PCH1 and PCHL Directly Interact with PIF1, Promote Its Degradation and Inhibit Its Transcriptional Function during Photomorphogenesis

Journal Pre-proof PCH1 and PCHL Directly Interact with PIF1, Promote Its Degradation and Inhibit Its Transcriptional Function during Photomorphogenesis Mei-Chun Cheng, Beatrix Enderle, Praveen Kumar Kathare, Rafya Islam, Andreas Hiltbrunner, Enamul Huq PII: DOI: Reference:

S1674-2052(20)30033-2 https://doi.org/10.1016/j.molp.2020.02.003 MOLP 891

To appear in: MOLECULAR PLANT Accepted Date: 7 February 2020

Please cite this article as: Cheng M.-C., Enderle B., Kathare P.K., Islam R., Hiltbrunner A., and Huq E. (2020). PCH1 and PCHL Directly Interact with PIF1, Promote Its Degradation and Inhibit Its Transcriptional Function during Photomorphogenesis. Mol. Plant. doi: https://doi.org/10.1016/ j.molp.2020.02.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. © 2020 The Author

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PCH1 and PCHL Directly Interact with PIF1, Promote Its Degradation and Inhibit Its

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Transcriptional Function during Photomorphogenesis

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Mei-Chun Cheng1, Beatrix Enderle2, Praveen Kumar Kathare1, Rafya Islam1, Andreas

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Hiltbrunner2,3 and Enamul Huq1,*

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100 East 24th St. Austin, TX 78712-1095.

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[email protected]

Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712 Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany 3 Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany 2

Running Title: PCH1/L regulate light responses by interacting with PIF1 and COP1 *

Corresponding author: Enamul Huq, University of Texas at Austin, NHB 2.616, Stop A5000, Tel: 512-471-9848, Fax: 512-471-1218, e-mail:

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Short summary: This study shows that PCH1 and PCHL promote the degradation of PIF1 both in

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dark and light, and inhibit PIF1 DNA binding activity, while both of them are being degraded in

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the dark by direct interaction with COP1. These data expand the molecular basis by which PCH1

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and PCHL regulate photomorphogenesis.

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ABSTRACT

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PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and PCH1-LIKE (PCHL) were

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shown to directly bind to phytochrome B (phyB) and suppress phyB thermal reversion, resulting

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in plants with dramatically enhanced light sensitivity. Here we show that PCH1 and PCHL also

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positively regulate many light responses including seed germination, hypocotyl gravitropism,

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and chlorophyll biosynthesis by physically interacting with PHYTOCHROME INTERACTING

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FACTOR 1 (PIF1) AND CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). PCH1 and

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PCHL interact with PIF1 both in the dark and light, and regulate PIF1 abundance. Moreover,

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PCH1 and PCHL facilitate the physical interaction between phyB and PIF1 in vivo to promote

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the light-induced degradation of PIF1. PCH1 and PCHL also inhibit the DNA binding ability of

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PIF1 to negatively regulate the expressions of PIF1 target genes. In addition, PCH1 and PCHL

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interact with COP1 and undergo degradation through the 26S proteasome pathway in the dark.

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Consistently, pch1 suppresses cop1 phenotype in darkness. Collectively, our study revealed a

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novel mechanism by which PCH1/L regulates diverse light responses not only by stabilizing

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phyB Pfr form but also by directly interacting with PIF1 and COP1, and also suggest a molecular

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basis for the control of hypocotyl growth by these proteins.

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Keywords: Arabidopsis, phytochrome, phytochrome interacting factor (PIF), photoperiodic

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growth, protein degradation.

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INTRODUCTION

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Phytochromes (phys) are evolutionarily conserved photoreceptors in bacteria, fungi, algae, and

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plants (Rockwell and Lagarias, 2019). Phys regulate almost all aspects of plant development and

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growth, including germination, de-etiolation, shade avoidance, plant defense, floral induction,

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and senescence (Casal, 2013; Legris et al., 2019; Paik and Huq, 2019). Phys are red (R) and

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far-red (FR) light photoreceptors that can be photo-converted between two relatively stable

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forms: the R light absorbing inactive Pr form localized in cytoplasm and the FR light absorbing

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biologically active Pfr form which migrates into nucleus (Huq and Quail, 2005; Legris et al.,

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2019). In addition to light-induced Pfr→Pr reversion, the active Pfr state can also revert to the

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inactive Pr state in a light-independent thermal relaxation process referred to as thermal

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reversion (Klose et al., 2020; Medzihradszky et al., 2013). Phytochrome A (phyA) and

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phytochrome B (phyB) play a major role in seed plants at the seedling stage. PhyA is required for

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sensing FR light and weak light of any wavelength, while phyB is the primary receptor for R

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light (Legris et al., 2019). The physiological activity of phyB, the primary phytochrome in

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light-grown and adult plants, is strongly affected by thermal reversion. Increased rates of phyB

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thermal reversion in Arabidopsis seedlings exposed tohigh temperature reduce both the

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abundance of the biologically active Pfr-Pfr dimer pool and the size of the associated nuclear

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bodies under daylight and dark conditions. Mathematical analysis of stem growth for seedlings

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expressing wild-type phyB or thermally stable variants under various combinations of light and

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temperature revealed that phyB is physiologically responsive to both signals (Jung et al., 2016;

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Legris et al., 2016; Quint et al., 2016). Therefore, thermal reversion is an important factor that

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determines how plants respond to light and temperature.

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Within the nucleus, the activated Pfr physically interacts with multiple proteins, including a

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small group of basic helix-loop-helix (bHLH) transcription factors called PHYTOCHROME

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INTERACTING FACTORS (PIFs; PIF1 to PIF8) (Leivar and Quail, 2011; Oh et al., 2019; Pham

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et al., 2018b). Phytochromes regulate light responses partly by inhibiting these PIFs which

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negatively regulate various light responses. PIFs constitutively accumulate in the nucleus in the

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dark and inhibit photomorphogenesis (Leivar and Quail, 2011; Oh et al., 2019; Pham et al.,

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2018b). Upon light exposure, the physical interaction between Pfr and PIFs triggers a cascade of

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events, including light-induced phosphorylation, ubiquitination, and 26S proteasome–mediated

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degradation of PIFs (Bauer et al., 2004; Paik et al., 2019; Shen et al., 2005). The removal of PIFs

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after light exposure results in large-scale changes in gene expression that promote

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photomorphogenic development (Leivar and Monte, 2014; Leivar et al., 2009; Shin et al., 2009).

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Consistently, the reduction in PIF level in the pifq (pif1 pif3 pif4 pif5) quadruple mutant or the

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overexpression of a truncated form of PIF1 results in photomorphogenic development in the dark

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(Leivar et al., 2008; Pham et al., 2018c; Shen et al., 2008; Shin et al., 2009).

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Among the negative regulators in light signaling, COP1 is a RING type E3 ubiquitin ligase

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that forms multiple complexes with SUPPRESSOR OF PHYA-105 family members (SPA1-4)

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(Deng et al., 1992; Lau and Deng, 2012; Xu et al., 2015). These complexes, either by themselves

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or by forming CUL4COP1–SPA E3 ubiquitin ligases, degrade the positively acting transcription

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factors through the ubiquitin proteasome system (UPS) to repress photomorphogenesis in the

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dark (Chen et al., 2010). Therefore, cop1 and spa quadruple mutant seedlings (spaq) exhibit

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constitutive photomorphogenesis when grown in darkness (Deng et al., 1992; Hoecker, 2017;

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Laubinger et al., 2004). In wild-type seedlings grown in the dark, nuclear-localized COP1

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ubiquitinates HY5, promoting its degradation (Lau and Deng, 2012; Osterlund et al., 2000),

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whereas light exposure reduces nuclear COP1 to a level that permits the accumulation of HY5

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(Pacín et al., 2014; Subramanian et al., 2004). Light also represses COP1 activity by activating

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the photoreceptors phyA, phyB, CRYPTOCHROME 1 (CRY1) and CRY2 to modulate the

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complex between COP1 and SPA proteins, ultimately leading to HY5 accumulation (Lian et al.,

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2011; Lu et al., 2015; Sheerin et al., 2015; Zuo et al., 2011). Phytochromes directly interact with

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SPA proteins and inhibit the function of the COP1/SPA ubiquitin E3 ligase complex by either

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disrupting the interaction between COP1 and SPAs, or by promoting the degradation of SPA2

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(Balcerowicz et al., 2011; Lu et al., 2015; Sheerin et al., 2015). In response to light, the inhibition

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of the COP1/SPA activity by phytochromes results in the accumulation of its target substrates

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such as ELONGATED HYPOCOTYL 5 (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1),

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and the B-BOX ZINC FINGER PROTEINs (BBXs) to accumulate (Jang et al., 2005; Osterlund

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et al., 2000; Pham et al., 2018c; Xu et al., 2017; Xu et al., 2015). These, in turn, reprogram gene

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expression to promote photomorphogenesis.

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PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) has been identified as one of the

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proteins interacting with the light-activated phyB (Enderle et al., 2017; Huang et al., 2016).

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Seedlings lacking functional PCH1 display elongated hypocotyls compared to the wild type

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under short days. Under these conditions, PCH1 transcript and protein levels peak at dusk,

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enhancing phyB-dependent inactivation of the growth-promoting transcription factor

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PHYTOCHROME INTERACTING FACTOR 4 (PIF4). In contrast, PCH1 levels are low

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towards the end of the night, leading to increased PIF4 activity and hypocotyl growth. Thus,

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PCH1 has been suggested to integrate clock and light signals through modulation of diurnal

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phyB activity (Huang et al., 2016). We showed in our previous report that the pch1 pchl double

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mutant, which lacks functional PCH1 and a homologue, PCH1-LIKE (PCHL), displays strongly

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accelerated phyB thermal reversion. Moreover, PCH1 and PCHL stabilize phyB in the active

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state and inhibit phyB thermal reversion. We also showed that PCH1/L accumulates in response

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to various lights and additional signaling pathways control the expression of PCH1 and PCHL,

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thereby affecting the activity of phyB (Enderle et al., 2017). Thus, PCH1 and PCHL fine-tuned

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light signaling by integrating environmental signals to regulate phyB thermal reversion.

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Recently, Huang et al further provided biochemical and genetic evidence that PCH1 is

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sufficient to slow down the process of thermal reversion of phyB from Pfr to Pr (Huang et al.,

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2019). They also showed that PCH1 enhances the assembly of phyB into the subnuclear foci

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known as photobodies, which are positively connected with phyB activity (Buskirk et al., 2014;

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Huang et al., 2019; Klose et al., 2015). Using the constitutively active phyB allele phyB Y276H

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tagged with YFP, they showed that the loss of photobodies in phyB Y276H-YFP pch1 seedlings

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represses the constitutive photomorphogenic phenotype of phyB Y276H, alleviating the

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thermo-insensitivity caused by the presence of phyB Y276H, and abolishes the phyB

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Y276H-mediated light input into the circadian clock of dark-grown seedlings (Huang et al.,

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2019). Collectively, these data indicate that PCH1 acts as a positive regulator of phyB photobody

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formation and multiple phyB-controlled physiological processes.

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Although it is known that PCH1 and PCHL positively regulate the function of phyB by

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promoting phyB photobody formation, their relationship and functions in regulating other light

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signaling components as well as the mechanism underlying their protein stability have not yet

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been shown. Here, we show that PCH1 and PCHL positively regulate various light responses,

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such as seed germination, negative gravitropism, and chlorophyll biosynthesis by directly

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interacting with PIF1 and COP1. In this process, PCH1 and PCHL inhibit PIF1 function by

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either directly inhibiting PIF1 DNA binding and/or promoting PIF1 degradation by facilitating 5

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the formation of phyB-PIF1 and COP1-PIF1 complexes. Moreover, like other positive

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components in light signaling, PCH1 and PCHL are also the substrates of COP1 and are targeted

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for degradation in darkness.

