Research BBX16 mediates the repression of seedling photomorphogenesis downstream of the GUN1/GLK1 module during retrograde signalling Nil Veciana1* , Guiomar Martın1* , Pablo Leivar2 and Elena Monte1,3 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, 08193 Barcelona, Spain; 2Laboratory of Biochemistry, Institut Quımic de Sarria, Universitat Ramon Llull, 08017 Barcelona, Spain; 3Consejo Superior de Investigaciones Cientıficas (CSIC), 08028 Barcelona, Spain Summary Author for correspondence:  Plastid-to-nucleus retrograde signalling (RS) initiated by dysfunctional chloroplasts impact Elena Monte photomorphogenic development. We have previously shown that the transcription factor Email: elena.monte@cragenomica.es GLK1 acts downstream of the RS regulator GUN1 in photodamaging conditions to regulate not only the well established expression of photosynthesis-associated nuclear genes (PhANGs) Received: 5 November 2021 but also to regulate seedling morphogenesis. Specifically, the GUN1/GLK1 module inhibits Accepted: 5 January 2022 the light-induced phytochrome-interacting factor (PIF)-repressed transcriptional network to suppress cotyledon development when chloroplast integrity is compromised, modulating the New Phytologist (2022) 234: 93–106 area exposed to potentially damaging high light. However, how the GUN1/GLK1 module doi: 10.1111/nph.17975 inhibits photomorphogenesis upon chloroplast damage remained undefined.  Here, we report the identification of BBX16 as a novel direct target of GLK1. BBX16 is Key words: B-box factor BBX16, chloroplast induced and promotes photomorphogenesis in moderate light and is repressed via GUN1/ integrity and retrograde signalling, cotyledon GLK1 after chloroplast damage. Additionally, we showed that BBX16 represents a regulatory opening, GLK1, GUN1, light signalling, branching point downstream of GUN1/GLK1 in the regulation of PhANG expression and photomorphogenesis, photoprotection. seedling development upon RS activation.  The gun1 phenotype in lincomycin and the gun1-like phenotype of GLK1OX are markedly suppressed in gun1bbx16 and GLK1OXbbx16.  This study identified BBX16 as the first member of the BBX family involved in RS, and defines a molecular bifurcation mechanism operated by GLK1/BBX16 to optimise seedling de- etiolation, and to ensure photoprotection in unfavourable light conditions. One of the most dramatic developmental transitions in plants Introduction is de-etiolation, in which a germinating seedling experiences light To cope with their sessile condition, plants need to optimise their for the first time (Arsovski et al., 2012; Gommers & Monte, growth and development in response to changes in their habitat. 2018). When germinating in the dark, skotomorphogenic Light is a critical environmental component necessary for photo- seedlings growing heterotrophically exhibit fast-growing synthesis and for the regulation of growth and development hypocotyls, unexpanded and appressed cotyledons with etioplas- (Arsovski et al., 2012). Required as a primary source of energy ts, and the formation of an apical hook to protect the apical and as an informative cue, light also represents a challenge for meristem from damage. In the light, de-etiolated or photomor- plant life when in excess. Plants have therefore evolved exquisite phogenic seedlings adapt their morphology to enhance light cap- methods for light sensing and signalling to allow the appropriate ture for photosynthesis, which involves inhibition of hypocotyl adaptive response. Light of different wavelengths is perceived by elongation, hook unfolding, stimulation of cotyledon separation different photoreceptors. Phytochromes sense red and far-red and expansion, and the formation of the photosynthetic appara- light (600–750 nm), whereas cryptochromes, phototropins, and tus and fully functional chloroplasts. Zeitlupes perceive blue and UVA (320–500 nm) and UVR8 Distinct transcriptomic landscapes underlay the skotomor- senses UVB (Galv~ao & Fankhauser, 2015). Light perception by phogenic and photomorphogenic programmes, regulated by a photoreceptors can be complemented by chloroplasts, which act suite of positive- and negative-acting factors (Ma et al., 2001; Jiao as sensors of environmental changes and contribute to responses et al., 2005; Pham et al., 2018; Shi et al., 2018; Jing & Lin, in high light (Chan et al., 2016). 2020). Major positive regulators are HFR1, HY5/HYH and LAF1 (Lau & Deng, 2012; Xu et al., 2015, 2016), whereas *These authors contributed equally to this work. phytochrome-interacting factors (PIFs) act as major negative- Ó 2022 The Authors New Phytologist (2022) 234: 93–106 93 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. New 94 Research Phytologist acting factors of photomorphogenesis (Castillon et al., 2007; 2016), and GLK1 subsequently promote photosynthetic appara- Leivar & Quail, 2011; Leivar & Monte, 2014). PIFs (PIF1, tus formation by directly inducing the expression of nuclear- PIF3-8) are basic helix–loop–helix (bHLH) transcription factors encoded photosynthetic genes (PhANGs) such as those from the (Toledo-Ortiz et al., 2003) that bind to G-box (CACGTG) and LHCb gene family (Waters et al., 2009). Under photodamaging PBE (CACATG) DNA elements in the dark to inhibit or activate conditions, however, RS is activated (Ruckle et al., 2007; the expression of light-induced or light-repressed genes, respec- Estavillo et al., 2011; Kindgren et al., 2012) leading to the repres- tively (Leivar et al., 2009; Zhang et al., 2013; Pfeiffer et al., sion of GLK1 expression and downregulation of PhANGs 2014). The quadruple mutant pifq lacking PIF1, PIF3, PIF4 and (Waters et al., 2009; Martın et al., 2016). The use of drugs such PIF5 displays a partial constitutively photomorphogenic pheno- as lincomycin specifically inhibits plastid translation and activates type in the dark, suggesting that PIFs promote skotomorphogen- RS and repression of PhANG expression (Oelm€ uller et al., 1986; esis (Leivar et al., 2008; Shin et al., 2009). Upon illumination, Sullivan & Gray, 1999). Genomes uncoupled (gun) mutants phytochromes become active and trigger PIF inactivation and exhibit PhANG derepression in response to these drugs, and have degradation through the 26S proteasome-mediated pathway, helped elucidate components of RS-like tetrapyrroles such as allowing seedlings to initiate light-regulated gene expression and heme, and GUN1 (Koussevitzky et al., 2007; Chan et al., 2016). follow a photomorphogenic programme of development (Leivar Importantly, RS has been shown to impact light-regulated et al., 2008, 2009; Pham et al., 2018). Additional transcription seedling development in high light environments to prevent pho- factors involved include the GOLDEN2-LIKE 1 (GLK1) and todamage, through a GUN1-mediated mechanism that is still GLK2 (Chen et al., 2016) and members of the B-box family not well defined (Ruckle et al., 2007; Martın et al., 2016). It is (BBX) (Khanna et al., 2009; Gangappa & Botto, 2014; Su et al., also currently unknown whether light regulation of seedling 2015; Song et al., 2020a). Whereas GLKs target genes involved development and PhANG expression after RS activation operate in chlorophyll biosynthesis, light harvesting and electron trans- through the same components. port are necessary for chloroplast development (Fitter et al., We have previously shown that the RS and phytochrome path- 2002; Waters et al., 2008, 2009; Oh & Montgomery, 2014; ways converge to antagonistically regulate the PIF-repressed Zubo et al., 2018), some BBX members have been described as light-induced transcriptional network (Martın et al., 2016). Our general positive regulators of photomorphogenesis (e.g. BBX4/ findings showed that GLK1 acts downstream of GUN1 to modu- COL3, BBX11, BBX20/BZS1, BBX21/STH2 and BBX22/ late not only PhANG expression but also seedling morphogenesis LZF1) (Datta et al., 2006, 2007, 2008; Chang et al., 2008; Fan in photodamaging conditions. Specifically, GUN1/GLK1- et al., 2012; Xu et al., 2018; Job & Datta, 2021), and some as mediated RS antagonise phytochrome/PIF signalling to inhibit negative regulators (e.g. BBX18/DBB1a, BBX19/DBB1b, cotyledon separation and expansion when chloroplast integrity is BBX24/STO, BBX25/STH, BBX28, BBX29, BBX30, BBX31 compromised, effectively reducing the area exposed to potentially and BBX32/EIP6) (Datta et al., 2006; Khanna et al., 2006; damaging high light. How this is achieved is still unclear, but Indorf et al., 2007; Kumagai et al., 2008; Holtan et al., 2011; does not involve the reaccumulation of PIF proteins in these con- Wang et al., 2011, 2015; Gangappa et al., 2013; Lin et al., 2018; ditions (Martın et al., 2016), therefore suggesting the participa- Heng et al., 2019b; Song et al., 2020b; Ravindran et al., 2021). tion of yet undefined components (Supporting Information In addition, the role in photomorphogenesis of BBX23/MIDA10 Fig. S1). Here, we address the question of how the GUN1/GLK1 appears to be organ specific (positive for hypocotyl elongation) module inhibits photomorphogenesis upon chloroplast damage, (Zhang et al., 2017) and negative for hook unfolding (Sentan- and report the identification and characterisation of BBX16 as a dreu et al., 2011). The protein stability of several of these tran- novel GLK1 