PHYTOCHROME INTERACTING FACTORs from Physcomitrella patens are active in Arabidopsis and complement the pif quadruple mutant

ABSTRACT

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development in response to changes in the ambient environment. An important mode of action of plant phytochromes depends on their light-regulated relocation from the cytosol into the nucleus and control of gene expression; in addition, there is also evidence for a cytosolic or plasma membrane associated function of phytochromes in different species. The PHYTOCHROME INTERACTING FACTORs (PIFs) form a subgroup of the bHLH transcription factors and it is well established that PIFs are key components of phytochrome downstream signalling in the nucleus of seed plants. Recent studies identified members of the PIF family also in the liverwort Marchantia polymorpha and the moss Physcomitrella patens. Here, we show that all four potential PIF homologs from Physcomitrella have PIF function when expressed in the Arabidopsis pifQ mutant, which is deficient in multiple PIFs. We propose that PIFs are ancient components of nuclear phytochrome signalling that have emerged in the last common ancestor of today's land plants.

Introduction

The light conditions in different terrestrial habitats are highly variable and often rapidly changing. The phytochrome family of red/far-red photoreceptors plays a key role in adaptation of growth and development to the ambient environment.¹ Canonical plant phytochromes are present in all land plants; in many species, they form small gene families that evolved through successive gene duplication events from a single phytochrome in the last common ancestor of land plants.^2,3 Rapid responses with a lag time in the range from seconds to a few minutes or responses depending on the association of phytochromes with the plasma membrane suggest that there are phytochrome mediated responses that do not depend on nuclear transport of phytochrome and regulation of gene expression.^4,5 However, the primary mode of action of phytochromes in seed plants requires light-induced nuclear accumulation of phytochromes.⁶ Light-activated phytochromes suppress the activity of the nuclear localised CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)/SUPPRESSOR OF PHYA-105 (SPA) E3-ubiquitin ligase complex and thereby induce light signalling.⁷ In parallel, phytochromes bind to members of the PHYTOCHROME INTERACTING FACTOR (PIF) family and inactivate them by promoting their degradation and inhibiting binding to target promoters.⁸ PIFs are bHLH transcription factors that negatively regulate photomorphogenesis and therefore inactivation of PIFs by phytochromes promotes light signalling. Also in the fern Adiantum capillus-veneris, the moss Physcomitrella patens, and the liverwort Marchantia polymorpha at least a subpool of phytochromes accumulates in the nucleus in response to light.^9–11 Moreover, potential homologs of key factors of phytochrome downstream signalling, such as COP1, SPAs, and PIFs, are possibly present in all land plants.^11–13 We have previously identified four potential Physcomitrella homologs of Arabidopsis PIFs (AtPIFs), which we named PpPIF1 – PpPIF4.¹³ Specific motifs in PIFs required for the interaction with phytochromes have been identified. The APA motif is essential for binding of Arabidopsis phytochrome A (phyA) to AtPIF1 and AtPIF3 as well as for the interaction of phytochromes and PIFs in Physcomitrella and Marchantia.^8,11,13 In contrast, binding of Arabidopsis phytochrome B (phyB) to AtPIFs depends on the APB motif.⁸ The Arabidopsis pifQ (pif1 pif3 pif4 pif5) mutant lacks four members of the PIF family and exhibits constitutive photomorphogenic growth.^14,15 Expression of PpPIF1 or PpPIF2 complements the mutant phenotype of dark-grown pifQ seedlings, suggesting that PpPIF1 and PpPIF2 have PIF activity when expressed in Arabidopsis.¹³ However, such analyses are still lacking for PpPIF3 and PpPIF4 as well as for PpPIFs unable to bind to phytochromes due to amino acid substitutions in the APA motif. Here, we show that – in addition to PpPIF1 and PpPIF2 – also PpPIF3 and PpPIF4 have PIF activity in Arabidopsis and that complementation of the pifQ mutant phenotype by expression of PpPIF1 and PpPIF2 does not require a functional APA motif.

