# Source paper archive - Source title: PHYRE, a Translated uORF, is Required for Phytochrome B Translation and Photomorphogenesis in Arabidopsis - Author(s): Naveen Shankar; Anne Van Humbeeck; Manuela Jurca; Ashleigh Edwards; Amir Mahboubi; Johannes Hanson; José Carlos Montanés; Chris Papadopoulos; M. Mar Albà; Stephan Wenkel - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.31.763305v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.10.31.763305v1.full.pdf - PDF SHA-256: edf1da46230e8f4c4d891fc640a0279b8527235ca07e5f42e0db64e1206bd543 - 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/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity.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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) # 1 PHYRE, a Translated uORF, is Required for Phytochrome B 2 Translation and Photomorphogenesis in Arabidopsis 3 4 Naveen Shankar [1], Anne Van Humbeeck [1], Manuela Jurca [1], Ashleigh Edwards [1,2], Amir 5 Mahboubi [1], Johannes Hanson [1], José Carlos Montanés [3], Chris Papadopoulos [3], M. 6 Mar Albà [3,4] and Stephan Wenkel [1] # 7 8 Affiliations: 1 9 Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 10 Linnaeus väg 6, 90736 Umeå, Sweden 11 2 12 Department of Plant & Environmental Sciences, University of Copenhagen, 13 Thorvaldsensvej 40, 1871 Frederiksberg, Denmark 14 3 15 Hospital del Mar Research Institute (HMRIB), Barcelona 08003, Spain 16 4 17 Catalan Institute for Research and Advanced Studies(ICREA), Barcelona 08010, 18 Spain 19 20 #corresponding author: stephan.wenkel@umu.se 21 # 22 ABSTRACT 23 Translational control allows cells to adjust protein output independently of 24 transcription, yet the mRNA-encoded mechanisms that govern this regulatory layer 25 remain poorly understood. Upstream open reading frames (uORFs) are among the 26 most prevalent translational regulatory elements in eukaryotic genomes; however, 27 characterised examples of activating uORFs that promote rather than inhibit 28 downstream translation are exceedingly rare. Here we show that efficient expression 29 of phytochrome B (phyB), the master red-light photoreceptor of Arabidopsis thaliana, 30 requires translation of a short uORF in its own 5′UTR, which we name 31 _PHYTOCHROME REGULATOR_ ( _PHYRE_ ). Loss of _PHYRE_ function phenocopies 32 _phyB_ null mutants across germination, hypocotyl growth, leaf development, and 33 flowering time. _PHYRE_ activity depends on its encoded peptide sequence, and the 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 spacing between _PHYRE_ and the _PHYB_ start codon is a critical determinant of 2 downstream translation efficiency, consistent with a position-dependent reinitiation 3 mechanism. _PHYRE_ -like elements are conserved across eudicots, establishing 4 uORF-mediated translational gating as a broadly maintained regulatory layer 5 controlling a master photoreceptor. 6 # 7 INTRODUCTION 8 Eukaryotic cells can respond rapidly to signals, and within seconds strong changes 9 in the transcriptome can be observed that rely predominantly on transcription [1] . 10 However, mRNA abundance and protein output are frequently decoupled, and 11 transcripts that accumulate rapidly are not necessarily translated. Systematic 12 comparisons of mRNA and protein levels across eukaryotes have established that 13 transcript abundance explains only approximately 40-50% of the variation in protein 14 levels [2,3] . The remainder reflects additional regulatory processes, including 15 translational control, post-translational modifications, and protein degradation, that 16 collectively shape protein abundance and activity but remain less understood. 17 Ribosome profiling, which captures the positions of translating ribosomes at 18 genome-wide scale, has revealed extensive translational regulation that is uncoupled 19 from changes in mRNA abundance [4,5] . Mechanistic models further demonstrate how 20 translation initiation, elongation and ribosome traffic jointly determine protein 21 synthesis rates [6,7] . Moreover, translational activity is dynamically reprogrammed in 22 response to environmental cues and temporal changes in plant physiology [8,9] . 23 Translational control thus constitutes an independent regulatory axis through which 24 cells modulate their protein output, and understanding its molecular basis remains a 25 central challenge in cell and developmental biology. 26 27 One mechanism by which this regulation is encoded directly within mRNA 28 sequences involves upstream open reading frames (uORFs) located in the 5′ 29 untranslated region (UTR). During ribosome scanning, initiation at an uORF can 30 cause the ribosome to stall, terminate, or reinitiate at a downstream position, thereby 31 modulating translation of the primary coding sequence [10] . uORFs are prevalent 32 across eukaryotic genomes, occurring in the 5′UTRs of 30-50% of mammalian and 33 plant transcripts [11,12] . One of the best-studied examples of uORF-mediated 34 translational regulation is of _GCN4_, where (uORFs) inhibit translation under non 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 stress conditions but allow increased _GCN4_ translation during amino acid 2 starvation [13] . Inhibitory uORFs that operate through nascent peptide-mediated 3 ribosome stalling have also been described [14] . By contrast, uORFs that directly 4 activate translation of a downstream ORF are exceedingly rare, and no clear 5 mechanistic paradigm for an activating uORF has been established in plants or 6 animals [12,15] . Consequently, uORF-encoded peptides as a class of functional 7 regulators that promote rather than inhibit downstream expression remain almost 8 entirely uncharacterised [16] . 9 10 Plants must continuously monitor their light environment and adjust development 11 accordingly, as light is both their primary energy source and a key positional signal. 12 Among the photoreceptors that mediate light perception, phytochrome B (phyB) 13 functions as a master developmental switch. The phyB protein is activated by red 14 light, which converts it to its active Pfr form, and inactivated by far-red light, reverting 15 phyB to its inactive Pr form [17,18] . Under conditions in which the ratio of red to far-red 16 light is reduced, as occurs when plants grow close to competitors or beneath a plant 17 canopy, active phyB levels fall and trigger the shade avoidance syndrome, a 18 coordinated developmental programme that includes hypocotyl elongation, altered 19 leaf morphology, and accelerated flowering [19] . These diverse developmental outputs 20 place phyB protein abundance at the centre of multiple critical life history decisions. 21 Notably, _PHYB_ mRNA is constitutively expressed and does not vary markedly with 22 light conditions, yet phyB protein abundance is tightly regulated across 23 developmental stages and in response to the light environment [20,21] . This discordance 24 between stable transcript levels and regulated protein output suggests that post 25 transcriptional mechanisms contribute substantially to governing phyB expression. 26 27 Despite decades of investigation, the mechanisms controlling phyB protein levels 28 have been studied almost exclusively at the transcriptional and post-translational 29 levels. At the post-translational level, phyB abundance and activity are regulated 30 through light-dependent nuclear accumulation, interactions with PHYTOCHROME 31 INTERACTING FACTOR (PIF) transcription factors, and ubiquitin-mediated 32 degradation [21,22,23] . Whether translational control contributes to the regulation of phyB 33 protein expression has not been examined. Ribosome profiling has recently been 34 applied in plants and revealed a complex translational landscape that includes 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 numerous small ORFs in 5′UTRs without known function [8,24,25] . Whether any of these 2 novel translated elements regulate the expression of major developmental regulators 3 has remained an open question. 