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RESULTS

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PCH1 and PCHL positively regulate many light responses and light-responsive gene

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expression

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To further understand the functions of PCH1 and PCHL in light signaling pathways, we

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examined multiple light-related phenotypes using the previously described pch1, pchl, pch1 pchl

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double mutants, as well as 35Spro:HA-YFP-PCH1 (PCH1OE) and 35Spro:HA-YFP-PCHL

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(PCHLOE) overexpression transgenic lines. As phyB is known to promote seed germination in

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response to red light, we examined whether PCH1 and PCHL positively regulate seed

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germination. As shown in Figure 1A, PCH1OE and PCHLOE showed higher levels of seeds

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germination under increasing amount of red light, whereas pch1, pchl and pch1 pchl mutants

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displayed much lower germination compared to wild type. To assess whether PCH1 and PCHL

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promote seed germination through inhibition of PIF1 function, we created pch1 pif1, pchl pif1

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and pch1 pchl pif1 mutant combinations and examined the seed germination phenotype. Results

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show that the reduced seed germination of pch1 and pchl mutants in response to light is

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eliminated in the pif1 background. The double and triple mutant seeds germinated similar to the

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pif1 single mutant (Supplemental Figure 1), suggesting that pif1 is epistatic to pch in regulating

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seed germination.

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Phytochromes suppress the hypocotyls negative gravitropism by inhibiting PIFs in red or

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far-red light (Kim et al., 2011). We next investigated how PCH1 and PCHL regulate hypocotyls

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negative gravitropism by examining whether gravity sensing is disrupted in PCH1OE and

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PCHLOE. Wild-type hypocotyls curved against the direction of gravity upon alteration of the

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gravity vector, whereas PCH1OE and PCHLOE seedlings grew randomly in all directions

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(Figure 1B; Supplementary Figure 2). Moreover, pch1, pchl, and pch1 pchl mutants displayed

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hypersensitive phenotypes in response to gravity as they have higher curvature compared to

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wild-type hypocotyls after changing the direction of gravity, and the degrees of the curvature

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gradually decreased along with the increase in the red light fluence (Figure 1B and C;

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Supplementary Figure 2). We also examined the gravitropism phenotype of pch1 and pchl

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mutants in pif1 mutant background. The hypersensitive phenotypes of pch1 and pchl mutants are

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slightly rescued by pif1 mutation (Supplemental Figure 3), suggesting that pif1 is epistatic to pch

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mutants. Starch granules containing amyloplasts of hypocotyl endodermal cells are responsible

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for gravity sensing in hypocotyls. We next examined endodermal amyloplasts by staining them

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with I2-KI. Consistent with the agravitropic phenotypes, PCH1OE and PCHLOE displayed little

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to no staining of amyloplasts in hypocotyl endodermal cells compared to dark stained hypocotyls

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of wild type seedlings. However, the stain intensity in pch1, pchl and pch1 pchl hypocotyls was

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comparable to that of wild type seedlings (Figure 1D). We also compared this gravitropism

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phenotype in transgenic seedlings overexpressing PCH1 and PCHL in phyB mutant backgrounds.

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As shown in Supplemental Figure 4, the negative gravitropism phenotype is completely rescued

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by phyB mutation, suggesting that the phenotype of PCH1OE and PCHLOE plants is

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phyB-dependent.

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To determine the function of PCH1 and PCHL at the molecular level, we examined the

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expression levels of a selected group of genes that are usually expressed in light-grown seedlings,

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including CHLOROPHYLL A/B BINDING PROTEIN 3 (CAB3), RIBULOSE BISPHOSPHATE

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CARBOXYLASE SMALL CHAIN 1A (RBCS) and FERREDOXIN 2 (Fd2). As expected, PCH1OE

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and PCHLOE seedlings displayed higher expression of these genes compared with the wild type

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under red light treatment (Figure 1E), whereas pch1, pchl and pch1 pchl mutant showed lower

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expression compared with wild-type seedlings. These data suggest that PCH1 and PCHL

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positively regulate many light-responses both at the morphological and molecular levels.

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PCH1 and PCHL regulate chlorophyll biosynthesis at the early stages

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phyB has been shown to positively regulate chlorophyll synthesis and cotyledon greening (Huq

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et al., 2004), whereas PIF1 and PIF3 play a negative role in these responses (Huq et al., 2004;

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Moon et al., 2008; Stephenson et al., 2009). To prepare for exposure to light, seedlings

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accumulate a small pool of the immediate precursor of chlorophyll, called protochlorophyllide,

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to permit rapid assembly of functional photosynthetic machinery. However, over-accumulation

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of free protochlorophyllide leads to lethal photooxidative damage and bleaching (Huq et al.,

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2004). As PCH1 and PCHL are positive regulator in phyB-mediated light signalling, we also

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examined their cotyledon greening and chlorophyll biosynthesis phenotypes. Interestingly, 4-d

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etiolated PCH1OE and PCHLOE seedlings showed delayed cotyledon greening, whereas pch1,

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pchl, and pch1 pchl double mutants showed enhanced greening phenotype (Supplemental

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Figures 5A, B). We also compared this greening phenotype in transgenic seedlings

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overexpressing PCH1 and PCHL in phyA and phyB mutant backgrounds. As shown in

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Supplemental Figure 6A, B, the delayed greening phenotype is partially rescued by phyA and

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phyB mutations. In other words, this delayed greening of PCH1OE and PCHLOE plants is phyA-

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and/or phyB-dependent. On the other hand, we also checked the greening phenotype in pch1 and

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pchl mutants in pif1 mutant background. Results showed that the enhanced greening phenotype

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of pch mutants are eliminated by pif1 mutation (Supplemental Figure 6A, B), meaning that pif1

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is also epistatic to pch in regulating cotyledon greening. To determine whether PCH1/LOE and

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mutants have altered levels of Pchlide, we performed spectrofluorometric analyses on acetone

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extracts of 4-day-old dark grown seedlings of each genotype. The results show that PCH1OE

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and PCHLOE seedlings have higher relative fluorescence peaks at 632 nm, indicative of Pchlide,

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and pch1, pchl, pch1 pchl mutants have lower relative fluorescence compared to WT seedlings

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(Supplemental Figure 7). These data suggest that the delayed cotyledon greening phenotypes of

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OE lines might result from photobleaching.

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To examine whether PCH1 and PCHL regulate the expressions of the key genes involved in

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the regulation of the tetrapyrrole pathway (Supplementary Figure 8), we performed quantitative

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RT-PCR (RT-qPCR) assays. As shown in Supplemental Figure 9A, B, the expressions of genes

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involved

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GLUTAMATE-1-SEMIALDEHYDE 2 (GSA2) were significantly up-regulated in PCH1OE and

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PCHLOE seedlings and down-regulated in pch1, pchl, and pch1 pchl mutants. However, the

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expression of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A, B, and C (PORA-C), which

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regulate the later steps of the pathway, is down-regulated in the overexpression lines and slightly

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up-regulated in the mutants (Supplemental Figure 9B).

in

the

early

steps

of

tetrapyrrole

pathway,

such

as

HEMA1

and

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To further understand whether the delayed cotyledon greening phenotype in PCH1OE and

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PCHLOE seedlings is caused by photobleaching, we tested several antioxidant-related genes in

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these backgrounds. COPPER/ZINC SUPEROXIDE DISMUTASE 2 (CSD2) and FE

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SUPEROXIDE DISMUTASE 1 (FSD1) are genes encoding superoxide dismutases; STROMAL

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ASCORBATE PEROXIDASE (SAPX) and CATIONIC AMINO ACID TRANSPORTER 2 (CAT2)

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encode enzymes catalysing the degradation of H2O2 (Mittler, 2002). As a result, all these genes 8

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showed higher expression in the dark and also after white light treatment in dark-grown

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PCH1OE and PCHLOE seedlings except for FSD1. FSD1 had lower expression in the dark but

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higher expression after light treatment in the overexpression seedlings. The expressions of these

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genes were similar or slightly lower in pch1 and pchl mutants compared with wild type

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(Supplemental Figure 10). Taken together, these results suggest that PCH1 and PCHL are subtle

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regulators that controls a small set of key genes involved in the early steps of chlorophyll

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biosynthetic and anti-oxidative pathways.

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PCH1 and PCHL promote degradation of PIF1 and negatively regulate PIF1 target genes

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expressions

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Because light-induced seed germination is mainly regulated by PIF1 (Oh et al., 2004), and PCH1

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and PCHL positively regulate the seed germination (Figure 1A), we examined the native PIF1

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levels in the wild-type, PCH1OE and PCHLOE, pch1, pchl and pch1 pchl double mutant seeds

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under dark and R light pulse treatment. Results show that the light-induced degradation of native

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PIF1 is strongly enhanced in PCH1OE and PCHLOE seeds (Figure 2E, F), where much weaker

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PIF1 band was detected in PCH1/LOE after Rp treatment compared with WT. Conversely, this

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degradation was reduced in pch1, pchl and pch1 pchl double mutants after Rp treatment where

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stronger PIF1 band was observed compared with wild-type seeds (Figure 2A, B). PIF1 mRNA

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levels remain unchanged under the same conditions in these genotypes (Supplemental Figure 11).

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To test if the reduction in PIF1 is phyB-dependent or -independent, we also examined the PIF1

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protein levels in PCH1/LOE and pch1 pchl in phyB-9 mutant backgrounds. Results show that the

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PIF1 levels were partly restored to the wild type levels in the PCH1OE/phyB-9 and

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PCHLOE/phyB-9 (Figure 2E, F). On the other hand, the reduced degradation of PIF1 in pch1

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pchl double mutant remained similar in phyB-9 mutant background (Figure 2C, D). These data

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suggest that PCH1 and PCHL regulate PIF1 degradation in part in a phyB-dependent manner.

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To confirm if the PCH1/L can induce degradation of PIF1 in a phyB-independent manner, we

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examined PIF1 levels in etiolated seedlings grown in true dark condition, where phyB remains in

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inactive Pr form. PIF1 level was still lower in the PCH1/LOE background compared to wild type

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(Figure 2G, H), suggesting that PCH1 and PCHL also regulate PIF1 abundance in darkness in a

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phyB-independent manner.

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PCHs interact with PIF1 independent of phyB and facilitate phyB-PIF1 interaction in vivo

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Because PCH1 and PCHL interact with phyB and phyB interacts with PIF1, we tested whether

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PCH1 and PCHL also interact with PIF1. Yeast two-hybrid assays show that PCH1 and PCHL

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robustly interact with four major PIFs (Supplemental Figures 12A, B). phyB interacts with the

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N-terminal 150 amino acids containing the active phytochrome binding (APB) domain of PIF1

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(Shen et al., 2008). Domain-mapping analyses show that PCH1 might interact with the

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N-terminal APB-APA domain of PIF1 while PCHL might interact with the C-terminal 328

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amino acids containing the bHLH domain of PIF1 (Supplemental Figures 13B, C). Conversely,

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the N-terminal 118 a.a. fragment of PCH1 might interact with PIF1 while the C-terminal

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(184-279 aa) fragment of PCHL might interact with PIF1 (Supplemental Figure 13D, E, F).

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However, these preliminary domain mapping results need further verification.

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To independently verify the physical interactions between PCH proteins and PIF1, we

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performed pull-down assays to test the interaction between the full-length PIF1 and PCH1/L. In

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vitro pull-down assays show that GST-PIF1 could be co-immunoprecipitated by PCH1-His, and

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His-PIF1 could be co-immunoprecipitated by GST-PCHL (Supplemental Figure 14A, B). These

272

data suggest that PCH1 and PCHL physically interact with PIF1 in vitro.

