# Source paper archive - Source title: The chromatin-associated PWO proteins interface with circadian and epigenetic signaling to regulate flowering time in Arabidopsis - Author(s): Haider, S., Riviere, Q., Shelton, A., Khan, A., Webb, A. A., Mozgova, I., Farrona, S. - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.03.763775v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.03.763775v1.full.pdf - PDF SHA-256: 631a52ae490fc0afa876b1b66b814e2789042fc95973ab30fbf5f473ed454ffc - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 1 **The chroma�n-associated PWO proteins interface with circadian and epigene�c signaling to** 2 **regulate flowering �me in Arabidopsis** 3 Saqlain Haider [1,2,a], Quen�n Rivière [3], Annie Shelton [4], Ahamed Khan [3], Alex Webb [4], Iva 4 Mozgova [3], Sara Farrona [1,2*] 5 1. School of Biological and Chemical Sciences, College of Science and Engineering, University 6 of Galway, H91 TK33, Galway, Ireland 7 2. Centre for Chromosome Biology, Ins�tute for Health Discovery and Innova�on, University 8 of Galway, H91 TK33, Galway, Ireland 9 3. Biology Centre of the Czech Academy of Sciences - Ins�tute of Plant Molecular Biology, 10 37005 České Budějovice, Czech Republic 11 4. Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom 12 a. Present address: Department of Biological Sciences, University of Alberta, Edmonton, 13 Alberta, Canada 14 *Author for correspondence: Email: sara.farrona@universityofgalway.ie 15 **Key words:** Circadian clock, Chroma�n, Flowering �me, Photoperiod pathway, PWO1/2, PRR7 16 **Abstract** 17 Flowering �me is a major determinant of reproduc�ve success in plants and is regulated by 18 several interconnected pathways which converge on few key flowering �me-related genes. In 19 the model plant _Arabidopsis thaliana_ (Arabidopsis), both the circadian clock and chroma�n 20 associated proteins integrate environmental cues to coordinate flowering (Staiger _et al._ 2013; 21 Nohales and Kay, 2016; Du _et al._ 2019; Haider and Farrona, 2024). However, how the circadian 22 epigene�c signaling crosstalk regulates this crucial developmental transi�on has s�ll been 23 underexplored. PWWP-DOMAIN INTERACTORS OF POLYCOMBS 1 and 2 (PWO1 and PWO2) 24 are chroma�n-associated proteins that have been linked to the regula�on of histone 2A (H2A) 25 mono-ubiqui�na�on (ub) (H2Aub) levels (Zheng _et al._ 2023). PWO1 and PWO2 also affect 26 flowering �me (Hohensta� _et al._ 2018; Khan _et al._ 2026), but the underlying molecular 27 mechanisms, their effects on clock genes, as well as their dis�nct contribu�ons to flowering 28 �me regula�on remain mostly unknown. 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 29 **Introduc�on** 30 Circadian clocks are endogenous �me-keeping systems that generate ~24-hour biological 31 rhythms and enable organisms to align their physiology with daily and seasonal environmental 32 cues. In Arabidopsis, the circadian oscillator includes two morning expressed MYB 33 transcrip�on factors encoding genes namely _CIRCADIAN CLOCK ASSOCIATED 1_ ( _CCA1_ ) and 34 _LATE ELONGATED HYPOCOTYL_ ( _LHY_ ), and the evening expressed _TIMING OF CAB EXPRESSION_ 35 _1_ ( _TOC1_ ) gene, also known as _PSEUDO RESPONSE REGULATOR 1_ ( _PRR1_ ) (Wang _et al._ 1998; 36 Schaffer _et al._ 1998; Strayer _et al._ 2000; Huang _et al._ 2012). During day�me, the _PRR_ gene 37 family is expressed in a sequen�al order, i.e. _PRR9_, _PPR7_, _PRR5_, _PRR3_, and _TOC1_, each peaking 38 at ~2hrs interval (Matsushika _et al._ 2000; Nakamichi _et al._ 2010; Nohales and Kay, 2016). The 39 evening complex (EC) comprises _EARLY FLOWERING 3_ ( _ELF3_ ), _EARLY FLOWERING 4_ ( _ELF4_ ), and 40 _LUX ARRHYTHMO_ ( _LUX_ ) (Nohales and Kay, 2016). Addi�onal clock associated proteins such as 41 GIGANTEA (GI), ZEITLUPE (ZTL), and FLAVIN-BINDING, KELCH REPEAT, F BOX 1 (FKF1) interact 42 with core clock components (Imaizumi _et al._ 2003; Mizoguchi _et al._ 2005; Song _et al._ 2012; 43 14). Altogether, the circadian oscillator regulates numerous physiological and metabolic 44 processes in plants (McClung, 2011; Staiger _et al._ 2013; Nohales and Kay, 2016; Oakenfull and 45 Davis, 2017). 46 The circadian clock plays a fundamental role in the regula�on of flowering �me through the 47 so-called photoperiodi pathway. The photoperiodism is the process by which plants measure 48 the changes in day and night cycles associated with the seasons, and make accordingly floral 49 transi�on (Samach _et al._ 2000; Blackman, 2017). Mechanis�cally, circadian oscillators regulate 50 photoperiodic response at both transcrip�onal and pos-transcrip�onal levels. In Arabidopsis, 51 the circadian clock rhythmically maintains the expression of the key posi�ve flowering gene 52 _CONSTANS_ ( _CO_ ) late, at a �me that corresponds to the night under non-induc�ve short day 53 (SD) (Suárez-López _et_ _al._ 2001; Yanovsky and Kay, 2002). CONSTITUTIVE 54 PHOTOMORPHOGENIC1 (COP1) degrades CO in the dark via the proteasomal pathway and the 55 reduced CO ac�vity limits _FLOWERING LOCUS T_ ( _FT)_ (key florigen) induc�on, delaying 56 flowering (Jang _et al._ 2008). CO keeps being degraded un�l the photoperiod becomes long 57 enough, and the peak of the CO protein coincides with daylight in long day (LD) (Yanovsky and 58 Kay, 2002, Valverde _et al._ 2004). 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 59 The circadian clock is mainly involved in that process through GI and PRRs. GI regulates _CO_ 60 mRNA by degrading CYCLING DOF TRANSCRIPTION FACTORS (CDFs) (Fornara _et al._ 2009). 