4 5 Here, we identify a translated upstream ORF in the _PHYB_ 5′UTR, which we name 6 _PHYTOCHROME REGULATOR_ ( _PHYRE_ ), through ribosome profiling of Arabidopsis 7 seedlings subjected to shade. Unlike the predominant class of inhibitory uORFs, 8 _PHYRE_ translation is required to promote phyB protein expression. Loss of _PHYRE_ 9 function phenocopies loss of phyB across multiple developmental contexts, including 10 seed germination, hypocotyl elongation, leaf development, and flowering time. 11 Genetic complementation and reporter analyses demonstrate that _PHYRE_ activity 12 depends on its encoded peptide sequence and that the spacing between the _PHYRE_ 13 stop codon and the _PHYB_ start codon is a critical determinant of downstream 14 translation efficiency. _PHYRE_ -like elements are conserved across angiosperms, 15 suggesting that translational activation of _PHYB_ by an upstream ORF is a broadly 16 conserved regulatory mechanism. Together, these findings identify _PHYRE_ as one of 17 very few characterised activating uORFs in any eukaryote and establish uORF 18 mediated translational gating as a new regulatory layer controlling a master 19 photoreceptor. 20 # 21 RESULTS 22 Despite their prevalence across eukaryotic genomes, short open reading frames 23 (sORFs) remain a largely unexplored layer of translational regulation. To 24 systematically identify translated sORFs in Arabidopsis seedlings exposed to light 25 and shade, we performed ribosome profiling on 7-day-old seedlings grown under 26 white light and subjected to 45 or 90 min of deep shade (Fig. 1A; see Methods). 27 Integrating all nine Ribo-seq datasets, we predicted translated non-canonical sORFs 28 using RibORF v.2.0 [26] (Figs. 1B, S1; see also Methods). This analysis identified 2,856 29 novel translated sORFs, comprising 2,393 uORFs, 366 ncRNA-associated ORFs, 30 and 97 downstream ORFs (Fig. 1C, Supplementary Table 1). Filtering for high 31 confidence candidates with greater than 50% read periodicity reduced this set to 208 32 sORFs, including 184 uORFs, 21 ncRNA ORFs, and 3 dORFs, which were 33 prioritised for further analysis (Fig. 1C, Supplementary Table 2). 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Among the high-confidence sORFs associated with photomorphogenesis genes, one 2 candidate immediately drew our attention. An uORF residing in the 5′UTR of _PHYB_, 3 the gene encoding the master red-light photoreceptor, was translated under both 4 white light and shade conditions (Figs. 1D, S1C). We named this uORF 5 _PHYTOCHROME REGULATOR_ ( _PHYRE_ ). The _PHYRE_ stop codon lies only 35 bp 6 upstream of the _PHYB_ start codon (Fig. S1D), placing it in unusually close proximity 7 to the downstream ORF and prompting us to investigate whether _PHYRE_ translation 8 regulates _PHYB_ expression. 9 10 To uncover the role of _PHYRE_, we generated a series of frameshift mutants, _phyre-_ 11 _cr,_ using a modular cloning-based CRISPR-Cas9 system [27] . These _phyre-cr_ mutants 12 either carried a single base insertion or 1-2 base deletion in the _PHYRE_ coding 13 sequence, resulting in frameshifts that abolished the native stop codon and led either 14 to premature termination or translational read-through into _PHYB_ (Fig. 1E; S2A). In 15 addition, we generated two _phyB-cr_ alleles carrying large deletions in the _PHYB_ 16 locus that disrupted both _PHYRE_ and _PHYB_ ORFs (Fig. 1E). We analysed the 17 hypocotyl lengths in white light and deep shade regimes to assess the shade 18 avoidance response of _phyB-cr_ and _phyre-cr_ mutants. Both _phyB-cr_ mutants 19 exhibited elongated hypocotyls under white light and hypocotyl elongation 20 comparable to the wild type under deep shade (Fig. 1F, H), similar to previously 21 reported _phyB_ loss-of-function mutants [28,29] . Strikingly, all five independent _phyre-cr_ 22 mutants displayed elongated hypocotyls under white light indistinguishable from 23 those of _phyB-cr_ mutants (Figs. 1F, 1H; S2B), establishing that _PHYRE_ is required to 24 suppress hypocotyl growth under these conditions. In contrast, _phyre-cr_ mutants had 25 about 30% shorter-than-wild-type hypocotyls under deep shade (Fig. 1F, H; S2C), 26 indicating _PHYRE_ may promote growth specifically in shade. These results suggest 27 that _PHYRE_ has opposite roles in regulating hypocotyl growth. In white light it 28 suppresses growth like _PHYB_ and in deep shade conditions it promotes growth. 29 30 Because phyB accumulates in dormant seeds and promotes germination in 31 darkness [30], we next asked whether _PHYRE_ also contributes to this developmental 32 response. We therefore quantified germination efficiency of _phyre-cr_ seeds in 33 darkness following a 15-min pulse of red light, which activates phyB, or far-red light, 34 which inactivates it (Fig. 2A). Following red-light treatment, germination efficiency in 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 _phyre-cr_ mutants was severely reduced (8–10%), closely resembling _phyB-cr1_ (5%) 2 and contrasting sharply with wild-type seeds (75%) (Fig. 2B,E), consistent with the 3 germination defects previously reported for phyB loss-of-function mutants [30,31] . 4 Following far-red light treatment, germination remained strongly impaired in _phyre-cr_ 5 and _phyB-cr_ mutants (0.5-1.5%), in contrast to wild-type seeds, which reached 15% 6 germination (Fig. 2C,E). Under white light, germination efficiency exceeded 95% in 7 both _phyre-cr_ and _phyB-cr_ mutants, indistinguishable from wild type (Fig. 2D,E). 8 Together, these results demonstrate that _PHYRE_ is required for efficient seed 9 germination under conditions that depend on phyB activity. 10 11 To investigate the impact of _PHYRE_ at later developmental stages, we examined 12 rosette development and flowering time in _phyB-cr_ and _phyre-cr_ mutants grown 13 under long-day white-light conditions. Both _phyre-cr1_ and _phyre-cr2_ displayed 14 elongated petioles and narrower leaves resembling _phyB-cr1_, although the 15 phenotype of _phyre-cr2_ was intermediate between _phyB-cr1_ and wild type (Fig. 2F). 16 Consistent with _phyB-cr1_, _phyre-cr1_ exhibited early flowering, whereas _phyre-cr2_ 17 displayed a more variable flowering-time phenotype that overall remained closer to 18 the wild type (Fig. 2G). Taken together, these findings indicate that _phyre-cr1_ and 19 _phyre-cr2_ represent strong and weak alleles of _PHYRE_, respectively. 20 21 The frameshift mutation in _phyre-cr1_ disrupts the normal _PHYRE_ reading frame, 22 causing translation to continue into the _PHYB_ locus in an alternative frame. 23 Translation terminates after only 31 amino acids because of a premature stop codon, 24 thereby preventing production of any peptide similar to phyB (Fig. S3A). The single 25 nucleotide insertion in _phyre-cr2_ increases the spacing between _PHYRE_ and _PHYB_ 26 to 36 nt. This eliminates the native _PHYRE_ stop codon, thereby enabling complete 27 translational read-through into the _PHYB_ ORF and potentially producing an extended 28 phyB protein containing additional extra N-terminal peptide. Given these distinct 29 molecular outcomes, we directly tested whether phyB protein abundance is affected 30 in the two mutants. 31 32 We first quantified phyB protein levels in 7-day-old seedlings by western blotting with 33 an anti-phyB antibody. phyB protein was undetectable in _phyre-cr1_, _phyB-9_ and 34 _phyB-cr1_, whereas _phyre-cr2_ seedlings had reduced levels of phyB under both 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 white-light and shade conditions compared with the wild type (Figs. 3A, S3B). To 2 investigate whether the lower phyB abundance in _phyre-cr_ lines could be due to 3 reduced mRNA levels, we determined _PHYB_ transcript levels in 7-day old white light 4 grown seedlings. RT-qPCR analysis with primers targeting N and C-terminus of 5 _PHYB_ (Fig. S3C) revealed that the mRNA level was reduced by 50% in the _phyre-cr1_ 6 mutant, possibly due to non-sense mediated decay [32], but remained unchanged in 7 _phyre-cr2_ (Fig. 3B). As expected, the _PHYB_ transcript was not detected in _phyB-cr1_ . 8 The transcript level of _PIL1_, a known gene target repressed by phyB [33], was 9 increased 10-fold in _phyre-cr1_, similar to _phyB-cr1_, and 2.5-fold in _phyre-cr2_ . The 10 reduction in _PHYB_ transcript levels in _phyre-cr1_ is insufficient to account for the near 11 complete loss of phyB protein and the strong shade-avoidance phenotype, 12 establishing that _PHYRE_ regulates phyB abundance primarily at the translational 13 level. 14 15 To test if _PHYRE_ translation is required for phyB expression and shade avoidance, 16 we complemented the _phyB-cr1_ mutant with _PHYB_ genomic fragments expressing 17 either 3x-FLAG-tagged wild-type _PHYRE_ or a translation-deficient _PHYRE_ variant in 18 which the start codons were mutated, together with 2x-Myc-tagged phyB under the 19 endogenous promoter (Fig. 3C). Two independent lines carrying the wild-type 20 construct ( _PHYBg_WT_ ) fully rescued the hypocotyl elongation phenotype of _phyB-_ 21 _cr1_ (Fig. 3D), demonstrating that the transgene was functional. However, two 22 independent lines carrying the translation deficient _PHYRE_ genomic fragment 23 ( _PHYBg_mut_ ) failed to restore the hypocotyl length of _phyB-cr1_ mutants to wild-type 24 level. Consistent with these phenotypes, phyB protein was restored to wild-type 25 levels in _PHYBg_WT_ lines but remained undetectable in _PHYBg_mut_ lines (Fig. 3E). 