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To further demonstrate that PCH proteins interact with PIF1 in vivo, we performed

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co-immunoprecipitation (Co-IP) assays using protein extracts from PCH1OE and PCHLOE

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transgenic seedlings. HA-YFP-PCH1 and HA-YFP-PCHL were immunoprecipitated and PIF1

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was

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co-immunoprecipitate PIF1 both under dark and red light conditions (Figure 3A). Moreover, this

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interaction between PCH1/PCHL and PIF1 is phyA- and/or phyB-independent, as both PCH1

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and PCHL also interact with PIF1 in phyB-9 and phyA-211 phyB-9 mutant backgrounds (Figure

280

3A, B).

detected

using

anti-PIF1

antibody.

Results

show

that

YFP-PCH1/L

could

281

Because phyB interacts with PCH1 and PCHL as well as PIF1, we further tested whether

282

phyB requires PCH1 and PCHL in order to interact with PIF1 in vivo. We performed Co-IP

283

assays with phyB-GFP transgenic seedlings in both wild-type and pch1 pchl double mutant

284

backgrounds and detected native PIF1. As shown in Figure 3C and D, phyB-GFP interacts with

285

PIF1 in the wild type but not efficiently in the pch1 pchl double mutant background, indicating

286

that phyB-GFP interacts with PIF1 preferentially in the presence of PCH1 and PCHL. Taken

287

together, these data suggest that PCH1 and PCHL interact with PIF1 and might function in

10

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facilitating the phyB-PIF1 interaction in vivo.

289 290

PCH1/L impaired the DNA-binding and transcriptional activity of PIF1

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To determine if PCH1 and PCHL can block the DNA binding ability of PIF1, we performed

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an in vitro DNA-immunoprecipitation assay using the DNA fragment of PIF1 binding region in

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PIL1 promoter. One microgram of biotin-labelled DNA was used for binding assay with

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GST-tagged recombinant PIF1 protein with or without PCH1 and PCHL. Results show that when

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increasing amount of PCH1 and PCHL were added, reduced level of PIF1 was

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immunoprecipitated by PIL1 promoter fragment compared to without PCH1 and PCHL. This

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means that PCH1/L prevents the binding of PIF1 to the PIL1 G-box fragment (Figure 4A). We

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also tested if PCH1 and PCHL affect the DNA-binding ability of PIF1 in vivo by using

299

chromatin immunoprecipitation (ChIP) assays in WT, PCH1OE, and pch1 pchl double mutant

300

seeds. Results show that PIF1 binding to the G-box region of RGA was decreased in PCH1OE,

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but increased in pch1 pchl double mutant compared with WT (Figure 4B). To further determine

302

whether PCH1 and PCHL inhibit the transcriptional activation activity of PIF1, we performed in

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vivo transient transcription assays as described previously (Moon et al., 2008; Shiet al., 2013;

304

Zhu et al., 2016). pPIL1:LUC and 35S:PIF1-HA were co-transformed with either GFP only,

305

PCH1-GFP, or PCHL-GFP driven by the constitutively active 35S promoter into tobacco leaves.

306

Renilla LUC driven by 35S promoter was used as an internal control. The results show that PIF1

307

activates pPIL1 driving LUC expression (Figure 4C); however, the addition of both PCH1- and

308

PCHL-GFP reduced the level of LUC activity, suggesting that PCH1/L blocks PIF1 transcription

309

activation from the PIL1 promoter. Together, our data suggest that PCH1/L impaired the DNA

310

binding and transcriptional activation activity of PIF1.

311

To examine whether the PIF1 target gene expression is altered in these backgrounds, we

312

performed RT-qPCR analyses of a few PIF1 target genes at the seed stage. Results show that the

313

expression of the PIF1 target genes both under far-red light and red light is decreased in

314

PCH1OE and PCHLOE seeds and increased in mutants compared with wild-type seeds,

315

consistent with the PIF1 level in these backgrounds (Figure 4D). These data suggest that PCH1

316

and PCHL inhibit the expression of PIF1 target gene possibly by reducingthe DNA binding and

317

transcriptional activation activity of PIF1.

318

11

319

PCH1 and PCHL are unstable in the dark and this degradation is mediated by COP1

320

PCH1 has been shown to be involved in photoperiodic control of hypocotyl length (Huang et al.,

321

2016). To examine if the protein stability of PCH1 and PCHL is regulated under dark and light

322

conditions, PCH1OE and PCHLOE seedlings were grown in darkness for 4 d and then

323

transferred to light over time or dark-grown seedling were exposed to light for 4 h and then

324

transferred back to darkness over time. Immunoblot analysis using anti-GFP antibody showed

325

that PCH1 and PCHL proteins were increased after exposure to light for 1 to 24 h. Conversely,

326

PCH1 and PCHL proteins became unstable when light-exposed plants were returned to darkness

327

(Figure 5A-C). To examine whether the 26S proteasome-dependent proteolysis is involved in the

328

degradation of YFP-PCH1/L under dark, we treated dark-grown seedlings with bortezomib, a

329

proteasome inhibitor and perform immunoblot analysis. Results show that both the YFP-PCH1

330

and YFP-PCHL fusion proteins remained abundant under dark when treated with bortezormib

331

(Supplemental Figure 15A, B). Although the expression of PCH1and PCHL transgenes was

332

minimally affected under these conditions (Supplemental Figure 15C), the corresponding

333

proteins were unstable and might be degraded by the 26S proteasome pathway.

334

Because COP1 is known to target various light signaling components in darkness, we

335

hypothesized that COP1 might be involved in regulating PCH1 and PCHL levels under dark

336

condition. To support this hypothesis, we crossed PCH1OE and PCHLOE with cop1-4 mutants

337

and detected the PCH1 and PCHL protein levels in these backgrounds. Both PCH1 and PCHL

338

proteins were more abundant in cop1-4 mutant background compared to controls under dark

339

conditions (Figure 5D), suggesting that COP1 might mediate PCH1 and PCHL’s degradation in

340

darkness.

341

To further test whether COP1 could interact with PCH1 and PCHL, we performed yeast

342

two-hybrid analysis. Our results show that the PCH1 and PCHL full-length proteins robustly

343

interact with COP1 (Supplemental Figure 16A). Domain-mapping analyses show that PCH1 has

344

the highest interaction activity with the ZINC and coil-coil domain of COP1. PCH1 and PCHL

345

also have similar and relatively high interaction with the WD40 repeat domain (Gβ) of COP1

346

and COP1 without the ZINC and coil-coil domains (Supplemental Figure 16A). Conversely, the

347

N-terminal 118 amino acids fragment of PCH1 is both necessary and sufficient for the

348

interaction with full-length COP1 (Supplemental Figure 16B). On the other hand, the C-terminal

349

fragment of PCHL displayed a strong interaction with COP1 (Supplemental Figure 16B). We

12

350

also performed pull-down assays to test the interaction between the full-length COP1 and

351

PCH1/L. Results show that both full-length PCH1-His and GST-PCHL could be pulled down by

352

MBP-COP1 (Supplemental Figures 17A, B). We also used PCH1OE and PCHLOE in COP1-HA

353

backgrounds to perform in vivo co-immunoprecipitation (co-IP) assays. Figure 5E shows that

354

COP1-HA robustly co-immunoprecipitates YFP-PCH1 and YFP-PCHL. Taken together, the

355

yeast two-hybrid and in vitro/vivo co-IP assays provide strong evidence that PCH1 and PCHL

356

can associate with COP1.

357

COP1 has been previously reported to be the E3 ligase of PIF1 (Zhu et al., 2015). As PCH1

358

and PCHL interact with both COP1 and PIF1, and PIF1 is relatively more abundant in pch1 and

359

pchl mutant seeds compared to wild type after Rp treatment (Figure 2), we further investigated

360

whether PCH1 and PCHL promote PIF1 degradation through regulating the interaction between

361

COP1 and PIF1. We performed in vitro co-IP assays between COP1 and PIF1 in the absence and

362

presence of increasing concentrations of PCH1/L. The results show that more PIF1 was

363

immunoprecipitated by COP1 after adding increasing amount of both PCH1 and PCHL

364

(Supplemental Figures 18). These data suggest that the enhanced interaction between COP1 and

365

PIF1 in the presence of PCHs might contribute to the enhanced degradation of PIF1 by COP1.

366 367

COP1 mediates the ubiquitination of both PCH1 and PCHL

368

To further assess whether COP1 mediates the ubiquitination of PCH1 and PCHL, we first

369

examined the in vivo ubiquitination levels of PCH1 and PCHL under dark and light conditions.

370

PCH1OE and PCHLOE were grown in the dark for 4 d and the seedlings were pretreated with

371

bortezormib for 4 h in darkness before being exposed to white light or remained in the dark for

372

additional 4 h. The YFP-PCH1 and YFP-PCHL were immunoprecipitated from these samples

373

and then detected with anti-GFP and anti-Ubi antibodies. Results show that the ubiquitination

374

levels of YFP-PCH1 and YFP-PCHL are higher in darkness compared to light samples (Figures

375

6A,

376

poly-ubiquitination-dependent. We also examined whether the ubiquitination of PCH1 and

377

PCHL in the dark is affected in cop1 4 mutant. Four-day old dark grown seedlings

378

overexpressing PCH1 and PCHL in both wild-type and cop1-4 mutant backgrounds were

379

pretreated with bortezormib for 4 h in darkness and then remained in the dark for additional 4 h.

380

Strikingly, the level of PCH1 and PCHL ubiquitination is drastically reduced in the cop1 4

B),

suggesting

that

PCH1

and

PCHL’s

13

degradation

in

the

dark

is

381

backgrounds compared with the wild type (Figure 6C).

382

To investigate the trans-ubiquitination activity of COP1 to PCH1 and PCHL, we performed in

383

vitro ubiquitination assays as described previously (Saijo et al., 2003; Seo et al., 2003; Xu et al.,

384

2014). In vitro, COP1 directly ubiquitinates PCH1 and PCHL (Figures 6D, E, left blots).

385

Immunoblotting with anti-GST antibody also displayed the ubiquitinated GST-PCH1 and

386

GST-PCHL (Figures 6D, E, right blots), suggesting that COP1 specifically mediates PCH1 and

387

PCHL’s ubiquitination in vitro.

388

pch1 partially suppress the constitutive photomorphogenic phenotypes in cop1-6

389

To examine the biological significance of regulation of PCH1 and PCHL protein levels by COP1,

390

we created pch1 cop1-6 double mutant and compared their phenotypes under dark conditions.

391

Results show that pch1 partially suppresses the short hypocotyl and open cotyledon phenotypes

392

of cop1-6 under darkness (Figure 6F-H). These data along with our biochemical evidence that

393

COP1 directly targets PCH1 and PCHL for degradation suggest that these two proteins are

394

functioning positively in light signaling pathways and that COP1 is targeting them to prevent

395

photomorphogenesis in darkness.

