Adjustment of the PIF7‐HFR1 transcriptional module activity controls plant shade adaptation

Article2 December 2020free access Source DataTransparent process Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation Sandi Paulišić Sandi Paulišić orcid.org/0000-0003-4696-5134 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Wenting Qin Wenting Qin orcid.org/0000-0002-7221-2067 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Harshul Arora Verasztó Harshul Arora Verasztó orcid.org/0000-0002-0702-6022 Structural Plant Biology Laboratory, Section of Biology, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland Search for more papers by this author Christiane Then Christiane Then orcid.org/0000-0001-8867-7080 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Benjamin Alary Benjamin Alary orcid.org/0000-0002-7845-0058 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Fabien Nogue Fabien Nogue orcid.org/0000-0003-0619-4638 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Miltos Tsiantis Miltos Tsiantis orcid.org/0000-0001-7150-1855 Department of Comparative Development and Genetics, Max Planck Institute from Plant Breeding Research, Cologne, Germany Search for more papers by this author Michael Hothorn Michael Hothorn orcid.org/0000-0002-3597-5698 Structural Plant Biology Laboratory, Section of Biology, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland Search for more papers by this author Jaime F Martínez-García Corresponding Author Jaime F Martínez-García [email protected] orcid.org/0000-0003-1516-0341 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Institute for Plant Molecular and Cellular Biology (IBMCP), CSIC-UPV, València, Spain Search for more papers by this author Sandi Paulišić Sandi Paulišić orcid.org/0000-0003-4696-5134 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Wenting Qin Wenting Qin orcid.org/0000-0002-7221-2067 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Harshul Arora Verasztó Harshul Arora Verasztó orcid.org/0000-0002-0702-6022 Structural Plant Biology Laboratory, Section of Biology, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland Search for more papers by this author Christiane Then Christiane Then orcid.org/0000-0001-8867-7080 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Benjamin Alary Benjamin Alary orcid.org/0000-0002-7845-0058 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Search for more papers by this author Fabien Nogue Fabien Nogue orcid.org/0000-0003-0619-4638 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France Search for more papers by this author Miltos Tsiantis Miltos Tsiantis orcid.org/0000-0001-7150-1855 Department of Comparative Development and Genetics, Max Planck Institute from Plant Breeding Research, Cologne, Germany Search for more papers by this author Michael Hothorn Michael Hothorn orcid.org/0000-0002-3597-5698 Structural Plant Biology Laboratory, Section of Biology, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland Search for more papers by this author Jaime F Martínez-García Corresponding Author Jaime F Martínez-García [email protected] orcid.org/0000-0003-1516-0341 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Institute for Plant Molecular and Cellular Biology (IBMCP), CSIC-UPV, València, Spain Search for more papers by this author Author Information Sandi Paulišić1, Wenting Qin1, Harshul Arora Verasztó2, Christiane Then1,†, Benjamin Alary1, Fabien Nogue3, Miltos Tsiantis4, Michael Hothorn2 and Jaime F Martínez-García *,1,5,6 1Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Campus UAB, Barcelona, Spain 2Structural Plant Biology Laboratory, Section of Biology, Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland 3Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France 4Department of Comparative Development and Genetics, Max Planck Institute from Plant Breeding Research, Cologne, Germany 5Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 6Institute for Plant Molecular and Cellular Biology (IBMCP), CSIC-UPV, València, Spain †Present address: Department for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut, Federal Research Institute for Cultivated Plants, Braunschweig, Germany *Corresponding author. Tel: +34 963 878 627; E-mail: [email protected] The EMBO Journal (2021)40:e104273https://doi.org/10.15252/embj.2019104273 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Shade caused by the proximity of neighboring vegetation