target. BBX16 promotes photomorphogenesis scription factors (e.g. HY5, LAF1, HFR1, BBX21, BBX22 and downstream of PIF and GLK1 in moderate light and is repressed others) is directly modulated by the COP1/SPA complex acting via the GUN1/GLK1 module after chloroplast damage. Addi- as an E3 ubiquitin ligase, which interacts and targets them for tionally, we showed that BBX16 represents a regulatory branch- degradation via the 26S proteasome pathway in darkness (Yi & ing point in the regulation of PhANG expression and seedling Deng, 2005; Hoecker, 2017). development upon RS activation. In Arabidopsis, chloroplast biogenesis during seedling de- etiolation depends on the expression of chloroplast proteins Materials and Methods encoded by the nuclear genome (c. 2000–3000) (Li & Chiu, 2010) (anterograde regulation) that are imported into the chloro- Plant material and growth conditions plast following synthesis in the cytosol (Jung & Chory, 2010). In turn, chloroplasts can communicate with the nucleus through Arabidopsis thaliana wild-type and mutant seeds used in this retrograde signalling (RS) to regulate nuclear gene expression study have been described previously. gun1 (gun1-201) (Martın according to chloroplast status (Kleine et al., 2009; Jarvis & et al., 2016), glk1 and glk1glk2 (Fitter et al., 2002), GLK1OX and Lopez-Juez, 2014). This coordination between the nucleus and GLK1OX-GFP (both on the glk1glk2 background) (Waters et al., chloroplast genomes ensures optimised photosynthetic capacity 2008) are on the Col-0 background; whereas col7, BBX16OX and growth (Ruckle et al., 2007; Hills et al., 2015; Martın et al., #10 and BBX16OX #11 (here renamed as bbx16-1, BBX16OX1 2016). Moderate light intensities during de-etiolation induce and BBX16OX2, respectively) (H. Wang et al., 2013) are on the expression of the PIF-repressed target gene GLK1 (Martın et al., Col-4 background. BBX16OX lines express the BBX16 open New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 95 reading frame under the control of the 35S promoter and were used for real-time PCR (Light Cycler 480; Roche) using SYBR described to overexpress BBX16 c. 250-fold (H. Wang et al., Premix Ex Taq (Roche) and primers at a 300 nM concentration. 2013). bbx16-1 is an insertional mutant from the GABI-Kat col- Gene expression was generally measured in three independent lection (GABI-639C04) with a T-DNA insertion in the second biological replicates, and at least two technical replicates exon of BBX16 (H. Wang et al., 2013). A new second BBX16 were used for each of the biological replicates. PP2A mutant allele (named bbx16-2) was obtained from the SALK col- (AT1G13320) was used for normalisation as described (Shin lection (SALK_036059), harbouring a T-DNA insertion in the et al., 2007). Primers sequences used for qRT-PCR are described first exon (Fig. S2). gun1bbx16-1 was obtained by crossing gun1- in Table S2. 201 to bbx16-1; wild-type (WT) (Col-0 9 Col-4 background), gun1 and bbx16 siblings from the cross were selected to be used Chromatin immunoprecipitation assay in the experiments shown in Fig. 4. GLK1OXbbx16-1 and GLK1OXbbx16-2 were generated by crossing GLK1OX to bbx16- Chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays 1 and to bbx16-2, respectively. The obtained mutants were were performed as described previously (Martın et al., 2018) selected to maintain the glk1glk2 background in GLK1OX; using the previously described 35S::GLK1OX-GFP line (Waters GLK1OX siblings from each cross were selected to be used as con- et al., 2008). Seedlings (3 g) were vacuum infiltrated with 1% trols. Seeds were surface sterilised in 20% bleach and 0.25% formaldehyde and cross-linking was quenched using vacuum sodium dodecyl sulfate (SDS) for 10 min and plated on half- infiltration with 0.125 M glycine for 5 min. The tissue was strength Murashige and Skoog (0.59 MS) medium without ground, and nuclei-containing cross-linked protein and DNA sucrose, stratified at 4°C in the dark for 4 d, exposed to white were purified using sequential extraction with extraction buffer 1 light for 3 h to induce germination, and then placed under the (0.4 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 5 mM specific light conditions indicated in each experiment. For experi- b-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride ments carried out under continuous conditions, plates were (PMSF), 50 mM MG132, proteinase inhibitor cocktail); buffer 2 placed under white light (5 µmol m 2 s 1) or darkness for 3 d (0.25 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 1% unless otherwise indicated. In the text we refer to low light Triton X-100, 5 mM b-mercaptoethanol, 0.1 mM PMSF, (< 25 µmol m 2 s 1), light (100–150 µmol m 2 s 1), and high 50 mM MG132, proteinase inhibitor cocktail); and buffer 3 light (> 300 µmol m 2 s 1), whereas the specific light intensity (1.7 M sucrose, 10 mM Tris-HCl pH 8, 0.15% Triton X-100, used in each experiment is specified in the corresponding figure 2 mM MgCl2, 5 mM b-mercaptoethanol, 0.1 mM PMSF, legend. For lincomycin treatments, the medium was supple- 50 mM MG132, proteinase inhibitor cocktail). Nuclei were mented with 0.5 mM lincomycin (Sigma L6004) (Sullivan & resuspended in nuclei lysis buffer (50 mM Tris-HCl pH 8, Gray, 1999). Primers sequences used for genotyping are provided 10 mM EDTA, 1% SDS, 50 mM MG132, proteinase inhibitor in Table S1. cocktail), sonicated 10 times for 30 s each, and diluted in 10 vol- umes of dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl). Overnight Phenotypic measurements incubation was performed with the corresponding antibody (or Hypocotyl length, cotyledon area and cotyledon aperture were with no antibody as control) at 4°C and immunoprecipitation measured as described previously (Sentandreu et al., 2011), using was performed using Dynabeads. Washes were done sequentially NIH Image software (IMAGEJ; National Institutes of Health). in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, The median was calculated from at least 20 seedlings and 20 mM Tris-HCl pH 8, 150 mM NaCl), high salt buffer (0.1% the experiments were repeated at least two times with similar SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, results. 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% deoxycholic acid sodium, 1 mM EDTA, 10 mM Tris-HCl pH 8), and 19 TE. Immunocomplexes were eluted in elution buffer Quantitative reverse transcriptase (1% SDS, 0.1 M NaHCO3), de-crosslinked overnight at 65°C in For quantitative reverse transcriptase (qRT-PCR) analysis, 10 mM NaCl, and then treated with proteinase K. DNA was seedlings were grown in the dark or in white light for the indi- purified using Qiagen columns and eluted in 100 µl of Qiagen cated time. qRT-PCR was performed as described previously elution buffer, and 2 µl were used for qPCR (ChIP-qPCR) analy- (Khanna et al., 2007) with variations. Briefly, 1 µg of total RNA sis using BBX16 promoter-specific primers spanning the regions was extracted using the RNeasy Plant Mini Kit (Qiagen) or using P1 (EMP1180-P1 and EMP1182-P1) and P2 (EMP1175-P2 the Maxwell® RSC Plant RNA Kit (Promega), treated with and EMP1176-P2) containing the predicted binding sites for DNase I (Ambion) according to the manufacturer’s instructions GLK1 (Waters et al., 2009; Franco-Zorrilla et al., 2014), and a (if extracted with Qiagen kit), and first-strand cDNA synthesis pair of primers inside the BBX16 gene body as control was performed using the SuperScript III reverse transcriptase (EMP869-P3 and EMP1177-P3). Three biological replicates (Invitrogen) and oligo(dT) as a primer (dT30) or the NZY First- were performed for 35S::GLK1-GFP (Waters et al., 2008) incu- Strand cDNA Synthesis Kit (NZYTech). In all cases, cDNA was bated with or without antibody. Wild-type controls were per- then treated with RNase Out (Invitrogen) before being sub- formed with one replicate of Col-0 seedlings with or without jected to a 1 : 20 dilution with water, and 2 µl of this mix was antibody. Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 96 Research Phytologist conditions, we aimed to identify genes downstream of GLK1 that Statistical analysis might be involved in the regulation of photomorphogenesis. We Cotyledon angle and hypocotyl length differences between all reasoned that plausible candidates would need to meet the fol- genotypes across the two conditions Light and Light Linc were lowing criteria: (1) be a light-induced gene in a GLK1-dependent analysed using the Kruskal–Wallis test to assess the significance manner and PIF repressed in the dark; (2) promote cotyledon of global variation in a nonparametric dataset. After significant development under moderate light; (3) be a high light- and result of the omnibus test (Kruskal–Wallis) was found, a post- lincomycin-repressed gene via the GUN1/GLK1 module; (4) dis- hoc Dunn test was performed to identify significantly different play reduced sensitivity to RS-inducing treatments when overex- pairs of genotypes taking into account the global variation across pressed in seedlings, preventing RS repression of cotyledon the two conditions. Significantly different pairs of genotypes were development. Additionally, to verify the importance of the represented by letters. Subsequently, we sought to find different selected candidate (represented as X); (5) genetic removal of X in genotypes focusing in the single condition Light Linc. Therefore, gun1 and GLK1OX mutants should suppress their phenotype in a pairwise Mann–Whitney test was used and the significant effect lincomycin at least partly (Fig. S1). was represented with asterisks. To begin our search, we made use of previous data describing To identify differences at the gene expression level between all genes directly targeted and upregulated by GLKs (Waters et al., genotypes taking into account the global variation across the two 2009). We observed that these targets (119 in total) not only conditions Light and Light with Linc (unless otherwise indi- included chloroplast-localised photosynthetic genes (the main cated), and given the parametric nature of the gene expression focus of Waters and colleagues’ work). Significantly, we observed measurements, data were analysed using ANOVA. Upon a signif- among them an enrichment of genes encoding for BBX transcrip- icant result of the omnibus test (ANOVA), a post-hoc Tukey test tion factors, with four of the described 32 BBX family members was performed to identify significant differences between pairs of being present in the list of 119 genes (P-value: 2.46 e-05). More- genotypes. Significantly different pairs of genotypes were repre- over, three of these BBX were members of subclass III, which is sented by letters. composed by four members (BBX14–BBX17). Different BBX To find different genotypes within the single condition Dark, proteins have been involved in several aspects of light-regulated Light or Light Linc, a t-test was performed and asterisks in speci- development (Gangappa & Botto, 2014). In particular, BBX16/ fic samples indicated statistically significant differences between COL7 has been described to play a role in shade responses (H. each mutant and its respective wild-type seedlings. Wang et al., 2013; Zhang et al., 2014), and was considered a good candidate for further characterisation. To start to evaluate this candidate, BBX16 expression was anal- Results ysed in dark-grown and light-grown wild-type, GLK1-deficient glk1 and glk1glk2 (Fitter et al., 2002), and GLK1-overexpression BBX16 is a PIF-repressed gene that is induced by light in a GLK1OX (Waters et al., 2008) seedlings. BBX16 was strongly GLK1-dependent manner upregulated in light-grown wild-type seedlings compared with To elucidate how the PIF/GLK1 and GUN1/GLK1 modules dark, and this induction required GLK1 (Fig. 1a). BBX16 is a regulated cotyledon development under different light PIF-repressed gene, although not described as a direct target (a) (b) BBX16 expression BBX16 expression 2.0 ** 30 *** Dark Light 1.5 ** 20 1.0 10 0.5 ns 0 0 l-0 fq -0 k1 -0 k2 X 1O pi ol ol gl gl Co C C k1 LK gl G Dark Light Dark Fig. 1 BBX16 is a phytochrome-interacting factor (PIF)-repressed gene whose expression is induced by light in a GOLDEN2-LIKE 1 (GLK1)-dependent manner. (a, b) Transcript levels of BBX16 analysed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in (a) 3-d-old Col-0, pifq and glk1 and (b) Col-0, glk1glk2 and GLK1OX Arabidopsis seedlings grown in the dark or in continuous white light (5 µmol m 2 s 1) as indicated. Values were normalised to PP2A, and expression levels are expressed relative to Col-0 light set at one. Data are the means  SE of biological triplicates (n = 3) and asterisks indicate statistically significant differences between each mutant and its respective wild-type (WT) seedlings (t-test; **, P < 0.01; ***, P < 0.001; ns, nonsignificant). New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 97 (Pfeiffer et al., 2014). As such, in pifq etiolated seedlings, BBX16 section and Fig. S2), and two overexpressing BBX16 lines (OX1 expression showed high levels of expression compared with the and OX2) (H. Wang et al., 2013). Under 3 d of continuous wild-type (Fig. 1a). Interestingly, the expression of the other BBX low light conditions, deficiency of BBX16 in the bbx16 mutants in the same clade showed a similar pattern except for BBX17 led to significantly reduced cotyledon area compared with the (Fig. S3), suggesting that BBX14 and BBX15 might share some wild-type, whereas cotyledons in BBX16-OX1 and OX2 were function with BBX16. Furthermore, GLK1 overexpression in the more expanded (Fig. 2a,b). BBX16-OX1 and OX2 also showed dark induced BBX16 expression (Fig. 1b). Together, these results slightly shorter hypocotyls (Fig. 2c). In addition, dark-grown indicated that, during seedling establishment, BBX16 is a PIF- OX lines displayed faster cotyledon aperture compared with the repressed gene in the dark that is light-induced in a GLK1- wild-type after light exposure (Fig. 2d). Together, these results mediated manner. Therefore, the identified BBX16 met our first indicated that BBX16 contributed to the promotion of early criterion (Fig. S1) and was considered for further genetic and photomorphogenesis with a role in cotyledon development molecular analyses. (and therefore fulfilled the second criterion, Fig. S1), and a possible minor contribution to the inhibition of hypocotyl elongation. BBX16 promotes cotyledon development during seedling de-etiolation Under lincomycin treatment, inhibition of cotyledon Next, to evaluate the role of BBX16 during de-etiolation, we separation involves GUN1-mediated repression of BBX16 analysed the previously described bbx16 T-DNA insertion mutant line col7 (referred here as bbx16-1 for clarity), a newly Next, BBX16 expression was analysed under conditions in which characterised bbx16-2 line (see the Materials and Methods chloroplast integrity was compromised by lincomycin treatment, (a) (b) (c) (d) Fig. 2 BBX16 regulates cotyledon development during early seedling development in continuous light. (a) Boxplot representation of the cotyledon area of BBX16 loss-of-function (bbx16) and gain-of-function (BBX16OX1 and OX2) Arabidopsis mutants grown for 3 d under continuous white light (5 µmol m 2 s 1). (b) Visual phenotypes of Arabidopsis seedlings grown as detailed in (a). Bar, 2.5 mm. (c) Boxplot representation of the hypocotyl length of seedlings grown as detailed in (a). (d) Quantification of the cotyledon angle of 2-d-old dark-grown wild-type (WT), bbx16 and two BBX16 Arabidopsis overexpressor lines transferred to white light (20 µmol m 2 s 1) for the indicated hours (h). The thick lines and shaded areas represent the median and the 95% confidence interval of at least 60 seedlings, respectively. Letters denote the statistically significant differences between genotypes using Dunn’s test at each time point (P < 0.05). (a, c) Boxplots indicate the median (centre line), interquartile range (box limits), and minimum and maximum values (whiskers). Data represent the median of at least 20 seedlings and asterisks indicate statistically significant differences between each mutant and its respective WT seedlings (Mann–Whitney test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant). Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 98 Research Phytologist an inhibitor of chloroplast translation that specifically damages of BBX16 in dark-grown pifq (Fig. 3a). Moreover, the light- the chloroplast under both dark and light conditions (Sullivan & induced expression of BBX16 shown in Fig. 1 was strongly inhib- Gray, 1999). When the chloroplast is perturbed, activation of RS ited in response to lincomycin in wild-type seedlings (Fig. 3b,c), induces downregulation of GLK1 expression in a GUN1- similarly to the reported inhibition of PhANGs and GLK1 expres- mediated manner, impacting cotyledon development (Martın sion (Martın et al., 2016). Importantly, the inhibition of BBX16 et al., 2016). We hypothesised that, under these conditions, expression in lincomycin was only partial in GLK1OX (Fig. 3b), repression of GLK1 should also result in the repression of BBX16 similar to the gun1 mutant (Fig. 3c). Compared with gun1, expression as a downstream effector of GLK1 (criterion 3, BBX16 expression in lincomycin was not significantly affected in Fig. S1). Notably, lincomycin treatment prevented de-repression a gun1glk1 double mutant (Fig. S4), suggesting that the (a) (b) (c) (d) (e) (f) Fig. 3 Downregulation of BBX16 mediated by the GUN1/GLK1 module is necessary to repress cotyledon development under lincomycin treatment. (a) Transcript levels of BBX16 from RNA sequencing of Arabidopsis wild-type (WT) Col-0 and pifq seedlings grown for 3 d in the dark in the absence or presence of lincomycin (Martın et al., 2016). (b, c) Transcript levels of BBX16 analysed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 3-d-old light-grown (5 µmol m 2 s 1) Arabidopsis Col-0 and GLK1OX seedlings (b), and Col-0 and gun1 seedlings (c), in the absence or presence of lincomycin. (d) BBX16 expression levels in 3-d-old dark-grown Arabidopsis WT and gun1 mutant seedlings (time 0 h) exposed to 3 h of high light (310 µmol m 2 s 1) compared with light (130 µmol m 2 s 1). (b–d) Values were normalised to PP2A, and expression levels are expressed relative to Col-0 light (b, c) or Col-0 light 3 h (d), set at one. Data are the means  SE of biological triplicates (n = 3). (a–d) Letters denote the statistically significant differences using Tukey’s test (P < 0.05), and asterisks in specific samples indicate