396 397 398 399

DISCUSSION

400

positively regulate phyB signalling by preventing its thermal reversion (Enderle et al., 2017;

401

Huang et al., 2016). Recently, it was also shown that PCH1 promotes phyB photobody formation

402

and interacts with phyB’s PASII domain to regulate light, temperature and circadian signalling

403

pathways (Huang et al., 2019). Here, we provide evidence that PCH1 and PCHL play a broader

404

role in regulating light responses including seed germination, hypocotyl negative gravitropism,

405

and chlorophyll biosynthesis not only by delaying phyB thermal reversion, but also by

406

interacting with other light signalling components, PIFs and COP1. Several lines of evidence

407

support this hypothesis that PCH1 and PCHL regulate these pathways by modulating either the

408

stability and/or activity of PIF1 and possibly other PIFs in a phyB-dependent and -independent

409

manner. By examining the PIF1 protein levels in PCH1/LOE and pch1 and pchl mutant seeds,

410

we provide molecular evidence that PCH1 and PCHL negatively regulate PIF1 level (Figure

411

2A-H). PCH1 and PCHL directly interact with PIF1 and possibly other PIFs and might facilitate

In previous reports, PCH1 and PCHL were identified as phyB-interacting proteins that

14

412

the interaction between phyB and PIF1, thereby promoting PIF1 degradation in response to light

413

(Figures 3). Moreover, PCH1/L impaired the DNA-binding and transcriptional activity of PIF1

414

(Figure 4A-C). Consistently, the PIF1 target genes expressions were down-regulated in

415

PCH1/LOE and up-regulated in pch1 and pchl mutants (Figure 4D). Thus, PCH1/PCHL and

416

PIF1 are functioning antagonistically to regulate seed germination, hypocotyl negative

417

gravitropism and chlorophyll biosynthesis. Finally, we also demonstrate that, like other positive

418

light signalling regulators, PCH1 and PCHL are ubiquitinated by COP1 and are degraded

419

through 26S proteasome pathway.

420

Chlorophyll and carotenoid biosynthesis are co-ordinately regulated in Arabidopsis in

421

response to light to avoid photooxidative damage of seedlings upon illumination, and PIFs play a

422

critical role in directly regulating both of these pathways (Moon et al., 2008; Stephenson et al.,

423

2009; Toledo-Ortíz et al., 2010). It has been demonstrated that PIF1 and PIF3 directly and

424

indirectly regulate the key genes to fine-tune the tetrapyrrole pathway (Moon et al., 2008;

425

Stephenson et al., 2009). In these pathways, phyB and PIF1/PIF3 work antagonistically to

426

regulate the greening process of seedlings. However, in our cotyledon greening analyses, PCH1

427

and PCHL did not promote the greening but instead lead to the pale green cotyledon phenotype

428

which is probably caused by photobleaching. Here, we provide molecular data that PCH1/L

429

positively regulates the expressions of HEMA1 and GSA2 gene which are enzymes catalysing the

430

early stages of chlorophyll biosynthesis (Supplemental Figure 8), while PCH1/L negatively

431

regulates the expression of POR genes which catalyse the final steps of chlorophyll biosynthesis

432

(Supplemental Figure 9). These genes are oppositely regulated by PIF1 and PIF3 (Moon et al.,

433

2008; Stephenson et al., 2009), resulting in accumulation of free protochlorophyllide in excess

434

due to an imbalance between the production of protochlorophyllide by HEMA1 and GSA2, and

435

catabolism of protochlorophyllide by the POR enzymes. Consistent with our hypothesis, PCH1/L

436

shows higher expression levels of antioxidant-related genes including CSD2, FSD1, SAPX, and

437

CAT2 compared to the wild type (Supplemental Figure 10). Because PCH1 and PCHL directly

438

interact with PIF1 and other PIFs and regulate PIF1 abundance, it is possible that PCH1/L might

439

also prevent PIF function to fine-tune chlorophyll biosynthesis.

440

It is well accepted that phytochromes and PIFs have a Yin Yang relationship. In response to

441

light signal, phytochromes interact with PIFs to induce its phosphorylation, polyubiquitination,

442

and subsequent degradation under both red and far-red light conditions (Leivar and Quail, 2011; 15

443

Pham et al., 2018b). In our previous reports, we showed that CUL4COP1–SPA complex functions as

444

a kinase-E3 ubiquitin ligase complex for the rapid light-induced degradation of PIF1 and PIF5

445

(Paik et al., 2019; Pham et al., 2018b; Zhu et al., 2015). Here, we show that PCH1 and PCHL

446

promote PIF1 degradation (Figure 2) and also directly interact with PIF1 to inhibit the DNA

447

binding and transcriptional activation activity of PIF1 (Figures 3-4). Previously, other signalling

448

components were shown to interact with PIFs through the bHLH domain and negatively regulate

449

PIF function. For example, DELLAs and ELF3 interact with PIFs through the bHLH domain and

450

prevent PIFs from binding to DNA (de Lucas et al., 2008; Feng et al., 2008; Nieto et al., 2014).

451

Thus, PCH1/L might regulate PIFs in multiple ways: (i) Under light, PCH1/L facilitates the

452

binding of phyB to PIF1 and increases the phosphorylation and subsequent degradation of PIF1

453

(Figure 3C); (ii) PCH1/L increases the interaction of PIF1 with COP1-SPA1 complex to mediate

454

PIF1’s polyubiquitination and subsequent degradation under both dark and light conditions

455

(Supplemental Figure 18); and/or (iii) PCH1 and PCHL interact with PIF1 to inhibit the

456

DNA-binding and transcriptional activation activities of PIF1 (Figures 3-4). While the

457

light-induced degradation of PIF1 is most likely a phyB-dependent process, the physical

458

interaction between PCH and PIF1 along with inhibition of DNA binding and transcriptional

459

activation activities of PIF1 might be phyB-independent.

460

Biochemical assays showed that PCH1 and PCHL proteins are unstable in the dark and are

461

stabilized in response to light (Figure 5A). In 2016, Huang et al. reported that PCH1 is an

462

evening-peaked protein and oscillate during a short-day time course (Huang et al., 2016). This is

463

probably because PCH1 and PCHL are stabilized under light condition and are degraded in the

464

dark as observed in this study. When light is given up to 24 h, PCH1 and PCHL gradually

465

accumulate regardless of the internal circadian clock (Figure 5A). Under the light conditions,

466

PCH1 and PCHL accumulate and act as stabilizers of phyB-photobodies and positively regulate

467

phyB-mediated light responses. However, in the dark, PCH1 and PCHL are recognized by COP1

468

and are degraded through the 26S proteasome system (Figures 5-6). Many light signalling

469

components are reported as the substrates of COP1, including the bZIP transcription factor HY5

470

(Osterlund et al., 2000), the myb transcription factor LAF1 (Seo et al., 2003), the bHLH

471

transcription factor HFR1 (Duek et al., 2004), and others (Lau and Deng, 2012; Xu et al., 2015).

472

In general, COP1 interacts with its substrates through the WD40 repeats to mediate their

473

degradation in plants (Holm and Deng, 1999; Holm et al., 2001). Our yeast two-hybrid results

16

474

showed that PCH1 and PCHL do have modest interaction activity with WD40 domain of COP1

475

(Supplemental Figure 16A). However, PCH1 also showed very high interaction activity with Zn

476

finger and coiled-coil domain only (Supplemental Figure 16A). It is possible that PCH1 has

477

other role(s) in regulating the function of COP1.

478

Based on our data and those of others, we propose a model that summarizes our findings

479

(Figure 7). In the dark, PCH1 and PCHL are associated with COP1 complex and are being

480

degraded through the 26S proteasome pathway. However, PCH1 and PCHL also interact with

481

PIF1 and regulate PIF1 either by inhibiting the DNA binding and transcriptional activation

482

activity of PIF1 and/or by inducing it’s degradation through the COP1/SPA complex as this

483

complex has been shown to degrade PIFs even in darkness (Pham et al., 2018a; Pham et al.,

484

2018c; Xu et al., 2017). In the light condition, PCH1 and PCHL facilitate phyB-PIF1 interaction

485

and accelerate PIF1 degradation through the 26S proteasome system. Taken together, these data

486

provide a novel mechanism by which PCH1 and PCHL fine-tune light responses under

487

continuous light and dark and/or diurnal conditions.

488 489

METHODS

490 491

Plant Materials and Growth Conditions

492

Arabidopsis thaliana plants were grown on Metro-Mix 200 soil (Sun Gro Horticulture) (Shen et

493

al., 2005). Light fluence rates were measured using a spectroradiometer (model EPP2000;

494

StellarNet) as described (Shen et al., 2005). Seeds were surface-sterilized and plated on

495

Murashige and Skoog (MS) growth medium containing 0.9% agar without sucrose as described

496

(Shen et al., 2005). After 3 to 4 d of moist chilling at 4°C in the dark, seeds were exposed to 3 h

497

of white light at room temperature before placing them in the dark for another 4 d.

498

All Arabidopsis thaliana lines used were in Col-0 background. The pch1 (T-DNA insertion line;

499

SALK_024229), pchl mutant (SALK_206946C; #N696800), pch1 pchl double mutant, phyB-9,

500

and phyA-211 phyB-9 double mutant mutants have been described previously (Enderle et al.,

501

2017).

502

(p35S:HA-YFP-PCH1:terRbcS;

503

(p35S:HA-YFP-PCHL:terRbcS; plasmid pBE52c) have also been described (Enderle et al.,

504

2017). phyB-GFP over-expression line (p35S:PHYB-GFP) and phyB-GFP phyA-211 pch1 pchl

The

PCH1

and

PCHL

overexpression plasmid

lines

pDS366)

17

expressing

HA-YFP-PCH1ox

and

HA-YFP-PCHLox

505

used for co-IP is in phyA-211 phyB-9 double mutant background and have been described

506

previously (Enderle et al., 2017).

507

For PIF1 CRISPR lines, we transformed pHEE2E-PIF1-PAM binary vector (Wang et al., 2015)

508

into pch1, pchl, and pch1 pchl mutant plants via the floral dip method. T0 plants were screened

509

on MS plates containing 25 mg/L hygromycin and transplanted to soil. We extracted genomic

510

DNA from T1 plants and amplified fragments surrounding the target sites of PIF1 by PCR using

511

PAM1-F and R primers, and the sequencing results showed an insertion of adenine (A) in pch1

512

or thymidine (T) in pchl and pch1 pchl backgrounds after G473 in PIF1 ORF (Supplementary

513

figure 19), making them frame shift mutations. Homozygous lines were selected for the

514

phenotypic assays. The CRISPR T-DNA has not been outcrossed yet in these lines.

515 516

Germination and Hypocotyl Negative Gravitropism assay

517

For the phyB-dependent germination assay, triplicate sets of 60 seeds for each genotype were

518

surface sterilized and plated on filter paper placed on agar medium (0.6% phytoagar, pH 5.7). At

519

1 h after the start of seed sterilization, the plated seeds were irradiated with far-red (34 µmol m-2

520

s-1) light for 5 min. Seeds were then either directly incubated in the dark or first exposed to

521

increasing amount of red light before being placed in the dark at 21°C. After 5 days of incubation

522

in the dark, germinated seeds were determined by the emergence of radicles.

523

For hypocotyl negative gravitropism assay, surface sterilized seeds were plated on MS agar (1/2

524

MS, 0.8% phytoagar, and 0.05% Mes, pH 5.7), imbibed for 3 d at 4 °C in the dark, and then the

525

germination was induced by incubation in white light (70 µmolm−2 s−1) at 22°C for 4–8h. Plates

526

were incubated vertically in the dark. Following 16 h in darkness at 22°C, seedlings were given a

527

far-red light pulse (5 min, 34 µmolm−2s−1), to inactivate phytochromes before transfer to

528

different fluences of red-light pulse, which were given at the same time each day. After 2 d

529

incubation, plates were turned by 90° counter-clockwise, and incubated 2 more days under the

530

same light conditions. Growth orientations of hypocotyls were determined by the degrees from

531

vertical axis.

532 533

Cotyledon Greening and Chlorophyll Measurement

534

Surface sterilized seeds were plated on MS agar (1/2 MS, 0.8% phytoagar, and 0.05% Mes, pH

535

5.7), imbibed for 3 d at 4 °C in the dark, then germination was induced by incubation in white

18

536

light (70 µmolm−2 s−1) at 22°C for 3 h. Following 16h in darkness at 22°C, seedlings were given

537

a far-red light pulse (5min, 34 µmolm−2 s−1), to inactivate phytochromes before transfer to dark

538

for 3 d. After dark incubation, seedlings were transferred to white light (70 µmolm−2 s−1) at 22°C.