26 As _PHYB_ transcript abundance was restored to wild-type levels in both _PHYBg_WT_ 27 and _PHYBg_mut_ lines (Fig. 3F), the failure of _PHYBg_mut_ to express phyB protein 28 cannot be attributed to reduced transcript levels, further demonstrating that _PHYRE_ 29 acts at the translational level. _PIL1_ expression was lower in _PHYBg_WT_ lines (3–4 30 fold above Col-0, compared with ~20-fold in _phyB-cr1_ ) but remained elevated in 31 _PHYBg_mut_ lines (12–15-fold above Col-0). The petiole and leaf blade phenotypes 32 of _PHYBg_WT_ plants were also similar to those of wild-type plants, whereas 33 _PHYBg_mut_ plants largely resembled _phyB-cr1_ (Fig. S4A). Similarly, flowering time 34 was restored in _PHYBg_WT_ plants, whereas _PHYBg_mut_ plants flowered early, as 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 did _phyB-cr1_ (Fig. S4B). Together, these results establish that _PHYRE_ translation is 2 both necessary and sufficient for phyB protein expression and for normal 3 photomorphogenesis across multiple developmental contexts, demonstrating that 4 translational rather than transcriptional control is the critical regulatory step. 5 6 The absolute dependence of phyB protein expression on _PHYRE_ translation raised 7 the question of whether this translational control is intrinsic to the _PHYB_ 5′UTR or 8 specific to the _PHYB_ ORF. To distinguish these possibilities, we replaced the _PHYB_ 9 coding sequence with _GUS_, placing it under the control of either a wild-type _PHYRE_ 10 containing 5′UTR ( _pPHYB::GUS_ ) or a start-codon-mutated version 11 ( _mutpPHYB::GUS_ ) driven by the native _PHYB_ promoter. GUS activity was 12 undetectable in seedlings carrying _mutpPHYB::GUS_ compared with _pPHYB::GUS_ 13 controls (Fig. 4A,B), demonstrating that _PHYRE_ -dependent translational activation is 14 a general property of the _PHYB_ 5′UTR and not specific to the _PHYB_ coding 15 sequence. 16 17 uORFs regulate downstream translation through three distinct mechanisms: 18 nucleotide sequence, nascent peptide identity, and intercistronic distance [34,35,36] . To 19 distinguish whether the regulatory activity of _PHYRE_ resides in its RNA sequence or 20 encoded peptide, we measured firefly luciferase activity from reporter constructs 21 carrying _PHYB 5′UTRs_ with frameshift, scrambled, or synonymous mutations in 22 _PHYRE_ (Fig. 4C). Scrambling the _PHYRE_ sequence altered the encoded amino acid 23 sequence whereas synonymous mutations altered the nucleotide sequence without 24 changing the encoded peptide sequence. The constructs were driven by the native 25 Arabidopsis _PHYB_ promoter and transiently expressed in tobacco leaves. Relative to 26 the wild-type _PHYRE_ control, a frameshift identical to that in _phyre-cr1_ caused a 27 marked reduction in luciferase activity, while scrambling the PHYRE peptide 28 sequence resulted in a partial reduction (Fig. 4E). Synonymous recoding, which 29 preserved the wild-type peptide while altering the RNA sequence, had no effect on 30 luciferase activity. Together, these results indicate that the PHYRE peptide 31 sequence, rather than the RNA sequence, contributes to downstream translational 32 activation. 33 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 To disentangle the effects of spacer sequence and length on downstream ORF 2 expression, we scrambled the native 35-bp region separating the _PHYRE_ stop codon 3 from the downstream _PHYB_ start codon and generated longer spacers of 140 and 4 280 bp by tandemly repeating the scrambled sequence, ensuring that no additional 5 ORFs were introduced (Fig. 4D). We also generated a construct with a 15 bp spacer 6 matching the shorter intercistronic distance found in _Arabidopsis lyrata_, whose 7 _PHYRE_ sequence is otherwise identical to that of _Arabidopsis thaliana_ . The reporter 8 carrying the scrambled 35 bp spacer exhibited luciferase activity comparable to that 9 of the wild-type control (Fig. 4F), indicating that spacer sequence has little influence 10 on downstream ORF expression. In contrast, shortening the spacer to 15 bp or 11 extending it to 280 bp significantly reduced luciferase activity. Interestingly, extending 12 the spacer to 140 bp did not affect luciferase activity, which remained comparable to 13 that of the 35-bp spacer control. Together, these findings establish that intercistronic 14 distance is a key determinant of _PHYRE_ -dependent translational activation, pointing 15 to a position-sensitive mechanism linking uORF translation to downstream ORF 16 expression. 17 # 18 DISCUSSION 19 _PHYRE_ defines one of the few characterised activating uORFs, and our results 20 establish its translation as an essential prerequisite for phyB protein expression and 21 normal photomorphogenesis across multiple developmental contexts (Fig. 1-3). 22 Disrupting _PHYRE_ translation through either a frameshift or start codon mutation 23 eliminates phyB protein expression without substantially affecting _PHYB_ mRNA 24 levels (Fig. 3), suggesting that _PHYRE_ acts primarily at the translational level. We 25 propose that translation of _PHYRE_ is required for translation of the downstream 26 _PHYB_ ORF (Fig. 5). The spacer experiments indicate that efficient _PHYB_ translation 27 depends on the distance between the _PHYRE_ stop codon and the _PHYB_ start 28 codon, with both shorter and longer spacers reducing downstream translation (Fig. 29 4). These findings support a model in which translation of _PHYRE_ facilitates 30 reinitiation at the downstream _PHYB_ start codon, with the efficiency of this process 31 depending on the intervening distance. 32 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Whether position-dependent uORF-mediated regulation of _PHYB_ translation is 2 evolutionarily conserved is an important question. A survey of the 150 bp genomic 3 region upstream of the _PHYB_ coding sequence across 49 land plant species 4 revealed short proximal uORFs of 5–20 aa across eudicots, while monocot and 5 gymnosperm representatives lacked such elements entirely (Fig. S5). In 6 _Brassicaceae_, a proximal uORF is maintained at a similar relative position across 7 major genera from _Eutrema_ to _Brassica_, consistent with functional constraint on 8 uORF position. However, peptide-level sequence conservation is restricted to 9 species closely related to _Arabidopsis thaliana_, suggesting that across eudicots more 10 broadly, the act of uORF translation rather than the identity of the encoded peptide 11 may be the primary conserved regulatory feature. Whether divergent PHYRE 12 peptides share a conserved three-dimensional structure that underlies their function 13 across species is an intriguing question that warrants further investigation. Together, 14 these observations are consistent with a model in which uORF-mediated 15 translational activation of _PHYB_ is a eudicot innovation, with peptide-level 16 contributions to this regulation arising more recently within the _Brassicaceae_ . 17 18 The partial reduction in luciferase activity observed with a scrambled PHYRE peptide 19 sequence (Fig. 4) supports the idea that peptide identity contributes to the efficiency 20 of downstream translational activation in _Arabidopsis thaliana_, consistent with the 21 more restricted conservation of the PHYRE peptide sequence within the 22 _Brassicaceae_ . Previous studies have shown that uORF-encoded peptides can 23 regulate downstream ORF translation in genes involved in metabolic processes, 24 including _bZIP11_ and _Polyamine Oxidase2_ [37,38] . A well-characterised precedent is the 25 sucrose control peptide encoded by a uORF in the _bZIP11_ 5′UTR, which induces 26 ribosome stalling under high-sucrose conditions to inhibit downstream translation. 27 _PHYRE_ represents a mechanistic inversion of this paradigm, promoting rather than 28 inhibiting downstream translation, yet whether this activity is mediated through direct 29 peptide-ribosome interactions during translation or termination, or simply through the 30 act of uORF translation itself, remains to be established. 