Nucleo‐plastidic PAP 8/ pTAC 6 couples chloroplast formation with photomorphogenesis
2020; Springer Nature; Volume: 39; Issue: 22 Linguagem: Inglês
10.15252/embj.2020104941
ISSN1460-2075
AutoresMonique Liebers, François‐Xavier Gillet, Abir Israel, Kévin Pounot, Louise Chambon, Maha Chieb, Fabien Chevalier, Rémi Ruedas, Adrien Favier, Pierre Gans, Elisabetta Boeri Erba, David Cobessi, Thomas Pfannschmidt, Robert Blanvillain,
Tópico(s)Photoreceptor and optogenetics research
ResumoArticle1 October 2020free access Source DataTransparent process Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis Monique Liebers Monique Liebers orcid.org/0000-0001-8363-2449 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author François-Xavier Gillet François-Xavier Gillet CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Abir Israel Abir Israel orcid.org/0000-0002-2662-5821 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Kevin Pounot Kevin Pounot CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Louise Chambon Louise Chambon CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Maha Chieb Maha Chieb orcid.org/0000-0002-3171-7253 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Fabien Chevalier Fabien Chevalier CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Rémi Ruedas Rémi Ruedas CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Adrien Favier Adrien Favier CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Pierre Gans Pierre Gans CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Elisabetta Boeri Erba Elisabetta Boeri Erba CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author David Cobessi David Cobessi orcid.org/0000-0002-5103-7976 CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Thomas Pfannschmidt Corresponding Author Thomas Pfannschmidt [email protected] orcid.org/0000-0002-7532-3467 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Leibniz Universität Hannover, Hannover, Germany Search for more papers by this author Robert Blanvillain Corresponding Author Robert Blanvillain [email protected] orcid.org/0000-0002-6320-7207 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Monique Liebers Monique Liebers orcid.org/0000-0001-8363-2449 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author François-Xavier Gillet François-Xavier Gillet CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Abir Israel Abir Israel orcid.org/0000-0002-2662-5821 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Kevin Pounot Kevin Pounot CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Louise Chambon Louise Chambon CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Maha Chieb Maha Chieb orcid.org/0000-0002-3171-7253 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Fabien Chevalier Fabien Chevalier CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Rémi Ruedas Rémi Ruedas CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Adrien Favier Adrien Favier CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Pierre Gans Pierre Gans CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Elisabetta Boeri Erba Elisabetta Boeri Erba CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author David Cobessi David Cobessi orcid.org/0000-0002-5103-7976 CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Thomas Pfannschmidt Corresponding Author Thomas Pfannschmidt [email protected] orcid.org/0000-0002-7532-3467 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Leibniz Universität Hannover, Hannover, Germany Search for more papers by this author Robert Blanvillain Corresponding Author Robert Blanvillain [email protected] orcid.org/0000-0002-6320-7207 CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France Search for more papers by this author Author Information Monique Liebers1,†,‡, François-Xavier Gillet1,‡, Abir Israel1, Kevin Pounot1, Louise Chambon1, Maha Chieb1, Fabien Chevalier1, Rémi Ruedas2, Adrien Favier2, Pierre Gans2, Elisabetta Boeri Erba2, David Cobessi2, Thomas Pfannschmidt *,1,3,† and Robert Blanvillain *,1 1CNRS, CEA, INRA, IRIG-LPCV, Univ. Grenoble-Alpes, Grenoble, France 2CEA, CNRS, IBS, Univ. Grenoble Alpes, Grenoble, France 3Leibniz Universität Hannover, Hannover, Germany †Present address: Institute of Plant Science and Microbiology, Molecular Plant Physiology, Hamburg Universität, Hamburg, Germany †Present address: Institute for Botany, Leibniz Universität Hannover, Hannover, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 511 762 26 32; E-mail: [email protected] *Corresponding author. Tel: +33 04 38 78 34 84; E-mail: [email protected] The EMBO Journal (2020)39:e104941https://doi.org/10.15252/embj.2020104941 [Correction added on February 2 after first online publication: Leibniz Universität Hannover was added for Thomas Pfannschmidt] 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 The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis. Synopsis PAP8, an essential subunit of the chloroplast-encoded RNA polymerase in Arabidopsis thaliana, is required for phytochrome-B-mediated photomorphogenesis by modulating both nuclear and chloroplast-encoded gene expression during chloroplast biogenesis. The bZIP transcription factor HY5 promotes PAP8 expression. PAP8 is localized and active in both plastids and the nucleus. PAP8 is essential for the formation of late photobodies. PAP8 is required for various PHYB-dependent photoresponses, e.g., regulation of PIF1/3 stability and expression of GLK1/2 transcription factors. PAP8 interacts with PAP5/HMR and drives photosynthesis-associated plastid-encoded gene (PhAPG) expression. Introduction Chloroplasts are the organelles conducting photosynthesis in plants and green algae (Jarvis & Lopez-Juez, 2013). In angiosperms, the perception of light is essential to trigger photomorphogenesis, during which the photosynthetic organelles differentiate from chlorophyll-free proplastids. In contrast, seedlings sheltered from light, perform skotomorphogenesis, a dark-specific developmental programme triggering hypocotyl elongation giving the shoot apex a chance to reach light. Meanwhile, the apical hook is preserved, maintaining non-developing cotyledons downward and close, serving as a protective shield for the quiescent shoot apical meristem. At the cellular level, the cotyledons generate chlorophyll-free etioplasts incapable of performing photosynthesis (Liebers et al, 2017). The lack of chloroplast development in the dark can be regarded as a way to optimize the use of limited resources stored in the seed to efficiently reach the surface. Then, illumination of the cotyledons causes the conversion of the phytochrome photoreceptors into an active state launching the photomorphogenic programme (Solymosi & Schoefs, 2010) and reviewed in Ref. Hernandez-Verdeja et al (2020). This programme involves also major morphological changes in the seedling including the repression of hypocotyl elongation and the opening of the cotyledons that are then rapidly engaged in chloroplast biogenesis (Pogson et al, 2015). As remnant of their endosymbiotic origin, plastids possess their own genetic system, which contributes to the construction of the photosynthetic apparatus after illumination (Jarvis & Lopez-Juez, 2013). A plastid-encoded RNA polymerase (PEP) is required for proper transcription of photosynthesis genes encoded by the plastid genome. This PEP complex is composed of a prokaryotic core of four distinct bacterial-like subunits (α2, β, β′, β″) surrounded by (at least) 12 additional nuclear-encoded subunits of eukaryotic origin (Pfannschmidt et al, 2015) known as PEP-associated proteins (PAPs). The association of PAPs to the prokaryotic core is strictly induced by light through the action of phytochromes (Pfannschmidt & Link, 1994; Yang et al, 2019; Yoo et al, 2019). Importantly, the PAP association to the core of the PEP appears to be one important bottleneck of chloroplast formation since genetic inactivation of any PAP results in albinism (Pfalz & Pfannschmidt, 2013). The genes for PAPs appear to represent a regulatory unit that exhibits very similar co-expression profiles albeit the involved genes encode proteins that belong to very different functional classes that could not be predictably united before. They all exhibit a basal expression in the dark followed by a rapid and transient peak after light exposure strongly suggesting a connection of their expression to the light regulation network (Liebers et al, 2018). In the dark, photomorphogenesis is actively inhibited by the negative regulatory module CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) (Sullivan et al, 2003). In particular, the E3 ubiquitin ligase COP1 was shown to destabilize two basic domain/leucine zipper (bZIP) transcription factors known to initiate photomorphogenesis (ELONGATED HYPOCOTYL 5, HY5 and its homologous protein HYH) (Osterlund et al, 2000; Holm et al, 2002). Upon illumination, the cytosolic pool of inactive phytochrome B (PHYBPr) is converted into an active state (PHYBPfr), triggering its translocation into the nucleus (Yamaguchi et al, 1999; Chen et al, 2003) where it physically interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) (Huq et al, 2004) leading to the emergence of a mutual negative feedback loop (Leivar & Monte, 2014). PIFs belong to a subset of the basic helix–loop–helix (bHLH) superfamily of transcription factors. Four of them in particular (PIF1, P1F3, P1F4 and P1F5) collectively act with some redundancy, as transcriptional repressors of photomorphogenesis in the dark (Leivar et al, 2008). They are destabilized by light upon their interaction with the photoactivated phytochrome molecules (Al-Sady et al, 2006) leading to a de-repression of the photomorphogenic programme (Jiao et al, 2007). The antagonistic role of phytochromes and the different PIFs is responsible for complex adaptive developmental responses of the seedling to changes in their light environment including de-etiolation and shade avoidance. PIF1 and PIF3, in particular, were identified as having a predominant role in chloroplast development (Stephenson et al, 2009). In the pursuit of greening, the phytochrome-mediated light signalling represses the COP1-mediated destabilization of HY5, thereby leading to its accumulation. Stabilized HY5 can then initiate expression of photomorphogenic factors (Lee et al, 2007). Meanwhile, light exposure triggers the transcriptional activation of GOLDEN2-LIKE 1 and 2 (GLK1 and GLK2) transcription factors that are responsible for the proper expression of nuclear photosynthesis genes (Waters & Langdale, 2009; Waters et al, 2009). The action of phytochrome within the nucleus was visualized using a GFP tag (PHYB-GFP or PBG) revealing that phytochrome B translocates into the nucleus and then aggregates into specific speckles within the nuclear matrix (Yamaguchi et al, 1999). Early speckles are numerous and small, while later speckles become larger and less abundant without changing the phytochrome content that remains rather stable. Late speckle formation, also designated “late photobodies”, requires the presence of HEMERA (HMR), a dually localized protein present in the nucleus and in plastids (Chen et al, 2010; Nevarez et al, 2017), that is able to physically interact with phytochromes (Galvao et al, 2012). In plastids, HMR is known as pTAC12/PAP5 representing a member of the PAP family that is essential for chloroplast biogenesis since genetic inactivation of the protein blocks plastid differentiation leading to albinism (Pfalz et al, 2006, 2015). For PAPs, different functions can be predicted from their amino acid sequences, but their precise roles either as single subunits or in complex are not yet understood. PAP8 is one of the most enigmatic members among the PAPs, as its deduced amino acid sequence does not harbour any known functional domain although recent findings suggest a role as a transcriptional enhancer of the PEP complex (Ding et al, 2019). Separate genetic studies based on the mapping of quantitative trait loci in natural accessions of Arabidopsis revealed that the overlapping loci ESPRESSO (Swarup et al, 1999) and LIGHT1 (Borevitz et al, 2002), both corresponding to PAP8, are responsible for major variation in natural circadian rhythms or hypocotyl elongation across a wide range of environments (Loudet et al, 2008). ESPRESSO transcripts cycle in a diurnal pattern and LIGHT1 responds to various wavelengths and has a significant epistatic interaction with LIGHT2/PHYB. A separate transcript analysis revealed that PAP8 responds to temperature (Danilova et al, 2018a) and phyto-hormones (Danilova et al, 2018b). In the following, PAP8 will be used as the gene name for At1g21600 also corresponding to ESPRESSO, LIGHT1 or pTAC6. Here, we show that PAP8 is a dually localized nucleo-plastidic protein with a nuclear pool important for the proper timing of chloroplast biogenesis. In particular PAP8 interacts with HMR, it is essential for phytochrome-mediated signal transduction, PIF1 and PIF3 degradation, HY5 stabilization and GLK transcript accumulation indicating that it represents a novel key regulator of the light-signalling network. Results PAP8 plays an essential role in chloroplast biogenesis PAP8 was identified by targeted proteomics as pTAC6, a component of the transcriptionally active chromosome of plastids, a biochemically defined DNA-protein structure capable of performing faithful transcription of plastid genes in vitro (Pfalz et al, 2006). The T-DNA insertion line “SALK_024431” of Arabidopsis, referred to as the pap8-1 mutant in this