triggers a set of acclimation responses to either avoid or tolerate shade. Comparative analyses between the shade-avoider Arabidopsis thaliana and the shade-tolerant Cardamine hirsuta revealed a role for the atypical basic-helix-loop-helix LONG HYPOCOTYL IN FR 1 (HFR1) in maintaining the shade tolerance in C. hirsuta, inhibiting hypocotyl elongation in shade and constraining expression profile of shade-induced genes. We showed that C. hirsuta HFR1 protein is more stable than its A. thaliana counterpart, likely due to its lower binding affinity to CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), contributing to enhance its biological activity. The enhanced HFR1 total activity is accompanied by an attenuated PHYTOCHROME INTERACTING FACTOR (PIF) activity in C. hirsuta. As a result, the PIF-HFR1 module is differently balanced, causing a reduced PIF activity and attenuating other PIF-mediated responses such as warm temperature-induced hypocotyl elongation (thermomorphogenesis) and dark-induced senescence. By this mechanism and that of the already-known of phytochrome A photoreceptor, plants might ensure to properly adapt and thrive in habitats with disparate light amounts. Synopsis Plant shade triggers distinct acclimation responses in neighboring vegetation. Here, comparison of shade-avoiding Arabidopsis thaliana and shade-tolerant Cardamine hirsuta reveals that enhanced repressor activity of the photomorphogenesis regulator HFR1 promotes shade tolerance in C. hirsuta via regulation of the transcription factor PIF7. HFR1 activity is enhanced and PIF7 activity attenuated in C. hirsuta compared to A. thaliana. Cardamine hirsuta HFR1 protein is more stable than its A. thaliana counterpart. Cardamine hirsuta HFR1 has lower binding affinity to COP1, which contributes to enhance its biological activity. Other PIF-mediated growth responses, such as temperature-induced hypocotyl elongation and dark-induced senescence, are attenuated in C. hirsuta. Introduction Acclimation of plants to adjust their development to the changing environment is of utmost importance. This acclimation relies on the plant’s ability to perceive many cues such as water, nutrients, temperature, or light. Conditions in nature often involve simultaneous changes in multiple light cues leading to an interplay of various photoreceptors to adjust plant growth appropriately (Pierik & Testerink, 2014; Mazza & Ballare, 2015; de Wit et al, 2016; Ballare & Pierik, 2017; Fiorucci & Fankhauser, 2017). Nearby vegetation can impact both light quantity and quality. Under a canopy, light intensity is decreased and its quality is changed as the overtopping green leaves strongly absorb blue and red light (R) but reflect far-red light (FR). As a consequence, plants growing in forest understories receive less light of a much lower R to FR ratio (R:FR) than those growing in open spaces. In dense plant communities, FR reflected by neighboring plants also decreases R:FR but typically without changing light intensity. We refer to the first situation as canopy shade (very low R:FR) and the second as proximity shade (low R:FR). In general, two strategies have emerged to deal with shade: avoidance and tolerance (Valladares & Niinemets, 2008; Gommers et al, 2013; Pierik & Testerink, 2014). Shade avoiders usually promote elongation of organs to outgrow the neighbors and avoid light shortages, reduce the levels of photosynthetic pigments to cope to light shortage, and accelerate flowering to ensure species survival (Casal, 2013). The set of responses to acclimate to shade is collectively known as the shade avoidance syndrome (SAS). In contrast, shade-tolerant species usually lack the promotion of elongation growth in response to shade and have developed a variety of traits to acclimate to low light conditions and optimize net carbon gain (Smith, 1982; Valladares & Niinemets, 2008). In Arabidopsis thaliana, a shade-avoider plant, low R:FR is perceived by phytochromes. Among them, phyA has a negative role in elongation, particularly under canopy shade, whereas phyB inhibits elongation inactivating PHYTOCHROME INTERACTING FACTORS (PIFs), members of the basic-helix-loop-helix (bHLH) transcription factor family that promote elongation growth. In particular, PIFs induce hypocotyl elongation by initiating an expression cascade of genes involved in auxin biosynthesis and signaling [e.g., YUCCA 8 (YUC8), YUC9, INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), IAA29], and