Emerging from the darkness: interplay between light and plastid signaling during chloroplast biogenesis

Chloroplast biogenesis is a highly complex process that requires carefully coordinated communication between the nucleus and the chloroplast to integrate light signaling and information about the state of the plastid through retrograde signals. Most studies on plastid development have been performed using dark-grown seedlings and have focused on the transition from etioplast to chloroplast in response to light. Some advances are now also being made to understand the transition directly from proplastids to chloroplasts as it occurs in the shoot apical meristems. Recent reports have highlighted the importance of repressive mechanisms to block premature chloroplast development in dark, both at the transcriptional and post-transcriptional level. A group of new proteins with dual plastid and nuclear localization were shown to take part in the light triggered degradation of PHYTOCHROME INTERACTING FACTORs (PIFs) in the nucleus and thereby release the suppression of the nuclear photosynthesis associated genes. These dually localized proteins are also required to activate transcription of photosynthesis genes in the plastid in response to light, emphasizing the close link between the nucleus and the plastids during early light response. Furthermore, development of a fully functional chloroplast requires a plastid signal but the nature of this signal(s) is still unknown. GENOMES UNCOUPLED1 (GUN1) is a plastid protein pivotal for retrograde signal(s) during early seedling development, and recent reports have revealed multiple interactors of GUN1 from different plastid processes. These new GUN1 interactors could reveal the true molecular function of the enigmatic character, GUN1, under naturally occurring adverse growth conditions.


Introduction
Light energy conversion into chemical energy is one of the most important processes to sustain life on Earth.Photosynthetic organisms, algae and plants, perform this conversion through the photosynthetic reactions, a task that takes place in specialized organelles: the chloroplasts.Chloroplasts are derived from ancient photosynthetic cyanobacteria that were engulfed by a eukaryotic cell in an endosymbiotic event (Gould et al. 2008).The photosynthetic bacteria were integrated in the cell, and during evolution the endosymbiont lost its independence and most of its genetic material was transferred to the nucleus.As a result of this gene transfer, the protein complexes in the chloroplast are encoded in two different compartments, the nucleus and the chloroplast.Thus, optimal function of the chloroplast relies on the precise communication between these two cellular compartments.Eukaryotic cells have developed a complex network of signals that allow the nucleus to control plastid biogenesis and function, via anterograde signals, and the plastids to signal their developmental and functional state to the nucleus via retrograde signals.
Plastids are inherited as undifferentiated proplastids, non-photosynthetically plastids without differentiated structures that undergo different developmental programs (Liebers et al. 2017).The development of functional chloroplast from proplastids is complex and the mechanistic details differ between plant lineages, but also between organs and tissues, and many specific molecular details are still unknown (Liebers et al. 2017).In dicotyledonous plants, such as Arabidopsis, seeds germinate under a layer of soil, seedlings grow in darkness, and the plastids develop into an intermediate form, the etioplast, characterized by the prolamellar bodies (PLB) a lattice-like structure composed of proteins and metabolites that will be required for photosynthesis (Liebers et al. 2017).In true leaves that develop from the shoot apical meristem, proplastids develop directly into chloroplast without the intermediate step (Liebers et al. 2017).Differentiation into photosynthetically active chloroplast is initiated upon light perception, that start changes in expression of nuclear and plastid genes coding for plastid proteins needed for the assembly and activation of plastid complexes, and the formation of the thylakoid membranes where the photosynthetic reactions take place.Although the variable conditions of chloroplast biogenesis suggest different underlying molecular mechanisms (López-Juez et al. 2008), recent research has revealed common regulatory phases during proplastid-tochloroplast development in different models that also appears to extend to the etioplast-to-chloroplast transition (Fig. 1) (Dubreuil et al. 2018, Armarego-Marriott et al. 2019).These phases will be discussed in detail in the next sections with focus on recent advances of the interplay between light and plastid retrograde signals during chloroplast biogenesis.