539

Photographs were taken after 12 h light incubation. Measurement of chlorophyll content was

540

performed as described previously (Runge et al., 1995). Briefly, Arabidopsis seedlings were

541

weighed and ground in liquid nitrogen. Chlorophyll was extracted from powdered samples with

542

80% acetone in water, and chlorophyll concentration was calculated after measuring the

543

absorption at 663 and 645 nm.

544

Pchlide were extracted as described (Runge et al., 1995) except 4-day-old dark-grown seedlings

545

for each genotype were used. Spectrofluorometery (TimeMaster Pro; Photon Technologies

546

International) was performed at an excitation wavelength of 440 nm and an emission wavelength

547

of 600-700 nm, and data were curve-fitted by using PeakFit, version 4.11 (Systat Software).

548 549

Visualization of Endodermal Amyloplasts.

550

To visualize endodermal amyloplasts by iodine staining, seedlings were fixed in FAA (5%

551

Formaldehyde, 45% Ethanol, 5% Acetic acid) solution for 24 h at 4 °C. After fixation, seedlings

552

were rinsed in 50% (vol/vol) ethanol once and stained in I2-KI solution [2% (wt/vol) iodine, 5%

553

(wt/vol) potassium iodine and 20% (wt/vol) chloral hydrate] for 1 min. Samples were de-stained

554

in trichloroacetic acid: phenol: lactic acid (1:1:1 ratio) for 5 min and carefully mounted on slides

555

with a drop of distaining solution for the light microscopic observation.

556 557

RNA Isolation, RT-PCR, and Quantitative RT-PCR Assays

558

Total RNA was isolated from materials indicated in the figure legends using the Sigma-Aldrich

559

plant RNA isolation kit as described (Pham et al., 2018c). One microgram of total RNA was

560

reverse transcribed using SuperScript III (Invitrogen) as described in the manufacturer’s protocol.

561

For the RT-PCR, gene-specific primers listed in Supplemental Table 1 were used to detect

562

mRNA levels. PP2A (At1g13320) was used as a control for normalization of the expression data.

563

The RT-qPCR assays used the Power SYBR Green RT-qPCR Reagents Kit (Applied

564

Biosystems).

565 566

Protein Extraction and Protein Gel Blotting

19

567

A total of 50 µg seeds were plated on wet filter paper, incubated under phyB-dependent

568

germination assay conditions (Oh et al., 2004; Zhu et al., 2015), and harvested at the times

569

indicated. All seed tissues were collected in the dark and ground into powder in liquid nitrogen.

570

For individual sample, total protein was extracted in 50 µl urea extraction buffer (0.1M

571

phosphate buffer pH 6.8, 0.01 M Tris-Cl, pH 6.8, 48% urea (w/v), 1mM PMSF, 40 µM

572

bortezomib and 1 x protease inhibitor cocktail (Sigma-Aldrich Co., St Louis, MO, Cat No:

573

P9599). Samples were centrifuged at 16,000g for 10min at 4°C. Supernatants were filtered

574

through Filtration columns (Sigma-Aldrich Co., St Louis, MO, Cat. No: C6866) and boiled for 3

575

min with 6 x SDS buffer added. Thirty µl supernatants of individual samples were loaded on an

576

8% SDS–PAGE gels. The total protein was blotted onto PVDF membranes and probed with

577

native anti-PIF1 (1:5,000 dilutions) (Shen et al., 2008) and anti-RPT5 (1:1000 dilutions; Enzo

578

Life Sciences, Farmingdale, NY, Cat. No: PW8375-0100) antibodies.

579 580

Yeast Two-Hybrid Analyses

581

The cloning of the full-length, different truncated forms and mutant versions of PIF1 and PCH1

582

have been described previously (Enderle et al., 2017; Xu et al., 2014). The full-length and

583

various truncated forms of PCHL were PCR amplified using the primers listed in Supplementary

584

Table 1. The PCR product and the vector pJG4.5 were digested with EcoRI and XhoI restriction

585

enzymes, and then ligated to produce AD-fusion constructs. All the constructs were verified by

586

restriction enzyme digestion and sequencing. For the yeast two-hybrid assays, different

587

combinations of prey and bait constructs were transformed into the yeast strain, EGY48-0 and

588

selected on His, -Ura, -Trp minimal synthetic medium at 30°C for 3–4 d. The quantitative

589

β-galactosidase assay was performed according to the manufacturer’s instructions (Matchmaker

590

Two-Hybrid System, Clonetech Laboratories Inc.,). Three independent repeats were performed

591

for the β-galactosidase assays and the average values are shown with standard deviation.

592 593

In Vitro and in Vivo Co-immunoprecipitation Assays

594

The in vivo co-immunoprecipitation assays were performed as described (Zhu et al., 2015).

595

Briefly, 4-d-old dark-grown seedlings were pre-treated with 40 µM bortezomib (LC Laboratories,

596

Woburn, MA) for at least 4h. Total proteins were extracted from 0.4g tissue with 800µl native

597

extraction buffer (100mM phosphate buffer, pH 7.8, 150mM NaCl, 0.1% NP40, 1x protease

20

598

inhibitor cocktail (Sigma-Aldrich Co., St Louis, MO, Cat. No: P9599), 1mM PMSF, 40µM

599

bortezomib, 25mM β-glycerophosphate, 10mM sodium fluoride (NaF) and 2mM Na

600

orthovanadate). After 15 min centrifugation at 16,000g at 4°C in darkness, supernatants were

601

incubated with Dynabeads Protein A (Life Technologies Co., Carlsbad, CA, Cat. No: 10002D)

602

bound with anti-GFP antibody (Abcam, Cambridge, MA, Cat. No: ab9110). Twenty-microlitre

603

Dynabeads with 1g antibody were used for individual sample. After 2 h incubation in the dark at

604

4°C, beads were washed three times with 1 ml extraction buffer with 0.2% NP40.

605

Immuno-precipitated proteins were eluted with 1x SDS loading buffer and incubated at 65°C for

606

5 min. Samples were loaded on an 8 % SDS–PAGE gel, blotted onto PVDF membranes and

607

probed with antibodies.

608

For in vitro co-immunoprecipitation assay, His-PCH1 and GST-PCHL were used as bait protein

609

to pull-down GST-PIF1 and His-PIF1 (Huq et al., 2004), respectively. His-PCH1 was expressed

610

from pDEST17 vector. The full-length open reading frame of PCH1 and PCHL were PCR

611

amplified using the primers indicated in Supplementary Table 1. The PCH1 PCR product was

612

cloned into pENTR and then recombined into pDEST17 vector. The PCHL PCR product was

613

digested along with pGEX4t-1 vector with EcoRI and XhoI and then ligated to produce

614

pGEX4t-1-PCHL construct. Bacterial extracts expressing His-PCH1 and GST-PCHL were

615

purified with amylose resin and glutathione agarose resin, respectively, in 1 x PBS buffer as

616

described in the manufacturer’s protocol. The samples were boiled and analysed by Western blot

617

onto PVDF membrane. Anti-His antibody (Santa Cruz Biotechnology, Cat. No: SC-803, 1:500

618

dilutions) and anti-GST antibodies were used to detect bait and prey proteins.

619 620

In Vitro DNA Pull-Down Assay Using Biotinylated DNA

621

One microgram of biotin-labeled DNAs was used for binding assay with GST-tagged

622

recombinant PIF1 protein (100 ng). His-PCH1 and GST-PCHL (2x, 5x and 10x) were used for

623

the inhibition of PIF1 DNA binding. After 2 hours incubation at 4°C in pull-down buffer [50mM

624

Tris-Cl (pH 7.5), 100mM NaCl, 2mM DTT, 0.05% NP-40 and 1X protease inhibitor],

625

streptavidin agarose beads (Roche, IN, USA) was added to each binding reaction and further

626

incubated for 30 min at 4°C. Beads were washed at 4°C briefly five times using pull-down buffer

627

and boiled in SDS loading buffer, and then loaded onto SDS-PAGE gel for further Western blot

628

analysis using anti-GST antibodies.

21

629 630

Chromatin Immunoprecipitation (ChIP) Assay

631

For the ChIP assay, WT, PCH1OE, and pch1 pchl double mutant seeds were irradiated with FR

632

light (3.2 µmolm-2s-1) for 5 min, incubated in the dark for 6 h, and then cross-linked in 1%

633

formaldehyde solution under a vacuum for 1 h. The seeds were then ground to powder in liquid

634

nitrogen, and chromatin complexes were isolated and sonicated as described (Oh et al., 2007).

635

The sonicated chromatin complexes were precipitated with anti-PIF1 antibody. The cross-linking

636

was then reversed, and DNA was extracted using the QIAEX II gel extraction kit (Qiagen;

637

catalog no.

638

immunoprecipitated at the different promoter regions of binding target genes (see Figure 4B).

20051).

RT-qPCR was performed

to

measure the amount of

DNA

639 640

In Vitro Ubiquitination Assays

641

The His-PCH1, GST-PCHL, MBP-COP1 (Saijo et al., 2003), and E2 At-UBC8 (Lee et al., 2009)

642

were prepared as described previously. All in vitro ubiquitination assay procedures were

643

performed as described (Saijo et al., 2003). Briefly, 2g of FLAG-ubiquitin (U120; Boston

644

Biochem), 25ng of E1 (UBE1, E-305; Boston Biochem), 100ng of E2 (At-UBC8), 600ng of

645

MBP-COP1, and 400ng of His-PCH1 or GST-PCHL were used in the reaction. The

646

FLAG-ubiquitin-conjugated PCH1, PCHL and COP1 were detected by immunoblot with

647

anti-FLAG antibody (F1804; Sigma-Aldrich). Anti-His-HRP conjugate and anti-GST-HRP

648

conjugate were used for His-PCH1 and GST-PCHL detection, respectively.

649 650 651

SUPPLEMENTARY MATERIAL

652

Supplementary material is available online.

653 654

Supplemental Figure 1.pif1 is epistatic to pch in regulating seed germination.

655

Supplemental Figure 2. Hypocotyl gravitropism in WT, PCH1OE, PCHLOE, pch1, pchl and

656

pch1 pchl double mutants.

657

Supplemental Figure 3.pif1mutation partially rescued the gravitropism phenotypes of pch

658

mutants.

659

Supplemental Figure 4. The gravitropism phenotypes of PCH1/LOE is phyB-dependent.

22

660

Supplemental Figure 5. Delayed cotyledon greening phenotype of PCH1 and PCHL

661

overexpression transgenic plants.

662

Supplemental Figure 6. Delayed cotyledon greening phenotype of PCH1 and PCHL

663

overexpression plants were rescued by phyB mutation.

664

Supplemental Figure 7. PCH1 and PCHL promote the accumulation of Pchlide.

665

Supplemental Figure 8. Tetrapyrrole pathway showing genes directly or indirectly regulated by

666

PIF1.

667

Supplemental Figure 9. Chlorophyll biosynthesis genes involved in the early stages are

668

upregulated by PCH1 and PCHL.

669

Supplemental Figure 10. The expressions of antioxidant-related genes are increased in PCH1

670

and PCHL overexpression lines.

671

Supplemental Figure 11. The PIF1 mRNA expression levels in Col-0, PCH1/LOE, pch1, pchl,

672

and pch1 pchl double mutant background.

673

Supplemental Figure 12.PCH1 and PCHL interact with four major PIFs.

674

Supplemental Figure 13. Yeast two-hybrid assay showing PCH1 and PCHL interact with PIF1

675

in vitro.

676

Supplemental Figure 14. Pull-down assays showing PCH1 and PCHL interact with PIF1 in

677

vitro.

678

Supplemental Figure 15. PCH1 and PCHL are degraded by the 26S proteasome pathway.

679

Supplemental Figure 16. Y2H showing PCH1 and PCHL interact with COP1 in vitro.