other processes related to cell elongation [e.g., XYLOGLUCAN ENDOTRANSGLYCOSYLASE 7 (XTR7)]. Genetic analyses indicated that PIF7 is the key PIF regulator of the low R:FR-induced hypocotyl elongation with PIF4 and PIF5 having important contributions. Indeed, pif7 mutant responds poorly to low R:FR compared to the pif4 pif5 double or pif1 pif3 pif4 pif5 quadruple (pifq) mutants, but the triple pif4 pif5 pif7 mutant is almost unresponsive to low R:FR (Lorrain et al, 2008; Li et al, 2012; de Wit et al, 2016; van Gelderen et al, 2018). PhyB-mediated shade signaling involves other transcriptional regulators, such as LONG HYPOCOTYL IN FR 1 (HFR1), PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), BIM1, ATHB4, or BBX factors, that either promote or inhibit shade-induced hypocotyl elongation (Sessa et al, 2005; Roig-Villanova et al, 2007; Sasidharan & Pierik, 2010; Cifuentes-Esquivel et al, 2013; Bou-Torrent et al, 2014; Gallemi et al, 2017; Yang & Li, 2017). HFR1, a member of the bHLH family, is structurally related to PIFs but lacks the phyB- and DNA-binding ability that PIFs possess (Galstyan et al, 2011; Hornitschek et al, 2012). HFR1 inhibits PIF activity by heterodimerizing with them, as described for PIF1 (Shi et al, 2013), PIF3 (Fairchild et al, 2000), PIF4, and PIF5 (Hornitschek et al, 2009), Heterodimerization with HFR1 prevents PIFs from binding to the DNA and altering gene expression. In this manner, HFR1 acts as a transcriptional cofactor that modulates SAS responses, e.g., it inhibits hypocotyl elongation in seedlings in a PIF-dependent manner, forming the PIF-HFR1 transcriptional regulatory module (Galstyan et al, 2011). What mechanistic and regulatory adjustments in shade signaling are made between species to adapt to plant shade is a topic that has not received much attention until now. This question has been recently addressed performing comparative analyses between phylogenetically related species. In two related Geranium species that showed petioles with divergent elongation responses to shade, transcriptomic analysis led to propose that differences in expression of three factors, FERONIA, THESEUS1, and KIDARI, shown to activate SAS elongation responses in A. thaliana, might be part of the adjustments necessary to acquire a shade-avoiding or tolerant habit (Gommers et al, 2017). When comparing two related mustard species that showed divergent hypocotyl elongation response to shade, A. thaliana and Cardamine hirsuta (Hay et al, 2014), molecular and genetic analyses indicated that phyA, and to a lesser extent phyB, contributed to establish this divergent response. In particular, the identification and characterization of the C. hirsuta phyA-deficient slender in shade 1 (sis1) mutant indicated that differential features of this photoreceptor in A. thaliana and C. hirsuta could explain their differential response to shade. Thus, stronger phyA activity in C. hirsuta wild-type plants resulted in a suppressed hypocotyl elongation response when exposed to low or very low R:FR (Molina-Contreras et al, 2019). These approaches indicated that the implementation of shade avoidance and shade tolerance involved the participation of shared genetic components. They also suggest that other responses co-regulated by these shared components will be accordingly affected. With this frame of reference, we asked whether the phyB-dependent PIF-HFR1 module was also relevant to shape the shade response habits in different plant species. We found that C. hirsuta plants deficient in ChHFR1 gained a capacity to elongate in response to shade. We also report that AtHFR1 and ChHFR1 are expressed at different levels and encode proteins with different protein stability, caused by their different binding affinities with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), known to affect AtHFR1 stability under shade (Pacin et al, 2016). We propose that adaptation to plant shade in A. thaliana and C. hirsuta relies on the PIF-HFR1 regulatory module. As PIFs regulate several other processes, we hypothesized that a set of responses co-regulated by the PIF-HFR1 module are also affected and associated with the shade-avoidance and shade-tolerant habits. After exploring this possibility, we found that thermoregulation of hypocotyl elongation and dark-induced senescence, two well-known PIF-regulated responses (Koini et al, 2009; Stavang et al, 2009; Sakuraba et al, 2014), is