61 CDFs, among which CDF1 and CDF2 play a major role, redundantly repress _CO_ transcrip�on by 62 directly binding to its promoter (Imaizumi _et al._ 2005; Imaizumi _et al._ 2003; Fornara _et al._ 63 2009; Hayama _et al._ 2017) and can also directly repress _FT_ expression (Corbesier _et al._ 2007; 64 Jaeger and Wigge, 2007; Song _et al._ 2012). In addi�on, PRRs, including PRR9, PRR7, and PRR5, 65 also directly repress _CDFs_ (primarily _CDF1_ ) transcrip�on thus promo�ng _CO_ expression 66 (Nakamichi _et al._ 2007). At post-transcrip�onal level, GI contributes to CO protein stability at 67 the end of the day in LD (Song _et al._ 2014; Hwang _et al._ 2019) and PRRs physically interact with 68 CO to prevent its degrada�on by COP1 (Hayama _et al._ 2017). On the other hand, FLOWERING 69 LOCUS C (FLC) represses _FT_ transcrip�on by directly binding to its promoter, thus preven�ng 70 precocious flowering (Searle _et al._ 2006). 71 Here, we conducted extensive phenotyping and data mining and found that PWOs play a 72 decisive role in the photoperiodic pathway in concert with the circadian clock. 73 **PWO2 has a stronger effect on flowering �me than PWO1** 74 To be�er understand the interac�on between PWO1 and PWO2 in flowering �me regula�on, 75 we scored the flowering �me phenotypes of the _pwo1-1_ ( _pwo1)_, _pwo2-2_ ( _pwo2_ ), and _pwo1-_ 76 _1;pwo2-2_ ( _pwo1;2_ ) mutants versus Col-0 plants under LD condi�ons (Figure 1a). The _pwo1_ 77 mutants had a weak flowering phenotype in terms of leaf number in line with a previous study 78 (Hohensta� _et al._ 2018) (Figure 1b). However, _pwo1_ mutant was similar to Col-0 in terms of 79 number of days (Figure 1c). Contrarily, the _pwo2_ mutant was considerably delayed compared 80 to Col-0 _i.e.,_ higher number of leaves alongside number of days (Figure 1b,c). Notably, the 81 flowering phenotype of _pwo1;2_ mostly resembled that of _pwo2_ both in terms of days to flower 82 and number of leaves (Figure 1b-c). A downward trend in terms of number of leaves in _pwo1;_ 2 83 in comparison with _pwo2_ was consistent with an addi�ve effect of the two muta�ons, but the 84 absence of sta�s�cal difference prevented us from reaching a defini�ve conclusion. 85 Nevertheless, our data clearly indicated that flowering is primarily controlled by PWO2. We 86 also analyzed the flowering phenotype of Col-0 vs _pwo1;2_ under SD condi�ons which revealed 87 a slow growth trend in _pwo1;2_ (Supplementary figure 1a). Interes�ngly under SD, the _pwo1;2_ 88 double mutant produced lesser number of leaves compared to Col-0 plants despite being 89 delayed in terms of number of days (Supplementary figure 1b-d). 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 90 _**PWO1/2**_ **regulate diel expression of flowering regulator genes** 91 To understand the impact of _PWO1/2_ on flowering regulators, we first examined the 92 expression profiles of _FLC_ and _FT_ (key flowering regulators) in Col-0 vs _pwo1;2_ from 6-day-old 93 and 10-day-old seedlings harvested at ZT16. Both _FLC_ and _FT_ were reduced in _pwo1;2_ 94 regardless of the developmental age of the plants (Supplementary figure 2). A reduced _FT_ 95 despite lower _FLC_ indicated a possible defect in the photoperiod pathway. To this end, we first 96 analyzed the binding of PWO1-FLAG using ChIP-seq data from Zheng _et al._ (2023) which 97 indicated PWO1-FLAG binding to _TOC1_, _PRR7_, _PRR5_, _ELF3_, and _GI_ (Supplementary figure 3) _._ 98 Expression analyses of these genes revealed a reduc�on in the expression of _PRR7_ and a non 99 significant decline in the other tested genes in _pwo1;2_ (Supplementary figure 4). As core clock 100 components have been implicated in regula�on of photoperiodic flowering (Fowler _et al._ 101 1999; Mizoguchi _et al._ 2005; Nakamichi _et al._ 5; 2007; Song _et al._ 2014), we hypothesized 102 PWO1/2 may control flowering �me through regula�on of circadian clock and photoperiod 103 pathway genes. 104 To inves�gate the possible role of PWOs in regula�ng the diel oscilla�ons of key clock and 105 flowering genes, we harvested samples from 10-day-old long day (LD)-grown seedlings every 106 4 hours from ZT0 to ZT24 (Figure 1d-k). To this end, we examined the expression profiles of 107 the _CCA1_, _LHY_, _PRR7_, _GI_, _CO_, _FLC_, _FT_, and _CDF1_ genes with a well-established role in control 108 of the photoperiod pathway (Schaffer _et al._ 1998; Suárez-López _et al._ 2001; Mizoguchi _et al._ 109 2005; Searle _et al._ 2006; Corbesier _et al._ 2007; Nakamichi _et al._ 2007; Hayama _et al._ 2017). 110 _CCA1_ had a reduced expression at ZT0 in _pwo1_, _pwo2_, and _pwo1;2_ mutants, although the 111 rhythmic profile was maintained across a diel cycle (Figure 1d). _LHY_ expression was similar to 112 Col-0 in _pwo1;2_ while it was visibly reduced in _pwo1_ and _pwo2_ at ZT0 (Figure 1e). _PRR7_ 113 expression was altered in all _pwo_ mutant backgrounds. In _pwo1_ mutants, _PRR7_ transcript 114 levels were reduced from ZT8 onwards. In _pwo2_ mutants, _PRR7_ had an altered pa�ern with 115 peaks at ZT4 and ZT12, although expression was strongly reduced at ZT8. Notably, _pwo1;2_ 116 mutants had a near-complete loss of _PRR7_ rhythms together with near loss of expression 117 (Figure 1f). For _GI,_ a pronounced reduc�on at ZT12 was observed in _pwo1_ and _pwo1;2_ mutants 118 while _pwo2_ had WT-like expression (Figure 1g). _CO_ expression remained largely unaffected in 119 all _pwo_ mutants across the diel cycle (Figure 1h). _FLC_ expression was not rhythmic in both 120 _pwo1_ and _pwo1;2_ mutants. By