The plastids in darkness
It is well established that the first step and main trigger for chloroplast development is light, irrespective of conditions and tissue type.Seedlings undergo a developmental program in the dark referred to as skotomorphogenesis (Seluzicki et al. 2017, Gommers andMonte 2018).During this heterotrophic phase, chloroplast biogenesis is repressed by a complex network of transcriptional and post-transcriptional mechanisms to avoid the overaccumulation of photosynthetic components that can cause photodamage when the seedlings are exposed to light.These repressive processes are largely controlled by the PHYTOCHROME INTERACTING FACTORs (PIFs) and the degradation of transcriptional regulators by the ubiquitin-proteasome CONSTITUTIVE PHOTO-MORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/ FUS) pathway (Fig. 1) (Seluzicki et al. 2017).PIF1 and PIF3 were described to act as main repressors of chloroplast development, primarily repressing nuclear genes that encode enzymes in chlorophyll biosynthesis (Gommers and Monte 2018).However, PIFs are not the only repressors of photosynthesis associated genes in the nucleus.ETHYLENE-INSENSITIVE3 (EIN3) has also been shown to play a role in repressing genes associated with chloroplast development in etiolated seedlings (Zhong et al. 2009, Liu et al. 2017, Shi et al. 2018).The brassinosteroid responsive BRASSINAZOLE-RESISTANT1 (BZR1) transcription factor has been reported to interact with PIF4 and form a module to regulate gene expression during skotomorphogenesis (Oh et al. 2012).The gibberellinregulated DELLA transcriptional regulators are present in etiolated cotyledons and interact with PIFs and thereby blocking their activity to regulate the expression of PIF target genes, and as a consequence, upregulate the expression of PROTOCHLOROPHYLLIDE-OXIDOREDUCTASE (POR) (Cheminant et al. 2011).

Emerging from the darkness
Upon light exposure a completely new developmental phase starts, and the plant shifts from heterotrophic to photoautotrophic growth, a process for which chloroplast development is essential (Gommers and Monte 2018).Plants have evolved a series of photoreceptors to sense light and initiate molecular signaling pathways to transduce the light information.Five classes of photoreceptors have been identified in Arabidopsis: (1) phytochromes (phy) that perceive red and far-red light, (2) cryptochromes and (3) phototropins that perceive blue and UV-A light, (4) UV RESISTANCE LOCUS 8 (UVR8) for UV-B and (5) the LOV-F-Box-Kelch domain families that perceive blue light (Seluzicki et al. 2017).Photomorphogenesis and chloroplast development are mainly orchestrated by phytochromes and cryptochromes.Cryptochromes upon perception of blue light, regulate chloroplast development through the suppression of COP1 activity (Ponnu et al. 2019).In response to red light the inactive form of phy (Pr) that resides in the cytosol changes conformation to the active form (Pfr) that is translocated to the nuclear to nuclear active form (Pfr) that triggers degradation of the PIFs.In the light COP1 is excluded from the nucleus and transcription factors like HY5 are stabilized.As a result, there is an increase in the expression of PhANGs, including the genes encoding PEP-associated proteins.In the plastid, the main transcriptional activity is taken over by PEP.The increase in plastid transcription and the correct progression of plastid development generates a retrograde signal(s) of unknown nature that boost PhANGs transcription leading to fully functional chloroplast and photoautotrophic growth.
photobodies.In the photobodies the active Pfr form interacts with multiple proteins, and as a result PIFs and EIN3 are targeted for degradation leading to massive transcriptional changes that will reprogram development (Gommers and Monte 2018).Nuclear photobodies are heterogeneous and dynamic in their protein composition and their formation is dependent on several proteins.Three recently characterized proteins with dual nuclear-plastid localization essential for the formation of photobodies and consequently also for light signaling and chloroplast biogenesis are: HEMERA (HMR), REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) and NUCLEAR CONTROL OF PEP ACTIVITY (NCP) (Fig. 2).HMR is required for the formation of photobodies via interaction with phyB and for degradation of PIF1 and PIF3 in light (Chen et al. 2010, Galvão et al. 2012).In the nucleus, and in response to light, RCB interacts with phyA and phyB and is required for the formation of photobodies and the subsequent degradation of PIF1 and PIF5 (Yoo et al. 2019).NCP is required for phy signaling, photobody formation and PIF1 and PIF3 degradation (Yang et al. 2019).