consistently affected in C. hirsuta. Results HFR1 is required for the shade tolerance habit of Cardamine hirsuta First, we wanted to determine if HFR1 has a role in the shade-tolerance habit of C. hirsuta, i.e., whether ChHFR1 contributes to inhibit hypocotyl elongation when this species is exposed to shade. For this purpose, we generated several C. hirsuta RNAi lines to downregulate HFR1 expression (RNAi-HFR1 lines). As expected, ChHFR1 expression was attenuated in seedlings of two RNAi-HFR1 selected lines (#01 and #21) compared to the wild type (ChWT) (Fig EV1A). When growing under white light (W) of high R:FR (> 1.5), hypocotyl length of these two RNAi-HFR1 lines was undistinguishable from ChWT (Fig 1A). By contrast, under W supplemented with increasing amounts of FR (W + FR) resulting in moderate (0.09), low (0.05–0.06), and very low (0.02) R:FR (that simulated proximity and canopy shade) (Martinez-Garcia et al, 2014), the hypocotyl elongation of RNAi-HFR1 seedlings was significantly promoted compared to ChWT, which was unresponsive (Fig 1A). Click here to expand this figure. Figure EV1. Characterization of RNAi-HFR1 and chfr1 mutants in Cardamine hirsuta A, B. Relative expression levels of ChHFR1 gene, normalized to EF1α in ChWT, (A) two RNAi-HFR1 lines (#01 and #21) and (B) the two chfr1 mutants of C. hirsuta. Seedlings were grown for 7 days in W. Expression values are the mean ± SE of three independent biological replicates relative to ChWT. Asterisks mark significant differences (Student t-test: **P-value < 0.01) relative to ChWT. C. The two identified chfr1-1 and chfr1-2 mutants have one nucleotide insertion at position 420 of the ChHFR1 ORF, which leads to a frame shift and a premature stop codon. Download figure Download PowerPoint Figure 1. Hypocotyls of Cardamine hirsuta seedlings with reduced levels of ChHFR1 strongly elongate in response to simulated shade A, B. Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic, and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR > 1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W + FR) for 4 more days, producing various R:FR. Aspect of representative 7-day-old ChWT, RNAi-HFR1 and chfr1-1 seedlings grown in W or W + FR (R:FR, 0.02), as indicated, is shown in lower panel. C, D. Effect of W + FR exposure on the expression of PIL1, YUC8, and XTR7 genes in seedlings of ChWT, (C) RNAi-HFR1, and (D) chfr1 mutant lines. Expression was analyzed in 7-day-old W-grown seedlings transferred to W + FR (R:FR, 0.02) for 0, 1, 4, 8, and 12 h. Transcript abundance is normalized to EF1α levels. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student's t-test: **P-value < 0.01; *P-value < 0.05) relative to ChWT value at the same time point. Download figure Download PowerPoint Using CRISPR-Cas9, we obtained two mutant lines of ChHFR1 (named chfr1-1 and chfr1-2) with a single nucleotide insertion in their sequence leading to a premature stop codon (Fig EV1C). These mutants showed a non-significant decrease of ChHFR1 expression in W-grown seedlings (Fig EV1B). Similar to the RNAi-HFR1 lines, their hypocotyls were undistinguishable from ChWT under W but elongated strongly in response to W + FR exposure (Fig 1B), showing a slender in shade (sis) phenotype. Together, we concluded that HFR1 represses hypocotyl elongation in response to shade in C. hirsuta. Exposure of A. thaliana wild-type (AtWT) and ChWT seedlings to low R:FR induces a rapid increase in the expression of various direct target genes of PIFs, including PIF3-LIKE 1 (PIL1), YUC8, and XTR7 (Fig 1C and D) (Ciolfi et al, 2013; Hersch et al, 2014; Molina-Contreras et al, 2019). The shade-induced expression of these genes was significantly higher in RNAi-HFR1 and chfr1 mutant lines compared to ChWT (Fig 1C and D), indicating that ChHFR1 might repress shade-triggered hypocotyl elongation in part by downregulating the rapid shade-induced expression of these genes in C. hirsuta, as it was observed with AtHFR1 in A. thaliana seedlings (Hornitschek et al, 2009). HFR1 expression is higher in Cardamine hirsuta than in Arabidopsis thaliana seedlings To test if the lack of elongation of ChWT hypocotyls in response to shade was caused by higher levels of ChHFR1 expression in this species, we used primer pairs that amplify HFR1 (Fig EV2A) and three housekeeping genes (EF1α, SPC25, YLS8) in both species (Molina-Contreras et al, 2019). As expected, expression of HFR1 was induced in shade-treated