study, displayed an albino phenotype with a strong depletion of PEP-dependent photosynthesis transcripts, a reduced pigments accumulation and developmentally arrested plastids (Pfalz et al, 2006; Appendix Fig S1). An orthologous protein of pTAC6 was then isolated from a highly purified Sinapis alba PEP complex and subsequently renamed PAP8 as being a bona fide component of this PEP complex (Steiner et al, 2011). The pap8-1 allele corresponds to the insertion of an inverted repeat of the T-DNA into the first intron of the gene (Fig 1A). Amplicon sequencing, after PCR-based genotyping (Fig 1B), showed that 11 bp of the second exon are missing so that the open reading frame (ORF) is destroyed notwithstanding possible events of T-DNA splicing. Besides, a PAP8 transcript spanning the insertion point could not be detected with RT–PCR in the homozygous pap8-1 mutant (Fig 1C), indicating that pap8-1 is a genuine null allele. The conceptually translated protein sequence is found in the terrestrial green lineage starting from mosses to Eudicots (Fig 1D), though absent in ferns, gymnosperms and a few basal angiosperms. A predicted N-terminal chloroplast transit peptide (cTP) rapidly diverged while a highly conserved region (HCR) of unknown function seems to be under a strong selection pressure, as it is almost unchanged since the last common ancestor of all terrestrial plants. Hence, the sporophytic lethality of the pap8 mutant triggers the assumption that the protein had brought an important function to the green lineage in its way to conquer dry lands, and then became essential to Eudicots and Monocots. Figure 1. Genetic analysis of the mutant pap8-1 A. Structure of the PAP8 locus, blue boxes: exons, lines: introns. Red box: inserted T-DNA as inverted repeats (IR1/IR2) in the first intron. White box with a diagonal red line: deletion at the left border of IR2 and part of second exon (italicized grey sequence). Green and red arrows represent forward and reverse primers, respectively, as o1: oPAP8_rtp_F; o2: oPAP8_E3_R; o3: oPAP8_rtp_R; o4:op8i2_R; and oLB: oLBb1.3. B. PCR performed on genomic DNA with indicated primers as shown: o1: oPAP8_rtpF, o2: oPAP8_E3R, o3: oPAP8_rtpR, oLB: oLBb1.3, EF1α: ELONGATION FACTOR 1α, WT: wild type, pap8-1: homozygous albino plant, Ht: heterozygous green plant; T: T-DNA, arrowhead: 670-bp contaminant amplification product used as loading control. C. RT–PCR on wild type and pap8-1 homozygous plants grown in the dark for 3 days followed with 72-h growth under white light to allow greening of the wild type; EF1α used as control. D. Sequence alignment of predicted full-length orthologous PAP8 protein found in representatives of major phylogenetic clades Arabidopsis thaliana, At1g21600; Oryza sativa Indica, EEC67529.1; Amborella trichopoda, XP_006827378.1; Selaginella moellendorffii, XP_002976643.2; Physcomitrella patens, XP_024396032.1. cTP, chloroplast transit peptide as predicted with ChloroP1.1 (www.cbs.dtu.dk) underlined in yellow; HCR shaded in grey, highly conserved region. (*), (:) or (.), conserved, strongly similar or weakly similar amino acid properties (standards from www.uniprot.org). Amino acids colours as in Clustal Omega (red (AVFPMILW): small + hydrophobic [includes aromomatic − Y]); blue (DE): acidic; magenta (RHK): basic; green (STYHCNGQ): hydroxly + sulfhydryl + amine + G.) bNLS, bipartite NLS as predicted with NLS mapper (http://nls-mapper.iab.keio.ac.jp). E. Half-open siliques of a heterozygous plant showing the embryo greening; scale bar equals 250 μm. The given number is the position rank of the silique from top to bottom of the inflorescence presenting the segregation of homozygous and heterozygous seeds based on their ability to transiently develop chloroplasts. F. Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 μmol m−2 s−1); scale bar equals 20 mm. Source data are available online for this figure. Source Data for Figure 1 [embj2020104941-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint In all orthologous proteins, a nuclear localization signal (NLS) could be predicted, pointing to a possible localization of the protein inside the nucleus. Functional complementation (Fig EV1) was carried out following the strategy presented in Appendix Fig S2. The full-length coding sequence of PAP8 driven by 1.1 kb of its own promoter (pPAP8::PAP8; Appendix Table S1) could fully restore the greening of the mutant with a chlorophyll content undistinguishable from that of the wild type (Fig EV1D and E). Heterozygotes