Unraveling diel regulation of cuticle biosynthesis

11 The plant cuticle is vital for growth and adaptation to environmental stresses. Although cuticle 12 biosynthesis is dynamically regulated by environmental cues, the molecular mechanisms 13 integrating these external signals with intracellular responses remain largely unknown. Here, 14 we discovered that cuticle biosynthesis is precisely orchestrated by two distinct regulatory 15 modules acting in synchrony with the diel cycle. Daylight is perceived by phytochrome B 16 (phyB), which suppresses cuticular wax biosynthesis by degradation of PIF4, a phytochrome-17 interacting bHLH factor that activates wax biosynthetic genes. This suppression is alleviated 18 when phyB itself is degraded by the E3 ubiquitin ligase LRB, leading to diurnal activation of 19 PIF4. In contrast, loss-of-function and transcriptional assays of CFLAP1 demonstrated its direct 20 negative role in cutin biosynthesis. At night, the E3 ubiquitin ligase COP1 mediates proteasomal 21 degradation of CFLAP1, thereby promoting cutin accumulation via activation of BDG1. 22 Together, these results reveal that two regulatory modules, LRB-phyB-PIF4 and COP1-23 CFLAP1 coordinate the diel regulation of cuticle formation, ensuring timely assembly of the 24 protective barrier. 25 26 Key words: Cuticle ; Cuticular wax; Cutin; Diel regulation ; E3 ligase ; Phytochrome ; 27 Transcription factor 28 29 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 3

30 Daily light-dark cycles have driven sessile plants to adapt to diurnal environmental changes that 31 regulate photosynthesis, flowering, and defence responses against biotic and abiotic stresses 32 (Dodd et al., 2005; Andres and Coupland, 2012; Roeber et al.,202 1). The plant cuticle, the 33 outermost physical barrier between plants and the environment, facilitates growth, development, 34 and tolerance to terrestrial stresses (Yeats and Rose, 2013; Ingram and Nawrath, 2017). This 35 hydrophobic layer is composed of cutin and cuticular waxes, deposited in the epidermis of aerial 36 tissues, root caps, and wounded surfaces (Suh et al., 2005; Berhin et al., 2019; Lee et al., 2025). 37 Cutin serves as a fundamental structural component, forming a polyester matrix, whereas 38 cuticular waxes are either embedded within the cutin or deposited on the surface as epicuticular 39 crystals (Kunst and Samuels, 2003; Yeats and Rose, 2013). Polyester cutin is a polymer 40 composed of C16- and C18-fatty acids (FAs) and their derivatives, including -hydroxy fatty 41 acids (HFAs) and 1, -dicarboxylic acids (DCAs), which are generated by cytochrome P450 42 family enzymes and oxidoreductases (Li-Beisson et al., 2013). Cuticular waxes mainly consist 43 of very long-chain fatty acids (VLCFAs) and their derivatives, including alkanes (AKs), 44 primary and secondary alcohols (PAs and SAs), aldehydes (ALs), ketones (KEs), and wax esters 45 (WEs), which are synthesized through alkane- and alcohol-forming pathways (Samuels et al., 46 2008; Bernard and Joubès, 2013; Lewandowska et al., 2020; Lee and Suh, 2022). 47 Light is one of the most influential environmental cues that drive diel changes in plant 48 growth and development, including cuticle formation (Shepherd and Griffiths, 2006). Light 49 enhances wax deposition in various plant species such as kale, swede, and Arabidopsis 50 (Shepherd et al., 1995; Go et al., 2014). Furthermore, the biosynthesis of cuticular waxes is 51 finely modulated by qualitative properties of light, such as light intensity and wavelength 52 (Huang et al., 2020). Interestingly, the expression of cutin biosynthetic genes such as BDG1 53 and LACS2 is upregulated during etiolation but markedly reduced during photomorphogenesis 54 (Ma et al., 2024). Together, these findings suggest a tight coupling between diel light signaling 55 and cuticle metabolism. On the other hand, loss-of-function mutants defective in cutin 56 biosynthesis, such as lacs2, bdg1, and cyp86a8, exhibit growth retardation and developmental 57 abnormalities, including organ fusion, suggesting that cutin production is a fundamental process 58 regulated throughout plant growth and development (Wellesen et al., 2001; Xiao et al., 2004; 59 Tang et al., 2007). Notably, cutin—but not wax— biosynthesis is particularly enhanced in the 60 rapidly elongating upper stem regions of Arabidopsis (Suh et al., 2005). Considering that plant 61 elongation growth is predominantly promoted during the night (Apelt et al., 2017; Urrea -62 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 4 Castellanos et al., 2022), these observations raise the possibility that cutin biosynthesis may be 63 positively associated with nocturnal growth. However, the molecular mechanisms underlying 64 the nighttime regulation of cutin biosynthesis remain largely unexplored. 65 Over the past decades, extensive studies have identified a network of transcriptional 66 regulators—including HDG1, CFLAP1, MYB96, WIN1, DEWAX, and SPL9—that control 67 cuticle biosynthesis during development and in response to environmental stimuli such as 68 drought and diurnal light cycles (Kannangara et al., 2007; Seo et al., 2011; Wu et al., 2011; Go 69 et al., 2014 ; Li et al., 2016; Li et al., 2019). Despite these advances, the upstream signaling 70 mechanisms that modulate the activity and stability of these transcription factors remain poorly 71 understood. The factors directly mediating environmental cues and plant responses include 72 photoreceptors, such as red/far-red light-sensing phytochrome B (phyB). phyB and its related 73 E3 ubiquitin ligases, COP1, SPA1, LRB, HOS1, and BOP2 mediate light responses under 74 continuous red light, including germination, de-etiolation, leaf expansion, inhibition of stem 75 and petiole elongation, and flowering (Rockwell et al., 2006 ; Christians et al., 2012; Lu et al., 76 2015; Kim et al., 2017a ; Zhang et al., 2017). However, how plants integrate light perception 77 and signal transduction to coordinate the diel regulation of cutin and cuticular wax biosynthesis 78 remains elusive. In this study, we demonstrate that plants regulate cuticle biosynthesis 79 according to light-dark cycles via two distinct modules, LRB-phyB-PIF4 and COP1-CFLAP1, 80 each stimulating diurnal wax and nocturnal cutin accumulation, respectively. These findings 81 expand our insights into diel regulation of cuticle biosynthesis in Arabidopsis. 82 83

84 phyB negatively regulates cuticular wax biosynthesis 85 To investigate how plants perceive and transduce light signals to regulate cuticular wax 86 biosynthesis, we focused on a previous report showing that maize phytochrome ௗB (phyB) 87 mutants exhibit increased total wax loads in leaves (Qiao ௗetௗal.,ௗ2020). We therefore examined 88 the role of phyB in Arabidopsis cuticular wax biosynthesis. The total wax load increased by 89 approximately 20%, 60%, and 50% in the leaves of phyA-211, phyB-9, and phyA-211 phyB-9 90 mutants, respectively, compared with the wild type (WT) (Figureௗ1A). Levels of alkanes (AKs) 91 and primary alcohols (PAs) were significantly elevated in phyB-9 and phyA-211 phyB-9 92 (Figureௗ1A and Supplemental ௗFigureௗ1A). Consistent with these findings in the Col-0 93 background, total wax amounts were higher by approximately 47% in phyA-201 phyB-5 and 94 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 5 lower to approximately 66% of WT levels in the PHYB-overexpressing (OX) line in the L er 95

(Figureௗ1B and Supplemental ௗFigureௗ1B). Expression of wax biosynthetic genes 96 was markedly upregulated in leaves of phyB-9, phyA-211 phyB-9 (Col-0), and phyA-201 phyB-97 5 (Ler), but downregulated in PHYBௗOX leaves (Figures ௗ1C andௗ1D). In particular, transcript 98 levels of CER1, CER4, KCS2, and SOH1 were increased in phyB and phyA phyB, whereas 99 decreased in PHYBௗOX leaves. Collectively, these results demonstrate that phyB acts as a 100 negative regulator of cuticular wax biosynthesis in Arabidopsis, modulating wax production in 101 response to light conditions. 102 103 PIF4 activates cuticular wax biosynthesis by directly binding to the promoters of KCS2, 104 CER1, and CER4, thereby upregulating their expression. 105 Alterations in the transcript levels of wax biosynthetic genes in phyB and PHYB OX suggest 106 that transcription factors downstream of phyB may activate cuticular wax biosynthesis. The PIF 107 family of transcription factors, which physically interact with phyB under light conditions 108 (Lorrainௗetௗal.,ௗ2008), were considered as potential candidates. To identify the most relevant 109 PIFs among the eight members in Arabidopsis, we considered two criteria: transcript abundance 110 and protein stability during the light period. The expression levels of PIF4 and PIF5 were 111 significantly higher than those of other PIFs during the light phase, although PIF5 expression 112 was also detected in darkness (SupplementalௗFigureௗ2A). Consistently, PIF4 and PIF5 proteins 113 accumulated during daytime until 14:30, but PIF5 remained detectable at night 114 (SupplementalௗFigureௗ2B). These temporal expression patterns suggest that PIF4 is the primary 115 mediator of light-responsive cuticular wax biosynthesis. 