contrast, _pwo2_ mutants maintained a largely WT-like 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 121 expression pa�ern, although a stronger peak was observed at ZT12 (Figure 1i). In _pwo1_ 122 mutants, _FT_ expression was transiently elevated at ZT4 possibly explaining the slight early 123 flowering of _pwo1_ mutant as a _FT_ expression peak at ZT4 is sufficient for floral induc�on under 124 natural light condi�ons (Song _et al._ 2018). In _pwo2_ mutants, _FT_ expression was strongly 125 reduced at ZT16 in line with the delayed flowering phenotype while _pwo1;2_ also had reduced 126 expression at ZT16 (Figure 1j). Finally, all the _pwo_ mutants had a higher _CDF1_ expression at 127 ZT0 with _pwo2_ uniquely showing a strong upregula�on at ZT16 and _pwo1;2_ also showing 128 higher _CDF1_ expression at ZT20 (Figure 1k). Taking together, these results indicate that the 129 diel oscilla�on pa�erns of key flowering regulatory genes are disrupted in _pwo1_, _pwo2_, and 130 _pwo1;2_ mutants. 131 The impact of PWO1/2 on rhythms of the circadian oscillator gene _PRR7_ prompted us to 132 inves�gate whether these mutants also affect the free running circadian period. We es�mated 133 the free-running period in wild-type and _pwo1, pwo2, pwo1;2_ mutant plants in constant light 134 and temperature using leaf movement (Herrero _et al._ 2026). However, no significant 135 differences were detected between these genotypes and Col-0, indica�ng that loss of _PWO1/2_ 136 func�on does not significantly alter circadian period under the condi�ons examined 137 (Supplementary figure 5). These findings are also supported by a similar WT-like trend for 138 _CCA1_ and _LHY_ genes in _pwo1;_ _pwo2_ ; _pwo1;2_ mutants (Figure 1d-e). Nevertheless, as in our 139 diel expression analysis, two key genes regula�ng flowering, _PRR7_ and _GI_, showed strong 140 perturba�ons in _pwo1;2_ compared to Col-0 (Figure 1f-g), we further tested whether PWO1/2 141 may control flowering via _PRR7_ and _GI_ transcrip�onal regula�on. 142 _**PRR7**_ **is epista�c to** _**PWO1/2**_ **for flowering �me control** 143 To be�er understand the gene�c rela�onship between _PWO1/2_ and _PRR7_ for flowering �me 144 control, we crossed _pwo1;2_ with _prr7-11 (prr7)_ to obtain the _pwo1;2;prr7_ triple mutant. 145 Flowering �me analysis of _pwo1;2;prr7_ triple mutant and _prr7_ revealed an epista�c 146 rela�onship between _PWO1/2_ and _PRR7_ both in terms of number of days and rose�e leaves 147 (Figure 2a). Briefly, _pwo1;2_ and _prr7_ flowered at 42 days and 59 days, respec�vely, while 148 flowering in _pwo1;2;prr7_ occurred at 60 days. On the other hand, _pwo1;2_ produced 17 leaves, 149 _prr7_ 40 leaves, and _pwo1;pwo2;prr7_ 38 leaves at flowering (Figures 2b-c) _._ Such results were 150 in line with an epista�c rela�onship between _PRR7_ and _PWO1/2_ for the control of flowering 151 �me. 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 152 We next wanted to clarify whether the observed epista�c interac�on between _PWO1/2_ and 153 _PRR7_ converges on _CO_ for flowering �me control (Nakamichi _et al._ 2007). To this end, we 154 crossed the _pwo1;2_ double mutant with the _co-101_ ( _co_ ) single mutant to obtain triple 155 _pwo1;2;co_ mutant plants (Figure 2a). While _co_ plants produced ~51 leaves, _pwo1;2;co_ plants 156 produced ~48 leaves at the �me of flowering (Figure 2b). These results confirmed that _CO_ is 157 epista�c to _PWO1/2_ for flowering in terms of leaf number. Nevertheless, _pwo1;2;co_ triple 158 mutant was significantly delayed when compared to _co_ considering days to flower: _co_ 159 flowered on average on day 69, while the _pwo1;2;co_ triple mutant flowered on day 84 (Figure 160 2c). Therefore, _CO_ and _PWO1/2_ showed a synergis�c interac�on for flowering in terms of 161 number of days. 162 As _GI_ is a dominant regulator of _CO_ expression and protein stability under LDs, we crossed 163 _pwo1;2_ double mutants with _gi-2 (gi)_ to obtain the _pwo1;2;gi_ triple mutant (Figure 2a). The 164 flowering phenotype revealed that _gi_ and _pwo1;2_ mutants flowered ~36 days and ~9 days 165 later than Col-0 respec�vely (Figure 2c). On the other hand, _pwo1;2;gi_ mutants flowered ~58 166 days later than Col-0, indica�ng that the _pwo1;2_ gene�c background enhanced the phenotype 167 of _gi_ (Figure 2c). While for rose�e leaf number, _pwo1;2_ and _gi_ produced ~4 and ~50 extra 168 leaves compared to Col-0, _pwo1;2;gi_ produced ~60 extra leaves (Figure 2b), indica�ng an 169 addi�ve or possible close to synergis�c gene�c interac�on between _gi_ and _pwo1;pwo2_ for 170 rose�e leaf number. 171 To determine whether the epista�c interac�ons between _PWO1/PWO2_ and _PRR7_ or _CO_ 172 converge on the expression of _FT_, _FT_ transcript levels were quan�fied in the Col-0, _pwo1;2_, 173 _prr7_, _pwo1;2;prr7_, _co_, _pwo1;2;co_, _gi_, and _pwo1;2;gi_ genotypes from samples harvested at 174 ZT16. Compared to WT, _prr7_ showed reduced _FT_ expression as reported previously (Nakamichi 175 _et al._ 2007), while it remained the same as _prr7_ in _pwo1;2;prr7,_ in line with a similar flowering 176 phenotype. A complete loss of _FT_ expression was observed in _co_ and _gi_ (Figure 2d), as 177 expected from previous reports (Suárez-López _et al._ 2001; Mizoguchi _et al._ 2005). Similar to 178 _co_ and _gi_, low _FT_ mRNA levels were also quan�fied in the _pwo1;2;co_ and _pwo1;2;gi_ mutant 179 (Figure 2d). 