Transcription factors like HY5 play a pivotal role downstream of multiple photoreceptors promoting chloroplast biogenesis by regulating the early transcriptional response to light.The HY5 expression is promoted by light, and HY5 is also post-transcriptionally regulated by phosphorylation and the COP/DET/FUS degradation machinery (Gangappa and Botto 2016).HY5 and PIFs regulate expression of genes involved in synthesis of photosynthetic pigments by competing for the same cis element, the G-box.These results suggest a model for the dynamic action of HY5 and PIFs to regulate chloroplast development in response to light and other environmental cues, such as temperature (Toledo-Ortiz et al. 2014).HY5 and ABSCISIC ACID INSENSITIVE4 (ABI4) have been described to form another antagonistic module to control seedling and chloroplast development during the transition from dark to light (Xu et al. 2016).The GLK transcription factors are also major regulators of chloroplast development controlling the expression of genes encoding chlorophyll biosynthesis enzymes and Fig. 2. Assembly and activation of PEP.The change in plastid from NEP to PEP driven transcription in light requires the assembly and activation of PEP.In the dark PIFs repress PEP complex assembly by a yet unknown mechanism.In light the phytochrome active form (Pfr) moves to nuclear foci, the photobodies, and trigger degradation of PIFs.The dual plastid-nuclear localized proteins HMR, NCP and RCB, are required in the nucleus for photobody formation, interaction with Pfr and PIF degradation in the nucleus during the early light response.In response to light, nuclear transcription of genes coding for PAPs, and other proteins required for PEP activity (SIGs, PRIN2, NCP, RCB) is induced.PAPs, among them HMR, and SIGs will assemble with the PEP core.NCP and RCB are also needed for PEP activity, although the mechanism is still unknown.The activity of the newly established photosynthetic electron transport (−e) will reduce PRIN2 dimers to monomers that promote full PEP activity.

light-harvesting chlorophyll binding proteins (Waters et al. 2009). GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and GNC-LIKE
(GNL) belong to the GATA family and regulate chloroplast development.Although the GLKs, GNC and GNL have overlapping functions, genetic analysis indicates that the GLKs have a prevalent role in chloroplast development (Zubo et al. 2018).In addition to the lightinduced transcriptional changes directed by different transcription factors, there is also an enhanced translation of selected mRNAs by a phy-dependent reduction in P-bodies that will release stalled mRNAs for their active translation (Jang et al. 2019).

Changes to plastids triggered by the light
A significant proportion of the light-induced transcriptional changes derive from chloroplast localized or photosynthesis-associated genes, indicating that the plastids rapidly start to change their protein composition, morphology and cellular position for the transition into functional chloroplast (Dubreuil et al. 2018, Armarego-Marriott et al. 2019).One of the initial plastid changes in response to light is the shift in plastid transcriptional activity.Plastid transcription is carried out by to different polymerase complexes: nuclear-encoded RNA polymerase (NEP) and plastid-encoded RNA polymerase (PEP).In plastids, NEP is the main RNA polymerase, while in mature chloroplast PEP represents the main transcriptional activity.PEP activity is dependent on PEP-ASSOCIATED PROTEINs (PAPs) and the SIGMA factors (SIGs) that are nuclear encoded (Liebers et al. 2017).The promoters of all of these nuclear genes, in addition to the promoter of PLASTID REDOX INSENSITIVE2 (PRIN2), encoding a plastid localized protein required for full PEP activity (Kindgren et al. 2012a), contain a PIF3 binding motif suggesting that their induction is regulated by light through the phy/PIF module (Dubreuil et al. 2017).Experimental data from an Arabidopsis cell culture where chloroplasts develop directly from proplastids similar to meristematic cells, in combination with mathematical modeling, indicated a role for phyB and PIF3 in the light-dependent transcription of these genes (Dubreuil et al. 2017(Dubreuil et al. , 2018)).