116 To test the hypothesis that PIF4 positively regulates wax biosynthesis, we analyzed 117 cuticular wax content in the leaves of WT (Col-0), pif4, pifQ ( pif1pif3pif4pif5), a PIF4 118 complementation line in pifQ background (PIF4/pifQ), and a PIF4 overexpression line (PIF4-119 OX). Total wax amounts were reduced by approximately 10% and 20% in pif4 and pifQ, 120 respectively, whereas they were increased by approximately 30% and 85% in PIF4/pifQ and 121 PIF4-OX relative to the WT, respectively (Figure 2A and 2B). In particular, levels of AKs and 122 PAs significantly decreased in pif4 and pifQ, but markedly elevated in PIF4/pifQ and PIF4-OX 123 (Figure 2A, 2B and Supplemental Figure 3). The transcript levels of KCS2, CER1, and CER4, 124 which are involved in AK and PA biosynthesis, were significantly reduced in pif4 and pifQ 125 leaves, but increased in PIF4/pifQ and PIF4-OX leaves compared with WT (Figure 2C). No 126 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 6 significant differences in total cutin monomer levels were detected between WT and pif4, pifQ 127 or PIF4/pifQ, except for moderate increases in DCAs and ω‑HFAs in PIF4/pifQ (Supplemental 128 Figure 4). Collectively, these results demonstrate that PIF4 acts as a positive regulator of 129 cuticular wax biosynthesis. 130 To identify target genes of PIF4 involved in AK and PA biosynthesis, we performed RT-131 qPCR analysis in WT and pif4 leaves over the diel cycle. In WT, KCS2, CER1, CER4, SOH1, 132 and LACS2 displayed typical diurnal expression patterns, peaking at 10:30 or 14:30 during the 133 light period and declining thereafter. In contrast, KCS6 exhibited only minor variation during 134 the day, showing a modest decrease during the light phase followed by a slight recovery (Figure 135 2D). In pif4 mutant, transcript levels of KCS2, CER1, and CER4 were noticeably reduced 136 compared with WT, whereas no significant differences were observed for SOH1, LACS2, and 137 KCS6 (Figure 2D). Notably, the diurnal oscillation of CER1 expression was completely 138 diminished in pif4 leaves. Therefore, these findings indicate that PIF4 regulates the transcription 139 of cuticular wax biosynthetic genes in a diurnal manner. We next examined whether PIF4 140 directly activates the expression of KCS2, CER1, and CER4. Transient dual-luciferase reporter 141 assays in tobacco (Nicotiana benthamiana) leaves revealed that LUC reporter activities driven 142 by the promoters of KCS2, CER1, and CER4 were significantly enhanced in the presence of 143 PIF4 compared with the control (Figure 2E). Consistently, chromatin immunoprecipitation 144 (ChIP) assays demonstrated that PIF4 directly binds to the promoter regions of KCS2 (region 145 A), CER1 (regions B and C), and CER4 (regions A, B, and C), each containing a canonical E-146 box motif (Figure 2F). 147 148 Disruption of phyB stabilizes PIF4, leading to upregulation of cuticular wax biosynthesis 149 during the daytime. 150 We next examined whether phyB functions upstream of PIF4 to inhibit upregulation of cuticular 151 wax biosynthesis. Immunoblot analysis revealed that PIF4 protein levels were markedly higher 152 in phyB-9 than in WT (Figure 3A). Consistent with the elevated PIF4 levels, the expression of 153 its direct target genes, KCS2, CER1 , and CER4, was significantly upregulated in phyB-9 154 compared with WT at 10:30 and/or 14:30, with SOH1 and KCS6 transcripts likewise increased 155 (Figure 3B). Given that light-activated phyB represses PIF4 activity (Lorrain et al., 2008), wax 156 biosynthesis is suppressed while phyB remains active. The enhanced wax production observed 157 under light therefore indicates that phyB must be degraded to relieve PIF4 from repression. 158 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 7 Because Light-Responsive BTB (LRB) proteins, adaptors of E3 ubiquitin ligase complexes, 159 promote the degradation of active phyB (Pfr form) together with PIF3 (Ni ௗetௗal.,ௗ2014), we 160 examined phyB and PIF4 abundance in WT and the lrb1lrb2‑2lrb3 (lrb123) triple mutant leaves. 161 In lrb123, phyB protein levels were substantially increased, whereas PIF4 protein levels were 162 reduced compared with WT (Figure 3C and 3D). In line with the lowered levels of PIF4, 163 daytime expression of KCS2, CER1 , CER4, and SOH1 were significantly downregulated in 164 lrb123 relative to WT, while the expression of LACS2 and KCS6 remained unchanged (Figure 165 3E). Cuticular wax analysis further showed that total wax loads in lrb123 were reduced to 166 approximately 60% of WT levels (Figure 3F). Significant decreases in the levels of AKs, 167 VLCFAs, and PAs, the major wax components, were detected in lrb123 leaves relative to WT 168 (Figure 3F and Supplemental Figure 5). These results indicate that LRB-dependent phyB 169 degradation is important for the upregulation of cuticular wax biosynthesis under light 170 conditions. 171 172 CFLAP1 negatively regulates cutin biosynthesis 173 Given that the cuticular structure where cutin is deposited underneath the wax layer (Yeats and 174 Rose, 2013), cuticular wax accumulation during the day should be preceded by cutin deposition. 175 Suh et al. (2005) reported that in Arabidopsis stems, cutin monomer loads were approximately 176 2-fold higher in the elongating upper region than in the basal region, whereas total wax loads 177 showed no significant difference. Comparative analysis of cuticle biosynthetic gene expression 178 between the upper and lower stem regions revealed that cutin-related genes were generally more 179 strongly upregulated than wax-related genes ( Suh et al., 2005; Supplemental Figure 6A), 180 suggesting the crucial role of cutin biosynthesis in highly expanding tissues. Considering that 181 plant elongation growth primarily occurs at night (Apelt et al., 2017), we hypothesized that 182 cutin biosynthesis is closely related with nocturnal growth. Supporting this idea, microarray 183 data from Blaesing ௗetௗal.ௗ(2005) showed that most cutin biosynthetic genes were upregulated 184 under dark conditions (SupplementalௗFigureௗ6B). Among the transcription factors implicated in 185 cuticle formation (Lee and Suh, 2022), the expression of the negative regulator CFLAP1 (Li et 186 al., 2016) was markedly decreased in Arabidopsis leaves in darkness relative to light, suggesting 187 that darkness-induced suppression of CFLAP1 may contribute to the enhanced expression of 188 cutin biosynthetic genes. 189 To examine the function of CFLAP1 in cuticle development, we isolated two loss-of-190 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 8 function mutants, cflap1-1 and cflap1-2 (Supplemental Figure 6C and 6D), and analyzed cutin 191 and wax composition and amounts by gas chromatography. Total cutin monomer content, 192 particularly that of ω‑HFAs and DCAs, was significantly increased in cflap1 mutant leaves 193 compared with WT, whereas cuticular wax content showed no significant change (Figure 4A, 194 4B and Supplemental Figure 7A and 7B). These results indicate that CFLAP1 acts as a negative 195 regulator in cutin deposition. 196 Based on the marked upregulation of cutin biosynthetic genes in response to darkness, 197 when nocturnal growth occurs, we examined whether CFLAP1 transcripts and protein levels 198 are diurnally regulated. CFLAP1 mRNA levels increased during the light period, showing two 199 distinct peaks at 10:30 and 18:30, and declined significantly at night (Supplemental Figure 8A 200 and 8B). In Arabidopsis seedlings overexpressing CFLAP1 fused with a MYC epitope under 201 the control of CaMV 35S promoter (CFLAP1 OX), the accumulation pattern of MYC-CFLAP1 202 closely paralleled with that of CFLAP1 mRNA (Supplemental Figure 8C), indicating that 203 CFLAP1 expression is subject to diurnal regulation. 204 To elucidate the regulatory role of CFLAP1 in cutin biosynthesis, we analyzed the 205 expression of cutin biosynthetic genes in WT, cflap1-1, and cflap1-2 leaves at 18:30, when 206 CFLAP1 expression peaks. The transcript levels of CYP86A8, GP AT4, GP AT8, and LACS2 were 207 reduced, whereas those of CYP86A2, CYP86A4 , and BDG1 were significantly increased in 208 cflap1 mutants relative to WT (Figure 4C), suggesting that CYP86A2, CYP86A4, and BDG1 are 209 potential downstream targets of CFLAP1. Because CFLAP1 is a bHLH transcription factor that 210 binds to E-box motif (5 -CANNTG-3), approximately 3‑kb promoter regions of CYP86A2, 211 CYP86A4 and BDG1 including E-box motifs were used for the generation of reporter constructs. 212 The effector construct contained MYC‑CFLAP1 driven by the CaMVௗ35Sௗpromoter. In a dual-213 luciferase assay, MYC-CFLAP1 significantly repressed the LUC/REN activity driven by the 214 pBDG1, pCYP86A2 and pCYP86A4 promoters by approximately 1.5- to 4-fold compared with 215 the control (Figure 4D). To test direct binding, electrophoretic mobility shift assays (EMSAs) 216 were performed using recombinant maltose‑binding protein (MBP)–CFLAP1 purified from 217 E.ࣟcoli ( SupplementalௗFigureௗ9) and promoter fragments from pCYP86A2, pCYP86A4 , and 218 pBDG1, each containing one or more E‑boxes ( Figureௗ4E). EMSAs demonstrated that MBP-219 CFLAP1 specifically interacts with these promoter fragments (Figureௗ4E). Consistently, ChIP-220 qPCR assay showed enrichment of CFLAP1 on the E-box regions of CYP86A2, CYP86A4, and 221 BDG1 promoters in MYC-CFLAP1 OX seedlings relative to the control (Figure 4F). These 222