180 **PWO1/PWO2 regulate H2Aub levels at the** _**PRR7**_ **locus** 181 Polycomb repressive complex 1 (PRC1) deposits the repressive H2Aub mark at its target genes 182 (Bratzel _et al._ 2010). On the other hand, PWO1/2 facilitate the removal of the H2Aub mark by 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 183 UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) (Zheng _et al._ 2023; Godwin _et al._ 2024). Therefore, 184 considering our expression and phenotypic data, we sought to clarify whether both _PRR7_ and 185 _GI_ show varia�on in H2Aub levels between WT and _pwo1;2_ (Figure 2e). We harvested samples 186 from 10-day-old seedlings at ZT8 and ZT12, when _PRR7_ and _GI_ expression reached their peak 187 in WT plants and showed dras�cally reduced expression in _pwo1;2_ (Figure 1f-g). A significant 188 increase in H2Aub enrichment was observed in _pwo1;2_ compared to Col-0 just downstream 189 of _PRR7_ TSS at both �mepoints (ZT8, ZT12) (Figure 2e). However, we did not observe any 190 changes for _GI_ between WT and _pwo1;2_ . Despite low _GI_ expression (Figure 1g), we could not 191 detect a change in H2Aub levels between WT and _pwo1;2_ (Figure 2e). Indeed, data mining 192 from Godwin _et al._ (2024) confirmed binding of UBP5 to _PRR7_ locus but not _GI_, despite a 193 possible binding of PWO1 to _GI_ (Supplementary figure 3; Zheng _et al._ 2023). 194 **Do** _**PWO1/2**_ **also regulate flowering independently of** _**CO**_ **-dependent photoperiod pathway?** 195 A central finding from our work is the fact that PWOs regulate the CO-dependent photoperiod 196 pathway through the transcrip�onal regula�on of _PRR7_ at the epigene�c level. However, the 197 photoperiod pathway is probably not the only flowering pathway impacted by PWOs, as 198 suggested by the synergis�c effects of _CO_ and _PWO1/2_ in the number of days for flowering 199 (Figure 2c). This hypothesis is further strengthened by the addi�ve and synergis�c interac�on 200 between _PWO1/2_ and _GI_ for rose�e leaf number and for number of days despite similar _FT_ 201 levels _._ The poten�al addi�onal flowering pathway(s) influenced by PWOs were likely 202 disrupted in the _prr7_ loss-of-func�on background, explaining why _PRR7_ appeared epista�c to 203 _PWO1/2_ (Figure 2a) despite though _PRR7_ reported to regulate flowering �me through _CO_ 204 (Figure 2b) (Nakamichi _et al._ 2007). With such considera�on, our results did not contradict the 205 previous observa�on by Hohensta� et al. (2018) that _PWO1_ interacts with _FLC_ to control the 206 floral transi�on. This prompted us to evaluate another circadian clock gene, which uses both 207 CO and alterna�ve routes to regulate the flowering �me, _ELF3_ (Kim _et al._ 2005). We observed 208 a misregula�on of _ELF3_ expression in all _pwo_ mutants (Supplementary figure 6), providing new 209 avenues for inves�ga�ng the interplay between PWOs, the circadian clock and the floral 210 transi�on. 211 One addi�onal candidate pathway that PWOs may interact with is the age-related one. The 212 _pwo1;2_ mutants grown under SD showed slower growth and leaf produc�on rate (number of 213 leaves at flowering/number of days at flowering), with Col-0 showing 0.79 leaves/days, while 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 214 _pwo1;2_ having a rate of 0.45 leaves/days (Supplementary figure 1a). A non-significant, yet 215 mild delay in juvenile-to-adult transi�on was also observed under LD condi�ons in _pwo1;2_ 216 (Supplementary figure 7). Interes�ngly, under LD condi�ons, introduc�on of _pwo1;2_ into the 217 _gi_ background slows the growth and delays the leaf produc�on of the _gi_ mutant 218 (Supplementary figure 8). 219 A non-photoperiodic role of _PWO1/2_ is also suggested by an early flowering phenotype of 220 _pwo1;2_ under SDs (Supplementary figure 1b,c). A complete reduc�on of _FLC_ expression in 221 _pwo1_ and a similar trend in _pwo1;2_ but not in _pwo2_ (Figure 1i) also pointed out to a possible 222 interac�on specifically between PWO1 and the autonomous pathway (Figure 2f) in which _FLC_ 223 is involved (Rouse _et al._ 2002; Qi _et al._ 2022). Interes�ngly, the expression of _FLC_ returned to 224 WT levels in _pwo1;2;prr7_ and _prr7_ (Supplementary figure 9). These results indicate the 225 broader impact of PWO1/2 on PRR7-associated pathways while also revealing the dis�nc�ve 226 interac�ons between PWO1/2 and circadian and photoperiod pathway genes. 227 **Material and Methods** 228 **Plant material and growth condi�ons** 229 _Arabidopsis thaliana_ accession Columbia-0 (Col-0) was used as the wild type (WT) in this study. 230 All the mutant genotypes used in this study were in Col-0 background; these include _pwo1-1_ 231 ( _pwo1_ ), _pwo2-2_ ( _pwo2_ ), _pwo1-1;pwo2-2_ ( _pwo1;2_ ), _gi-2_ ( _gi_ ), _co-101_ ( _co_ ), and _prr7-11_ ( _prr7_ ). 232 These mutants have been previously described (Hohensta� _et al._ 2018; Khan _et al._ 2026; 233 Fowler _et al._ 1999; Takada _et al._ 2003; Nakamichi _et al._ 2005; 7). The triple mutants of 234 _pwo1;pwo2;gi_, _pwo1;pwo2;co_, and _pwo1;pwo2;prr7_ were obtained by crossing _pwo1;2_ with 235 the single mutants of _gi_, _co_, _prr7_ . Homozygous F3 lines were further used for subsequent 236 analysis. Primers used to confirm the mutant genotypes are men�oned in supplementary 237 table 1. 