It appears that the mere presence of the PEP-associated proteins and sigma factors in the plastids is not enough for the activation of PEP.The activation most probably requires correct assembly of PAPs and sigma factors into the complex, but also PEP-associated proteins such as PRIN2 play an important role.The assembly and activation of PEP is triggered by light through phy.Full PEP assembly was detected in a constitutively active phyB line and in a PIF quadruple mutant (pifq) in 3-day-old dark-grown seedlings, while less or no complex was detected in phy mutants.These results clearly indicate that light/phy/PIF are needed for full assembly of the PEP complex in the plastids (Yoo et al. 2019).The recent identification of the three nuclear encoded proteins with dual plastid-and nuclear-localization mentioned above: HMR, RCB and NCP (Fig. 2) has further advanced our understanding of PEP activation.Lack-of-function seedlings for these proteins share a long hypocotyl phenotype in red light, which is an indication of deficient phy signaling.Furthermore, the mutants are albino with severely defective chloroplast development.HMR is one of the PEP associated proteins (PAP5) in plastid nucleoids (Chen et al. 2010, Galvão et al. 2012).RCB was first identified as a plastid protein, which was loosely associated but essential for PEP transcriptional activity.Although RCB interacts with two PAPs (THIOREDOXINZ and FE SUPEROXIDE DISMUTASE3), and has potential thioredoxin activity in vitro, it is not clear whether it has a direct role in PEP assembly or in activation, especially as its role in PIF degradation is required for PEP assembly and photosynthetic plastid gene expression (Yoo et al. 2019).The third PEP-activating protein is an RCB-paralog named NCP, which also has a thioredoxin domain.Interestingly, the albino phenotype and the failure to assemble the PEP complex in the ncp mutant, was not rescued by the pifq mutant, indicating that the role of NCP in PEP assembly and photosynthetic plastid gene expression is PIF independent (Yang et al. 2019).Thus, the major role of NCP might be in the plastids.A recent screen has identified an additional 23 tall-and-albino mutants (Yoo et al. 2019), suggesting a promising future for the identification of new components of light and plastid signaling.An additional component required for full PEP activation in response to light is PRIN2, a plastid-localized and redox-regulated protein required for PEP-dependent transcription.PRIN2 constitutes the first described mechanism of redox-regulation of PEP activity (Kindgren et al. 2012a, Díaz et al. 2018).PRIN2 forms a dimer via disulphide bonds in the dark that in response to light are reduced, increasing the ratio of PRIN2 monomers that boost PEP activity, potentially via the interaction with THIOREDOXINZ, a PEP-associated protein (Fig. 2) (Díaz et al. 2018).