indicate that CFLAP1 directly binds to the E‑box in the promoters of CYP86A2, 223 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 9 CYP86A4, and BDG1, thereby repressing their transcription to negatively regulate cutin 224 biosynthesis. 225 226 COP1 interacts with and degrades CFLAP1 proteins via 26S proteasome pathway 227 Because CFLAP1 protein levels were markedly lower at night than those during the day 228 (Supplemental Figure 8C), we investigated whether COP1, which acts in the nucleus under dark 229 conditions, is involved in regulating CFLAP1 stability. Luciferase complementation imaging 230 (LCI) assays showed a physical interaction between COP1 and CFLAP1 when co-expressed in 231 tobacco leaves (Figure 5A). Bimolecular fluorescence complementation (BiFC) analysis further 232 showed nuclear YFP signals in tobacco epidermal cells coexpressing COP1-eYFPN and 233 CFLAP1-eYFPC (Figure 5B). Co-immunoprecipitation (Co-IP) assays confirmed this 234 interaction: COP1-HA proteins were detected in extracts from tobacco leaves co-expressing 235 MYC-CFLAP1 and COP1-HA and immunoprecipitated with anti-MYC antibody, but not in 236 extracts co-expressing with empty MYC and COP1‑HA (Figure 5C). Immunoblot analyses 237 demonstrated that the COP1–CFLAP1 interaction promotes CFLAP1 ubiquitination and 238 subsequent 26S proteasome-mediated degradation, as indicated by the increased accumulation 239 of ubiquitinated CFLAP1 following MG132 treatment (Figure 5D and 5E). To examine 240 COP1‑dependent degradation in planta, MYC-CFLAP1 OX lines were crossed with the cop1‑4 241 mutant. Immunoblot analysis revealed markedly higher levels of MYC‑CFLAP1 protein in the 242 cop1‑4 background than in WT (Col‑0), whereas transcript levels of MYC‑CFLAP1 were 243 comparable between the two genotypes (Figure 5F). These results demonstrate that COP1 244 mediates ubiquitination and 26S proteasome-dependent degradation of CFLAP1. 245 Next, we examined whether CFLAP1 protein levels depend on COP1 activity and 246 subcellular localization in CaMV35S:MYC-CFLAP1/WT and CaMV35S:MYC-CFLAP1/cop1-247 4 seedlings across the day-night cycle. In the WT background, MYC-CFLAP1 levels peaked 248 during the daytime and declined rapidly at night. By contrast, in cop1-4 background, MYC–249 CFLAP1 proteins accumulated continuously throughout all examined time points, with no 250 decrease detected at night (Figure 5G). To further examine whether COP1 mediates nocturnal 251 degradation of CFLAP1 via the 26S proteasome pathway, seedlings were treated with 252 cycloheximide (CHX) in the presence or absence of MG132. In CFLAP1 OX/Col-0, 253 degradation of MYC-CFLAP1 at night under CHX treatment was abolished by MG132, 254 whereas MYC-CFLAP1 levels remained largely unchanged in CFLAP1 OX/cop1-4 under both 255 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 10 CHX and CHX ௗ+ௗMG132 treatments (Figure 5H). These results indicate that COP1 mediates 256 the nocturnal ubiquitination and subsequent 26S ௗproteasome‑dependent degradation of 257 CFLAP1. 258 259 The COP1-CFLAP1 module positively regulates cutin biosynthesis during nighttime 260 COP1-mediated degradation of CFLAP1 prompted examination of cuticle phenotypes in two 261 loss-of-function cop1 alleles ( cop1-4 and cop1-6). Cuticle permeability to toluidine blue O 262 (TBO) was dramatically increased in leaves of cop1 mutants compared with WT (Figure 6A). 263 Transmission electron microscopy (TEM) analysis revealed a reduced cuticular proper layer 264 and thinner cell wall and cuticle in cop1-4 relative to WT (Figure 6B). Consistent with these 265 structural defects, cuticular transpiration and chlorophyll leaching assays showed enhanced 266 water loss and faster diffusion of chlorophylls into 80% ethanol from leaves of cop1-4 and 267 cop1-6 mutants compared with WT (Figure 6C and 6D). GC-FID analysis further revealed that 268 leaves of cop1-4 and cop1-6 mutants exhibited a marked decrease in total cutin monomer levels, 269 particularly ω-HFAs and DCAs, whereas total wax composition and content were not 270 significantly altered (Supplemental Figure 10). These results demonstrate that COP1 is essential 271 for normal cuticle formation by maintaining proper cutin deposition. 272 To investigate the functional relationship between COP1 and CFLAP1 in cuticle 273 formation, cflap1-1 and cflap1-2 were crossed with cop1-4 (Supplemental Figure 11) and the 274 resulting cflap1-1 cop1-4 and cflap1-2 cop1-4 double knockout mutants were analyzed for cutin 275 monomer and cuticular wax composition and amounts by GC–FID. Leaves of both double 276 mutants exhibited partial restoration of the reduced ω‑HFA and DCA contents observed in 277 cop1‑4 (Figure 6E and Supplemental Figure 12A). Total wax load and composition were largely 278 unchanged, except for minor differences in C28– C30ௗVLCFAs and C30 ௗPAs (Figure 6F and 279 Supplemental Figure 12B). We next examined the transcript levels of CYP86A2, CYP86A4 and 280 BDG1 identified as direct CFLAP1 target genes in WT, cflap1-1, cflap1-2, cop1-4, cflap1-1 281 cop1-4, and cflap1-2 cop1-4 seedlings at night (06:30) and during the day (18:30). Transcript 282 levels of BDG1 were markedly elevated in cflap1-1 and cflap1-2 and the downregulation of 283 BDG1 observed in cop1-4 was substantially restored in cflap1-1 cop1-4 and cflap1-2 cop1-4 284 mutants at both time points (Figure 6G). Expression of CYP86A2 and CYP86A4 did not show 285 consistent patterns across the mutant lines (Figure 6G). Together, these results indicate that 286 elevated CFLAP1 levels in cop1 mutants primarily cause cuticle defects through repression of 287 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 11 BDG1, indicating that the COP1-CFLAP1 regulatory module coordinates nocturnal activation 288 of cutin biosynthesis. 289 290

291 The plant cuticle is a crucial surface barrier that supports growth and confers tolerance to 292 external stresses. Cuticle-defective mutants display pronounced developmental retardation and 293 reduced stress resistance, underscoring its protective function (Wellesen et al., 2001; Xiao et 294 al., 2004; Tang et al., 2007; Seo et al., 2011). To maintain appropriate surface properties, plants 295 must temporally regulate cuticle biosynthesis. Our study reveals an intricate diel regulation of 296 cuticle biosynthesis mediated by two distinct modules, the LRB-phyB-PIF4 and COP1-297 CFLAP1 pathways. Light-perceiving phyB inhibits PIF4 activity, which promotes cuticular 298 wax biosynthesis by direct upregulation of KCS2, CER1 , and CER4. LRB destabilizes phyB 299 during the daytime, leading to wax biosynthesis through active PIF4. CFLAP1 negatively 300 controls cutin biosynthesis by repressing BDG1 expression. COP1 is nuclear-localized at night 301 and gains access to CFLAP1, inducing its degradation via the 26S proteasomal pathway and 302 consequently upregulating cutin biosynthesis. Nocturnal stimulation of cutin formation is 303 followed by diurnal enhancement of cuticular wax biosynthesis. Structurally, the cuticle 304 consists of a polyester cutin matrix associated with the cell wall, which is subsequently 305 complemented by an outer wax layer (Yeats and Rose, 2013). Given that cell expansion often 306 peaks at night (Urrea-Castellanos et al., 2022), we propose that rapid nocturnal cutin deposition 307 safeguards newly exposed cell surfaces, while daytime wax biosynthesis further reinforces the 308 barrier (Figure 7). Our findings provide a mechanistic framework for cuticle development 309 through time-of-day coordination. 310 311 LRB-phyB-PIF4 module upregulates cuticular wax biosynthesis during the daytime 312 Diurnal regulation of wax biosynthesis has been widely investigated, particularly at 313 transcriptional levels. Two physically interacting transcription factors, SPL9 and DEWAX, act 314 antagonistically to regulate CER1 expression, optimizing wax biosynthesis during daily light-315 dark cycles (Go et al., 2014; Li et al., 2019). Here, we further identified PIF4 as a transcriptional 316 activator of cuticular wax biosynthesis. PIF4 directly promotes the expression of cuticular wax 317 biosynthetic genes KCS2, CER1, and CER4 during the daytime (Figure 2D-2F), highlighting 318 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 12 KCS2 and CER4 as additional diurnally regulated wax-related genes. CER1 encodes an 319 aldehyde decarbonylase that produces AKs whereas CER4 encodes a fatty acyl-CoA reductase 320 that converts acyl-CoA into PAs. AKs function as hydrophobic barriers that enhance drought 321 tolerance by reducing cuticular permeability, whereas PAs increase cuticle permeability to 322 facilitate evaporative cooling under heat stress, revealing a dynamic balance between the two 323 pathways for environmental adaptation (Li et al., 2025). PIF4 thus acts as a critical regulator 324 fine-tuning wax biosynthesis by contributing to the expression of both genes. Although SOH1 325 expression does not appear to be regulated by PIF4 (Figure 2D), pifQ mutant displayed 326 significantly reduced SOH1 transcript levels (Figure 2C), accompanied by lower cuticular wax 327 amounts than the pif4 single mutant (Figure 2A). PIFs are known to act both redundantly and 328 independently as key integrators of plant light signal responses (Leivar and Monte, 2014). Thus, 329 in addition to PIF4, other PIFs may also contribute to the regulation of cuticular wax 330 biosynthesis. PIF5 accumulates both transcriptionally and translationally during the daytime, 331 in parallel with PIF4 (Supplemental Figure 2), which further supports its potential additive role 332 in PIF4-mediated regulation of cuticular wax biosynthesis. 333 Despite advances in transcriptional regulation of diurnal wax accumulation, how 334 upstream light signals are transduced to control the expression of wax biosynthetic genes 335 remains poorly understood. Our findings demonstrate that defective phyB results in increased 336 PIF4 protein abundance and enhanced expression of its target genes (Figure 3A and 3B). These 337