680

Supplemental Figure 17. Pull-down assays showing PCH1 and PCHL interact with COP1 in

681

vitro.

682

Supplemental Figure 18. PCH1/Lpromotes the interaction between COP1 and PIF1 in an in

683

vitropull-down assay.

684

Supplemental Figure 19. Sequencing results showing the additional nucleotide insertion of the

685

PIF1 CRISPR lines in pch1, pchl, and pch1 pchl double mutant backgrounds.

686 687 688

FUNDING

23

689

This work was supported by grants from the National Institute of Health (NIH) (GM-114297)

690

and National Science Foundation (MCB-1543813) to E.H, and by a grant from the German

691

Research Foundation (DFG) to A.H.(HI 1369/7-1), and by the DFG under Germany's Excellence

692

Strategy (CIBSS – EXC-2189 – Project ID 390939984).

693 694

AUTHOR CONTRIBUTIONS

695

M.C.C., B.E., P.K.K., A.H. and E.H. designed experiments. M.C.C., B.E., P.K.K. and R.I. carried

696

out experiments. M.C.C. and E.H. analyzed data and interpreted the results. M.C.C. wrote the

697

article. M.C.C., B.E., A.H. and E.H. edited the manuscript.

698 699 700

ACKNOWLEDGMENTS

701

We thank Ms. Madeleine Nagle for technical assistance, and the Huq lab members for the

702

technical support and critical reading of the manuscript.

703 704

COMPETING INTERESTS

705

The authors declare no competing financial interests.

706 707

24

708 709 710 711 712 713

REFERENCES

714 715 716 717

Bauer, D., Viczian, A., Kircher, S., Nobis, T., Nitschke, R., Kunkel, T., Panigrahi, K.C., Adam, E., Fejes, E., Schafer, E., et al. (2004). Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis. Plant Cell 16:1433-1445.

718 719 720

Buskirk, E.K.V., Reddy, A.K., Nagatani, A., and Chen, M. (2014). Photobody localization of phytochrome B is tightly correlated with prolonged and light-dependent inhibition of hypocotyl elongation in the dark. Plant Physiol 165:595-617.

721 722

Casal, J.J. (2013). Photoreceptor signaling networks in plant responses to shade. Annu Rev Plant Biol 64:403-427.

723 724 725 726 727

Chen, H.D., Huang, X., Gusmaroli, G., Terzaghi, W., Lau, O.S., Yanagawa, Y., Zhang, Y., Li, J.G., Lee, J.H., Zhu, D.M., et al. (2010). Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time. Plant Cell 22:108-123.

728 729 730

de Lucas, M., Davière, J.M., Rodríguez-Falcón, M., Pontin, M., Iglesias-Pedraz, J.M., Lorrain, S., Fankhauser, C., Blaezquez, M.A., Titarenko, E., and Prat, S. (2008). A molecular framework for light and gibberellin control of cell elongation Nature 451:480-484.

731 732 733

Deng, X.-W., Matsui, M., Wei, N., Wagner, D., Chu, A.M., Feldman, K.A., and Quail, P.H. (1992). COP1, an arabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and a Gβ homologous domain. Cell 71:791-801.

734 735 736

Duek, P.D., Elmer, M.V., van Oosten, V.R., and Fankhauser, C. (2004). The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1. Curr Biol 14:2296-2301.

737 738 739

Enderle, B., Sheerin, D.J., Paik, I., Kathare, P.K., Schwenk, P., Klose, C., Ulbrich, M.H., Huq, E., and Hiltbrunner, A. (2017). PCH1 and PCHL promote photomorphogenesis in plants by controlling phytochrome B dark reversion. Nature Communications 8:2221.

740 741 742

Feng, S.H., Martinez, C., Gusmaroli, G., Wang, Y., Zhou, J.L., Wang, F., Chen, L.Y., Yu, L., Iglesias-Pedraz, J.M., Kircher, S., et al. (2008). Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature 451:475-U479.

743 744

Hoecker, U. (2017). The activities of the E3 ubiquitin ligase COP1/SPA, a key repressor in light signaling. Current Opinion in Plant Biology 37:63-69.

745 746

Holm, M., and Deng, X.W. (1999). Structural organization and interactions of COP1, a light-regulated developmental switch. Plant Mol Biol. 41:151-158.

747 748

Holm, M., Hardtke, C.S., Gaudet, R., and Deng, X.W. (2001). Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1.

Balcerowicz, M., Fittinghoff, K., Wirthmueller, L., Maier, A., Fackendahl, P., Fiene, G., Koncz, C., and Hoecker, U. (2011). Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2. The Plant Journal 65:712-723.

25

749

EMBO J 20:118-127.

750 751 752 753

Huang, H., McLoughlin, K.E., Sorkin, M.L., Burgie, E.S., Bindbeutel, R.K., Vierstra, R.D., and Nusinow, D.A. (2019). PCH1 regulates light, temperature, and circadian signaling as a structural component of phytochrome B-photobodies in Arabidopsis. Proceedings of the National Academy of Sciences 116:8603-8608.

754 755 756 757

Huang, H., Yoo, C.Y., Bindbeutel, R., Goldsworthy, J., Tielking, A., Alvarez, S., Naldrett, M.J., Evans, B.S., Chen, M., and Nusinow, D.A. (2016). PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis. eLife 5:e13292.

758 759 760

Huq, E., Al-Sady, B., Hudson, M., Kim, C., Apel, K., and Quail, P.H. (2004). Phytochrome-interacting factor 1 is a critical bHLH regulator of chlorophyll biosynthesis. Science 305:1937-1941.

761 762 763

Huq, E., and Quail, P.H. (2005). Phytochrome signaling. In: Handbook of Photosensory Receptors--Briggs, W.R., and Spudich, J.L., eds. Weinheim, Germany: Wiley-VCH. 151-170.

764 765

Jang, I.C., Yang, J.Y., Seo, H.S., and Chua, N.H. (2005). HFR1 is targeted by COP1 E3 ligase for post-translational proteolysis during phytochrome A signaling. Genes Dev 19:593-602.

766 767 768

Jung, J.-H., Domijan, M., Klose, C., Biswas, S., Ezer, D., Gao, M., Khattak, A.K., Box, M.S., Charoensawan, V., Cortijo, S., et al. (2016). Phytochromes function as thermosensors in Arabidopsis. Science 354:886-889.

769 770 771 772

Kim, K., Shin, J., Lee, S.H., Kweon, H.S., Maloof, J.N., and Choi, G. (2011). Phytochromes inhibit hypocotyl negative gravitropism by regulating the development of endodermal amyloplasts through phytochrome-interacting factors Proc Natl Acad Sci U S A 108:1729-1734.

773 774

Klose, C., Nagy, F., and Schäfer, E. (2020). Thermal reversion of plant phytochromes. Molecular Plant.

775 776 777

Klose, C., Viczián, A., Kircher, S., Schäfer, E., and Nagy, F. (2015). Molecular mechanisms for mediating light‐dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors. New Phytologist 206:965-971.

778 779

Lau, O.S., and Deng, X.W. (2012). The photomorphogenic repressors COP1 and DET1: 20 years later. Trends Plant Sci 17:584-593.

780 781 782

Laubinger, S., Fittinghoff, K., and Hoecker, U. (2004). The SPA Quartet: A Family of WD-Repeat Proteins with a Central Role in Suppression of Photomorphogenesis in Arabidopsis. Plant Cell 16:2293-2306.

783 784 785 786

Lee, H.K., Cho, S.K., Son, O., Xu, Z., Hwang, I., and Kim, W.T. (2009). Drought Stress-Induced Rma1H1, a RING Membrane-Anchor E3 Ubiquitin Ligase Homolog, Regulates Aquaporin Levels via Ubiquitination in Transgenic Arabidopsis Plants. The Plant Cell 21:622-641.

787 788

Legris, M., Ince, Y.Ç., and Fankhauser, C. (2019). Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nature Communications 10:5219.

26

789 790 791

Legris, M., Klose, C., Burgie, E.S., Costigliolo, C., Neme, M., Hiltbrunner, A., Wigge, P.A., Schäfer, E., Vierstra, R.D., and Casal, J.J. (2016). Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354:897-900.

792 793

Leivar, P., and Monte, E. (2014). PIFs: Systems Integrators in Plant Development Plant Cell 26:56-78.

794 795 796

Leivar, P., Monte, E., Oka, Y., Liu, T., Carle, C., Castillon, A., Huq, E., and Quail, P.H. (2008). Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness. Curr Biol 18:1815-1823.

797 798

Leivar, P., and Quail, P.H. (2011). PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16:19-28.

799 800 801 802

Leivar, P., Tepperman, J.M., Monte, E., Calderon, R.H., Liu, T.L., and Quail, P.H. (2009). Definition of Early Transcriptional Circuitry Involved in Light-Induced Reversal of PIF-Imposed Repression of Photomorphogenesis in Young Arabidopsis Seedlings. Plant Cell 21:3535-3553.

803 804 805

Lian, H.-L., He, S.-B., Zhang, Y.-C., Zhu, D.-M., Zhang, J.-Y., Jia, K.-P., Sun, S.-X., Li, L., and Yang, H.-Q. (2011). Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes & Development 25:1023-1028.

806 807 808 809

Lu, X.-D., Zhou, C.-M., Xu, P.-B., Luo, Q., Lian, H.-L., and Yang, H.-Q. (2015). Red-Light-Dependent Interaction of phyB with SPA1 Promotes COP1–SPA1 Dissociation and Photomorphogenic Development in Arabidopsis. Molecular Plant 8:467-478.

810 811 812 813

Medzihradszky, M., Bindics, J., Ádám, É., Viczián, A., Klement, É., Lorrain, S., Gyula, P., Mérai, Z., Fankhauser, C., Medzihradszky, K.F., et al. (2013). Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis. The Plant Cell 25:535-544.

814 815

Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7:405-410.

816 817 818

Moon, J., Zhu, L., Shen, H., and Huq, E. (2008). PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis. Proc Natl Acad Sci U S A 105:9433-9438.

819 820 821

Nieto, C., López-Salmerón, V., Davière, J.-M., and Prat, S. (2014). ELF3-PIF4 Interaction Regulates Plant Growth Independently of the Evening Complex. Current Biology 25:187-193.

822 823 824

Oh, E., Kim, J., Park, E., Kim, J.I., Kang, C., and Choi, G. (2004). PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana. Plant Cell 16:3045-3058.

825 826 827 828

Oh, E., Yamaguchi, S., Huc, J., Yusukeb, J., Jung, B., Paik, I., Leed, H.-S., Sun, T.-P., Kamiya, Y., and Choi, G. (2007). PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by directly binding to the GAI and RGA promoters in Arabidopsis seeds Plant Cell 19:1192-1208.

829

Oh, J., Park, E., Song, K., Bae, G., and Choi, G. (2019). PHYTOCHROME INTERACTING

27

830 831

FACTOR 8 inhibits phytochrome A-mediated far-red light responses in Arabidopsis. The Plant Cell:tpc.00515.02019.

832 833

Osterlund, M.T., Hardtke, C.S., Wei, N., and Deng, X.W. (2000). Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405:462-466.

834 835 836

Pacín, M., Legris, M., and Casal, J.J. (2014). Rapid Decline in Nuclear COSTITUTIVE PHOTOMORPHOGENESIS1 Abundance Anticipates the Stabilization of Its Target ELONGATED HYPOCOTYL5 in the Light. Plant Physiol 164:1134-1138.

837 838 839

Paik, I., Chen, F., Pham, V.N., Zhu, L., Kim, J.-I., and Huq, E. (2019). A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis. Nature Communications in press.

840 841

Paik, I., and Huq, E. (2019). Plant photoreceptors: Multi-functional sensory proteins and their signaling networks. Seminars in Cell & Developmental Biology 92:114-121.