were phenotypically undistinguishable from wild type except within the developing silique where one quarter of the embryos were unable to green (Fig 1E) following, without gametic distortion, Mendel's ratio for the segregation of recessive alleles (Appendix Fig S1B). Mutant homozygotes, however, were albino and sporophytic lethal, with a strong reduction in cotyledon size (Appendix Fig S1C). pap8-1 dies quickly after light exposure unless grown in vitro on a carbon source in dimmed light (Fig 1F). Albeit their heterotrophic growth, plants pursued a rather normal development until reproduction. PAP8 is, therefore, a specific factor essential for chloroplast biogenesis without noticeably affecting other plastid functions non-related to photosynthesis or the apparent photomorphogenic programme that is associated with de-etiolated plants (such as ceased hypocotyl elongation, apical hook unfolding and cotyledon opening). Click here to expand this figure. Figure EV1. (Related to Fig 1) Functional complementation of pap8-1The construction used for complementation is pPAP8::PAP8cds (pP8::P8 in short): PAP8 coding sequence under control of a 1.1-kb upstream region used as promoter (see pBB389 in Appendix Table S1 and Fig 2A for the description of the regulatory region used as promoter). A. PCR on genomic DNA; L35, L49: Two independent “pBB389” transgenic lines; primers are the same as in Fig 1B and o4: op8i2_R. B. Greening assay on wild type and rescued pap8-1 homozygous plants from third generation transgenic lines (T3) grown in vitro 3 days in the dark followed with a 30-h light treatment. L35 and L49 are two independent rescued lines. C. Phenotypes of pap8-1 homozygous plant grown in vitro, and two representative plants of wild type or pap8-1/pP8::PAP8 (line L35 or line L49) grown on soil. D. Content of total chlorophylls (Chl(a+b)) normalized to fresh weight and relative to wild type in the given genotypes grown in the dark (D) or grown in the dark followed with 30 h of white light treatment (+L); n.a. not applicable. E. Spectrophotometric analysis of pigments: absorption spectra of acetone-soluble extracts from seedling grown in vitro 3 days in the dark (D) or 3 days in the dark plus 30 h of white light (L) Col-0, wild type; p8-1/p8-1, homozygous mutant pap8-1; L35 and L49, two lines of pap8-1/pPAP8::PAP8; n.a., not applicable. Absorbance was normalized to fresh weight (FW); Chla, chlorophyll a; Chlb, chlorophyll b; Car, carotenoids. Source data are available online for this figure. Download figure Download PowerPoint The PAP8 promoter involves typical light-responsive cis-elements PAP genes are co-regulated, at least for a significant part of their transcriptional response (Pfannschmidt et al, 2015; Liebers et al, 2018); as a canonical example, the promoter activity of PAP8 is transitorily specific to tissues with photosynthetic potential such as the cotyledons and leaf primordia. It is first restricted to the epidermis during skotomorphogenesis, induced in the palisade after light exposure and then slowly diminished (Liebers et al, 2018). Searching for cis-regulatory elements by a deletion series of the PAP8 promoter, a short sequence starting at −97 from the transcriptional initiation start (tis) was found to be sufficient to retain cotyledon specificity while a construct starting at position +1 completely lost its reporter activity (Fig 2A and B). The two short versions of the promoter (−257 and −97) driving PAP8 expression were able to complement pap8-1 (Appendix Table S1). Within the 97-bp region (Fig 2C), a nearly palindromic element (GAcGCTC) was predicted to be a putative non-symmetrical element recognized by proteins with basic leucine zipper domains (bZIP). Site-directed mutagenesis of this element resulted in a disturbed GUS expression (Fig 2B). Using PlantPAN3 (Chow et al, 2019), three bona fide elements for bZIP transcription factors (TF) were predicted in both strands of the DNA (Appendix Fig S3 and Table S2). Interestingly, the two bZIP TFs, HY5 and HYH are known to be involved in the early steps of photomorphogenesis (Holm et al, 2002; Li et al, 2017). Hence, a few bZIP TFs, TGA2 as the best prediction according to the two elements found on the plus strand, HY5 and HYH as educated guesses, and bZIP60 as an out-group related to stress response (Iwata et al, 2008) were tested in a dual-luciferase reporter assay (Appendix Fig S4). HY5 proved to be the most efficient, enhancing transcriptional activity of the long (−1,133 bp) PAP8 