identify phyB as a principal light transducer that negatively regulates wax activator PIF4, 338 likely by promoting its phosphorylation-dependent proteasomal degradation (Huq and Quail, 339 2002; Park et al., 2018). Among the five phytochromes (phyA-E) in Arabidopsis, phyA is a 340 light-unstable phytochrome, unlike the other light-stable phytochromes (Pratt, 1995). Minor 341 differences in cuticular wax amounts between phyA-201 and wild type (Figure 1A) may reflect 342 insufficient phyA protein abundance in wild-type plants. The lrb123 mutant exhibits marked 343 accumulation of phyB accompanied by reduced expression of wax biosynthetic genes and 344 decreased wax deposition (Figure 3E and 3F). Since precise quantitative regulation of phyB 345 abundance is required to maintain proper wax production, LRB acts as a direct repressor of 346 phyB to ensure its optimal level. Although COP1 was identified as another E3 ligase targeting 347 phyB (Jang et al., 2010), phyB abundance changes little during the nighttime in WT (Figure 348 3C). A likely explanation is that during light period, phyB is imported into the nucleus whereas 349 COP1 is exported to the cytosol, limiting COP1 access to phyB (von Arnim and Deng, 1994 ; 350 Yamaguchi et al., 1999). Accordingly, wax biosynthesis is only modestly affected in cop1 351 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 13 mutants, presumably because phyB levels remain unchanged (Figure 6F and Supplemental 352 figure 10B). Although LRB is indispensable for phyB degradation, PIF4 turnover appears to 353 involve LRB-independent mechanisms, as its protein levels were not significantly affected in 354 lrb123 (Figure 3D). Under red light, BLADE-ON-PETIOLE1 (BOP1) and BOP2 assemble 355 CUL3BOP1/BOP2 E3 ligases that target PIF4 for ubiquitination (Zhang et al., 2017). It would be 356 intriguing to investigate whether BOPs affect cuticular wax biosynthesis through the regulation 357 of PIF4 stability. Previous studies have shown that most terrestrial plants possess multiple 358 functional LRB proteins that cooperatively regulate phytochrome-mediated light signaling 359 (Christians et al., 2012). This evolutionary conservation suggests that LRB-dependent 360 regulation of phyB abundance may also represent a fundamental mechanism controlling 361 cuticular wax biosynthesis in land plants. 362 363 The COP1-CFLAP1 module activates cutin biosynthesis at night 364 Previous studies have identified CFLAP1, also known as ABA-RESPONSIVE KINASE 365 SUBSTRATE1 (AKS1), as a regulator of stomatal opening (Takahashi et al., 2017). Under high 366 ABA conditions, SnRK2.6/OST1 phosphorylates AKS1, preventing its binding to the KAT1 367 promoter and thereby suppressing potassium channel accumulation in guard cells, which leads 368 to stomatal closure (Takahashi et al., 2017). Our research further uncovered a distinct role of 369 CFLAP1 in regulating cutin biosynthesis. CFLAP1 acts as a transcriptional repressor of cutin 370 related genes by directly binding to their promoters (Figure 4). At night, COP1-mediated 371 degradation of CFLAP1 (Figure 5A-5H) releases this repression and promotes cutin 372 biosynthesis, particularly through the activation of BDG1 (Figure 6E-6G). COP1 has been 373 established as the central regulator of dynamic responses to light-dark cycles, controlling 374 processes such as photomorphogenesis, stomatal development, and conductance (Yang et al., 375 2005; Liu et al., 2008; Kim et al., 2017b; Lee et al., 2017). The identification of CFLAP1 as a 376 direct target of COP1 provides the missing link explaining how COP1 coordinates diel 377 adaptation in the epidermal cells at both transcriptional and post-translational levels. The dual 378 regulation of cutin biosynthesis and stomatal movement by the COP1-CFLAP1 module 379 suggests a regulatory hub that facilitates protection against water loss, ensuring plant 380 homeostasis across day-night cycles under terrestrial conditions. 381 Under low-light conditions or darkness, plants exhibit enhanced growth such as 382 hypocotyl elongation and leaf expansion (Apelt et al., 2017). This aerial surface expansion 383 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 14 increases the demand for cuticle deposition on newly developed tissues to maintain cellular 384 homeostasis (Ma et al., 2024). The physical properties of cutin support its role as the primary 385 protective layer covering emerging cell surfaces. Lopez-Casado et al. (2007) reported that the 386 cutin matrix displays a low elastic modulus and high strain values relative to the whole cuticle, 387 indicating its flexible and extensible nature. However, the cutin layer alone cannot achieve 388 mechanical stability without the presence of waxes. The incorporation of wax into the cutin 389 matrix converts reversible elastic deformation into irreversible plastic strain, thereby enhancing 390 rigidity. Indeed, the removal of wax from tomato fruit surfaces increased plasticity, indicating 391 that waxes act as structural fillers that reinforce the cutin framework (Petracek and Bukovac, 392 1995; Domínguez et al., 2011). Such biomechanical characteristics establish a robust cuticle by 393 strengthening the cutin matrix and limiting further surface extension (Khanal et al., 2013). 394 Together, these observations suggest a sequential model in which cutin is initially deposited to 395 protect expanding tissues without impeding cell elongation, followed by wax accumulation that 396 strengthens the cuticle and stabilizes tissue morphology. Considering the provocation of cell 397 elongation during nighttime, concurrent cutin biosynthesis and the following diurnal wax 398 accumulation is critically required. We propose that this mechanism is precisely regulated via 399 COP1-CFLAP1 and LRB-phyB-PIF4 pathways, respectively. 400 Chronotherapy is the use of circadian rhythm in medicine, which considers 401 environmental and biological rhythms to improve drug treatment efficacy (Selfridge et al., 2016; 402 Kelly et al., 2018). This concept can also be applied to agriculture by aligning genetics and field 403 practices with plants’ daily rhythms (Hotta, 2021). For example, aquaporin expression in roots 404 shows higher abundance during the day, accompanied by stomatal opening (Caldeira et al., 405 2014; Maurel et al., 2016) which together suggest enhanced daytime water uptake. Our study 406 on diel regulation of cuticle formation may support chronotherapy-inspired crop management. 407 If wax and cutin deposition peaks or off-peaks at predictable time phases, applying pesticides, 408 foliar nutrients, or other external resources at those optimal times may increase uptake 409 efficiency while reducing cost and contamination. Furthermore, breeding or engineering clock 410 and photoreceptor modules to shift the timing of cuticle reinforcement towards periods of 411 maximal drought, heat or UV stress could offer a novel strategy to improve crop resilience and 412 yield. 413 414

415 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 15 Plant materials and growth conditions 416 Arabidopsis mutants and transgenic lines used in this study were listed in Supplemental Table 417 1. Arabidopsis seeds were sterilized and sown on half-strength Murashige and Skoog (MS) agar 418 plates (pH 5.7) supplemented with 1% sucrose. After stratification for 2 days at 4 oC, plates 419 were placed in growth room under long-day conditions (16 h/8 h, light/dark) at 23 oC and ~50% 420 humidity. Seven-day-old seedlings were transferred to soil for further experiments. Tobacco 421 (Nicotiana benthamiana ) were also grown under the same growth conditions as described 422 above. 423 424 Genomic DNA analysis 425 Genomic DNA was extracted from Arabidopsis seedlings or leaves using extraction buffer (200 426 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) and performed PCR to verify 427 the CFLAP1 locus/transgene using gene-specific primers listed in Supplemental Table S2. 428 429 Gene expression analysis 430 Total RNA was isolated from 12-day-old seedlings or 3-week-old Arabidopsis leaves using the 431 Total RNA Purification Kit following manufacturer’s instructions (Xenohelix). About 2 μg of 432 total RNA was used for reverse transcription using GoScript™ Reverse Transcriptase 433 (Promega). RT-PCR was performed using 2x Prime Taq Premix (GeNetBio). Real-Time 434 Quantitative Polymerase Chain Reaction (RT-qPCR) was performed using TOPreal™ SYBR 435 Green qPCR PreMIX (Enzynomics) by CFX Opus 96 Real-Time PCR system (Bio-Rad). 436 Transcript levels were normalized to that of PP2AA3 (At1g13320). The used primers are listed 437 in Supplemental Table 2. 438 439 Dual luciferase reporter assay 440 Effector constructs were made by insertion of the CFLAP1 coding sequence (CDS) into 441 pBA002 to express CaMV35S:MYC-CFLAP1. The PIF4 CDS was inserted into pSPYCE 442 vector to generate a PIF4-eYFPC. For reporter constructs, the promoter regions of CYP86A2, 443 CYP86A4, CYP86A8, HTH, BDG1, GP AT4, KAT1, KCS2, CER1, and CER4 were cloned into 444 pGreenII 0800-LUC vector upstream of LUC gene. The effector and reporter constructs were 445 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 16 introduced into Agrobacterium tumefaciens strain GV3101 (pSOUP). Agrobacterium strains 446 harboring the effector or reporter constructs were cultured to OD 600 = 0.8, pelleted, and 447 resuspended in infiltration buffer (10 mM MgCl 2, 10 mM MES, pH 5.7, 200 μM 448 acetosyringone), adjusted to an OD 600 of 0.5, and infiltrated into 4-week-old tobacco leaves. 449 Leaf disks were harvested 2 days after infiltration and then dual-luciferase assays were 450 performed using the Dual Luciferase Assay System following the manufacturer’s instructions 451 (Promega). 