seedlings of both species, in agreement with the presence of canonical PIF-binding sites (G-box, CACGTG) in the HFR1 promoters (Martinez-Garcia et al, 2000; Hornitschek et al, 2009; Fig EV3A). More importantly, ChHFR1 transcript levels were always higher than those of AtHFR1 during the whole period analyzed (from days 3 to 7) (Fig 2). Because HFR1 is part of the PIF-HFR1 regulatory module, we next compared transcript levels of PIF genes in both species. PIF7 expression was significantly lower in C. hirsuta than in A. thaliana in either W or W + FR during the period analyzed (Fig 2). By contrast, PIF4 expression was higher in C. hirsuta than in A. thaliana, whereas that of PIF5 was similar in both species (Fig EV2B). Together, these results indicated that whereas HFR1 expression is enhanced, that of PIF7 is globally attenuated in ChWT compared to AtWT seedlings. As a consequence, the PIF-HFR1 transcriptional module might be differently balanced in these species, with HFR1 imposing a stronger suppression on the PIF7-driven hypocotyl elongation in the shade-tolerant C. hirsuta seedlings. Click here to expand this figure. Figure EV2. Alignments of HFR1, PIF4, PIF5, and PIF7 partial DNA sequences in Arabidopsis thaliana and Cardamine hirsuta Location of shared primers and amplicons used for comparison of expression levels by RT-qPCR between species. Transcript abundance of PIF4 and PIF5, normalized to YLS8, SPC25, and EF1α in ChWT and AtWT grown as in Fig 2. Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: **P-value < 0.01, ***P-value < 0.001) between ChWT and AtWT when grown under W (black asterisks) or W + FR (red asterisks). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. ChHFR1 and AtHFR1 complement the Arabidopsis thaliana hfr1-5 mutant long hypocotyl phenotype Cartoon of HFR1 promoters from A. thaliana (pAtHFR1) and C. hirsuta (pChHFR1). These promoters cover 2,000 bp from the beginning of the translation start of the two HFR1 genes. The positions of G-boxes (CACGTG) are indicated with arrows. GUS staining of representative A. thaliana seedlings expressing GUS under the pAtHFR1 (line #03). Seven-day-old W-grown seedlings were treated with W + FR for the indicated amount of time. Correlation between HypW+FR-HypW (means ± SE of at least four biological replicates, data shown in Fig 3C) and relative levels of ChHFR1 or AtHFR1 expression (means ± SE of three biological replicates, data shown in Fig 3B). The estimated regression equations and the R2 values are shown for each plot. Download figure Download PowerPoint Figure 2. Levels of HFR1 transcript are higher in Cardamine hirsuta than Arabidopsis thaliana seedlings Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W + FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: **P-value < 0.01, ***P-value < 0.001) between ChWT and AtWT when grown under W (black asterisks) or W + FR (red asterisks). Download figure Download PowerPoint ChHFR1 protein is more stable than AtHFR1 A higher specific activity of ChHFR1 compared to its orthologue AtHFR1 might also contribute to the role of this transcriptional cofactor in maintaining the shade tolerance habit of C. hirsuta. To test this possibility, we transformed A. thaliana hfr1-5 plants with constructs to express either AtHFR1 or ChHFR1 fused to the 3x hemagglutinin tag (3xHA). These genes were expressed under the transcriptional control of the 2 kb of the AtHFR1 promoter (pAt), generating hfr1>pAt:ChHFR1 and hfr1>pAt:AtHFR1 lines (Fig 3A). Fusion of pAt to the GUS reporter gene resulted in GUS activity in cotyledons and roots of transgenic lines, with increased levels in hypocotyls of seedlings exposed for 2–4 h to W + FR (Fig EV3B). Several independent transgenic lines of each construct were analyzed for hypocotyl length (Appendix Fig S1), HFR1 transcript levels and 3xHA-tagged protein abundance. In these lines, HFR1 biological activity was estimated as the difference in hypocotyl length of seedlings grown under W + FR (HypW+FR) and W (HypW) (HypW+FR-HypW) (Molina-Contreras et al, 2019). The potential to suppress the hypocotyl elongation in shade below that of hfr1-5 seedlings would depend on the transcript level of HFR1 and/or its protein levels. The hfr1>pAt:ChHFR1 lines had shorter hypocotyls in shade (i.e., stronger global HFR1 activity) compared to hfr1>pAt:AtHFR1 lines of similar HFR1 expression levels (Figs 3B and C, and EV3C), suggesting that total HFR1 activity was