statistically significant differences between each mutant and its respective WT seedlings (t-test; *, P < 0.05; **, P < 0.01). (e) Visual phenotypes (top) and cotyledon angle quantification (of at least 40 seedlings) (bottom) of Arabidopsis WT and BBX16OX seedlings grown as in (b). Representative seedlings grown in presence of lincomycin are shown in the photograph. Bar, 2.5 mm. Boxplots indicate the median (centre line), interquartile range (box limits), and minimum and maximum values (whiskers). Letters denote the statistically significant differences among genotypes by Dunn’s test (P < 0.05). (f) Quantification of the cotyledon angle of 2-d-old dark-grown Arabidopsis WT Col-0, gun1, WT Col-4, bbx16-1 and two BBX16OX lines transferred to white light (10 µmol m 2 s 1) for the indicated times in the presence of lincomycin. The thick lines and shaded areas represent the median and the 95% confidence interval of at least 20 seedlings, respectively. Different letters denote statistically significant differences between genotypes by Dunn’s test at each time point (P < 0.05). Linc, lincomycin. New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 99 inhibition of BBX16 expression downstream of GUN1 might cotyledon aperture that was similar to that of wild-type seedlings require GLK1 and additional factors. without lincomycin (Fig. 3e). Similarly, in a de-etiolation experi- The biological relevance of these findings using lincomycin ment using 2-d-old dark-grown seedlings transferred to light in was assessed by testing BBX16 expression under high light condi- the presence of lincomycin, BBX16OX lines showed reduced tions, which causes GUN1-mediated inhibition of cotyledon sep- sensitivity to lincomycin like gun1, and displayed higher cotyle- aration (Martın et al., 2016). Induction of BBX16 in high light in don angles compared with the wild-type (Fig. 3f). These results the wild-type was reduced compared with normal light (Fig. 3d), indicated that BBX16 also fulfilled criteria 3 (high light and suggesting that high light damage partially inhibits BBX16 induc- lincomycin-repressed (via GUN1/GLK1)) and 4 (OX seedlings tion, in agreement with recent transcriptomic data obtained display reduced sensitivity to RS) (Fig. S1), and provided strong under high light stress (Huang et al., 2019). This effect was not support that RS-imposed GUN1/GLK1-mediated repression of observed in gun1 mutants (Fig. 3d), indicating that this repres- BBX16 was necessary for the inhibition of cotyledon develop- sion was mediated by GUN1. These results are in accordance ment under conditions in which the chloroplast is damaged. with previously observed inhibition of GLK1 under similar con- Importantly, to provide conclusive support for this pathway, ditions (Martın et al., 2016) and suggested that the light induc- we next tested the genetic interactions between GLK1, GUN1 tion of BBX16 downstream of GLK1 is repressed in conditions and BBX16 (criterion 5, gun1-X and GLK1OX-X mutants con- in which RS is active and inhibits GLK1 function. firm X contribution to the pathway) (Fig. S1). Genetic removal Next, we tested whether the transcriptional repression of of BBX16 in GLK1OXbbx16 and gun1bbx16-1 mutants allowed BBX16 in response to RS might contribute to the inhibition of us to determine the contribution of the endogenous BBX16 to seedling deetiolation upon chloroplast damage previously the cotyledon phenotypes of GLK1OX and gun1 in lincomycin observed (Martın et al., 2016). Indeed, BBX16OX lines grown (Figs 4, S5). Markedly, the gun1-like phenotype of GLK1OX in for 3 d in plates containing lincomycin under light were less sen- lincomycin was clearly suppressed in GLK1OXbbx16 (Figs 4a, sitive to lincomycin and were able to de-etiolate, showing a S5c). Similarly, the gun1bbx16-1 double mutant showed strong (a) (b) Fig. 4 Genetic removal of BBX16 partially suppresses the gun1 and GLK1OX open cotyledon phenotype in the presence of lincomycin. (a) Visual phenotypes (left) and quantification of cotyledon angle (right) of 3-d-old light-grown (5 µmol m 2 s 1) Arabidopsis Col-0, Col-4, bbx16-1, GLK1OX and GLK1OX bbx16-1 seedlings in the presence or absence of lincomycin. (b) Visual phenotypes (left) and quantification of cotyledon angle (right) of 2-d-old dark-grown Arabidopsis WT, bbx16-1, gun1, and gun1bbx16-1 seedlings transferred to light (10 µmol m 2 s 1) for 24 h in the presence or absence of lincomycin. (a, b) Bars, 2.5 mm. Letters denote the statistically significant differences among genotypes by Dunn’s test (P < 0.05), and asterisks indicate statistically significant differences between each GLK1OX bbx16-1 mutant and GLK1OX seedlings (Mann–Whitney test; **, P < 0.01; ***, P < 0.001). Boxplots indicate the median (centre line), interquartile range (box limits), and minimum and maximum values (whiskers). Linc, lincomycin. Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 100 Research Phytologist suppression of the open cotyledon phenotype of gun1 (Fig. 4b). downregulation of PhANG expression in response to retrograde Together, we concluded that BBX16 is a promoter of cotyledon signals, we next studied the expression of the described RS- photomorphogenesis in moderate light that is targeted by the regulated PhANGs LCHB1.4, LHCB.2.2, CA1, RBCS1A and GUN1/GLK1 module under high light conditions to protect the RBCS3B (Waters et al., 2009), in low light-grown wild-type, seedling by reducing the exposed cotyledon surface. bbx16, BBX16OX, gun1, GLK1OX and GLK1OXbbx16-1 seedlings. In the absence of lincomycin, LCHB1.4 and LHCB.2.2 expression was similar to that of the wild-type in all GLK1 associates with the promoter of BBX16 lines tested except in GLK1OX, in which expression of both To further understand the mechanism by which the light environ- genes was upregulated as described (Waters et al., 2009), and in ment impacts development through the GLK1 regulation of BBX16-OX, in which LHCB.2.2 expression was approximately BBX16 expression, we aimed to test whether BBX16 was a direct two-fold higher compared with the wild-type (Fig. 6). In downstream target of GLK1 during de-etiolation. Interestingly, response to lincomycin, expression levels in gun1 and GLK1OX analysis of the promoter region of BBX16 revealed two CCAATC lines were derepressed in accordance with Waters et al. (2009), motifs, described as putative GLK1 binding sequences using whereas expression in BBX16-OX seedlings was similar to that Waters et al. (2009) based on the enrichment in the promoter of the wild-type (Fig. 6). In clear contrast, expression of CA1, regions of GLK1 targets. These two motifs were 2101 bp (Motif RBCS1A and RBCS3B was similar to that of the wild-type in all 1) and 767 bp (Motif 2) upstream of the transcriptional start site lines in the absence of lincomycin, but interestingly their (TSS) (Fig. 5a). Chromatin immunoprecipitation (ChIP) fol- expression in BBX16OX in the presence of lincomycin was dere- lowed by qPCR in light-grown seedlings expressing GLK1-GFP pressed compared with the wild-type, similarly to gun1 (Fig. 6). (Waters et al., 2008) detected strong specific binding of GLK1 to Together, these results can be interpreted to suggest that the BBX16 promoter specifically in the region that spanned Motif BBX16 does not mediate the regulation of the LCHB1.4 and 2 (P2), whereas no binding was detected in the region containing LHCB.2.2 upon chloroplast damage, whereas BBX16OX Motif 1 (P1) or a control sector within the gene body (P3) exhibits a gun-like phenotype for some PhANGs such as CA1, (Fig. 5b). This result indicated that BBX16 is indeed a direct target RBCS1A and RBCS3B. This difference may be indicative of of GLK1 during seedling de-etiolation. Interestingly, we observed branching in signalling downstream of GLK1, whereby GLK1- that the region spanning Motif 2 also contained an AGATTCT mediated regulation of some PhANGs might be indirect sequence in the reverse strand, identified as a potential GLK1 through transcriptional regulation of BBX16 and possibly other binding site using protein-binding microarrays (Franco-Zorrilla factors. Indeed, whereas LCHB1.4 and LHCB.2.2 were et al., 2014). It is currently unknown whether the two binding ele- described as GLK1 primary targets, CA1, RBCS1A and RBCS3B ments in the region spanning Motif 2 are necessary for GLK1 failed to meet the criteria to be considered in this group association with the BBX16 promoter. (Waters et al., 2009). Importantly, CA1, RBCS1A and RBCS3B transcript levels in lincomycin were similar in GLK1OX and GLK1OXbbx16 (Fig. 6). This was in contrast with the clear sup- BBX16 mediates regulation of only some GLK1-regulated pression of the GLK1OX cotyledon phenotype in PhANG genes GLK1OXbbx16 shown above (Fig. 4), suggesting that for GLKs are key regulators of PhANGs (Waters et al., 2009; Zubo PhANG expression the contribution of endogenous BBX16 et al., 2018). To test whether BBX16 participates in the under these conditions might be relatively small. (a) (b) Fig. 5 GOLDEN2-LIKE 1 (GLK1) binds to the BBX16 promoter. (a) Schematic representation of the BBX16 promoter and gene body. GLK1 binding sites (CCAATC and AGAATCT) (Waters et al., 2009; Franco-Zorrilla et al., 2014) are indicated with vertical lines in the promoter, and the regions recognised by primer