31 32 An intriguing observation is that _phyre-cr_ mutants consistently exhibited shorter 33 hypocotyls than wild-type plants under deep shade, a condition under which phyB 34 activity is strongly reduced and _phyre-cr_ mutants are therefore expected to 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 phenocopy _phyB-cr_ lines (Fig. 1). This difference raises the possibility that the 2 PHYRE peptide has an additional role in regulating hypocotyl elongation under 3 shade, independent of its function in promoting phyB translation. 4 5 Why would plants evolve _PHYRE_ -mediated translational control of a constitutively 6 transcribed photoreceptor? phyB, along with phyA, contribute to shade perception 7 and the regulation of growth under changing light conditions, with phyB playing a 8 prominent role in sensing changes in the R:FR ratio [39,40] . Moreover, phyB acts as 9 both a photoreceptor and a thermosensor, integrating light and temperature signals 10 to regulate plant growth and development [41,42] . Recent evidence indicates that phyB 11 abundance influences photobody formation and Phytochrome Interacting Factor 12 (PIF) activity, thereby shaping downstream signalling [43] . Thus, _PHYRE_ -mediated 13 translational control provides an additional mechanism for tuning phyB abundance 14 as environmental conditions change. The detection of _PHYRE_ translation under both 15 white light and shade indicates that this mechanism operates continuously across 16 distinct light regimes, positioning it to act as a constitutive translational amplifier of 17 phyB output. Such regulation is likely to be particularly important when rapid shifts in 18 light quality or temperature alter the balance between active and inactive phyB, 19 requiring proportionate adjustments in photoreceptor abundance that cannot be 20 achieved through transcriptional regulation of a constitutively expressed gene. 21 # 22 MATERIALS AND METHODS 23 **Plant materials and growth conditions** 24 _Arabidopsis thaliana_ ecotype Columbia-0 (Col-0) was used as the wild-type control. 25 All the lines used in this study including _phyB-9;ven4+/+_ (Reed et al. [44] ; Gift from Dr. 26 Andreas Hiltbrunner, University of Freiburg) are in Col-0 genetic background. Seeds 27 were surface sterilized using sodium hypochlorite (1.4%) in 70% ethanol containing 28 0.001% TritonX-100 for 2-3 minutes. Surface sterilized seeds were spotted on plates 29 with 0.5x MS medium including vitamins (Duchefa Biochemie M0222) supplemented 30 with 0.1% Sucrose (Duchefa Biochemie S0809), and 0.8% agar (Duchefa Biochemie 31 P1001). Seeds were stratified for 3 days in dark at 4°C and shifted to Percival LED 32 growth chamber (Model LED-30SB10) for seed germination, hypocotyl growth, and 33 ribosome profiling experiments. 34 35 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 **DNA constructs** 2 Genome editing constructs for _phyB-cr_ and _phyre-cr_ generation were assembled using 3 the modular cloning (MoClo) approach described in Grutzner et al. [45] ; Stuttmann et al. [27] 4 Briefly, sgRNAs targeting _PHYB_ or _PHYRE_ were designed using CRISPR-P 2.0 5 (http://crispr.hzau.edu.cn/CRISPR2/) and cloned individually in shuttle vectors to 6 generate sgRNA TUs. Multiple sgRNA TUs were assembled in pDGE347 recipient 7 binary vector containing zCas9i and FAST selection cassettes (zCas9i cloning kit, 8 Addgene Kit # 1000000171). Protocols for MoClo assembly reactions were followed 9 exactly as described in zCas9i cloning manual v5 from Addgene Kit # 1000000171. 10 _PHYBg_WT_ and _PHYBg_mut_ complementation constructs were assembled using 11 NEBuilder ® HiFi DNA Assembly Reaction Protocol (NEB #E2621) according to 12 manufacturer’s instructions. Briefly, synthetic fragments containing C-term 3x-FLAG 13 tagged wild-type or start-codons mutated version of _PHYRE_ and N-term 2x-Myc 14 tagged _PHYB_ region (Twist Bioscience) was assembled with gel purified PCR 15 fragments spanning 7.2 kb _PHYB_ intergenic region into pFAST-R07 16 (https://vectorvault.vib.be/collection/pfast-r07) binary vector containing FAST cassette 17 for seed coat RFP selection. 18 For generating _pPHYB::GUS_ and _mutPHYB::GUS_ constructs, 2 kb _PHYB_ promoter 19 was assembled along with 300 bp 5’UTR containing wild-type or start-codons mutated 20 version of _PHYRE_ and _GUS_ coding sequence into the gateway multisite destination 21 vector pH7m34GW (https://vectorvault.vib.be/collection/ph7m34gw) using Gateway 22 LR Clonase II enzyme mix (Invitrogen). 23 Dual luciferase reporter constructs were generated by GreenGate cloning system. 24 _PHYB_ 5’UTR variants were synthesized from Twist bioscience and assembled along 25 with 2 kb _PHYB_ promoter, firefly luciferase coding sequence and p35S renilla 26 luciferase modules into the binary destination vector pGGZ003 (GreenGate Cloning 27 System, Addgene Kit # 1000000036). Protocols for GreenGate assembly reactions 28 were set up as described in Lampropoulos et al. [46] Gene fragments were assembled 29 in single cut/ligation reactions containing 1µl of FastDigest Eco31I (Thermo Scientific 30 FD0293), 15 U of T4 DNA Ligase, HC (Thermo Scientific EL0013) and 1mM ATP 31 (Thermo Scientific R0441). 32 Genome editing, complementation, and GUS constructs were transformed into 33 _Agrobacterium tumefaciens_ GV3101, while dual luciferase constructs were 34 transformed into _Agrobacterium tumefaciens_ GV3101 (pSoup) strain. Colonies were 35 screened for insert by PCR before Col-0 transformation. Oligos used for cloning are 36 included in the Supplementary table 3. 37 38 **Plant transformation and line establishment** 39 _Agrobacterium tumefaciens_ carrying DNA constructs were transformed into 4 week old 40 _Arabidopsis thaliana_ Col-0 plants by floral dipping as described in Clough and 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Bent [47] .The transformants were screened for seed coat RFP fluorescence under Leica 2 MZ FLIII fluorescence stereomicroscope fitted with CoolLED pE-300 [lite] illumination 3 system. For _phyB-cr_ and _phyre-cr_ line establishment, RFP negatives were selected 4 from T2 segregating population and genotyped for deletions and point mutations by 5 PCR, followed by sanger sequencing. Positive T2 plants were selfed to establish T3 6 homozygous lines. T3 lines were used for all the experiments described in this study. 7 For _PHYBg_WT_ and _PHYBg_mut_ complementation, T1 RFP positive plants were 8 selected and transgene insertion was confirmed by PCR. T2 segregating population 9 obtained from T1 RFP positive plants was used for qPCR and western blotting 10 experiments. For hypocotyl assays under white light and deep shade, RFP positives 11 (homozygous/heterozygous for transgene) were selected from T2 segregating 12 population and spotted on 0.5X MS plates. For GUS assays, hygromycin resistant T1 13 plants were selected on 0.5X MS agar plates supplemented with 25µg/ml hygromycin 14 B (Duchefa Biochemie H0192) and 100 µg/ml cefotaxime sodium (Duchefa Biochemie 15 C0111). Insertion of GUS-transgene in hygromycin resistant T1 plants was verified by 16 genotyping PCR. T2 segregating population obtained from T1 positive plants was used 17 for GUS assay. All genotyping PCR primers are listed in Supplementary table 3. 18 19 **Hypocotyl measurements** 20 Following 3 days of stratification in dark at 4°C, plates were shifted to Percival LED 21 growth chamber (Model LED-30SB10) set under white light condition 22 (WL; PAR- 6.5 µmolm [-2] s [-1] ; R:FR= 12.1; 16-h light/8-h dark photoperiod) at 22°C, 65% 23 relative humidity for 2 days for germination and establishment. Seedlings were grown 24 further for 4 days under white light or deep shade (WL+ FR; PAR- 6.5 µmolm [-2] s [-1] ; 25 R:FR= 0.15; 16-h light/8-h dark photoperiod) conditions at 22°C and 65% relative 26 humidity. 6-day old seedlings were imaged using Epson scanner expression 12000XL 27 equipped with SilverFast9 (Laser Software Imaging). Hypocotyls were measured 28 using segmented line tool from ImageJ v1.54t software. Representatives images were 29 compiled by removing background using online background remover tool from Canva 30 and performing brightness/contrast adjustments using in-built Microsoft Powerpoint 31 function. 32 33 **Seed germination** 34 Seed germination assays were performed as described in Gu et al. [31] with minor 35 modifications. Following 3 days of stratification in dark at 4°C, plates were placed in 36 Percival LED growth chamber (Model LED-30SB10) under white light 37 (PAR- 100 µmolm [-2] s [-1] ; R:FR= 12.1) for 1 hour at 22°C, 65% relative humidity, followed 38 by 15 min of monochromatic red light (35 µmolm [-2] s [-1] ) or far-red light (38 µmolm [-2] s [-1] ) 39 treatment. After light treatment, plates were maintained at 22°C in darkness for 4 days 40 before germination rates were calculated. 