higher in hfr1>pAt:ChHFR1 than in hfr1>pAt:AtHFR1 lines. However, we observed much higher abundance of HFR1-3xHA protein after shade exposure in hfr1>pAt:ChHFR1 lines than in hfr1>pAt:AtHFR1 lines with comparable levels of HFR1 expression (Fig 3D), suggesting that the ChHFR1 protein might be much more stable. Together, these results point to differences in protein stability (rather than in specific activity) as the main cause for the enhanced HFR1 total activity of ChHFR1 compared to AtHFR1 in complemented lines. Figure 3. The activity of ChHFR1 is higher than that of AtHFR1 in Arabidopsis thaliana seedlings Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 promoter of Arabidopsis thaliana (pAtHFR1) used to complement hfr1-5 mutant of A. thaliana (At hfr1-5). Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue), and hfr1>pAt:AtHFR1 (in red) lines grown under W + FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels. Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W + FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W + FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE. Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~ 20 µmol/m2·s1) after which they were incubated for 3 h in high W (~ 100 µmol/m2·s1) and transferred to W + FR (R:FR, 0.06) for 3 h. Data information: Different letters denote significant differences (one-way ANOVA with the Tukey test, P-value < 0.05) among means. Source data are available online for this figure. Source Data for Figure 3 [embj2019104273-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint AtHFR1 stability is affected by light conditions. In etiolated seedlings, exposure to W promotes stabilization and accumulation of AtHFR1, whereas in W-grown seedlings, high intensity of W increases its abundance (Duek et al, 2004; Yang et al, 2005). Importantly, AtHFR1 stability has a strong impact on its biological activity as overexpression of stable forms of this protein leads to phenotypes resulting from enhanced HFR1 activity (Yang et al, 2005; Galstyan et al, 2011). As AtHFR1 and ChHFR1 primary structures are globally similar (Fig EV4A), we aimed to test if ChHFR1 stability is also light-dependent. We first examined ChHFR1 protein accumulation in response to different W intensities in seedlings of an A. thaliana hfr1-5 line that constitutively express ChHFR1 (hfr1>35S:ChHFR1) (Fig EV4B). When grown in our normal W conditions (~ 20 µmol/m2·s1), these seedlings accumulated low but detectable levels of ChHFR1; when transferred to higher W intensity (~ 100 µmol/m2·s1), ChHFR1 levels increased 10-fold (Fig EV4C). As ChHFR1 is expressed under the constitutive 35S promoter, these results indicate that ChHFR1 protein accumulation is induced by high W intensity, as it has been described for AtHFR1 (Yang et al, 2005). This prompted us to pretreat W-grown seedlings with 3 h of high W intensity in all our subsequent experiments to analyze ChHFR1 levels. Click here to expand this figure. Figure EV4. ChHFR1 protein accumulates in high W Alignment of AtHFR1 and ChHFR1 protein sequences. Putative COP1 interacting motifs, defined in AtHFR1, are highlighted with a light gray box. VP motifs are highlighted with blue letters. Amino acid sequences inside the blue line rectangles correspond to the synthetic AtHFR1, ChHFR1, and At/ChHFR1 VP peptides used in the microscale thermophoresis assays (Appendix Table S3). Cartoon representing the light treatments given to seedlings to estimate relative HFR1-3xHA levels. Seedlings grown for 7 days in low W (~ 20 µmol/m2·s1, R:FR ≈ 6.4) were first moved to high W (~ 100 µmol/m2·s1, R:FR ≈ 3.9) for 3 h and then either transferred to high W (control) or high W + FR (R:FR ≈ 0.06) for 3 h. Seedling samples were collected at the time points indicated with asterisks. Relative HFR1-3xHA protein levels of hfr1>35S:ChHFR1 seedlings (line #16) grown as indicated in B, with a representative immunoblot in a lower panel. Relative protein levels are the mean ± SE of three independent biological replicates relative to the data point of 0 h in high W (0 h W). Asterisks mark significant differences in protein levels (Student t-test: **P-value < 0.01; *P-value < 0.05) relative to the 0 h W value. Source data are available online for this figure. Download figure Download PowerPoint Next, we exposed hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13) seedlings to W + FR (Fig 4A). Although HFR1 expression in both lines was simil