pairs P1, P2 and P3 used in chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) are underlined (Supporting Information Table S2). (b) GLK1 binding to the BBX16 promoter in 3-d-old white light (5 µmol m 2 s 1) grown Arabidopsis Col-0 and GLK1OX-GFP seedlings. Data for GLK1OX-GFP correspond to three independent ChIP experiments and error bars indicate the SE. Col-0 controls correspond to one biological replicate. Letters denote the statistically significant differences among GLK1OX-GFP samples by Tukey’s test (P < 0.05). Ab, samples immunoprecipitated with antibody; No Ab, control samples immunoprecipitated without antibody. New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 101 Fig. 6 BBX16 regulation of PhANG genes in response to lincomycin. Expression of LHCB2.2, LHCB1.4, RBCS3B, RBCS1A and CA1 was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in Arabidopsis wild-type (WT), bbx16, BBX16OX, gun1, GLK1OX and GLK1OXbbx16 seedlings grown for 3 d in white light (5 µmol m 2 s 1) in the absence or presence of lincomycin. Expression levels relative to Col-0 light are shown. Data are the means  SE of biological triplicates. Letters denote the statistically significant differences among genotypes by Tukey’s test at each condition (P < 0.05). Linc, lincomycin. Labelling is indicated by colour, and sequence of represented genotypes is the same within each graph in the Light and Light Linc sections. unknown (Martın et al., 2016). Here, we show that GLK1 Discussion directly induces BBX16 to promote cotyledon development dur- The establishment of young seedlings after germination is a ing seedling de-etiolation in light conditions, sustaining normal highly vulnerable process regulated by a myriad of factors, light photosynthetic activity. By contrast, activation of RS under high being one of the most important (Gommers & Monte, 2018). light prevents BBX16 upregulation through GUN1-mediated Light induces transcriptional changes of hundreds of genes repression of GLK1, and probably other factors, and this keeps involved in de-etiolation (Ma et al., 2001), many of them directly the cotyledons underdeveloped to reduce the photosynthetic tis- regulated by the phytochrome/PIF system, including GLK1 sues exposed to light. Therefore, the identification of BBX16 as a (Leivar et al., 2009; Pfeiffer et al., 2014). However, because too direct target of GLK1 in the regulation of photomorphogenesis much light is detrimental for chloroplast function and can hinder defines a new molecular mechanism to optimise development establishment, seedlings in potentially photodamaging light initi- during seedling de-etiolation and to ensure photoprotection of ate RS and inhibit de-etiolation (Ruckle et al., 2007; Martın the organism in unfavourable light conditions (Fig. 7). et al., 2016). This process is mediated by the nuclear-encoded chloroplast-localised PRR protein GUN1, which accumulates BBX16 defines a signal branching hub in chloroplast-to- preferentially during the early stages of chloroplast biogenesis and nucleus RS downstream of the GUN1/GLK1 module under RS conditions (Wu et al., 2018), through a process that is not yet well understood but may require physically interaction Our finding that GLK1 targets BBX16 to regulate cotyledon with a large number of proteins (Pesaresi & Kim, 2019; Jiang & development and to possibly regulate some PhANGs indirectly, Dehesh, 2021; Wu & Bock, 2021) involved in plastid translation whereas other PhANGs are directly regulated by GLK1, estab- machinery (Tadini et al., 2016; Marino et al., 2019), tetrapyrrole lishes a branching point in the regulation of seedling morphology biosynthesis (Shimizu et al., 2019), RNA editing (Zhao et al., downstream of the GUN1/GLK1 module. This indicates that 2019), and plastidial import (Khanna et al., 2009; Wu et al., the signal that GLK1 relays diversifies to specifically regulate dif- 2019; Tadini et al., 2020). Given all these putative interactions, ferent processes central to seedling de-etiolation. Signalling net- GUN1 has been proposed to act as a scaffold protein that pro- work branching is common in all organisms and contributes to motes protein complex formation (Colombo et al., 2016), and establishing complex responses to a given unique stimulus (Purvis may allow GUN1 to function as an integrator of signals from sev- et al., 2008). Interestingly, signal branching was previously eral RS pathways. Downstream of GUN1, the nuclear-localised described downstream of the PIFs to regulate different organ- GLKs directly regulate PhANG expression to inhibit chloroplast specific pathways during seedling de-etiolation (Sentandreu et al., development (Waters et al., 2009). The GUN1/GLK1 module 2011), in which the BBX protein BBX23/MIDA10 was shown has also been shown to be central to the regulation of seedling to predominantly regulate hook unfolding. Here, whereas direct morphology, although how this takes place was previously GLK1 targeting of some PhANG genes might allow for fast Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 102 Research Phytologist 2018), or BBX7 and BBX8 to cold stress (Li et al., 2021). In addition, altered expression levels of BBX19 were found in ceh1, a mutant with high levels of the MEcPP retrograde signal (Xiao et al., 2012), although the significance is still unclear (Wang et al., 2014). Interestingly, a recent bioinformatics analy- sis of the BBX family identified that the promoter region of BBX16 contains cis elements predicted to be abscisic acid, low temperature and drought responsive (Lyu et al., 2020), which could indicate a role for BBX16 in the cross-talk between differ- ent stress pathways. The BBX family in Arabidopsis thaliana consists of 32 proteins arranged into five structural groups (I–V) based on the number of B-box motifs (one or two) and the presence or absence of a CCT domain and a VP motif (Robson et al., 2001; Kumagai et al., 2008; Khanna et al., 2009; Gangappa & Botto, 2014). BBX16/COL7 belongs to the Class III clade, the least charac- terised of the BBX groups, together with BBX14/COL6, BBX15/ COL16 and BBX17/COL8, defined by having only one B-box motif (B-box 1) in combination with a CCT domain. The expression patterns shown in Fig. S3 indicated that BBX14 and BBX15 respond similarly to BBX16. Because functional redun- dancy is common among members of the same clade within tran- scription factor families (Soy et al., 2014; Pfeiffer et al., 2014; Fig. 7 The GUN1/GLK1 module regulates BBX16 expression during Zhang et al., 2017; Leivar et al., 2020; Martın et al., 2020) this retrograde signalling. Downstream branching of GOLDEN2-LIKE 1 (GLK1) led us to speculate that BBX14 and BBX15 might share some signalling directly induces two independent transcriptional pathways to functional aspects with BBX16. Redundancy within this clade regulate expression of (1) photosynthesis-associated nuclear genes (PhANGs) such as LHCB2.2 and LHCB1.4; and (2) BBX16 to implement would imply that the bbx16 mutant still retains functionality and, cotyledon development, and indirect regulation of PhANGs such as CA1, accordingly, we detected more prominent cotyledon phenotypes RBCS1A and RBCS3B, possibly with involvement of other factors (denoted in BBX16-OX compared with bbx16. Future genetic characterisa- as ?). In the dark, phytochrome-interacting factors (PIFs) bind to the GLK1 tion of single and high order mutant combinations in bbx14, promoter to directly repress GLK1 expression. In response to normal light, bbx15 and bbx16 will shed light on possible functional redun- activated phytochromes (Phys) release PIF repression on the GLK1 promoter, which triggers GLK1 transcription. If chloroplast integrity is dancy and address whether BBX14 and BBX15 might also play a disrupted by lincomycin or high light, retrograde signals emitted by regulatory role in response to chloroplast damage. Interestingly, a dysfunctional chloroplasts induce GUN1-mediated repression of GLK1 recent transcriptomic study identified the Class III clade as a expression (and possibly other factors not depicted in the model) by a yet potential player in response to high light (Huang et al., 2019). unknown mechanism, preventing BBX16 and PhANGs transcription to Of future interest will be, as well, to explore whether the BBX block the progression of photomorphogenesis. Arrows and blunt arrows represent positive and negative regulation, respectively, and the dashed family of transcription factors has functionally evolved and arrow represents indirect effects through an unknown intermediate diverged to specialise only in the Class III clade in RS regulation, factor(s). or whether BBX factors from other clades might also be involved. The domain-function structure of BBX16, a promoter of regulation of chloroplast protection to, for example, fluctuations photomorphogenesis in light conditions, branching of the signal to repress BBX16 and its target effectors would entail a slower response to arrest cotyle- The domain structure of BBX proteins has important functional don development only in more sustained high light conditions, a implications. B-box domains have been involved in protein–pro- possibility that needs further investigation. tein interactions and transcriptional regulation, whereas the CCT harbours a nuclear