Completion of chloroplast biogenesis requires a retrograde signal
The development of fully functional chloroplasts in light is dependent on a plastid retrograde signal, but the nature of this signal, or signals, is still not clear (Martín et al. 2016, Dubreuil et al. 2018).We know that interfering with plastid function during the early stages of Physiol.Plant.169, 2020 development, either genetically or chemically, generates one or more signals that represses PHOTOSYNTHESIS-ASSOCIATED NUCLEAR GENE (PhANG) expression (Hernández-Verdeja and Strand 2018).This specific molecular phenotype, i.e. the repression of LIGHT HAR-VESTING CHLOROPHYLL A/B BINDING PROTEIN (LHCB) genes in response to norflurazon (NF), a carotenoid biosynthesis inhibitor, led to the identification of the genomes uncoupled (gun) mutants, which have defective communication between plastid and nucleus (Susek et al. 1993).Five of the GUN proteins (GUN2-6) are enzymes of the tetrapyrrole biosynthesis pathway (TBP) and control the branched pathways downstream of Protoporphyrin IX.HEME OXYGENASE1 (GUN2), PHYTOCHROMOBILIN SYNTHASE (GUN3) and FER-ROCHELATASE1 (FC1; GUN6) are in the heme-branch, which produces phytochromobilin, while the activator of Mg-CHELATASE GUN4 and Mg-CHELATASE (GUN5) are in the chlorophyll-producing Mg-branch (Larkin 2016).Unlike the other GUN proteins, GUN1 protein is not an enzyme of the TBP, but GUN1 is required for retrograde signals triggered by defective TBP and inhibition of plastid gene expression and plastid translation (PGE) (Koussevitzky et al. 2007).The gun1 mutants have been described as hypersensitive to NF and lincomycin (LIN) treatment (Zhao et al. 2018), and sensitive to growth at high light at the seedling stage (Ruckle et al. 2007).GUN1 is a nuclear encoded plastid-localized protein with pentatricopeptide repeat (PPR) and small MutS-related (SMR) domains, which are associated with RNA binding and DNA repair, respectively.However, binding to DNA has been shown only in in vitro experiments (Koussevitzky et al. 2007) and rather a role of GUN1 in several plastid processes has been revealed in recent work (Fig. 3).
The well documented role of GUN1 in transmitting LIN-induced retrograde signal and the reported interactions and functional connections with proteins involved in transcription and translation, emphasize a functional role of GUN1 in plastid gene expression.The indispensable role of GUN1 in these processes is supported by the observation that the combination of gun1 with mutants defective in ribosomal, RNA processing or protease activities are seedling lethal (Tadini et al. 2016, Llamas et al. 2017).GUN1 was recently shown to interact with NEP and to be required for increased NEP activity upon LIN-induced inhibition of PEP-driven transcription (Tadini et al. 2019).The importance of GUN1 in transmitting signals of defective PEP activity was further shown as the repression of PhANGs in sig2, sig6 and prin2 mutants, all defective in PEP activity, is GUN1 dependent (Kindgren et al. 2012a, Woodson et al. 2013).Strangely, the scabra3-1 (sca3-1) mutant, defective in NEP (RpoTp), does not have a gun phenotype, demonstrating the complexity behind the GUN1-based plastid signaling (Tadini et al. 2019).GUN1 was also shown to be involved in editing of transcripts through interaction with the RNAeditosome component MULTIPLE ORGANELLAR RNA EDITING FACTOR2 (MORF2), especially under stress conditions (Zhao et al. 2019).It was reported that defects in this interaction, both in absence of GUN1 or due to overexpression of MORF2, reduce the editing efficiency and lead to a gun phenotype.While it is not known what determines the editing activity of the GUN1-MORF2 complex at each specific site, the decreased rate of RNA-editing of the rpoB and rpoC1 transcripts upon LIN and NF treatments in gun1 mutants suggests that proper editing of the components of the PEP complex is dependent on GUN1, and this regulation, may be part of GUN1-dependent retrograde signaling.The role of GUN1 extends also to the regulation of translation initiation.This link has been suggested via interactions with several ribosomal proteins including PLASTID RIBO-SOMAL PROTEIN S1 (PRPS1) and CHLOROPLAST TRANSLATION INITIATION FACTOR IF-2/FU-GAERI1 (cpIF2/FUG1) (Tadini et al. 2016).While interaction with GUN1 has been suggested to result in destabilization of PRPS1 monomer and stabilization of PRPS1-containing protein complexes in adult plants, genetic analysis suggest that GUN1 interaction with FUG1 seems to be most important during early seedling development (Marino et al. 2019).