Results and discussion

We have previously shown that PpPIF1 – PpPIF4 interact with Physcomitrella phytochromes in a light regulated manner, suggesting that they might control gene expression in Physcomitrella in response to light perceived by phytochromes.¹³ In line with this idea, PpPIF1 and PpPIF2 have PIF activity when expressed in the Arabidopsis pifQ mutant.¹³ Here, we complemented these previous analyses by investigating whether also PpPIF3 and PpPIF4 are active in Arabidopsis pifQ mutant background (Fig. 1). Similar to pifQ Pro35S:PpPIF1-YFP-HA and pifQ Pro35S:PpPIF2-YFP-HA, also pifQ seedlings expressing PpPIF3- or PpPIF4-YFP-HA under the control of the 35S promoter are fully etiolated, which is in stark contrast to pifQ and pifQ expressing YFP-HA alone. Yet, despite inhibition of de-etiolation in the dark, the hypocotyls of dark-grown pifQ seedlings expressing PpPIFs are slightly shorter than those of the wild type. AtPIF1 inhibits germination,¹⁶ and therefore we hypothesise that overexpression of PpPIFs might delay germination and thereby result in shorter hypocotyls than in the wild type; alternatively, PpPIFs might have a lower capacity to promote hypocotyl growth than AtPIFs. We also found that the APA and APB motifs of PpPIF1 and PpPIF2 are not required for complementation of the constitutive photomorphogenic phenotype of the pifQ mutant. In line with this observation, also PpPIF2 and PpPIF4 splicing variants lacking the APB motif (PpPIF2^ΔAPB and PpPIF4^ΔAPB) are active in pifQ mutant background (Fig. 1; Ref. 13).

Figure 1.

PpPIFs are active in Arabidopsis pifQ mutant background. Arabidopsis pifQ seedlings expressing different PpPIF-YFP-HA versions under the control of the 35S promoter were grown for four days in the dark. Representative seedlings are shown.

To further confirm that PpPIF3 and PpPIF4 complement the pifQ mutant phenotype, we measured transcript levels AtPIF-regulated genes.¹⁷ Expression of XTR7, HB-2, and PIL1 in dark-grown pifQ Pro35S:PpPIF1/2/3/4-YFP-HA seedlings was strongly increased compared to pifQ and pifQ Pro35S:YFP-HA (Fig. 2). In several PpPIF expressing lines the transcript levels of AtPIF-regulated genes were even higher than in the wild type. Moreover, expression of XTR7, HB-2, and PIL1 was not dependent on the APA and APB motifs, which is expected given that binding of phytochromes to PIFs downregulates their activity and that phytochromes do not bind to PIFs in dark-grown seedlings.⁸

Figure 3.

PpPIF protein stability in Arabidopsis pifQ background. Four day-old dark-grown pifQ seedlings expressing different YFP-HA-tagged PpPIF versions under the control of the 35S promoter and Col-0 seedlings expressing Pro35S:AtPIF3-CFP were exposed to red light (R, 22 μmol m⁻² s⁻¹) for 24 h or kept in the dark; etiolated pifQ Pro35S:YFP-HA seedlings were used as negative control. YFP/CFP-tagged proteins were detected in total protein extracts by immunoblot using a GFP antibody. Actin was used as loading control.

Figure 2.

Quantification of PIF-regulated transcripts in Arabidopsis pifQ mutants expressing PpPIFs. (A) and (B) Expression of XTR7, HB-2, and PIL1 was analysed by quantitative RT-PCR in 4 d old dark-grown seedlings of Col-0, pifQ, and pifQ expressing Pro35S:YFP-HA (empty vector) or different Pro35S:PpPIF-YFP-HA constructs. Data were normalised to PP2AA3 transcript levels. (A) and (B) show biological replicates. Each biological replicate includes three technical replicates. Error bars indicate ±SE.

Light-induced phosphorylation and subsequent degradation is an important mechanism for the inactivation of PIFs.^8,18 All PIFs in Arabdidopsis – except for PIF7 – are targeted for degradation in seedlings or plants exposed to light.⁸ Also the single PIF present in Marchantia polymorpha is rapidly degraded upon activation of phytochrome by light.¹¹ In contrast, PpPIF1 and PpPIF2 expressed in Arabidopsis pifQ mutant background are not degraded in light.¹³ Here, we tested light-regulated protein turnover for PpPIF3 and PpPIF4 as well as for APA and/or APB mutant versions of PpPIF1 and PpPIF2. Immunoblot analyses of dark-grown seedlings either exposed to red light for 24 h or incubated in the dark show that also PpPIF3 and PpPIF4 are light stable in Arabidopsis pifQ seedlings, similar to PpPIF1 and PpPIF2, while AtPIF3 is degraded. Furthermore, we also found that the lack of functional APA and APB motifs does not alter PpPIF1 and PpPIF2 turnover. Arabidopsis phyA, which contributes to AtPIF3 degradation in red light,¹⁸ interacts with PpPIF1 and therefore it appears unlikely that lack of binding to phytochromes is responsible for the different stability of AtPIFs and PpPIFs when expressed in Arabidopsis.¹³ We hypothesise that either targeting for degradation of PpPIFs and AtPIFs depends on a different mechanism and that the mechanism employed by PpPIFs is present in Physcomitrella but not in Arabidopsis, or that – similar to AtPIF7¹⁹– the light-regulation of PpPIFs depends on other mechanisms than on light-regulated turnover. Investigating the stability of PpPIFs in light- and dark-grown Physcomitrella plants will be important to distinguish between the two hypotheses.