238 Plants were grown in pots containing peat, vermiculite, and perlite in a 5:1:1 ra�o, in long day 239 (LD) (16/8-light/dark) condi�ons under fluorescent lamps at 120 μmol m−2 s−1 at a 240 temperature regime of 21°C. For short days (SD) experiments, plants were grown under similar 241 temperature regime with SD (8/16-light/dark) condi�ons. For _in vitro_ growth, seeds were 242 sowed on ½ Murashige & Skoog (MS) medium supplemented with 0.8% agar and 0.5% 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 243 sucrose. Plates were then stra�fied at 4°C for 3 days for breaking dormancy and synchronizing 244 germina�on. 245 **Flowering �me analysis** 246 Flowering �me was scored by coun�ng both the number of rose�e leaves (excluding 247 cotyledons) and number of days when the first floral bud opened. All the experiments were 248 repeated twice independently, and similar results were obtained (Supplementary table 2-7). 249 **Sta�s�cal analysis** 250 Differences in flowering �me and leaf numbers among genotypes were analyzed separately 251 for each experimental replicate using Welch’s one-way ANOVA followed by Games–Howell 252 post-hoc comparisons. Sta�s�cal significance was defined as adjusted _p_ < 0.05. 253 **Free running period es�ma�on** 254 Circadian period was measured using leaf movement as described in Herrero _et al._ (2026). 255 Plants were grown in 12L:12D un�l 3 days a�er germina�on (DAG), transferred to LL, and 256 monitored for five more days. The free-running circadian period was es�mated from leaf 257 movement �me series using BioDare2 (Zieliński _et al._ 2024). Individual traces were assessed 258 for period, rela�ve amplitude error (RAE), and oscilla�on amplitude. Traces with poor fits (RAE 259 - 0.5) or es�mated periods outside 18–30 h were excluded before genotype means were 260 calculated (Supplementary table 8). 261 **RNA extrac�on, cDNA synthesis, and qPCR** 262 RNA was extracted from 25-50 Arabidopsis seedlings for each biological replicate from each 263 line grown on an ½ MS agar plate by Monarch Total RNA Miniprep Kit (NEB) with on column 264 DNase treatment. 1 μg of total RNA was used to synthesize first strand cDNA using RevertAid 265 First strand cDNA synthesis kit by Thermo Scien�fic. The PCR amplifica�on from cDNA was 266 carried out using Takyon™ No ROX SYBR 2X MasterMix blue dTTP (Eurogentec). Expression 267 analyses were performed by real-�me PCR (Roche LightCycler). _TAP42 INTERACTING PROTEIN_ 268 _OF 41 kDa_ ( _TIP41_ ) was used as housekeeping gene. Error bars show standard devia�on of 269 three biological replicates. Student’s t-test was used to calculate the sta�s�cal significance of 270 results. A list of primer sets used to amplify genes is presented in supplementary table 9. 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 271 **Chroma�n-immunoprecipita�on coupled to qPCR (ChIP-qPCR)** 272 ChIP assays were performed as published recently (Khan _et al._ 2026). Briefly, plants were 273 grown for 10 days in LDs and harvested at ZT8 and ZT12. Plant �ssues were cross-linked using 274 1% formaldehyde under vacuum. Chroma�n immunoprecipita�on of WT and _pwo1;2_ was 275 performed by using _an�-H2Aub_ an�body (Cell Signalling Technology D27C4). Input DNA was 276 diluted 1:10 and IP by 1:5, and 2 μL of immunoprecipitated DNA was used for quan�ta�ve PCR 277 (qPCR). ChIP-qPCR was performed using a LightCycler® 480 Instrument (Roche) with Takyon™ 278 No ROX SYBR 2× MasterMix Blue dTTP (Eurogentec). Primers used to amplify the regions are 279 men�oned in supplementary table 10. Two biological replicates and three technical replicates 280 were used for ChIP-qPCR. Student’s t-test was used to calculate the sta�s�cal significance of 281 results. 282 **Author contribu�ons** 283 SH and SF conceptualized the study, experimental approach, and methodology. SH performed 284 the gene�c crossing, genotyping, phenotyping, expression profiling, and ChIP-assays. QR 285 provided cri�cal insights during all stages of manuscript prepara�on. AS performed the leaf 286 movement assay. AK performed ini�al gene�c analyses with _pwo_ mutant lines. SH, QR, and 287 SF analyzed the data. SH and SF wrote the manuscript. QR, IM, and AW reviewed the 288 manuscript. 289 **Acknowledgments** 290 SF and SH would like to thank Research Ireland (grant number 20/ FFP-P/8693) for funding this 291 work. IM would like to thank the Czech Academy of Sciences (Fellowship _Lumina quaeruntur_ 292 _–_ LQ200961901) and QR is grateful to MSCA Fellowship CZ (EUROTALENTS 293 CZ.02.01.01/00/22_010/0008764) for funding. AS is grateful to the Gates Cambridge Trust for 294 funding. SH and SF would like to thank Professor Rossana Henriques from University College 295 Cork (Ireland) for providing the _gi-2_ and _co-101_ seeds. The _prr7-11_ seeds were kindly provided 296 by Professor Paloma Mas from Center for Research in Agricultural Genomics (Spain). 297 **Compe�ng interests** 298 None declared 299 **Supplementary figure legends** 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 300 **Supplementary figure 1** . _pwo1;2_ mutants are early flowering short day (SD) condi�ons. 301 **Supplementary figure 2** . Expression analysis of _FLC_ and _FT_ in 6- and 10-day-old WT vs _pwo1;2_ 302 seedlings. 303 **Supplementary figure 3** . PWO1-FLAG binding to circadian clock genes. 304 **Supplementary figure 4** . Expression of selected PWO1 target genes in wild-type and _pwo1;2_ 305 plants. 306 **Supplementary figure 5** . Circadian period length based on rhythmic leaf movement in _pwo1,_ 307 _pwo2, pwo1;2_ mutants compared to WT. 308 **Supplementary figure 6** . Diel expression profiles of _ELF3_ in _pwo1_, _pwo2_, _pwo1;2_ compared to 309 Col-0. 310 **Supplementary figure 7** . Abaxial trichome analysis of Col-0 and _pwo1;2_ under LD condi�ons. 311 **Supplementary figure 8** . Vegeta�ve development and leaf produc�on in _pwo;1;2;gi_ compared 312 to _gi_ under LD condi�ons. 313 **Supplementary figure 9** . _FLC_ expression analysis 10-day-old seedlings in Col-0 and mutant 314 genotypes. 