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 2 **Flowering time** 3 6 day-old white light grown seedlings (PAR- 6.5 µmolm [-2] s [-1] ; R:FR= 12.1; 16-h light/84 h dark photoperiod) were transferred to pots containing a 3:1 soil:vermiculite mixture 5 (Hasselfors garden specialjord; Sibelco vermiculite) supplemented with 1% SW 6 Bouyant RIKA S 7-1-5 fertilizer and nematode mixture (NEMAblom; BioNEMA). Pots 7 were maintained in invitro growth rooms under white light irradiation (PAR- 180 8 µmolm [-2] s [-1] ; 16-h light/8-h dark photoperiod) at 22°C, 65% relative humidity until 9 bolting. Flowering time was determined by counting the number of rosette leaves when 10 the bolt reached a height of 5 cm. 11 12 **Ribosome profiling and library preparation** 13 Seven-day-old white light (WL; PAR- 6.5 µmolm [-2] s [-1] ; R:FR= 12.1; 16-h light/8-h dark 14 photoperiod) grown seedlings were subjected to deep-shade treatment (WL+ FR; 15 PAR- 6.5 µmolm [-2] s [-1] ; R:FR= 0.15) for 45 or 90 min, while control seedlings were 16 maintained under white light for an equivalent 90-min period. All samples were 17 harvested simultaneously at the end of the 90-min experimental period and 18 immediately flash-frozen in liquid nitrogen. Frozen tissue was ground to a fine powder 19 in liquid nitrogen, and ribosome-protected fragments (RPFs) were isolated and 20 processed for ribosome profiling (Ribo-seq) as described in Mahboubi et al [48] . Briefly, 21 powdered tissue was extracted in ribosome extraction buffer containing 22 cycloheximide, and the lysates were digested with RNase I to remove unprotected 23 RNA. RPFs were purified by TRIzol extraction and isopropanol precipitation, size24 selected (27–31 nt) by denaturing polyacrylamide gel electrophoresis, and used for 25 library preparation. 26 Libraries were constructed by 3′ linker ligation, reverse transcription, cDNA 27 circularization, rRNA depletion, and PCR amplification as described in Mahboubi et 28 al [48] . Purified libraries were quantified and subjected to single-end sequencing. 29 30 **Riboseq analysis** 31 Raw Ribo-seq libraries were processed independently. Adapter trimming and quality 32 control were performed separately for each library, followed by alignment to the 33 _Arabidopsis thaliana_ reference genome using STAR and the Araport11 annotation [49] . 34 P-site offsets were estimated independently for each library and read length using 35 the bioconductor package ribosomeProfilingQC 1.23.0 36 [(https://bioconductor.org/packages/release/bioc/html/ribosomeProfilingQC.html).](https://bioconductor.org/packages/release/bioc/html/ribosomeProfilingQC.html) 37 After offset correction, the alignments were pooled into a single coordinate-sorted 38 BAM file containing 126,198,671 primary mapped P-site alignments. 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Candidate ORFs were evaluated using RibORF v.2.0 [26] . We employed a sensitive 2 detection strategy (see Fig. S1) and considered ORFs with a RibORF score of at 3 least 0.6 to show evidence of translation. The final non-canonical ORF catalogue 4 was restricted to uORFs, dORFs and ORFs located in transcripts annotated as non5 coding (ncRNA ORFs). Candidates overlapping annotated coding sequences on the 6 same strand were excluded. Redundant predictions sharing the same chromosome, 7 strand and genomic ORF boundaries were collapsed. We identified 2,856 novel 8 translated sORFs using this procedure. 9 We applied a subsequent filter to focus on a set of 208 highly translated sORFs. 10 These sORFs had to be supported by more than 30 reads and show high signal 11 homogeneity along the ORF (more than 30% of codons covered by reads) and 12 display >50% periodicity. 13 14 **Western blotting** 15 Total protein was extracted from finely ground tissue of 7-day-old seedlings grown 16 under white light or deep shade using 100 µL of extraction buffer (50 mM Tris-HCl pH 17 7.5; 150mM NaCl; 1mM EDTA pH 8.0; 10%, glycerol; 2mM PMSF; 1% Triton X-100; 18 5 mM DTT; 1x cOmplete™ Protease Inhibitor Cocktail). Prior to separation on SDS 19 PAGE, extracts were quantified by Bradford assay and 60 µg of total protein was 20 resuspended in 1X loading dye (0.05% bromophenol blue, 0.1 M DTT, 2% glycerol, 21 2% SDS, 0.05M Tris-HCl pH 6.8) and incubated for 5 min at 95°C. Samples were 22 separated on 9% SDS-PAGE and blotted to PVDF membrane (Thermoscientific 23 88518; pore size 0.45 µm). Blot was blocked with 5% milk powder in TBS-T for 1 h/RT 24 with agitation. Blot was incubated in the primary antibody (Anti-PhyB, Agrisera AS21 25 4566) at a dilution of 1:1000 in TBS-T ON/4°C with agitation. The antibody solution 26 was decanted and the blot was rinsed with TBS-T briefly twice, then washed 3 times 27 for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary 28 antibody (anti-rabbit IgG horse radish peroxidase conjugated, Agrisera AS09 602) 29 diluted to 1:25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above, 30 developed for 5 min with ECL™ Western Blotting Reagents (Cytiva RPN2106) and 31 imaged using C600 Azure biosystems cSeries luminescence camera system under 32 auto exposure settings. Representative blot images were compiled after performing 33 brightness/contrast adjustments using in-built Microsoft Powerpoint function. 34 35 **RT-qPCR** 36 Total RNA was extracted from 100mg finely ground tissue of 7-days old white light 37 grown seedlings using RNAeasy Mini extraction kit (Qiagen 74104) according to 38 manufacturer’s protocol. 1µg of DNAse free RNA was converted to cDNA using iScript 39 cDNA synthesis kit (Biorad 1708891) according to manufacturer’s instructions. qPCR 40 was performed in Biorad CFX384 Touch Real-Time PCR Detection System using 41 1.2µL of cDNA reaction template with 0.5µM primers in 1x KAPA SYBR FAST qPCR 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Master Mix (KK4607). Fold change (2 [-ΔΔCq] ) in transcript levels was determined using 2 in-built analysis function of the Click-qPCR tool (Kubota and Tajima. [50] ). qPCR primers 3 are listed in Supplementary table 3. 4 5 **GUS assay** 6 Samples were incubated in GUS staining solution (50 mM sodium phosphate buffer 7 pH 7.2, 1 mM K 3 Fe(CN) 6, 1 mM K 4 Fe(CN) 6, 0.1% Triton X-100, 1 mM X-gluc) for 4h at 8 37°C with gentle shaking in the dark. Following staining, samples were fixed in FAA 9 (50% ethanol, 5% glacial acetic acid, 5% formaldehyde) for 10 min at room 10 temperature and decolorized in 100% ethanol with gentle shaking, replacing the 11 ethanol as needed until chlorophyll was removed. Samples were then rehydrated in 12 100%, 90%, and 80% ethanol for 5 min each, incubated in clearing solution I (20% 13 methanol and 0.24 M HCl) for 15 min at 57°C, and transferred to clearing solution II 14 (7% NaOH in 60% ethanol) for 15 min at room temperature. Samples were 15 subsequently incubated in 50% ethanol overnight and stored in 70% ethanol until 16 imaging. Before imaging, samples were rehydrated in 40%, 20%, and 10% ethanol for 17 5 min each. GUS-stained seedlings were imaged using Leica M205 FCA fluorescence 18 stereo microscope equipped with Leica DFC9000 GT camera and LAS X image 19 analysis software. Images were processed by removing background and adjusting 20 brightness/contrast using in-built Microsoft Powerpoint function. 21 22 **Dual Luciferase assay** 23 The reporter constructs were transformed into _Agrobacterium tumefaciens_ strain 24 GV3101(pSoup). A single positive colony was cultured overnight in 5 mL LB medium 25 containing the appropriate antibiotics at 28°C. The culture was diluted 1:100 into 50 26 mL fresh LB medium supplemented with antibiotics and grown overnight at 28°C. Cells 27 were resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES, pH 5.6, and 150 28 µM acetosyringone) to an OD₆₀₀ of 0.8 and incubated in the dark for 2 h. Fully 29 expanded leaves of 4-5-week-old _Nicotiana benthamiana_ plants were infiltrated using 30 a needleless syringe. For each construct, two leaves from each of two plants (four 31 leaves total) were infiltrated. Plants were maintained at 22°C under a 16-h light/8-h 32 dark photoperiod, with low light and covering for the first 24 h after infiltration. After 33 72 h, twelve leaf discs (5 mm diameter) were collected from the four infiltrated leaves 34 per construct and stored individually in liquid nitrogen. Non-infiltrated leaf discs were 35 collected as controls for background luminescence. 