localisation signal (NLS) to mediate nuclear protein transport (Robson et al., 2001), and has also been shown BBX16 is the first described BBX protein involved in RS to participate in the association with DNA (Ben-Naim et al., Our finding that BBX16 is a downstream target of the GUN1/ 2006; Tiwari et al., 2010). CCT-containing BBX proteins GLK1 module in RS-regulated development identifies the first include CONSTANTS (BBX1/CO), one of the best studied BBX protein involved in the response to chloroplast damage. BBX proteins and the founder of the family. In cotyledons, CCT This adds to previously described members of the BBX family is required to interact with the E3 ubiquitin ligases COP1 and with regulatory roles in stress-induced signalling pathways, such SPA proteins (Laubinger et al., 2006; Jang et al., 2008), whereas as BBX24/STO in responses to salt (Nagaoka & Takano, 2003), the B-box1 domain mediates interaction with BBX19 (Wang BBX18 and BBX23 to heat (Q. Wang et al., 2013; Ding et al., et al., 2014). In the regulation of seedling photomorphogenesis, New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 103 some BBX proteins are related to the COP1/SPA-HY5 regulatory supported by grants from FEDER/Ministerio de Ciencia, Inno- hub (Gangappa & Botto, 2014; Song et al., 2020a; Xu, 2020). vacion y Universidades – Agencia Estatal de Investigacion (Pro- Several of these BBX proteins interact with COP1 and are regu- ject References BIO2015-68460-P and PGC2018-099987-B- lated in a COP1-dependent manner, and/or regulate HY5 tran- I00) and from the CERCA Programme/Generalitat de Catalunya scription, stability or activity (Datta et al., 2006; Chang et al., (Project Reference 2017SGR-718) to EM. We acknowledge 2011; Holtan et al., 2011; Jiang et al., 2012; Gangappa et al., financial support from the Spanish Ministry of Economy and 2013; Huang et al., 2014; Wei et al., 2016; Xu et al., 2016; Competitiveness, through the ‘Severo Ochoa Programme for Zhang et al., 2017; Ding et al., 2018; Job et al., 2018; Lin et al., Centres of Excellence in R&D’ 2016–2019 (SEV-2015-0533). 2018; Bursch et al., 2020). Furthermore, BBX4 has been shown to interact with PIF3 and repress its activity in red light (Heng Author contributions et al., 2019a), whereas BBX18 and BBX23 have been shown to interact with ELF3 and regulate thermomorphogenesis in Ara- EM, PL, GM and NV conceived the project and planned the bidopsis (Ding et al., 2018). Whether BBX16 is regulated by the experiments. GM and NV performed experiments and analysed COP/SPA system, and whether BBX16 regulation of cotyledon the data. All authors wrote the manuscript. NV and GM con- development downstream of the GUN1/GLK1 module involves tributed equally to this work. HY5 or other interacting proteins, are matters that await future research. Interestingly, the CCT domain of BBX16/COL7 has ORCID been shown to mediate binding to the promoter of the auxin biosynthesis repressor SUR2 in the regulation of plant architec- Pablo Leivar https://orcid.org/0000-0003-4878-3684 ture under shade conditions in Arabidopsis adult plants (Zhang Guiomar Martın https://orcid.org/0000-0001-6642-1666 et al., 2014). In addition, other BBX factors such as BBX20 and Elena Monte https://orcid.org/0000-0002-7340-9355 BBX32 have been shown to regulate photomorphogenesis Nil Veciana https://orcid.org/0000-0002-1761-6616 through mediating brassinosteroid and strigolactone homeostasis (Wei et al., 2016; Ravindran et al., 2021). Because auxin and Data availability other hormones are well known key regulators of photomorpho- genesis, and integration of retrograde and hormonal signalling is The data that support the findings of this study are available from essential in the adaptation to a myriad of stresses (Jiang & the corresponding author upon reasonable request. Dehesh, 2021), it will be of interest in the future to explore a connection of RS-mediated control of BBX16 with key regula- References tory genes in diverse hormone pathways that could impact cotyle- Arsovski AA, Galstyan A, Guseman JM, Nemhauser JL. 2012. don development. Photomorphogenesis. The Arabidopsis Book 10: e0147. To conclude, this study supports a model whereby BBX16 is Ben-Naim O, Eshed R, Parnis A, Teper-Bamnolker P, Shalit A, Coupland G, Samach A, Lifschitz E. 2006. The CCAAT binding factor can mediate directly targeted by GLK1 to induce cotyledon photomorpho- interactions between CONSTANS-like proteins and DNA. The Plant Journal genesis under light conditions favourable for seedling de- 46: 462–476. etiolation. By contrast, when GUN1-mediated RS is activated, Bursch K, Toledo-Ortiz G, Pireyre M, Lohr M, Braatz C, Johansson H. 2020. the inhibition of GLK1, BBX16 and PhANG expression limits Identification of BBX proteins as rate-limiting cofactors of HY5. Nature Plants cotyledon and chloroplast development to minimise light damage 6: 921–928. Castillon A, Shen H, Huq E. 2007. Phytochrome interacting factors: central and optimise photoprotection. The importance of this response players in phytochrome-mediated light signaling networks. Trends in Plant is illustrated by studies with gun1 seedlings exposed to high light Science 12: 514–521. that exhibited more photobleached areas in their cotyledons com- Chan KX, Phua SY, Crisp P, McQuinn R, Pogson BJ. 2016. Learning the pared with the wild-type controls (Ruckle et al., 2007). This languages of the chloroplast: retrograde signaling and beyond. Annual Review of adaptive mechanism would protect an etiolated seedling, which is Plant Biology 67: 25–53. Chang CJ, Li Y-H, Chen L-T, Chen W-C, Hsieh W-P, Shin J, Jane W-N, Chou extremely vulnerable, emerging into excess light such as found on S-J, Choi G, Hu J-M et al. 2008. LZF1, a HY5-regulated transcriptional a hot sunny day. This could take place transiently during estab- factor, functions in Arabidopsis de-etiolation. The Plant Journal 54: 205–219. lishment, allowing the seedling to prevent damage and wait safely Chang C-SJ, Maloof JN, Wu S-H. 2011. COP1-mediated degradation of for the light to become less strong due to shading or the natural BBX22/LZF1 optimizes seedling development in Arabidopsis. Plant Physiology shift in the position of the sun. 156: 228–239. Chen M, Ji M, Wen B, Liu L, Li S, Chen X, Gao D, Li L. 2016. GOLDEN 2- LIKE transcription factors of plants. Frontiers in Plant Science 7: 1509. Acknowledgements Colombo M, Tadini L, Peracchio C, Ferrari R, Pesaresi P. 2016. GUN1, a jack- of-all-trades in chloroplast protein homeostasis and signaling. Frontiers in Plant We are grateful to Bin Liu (College of Life Sciences, Hunan Science 7: 1427. University, China) for generously sharing the bbx16-1/col7 and Datta S, Hettiarachchi C, Johansson H, Holm M. 2007. SALT TOLERANCE HOMOLOG2, a B-box protein in Arabidopsis that activates transcription and BBX16-OX lines, to Jane Langdale (University of Oxford) for positively regulates light-mediated development. Plant Cell 19: 3242–3255. GLK1 lines, and to ABRC/NASC for bbx16-2 seeds. We thank Datta S, Hettiarachchi GHCM, Deng X-W, Holm M. 2006. Arabidopsis Martı Bernardo-Faura (Molecular Data Analysis Area, CRAG) CONSTANS-LIKE3 is a positive regulator of red light signaling and root for his support with the statistical analysis. This work was growth. Plant Cell 18: 70–84. Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 104 Research Phytologist Datta S, Johansson H, Hettiarachchi C, Irigoyen ML, Desai M, Rubio V, Holm Jiang L, Wang Y, Li Q-F, Bj€orn LO, He J-X, Li S-S. 2012. Arabidopsis STO/ M. 2008. LZF1/SALT TOLERANCE HOMOLOG3, an Arabidopsis B-box BBX24 negatively regulates UV-B signaling by interacting with COP1 and protein involved in light-dependent development and gene expression, repressing HY5 transcriptional activity. Cell Research 22: 1046–1057. undergoes COP1-mediated ubiquitination. Plant Cell 20: 2324–2338. Jiao Y, Ma L, Strickland E, Deng XW. 2005. Conservation and divergence of Ding L, Wang S, Song Z-T, Jiang Y, Han J-J, Lu S-J, Li L, Liu J-X. 2018. Two light-regulated genome expression patterns during seedling development in rice B-box domain proteins, BBX18 and BBX23, interact with ELF3 and regulate and Arabidopsis. Plant Cell 17: 3239–3256. thermomorphogenesis in Arabidopsis. Cell Reports 25: 1718–1728.e4. Jing Y, Lin R. 2020. Transcriptional regulatory network of the light signaling Estavillo GM, Crisp PA, Pornsiriwong W, Wirtz M, Collinge D, Carrie C, pathways. New Phytologist 227: 683–697. Giraud E, Whelan J, David P, Javot H et al. 2011. Evidence for a SAL1-PAP Job N, Datta S. 2021. PIF3/HY5 module regulates BBX11 to suppress chloroplast retrograde pathway that functions in drought and high light protochlorophyllide levels in dark and promote photomorphogenesis in light. signaling in Arabidopsis. Plant Cell 23: 3992–4012. New Phytologist 230: 190–204. Fan X-Y, Sun Y, Cao D-M, Bai M-Y, Luo X-M, Yang H-J, Wei C-Q, Zhu S-W, Job N, Yadukrishnan P, Bursch K, Datta S, Johansson H. 2018. Two B-box Sun Y, Chong K et al. 2012. BZS1, a B-box protein, promotes proteins regulate photomorphogenesis by oppositely modulating HY5 through photomorphogenesis downstream of both brassinosteroid and light signaling their diverse C-terminal domains. Plant Physiology 176: 2963–2976. pathways. Molecular Plant 5: 591–600. Jung H-S, Chory J. 2010. Signaling between chloroplasts and the nucleus: can a Fitter DW, Martin DJ, Copley MJ, Scotland RW, Langdale JA. 2002. GLK gene systems biology approach bring clarity to a complex and highly regulated pairs regulate chloroplast development in diverse plant species. The Plant pathway? Plant Physiology 152: 453–459. Journal 31: 713–727. Khanna R, Kronmiller B, Maszle DR, Coupland G, Holm M, Mizuno T, Wu S- Franco-Zorrilla JM, L opez-Vidriero I, Carrasco JL, Godoy M, Vera P, Solano R. H. 2009. The Arabidopsis B-box zinc finger family. Plant Cell 21: 3416–3420. 