurements, which were performed by P. A. and O. N.. Figures were prepared by T. L.. The manuscript was written by T. L. with input from R. O. and S. B., and the manuscript was read and approved by all authors. Supporting Information Download figure Open in new tab Figure S1: AHL15 delays age-dependent rosette leaf senescence. A: Images of 42-day-old wild-type (Col-0), ahl15 , and AHL15 OX ( p35S:3xFLAG-AHL15 ) plants grown in 5 cm-wide pots under long-day conditions. B: Progression of senescence in the fifth leaf of wild-type (Col-0), ahl15 , and AHL15 OX ( p35S:3xFLAG-AHL15 ) plants at different days after emergence. White bars represent 1 cm. C: Chlorophyll content was quantified based on the images in panel B as the normalized Red value (Rn), with four leaves per time point (only three leaves are shown in B to preserve space). Groups were compared at each time point by Kruskall-Wallis test, and letters indicate significance groups; groups with the same letter do not differ significantly from one another (p<0.05). Similar results were obtained in four independent experiments in different growth chambers. Download figure Open in new tab Figure S2: AHL15 is expressed in young developing juvenile leaves. A,B: Images of 14-day-old ( A ) or 21-day-old ( B ) pAHL15:GUS GUS activity stained plants grown in long day (16h photoperiod) conditions. The leaf number (in order of appearance) is indicated with white numbers; cotyledons are marked with c. Size bar represents 5 mm. Download figure Open in new tab Figure S3. AHL15 only mildly reduces dark-induced SEN1 expression in leaves. Fifth leaves were detached from 23-day-old p35S:AHL15-GR plants and incubated in the dark on 5 mM MES buffer with or without 10 µM DEX for the indicated number of days. Expression was determined by qRT-PCR in three biological replicates (n=3), each composed of leaf material of three plants, using ACTIN2 as reference. The Kruskall-Wallis test was used to compare conditions, and significance groups are indicated by different letters (p < 0.05). Download figure Open in new tab Figure S4: AHL15 binds upstream of the SPL9 gene. ChIP seq performed on chromatin isolated from 31-day-old p35S:3xFLAG-AHL15 plants using an anti-FLAG antibody for IP. The SPL9 gene is shown in black (black blocks are exons, black lines are introns, transcription direction is indicated by a double arrow head), ChIP seq read alignments from the samples are shown in purple (bigwig track), and the location of significant peaks in blue, with a light blue bar indicating the peak summit (narrowpeak track) at approximately 500 bp upstream of the transcription start. Download figure Open in new tab Figure S5. AHL15 binds to cytokinin biosynthesis genes to differentially regulate their expression. A: ChIP seq performed on chromatin isolated from 31-day-old p35S:3xFLAG-AHL15 plants using an anti-FLAG antibody for IP. Genes are indicated black (black blocks are exons, black lines are introns), read alignments in purple (bigwig track), and the location of significant peaks in blue, with a light blue bar indicating the peak summit (narrowpeak track) at 1.4 kb and 3 kb upstream and in the 3’ UTR of IPT7 , at 1 kb and 2.1 kb upstream of LOG1 , directly upstream of LOG7 , and at 1.6 kb upstream of LOG8 . B: IPT7, LOG1,LOG7, and LOG8 expression in the fifth leaf detached from 23-day old p35S:AHL15-GR plants and incubated in the dark on 5 mM MES buffer with or without 10 µM DEX for the indicated number of days. Expression was determined by qRT-PCR on three biological replicates (n=3), each composed of leaf material of three plants, using ACTIN2 as reference. The Kruskall-Wallis test was used to compare conditions, and significance groups are indicated by different letters (p < 0.05). Download figure Open in new tab Figure S6. AHL15 delays the dark-induced decline in iP-type CK levels in leaves. The fifth leaf was collected from 23-day-old plants and floated on 5 mM MES with or without 10 µM DEX and incubated in the dark for the indicated number of days. The material of three leaves was pooled for each sample, and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). The iP base was not present at detectable levels. Download figure Open in new tab Figure S7. AHL15 delays the dark-induced decline in tZ- and tZR-type CK levels in leaves. The fifth leaf was collected from 23-day-old plants and floated on 5 mM MES with or without 10 µM DEX and stored in the dark for the indicated number of days. The material of three leaves was pooled for each sample, and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). Download figure Open in new tab Figure S8. The effect of AHL15 on cZ-type CK levels in dark-incubated leaves. The fifth leaf was collected from 23-day-old plants and floated on 5 mM MES with or without 10 µM DEX and stored in the dark for the indicated number of days. The material of three leaves was pooled for each sample, and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). Download figure Open in new tab Figure S9. The effect of AHL15 on DHZ-type CK levels in dark-incubated leaves. The fifth leaf was collected from 23-day-old plants and floated on 5 mM MES with or without 10 µM DEX and stored in the dark for the indicated number of days. Material of three leaves was pooled for each sample, and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). DHZ base was not present at detectable levels in these samples. Download figure Open in new tab Figure S10. AHL15 delays the dark-induced decline in active CK levels in leaves. The fifth leaf was collected from 23-day-old plants and floated on 5 mM MES with or without 10 µM DEX and incubated in the dark for the indicated number of days. The material of three leaves was pooled for each sample, and five samples were measured for each condition (n = 5). Groups were compared using the Kruskall-Wallis test, and letters indicate significance groups (p < 0.05). Download figure Open in new tab Figure S11. AHL15 preserves the chlorophyll content in leaves during dark incubation. A: Normalized Red values (Rn) based on RGB images of leaves just before collecting material for CK measurements. B: Total chlorophyll content determined by acetone-based chlorophyll extraction in leaf disks taken from the same samples as used for CK measurements. For each condition in A and B , five samples were measured (n = 5), each consisting of material from the fifth leaf harvested from three individual 23-day-old p35S:AHL15-GR plants. Groups were compared with the Kruskall-Wallis test, and letters indicate significance groups (p <0.05). View this table: View inline View popup Download powerpoint Table S1: Primers used for RT-qPCR. Acknowledgements We thank Jan Vink and Altay Temel for plant care, and Ward de Winter, Ellora Basu, and Mariël Lavrijsen for technical support. We also thank Jelmer van Lieshout for feedback on the project and Marion Larue for help with pilot experiments. This work is part of the REJUVENATOR project with file number GSGT.2019.024 (to TL) of the Graduate School Green Top sectors research programme, which is financed by the Dutch Research Council (NWO). Funder Information Declared Dutch Research Council, https://ror.org/04jsz6e67 , GSGT.2019.024 Literature ↵ Antoniadi , I. et al. 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Share The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation Thalia Luden , Petra Amakorová , Ondřej Novák , Salma Balazadeh , Remko Offringa bioRxiv 2025.11.23.689984; doi: https://doi.org/10.1101/2025.11.23.689984 Share This Article: Copy Citation Tools The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation Thalia Luden , Petra Amakorová , Ondřej Novák , Salma Balazadeh , Remko Offringa bioRxiv 2025.11.23.689984; doi: https://doi.org/10.1101/2025.11.23.689984 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41951) Biophysics (21456) Cancer Biology (18594) Cell Biology (25520) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22510) Immunology (17737) Microbiology (40401) Molecular Biology (17183) Neuroscience (88622) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)