More emphasis on the regulatory role of GUN1 in maintenance of chloroplast protein homeostasis comes from the impact of GUN1 on the translocon complexes TRANSLOCON AT THE INNER ENVELOPE MEMBRANE OF CHLOROPLASTS (TIC) and TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS (TOC) embedded in the chloroplast envelope.Recently, GUN1 was shown to interact with TIC-complexassociated chloroplast chaperone HEAT SHOCK COG-NATE PROTEIN 70-1 (HSC70-1), which facilitates the import of chloroplast proteins, and probably also stabilizes GUN1 (Wu et al. 2019).GUN1 regulates the expression of the genes encoding the subunits of TICand TOC-complexes upon LIN treatment, connecting the defective chloroplast protein synthesis to the protein import capacity (Tadini et al. 2019).The defective protein import capacity in gun1 has been further linked to an increase in unimported preproteins, and markedly increased overall level of protein ubiquitination in the cytosol (Tadini et al. 2019, Wu et al. 2019).These observations coincide with increased accumulation of HEAT SHOCK PROTEIN 90 (HSP90), HEAT SHOCK PROTEIN 70 (HSP70) and the HEAT SHOCK COGNATE PROTEIN 70-4 (HSC70-4) chaperones in the cytosol (Tadini et al. 2019, Wu et al. 2019).Besides participating in the GUN1-dependent retrograde signaling pathway, the HSP90 chaperone has emerged as an important factor also in the GUN5-dependent pathway (Kindgren et al. 2012b, Wu et al. 2019).The suppression of gun phenotype of gun1 and gun5 by the hsp90 knockdown line upon LIN and NF treatment and oxidative stress, indicates that HSP90 is required to transmit retrograde signals from both PGE and TBP (Wu et al. 2019).
Although GUN1 has been connected to the tetrapyrrole biosynthesis pathway through several interaction partners (Tadini et al. 2016), the consequences of these interactions have remained unclear.Recently, the PPR domain of GUN1 was shown to bind heme and other porphyrins, and it was further implied that GUN1 would increase the FC1 activity, but also to bind the produced heme, to prevent positive retrograde signaling in darkness or upon plastid dysfunction (Shimizu et al. 2019).Upon illumination, GUN1 is degraded by the caseinolytic protease complex (CLP) (Wu et al. 2018), and the free heme would act as a positive retrograde signal.The binding to heme is not specific as GUN1 also binds several other tetrapyrroles, and it is unclear if other PPR proteins also bind heme and tetrapyrroles.Thus, this proposed model requires some further investigations.GUN1 has also been suggested to reduce the accumulation of the phototoxic chlorophyll precursor, protochlorophyllide (Pchlide) (Xu et al. 2016, Shimizu et al. 2019) and this effect could explain the delayed greening and poor survival of etiolated gun1 seedlings (Susek et al. 1993).
Given the diversity of proteins reported as GUN1 interactors interpretation of the true function of GUN1 is difficult.By using artificial stress conditions such as norflurazon and lincomycin, GUN1 was assigned a role in communicating stress conditions in the plastids to the nucleus.Furthermore, the gun1 phenotypes described are all observed at the seedling stage and not at the adult stage of the plant.One of the first phenotypes described for gun1 were from seedlings grown in the dark and shifted to light (Susek et al. 1993) but the potential role of GUN1 during the deetiolation process has since then been overlooked.Possibly the true function of GUN1 is during seedling deetiolation and the inhibitors traditionally used to study GUN1 lock the seedlings in the plastid developmental state of darkness.Thus, the role of the GUN1-dependent retrograde signal and the interplay between light and GUN1-mediated signals during seedling development warrant further investigations.