This work, together with our previous study,¹³ shows that all four PpPIFs have PIF activity when expressed in Arabidopsis pifQ background, suggesting that they might play a role in phytochrome downstream signalling in Physcomitrella. The cross-species complementation indicates that the basic properties of PIFs essential for their activity are conserved between AtPIFs and PpPIFs despite the obvious difference regarding the light-regulation of protein turnover. It is interesting that cross-species complementation has also been observed for PpCOP1a, which at least partially complements the Arabidopsis cop1 mutant, while PpSPAs are not active in Arabidopsis.¹² Overall, it appears possible that the basic light signalling components identified in Arabidopsis are present in all land plants, though subtle differences may preclude cross-species complementation in some cases (e.g. for SPAs). These differences might be the result of adaptive changes during evolution that allow different plant species to respond to specific light conditions and survive in their natural habitats.

Material and methods

All Arabidopsis thaliana lines and mutants used in this study are in Columbia-0 (Col-0) ecotype; the pif1-1 pif3-3 pif4-2 pif5-3 quadruple (pifQ) mutant has been described previously.¹⁴ For transformation of Arabidopsis, we cloned PpPIF coding sequences into pPPO30v1HA, which contains a Pro35S:BamHI-XbaI-YFP-HA:TerRbcS expression cassette.¹³ The following lines have been described previously ¹³: pifQ mutant seedlings expressing Pro35S:YFP-HA (pPPO30v1HA, empty vector control) or Pro35S-driven PpPIF1-YFP-HA, PpPIF2-YFP-HA, PpPIF2^ΔAPB-YFP-HA, as well as Col-0 expressing Pro35S:AtPIF3-CFP.

T-DNA vectors for generation of pifQ lines expressing PpPIF3-YFP-HA (pPPO30v1HA-PpPIF3), PpPIF4-YFP-HA (pPPO30v1HA-PpPIF4), or PpPIF4^ΔAPB-YFP-HA (pPPO30v1HA-PpPIF4^ΔAPB) have been described previously.¹³

To generate pifQ lines expressing PpPIF1 or PpPIF2 lacking functional APA and/or APB motifs, we cut fragments containing the respective coding sequence from pJET constructs (pJET-PpPIF1^mAPB, pJET-PpPIF1^mAPA, pJET-PpPIF1^{mAPB mAPA}, pJET-PpPIF2^mAPA, and pJET-PpPIF2^ΔAPB mAPA) and inserted them into pPPO30v1HA.¹³ PpPIF1 mutant versions were cut using BamHI/SpeI and ligated into the BamHI/XbaI site of pPPO30v1HA; PpPIF2^mAPA and PpPIF2^{ΔAPB mAPA} were cut using SpeI and inserted into pPPO30v1HA-beta-Gal alpha digested with AvrII/XbaI to replace the beta-Gal alpha fragment.¹³

The pPPO30v1HA vectors containing PpPIF3, PpPIF4, PpPIF4^ΔAPB, or PpPIF1/PpPIF2 mutant versions were introduced into Agrobacterium tumefaciens which then was used for transformation of pifQ by floral dip.^20,21 Inspire/Butafenacil was used for selection of transgenic plants.²²

Western blot and qPCR analyses were performed as previously described.¹³ Details regarding antibodies and primers can be found in Ref. 13.

Fundings

This work was supported by the Excellence Initiative of the German Federal and State Governments (EXC294 BIOSS, Project C20) and the Human Frontier Science Program Organization (HFSP Research Grant RGP0025/2013) to A.H.; T.X. was supported by fellowships from the China Scholarship Council (CSC) and the Landesgraduiertenförderung (LGFG).