452 453 The electrophoretic mobility shift assay (EMSA) 454 To produce MBP-CFLAP1 the CFLAP1 CDS was cloned into pMAL-c2 vector (NEB). After 455 transformation into Escherichia coli (BL21), the cells were grown on in LB media containing 456 100 μg mL-1 ampicillin. Protein expression was induced with 0.1 mM IPTG (isopropyl β-D-1-457 thiogalactopyranoside) for 3 h at 37 oC. Total proteins were extracted in column buffer (20 mM 458 Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) from the harvested cells by 459 sonication, purified using amylose resin (NEB), and eluted with column buffer containing 10 460 mM maltose. Protein concentration and purity were verified using bradford assay (Bio-Rad) 461 and SDS-PAGE (8%). Biotin-labelled single-strand DNA oligonucleotides corresponding to 462 putative cis-elements in the promoter regions of CYP86A2, CYP86A4, and BDG1 were 463 synthesized (Bionics) and annealed by heating at 95 °C for 5 min and slowly cooling to RT. 464 EMSA was performed using the LightShift® Chemiluminescent EMSA Kit (Thermo Scientific) 465 following the manufacturer’s protocol with nylon membrane (Hybond). Signal visualization 466 was performed in AI600 Chemidoc Imaging System (GE Healthcare). 467 468 Chromatin immunoprecipitation (ChIP) assay 469 ChIP assays were performed as described in Gendrel et al., (2005). 35S:MYC-CFLAP1/Col-0 470 (14-day-old) and 35S:PIF4-MYC/Col-0 (12-day-old) seedlings were analyzed in separate 471 experiments, each with 35S:MYC/Col-0 as a control. Seedlings were crosslinked with 1% 472 formaldehyde under vacuum infiltration for 15 min, and the reaction was quenched with 0.125 473 M glycine. After washing, samples were frozen, and ground. Nuclei were isolated using 474 sequential extraction buffers, and chromatin was sheared by sonication (Bioruptor® Pico, 475 Diagenode) to an average DNA fragment size of approximately 0.2-1 kb. Immunoprecipitation 476 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 17 was conducted using an anti-MYC antibody (Millipore) to capture MYC-, MYC-CFLAP1- or 477 PIF4-MYC-bound chromatin complexes. Following reverse crosslinking and DNA purification, 478 the enrichment of promoter regions of target genes was analyzed by qPCR. The results were 479 normalized to the amplification of the PP2AA3 gene as an internal control. The primers used in 480 ChIP-qPCR were listed in Supplemental Table 2. 481 482 Luciferase complementation imaging (LCI) assay 483 The CDSs of CFLAP1 and COP1 were cloned into cLUC and nLUC vectors, respectively. 484 Agrobacterium harboring these constructs were grown until their OD600 reached 1.0, infiltrated 485 into tobacco leaves together with p19 helper strain. After 2 days, the infected leaf areas were 486 infiltrated with luciferin (100 mM) dissolved in 0.1% Triton X-100. After 5 min of dark 487 incubation, the signals were detected in AI600 Chemidoc Imaging System (GE Healthcare). 488 489 Bimolecular fluorescence complementation (BiFC) assay 490 The CDSs of CFLAP1, HY5, and DEWAX were cloned into the pSPYCE vector and the COP1 491 CDS was cloned into pSPYNE vector. Agrobacterium cells were co-infiltrated into tobacco 492 leaves together with the p19 helper strain. After 2 days, the fluorescence signals from tobacco 493 epidermal cells were detected using a confocal laser scanning microscope (TCS SPE, Leica) 494 with a yellow fluorescent protein (YFP) filter (519 nm excitation, 555 nm emission). 495 496 Immunoblot assays 497 Agrobacterium cells carrying COP1-HA or MYC-CFLAP1 were infiltrated into tobacco leaves, 498 treated with 100 µM MG132 8 h, and harvested 24 h after infiltration. Protein was extracted 499 from liquid nitrogen-ground tissues in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% 500 NP-40, 1 mM EDTA, 10 mM DTT, 2 mM NaVO3, 2 mM NaF, 2 mM PMSF, 3 μg/ml pepstatin 501 A, 3 μg/ml aprotinin, 5 μg/ml leupeptin, 10 μM MG132), separated by 8% SDS-PAGE, 502 transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), and detected using 503 anti-MYC (1:2000, Millipore) or anti-HA (1:4000, Invitrogen) primary antibodies (12 h at 4 504 oC), and anti-mouse IgG (1:5000, Millipore) secondary antibody (1 h at RT). Signal 505 visualization was performed with Pierce TM ECL Plus Western Blotting Substrate (Thermo 506 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 18 Scientific) in AI600 Chemidoc Imaging System (GE Healthcare). 507 For co-immunoprecipitation (Co-IP) and ubiquitination assays, protein from tobacco 508 leaves co-infiltrated with Agrobacterium cells COP1-HA or MYC-CFLAP1 with 100 μM 509 MG132 treatment were extracted in IP buffer. The extracts were incubated with anti-c-MYC 510 conjugated agarose beads (Sigma) on a rotator at 4 °C for 2 h, washed with IP buffer, 511 resuspended in sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, 512 0.025% bromophenol blue), and analyzed using anti-MYC (1:2000, Millipore), anti-HA 513 (1:4000, Invitrogen) and anti-Ub (1:2000, Santa Cruz Biotechnology) antibodies. 514 To assess MYC-CFLAP1 stability, 4-day-old 35S:MYC-CFLAP1/Col-0 or 35S:MYC-515 CFLAP1/cop1-4 seedlings were incubated in 1/2 MS liquid media with 100 mM cycloheximide 516 (CHX), 100 mM MG132 or DMSO. To assess MYC-CFLAP1 stability over the diurnal cycle, 517 10-day-old transgenic Arabidopsis seedlings 35S:MYC-CFLAP1/Col-0 or 35S:MYC-518 CFLAP1/cop1-4 were harvested at indicated time points. Sample was analyzed by western blot 519 with anti-MYC (1:2000, Millipore). 520 To assess PIF1, PIF3, PIF4, and PIF5 stability, 3-week-old Arabidopsis leaves were 521 harvested at indicated time points. Proteins were extracted using an extraction buffer A (125 522 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM DTT, 10% glycerol, and protease inhibitors including 523 2 mM PMSF, 3 μg/ml pepstatin A, 3 μg/ml aprotinin, 5 μg/ml leupeptin, 10 μM MG132) and 524 detected using anti-PIF1, anti-PIF3, anti-PIF4 (1:1000) and anti-PIF5 (1:1000, Agrisera) 525 antibodies. To assess phyB stability, 2-week-old Arabidopsis seedlings were harvested at 526 indicated time points. Proteins were extracted using an extraction buffer B [70 mM Tris-HCl, 527 pH 8.3, 35% ethylene glycol, 98 mM (NH 4)2SO4, 7 mM EDTA, 14 mM sodium metabisulfite, 528 0.07% polyethyleneimine, protease inhibitors (Roche)] and detected using anti-phyB (1:1000, 529 Agrisera) and anti-TCTP antibodies (1:10000). 530 531 TBO Staining 532 Three-week old Arabidopsis plants were immersed into TBO solution (0.1% TBO, 0.01% 533 Tween-20, Sigma) for 3 minutes, then washed three times in distilled water and photographed 534 using Samsung NX300 Digital camera. 535 536 Cuticular transpiration assay 537 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 19 Three-week-old plants were incubated in darkness for 12 h. The aerial parts were excised and 538 soaked in water for 1 h to equilibrate hydration. After surface water removal, the shoots were 539 weighed every 15 min for up to 3 h using a microbalance in darkness. 540 541 Chlorophyll leaching assay 542 The aerial parts of three-week-old plants were soaked in 80% ethanol. The extracted chlorophyll 543 amounts according to individual time points (up to 24 h) were displayed as percentages. The 544 amount of extracted chlorophyll was quantified by measuring the absorbance at 647 and 664 545 nm using a diode array spectrophotometer (Ultrospec 3100 pro; Amersham Biosciences). 546 547 TEM analysis 548 3-week-old rosette leaves from Col-0 and cop1-4 were harvested which were subsequently 549 fixed and analyzed as previously described (Kim et al., 2025). Shortly, the leaves were fixed 550 o/n at 4 oC using Karnovsky’s fixation solution (2% paraformaldehyde, 2.5% glutaraldehyde, 551 0.1 M sodium cacodylate buffer (pH 7.4)) and post-fixed using 1% osmium tetroxide (OsO4) at 552 4oC for 1 hour. Samples were dehydrated through a graded ethanol series and gradually 553 embedded with Spurr’s epoxy resin (medium hardness, Ted Pella) with sequential exchange of 554 EtOH:resin=2:1, 1:1, 1:2, and finally 100% resin o/n. Samples were hardened for 36~48 hours 555 at 60 ℃. Resin-embedded samples were sectioned into 80–100 nm slices using an 556 ultramicrotome (RMC Products), placed on a grid and stained with uranyl acetate and lead 557 citrate prior to observation with TEM (Jeol, JEM-2100F). 558 559 Cuticular wax and cutin analysis 560 Three-week-old Arabidopsis leaves were used for cuticular wax and cutin analyses. Cuticular 561 waxes were extracted by 4 ml chloroform for 30 sec. Internal standards (2 μg n-Octacosane, 1 562 μg 1-tricosanol, and 2 μ g heptadecanoic acid; Sigma) were added to the extracts, evaporated 563 under a stream of nitrogen gas, and trimethylsilylated by heating with 100 μl of N, O-Bis 564 (trimethylsilyl) trifluoroacetamide (Sigma) and 100 μl of pyridine (Sigma) at 100 oC for 30 min. 565 After evaporation under nitrogen, samples were dissolved in heptane:toluene (1:1, v/v). Wax 566 compounds were quantified using GC-FID (GC-2010 Plus, Shimazu) using DB-5 (60 m) 567 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 20 column (Agilent) as previously described (Kim et al., 2021). For cutin analysis, leaves were 568 delipidated and depolymerized. C17:0 FAME and ω-pentadecalactone (Sigma) were used as 569 internal standards, as previously described (Lee et al., 2019). Derivatization of cutin monomers 570 was performed with 100 μl of pyridine (Sigma) and 100 μl of acetic anhydride (J.T.Baker) at 571 60 oC for 2 h. Solvent was evaporated under nitrogen gas and redissolved in heptane/toluene 572 (1:1, v/v). Cutin monomers were quantified using the same equipment described above. The 573 oven temperature increased to 300°C at a rate of 2.5 °C/min and maintained at 300 °C for 3 574 min. 575 576 FUNDING 577 This work was supported by grants (RS-2022-NR070837 and RS-2021-NR058215) from the 578 National Research Foundation of the Republic of Korea. 579 580 AUTHOR CONTRIBUTIONS 581 Q.H.D., H.J.K., J.C., and M.C.S. conceived the study. Q.H.D., H.J.K., D.-M.C., and S.-H.K. 582 performed the experiments and jointly analyzed the data with J.C., J.-I.K., and M.C.S. Q.H.D., 583 H.J.K., J.C., and M.C.S. wrote the manuscript, and J.-I.K. contributed to scientific discussions 584 and provided a critical review of the manuscript. 585 586