Nuclear response and the impact of plastid signals on plant development
The site of action for retrograde signals is ultimately in the nucleus, regulating expression of PhANGs through the action of transcriptional regulators.On the nuclear side, only HY5 and the GLKs have been clearly associated with the response to retrograde signals.Different studies have shown that hy5 has a gun phenotype in response to LIN and oxidative stress (Ruckle et al. 2007, Ruckle and Larkin 2009, Kindgren et al. 2012b).So far HY5 has been involved in the GUN5 retrograde signal mediated by HSP90 and Mg-Protoporphyrin IX (Kindgren et al. 2012b).Further research is needed to reveal if HY5 is also involved in the retrograde signal-mediated by GUN1 and HSP90 in response to accumulation of preproteins in the cytosol.GLKs are known to respond to retrograde signaling at the transcriptional and posttranscriptional level, in a GUN1-dependent, and independent manner, respectively, and overexpression of GLKs has been shown to cause a gun phenotype (Kakizaki et al. 2009, Leister and Kleine 2016, Martín et al. 2016, Tokumaru et al. 2017).Recent studies revealed GLK1 as the link between photomorphogenic development and retrograde signaling pathways (Martín et al. 2016).A model was proposed where GLK1 is induced in seedlings grown under moderate light conditions, but upon high-light illumination a GUN1-dependent retrograde signal results in repression of GLK1, providing a mechanism to prevent seedling damage (Martín et al. 2016).
Interplay between plastid retrograde signals and light signals plays an important role during plant development.Flowering time, a trait that depends on light perception, was shown to be affected by GUN1 suggesting that vegetative-to-reproductive phase transition could be modulated by retrograde signals.However, the mechanism of the GUN1-mediated regulation of flowering time is unknown (Wu et al. 2018).Treatment with inhibitors of chloroplast development such as LIN or NF has led to the discovery that functional chloroplasts are required for phyB inactivation and stabilization of PIF3 during the shade-avoidance syndrome in seedlings.The retrograde signal that regulates the response to shade is however GUN1 independent and seems to be mediated independently by HY5 and carotenoid derived products, like abscisic acid (Ortiz-Alcaide et al. 2019).These results suggest a more close and complex interaction between plastid retrograde signals and light signaling to shape plant development and to respond correctly to environmental changes.Thus, to explore the impact of chloroplast function and integrity on leaf and plant development throughout the entire plant life cycle are exciting challenges for the future.

Fig. 1 .
Fig. 1.Overview of chloroplast development.Chloroplast biogenesis starts from proplastids, or the intermediate etioplast in cotyledons of dark-grown seedlings.The plastid transcription in dark is mainly driven by NEP.In the nucleus transcription of PhANGs is repressed by PIFs in cooperation with other proteins like EIN3.COP1 stabilizes PIF/EIN3 and degrades photomorphogenesis promoting transcription factors like HY5 in the dark.Perception of light by photoreceptors starts chloroplast biogenesis and the transition to photoautotrophic growth.Light changes the conformation of phy, from inactive (Pf)to nuclear active form (Pfr) that triggers degradation of the PIFs.In the light COP1 is excluded from the nucleus and transcription factors like HY5 are stabilized.As a result, there is an increase in the expression of PhANGs, including the genes encoding PEP-associated proteins.In the plastid, the main transcriptional activity is taken over by PEP.The increase in plastid transcription and the correct progression of plastid development generates a retrograde signal(s) of unknown nature that boost PhANGs transcription leading to fully functional chloroplast and photoautotrophic growth.

Fig. 3 .
Fig.3.GUN1-dependent retrograde signaling in response to plastid dysfunction.GUN1 has widespread impact on maintenance of protein homeostasis in chloroplast under conditions, which induce retrograde signaling.GUN1 affects plastid protein homeostasis by controlling plastid gene expression by interacting with NEP, MORF2, FUG1 and PRPS1, and plastid protein import by interacting with HSC70-1.These interactions favor the transcriptional activity of NEP, ensure proper RNA-editing of certain plastid encoded transcripts, support translation initiation and protein import to chloroplast.In the absence of GUN1, the defects in PGE and protein import result in accumulation of preproteins in cytosol, consequently increasing the accumulation of cytosolic chaperones (HSP90 and HSP70), which further modulate PhANG expression.GUN1 interacts with enzymes of the TBP affecting flux through the pathway, and can directly bind tetrapyrroles supporting a role of these molecules in mediating the retrograde signal.The majority of the research carried out to investigate the molecular function of GUN1 has involved the use of plastid translation inhibitor lincomycin (LIN) or inhibitor of the carotenoid pathway, norflurazon (NF).