315 **Supplementary table legends** 316 **Supplementary Table 1** . Genotyping primers used in this study to confirm Col-0 and mutant 317 alleles. 318 **Supplementary table 2.** Raw data and sta�s�cal analysis for flowering in terms of number of 319 days (Replicate 1). 320 **Supplementary table 3.** Raw data and sta�s�cal analysis for flowering in terms of number of 321 leaves (Replicate 1). 322 **Supplementary table 4.** Raw data and sta�s�cal analysis for flowering in terms of number of 323 days (Replicate 2). 324 **Supplementary table 5.** Raw data and sta�s�cal analysis for flowering in terms of number of 325 leaves (Replicate 2). 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 326 **Supplementary table 6.** Raw data and sta�s�cal analysis for flowering in terms of number of 327 leaves under SD (Rep 1 and 2). 328 **Supplementary table 7.** Raw data and sta�s�cal analysis for flowering in terms of number of 329 days under SD (Rep 1 and 2). 330 **Supplementary table 8.** qPCR primers used in this study to analyze the expression profiles of 331 genes 332 **Supplementary table 9.** ChIP-qPCR primers used in this study to analyze H2Aub enrichment. 333 **Supplementary table 10.** Free running circadian period raw values. 334 **References** 335 Blackman, B. K. (2017). Changing responses to changing seasons: natural varia�on in the 336 plas�city of flowering �me. _Plant physiology_, _173_ (1), 16-26. 337 Bratzel, F., López-Torrejón, G., Koch, M., Del Pozo, J. C., & Calonje, M. (2010). Keeping cell 338 iden�ty in Arabidopsis requires PRC1 RING-finger homologs that catalyze H2A 339 monoubiqui�na�on. _Current biology_, _20_ (20), 1853-1859. 340 Corbesier, L., Vincent, C., Jang, S., Fornara, F., Fan, Q., Searle, I., ... & Coupland, G. (2007). FT 341 protein movement contributes to long-distance signaling in floral induc�on of 342 Arabidopsis. _science_, _316_ (5827), 1030-1033. 343 Du, S., Chen, L., Ge, L., & Huang, W. (2019). A novel loop: mutual regula�on between 344 epigene�c modifica�on and the circadian clock. _Fron�ers in Plant Science_, _10_, 22. 345 Farré, E. M., Harmer, S. L., Harmon, F. G., Yanovsky, M. J., & Kay, S. A. (2005). Overlapping and 346 dis�nct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. _Current Biology_, _15_ (1), 47 347 54. 348 Fowler, S., Lee, K., Onouchi, H., Samach, A., Richardson, K., Morris, B., ... & Pu�erill, J. (1999). 349 _GIGANTEA_ : a circadian clock-controlled gene that regulates photoperiodic flowering in 350 Arabidopsis and encodes a protein with several possible membrane-spanning domains. _The_ 351 _EMBO journal_, _18_ (17), 4679-4688. 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 352 Fujiwara, S., Nakagawa, M., Oda, A., Kato, K., & Mizoguchi, T. (2010). Photoperiod pathway 353 regulates expression of MAF5 and FLC that encode MADS-box transcrip�on factors of the FLC 354 family in Arabidopsis. _Plant Biotechnology_, _27_ (5), 447-454. 355 Godwin, J., Govindasamy, M., Nedounsejian, K., March, E., Halton, R., Bourbousse, C., ... & 356 Farrona, S. (2024). The UBP5 histone H2A deubiqui�nase counteracts PRCs-mediated 357 repression to regulate Arabidopsis development. _Nature Communica�ons_, _15_ (1), 667. 358 Haider, S., & Farrona, S. (2024). Decoding histone 3 lysine methyla�on: Insights into seed 359 germina�on and flowering. _Current Opinion in Plant Biology_, _81_, 102598. 360 Hayama, R., Sarid-Krebs, L., Richter, R., Fernández, V., Jang, S., & Coupland, G. (2017). PSEUDO 361 RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day 362 length. _The EMBO journal_, _36_ (7), 904-918. 363 Helliwell, C. A., Wood, C. C., Robertson, M., James Peacock, W., & Dennis, E. S. (2006). The 364 Arabidopsis FLC protein interacts directly in vivo with _SOC1_ and _FT_ chroma�n and is part of a 365 high-molecular-weight protein complex. _The Plant Journal_, _46_ (2), 183-192. 366 Herrero, E., Gill, A. R., Wijeweera, S., Ginzburg, D. N., Stamford, J. D., Antoniades, A., ... & 367 Webb, A. A. (2026). Dim green light enables day-and-night monitoring of leaf movements. _The_ 368 _Plant Journal_, _127_ (5), e71119. 369 Hohensta�, M. L., Mikulski, P., Komarynets, O., Klose, C., Kycia, I., Jeltsch, A., ... & Schubert, D. 370 (2018). PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 interacts with polycomb-group 371 proteins and histones and regulates Arabidopsis flowering and development. _The Plant_ 372 _Cell_, _30_ (1), 117-133. 373 Huang, W., Pérez-García, P., Pokhilko, A., Millar, A. J., Antoshechkin, I., Riechmann, J. L., & Mas, 374 P. (2012). Mapping the core of the Arabidopsis circadian clock defines the network structure 375 of the oscillator. _Science, 336_ (6077), 75-79. 376 Hwang, D. Y., Park, S., Lee, S., Lee, S. S., Imaizumi, T., & Song, Y. H. (2019). GIGANTEA regulates 377 the �ming stabiliza�on of CONSTANS by altering the interac�on between FKF1 and ZEITLUPE. 378 _Molecules and cells_, _42_ (10), 693-701. 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 379 Imaizumi, T., Schultz, T. F., Harmon, F. G., Ho, L. A., & Kay, S. A. (2005). FKF1 F-box protein 380 mediates cyclic degrada�on of a repressor of CONSTANS in Arabidopsis. _Science_, _309_ (5732), 381 293-297. 382 Imaizumi, T., Tran, H. G., Swartz, T. E., Briggs, W. R., & Kay, S. A. (2003). FKF1 is essen�al for 383 photoperiodic-specific light signalling in Arabidopsis. _Nature_, _426_ (6964), 302-306. 