promoter region by more than fivefold over the control (Fig 2D). For the shorter though functional −97-bp promoter, HY5 promoted transcriptional activity with a twofold increase while a 3-bp replacement in the core of the element yielded significantly reduced activation. Moreover, recombinant HY5 was able to specifically bind the cis-regulatory element in vitro (Fig 2E) in strength comparable to that of the canonical G-box element used as competitor (Yoon et al, 2006). In addition, the release of chromatin-immuno-precipitation (ChIP) sequencing data using “GFP” antibody on a hy5/HY5::HY5-YFP genetic background (Hajdu et al, 2018) allowed the detection of HY5 on the 5′-region containing the identified regulatory element and the 3′-region of PAP8 after blue light or red light exposure (Fig 2F). While the expression of PAPs is essential for greening, hy5 mutants display slight greening defects indicating that functional redundancies and compensations occur in the regulation of its target genes (Gangappa & Botto, 2016). For example, the paralogous transcription factor HYH (Holm et al, 2002) is also active on the PAP8 promoter (Appendix Fig S4). In conclusion, ChIP and EMSA indicate that HY5 can bind the PAP8 promoter and that it can activate the promoter in a heterologous system, but given that no expression changes were seen in a hy5-1 mutant, possibly due to functional redundancy, the ChIP-seq/EMSA/transactivation data remain to be challenged in more sophisticated genetic backgrounds. Moreover, the epidermal specificity of the PAP-promoter activity during skotomorphogenesis may result from a separate pathway linked to cell identity in relation to development. In this context though, it is of interest to note that PHYB promoter activity in the dark shows a pattern similar to that of the PAP8 promoter (Somers & Quail, 1995). It is, thus, unlikely that the −97-element is solely responsible for the transcriptional regulation of PAP8. Future investigations will focus on the network that may regulate PAP8 and the PAPs in general. It would be of great interest to test (i) the role of the 3′-UTR element of PAP8, where HY5 is also sitting, and (ii) whether the PIFs play a role in the dark-dependent expression of the PAPs in the epidermal cell layer and/or as repressors in the palisade. Figure 2. HY5 is a potential regulator of PAP8 expression A. PAP8 promoter deletion strategy. ERI, EcoRI site; HIII, HindIII site; indicated positions are given relative to the transcription start noted as +1; red boxes represent untranslated regions; pink boxes, ORF of an upstream gene; nearly palindromic element is given in blue. −97m3: mutated promoter as described in C with three mutations (m3) indicated with “***”. B. Two or six (−97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm. C. Proximal PAP8 promoter region; m3, 3-bp substitutions within the −97-bp promoter; 5′-UTR in red; ATG, start codon of PAP8. D. Dual-luciferase reporter assay; Renilla luciferase (Rluc) used as internal control and GFPer used as control for the transfected area; the promoters driving Firefly luciferase (Fluc) were transfected in onion epidermis cells without or with constitutively expressed HY5. The Fluc/Rluc activity was set to 1 for the minus-HY5 control; mean ± standard error corresponding to 3 replicates; photon counts are given in source data. *ε-test = 3.43 > 1.96 corresponding to P-value < 0.001. E. Electromobility shift assay of a probe corresponding to the near palindromic PAP8 element (GAcGCTC) with recombinant HY5 protein; a probe containing a canonical G-box element (CACGTG) recognized by HY5 was used as cold competitor. F. Integrative genomics viewer (IGV) images of the chromatin immunoprecipitation (ChIP) sequencing data30 at the PAP8 locus; TAIR10, annotation according to the Arabidopsis thaliana information resource orange box indicates the PAP8 locus. ChIP on hy5-ks50; 35S:HY5-YFP exposed to blue light or red light using GFP antibody and compared with mock corresponding to ChIP control experiment done without antibody. Each treatment is presented as track overlay of triplicates: the read count is given within the “group autoscale” range in brackets. Close up on the 5′-UTR region centred on the −95-promoter element in yellow. Source data are available online for this figure. Source Data for Figure 2 [embj2020104941-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint PAP8 functions in plastids and in the nucleus PAP8 disp
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