2014. DNA-binding specificities of plant transcription factors and their Khanna R, Shen Y, Toledo-Ortiz G, Kikis EA, Johannesson H, Hwang Y-S, potential to define target genes. Proceedings of the National Academy of Sciences, Quail PH. 2006. Functional profiling reveals that only a small number of USA 111: 2367–2372. phytochrome-regulated early-response genes in arabidopsis are necessary for Galv~a o VC, Fankhauser C. 2015. Sensing the light environment in plants: optimal deetiolation. Plant Cell 18: 2157–2171. photoreceptors and early signaling steps. Current Opinion in Neurobiology 34: Khanna R, Shen Y, Marion CM, Tsuchisaka A, Theologis A, Sch€afer E, Quail 46–53. PH. 2007. The basic helix-loop-helix transcription factor PIF5 acts on ethylene Gangappa SN, Botto JF. 2014. The BBX family of plant transcription factors. biosynthesis and phytochrome signaling by distinct mechanisms. Plant Cell 19: Trends in Plant Science 19: 460–470. 3915–3929. Gangappa SN, Crocco CD, Johansson H, Datta S, Hettiarachchi C, Holm M, Kindgren P, Kremnev D, Blanco NE, de Dios Barajas Lopez J, Ferna ndez AP, Botto JF. 2013. The Arabidopsis B-BOX protein BBX25 interacts with HY5, Tellgren-Roth C, Small I, Strand  A. 2012. The plastid redox insensitive 2 negatively regulating BBX22 expression to suppress seedling mutant of Arabidopsis is impaired in PEP activity and high light-dependent photomorphogenesis. Plant Cell 25: 1243–1257. plastid redox signalling to the nucleus. The Plant Journal 70: 279–291. Gommers CMM, Monte E. 2018. Seedling establishment: a dimmer switch-regulated Kleine T, Maier UG, Leister D. 2009. DNA transfer from organelles to the process between dark and light signaling. Plant Physiology 176: 1061–1074. nucleus: the idiosyncratic genetics of endosymbiosis. Annual Review of Plant Heng Y, Jiang Y, Zhao X, Zhou H, Wang X, Deng XW, Xu D. 2019a. BBX4, a Biology 60: 115–138. phyB-interacting and modulated regulator, directly interacts with PIF3 to fine Koussevitzky S, Nott A, Mockler TC, Hong F, Sachetto-Martins G, Surpin M, tune red light-mediated photomorphogenesis. Proceedings of the National Lim J, Mittler R, Chory J. 2007. Signals from chloroplasts converge to Academy of Sciences, USA 116: 26049–26056. regulate nuclear gene expression. Science 316: 715–719. Heng Y, Lin F, Jiang Y, Ding M, Yan T, Lan H, Zhou H, Zhao X, Xu D, Deng Kumagai T, Ito S, Nakamichi N, Niwa Y, Murakami M, Yamashino T, Mizuno XW. 2019b. B-box containing proteins BBX30 and BBX31, acting T. 2008. The common function of a novel subfamily of B-box zinc finger downstream of HY5, negatively regulate photomorphogenesis in Arabidopsis. proteins with reference to circadian-associated events in Arabidopsis thaliana. Plant Physiology 180: 497–508. Bioscience, Biotechnology, and Biochemistry 72: 1539–1549. Hills AC, Khan S, Lopez-Juez E. 2015. Chloroplast biogenesis-associated nuclear Lau OS, Deng XW. 2012. The photomorphogenic repressors COP1 and DET1: genes: control by plastid signals evolved prior to their regulation as part of 20 years later. Trends in Plant Science 17: 584–593. photomorphogenesis. Frontiers in Plant Science 6: 1078. Laubinger S, Marchal V, Gentilhomme J, Wenkel S, Adrian J, Jang S, Kulajta Hoecker U. 2017. The activities of the E3 ubiquitin ligase COP1/SPA, a key C, Braun H, Coupland G, Hoecker U. 2006. Arabidopsis SPA proteins repressor in light signaling. Current Opinion in Plant Biology 37: 63–69. regulate photoperiodic flowering and interact with the floral inducer Holtan HE, Bandong S, Marion CM, Adam L, Tiwari S, Shen Y, Maloof JN, CONSTANS to regulate its stability. Development 133: 3213–3222. Maszle DR, Ohto M, Preuss S et al. 2011. BBX32, an Arabidopsis B-box Leivar P, Martın G, Soy J, Dalton-Roesler J, Quail PH, Monte E. 2020. protein, functions in light signaling by suppressing HY5-regulated gene Phytochrome-imposed inhibition of PIF7 activity shapes photoperiodic growth in expression and interacting with STH2/BBX21. Plant Physiology 156: 2109– Arabidopsis together with PIF1, 3, 4 and 5. Physiologia Plantarum 169: 452–466. 2123. Leivar P, Monte E, Oka Y, Liu T, Carle C, Castillon A, Huq E, Quail PH. Huang J, Zhao X, Chory J. 2019. The Arabidopsis transcriptome responds 2008. Multiple phytochrome-interacting bHLH transcription factors repress specifically and dynamically to high light stress. Cell Reports 29: 4186–4199.e3. premature seedling photomorphogenesis in darkness. Current Biology 18: Huang X, Ouyang X, Deng XW. 2014. Beyond repression of 1815–1823. photomorphogenesis: role switching of COP/DET/FUS in light signaling. Leivar P, Monte E. 2014. PIFs: systems Integrators in plant development. Plant Current Opinion in Plant Biology 21: 96–103. Cell 26: 56–78. Indorf M, Cordero J, Neuhaus G, Rodrıguez-Franco M. 2007. Salt tolerance Leivar P, Quail PH. 2011. PIFs: pivotal components in a cellular signaling hub. (STO), a stress-related protein, has a major role in light signalling. The Plant Trends in Plant Science 16: 19–28. Journal 51: 563–574. Leivar P, Tepperman JM, Monte E, Calderon RH, Liu TL, Quail PH. 2009. Jang S, Marchal V, Panigrahi KCS, Wenkel S, Soppe W, Deng X-W, Valverde Definition of early transcriptional circuitry involved in light-induced reversal of F, Coupland G. 2008. Arabidopsis COP1 shapes the temporal pattern of CO PIF-imposed repression of photomorphogenesis in young Arabidopsis accumulation conferring a photoperiodic flowering response. EMBO Journal seedlings. Plant Cell 21: 3535–3553. 27: 1277–1288. Li H, Chiu C-C. 2010. Protein transport into chloroplasts. Annual Review of Jarvis P, Lopez-Juez E. 2014. Biogenesis and homeostasis of chloroplasts and Plant Biology 61: 157–180. other plastids. Nature Reviews Molecular Cell Biology 15: 147. Li Y, Shi Y, Li M, Fu D, Wu S, Li J, Gong Z, Liu H, Yang S. 2021. The CRY2– Jiang J, Dehesh K. 2021. Plastidial retrograde modulation of light and hormonal COP1–HY5–BBX7/8 module regulates blue light-dependent cold acclimation signaling: an odyssey. New Phytologist 230: 931–937. in Arabidopsis. Plant Cell 33: 3555–3573. New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation New Phytologist Research 105 Lin F, Jiang Y, Li J, Yan T, Fan L, Liang J, Chen ZJ, Xu D, Deng XW. 2018. B- Shin J, Kim K, Kang H, Zulfugarov IS, Bae G, Lee C-H, Lee D, Choi G. 2009. BOX DOMAIN PROTEIN28 negatively regulates photomorphogenesis by Phytochromes promote seedling light responses by inhibiting four negatively- repressing the activity of transcription factor HY5 and undergoes COP1- acting phytochrome-interacting factors. Proceedings of the National Academy of mediated degradation. Plant Cell 30: 2006–2019. Sciences, USA 106: 7660–7665. Lyu G, Li D, Li S. 2020. Bioinformatics analysis of BBX family genes and its Shin J, Park E, Choi G. 2007. PIF3 regulates anthocyanin biosynthesis in an response to UV-B in Arabidopsis thaliana. Plant Signaling & Behavior 15: HY5-dependent manner with both factors directly binding anthocyanin 1782647. biosynthetic gene promoters in Arabidopsis. The Plant Journal 49: 981– Ma L, Li J, Qu L, Hager J, Chen Z, Zhao H, Deng XW. 2001. Light control of 994. Arabidopsis development entails coordinated regulation of genome expression Song Z, Bian Y, Liu J, Sun Y, Xu D. 2020a. B-box proteins: pivotal players in and cellular pathways. Plant Cell 13: 2589–2607. light-mediated development in plants. Journal of Integrative Plant Biology 62: Marino G, Naranjo B, Wang J, Penzler J-F, Kleine T, Leister D. 2019. 1293–1309. Relationship of GUN1 to FUG1 in chloroplast protein homeostasis. The Plant Song Z, Yan T, Liu J, Bian Y, Heng Y, Lin F, Jiang Y, Deng XW, Xu D. 2020b. Journal 99: 521–535. BBX28/BBX29, HY5 and BBX30/31 form a feedback loop to fine-tune Martın G, Leivar P, Ludevid D, Tepperman JM, Quail PH, Monte E. 2016. photomorphogenic development. The Plant Journal 104: 377–390. Phytochrome and retrograde signalling pathways converge to antagonistically Soy J, Leivar P, Monte E. 2014. PIF1 promotes phytochrome-regulated growth regulate a light-induced transcriptional network. Nature Communications 7: under photoperiodic conditions in Arabidopsis together with PIF3, PIF4, and 11431. PIF5. Journal of Experimental Botany 65: 2925–2936. Martın G, Rovira A, Veciana N, Soy J, Toledo-Ortiz G, Gommers CMM, Boix Su L, Hou P, Song M, Zheng X, Guo L, Xiao Y, Yan L, Li W, Yang J. 2015. M, Henriques R, Minguet EG, Alabadı D et al. 2018. Circadian waves of Synergistic and antagonistic action of phytochrome (Phy) A and PhyB during transcriptional repression shape PIF-regulated photoperiod-responsive growth seedling de-etiolation in Arabidopsis thaliana. International Journal of Molecular in Arabidopsis. Current Biology 28: 311–318.e5. Sciences 16: 12199–12212. Martın G, Veciana N, Boix M, Rovira A, Henriques R, Monte E. 2020. The Sullivan JA, Gray JC. 1999. Plastid translation is required for the expression of photoperiodic response of hypocotyl elongation involves regulation of CDF1 nuclear photosynthesis genes in the dark and in roots of the pea lip1 mutant. and CDF5 activity. Physiologia Plantarum 169: 480–490. Plant Cell 11: 901–910. Nagaoka S, Takano T. 2003. Salt tolerance-related protein STO binds to a Myb Tadini L, Peracchio C, Trotta A, Colombo M, Mancini I, Jeran N, Costa A, transcription factor homologue and confers salt tolerance in Arabidopsis. Faoro F, Marsoni M, Vannini C et al. 2020. GUN1 influences the Journal of Experimental Botany 54: 2231–2237. accumulation of NEP-dependent transcripts and chloroplast protein import in Oelm€ uller R, Levitan I, Bergfeld R, Rajasekhar VK, Mohr H. 1986. Expression Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis. of nuclear genes as affected by treatments acting on the plastids. Planta 168: The Plant Journal 101: 1198–1220. 