36 Each leaf disc was ground individually in liquid nitrogen, and 100 µL of 1x Passive 37 Lysis Buffer (Promega) was added. Lysates were incubated at room temperature for 38 15 min and centrifuged at 16,000 x g for 20 min at 4°C. Firefly and Renilla luciferase 39 activities were measured using the Dual-Luciferase Reporter Assay System 40 (Promega, E1960) according to the manufacturer's instructions. For each sample, 10 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 µL of lysate was assayed in three technical replicates using a GloMax Navigator 2 luminometer (Promega, Madison, WI, USA) with default settings. Background 3 luminescence from non-infiltrated leaf discs was subtracted, and relative promoter 4 activity was calculated as the ratio of firefly (LUC) to Renilla (REN) luciferase activity. 5 The experiment was repeated twice. 6 7 **PHYB 5’UTR survey** 8 Genomic sequences 150 nucleotides immediately upstream of the _PHYB_ translation 9 start site were retrieved for 49 land plant species spanning gymnosperms, monocots, 10 basal eudicots, asterids, and rosids, with emphasis on _Brassicaceae_ . For each 11 species, a RefSeq mRNA accession for _PHYB_ was used as the entry point: the 12 corresponding NCBI Gene record was retrieved via the Entrez API (Biopython 1.8x), 13 and the chromosomal accession and gene coordinates were obtained from the Gene 14 esummary. The annotated gene locus plus 350 bp of upstream padding was then 15 fetched from the chromosomal record, the _PHYB_ coding sequence (CDS) feature was 16 identified by keyword matching of gene/product annotations (keywords: phyb, 17 phytochrome b, phytochrome-b and variants), and exactly 150 bp immediately 18 upstream of the CDS ATG was extracted, with strand orientation handled by reverse19 complementing minus-strand loci. For species where the genomic CDS feature could 20 not be resolved automatically, the analysis fell back to the annotated 5′ UTR of the 21 mRNA record. The 150 bp window was scanned in-frame for all ATG codons followed 22 by in-frame stop codons (TAA, TAG, TGA) within the window; uORF positions are 23 reported as nucleotide offsets relative to the main PHYB ATG. A uORF was classified 24 as _PHYRE_ -like if it encoded 5-20 amino acids and its stop codon terminated ≤ 100 nt 25 upstream of the _PHYB_ ATG, matching the criteria of the _Arabidopsis thaliana_ _PHYRE_ 26 element (12 aa, gap of 38 nt). Tandem ATG pairs (two ATGs separated by ≤ 6 nt) were 27 also recorded. The Arabidopsis _PHYRE_ uORF position and sequence 28 (MMIHPKSFLVSR, ATG at −77, stop end at −39) were taken from TAIR. All scripts 29 were written in Python 3 using Biopython V.1.88. 30 31 **Data visualization and statistical analysis** 32 For hypocotyl, germination, flowering time and luciferase assays, plots and statistical 33 analysis were conducted using custom R-scripts (R version 4.4.1) in RStudio (Version 34 2026.09.0+174). For qPCR data, Click-qPCR tool (Kubota and Tajima. [50] ) was used to 35 generate bar plots and perform statistical analysis. Details about the statistical analysis 36 are as follows: 37 For hypocotyl measurements, flowering time, and luciferase assays, statistical 38 differences were assessed using one-way ANOVA followed by Tukey’s HSD test when 39 all groups satisfied the Shapiro-Wilk normality criterion (P > 0.05); otherwise, the 40 Kruskal-Wallis test followed by Dunn’s test with Benjamini-Hochberg adjustment was 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 used. 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It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 from Arabidopsis cell culture. Plant Methods _17_, 92. 2 https://doi.org/10.1186/s13007-021-00791-w. 3 49. Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, 4 P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA5 seq aligner. Bioinformatics _29_, 15–21. 6 https://doi.org/10.1093/bioinformatics/bts635. 7 50. Kubota, A., and Tajima, A. (2025). Click-qPCR: A Simple Tool for Interactive 8 qPCR Data Analysis. Bio. Protoc. _15_ . https://doi.org/10.21769/BioProtoc.5513. 9 10 # 11 FIGURE LEGENDS 12 **Figure 1. Ribo-seq analysis and hypocotyl phenotypes of** _**phyB**_ **and** _**phyre**_ **mutants.** **(A)** 13 Schematic of the Ribo-seq workflow. WL = white light, DS = deep shade. **(B)** Schematic of the 14 data analysis pipeline used to discover novel small ORFs. **(C)** Lollipop plot showing the 15 numbers of novel uORFs, non-coding RNA ORFs, and dORFs identified using sensitive and 16 strict analyses. The total number of p-sites determined by sensitive and strict analysis are 17 indicated above the plot. **(D)** Ribosome profile of _PHYRE_ and start of the _PHYB_ coding region. 18 Transcript gene models in blue are shown below the chromosome coordinates. Bars indicate 19 non-coding regions and rectangles indicate coding regions on the transcript models. The 20 signal corresponds to the P site of the codon being translated, and it is shown for all the 21 datasets taken together. **(E)** Schematic showing the coding sequence of the _PHYRE_ uORF in 22 the wild-type, _phyre-cr1_, and _phyre-cr2_ mutants (top). The start and the stop codons are in 23 bold. The deletion in _phyre-cr1_ is indicated by a red dashed line, and the insertion in _phyre-_ 24 _cr2_ is highlighted in green. The schematic of the _PHYB_ locus is shown in the middle, and the 25 _phyB-cr_ alleles are shown at the bottom. The promoter and downstream regulatory regions 26 are shown as black bars, 5' and 3' UTRs as grey boxes, and coding region as yellow boxes 27 separated by introns (black dotted lines). The _PHYRE_ uORF, _PHYB_ start codon, and stop 28 codon are indicated by a magenta rectangle, black arrowhead, and black asterisk, 29 respectively. The binding sites of sgRNAs 1-5, used to generate the _phyre-cr_ and _phyB-cr_ 30 mutants, are indicated by arrows. Deleted regions in the _phyB-cr_ mutants are indicated by red 31 dashed lines. **(F-H)** Representative images and the hypocotyl length quantification of 6-day32 old seedlings of the indicated genotypes grown under **(F)** long-day white light or **(G)** long-day 33 deep shade conditions. Scale bars = 5 mm. **(H)** Violin plots show the distribution of individual 34 observations, with overlaid box plots indicating the median and interquartile range. Different 35 letters indicate statistically significant differences among the genotypes (adjusted P < 0.05), 36 determined using the Kruskal-Wallis test followed by Dunn’s post hoc test with Benjamini37 Hochberg correction. n = 31-51 and 28-48 seedlings for white light and deep shade treatments 38 respectively. 39 40 **Figure 2. Seed germination, rosette, and flowering time phenotypes of** _**phyB-cr**_ **and** 41 _**phyre-cr**_ **mutants. (A)** Schematic of the light treatment used in the germination experiments. 42 Abbreviations- D = Dark, WL= white light, FR= far-red light, R= red light. **(B-D)** Representative 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 images of seeds/germinated seedlings of the indicated genotypes and light treatments. **(E)** 2 Bar plots showing the mean ± standard error (SE) of the percentage of germination, with 3 individual replicate measurements shown as points, for the indicated genotypes and light 4 treatments. Different letters above the bar plots indicate statistically significant differences 5 among genotypes (adjusted P < 0.05), determined using One-way ANOVA followed by Tukey’s 6 HSD (Honestly Significant Difference) post hoc test. N = 3 (32-75 seeds per biological 7 replicate). **(F)** Representative rosette images of 21 day-old white light-grown plants of 8 indicated genotypes. **(G)** Flowering time quantification indicated by the number of rosette 9 leaves at bolting in the corresponding genotypes on the X-axis. Violin plots show the 10 distribution of individual observations, with overlaid box plots indicating the median and 11 interquartile range. Different letters indicate statistically significant differences among 12 genotypes (adjusted P < 0.05), determined by Kruskal-Wallis test followed by Dunn's post hoc 13 test with Benjamini-Hochberg correction. n = 13-23 plants. 