384 Jaeger, K. E., & Wigge, P. A. (2007). FT protein acts as a long-range signal in 385 Arabidopsis. _Current biology_, _17_ (12), 1050-1054. 386 Jang, S., Marchal, V., Panigrahi, K., Wenkel, S., Soppe, W., Deng, X. W., ... & Coupland, G. (2008). 387 Arabidopsis COP1 shapes the temporal pa�ern of CO accumula�on conferring a photoperiodic 388 flowering response. _The EMBO journal_, _27_ (8), 1277-1288. 389 Khan, A., Haider, S., Sharaf, A., Kusová, A., Skalák, J., Jourdain, C., ... & Mozgová, I. (2026). PWO 390 proteins are associated with PRC2 since their emergence in vascular plants. _New Phytologist_, 391 _250_ (6), 4002-4021. 392 Kim, W. Y., Hicks, K. A., & Somers, D. E. (2005). Independent roles for EARLY FLOWERING 3 and 393 ZEITLUPE in the control of circadian �ming, hypocotyl length, and flowering �me. _Plant_ 394 _physiology_, _139_ (3), 1557-1569. 395 Matsushika, A., Makino, S., Kojima, M., & Mizuno, T. (2000). Circadian waves of expression of 396 the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana: insight into the 397 plant circadian clock. _Plant and Cell Physiology_, _41_ (9), 1002-1012. 398 McClung, C. R. (2011). The gene�cs of plant clocks. _Advances in gene�cs_, _74_, 105-139. 399 Mizoguchi, T., Wright, L., Fujiwara, S., Cremer, F., Lee, K., Onouchi, H., ... & Coupland, G. (2005). 400 Dis�nct roles of GIGANTEA in promo�ng flowering and regula�ng circadian rhythms in 401 Arabidopsis. _The Plant Cell_, _17_ (8), 2255-2270. 402 Nakamichi, N., Kiba, T., Henriques, R., Mizuno, T., Chua, N. H., & Sakakibara, H. (2010). 403 PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcrip�onal repressors in the Arabidopsis 404 circadian clock. _The Plant Cell_, _22_ (3), 594-605. 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 405 Nakamichi, N., Kita, M., Ito, S., Yamashino, T., & Mizuno, T. (2005). PSEUDO-RESPONSE 406 REGULATORS, PRR9, PRR7 and PRR5, together play essen�al roles close to the circadian clock 407 of Arabidopsis thaliana. _Plant and Cell Physiology_, _46_ (5), 686-698. 408 Nakamichi, N., Kita, M., Niinuma, K., Ito, S., Yamashino, T., Mizoguchi, T., & Mizuno, T. (2007). 409 Arabidopsis clock-associated pseudo-response regulators PRR9, PRR7 and PRR5 coordinately 410 and posi�vely regulate flowering �me through the canonical CONSTANS-dependent 411 photoperiodic pathway. 412 Nohales, M. A., & Kay, S. A. (2016). Molecular mechanisms at the core of the plant circadian 413 oscillator. _Nature structural & molecular biology_, _23_ (12), 1061-1069. 414 Oakenfull, R. J., & Davis, S. J. (2017). Shining a light on the Arabidopsis circadian clock. _Plant,_ 415 _cell & environment_, _40_ (11), 2571-2585. 416 Qi, P. L., Zhou, H. R., Zhao, Q. Q., Feng, C., Ning, Y. Q., Su, Y. N., ... & He, X. J. (2022). 417 Characteriza�on of an autonomous pathway complex that promotes flowering in Arabidopsis. 418 _Nucleic Acids Research_, _50_ (13), 7380-7395. 419 Rouse, D. T., Sheldon, C. C., Bagnall, D. J., Peacock, W. J., & Dennis, E. S. (2002). FLC, a repressor 420 of flowering, is regulated by genes in different induc�ve pathways. _The Plant Journal_, _29_ (2), 421 183-191. 422 Samach, A., Onouchi, H., Gold, S. E., Di�a, G. S., Schwarz-Sommer, Z., Yanofsky, M. F., & 423 Coupland, G. (2000). Dis�nct roles of CONSTANS target genes in reproduc�ve development of 424 Arabidopsis. _Science_, _288_ (5471), 1613-1616. 425 Schaffer, R., Ramsay, N., Samach, A., Corden, S., Pu�erill, J., Carré, I. A., & Coupland, G. (1998). 426 The late elongated hypocotyl muta�on of Arabidopsis disrupts circadian rhythms and the 427 photoperiodic control of flowering. _Cell_, _93_ (7), 1219-1229. 428 Searle, I., He, Y., Turck, F., Vincent, C., Fornara, F., Kröber, S., ... & Coupland, G. (2006). The 429 transcrip�on factor FLC confers a flowering response to vernaliza�on by repressing meristem 430 competence and systemic signaling in Arabidopsis. _Genes & development_, _20_ (7), 898-912. 431 Song, Y. H., Estrada, D. A., Johnson, R. S., Kim, S. K., Lee, S. Y., MacCoss, M. J., & Imaizumi, T. 432 (2014). Dis�nct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regula�on of 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 433 CONSTANS stability in Arabidopsis photoperiodic flowering. _Proceedings of the Na�onal_ 434 _Academy of Sciences_, _111_ (49), 17672-17677. 435 Song, Y. H., Kubota, A., Kwon, M. S., Covington, M. F., Lee, N., Taagen, E. R., ... & Imaizumi, T. 436 (2018). Molecular basis of flowering under natural long-day condi�ons in Arabidopsis. _Nature_ 437 _plants_, _4_ (10), 824-835. 438 Song, Y. H., Smith, R. W., To, B. J., Millar, A. J., & Imaizumi, T. (2012). FKF1 conveys �ming 439 informa�on for CONSTANS stabiliza�on in photoperiodic flowering. _Science_, _336_ (6084), 1045 440 1049.Staiger, D., Shin, J., Johansson, M., & Davis, S. J. (2013). The circadian clock goes 441 genomic. _Genome biology_, _14_ (6), 208. 442 Strayer, C., Oyama, T., Schultz, T. F., Raman, R., Somers, D. E., Más, P., ... & Kay, S. A. (2000). 443 Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. 444 _Science_, _289_ (5480), 768-771. 445 Suárez-López, P., Wheatley, K., Robson, F., Onouchi, H., Valverde, F., & Coupland, G. (2001). 446 CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. 447 _Nature_, _410_ (6832), 1116-1120. 448 Takada, S., & Goto, K. (2003). TERMINAL FLOWER2, an Arabidopsis homolog of 449 HETEROCHROMATIN PROTEIN1, counteracts the ac�va�on of _FLOWERING LOCUS T_ by 450 CONSTANS in the vascular �ssues of leaves to regulate flowering �me. _The Plant Cell_, _15_ (12), 451 2856-2865. 