482–492. Tadini L, Pesaresi P, Kleine T, Rossi F, Guljamow A, Sommer F, M€ uhlhaus T, Oh S, Montgomery BL. 2014. Phytochrome-dependent coordinate control of Schroda M, Masiero S, Pribil M et al. 2016. GUN1 controls accumulation of distinct aspects of nuclear and plastid gene expression during anterograde the plastid ribosomal protein s1 at the protein level and interacts with proteins signaling and photomorphogenesis. Frontiers in Plant Science 5: 171. involved in plastid protein homeostasis. Plant Physiology 170: 1817–1830. Pesaresi P, Kim C. 2019. Current understanding of GUN1: a key mediator Tiwari SB, Shen Y, Chang H-C, Hou Y, Harris A, Ma SF, McPartland M, involved in biogenic retrograde signaling. Plant Cell Reports 38: 819– Hymus GJ, Adam L, Marion C et al. 2010. The flowering time regulator 823. CONSTANS is recruited to the FLOWERING LOCUS T promoter via a Pfeiffer A, Shi H, Tepperman JM, Zhang Y, Quail PH. 2014. Combinatorial unique cis-element. New Phytologist 187: 57–66. complexity in a transcriptionally centered signaling hub in Arabidopsis. Toledo-Ortiz G, Huq E, Quail PH. 2003. The Arabidopsis basic/helix-loop- Molecular Plant 7: 1598–1618. helix transcription factor family. Plant Cell 15: 1749–1770. Pham VN, Xu X, Huq E. 2018. Molecular bases for the constitutive Wang C-Q, Guthrie C, Sarmast MK, Dehesh K. 2014. BBX19 interacts with photomorphogenic phenotypes in Arabidopsis. Development 145: dev169870. CONSTANS to repress FLOWERING LOCUS T transcription, defining a Purvis J, Ilango V, Radhakrishnan R. 2008. Role of network branching in flowering time checkpoint in Arabidopsis. Plant Cell 26: 3589–3602. eliciting differential short-term signaling responses in the hypersensitive Wang C-Q, Sarmast MK, Jiang J, Dehesh K. 2015. The transcriptional regulator epidermal growth factor receptor mutants implicated in lung cancer. BBX19 promotes hypocotyl growth by facilitating COP1-mediated EARLY Biotechnology Process 24: 540–553. FLOWERING3 degradation in Arabidopsis. Plant Cell 27: 1128–1139. Ravindran N, Ramachandran H, Job N, Yadav A, Vaishak KP, Datta S. 2021. Wang H, Zhang Z, Li H, Zhao X, Liu X, Ortiz M, Lin C, Liu B. 2013. B-box protein BBX32 integrates light and brassinosteroid signals to inhibit CONSTANS-LIKE 7 regulates branching and shade avoidance response in cotyledon opening. Plant Physiology 187: 446–461. Arabidopsis. Journal of Experimental Botany 64: 1017–1024. Robson F, Costa MMR, Hepworth SR, Vizir I, Pi~ neiro M, Reeves PH, Putterill Wang Q, Tu X, Zhang J, Chen X, Rao L. 2013. Heat stress-induced BBX18 J, Coupland G. 2001. Functional importance of conserved domains in the negatively regulates the thermotolerance in Arabidopsis. Molecular Biology flowering-time gene CONSTANS demonstrated by analysis of mutant alleles Reports 40: 2679–2688. and transgenic plants. The Plant Journal 28: 619–631. Wang Q, Zeng J, Deng K, Tu X, Zhao X, Tang D, Liu X. 2011. DBB1a, Ruckle ME, DeMarco SM, Larkin RM. 2007. Plastid signals remodel light involved in gibberellin homeostasis, functions as a negative regulator of blue signaling networks and are essential for efficient chloroplast biogenesis in light-mediated hypocotyl elongation in Arabidopsis. Planta 233: 13–23. Arabidopsis. Plant Cell 19: 3944–3960. Waters MT, Moylan EC, Langdale JA. 2008. GLK transcription factors regulate Sentandreu M, Martın G, Gonza lez-Schain N, Leivar P, Soy J, Tepperman JM, chloroplast development in a cell-autonomous manner. The Plant Journal 56: Quail PH, Monte E. 2011. Functional profiling identifies genes involved in 432–444. organ-specific branches of the PIF3 regulatory network in Arabidopsis. Plant Waters MT, Wang P, Korkaric M, Capper RG, Saunders NJ, Langdale JA. Cell 23: 3974–3991. 2009. GLK transcription factors coordinate expression of the photosynthetic Shi H, Lyu M, Luo Y, Liu S, Li Y, He H, Wei N, Deng XW, Zhong S. 2018. apparatus in Arabidopsis. Plant Cell 21: 1109–1128. Genome-wide regulation of light-controlled seedling morphogenesis by three Wei C-Q, Chien C-W, Ai L-F, Zhao J, Zhang Z, Li KH, Burlingame AL, Sun families of transcription factors. Proceedings of the National Academy of Sciences, Y, Wang Z-Y. 2016. The Arabidopsis B-box protein BZS1/BBX20 interacts USA 115: 6482–6487. with HY5 and mediates strigolactone regulation of photomorphogenesis. Shimizu T, Kacprzak SM, Mochizuki N, Nagatani A, Watanabe S, Shimada T, Journal of Genetics and Genomics 43: 555–563. Tanaka K, Hayashi Y, Arai M, Leister D et al. 2019. The retrograde signaling Wu G-Z, Bock R. 2021. GUN control in retrograde signaling: how GENOMES protein GUN1 regulates tetrapyrrole biosynthesis. Proceedings of the National UNCOUPLED proteins adjust nuclear gene expression to plastid biogenesis. Academy of Sciences, USA 116: 24900–24906. Plant Cell 33: 457–474. Ó 2022 The Authors New Phytologist (2022) 234: 93–106 New Phytologist Ó 2022 New Phytologist Foundation www.newphytologist.com New 106 Research Phytologist Wu G-Z, Chalvin C, Hoelscher M, Meyer EH, Wu XN, Bock R. 2018. Control Zubo YO, Blakley IC, Franco-Zorrilla JM, Yamburenko MV, Solano R, Kieber of retrograde signaling by rapid turnover of GENOMES UNCOUPLED1. JJ, Loraine AE, Schaller GE. 2018. Coordination of chloroplast development Plant Physiology 176: 2472–2495. through the action of the GNC and GLK transcription factor families. Plant Wu G-Z, Meyer EH, Richter AS, Schuster M, Ling Q, Sch€ottler MA, Walther D, Physiology 178: 130–147. Zoschke R, Grimm B, Jarvis RP, et al. 2019. Control of retrograde signalling by protein import and cytosolic folding stress. Nature Plants 5: 525–538. Xiao Y, Savchenko T, Baidoo EEK, Chehab WE, Hayden DM, Tolstikov V, Supporting Information Corwin JA, Kliebenstein DJ, Keasling JD, Dehesh K. 2012. Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear Additional Supporting Information may be found online in the stress-response genes. Cell 149: 1525–1535. Supporting Information section at the end of the article. Xu D. 2020. COP1 and BBXs-HY5-mediated light signal transduction in plants. New Phytologist 228: 1748–1753. Fig. S1 Model depicting the criteria we followed to identify puta- Xu D, Jiang Y, Li J, Lin F, Holm M, Deng XW. 2016. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S tive regulators of cotyledon development downstream of GLK1. proteasome-mediated degradation. Proceedings of the National Academy of Sciences, USA 113: 7655–7660. Fig. S2 Molecular characterisation of bbx16-2. Xu D, Jiang Y, Li J, Holm M, Deng XW. 2018. The B-box domain protein BBX21 promotes photomorphogenesis. Plant Physiology 176: 2365–2375. Fig. S3 BBX14, BBX15 and BBX16 are similarly regulated by Xu X, Paik I, Zhu L, Huq E. 2015. Illuminating progress in phytochrome- mediated light signaling pathways. Trends in Plant Science 20: 641–650. PIFs and GLK1 in dark and light. Yi C, Deng XW. 2005. COP1 – from plant photomorphogenesis to mammalian tumorigenesis. Trends in Cell Biology 15: 618–625. Fig. S4 Characterisation of gun1glk1. Zhang X, Huai J, Shang F, Xu G, Tang W, Jing Y, Lin R. 2017. A PIF1/PIF3- HY5-BBX23 transcription factor cascade affects photomorphogenesis. Plant Fig. S5 Characterisation of GLK1OXbbx16. Physiology 174: 2487–2500. Zhang Y, Mayba O, Pfeiffer A, Shi H, Tepperman JM, Speed TP, Quail PH. 2013. A quartet of PIF bHLH factors provides a transcriptionally centered Table S1 List of primers used for genotyping. signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS Genetics 9: Table S2 List of primers used for quantitative reverse transcrip- e1003244. tase (qRT-PCR). Zhang Z, Ji R, Li H, Zhao T, Liu J, Lin C, Liu B. 2014. CONSTANS-LIKE 7 (COL7) is involved in phytochrome B (phyB)-mediated light-quality regulation of auxin homeostasis. Molecular Plant 7: 1429–1440. Please note: Wiley Blackwell are not responsible for the content Zhao X, Huang J, Chory J. 2019. GUN1 interacts with MORF2 to regulate or functionality of any Supporting Information supplied by the plastid RNA editing during retrograde signaling. Proceedings of the National authors. Any queries (other than missing material) should be Academy of Sciences, USA 116: 10162–10167. directed to the New Phytologist Central Office. New Phytologist (2022) 234: 93–106 Ó 2022 The Authors www.newphytologist.com New Phytologist Ó 2022 New Phytologist Foundation