842 843

Pham, V.N., Kathare, P.K., and Huq, E. (2018a). Dynamic regulation of PIF5 by COP1-SPA complex to optimize photomorphogenesis in Arabidopsis. Plant Journal 96:260-273.

844 845

Pham, V.N., Kathare, P.K., and Huq, E. (2018b). Phytochromes and Phytochrome Interacting Factors. Plant Physiology 176:1025-1038.

846 847

Pham, V.N., Xu, X., and Huq, E. (2018c). Molecular bases for the constitutive photomorphogenic phenotypes in Arabidopsis. Development 145:dev169870.

848 849

Quint, M., Delker, C., Franklin, K.A., Wigge, P.A., Halliday, K.J., and van Zanten, M. (2016). Molecular and genetic control of plant thermomorphogenesis. Nature Plants 2:15190.

850 851

Rockwell, N., and Lagarias, J. (2019). Phytochrome evolution in 3D: deletion, duplication, and diversification. New Phytologist doi: 10.1111/nph.16240.

852 853 854

Runge, S., van Cleve, B., Lebedev, N., Armstrong, G., and Apel, K. (1995). Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana. Planta 197:490-500.

855 856 857

Saijo, Y., Sullivan, J.A., Wang, H., Yang, J., Shen, Y., Rubio, V., Ma, L., Hoecker, U., and Deng, X.W. (2003). The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes Dev 17:2642-2647.

858 859 860

Seo, H.S., Yang, J.Y., Ishikawa, M., Bolle, C., Ballesteros, M.L., and Chua, N.H. (2003). LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature 423:995-999.

861 862 863 864 865

Sheerin, D.J., Menon, C., zur Oven-Krockhaus, S., Enderle, B., Zhu, L., Johnen, P., Schleifenbaum, F., Stierhof, Y.-D., Huq, E., and Hiltbrunner, A. (2015). Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell 27:189-201.

866 867 868 869

Shen, H., Ling, Z., Castillon, A., Majee, M., Downie, B., and Huq, E. (2008). Light-induced phosphorylation and degradation of the negative regulator PHYTOCHROME INTERACTING FACTOR 1 depends upon its direct physical interactions with photoactivated phytochromes. Plant Cell 20:1586-1602.

28

870 871 872

Shen, H., Moon, J., and Huq, E. (2005). PIF1 is regulated by light-mediated degradation through the ubiquitin-26S proteasome pathway to optimize seedling photomorphogenesis in Arabidopsis. Plant J 44:1023-1035.

873 874 875

Shin, J., Kim, K., Kang, H., Zulfugarov, I.S., Bae, G., Lee, C.H., Lee, D., and Choi, G. (2009). Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors. Proc. Nat. Acad. Sci. U S A 106:7660-7665.

876 877

Stephenson, P.G., Fankhauser, C., and Terry, M.J. (2009). PIF3 is a repressor of chloroplast development. Proc Natl Acad Sci U S A 106:7654-7659.

878 879 880 881

Subramanian, C., Kim, B.H., Lyssenko, N.N., Xu, X., Johnson, C.H., and von Arnim, A.G. (2004). The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: mutational analysis by bioluminescence resonance energy transfer. Proc Natl Acad Sci USA. 101:6798-6802.

882 883 884

Toledo-Ortíz, G., Huq, E., and Rodríguez-Concepción, M. (2010). Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by Phytochrome-Interacting Factors. Proc Natl Acad Sci U S A 107:11626-11631.

885 886 887 888

Wang, Z.-P., Xing, H.-L., Dong, L., Zhang, H.-Y., Han, C.-Y., Wang, X.-C., and Chen, Q.-J. (2015). Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biology 16:144.

889 890 891

Xu, X., Kathare, P.K., Pham, V.N., Bu, Q., Nguyen, A., and Huq, E. (2017). Reciprocal proteasome-mediated degradation of PIFs and HFR1 underlies photomorphogenic development in Arabidopsis. Development 144:1831-1840.

892 893 894 895

Xu, X., Paik, I., Zhu, L., Bu, Q., Huang, X., Deng, X.W., and Huq, E. (2014). PHYTOCHROME INTERACTING FACTOR1 Enhances the E3 Ligase Activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to Synergistically Repress Photomorphogenesis in Arabidopsis. Plant Cell 26:1992-2006.

896 897

Xu, X., Paik, I., Zhu, L., and Huq, E. (2015). Illuminating Progress in Phytochrome-Mediated Light Signaling Pathways. Trends in plant science 20:641-650.

898 899 900

Zhu, L., Bu, Q., Xu, X., Paik, I., Huang, X., Hoecker, U., Deng, X.W., and Huq, E. (2015). CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1. Nature communications 6:7245.

901 902 903

Zuo, Z., Liu, H., Liu, B., Liu, X., and Lin, C. (2011). Blue Light-Dependent Interaction of CRY2 with SPA1 Regulates COP1 activity and Floral Initiation in Arabidopsis. Current Biology 21:841-847.

904

29

FIGURE LEGENDS:

905 906 907

Figure 1: PCH1 and PCHL positively regulate many light responses and light-regulated

908

gene expression.

909 910

(A) Seed germination phenotypes of Col-0, PCH1 and PCHL overexpression lines, pch1, pchl,

911

and pch1 pchl double mutants in response to an increasing fluence of red light. Col-0 and pifQ

912

were used as controls. Error bars indicate s.e.m. (n=3 biological repeats).

913

(B) Photograph showing the hypocotyls of PCH1 and PCHL overexpression seedlings do not

914

respond to changes in the direction of gravity. The direction of gravity was altered by turning

915

plates 90° after the seedlings were grown for 2 d in pulse red light on vertical agar plates. The

916

plates were incubated for another 2 d under the same light conditions.

917

(C) Quantification of hypocotyl negative gravitropism in response to an increasing fluence of red

918

light pulse. Data are mean with 95% confidence intervals indicated (n = 20 seedlings).

919

(D) I2-KI staining patterns of starch granules for the wild-type (Col-0), PCH1 and PCHL

920

overexpression lines (PCH1OE and PCHLOE), pch1, pchl, and pch1 pchl double mutant.

921

(E) Light-responsive gene expression in Col-0, PCH1 and PCHL overexpression lines, pch1,

922

pchl, and pch1 pchl double mutants in response to an increasing fluence of red light. Error bars

923

indicate s.e.m. (n=3 biological repeats). *P < 0.01 by Student’s t-test. Asterisk indicates

924

significant difference of each genotype compared with Col-0 in each condition.

925 926

Figure 2: PCH1 and PCHL promote the degradation of PIF1.

927 928

(A) and (C) Immunoblot shows the light-induced degradation of native PIF1 is reduced in pch1,

929

pchl and pch1 pchl double mutants compared with wild-type seeds under far-red (FR) and red (R)

930

light treatment (A). And this effect remained similar in phyB-9 mutant background (C).

931

(E) Immunoblot shows the light-induced degradation of native PIF1 is increased in PCH1 and

932

PCHL overexpression lines compared with wild-type seeds under far-red (FR) and red (R) light

933

treatment. But this effect is diminished in phyB-9 mutant background. Seeds (0.02g) of all

934

genotypes were surface sterilized within 1h of imbibition and plated on MS plates (0.1g/1 MS

935

salt) with filter paper, exposed to 5 min of FR light (3.2 µmolm-2s-1) or red light pulse (Rp,

936

20µmolm-2s-1), these plates were kept in the dark for 24 h before harvesting. 30

937

(G) Immunoblot shows that the PCH1 and PCHL overexpression induce the degradation of PIF1

938

compared with wild-type in etiolated seedlings under true dark conditions. Seeds of all genotypes

939

were surface sterilized, plated on MS mediaand stratified at 4°C for four days. The plates were

940

then exposed to white light for 3 h to induce the germination and then kept in the dark for 21 h at

941

22°C. The plates were then exposed to saturated FR light (FRp, 20µmolm-2s-1) for 5 min,

942

wrapped in aluminum foil and kept in darkness at 22°C for additional 3 days before being

943

harvested for protein extraction. Total protein was extracted from all the samples and separated

944

on a 10 % SDS–PAGE gel, transferred to PVDF membrane and probed with anti-PIF1 and

945

anti-RPT5 antibodies.

946

(B), (D), (F) and (H) Quantification of PIF1 levels according to (A), (C), (E) and (G) using

947

Western blot results from three independent experiments. RPT5 blots were used for

948

normalization. The error bars indicate s.e.m. (n=4 biological repeats). *P < 0.01 by Student’s

949

t-test. Asterisk indicates significant difference of each genotype compared with Col-0 in each

950

condition.

951 952 953 954

Figure 3: PCHs interact with PIF1 independent of phyB and facilitate phyB-PIF1 interaction in vivo.

955

(A) and (B) PIF1 co-purified with PCH1 and PCHL from native plant extracts. Four-day-old

956

dark-grown seedlings over-expressing HA-YFP-PCH1 (YFP-PCH1, A) or HAYFP-PCHL

957

(YFP-PCHL, B) in both Col-0 wild type, phyB-9 and phyA-211 phyB-9 double mutant

958

backgrounds were either treated with red light (R, 7 µmolm-2s-1) for 10 min or kept in darkness

959

(D).

960

(C) PIF1 co-purified with phyB from native plant extracts. Four-day-old dark-grown seedlings

961

over-expressing phyB-GFP in both Col-0 wild type and pch1 pchl double mutant background

962

were either treated with red light (R, 7 µmolm-2s-1) for 10 min or kept in darkness (D). Total

963

protein extracts were used for co-immunoprecipitation (Co-IP) assays. IP was performed using

964

mouse α-GFP antibody. Rabbit α-PIF1 and rabbit α-GFP antibodies were used to detect

965

endogenous PIF1 and YFP-tagged PCH1/L or GFP-tagged phyB, respectively.

966

(D) Bar graph shows the quantitative results of the Western blots shown in (C). The error bars

967

indicate s.e.m. Relative levels of PIF1 proteins immunoprecipitated by phyB were normalized

968

with the amount of phyB immunoprecipitated in the same experiments.

31

969 970

Figure 4: PCH1/L impaired the DNA-binding ability and the transcriptional activity of

971

PIF1 in vitro and in vivo.

972 973

(A) DNA-IP assay. Left: PIF1 immunoprecipitated by PIL1 promoter fragment is gradually

974

reduced when adding increasing amount of PCH1 and PCHL. Three biological repeats were

975

performed with similar results. Right: Quantitative results of the left figure. Error bars represent

976

SE (n = 5 biological repeats).

977

(B) Chromatin immunoprecipitation (ChIP) assays show PIF1 binding to the G-box motif of

978

PIF1 target promoters.

979

mutant seeds exposed to 5min of FR light (3.2 µmolm-2s-1) and kept in the dark for 6 h as

980

described by Oh et al. (2007). Anti-PIF1 antibodies were used to immunoprecipitate native PIF1

981

and associated DNA fragment. DNA was amplified using primers specific to the G-box

982

fragments or control regions in RGA promoters as indicated by the arrows in the promoter

983

structure above as designed by Oh et al. (2007). Error bars represent SE (n = 3 biological

984

repeats).

985

(C) Transient transcriptional activity assay. 4-week-old tobacco plants were transiently

986

transformed with the different combinations of constructs indicated below. Relative expression

987

of LUC activity was observed and measured. The data were normalized by protein concentration.

988

Error bars represent SE (n = 6 biological repeats).

989

(D) The expression of PIF1 target genes is decreased in PCH1 and PCHL overexpression lines

990

and increased in pch1 and pchl mutants compared with wild-type seeds under far-red and red

991

light. Bar graph shows expression of various PIF1 target genes in the indicated genotypes in

992

far-red and red light. The error bars indicate s.e.m. (n=3 biological repeats).