587 The authors thank Takato Imaizumi (University of Washington) and Woe-Yeon Kim 588 (Gyeongsang National University) for sharing Arabidopsis mutant seeds listed in Supplemental 589 Table 1. The authors are grateful to Ryeo Jin Kim for TEM analysis. 590 591 DECLARATION OF INTERESTS 592 Authors declare that they have no competing interests. 593 594 SUPPLEMENTAL INFORMATION 595 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 21 The following materials are available in the online version of this article. 596 Supplemental Figure S1. Cuticular wax composition and amounts from wild type (Col-0 /Ler), 597 phytochrome mutants, and a phyB overexpressing line (PHYB OX). 598 Supplemental Figure S2. Expression patterns of PIFs transcripts and proteins in Arabidopsis 599 WT during the daytime and nighttime 600 Supplemental Figure S3. Cuticular wax composition and amounts from wild type (Col-0), pif4, 601 pifQ, and PIF4/pifQ, and PIF4 OX. 602 Supplemental Figure S4. Cutin monomer composition and amounts from wild type (Col-0) , 603 pif4, pifQ, and PIF4/pifQ leaves. 604 Supplemental Figure S5. Cuticular wax composition and amounts from wild type (Col-0) and 605 lrb123. 606 Supplemental Figure S6. Isolation of the cflap1 mutant. 607 Supplemental Figure S7. Cutin monomer and cuticular wax composition and amounts from 608 wild type (Col-0), cflap1-1, and cflap1-2 leaves. 609 Supplemental Figure S8. Expression patterns of CFLAP1 transcripts and proteins in 610 Arabidopsis WT and CFLAP1 OX/Col-0 during the daytime and nighttime 611 Supplemental Figure S9. IPTG induction test and purification of recombinant MBP-CFLAP1 612 protein 613 Supplemental Figure S10. Cutin monomer and cuticular wax composition and amounts from 614 wild type (Col-0), cop1-4, and cop1-6 leaves. 615 Supplemental Figure S11. Genotyping of cop1-4, cflap1-1, cflap1-2, and cflap1cop1-4. 616 Supplemental Figure S12. Cutin monomer and cuticular wax composition and amounts from 617 wild type (Col-0), cflap1-1, cflap1-2, cop1-4, cflap1-1 cop1-4 and cflap1-2 cop1-4 leaves. 618 Supplemental Table S1. The list of Arabidopsis mutant and transgenic lines used in this study. 619 Supplemental Table S2. Oligonucleotide primers used in this study. 620 621

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Phytochrome B negatively regulates cuticular wax biosynthesis 804 (A) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Col-0), phyA-211, 805 phyB-9, and phyA-211 phyB-9. 806 (B) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Ler), phyA-201 807 phyB-5, and PHYB OX. 808 (C) Heatmap visualizing expression of cuticular wax biosynthesis-related genes in 3-week-old 809 leaves of Col-0, phyA-211, phyB-9, and phyA-211 phyB-9 harvested at 10:30. 810 (D) Heatmap visualizing expression of cuticular wax biosynthesis-related genes in leaves of 811 Ler, phyA-201 phyB-5, and PHYB OX harvested at 10:30. 812 (A and B) Each value represents the mean ±SD of three individual replicates. AK, alkanes; AL, 813 aldehydes; FA, fatty acids; PA, primary alcohols; UN, unidentified. Different letters indicate 814 statistically significant differences using one-way ANOV A with Tukey’s test (P < 0.01). 815 (C and D) Asterisks indicate statistically significant differences determined by Student’s t-test 816 (*, P < 0.05; **, P < 0.01). 817 See also Figure S1. 818 819 Figure 2. PIF4 activates cuticular wax biosynthesis by its direct binding to the promoter 820 regions of KCS2, CER1, and CER4 during the daytime 821 (A) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Col-0), pif4, and 822 pifQ. 823 (B) Quantification of cuticular wax loads in 3-week-old leaves of Col-0, PIF4pro:PIF4-824 MYC/pifQ (PIF4/pifQ), and 35Spro:PIF4-MYC/Col-0 (PIF4 OX). 825 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 28 (C) Heatmap showing expression of cuticular wax biosynthesis-related genes in 3-week-old 826 leaves of Col-0, pif4, pifQ, PIF4/pifQ, and PIF4 OX harvested at 10:30. 827 (D) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and 828 pif4. Leaves of 3-week-old plants grown under long-day conditions were harvested at indicated 829 time points. Transcript levels were examined by RT–qPCR. 830 (E) Dual-luciferase assays were performed in N. benthamiana to examine transcriptional 831 activities of PIF4-eYFPC on the promoter regions of KCS2, CER1, and CER4. LUC activity 832 values were normalized to Renilla (REN) luciferase activity to represent relative promoter 833 activities. 834 (F) ChIP-qPCR assays showed that PIF4 associates with cis-elements within the promoters of 835 KCS2, CER1 and CER4 in vivo. UTR, Untranslated region. 836 (A, B, D, E, and F) Values represent mean ±SD from three replicate experiments. Different 837 letters indicate statistically significant differences using one-way ANOV A with Tukey’s test (P 838 < 0.01). AK, alkanes; AL, aldehydes; FA, fatty acids; PA, primary alcohols; UN, unidentified. 839 (C, E, and F) Asterisks indicate statistically significant differences determined by Student’s t-840 test (*, P < 0.05; **, P < 0.01). 841 See also Figure S2-S4. 842 843 Figure 3. LRB-mediated destabilization of phyB results in PIF4 stabilization, thereby 844 promoting cuticular wax biosynthesis during the daytime 845 (A) Immunoblot analysis of PIF4 protein in 3-week-old wild type (Col-0) and phyB-9 using an 846 anti-PIF4 antibody. 847 (B) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and 848 phyB-9. 849 (C) Immunoblot analysis of phyB protein in 2-week-old Col-0 and lrb123 plants using an anti-850 phyB antibody. Total protein loading was assessed by anti-TCTP antibody. 2-week-old plants 851 were harvested at indicated time points. 852 (D) Immunoblot analysis of PIF4 protein in 3-week-old Col-0 and lrb123 using an anti-PIF4 853 antibody. 854 (E) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and 855 lrb123. 856 (F) Quantification of cuticular wax loads in 3-week-old leaves of Col-0 and lrb123. Values 857 represent mean ±SD of three replicate experiments. Asterisks indicate statistically significant 858 differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). AK, alkanes; AL, 859 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 29 aldehydes; FA, fatty acids; PA, primary alcohols; UN, unidentified. 860 (A, B, D, and E) Leaves of 3-week-old plants were harvested at indicated time points. 861 (A and D) Total protein loading was assessed by Ponceau S staining. 862 (B and E) Transcript levels were examined by RT–qPCR. Values represent mean ±SD of three 863 replicate experiments. 864 See also Figure S5. 865 866 Figure 4. CFLAP1 negatively regulates cutin biosynthesis by its direct binding to the 867 promoter regions of CYP86A2, CYP86A4 and BDG1. 868 (A and B) Quantification of cutin monomer (A) and cuticular wax (B) loads in 3-week-old 869 leaves of wild type (Col-0), cflap1-1, and cflap1-2. Each value represents the mean ±SD of 870 three individual replicates. FA, fatty acids; HFA, ⍵-hydroxy fatty acids; DCA, dicarboxylic 871 acids; AK, alkanes; PA, primary alcohols; AL, aldehydes. Different letters indicate statistically 872 significant differences using one-way ANOV A with Tukey’s test (P < 0.01). 873 (C) Relative expression levels of cutin biosynthetic genes in 12-day-old seedlings of Col-0, 874 cflap1-1, and cflap1-2 harvested at 18:30 are visualized as a heatmap. Scale bar, log 2 fold 875 change. 876 (D) Dual-luciferase assays were performed in N. benthamiana to examine transcriptional 877 activities of MYC-CFLAP1 on the promoter regions of KAT1, CYP86A2, CYP86A4, and BDG1. 878 LUC activity values were normalized to Renilla (REN) luciferase activity to represent relative 879 promoter activities. Data represent means ±SD from three biological replicates. 880 (E) EMSA assay showing binding of recombinant CFLAP1 protein to the promoters of 881 CYP86A2 (P1, P2, and P3), CYP86A4 (P1 and P2), and BDG1 (P1 and P2), and competition of 882 binding with increasing concentration of cold DNA probes. Diagram depicts the promoters of 883 pCYP86A2, pCYP86A4, and pBDG1 with putative binding E-box motifs. 884 (F) ChIP-qPCR assays showed that CFLAP1 associates with cis-elements within the promoters 885 of CYP86A2, CYP86A4 and BDG1 in vivo. Data are means ±SD (n = 3). 886 (D and F) Asterisks indicate statistically significant differences determined by Student’s t-test 887 (*, P < 0.05; **, P < 0.01). 888 See also Figure S6-S9. 889 890 Figure 5. COP1 interacts with CFLAP1 and facilitates its ubiquitination and degradation 891 via 26S proteasome system during the nighttime 892 (A) LCI assay of interaction between nLUC-COP1 and cLUC-CFLAP1 in vivo. 893 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 30 (B) BiFC assay of nuclear interaction between COP1-eYFPN and CFLAP1-eYFPC. YFP 894 fluorescence (green) indicates physical interaction between COP1 and each target protein. 895 DAPI staining (blue) marks nuclei. Scale bars, 100 µm. 896 (C) Co-immunoprecipitation (Co-IP) of the interaction between COP1-HA and MYC-CFLAP1. 897 Proteins were immunoprecipitated with an anti-MYC (α-MYC) antibody and detected using α-898 MYC and α-HA antibodies. 