452 Valverde, F., Mouradov, A., Soppe, W., Ravenscro�, D., Samach, A., & Coupland, G. (2004). 453 Photoreceptor regula�on of CONSTANS protein in photoperiodic flowering. _Science_, 454 _303_ (5660), 1003-1006. 455 Wang, Z. Y., & Tobin, E. M. (1998). Cons�tu�ve expression of the CIRCADIAN CLOCK 456 ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. _Cell_, 457 _93_ (7), 1207-1217. 458 Wu, G., Park, M. Y., Conway, S. R., Wang, J. W., Weigel, D., & Poethig, R. S. (2009). The 459 sequen�al ac�on of miR156 and miR172 regulates developmental �ming in Arabidopsis. _cell_, 460 _138_ (4), 750-759. 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 461 Yanovsky, M. J., & Kay, S. A. (2002). Molecular basis of seasonal �me measurement in 462 Arabidopsis. _Nature_, _419_ (6904), 308-312. 463 Zheng, S. Y., Guan, B. B., Yuan, D. Y., Zhao, Q. Q., Ge, W., Tan, L. M., ... & He, X. J. (2023). Dual 464 roles of the Arabidopsis PEAT complex in histone H2A deubiqui�na�on and H4K5 acetyla�on. 465 _Molecular Plant, 16(11), 1847-1865._ 466 Zieliński, T., Hay, J., & Millar, A. J. (2021). Period es�ma�on and rhythm detec�on in �meseries 467 data using BioDare2, the free, online, community resource. In _Plant Circadian Networks:_ 468 _Methods and Protocols_ (pp. 15-32). New York, NY: Springer US. 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 486 **Figures** 487 488 489 **Figure 1.** _**PWO1**_ **/** _**2**_ **regulate flowering �me and the expression of clock and photoperiod** 490 **genes under LD condi�ons** . (a) Representa�ve images of 40-day-old Col-0 (WT), _pwo1_, _pwo2_ 491 and _pwo1;2_ plants. Scale bar = 5cm. (b) Rose�e leaf number of Col-0, _pwo1_, _pwo2_ and _pwo1;2_ 492 double mutants grown under LD condi�ons. (c) Number of days �ll flowering for Col-0, _pwo1_, 493 _pwo2_ and _pwo1;2_ plants grown under LD condi�ons. At least 10 plants per genotype were 494 scored. (d-K) Expression levels of (d) _CCA1_, (e) LHY, (f) _PRR7_, (g) _GI_, (h) _CO_, (i) _FLC_, (j) _FT_ and 495 (k) _CDF1_ analyzed in 10-day-old Col-0, _pwo1_, _pwo2_, _pwo1;2_ seedlings grown under LD 496 condi�ons harvested every 4-hours. White and black bars represent day and night �mes 497 respec�vely. _TIP41_ was used as an internal control. At least 3 biological replicates were used. 498 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 499 500 **Figure 2.** _**PWO1/2**_ **modulates flowering �me through the regula�on of** _**PRR7**_ **.** (a) 501 Representa�ve images of 39-day-old WT and _pwo1;2_ plants, 62-day-old _prr7_, and _pwo1;2;prr7_ 502 mutants, 96-day-old _gi_ and _pwo1;2;gi_ mutants, and 95-day-old _co_, and _pwo1;2;co_ triple 503 mutants. Scale bars = 5cm. (b) Rose�e leaf number of Col-0, _pwo1;2, prr7_, _pwo1;2;prr7_, _gi_, 504 _pwo1;2;gi_, _co,_ and _pwo1;2;co_ mutants at the �me of flowering. (c) Number of days �ll 505 flowering for Col-0 _pwo1;2, prr7_, _pwo1;2;prr7_, _gi_, _pwo1;2;gi_, _co,_ and _pwo1;2;co_ mutants 506 grown under LD condi�ons. (d) Expression analysis of _FT_ in 10-day-old seedlings in Col-0 507 _pwo1;2, prr7_, _pwo1;2;prr7_, _co, pwo1;2;co gi,_ and _pwo1;2,gi_ mutants. Samples were harvested 508 at the end of long day (ZT16). Error bars represent SD of three biological replicates. Student t 509 test was used to calculate the significance level. (e) Primers spanning the TSS of _PRR7_ and 510 proximal promoter of _GI_ locus. Red bar: analyzed region. (f) ChIP-assays from samples 511 harvested at ZT8 and ZT12. Increased H2Aub levels downstream of _PRR7_ TSS was detected. 512 An intergenic region ( _AG.i_ ) which was previously shown to harbor no H2Aub enrichment was 513 selected as nega�ve control (Godwin _et al._ 2024). ChIP-qPCR results shown as % of input DNA. 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.03.763775; this version posted November 4, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.03.763775) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made [available under a CC-BY 4.0 International license.](http://creativecommons.org/licenses/by/4.0/) 514 Student t-test was used to calculate the significance level. Two biological and three technical 515 replicates were used. (g) A model for the role of _PWO1/2_ in control of flowering �me through 516 _PRR7-CO_ and an addi�onal unknown pathway. In this model, PWOs regulate the expression 517 of _PRR7_ via H2Aub removal. Once _PRR7_ mRNA is synthesized and translated, the PRR7 protein 518 may interact with CO to prevent its degrada�on and promote flowering through the _CO-FT_ 519 route (Nakamichi _et al._ 2007; Huang _et al._ 2017). Although further work is required to confirm 520 CO protein ac�vity and abundance in _pwo1;2_ mutant. PRR7 mediated _CDF1_ repression is 521 essen�al to promote flowering under LDs (Nakamichi _et al._ 2007). Here, PWO2 may uniquely 522 promote _FT_ expression by repressing _CDF1_ expression (in a PRR7 associated pathway), thus 523 limi�ng CDF1-mediated _FT_ repression (Song _et al._ 2012). On the other hand, an addi�ve 524 interac�on for leaves and synergis�c interac�on for days between _PWO1/2-GI_ indicates that 525 the proteins possibly work in parallel and may converge on similar pathway(s), although 526 independent of the _CO-FT_ branch, possibly through age- or growth-related pathways. PWO1 527 may interact with autonomous pathway genes to regulate _FLC_ expression (Hohensta� _et al._ 528 2018), providing an addi�onal layer of flowering �me regula�on. Solid lines: interac�on 529 known or proved through this study. Dashed lines: Proposed interac�ons. 530 531 532 533 534 535 536 537 538 539 540 20