The ChIP assay was performed using WT, PCH1OE, and pch1 pchl

993 994

Figure 5: PCH1 and PCHL are unstable in the dark and this degradation is mediated by

995

COP1.

996 997

(A) and (B) Western blot analyses of PCH1 (A) and PCHL (B) protein (by GFP antibody) in

998

PCH1OE and PCHLOE plants. The plants grown on MS were first illuminated for 4 h to induce

999

germination. Four-d-old dark-grownseedlings were exposed to light for up to 24 h (both left

32

1000

panels) or first incubated in the light for 4 h and then left in darkness up to 8 h (both right panels).

1001

RPT5 was detected as loading control. Experiments were repeated 3 times with the same results,

1002

and representative experiment is shown.

1003

(C) Bar graph shows quantitative data from the Western blots shown above. Error bars indicate

1004

s.e.m. (n=3 biological repeats).

1005

(D) Western blots show that PCH1 (Left) and PCHL (Right) are degraded slower in cop1-4

1006

mutants compared to wild type background. Four-d-old dark-grown seedlings were illuminated

1007

for 4 h (L) or first illuminated for 4 h and then incubated in the dark for another 4 h (D).

1008

(E)YFP-PCH1/L interacts with COP1-HA in vivo. The input and pellet fractions are indicated.

1009

Co-IP was carried out using the anti-GFP antibody and then probed with anti-GFP and anti-HA

1010

antibodies.

1011 1012

Figure 6: COP1-mediate PCH1/L degradation is 26S proteasome-dependent.

1013 1014

(A) and (B) Immunoblots show the ubiquitinated-PCH1/L level under both dark and light.

1015

(C) Immunoblots showing the relative ubiquitination status of YFP-PCH1 (left) and YFP-PCHL

1016

(right) in response to dark in cop1‐4 mutants, compared with the wild type. Total proteins were

1017

extracted from 4-day-old dark-grown seedlings and then immunoprecipitated with anti-GFP

1018

antibody (rabbit). The immunoprecipitated samples were then separated on 6.5% SDS-PAGE

1019

gels and probed with anti-GFP (mouse, left) or anti-Ubi antibodies (right). The upper smear

1020

bands are polyubiquitinated PCH1 and PCHL. Arrows indicates YFP-PCH1 and YFP-PCHL.

1021

Experiments were done 3 times with similar results, and a representative blot is shown.

1022

(D) and (E) COP1 promotes ubiquitination of PCH1 and PCHL in vitro. An in vitro

1023

ubiquitination assay was performed for both PCH1 (D) and PCHL (E). FLAG-tagged ubiquitin

1024

was used. Left, immunoblot detected by anti-FLAG antibody. Right, immunoblot using anti-His

1025

for PCH1 and GST antibody for PCHL. Arrows indicate His-PCH1 and GST-PCHL.

1026

(F) pch1 suppresses cop1-6 phenotype in darkness. Seedling morphology of seedlings grown

1027

under continuous darkness for 3 d. Bar = 5 mm.

1028

(G) and (H) Bar graphs show the quantification of hypocotyl length (G) and the cotyledon

1029

opening angle (H) in figure (F). Error bars represent SD (n >30 seedlings). Statistical

1030

significance among different genotypes was determined using one-way analysis of variance

33

1031

(ANOVA) and Tukey’s honestly significant difference (HSD) tests, and is indicated with

1032

different letters.

1033 1034

Figure 7: A model showing PCH1 and PCHL-mediated regulation of photomorphogenesis.

1035 1036

Left, in the dark, the biologically inactive Pr form of phytochrome is localized in the cytosol.

1037

The nuclear localized PIF1 homodimers bind to the promoter region of light-regulated target

1038

genes and repress their expression to prevent photomorphogenesis. On the other hand, PCH1 and

1039

PCHL interact with PIF1 and negatively regulate PIF1 level and its downstream target genes

1040

expression. Right, upon light exposure, the biologically active Pfr form of phytochrome

1041

translocates into nucleus and interacts with PCH1 and PCHL. This interaction enables Pfr phyB

1042

to further interact with PIF1 and thus triggers the rapid light-induced phosphorylation of PIF1.

1043

The phosphorylated form of PIF1 is then recruited to the COP1–SPA1 complex for rapid

1044

ubiquitination and subsequent degradation through the 26S proteasome pathway. The destruction

1045

of the negative regulator, PIF1, derepresses the light-regulated gene expression and thereby

1046

promotes photomorphogenesis.

34

A

Col-0 pch1 pifQ

Germination rate (%)

120

PCH1OE pchl

B

PCHLOE pch1pchl

Col-0

PCH1OE

PCHLOE

pchl

pch1 pchl

100 80

pch1 60 40 20 0 0

15

30

60

Red light fluence (µmol m-2)

C

Col-0

70

pch1

pchl

D

pch1pchl

Col-0

PCH1OE

PCHLOE

pch1

pchl

pch1 pchl

Degrees from vertical

60 50 40 30 20 10 0

50

100

150

200

Red light fluence (µmol m-2)

Col-0

E Relative expression

8 7

PCH1OE

CAB3

12

*

5

*

*

4 3

*

2

* **

pch1 pchl

RBCS * **

6

**

pchl 20

8

** **

pch1

10

*

6

PCHLOE

4

*

*

0 3

6

9

12

**

12

*

* 8

*

*

*

*

4

Dark

3

6

9

*

*

0 Dark

*

*

*

2

1

*

16

*

*

Fd2

12

Time incubated under red light (hour)

0 Dark

3

6

9

12

A

Col-0 FR

Rp

pchl

pch1 FR

Rp

FR

C

pch1pchl Rp

FR

Col-0

Rp

FR

Rp

pch1 pchl

pch1 pchl phyB-9

FR

FR

Rp

Rp

PIF1 PIF1 RPT5

Rp

FR

Rp

FR

hl

RPT5

RPT5 1.4

1.4

H

1.2

Relative protein level

Relative protein level

Rp

pch1 pchl phyB-9

PIF1

PIF1

F

G

Rp

FR

pc

FR

PCHLOE/ phyB-9

Rp

h1

FR

PCH1OE/ phyB-9

FR

pch1 pchl

-9

PCHLOE

Rp Col-0

pch1pchl

PCH1OE Rp

FR

pc

Rp

0

Rp

f1

FR

pchl

pch1

FR

pi

Col-0

Rp

X

E

FR

X

Col-0

Rp

-O

FR

H1

Rp

PC

FR

l -0

0

-O

0.5

* 0.5

yB

*

Co

*

*

ph

*

1

* 1

HL

Relative protein level

Relative protein level

*

1.5

1.5

PC

D

2

1 0.8

*

0.6

*

0.4 *

0.2

*

0 FR

Rp

Col-0

FR

Rp

PCH1OE

FR

Rp

FR

Rp

PCHLOE PCH1OE phyB-9

FR

Rp

PCHLOE phyB-9

1.2 1 0.8 0.6 0.4

* *

0.2 0 CCo oll -0-0 P C H 1O X P C H LO X ph yB -9 pc h1 pc h l

B

RPT5

B

A YFP-PCH1 YFP-PCH1 YFP-PCH1 phyB-9 phyA phyB D

R

D

R

D

R

R

α-PIF1

D

R

D

R

R Input

α-GFP

D

phyB-GFP D

R

phyB-GFP pch1pchl D

R

Col-0 D

R

pif1 R Input

IP α-GFP

IP α-GFP

α-PIF1

Relative PIF1 binding

C

α-PIF1

R

YFP-PCHL YFP-PCHL phyB-9 phyA phyB Col-0

α-PIF1

IP α-GFP

α-PIF1

α-GFP

D Input

α-GFP

α-PIF1

YFP-PCHL

Col-0

4 3.5 3 2.5 2 1.5 1 0.5 0

D

R phyB-GFP

D

R

phyB-GFP pch1pchl

A PCH1/PCHL GST-PIF1 -

-

-

+

+

+

-

+

+

GST +

-

+

-

-

-

25

GST-PCHL

+

+ - +

+

+

-

-

-

-

-

GST-PIF1 a-GST

Relative protein level

His-PCH1

PIF1

20 15 10 5 0 Mock GST

GST-PCHL

His-PCH1

C

D

Relative expression

4

14 12 10 8 6 4 2 0

PIF1 /PC H1

control G-box

WT

PCH1OE

PIF1/ GFP

PIF1/ PCH L

pch1 pchl

18 16 14 12 10 8 6 4 2 0

Relative LUC activity PI F1 /G FP PI F1 /P CH 1 PI F1 /P CH L

Fold enrichment

B

Col-0 F R

Col-0 Rp

PCH1OE FR

PCH1OE Rp

PCHLOE FR

PCHLOE Rp

pch1 FR

pch1 Rp

pchl FR

pchl Rp

pch1 pchl FR

pch1 pchl Rp

3

2

1

0

PIL2

FHL

RGA

GAI

YFP-PCH1

A D to L

0

3

1

YFP-PCH1

6

24 h

WL4h to D

95

B

1

2

3

1

a-RPT5

a-RPT5 YFP-PCHL

6

24 h

Relative protein level

95

WL4h to D

PCH1

15

PCHL

6

24 h

PCH1 1

PCHL

0.5 0

0

1

L a-GFP a-RPT5

D

E

YFP-PCHL Col-0

cop1-4 L

2

4

8h

WL4h to D

YFP-PCH1 Col-0

8h

1.5

D to L

D

4

aRPT5

0

3

2

a-RPT5

5

1

1

a-GFP

10

0

0

a-GFP

25 20

8h a-GFP

Relative protein level

0

4

a-GFP

YFP-PCHL D to L

C

0

L

D a-GFP

D

cop1-4 L

Input

IP: anti-GFP

PCH1/ PCHL/ COP1-HA COP1-HA COP1-HA

D

D YFP-PCH1/L

a-RPT5 COP1-HA

L

D

L

D L

PCH1/ PCHL/ COP1-HA COP1-HA COP1-HA

D

L

D

L

D L

D

L

D

L

IP: GFP

D

L

Input

Input IB: a-GFP

COP1 +

-

+

+

-

+

PCH1 -

+

+

-

+

+

YFP-PCHL

WT cop1-4

WT cop1-4

IP: GFP IB: aUbi

IB: a-GFP IB: a-GFP

IB: a-Ubi

YFP-PCH1

Input

IB: a-Ubi

E COP1+ PCHL -

-

+

+

-

+

+

+

-

+

+

a-FLAG

a-His

a-GST

G

F pch1

cop1-6 pch1 cop1-6 Hypocotyl length (mm)

Col-0

H 15

10

140 a

a

b c

5

opening angle ( ° )

a-FLAG

PCHL-YFP-Ubi

PCH1-YFP-Ubi

D

L

IP: GFP

YFP-PCH1-Ubi

D

C

YFP-PCHL YFP-PCHL

YFP-PCH-Ubi

B

YFP-PCH1 YFP-PCH1

YFP-PCHL-Ubi

A

a

120 100 80

b

60 40 20

c

c

0

0

l Co

-0

pc

h1

p co

1-

6

/ h 1 -6 pc p1 co

Col-0

pch1 cop1-6 pch1 cop1-6

Dark

Light

phyB in Pr

phyB in Pfr Ubi

Ubi

PCH1 COP1 PCHL Ubi SPA1 Ubi PI PIF Ubi F1 1 Ubi

P H C P H H C1 26S LC

I

P 1ProteaI some F 1 FP

I pp p pp p PIF1 PIF1

PCH1 PCH1PCHL PCHL

Ubi Ubi Ubi

PI PIF F1 1

PIF1 PIF1

Ubi

+++ Photomorphogenesis

PIF1 targets

26S Proteasome

p p p COP1 pp p PIF1 PIF1 SPA1 Ubi

+

P 1 I F 1 FP

Photomorphogenesis PIF1 targets