14 **Figure 3. PhyB protein and transcript levels in** _**phyB-cr**_ **,** _**phyre-cr**_ **mutants and** _**PHYB**_ 15 **complementation lines. (A) and (E)** Western blot images showing phyB protein levels (129 16 kDa) in the total protein extracts from 7-day white light-grown seedlings of the indicated 17 genotypes. Image of the corresponding ponceau stained blots (loading control) are shown 18 below. **(B) and (F)** Bar plots showing relative transcript levels of _PHYB_ (primer pair- 1 and 2) 19 and _PIL1_ transcripts in the genotypes indicated in the legend above the plot. Asterisks denote 20 statistical differences of the genotypes compared to wild-type as determined by One-way 21 ANOVA and Dunnett’s post hoc test. * p<0.05; ** p<0.01; *** p<0.001; ns p>0.05, nd. not 22 detected. Error bars denote SD of 3 biological replicates. Red dotted line indicates wild-type 23 transcript levels (normalized to 1). **(C)** Schematics of the wild-type ( _PHYBg_WT_ ) and start 24 codon mutated ( _PHYBg_mut_ ) versions of transgene constructs used for complementing _phyB-_ 25 _cr1_ mutant. The promoter and downstream regulatory regions are shown as black bars, 5' and 26 3' UTRs as grey boxes, and coding region as yellow boxes separated by introns (black dotted 27 lines). The _PHYRE_ uORF, _PHYB_ start codon, and stop codon are indicated by a magenta 28 rectangle, black arrowhead, and black asterisk, respectively. The C-term 3x-FLAG of _PHYRE_ 29 and the N-term 2x-Myc tag of _PHYB_ are indicated by green and purple rectangles, 30 respectively. **(D)** Representative images and the hypocotyl length quantification of 6-day-old 31 seedlings of the indicated genotypes grown under long-day white light conditions. Scale bar = 32 5 mm. Violin plots show the distribution of individual observations, with overlaid box plots 33 indicating the median and interquartile range. Different letters indicate statistically significant 34 differences among genotypes (adjusted P < 0.05), determined using Kruskal-Wallis test 35 followed by Dunn's post hoc test with Benjamini-Hochberg correction. n = 19-23 seedlings. 36 **Figure 4. Results of GUS and luciferase reporter assays for** _**PHYB**_ **5' UTR variants. (A-** 37 **B)** Representative images of GUS-stained 7-day-old seedlings from three independent lines 38 each of **(A)** _pPHYB::GUS_ (wild type) and **(B)** _mutpPHYB::GUS_ ( _PHYRE_ start codon mutant) 39 genotypes. Scale bar = 2 mm. **(C)** Schematic representation of the DNA and the amino acid 40 sequence of the _PHYRE_ variants used for lucifrase reporter assays in **(E)** . WT- wild type, 41 FS- frameshift, SCR- scrambled, SYNON- synonymous mutation. Deletion in FS is indicated 42 by red dashed line. Changes in coding sequence in SCR and SYNON constructs are indicated 43 in red. Changes in amino acid sequence in FS and SCR constructs are indicated in grey. **(D)** 44 Schematic of the spacer variants used for luciferase reporter assays in **(F)** . Magenta and 45 orange rectangles represent coding sequences of _PHYRE_ and firefly luciferase ( _LUC_ ) 46 respectively. **(E-F)** Quantification of relative firefly luciferase activity in tobacco leaf extracts 47 infiltrated with the indicated reporter constructs. Box plots indicate the median and interquartile 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 range (IQR), with whiskers extending to 1.5 × IQR. Individual points represent leaf discs. 2 Different letters above the violin plots indicate statistical differences among reporter constructs 3 (adjusted P < 0.05), determined using Kruskal-Wallis test followed by Dunn's post hoc test 4 with Benjamini-Hochberg correction. n= 12-24 leaf discs from 4-8 mature leaves. WT- wild 5 type, FS- frameshift, SCR- scrambled, SYNON- synonymous mutation. 6 **Figure 5: Proposed model for** _**PHYRE**_ **-mediated regulation of** _**PHYB**_ **translation.** Position7 dependent _PHYRE_ uORF translation maintains optimal phyB protein expression, possibly by 8 facilitating translation re-initiation at the _PHYB_ start codon. Impaired _PHYRE_ translation, for 9 example due to mutation of its start codon, results in reduced phyB protein levels, possibly 10 because ribosomes bypass _PHYRE_ and continue scanning, thereby reducing translation 11 initiation at the downstream _PHYB_ start codon. 12 **Figure S1. Results from the riboseq analysis. (A)** Periodicity of reads of different length 13 for the different experiments. The fraction of reads of a given read length (kmer) in the correct 14 frame, in the different experiments, is indicated as f0. A f0 value higher than 0.5, indicating 15 that the majority of reads are in the correct frame, was used as a cut-off to select a given read 16 length in an experiment. **(B)** Read length distribution. The plot shows the percentage of reads 17 of different length, considering only reads that passed the threshold indicated in **(A)** . **(C)** 18 Ribosome profile of _PHYRE_ and start of the _PHYB_ coding region under different conditions. 19 Transcript gene models in blue are shown below the chromosome coordinates. Bars indicate 20 non-coding regions and rectangles indicate coding regions on the transcript models. The 21 signal corresponds to the P site in the codon that is being translated. The accumulated signal 22 from the three replicates of each condition (white light, deep shade 45’ and deep shade 90’) 23 is shown. **(D)** Sequence information of 334 bp region of _PHYB_ gene including the annotated 24 5'UTR (underlined) and the start of the phyB coding sequence (highlighted in yellow). _PHYRE_ 25 coding sequence is in red and the 35 base spacer separating the _PHYRE_ stop codon and the 26 _PHYB_ start codon is in bold. 27 **Figure S2. Hypocotyl phenotypes and phyB protein levels in** _**phyB-cr**_ **and** _**phyre-cr**_ 28 **mutants. (A)** Schematic showing the sequence information of the _PHYRE_ uORF in the wild29 type, _phyre-cr3_, _phyre-cr4_, and _phyre-cr5_ mutants. The insertion in _phyre-cr3_ is highlighted in 30 green, and the deletions in _phyre-cr4_ and _phyre-cr5_ are indicated by blue highlighted red 31 dashed lines. **(B-C)** Representative images and hypocotyl length quantification of 6-day-old 32 seedlings of the indicated genotypes grown under **(B)** long-day white light or **(C)** long-day 33 deep shade. Scale bars = 5 mm. Violin plots show the distribution of individual observations, 34 with overlaid box plots indicating the median and interquartile range. Different letters indicate 35 statistically significant differences among genotypes (adjusted P < 0.05), as determined using 36 the Kruskal-Wallis test followed by Dunn’s post hoc test with Benjamini-Hochberg correction. 37 n = 9-20 seedlings in **(B)** and 14-20 seedlings in **(C)** . **Note:** The representative image and 38 violin plot data for Col-0 in **(B)** are reproduced from **Figure 3D**, as the genotypes in **(B)** were 39 analyzed together with Col-0 as the reference control in the same experiment. 40 **Figure S3.** _**PHYRE**_ **and** _**PHYB**_ **translation status in** _**phyre-cr**_ **mutants. (A)** Schematic 41 showing translation status of _PHYRE_ uORF and _PHYB_ main ORF in wild type, _phyre-cr1_ and 42 _phyre-cr2_ . Magenta and yellow rectangles represent the _PHYRE_ and _PHYB_ ORFs, 43 respectively. Start and stop codons are indicated above the rectangles, and the corresponding 44 amino acid sequences are shown below in black. Frameshift mutations in _phyre-cr1_ and _phyre-_ 45 _cr2_ eliminate the _PHYRE_ stop codon and alter the spacing between the _PHYRE_ stop codon 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 and the downstream _PHYB_ start codon. In _phyre-cr1_, the new reading frame extends into the 2 _PHYB_ coding sequence, resulting in loss of phyB protein due to the introduction of a premature 3 stop codon (indicated in red). In _phyre-cr2_, the _PHYRE_ and _PHYB_ ORFs are brought into the 4 same reading frame, allowing translation to extend into the downstream _PHYB_ coding 5 sequence and resulting in the production of an N-terminally extended phyB fusion protein. The 6 altered amino acid sequences in _phyre-cr_ mutants are shown in grey. **(B)** Western blot images 7 showing phyB protein levels (129 kDa) in the total protein extracts from 7-day deep shade8 grown seedlings of corresponding genotypes as indicated. Image of the corresponding 9 ponceau stained blot (loading control) is shown below. **(C)** Schematic of the qPCR primer 10 binding regions on the _PHYB_ locus. The promoter and downstream regulatory regions are 11 shown as black bars, 5' and 3' UTRs as grey boxes, and coding region as yellow boxes 12 separated by introns (black dotted lines). The _PHYRE_ uORF, _PHYB_ start codon, and stop 13 codon are indicated by a magenta rectangle, black arrowhead, and black asterisk, 14 respectively. 15 **Figure S4. Flowering time phenotypes of** _**PHYB**_ **complementation lines. (A)** 16 Representative rosette images of 21 day-old white light-grown plants of indicated genotypes. 