899 (D) Immunoblot analysis of the co-expression of MYC-CFLAP1 with COP1-HA with the 900 proteasome inhibitor MG132. Proteins were detected using α-MYC and α-HA antibodies. 901 (E) Ubiquitination assay of MYC-CFLAP1 upon co-expression with COP1-HA within 100 μM 902 MG132 treatment. Proteins were immunoprecipitated with an anti-MYC (α-MYC) antibody 903 and detected using α-MYC and anti-Ubiquitin (α-Ub) antibodies. 904 (F) Immunoblot (top) and RT-PCR (bottom) of MYC-CFLAP1 expression in Arabidopsis 905 CFLAP1-overexpressing (CFLAP1 OX) lines in the wild type (Col-0) or cop1-4 background. 906 (G) Diurnal accumulation patterns of CFLAP1 protein in CFLAP1 OX /Col-0 and CFLAP1 907 OX/cop1-4 seedlings collected at various time points. 908 (H) 100 μM cycloheximide (CHX) and 100 μM CHX + 100 μM MG132 treatment assays of 909 CFLAP1 protein stability in CFLAP1 OX/Col-0 and CFLAP1 OX/cop1-4 seedlings at various 910 time points. 911 (F-H) Proteins were detected using α-MYC. 912 (C-H) Ponceau S staining indicates equal protein loading. 913 914 Figure 6. The COP1-CFLAP1 module upregulates cutin biosynthesis during the nighttime 915 (A) Representative images of 3-week-old rosettes from Col-0, cop1-4 and cop1-6 before 916 staining, and adaxial or abaxial leaf surfaces after staining with 0.1% TBO containing 0.01% 917 Tween 20 for 3 min. Scale bars, 1 cm. 918 (B) Transmission electron microscopy (TEM) images of cuticle ultrastructure in the adaxial 919 epidermis of 3-week-old leaves. The cell wall and cuticle thickness were measured using 920 ImageJ at multiple positions. Values represent the mean ±SD of 15 (cell wall) and 20 (cuticle) 921 measurements. CP, cuticle proper; CL, cuticular layer; CW, cell wall; PM, plasma membrane. 922 Scale bars, 200 nm. 923 (C) Cuticular transpiration assay showing time courses of water loss (%) in 3-week-old Col-0, 924 cop1-4 and cop1-6 leaves. Values represent the mean ±SD of 3 individual replicates. 925 (D) Chlorophyll leaching assay showing time courses of chlorophyll extraction (%) from 3-926 week-old Col-0, cop1-4 and cop1-6 leaves. Values represent the mean ±SD of 3 individual 927 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 31 replicates. 928 (E and F) Quantification of cutin monomer (E) and cuticular wax (F) loads in 3-week-old rosette 929 leaves wild type (Col-0), cflap1-1, cflap1-2, cflap1-1 cop1-4, and cflap1-2 cop1-4 . Values 930 represent the mean ±SD of 3 individual replicates. FA, fatty acids; HFA, ⍵-hydroxy fatty acids; 931 DCA, dicarboxylic acids; AK, alkanes; PA, primary alcohols; AL, aldehydes. 932 (C-F) Different letters indicate statistically significant differences using one-way ANOV A with 933 Tukey’s test (P < 0.01). 934 (G) Heatmap showing relative expression levels of CYP86A2, CYP86A4 and BDG1 in 12-day-935 old seedlings of Col-0, cflap1-1, cflap1-2, cflap1-1 cop1-4, and cflap1-2 cop1-4 harvested at 936 6:30 and 18:30. Scale bar, log2 fold change. 937 (B and G) Asterisks indicate statistically significant differences determined by Student’s t-test 938 (*, P < 0.05; **, P < 0.01). 939 See also Figure S10-S12. 940 941 Figure 7. Diel regulation of cutin and cuticular wax biosynthesis 942 At night, nuclear-localized COP1 ubiquitinates CFLAP1, facilitating its degradation via the 26S 943 proteasome system. Repression of CFLAP1 leads to enhanced cutin biosynthesis. During the 944 day, light activates phyB to biologically active Pfr form. Photoactivated phyB is ubiquitinated 945 and degraded by LRB proteins. LRB-driven depletion of active phyB releases phyB-mediated 946 degradation of PIF4, allowing PIF4 accumulation. Accumulated PIF4 directly activates 947 cuticular wax biosynthesis. 948 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint Figure 1. Phytochrome B negatively regulates cuticular wax biosynthesis (A ) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Col-0), phyA-211, phyB-9, and phyA-211 phyB-9 . (B) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Ler), phyA-201 phyB-5 , and PHYB OX . (C) Heatmap visualizing expression of cuticular wax biosynthesis-related genes in 3-week-old leaves of Col-0, phyA-211, phyB-9, and phyA-211 phyB-9 harvested at 10:30. (D) Heatmap visualizing expression of cuticular wax biosynthesis-related genes in leaves of Ler, phyA-201 phyB-5 , and PHYB OX harvested at 10:30. (A and B) Each value represents the mean ±SD of three individual replicates. AK, alkanes; AL, aldehydes; FA, fatty acids; P A, primary alcohols; UN, unidentified. Different letters indicate statistically significant differences using one-way ANOV A with Tukey’s test (P < 0.01). (C and D) Asterisks indicate statistically significant differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). b ab a a a c a a b aa b a a a 0 0.1 0.2 0.3 0.4 0.5 AK AL FA PA UN b c a 0 0.2 0.4 0.6 0.8 Total Wax Load (𝜇g/cm2) Total Wax Load (𝜇g/cm2) a a b b 0 0.2 0.4 0.6 0.8 a b a a a a b b a a b b ab b a b a a b a 0 0.1 0.2 0.3 0.4 0.5 AK AL FA PA UN Wax Load (𝜇g/cm2) phyA-211 phyB-9phyA-211Col-0 phyB-9 ***LACS2 ****KCS2 ****KCS6 ****KCR1 **PAS2 ****ECR ******CER1 *CER3 ******CER4 ******SOH1 (Log 2) +2 -2 PHYB OXphyA-201 phyB-5Ler Wax Load (𝜇g/cm2) ***LACS2 ****KCS2 ***KCS6 **KCR1 ****PAS2 **ECR ***CER1 ****CER3 **CER4 *SOH1 (Log 2) +2 -2 A B C D preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint (Log 2) +2 -2 *****LACS2 ********KCS2 ******KCS6 *****KCR1 ******PAS2 *ECR ********CER1 ****CER3 ********CER4 *****SOH1 Effectors 35S PIF4 eYFPC 35S eYFPC Reporters proKCS2 LUCREN proCER1 LUCREN proCER4 LUCREN LUC/REN Raito **** 0 0.01 0.02 0.03 0.04 proCER4:LUC 0 0.02 0.04 0.06 proCER1:LUC 0 0.01 0.02 0.03 0.04 proKCS2:LUC ** 35Spro:MYC/Col-0 35Spro:PIF4-MYC/Col-0 Relative enrichment 0 1 2 3 A B C KCS2 ** 0 2 4 6 8 A B C CER4 ** ** ** 0 1 2 3 4 5 A B C CER1 ** ** A B C CER1-923 A B C CER4-1505 0.5 kb G-box 5-UTR E-box A B C KCS2-2980 pif4Col-0 pifQ PIF4/pifQCol-0 PIF4 OX Total Wax Load (𝜇g/cm2) Wax Load (𝜇g/cm2) a b c 0 0.2 0.4 0.6 0.8 c b a 0 0.1 0.2 0.3 0.4 0.5 c a a b a b a a a a a a a a a 0 0.05 0.1 0.15 0.2 0.25 AK AL FA PA UN Wax Load (𝜇g/cm2) Total Wax Load (𝜇g/cm2) pif4Col-0 Relative expression0 0.5 1 1.5 0 0.5 1 1.5 2 0 0.5 1 1.5 2 KCS2 CER1 CER4 LACS2 KCS6SOH1 0 1 2 3 4 0 2 4 6 8 0 0.5 1 1.5 2 2.5 A B C D E F a b a a a b a a b a c a a c a 0 0.1 0.2 0.3 0.4 0.5 AK AL FA PA UN 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 Figure 2. PIF4 activates cuticular wax biosynthesis by its direct binding to the promoter regions of KCS2, CER1, and CER4 during the daytime (A) Quantification of cuticular wax loads in 3-week-old leaves of wild type (Col-0), pif4, and pifQ. (B) Quantification of cuticular wax loads in 3-week-old leaves of Col-0, PIF4pro:PIF4- MYC/pifQ (PIF4/pifQ), and 35Spro:PIF4-MYC/Col-0 ( PIF4 OX ). (C) Heatmap showing expression of cuticular wax biosynthesis-related genes in 3-week-old leaves of Col-0, pif4, pifQ, PIF4/pifQ, and PIF4 OX harvested at 10:30. (D) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and pif4. Leaves of 3-week-old plants grown under long-day conditions were harvested at indicated time points. Transcript levels were examined by RT–qPCR. (E) Dual-luciferase assays were performed in N. benthamiana to examine transcriptional activities of PIF4-eYFPC on the promoter regions of KCS2, CER1, and CER4. LUC activity values were normalized to Renilla (REN) luciferase activity to represent relative promoter activities. (F) ChIP-qPCR assays showed that PIF4 associates with cis-elements within the promoters of KCS2, CER1 and CER4 in vivo . UTR, Untranslated region. (A, B, D, E, and F) V alues represent mean ±SD from three replicate experiments. Different letters indicate statistically significant differences using one-way ANOV A with Tukey’s test ( P < 0.01). AK, alkanes; AL, aldehydes; FA, fatty acids; PA, primary alcohols; UN, unidentified. (C, E, and F) Asterisks indicate statistically significant differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). 35S 35S 35S eYFPC PIF4-eYFPC preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint 0 2 4 6 CER4 phyB-9Col-0 KCS2 Relative expression0 1 2 3 4 CER1 0 10 20 30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 F Total Wax Load (𝜇g/cm2) ** 0 0.1 0.2 0.3 0.4 ** ** ** 0 0.04 0.08 0.12 0.16 AK AL FA PA UN Wax Load (𝜇g/cm2) lrb123Col-0 A B KCS6 0 0.5 1 1.5 2 LACS2 0 0.5 1 1.5 2 Relative expression SOH1 0 2 4 6 8 10 E 0 0.5 1 1.5 2 LACS2 Relative expression SOH1 0 1 2 3 4 5 0 0.5 1 1.5 2 KCS6 0 1 2 3 4 CER1 0 1 2 3 4 CER4 0 0.5 1 1.5 2 KCS2 Relative expression Figure 3. LRB-mediated destabilization of phyB results in PIF4 stabilization, thereby promoting cuticular wax biosynthesis during the daytime (A) Immunoblot analysis of PIF4 protein in 3-week-old wild type (Col-0) and phyB-9 using an anti-PIF4 antibody. (B) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and phyB-9. (C) Immunoblot analysis of phyB protein in 2-week-old Col-0 and lrb123 plants using an anti-phyB antibody. Total protein loading was assessed by anti-TCTP antibody. 