17 **(B)** Flowering time quantification indicated by the number of rosette leaves at bolting in the 18 corresponding genotypes on the X-axis. Violin plots show the distribution of individual 19 observations, with overlaid box plots indicating the median and interquartile range. Different 20 letters indicate statistically significant differences among genotypes (adjusted P < 0.05), 21 determined by Kruskal-Wallis test followed by Dunn's post hoc test with Benjamini-Hochberg 22 correction. n = 13-23 plants. **Note:** The representative image and violin plot data for Col-0 and 23 _phyB-cr1_ in **(A) and (B)** are reproduced from **Figure 2F and 2G** respectively, as the genotypes 24 in **(B)** were analyzed together with Col-0 and _phyB-cr1_ in the same experiment. 25 **Figure S5. Comparative analysis of the** _**PHYB**_ **5′ untranslated regions across 49 land** 26 **plant species.** Genomic sequences spanning 150 nt upstream of the _PHYB_ translation start 27 site are shown as bars for 49 land plant species representing gymnosperms, monocots, basal 28 eudicots, asterids, and rosids, with emphasis on _Brassicaceae_ . Positions relative to the _PHYB_ 29 ATG are indicated above (0 to -150). The _Arabidopsis thaliana_ _PHYRE_ region (-74 to -36) is 30 shaded green across all sequences to indicate the corresponding positional window. The 31 experimentally validated _A. thaliana_ _PHYRE_ (39 bp) is highlighted in purple. _PHYRE-_ like 32 complete uORFs encoding 5-20 amino acids with a stop codon ≤100 nt upstream of the _PHYB_ 33 ATG are highlighted in orange, other complete uORFs encoding peptides shorter than 5 or 34 longer than 20 amino acids are shown in grey. 35 # 36 ACKNOWLEDGEMENTS 37 Financial support was provided by the European Research Council (ERC) Synergy 38 grant RESYDE (101167406), the NovoCrops Centre (Novo Nordisk Foundation grant 39 2019OC53580), the Novo Nordisk Foundation (grants NNF18OC0034226, 40 NNF20OC0061440, and NNF23OC0084195), the Independent Research Fund 41 Denmark (grants 0136-00015B and 0135-00014B), and Vetenskapsrådet (VR; grant 42 2024-04792), all to S.W. Additional support was received from the Wallenberg 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.31.763305) preprint (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-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) 1 Initiatives in Forest Research (WIFORCE), funded by the Knut and Alice Wallenberg 2 Foundation. We are grateful to Jaume Martínez-García for his insightful comments 3 and critical reading of the manuscript. 4 5 6 # 7 AUTHOR CONTRIBUTIONS 8 N.S. drove the experimental work and wrote the manuscript. A.V.H., M.J., A.E. and 9 A.Ma. performed experiments. A.Ma. and J.H. contributed to data analysis. J.C.M., 10 C.P. and M.M.A. performed bioinformatic data analysis. S.W. contributed to data 11 analysis, wrote the manuscript, supervised the project, and acquired funding. All 12 authors commented on the manuscript. 27 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) **Wild-type** **Start** **codon** **mutation** **Figure 5: Proposed model for** _**PHYRE**_ **-mediated regulation of** _**PHYB**_ **translation.** Position-dependent _PHYRE_ uORF translation maintains optimal phyB protein expression, possibly by facilitating translation re-initiation at the _PHYB_ start codon. Impaired _PHYRE_ translation, for example due to mutation of its start codon, results in reduced phyB protein levels, possibly because ribosomes bypass _PHYRE_ and continue scanning, thereby reducing translation initiation at the downstream _PHYB_ start codon. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) A B Col-0 _phyre-cr3_ _phyre-cr4_ _phyre-cr5_ preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the pr [perpetuity. It is made available under a CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) C 10.0 7.5 5.0 Col-0 _phyre-cr3_ _phyre-cr4_ _phyre-cr5_ 15 10 5 0 2.5 0 **Figure S2. Hypocotyl phenotypes and phyB protein levels in** _**phyB-cr**_ **and** _**phyre-cr**_ **mutants.** **(A)** Schematic showing the sequence information of the _PHYRE_ uORF in the wild-type, _phyre-cr3_, _phyre-cr4_, and _phyre-cr5_ mutants. The insertion in _phyre-cr3_ is highlighted in green, and the deletions in _phyre-cr4_ and _phyre-cr5_ are indicated by blue highlighted red dashed lines. **(B-C)** Representative images and hypocotyl length quantification of 6day-old seedlings of the indicated genotypes grown under **(B)** long-day white light or **(C)** long-day deep shade. Scale bars = 5 mm. Violin plots show the distribution of individual observations, with overlaid box plots indicating the median and interquartile range. Different letters indicate statistically significant differences among genotypes (adjusted P < 0.05), as determined using the Kruskal-Wallis test followed by Dunn’s post hoc test with Benjamini-Hochberg correction. n = 9-20 seedlings in **(B)** and 14-20 seedlings in **(C)** . **Note:** The representative image and violin plot data for Col-0 in **(B)** are reproduced from **Figure 3D**, as the genotypes in **(B)** were analyzed together with Col-0 as the reference control in the same experiment. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) A phyB B phyB preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the pr **TAA** **ATG** perpetuity. It is made available under a **TAA** [CC-BY-NC 4.0 International license.](http://creativecommons.org/licenses/by-nc/4.0/) **ATG** **TAA** **ATG** **TAA** phyB 180 130 100 **MMIHPKSFLVSR-** **MVSGVGGS........................MMPY-** **ATG** **TAA*** **TAA** **MMIHPKSSLSQGN................RSRG-** **ATG** **ATG** **TAA** **MMIHPKILPCLKV..................MVSGVGGS........................MMPY-** No phyB C _PHYRE_ _PHYB_ _**PHYB_1F PHYB_1R**_ _**PHYB_2F**_ _**PHYB_1R**_ **Figure S3.** _**PHYRE**_ **and** _**PHYB**_ **translation status in** _**phyre-cr**_ **mutants. (A)** Schematic showing translation status of _PHYRE_ uORF and _PHYB_ main ORF in wild type, _phyre-cr1_ and _phyre-cr2_ . Magenta and yellow rectangles represent the _PHYRE_ and _PHYB_ ORFs, respectively. Start and stop codons are indicated above the rectangles, and the corresponding amino acid sequences are shown below in black. Frameshift mutations in _phyre-cr1_ and _phyre-cr2_ eliminate the _PHYRE_ stop codon and alter the spacing between the _PHYRE_ stop codon and the downstream _PHYB_ start codon. In _phyre-cr1_, the new reading frame extends into the _PHYB_ coding sequence, resulting in loss of phyB protein due to the introduction of a premature stop codon (indicated in red). In _phyre-cr2_, the _PHYRE_ and _PHYB_ ORFs are brought into the same reading frame, allowing translation to extend into the downstream _PHYB_ coding sequence and resulting in the production of an N-terminally extended phyB fusion protein. The altered amino acid sequences in _phyre-cr_ mutants are shown in grey. **(B)** Western blot images showing phyB protein levels (129 kDa) in the total protein extracts from 7-day deep shadegrown seedlings of corresponding genotypes as indicated. Image of the corresponding ponceau stained blot (loading control) is shown below. **(C)** Schematic of the qPCR primer binding regions on the _PHYB_ locus. The promoter and downstream regulatory regions are shown as black bars, 5' and 3' UTRs as grey boxes, and coding region as yellow boxes separated by introns (black dotted lines). The _PHYRE_ uORF, _PHYB_ start codon, and stop codon are indicated by a magenta rectangle, black arrowhead, and black asterisk, respectively. [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.31.763305; this version posted November 4, 2026. The copyright hold](https://doi.org/10.64898/2026.10.31.763305) **Figure S5. Comparative analysis of the** _**PHYB**_ **5′ untranslated regions across 49 land plant species.** Genomic sequences spanning 150 nt upstream of the _PHYB_ translation start site are shown as bars for 49 land plant species representing gymnosperms, monocots, basal eudicots, asterids, and rosids, with emphasis on _Brassicaceae_ . Positions relative to the _PHYB_ ATG are indicated above (0 to -150). The _Arabidopsis thaliana PHYRE_ region (-74 to -36) is shaded green across all sequences to indicate the corresponding positional window. The experimentally validated _A._ _thaliana_ _PHYRE_ (39 bp) is highlighted in purple. _PHYRE-_ like complete uORFs encoding 5-20 amino acids with a stop codon ≤100 nt upstream of the _PHYB_ ATG are highlighted in orange, other complete uORFs encoding peptides shorter than 5 or longer than 20 amino acids are shown in grey.