2-week-old plants were harvested at indicated time points. (D) Immunoblot analysis of PIF4 protein in 3-week-old Col-0 and lrb123 using an anti-PIF4 antibody. (E) Diurnal expression patterns of KCS2, CER1, CER4, SOH1, LACS2 and KCS6 in Col-0 and lrb123. (F) Quantification of cuticular wax loads in 3-week-old leaves of Col-0 and lrb123. V alues represent mean ±SD of three replicate experiments. Asterisks indicate statistically significant differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). AK, alkanes; AL, aldehydes; FA, fatty acids; P A, primary alcohols; UN, unidentified. (A, B, D, and E) Leaves of 3-week-old plants were harvested at indicated time points. (A and D) Total protein loading was assessed by Ponceau S staining. (B and E) Transcript levels were examined by RT–qPCR. V alues represent mean ±SD of three replicate experiments. PIF4 PIF4 Ponceau S phyB-9 Col-0 Ponceau S C D PIF4 Ponceau S PIF4 Ponceau S Col-0lrb123 lrb123 Col-0 phyB TCTP phyB TCTP 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 06:30 02:30 22:30 18:30 14:30 10:30 06:30 lrb123Col-0 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint Figure 4. CFLAP1 negatively regulates cutin biosynthesis by its direct binding to the promoter regions of CYP86A2, CYP86A4 and BDG1. (A and B) Quantification of cutin monomer (A) and cuticular wax (B) loads in 3-week-old leaves of wild type (Col-0), cflap1-1, and cflap1-2. Each value represents the mean ±SD of three individual replicates. FA, fatty acids; HFA, ⍵-hydroxy fatty acids; DCA, dicarboxylic acids; AK, alkanes; PA, primary alcohols; AL, aldehydes. Different letters indicate statistically significant differences using one-way ANOV A with Tukey’s test ( P < 0.01). (C) Relative expression levels of cutin biosynthetic genes in 12-day-old seedlings of Col-0, cflap1-1, and cflap1-2 harvested at 18:30 are visualized as a heatmap. Scale bar, log 2 fold change. (D) Dual-luciferase assays were performed in N. benthamiana to examine transcriptional activities of MYC-CFLAP1 on the promoter regions of KAT1, CYP86A2, CYP86A4, and BDG1. LUC activity values were normalized to Renilla (REN) luciferase activity to represent relative promoter activities. Data represent means ±SD from three biological replicates. (E) EMSA assay showing binding of recombinant CFLAP1 protein to the promoters of CYP86A2 (P1, P2, and P3), CYP86A4 (P1 and P2), and BDG1 (P1 and P2) , and competition of binding with increasing concentration of cold DNA probes. Diagram depicts the promoters of pCYP86A2, pCYP86A4, and pBDG1 with putative binding E-box motifs. (F) ChIP-qPCR assays showed that CFLAP1 associates with cis-elements within the promoters of CYP86A2, CYP86A4 and BDG1 in vivo . Data are means ±SD (n = 3). (D and F) Asterisks indicate statistically significant differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). a a a a a b b b a b b b 0 0.5 1 1.5 2 Cutin Load (μg/g DW) a a a a a a a a a a a a a a a 0 0.1 0.2 0.3 0.4 0.5Wax Load (μg/cm2) A B CYP86A2 CYP86A4 CYP86A8 HTH BDG1 GPAT4 GPAT8 LACS2 ** ** ** ** ** **** ** Competitors MBP-empty MBP-CFLAP1 ATGpBDG1 P1 P2 #1 #2 ATGpCYP86A4 P1 P2 #1 #2 ATGpCYP86A2 P1 P2 P3 #1 #2 #3 : E-box (CANNTG) : PCR fragments P3P2P1 pCYP86A2 E P2P1 pCYP86A4 P2P1 pBDG1 * ** ** ** * ** ** 0 2 4 6 8 10Relative enrichment F TotalDCAHFAFA TotalFAALPAAK 1000x 100x 10x-- ----+ ++++- Col-0 cflap1-1 cflap1-2 Col-0 cflap1-1 cflap1-2 (Log 2) +1 -1 ** ** ** ** 0 0.04 0.08 0.12 LUC/REN Ratio Effectors Reporters C D 35S MYC 35S MYC CFLAP1 35S pKAT1 LUCREN 35S pCYP86A2 LUCREN 35S pCYP86A4 LUCREN 35S pBDG1 LUCREN 35Spro:MYC/Col-0 35Spro:MYC-CFLAP1/Col-0 #1 #2 #3 #1 #2 #1 #2 pCYP86A2 pCYP86A4 pBDG1 MYC MYC-CFLAP1 1000x 100x 10x-- ----+ ++++- 1000x 100x 10x-- ----+ ++++- 1000x 100x 10x-- ----+ ++++- 1000x 100x 10x-- ----+ ++++-1000x 100x 10x-- ----+ ++++-1000x 100x 10x-- ----+ ++++- preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint Figure 5. COP1 interacts with CFLAP1 and facilitates its ubiquitination and degradation via 26S proteasome system during the nighttime (A) LCI assay of interaction between nLUC-COP1 and cLUC-CFLAP1 in vivo . (B) BiFC assay of nuclear interaction between COP1-eYFPN and CFLAP1-eYFPC. YFP fluorescence (green) indicates physical interaction between COP1 and each target protein. DAPI staining (blue) marks nuclei. Scale bars, 100 µm. (C) Co-immunoprecipitation (Co-IP) of the interaction between COP1-HA and MYC- CFLAP1. Proteins were immunoprecipitated with an anti-MYC (α-MYC) antibody and detected using α-MYC and α-HA antibodies. (D) Immunoblot analysis of the co-expression of MYC-CFLAP1 with COP1-HA with the proteasome inhibitor MG132. Proteins were detected using α-MYC and α-HA antibodies. (E) Ubiquitination assay of MYC-CFLAP1 upon co-expression with COP1-HA within 100 μM MG132 treatment. Proteins were immunoprecipitated with an anti-MYC (α-MYC) antibody and detected using α-MYC and anti-Ubiquitin (α-Ub) antibodies. (F) Immunoblot (top) and RT-PCR (bottom) of MYC-CFLAP1 expression in Arabidopsis CFLAP1-overexpressing ( CFLAP1 OX ) lines in the wild type (Col-0) or cop1-4 background. (G) Diurnal accumulation patterns of CFLAP1 protein in CFLAP1 OX /Col-0 and CFLAP1 OX /cop1-4 seedlings collected at various time points. (H) 100 μM cycloheximide (CHX) and 100 μM CHX + 100 μM MG132 treatment assays of CFLAP1 protein stability in CFLAP1 OX /Col-0 and CFLAP1 OX /cop1-4 seedlings at various time points. (F-H) Proteins were detected using α-MYC. (C-H) Ponceau S staining indicates equal protein loading. A MergedBrightDAPIYFP COP1-eYFPN HY5-eYFPC COP1-eYFPN DEWAX-eYFPC COP1-eYFPN CFLAP1-eYFPC B C ++ +- MYC-CFLAP1 COP1-HA MYC-CFLAP1 Input Ponceau S COP1-HA Ponceau S MYC-CFLAP1 IP:MYC Ubiquitinated CFLAP1 ++ ++ +- MYC-CFLAP1 COP1-HA MG132 E +++- ++-- +--- CFLAP1 OX/ cop1-4 CFLAP1 OX/ Col-0 cop1-4 Col-0 MYC-CFLAP1 Ponceau S COP1-HA Ponceau S MYC-CFLAP1 Ponceau S MYC-CFLAP1 PP2AA3 MYC-CFLAP1 COP1-HA MG132 D F CFLAP1 OX/Col-0 MYC-CFLAP1 Ponceau S MYC-CFLAP1 Ponceau S G H 02:3022:3018:3014:3010:3006:30 02:3000:3022:3020:3018:30 nLUC-COP1 cLUC nLUC-COP1 cLUC-CFLAP1 nLUC cLUC nLUC cLUC-CFLAP1 +CHX MYC-CFLAP1 Ponceau S MYC-CFLAP1 Ponceau S +CHX and MG132 CFLAP1 OX/Col-0 02:3000:3022:3020:3018:30 02:3000:3022:3020:3018:30 CFLAP1 OX/cop1-4 02:3000:3022:3020:3018:30 +CHX +CHX and MG132 MYC-CFLAP1 Ponceau S COP1-HA Ponceau S MYC-CFLAP1 COP1-HA InputIP:MYC CFLAP1 OX/cop1-4 02:3022:3018:3014:3010:3006:30 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint a a a a a a a a a a a a a b b b b b b b b b b b b b b b b b b b b b b b b b ab 0 20 40 60 80 100 120 Figure 6. The COP1-CFLAP1 module upregulates cutin biosynthesis during the nighttime (A) Representative images of 3-week-old rosettes from Col-0, cop1-4 and cop1-6 before staining, and adaxial or abaxial leaf surfaces after staining with 0.1% TBO containing 0.01% Tween 20 for 3 min. Scale bars, 1 cm. (B) Transmission electron microscopy (TEM) images of cuticle ultrastructure in the adaxial epidermis of 3-week-old leaves. The cell wall and cuticle thickness were measured using ImageJ at multiple positions. V alues represent the mean ±SD of 15 (cell wall) and 20 (cuticle) measurements. CP , cuticle proper; CL, cuticular layer; CW , cell wall; PM, plasma membrane. Scale bars, 200 nm. (C) Cuticular transpiration assay showing time courses of water loss (%) in 3-week-old Col-0, cop1-4 and cop1-6 leaves. V alues represent the mean ±SD of 3 individual replicates. (D) Chlorophyll leaching assay showing t ime courses of chlorophyll extraction (%) from 3-week-old Col- 0, cop1-4 and cop1-6 leaves. V alues represent the mean ±SD of 3 individual replicates. (E and F) Quantification of cutin monomer (E) and cuticular wax (F) loads in 3-week-old rosette leaves wild type (Col-0), cflap1-1, cflap1-2, cflap1-1 cop1-4, and cflap1-2 cop1-4 . V alues represent the mean ±SD of 3 individual replicates. FA, fatty acids; HFA, ⍵-hydroxy fatty acids; DCA, dicarboxylic acids; AK, alkanes; PA, primary alcohols; AL, aldehydes. (C-F) Different letters indicate statistically significant differences using one-way ANOV A with Tukey’s test ( P < 0.01). (G) Heatmap showing relative expression levels of CYP86A2, CYP86A4 and BDG1 in 12-day-old seedlings of Col-0, cflap1-1, cflap1-2, cflap1-1 cop1-4, and cflap1-2 cop1-4 harvested at 06:30 and 18:30. Scale bar, log 2 fold change. (B and G) Asterisks indicate statistically significant differences determined by Student’s t-test (*, P < 0.05; **, P < 0.01). Col-0 cop1-4 cop1-6 Water loss (%) b c c c c c c c c c c ca a a a a a a a a a a a ab b b b b b b b b b b b 0 20 40 60 80 100 120 180 165 150 135 120 105 90 75 60 45 30 15 0 Time (min) Extracted chlorophyll (%) Col-0 CW cop1-4 CW PM PM CL CP CL 0 100 200 300 400 500 600 0 5 10 15 20 25 30 Cell wall thickness (nm) Cuticle thickness (nm) * * cop1-4Col-0AbaxialAdaxialBefore cop1-4Col-0 180 165 150 135 120 105 90 75 60 45 30 15 0 Time (min) A B C D a c c b a d d c a d d c a a a a a b b b a b b b 0 0.5 1 1.5 2 2.5 Cutin Load (μg/g DW) E G a a bc a a a a c a a a a c a a a a a a a a a ab a a a a c a a 0 0.1 0.2 0.3 Wax Load (𝜇g/cm2) F TotalDCAHFAFA TotalFAALPAAK CYP86A2 CYP86A4 BDG1 ***** ** ****** ** **** CYP86A2 CYP86A4 BDG1 06:30 (Log 2) +1 -1 (Log 2) +1 -1 Col-0 cflap1-1 cflap1-2 cop1-4 cflap1-1 cop1-4 cflap1-2 cop1-4 Col-0 cflap1-1 cflap1-2 cop1-4 cflap1-1 cop1-4 cflap1-2 cop1-4 Col-0 cop1-4 cop1-6 Col-0 cop1-4 cop1-6 18:30 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint Figure 7. Diel regulation of cutin and cuticular wax biosynthesis At night, nuclear-localized COP1 ubiquitinates CFLAP1, facilitating its degradation via the 26S proteasome system. Repression of CFLAP1 leads to enhanced cutin biosynthesis. During the day, light activates phyB to biologically active Pfr form. Photoactivated phyB is ubiquitinated and degraded by LRB proteins. LRB-driven depletion of active phyB releases phyB-mediated degradation of PIF4, allowing PIF4 accumulation. Accumulated PIF4 directly activates cuticular wax biosynthesis. KCS2, CER1, CER4 CFLAP1 BDG1 PIF4 Cutin Cuticular wax CFLAP1 CFLAP1 PIF4 LRBCOP1 COP1 phyB (Pr) PIF4 phyB (Pfr) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 28, 2025. ; https://doi.org/10.1101/2025.10.28.684701doi: bioRxiv preprint