Arabidopsis thaliana atcrp1-1 (SALK_035048) (Alonso et al., 2003) and atcrp1-2 (SAIL_916A02) (Sessions et al., 2002) T-DNA insertion lines were identified by searching the T-DNA Express database². For promoter analyses, the putative AtCRP1 promoter region (AtCRP1p, -1062 to -2 upstream the translation starting codon) was cloned into pBGWFS7 destination vector and introduced into Arabidopsis wild type background, ecotype Columbia-0 (Col-0), by Agrobacterium tumefaciens-mediated transformation. AtCRP1-GFP transgenic lines were obtained by transformation of AtCRP1/atcrp1-1 heterozygous plants with either the AtCRP1 coding sequence fused to GFP under the control of 35S-CaMV promoter, cloned into pB7FWG2 vector, or the genomic locus fused to GFP under the control of the native promoter, cloned into a modified pGreenII vector (Gregis et al., 2009). The GUN1 coding sequence, devoid of the stop codon, was cloned into pB7RWG2 vector, carrying an RFP reporter gene. pB7FWG2, pBGWFS7, and pB7RWG2 plasmids were obtained from Flanders Interuniversity Institute for Biotechnology of Gent (Karimi et al., 2002). Primers used for amplification of the DNA fragments cloned into the vectors, reported above, are listed in Supplementary Table S2. Arabidopsis Col-0 and mutant plants were grown on soil under controlled growth chamber conditions with a 16 h light/8 h dark cycle at 22°C/18°C. In the case of mesophyll protoplast preparation, Arabidopsis plants were also grown on soil in a growth chamber under the above reported conditions. Moreover, phenotypic characterization and molecular biology analyses were also conducted on plants grown on Murashige and Skoog (MS) medium (Duchefa)³, supplemented with or without 1% (w/v) sucrose. Tobacco plants, employed for transient gene expression, were cultivated for 5–6 weeks in a greenhouse under a 12 h light/12 h dark cycle at 22°C/18°C.

Mesophyll protoplasts of Arabidopsis thaliana (Col-0) were isolated and transiently transformed according to Yoo et al. (2007) and Costa et al. (2012). Briefly, well-expanded rosette leaves from 3-to-5 week-old plants were cut into strips of 0.5–1 mm with a fresh razor blade. Leaf tissue was digested using an enzyme solution containing 1.25% cellulase Onozuka R-10 (Duchefa) and 0.3% Macerozyme R-10 (Duchefa) for 3 h at 23°C in the dark. The protoplast suspension was filtered through a 50 μm nylon mesh washed three times with W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.7 adjusted with KOH) and used for PEG-mediated transformation. For each protoplast transformation 10 μg of a MidiPrep purified DNA (QIAGEN) plasmid harboring the 35S-CaMV::AtCRP1-GFP cassette was used. Protoplasts were maintained for 16–24 h at 23°C in the dark, before performing epifluorescent microscopy.

Tobacco leaf infiltration was performed using A. tumefaciens strain GV3101/pMP90 carrying the specified constructs (see results for details) together with the p19-enhanced expression system (Voinnet et al., 2003), according to the method described by Waadt and Kudla (2008). The final OD₆₀₀ for A. tumefaciens strains harboring 35S-CaMV::AtCRP1-GFP and 35S-CaMV::GUN1-RFP was 0.2 and 0.3, respectively. After infiltration, plants were incubated for 3–5 days under the conditions described above.

Confocal Scanning Laser Microscopy analyses were performed using an inverted microscope, Leica DMIRE2, equipped with a Leica TCS SP2 laser scanning device (Leica). For the simultaneous detection of GFP and chlorophyll auto-fluorescence the cells were excited (Arabidopsis mesophyll protoplasts or tobacco leaf cells) with the 488 nm line of the Argon laser and the emissions were collected between 515/535 and 650/750 nm, respectively. For RFP detection the cells were excited at 561 nm from a He/Ne laser and the emission was collected between 575/625 nm. Image analyses were performed with Fiji⁴: an open-source platform for biological-image analysis (Schindelin et al., 2012).

Arabidopsis DNA was isolated according to Ihnatowicz et al. (2004). Isolation of total RNA from homozygous atcrp1-1 plants at four-leaf rosette stage and RNA gel blot analyses were performed as described by Meurer et al. (2002), using 10 μg of total RNA for each sample. For the RNA slot blot hybridization experiments, one-fourth of the RNA purified from each immunoprecipitation pellet and one-tenth of the RNA purified from the corresponding supernatant were applied to a nylon membrane with a slot-blot manifold and hybridized to specific radiolabeled probes (see Supplementary Table S2). ³²P-labeled DNA probes, complementary to chloroplast genes, were amplified using the primer pairs listed in Supplementary Table S2. Four micrograms of total RNA, treated with TURBO DNA-free (Ambion by Life Technologies), were employed for first-strand cDNA synthesis using GoScript Reverse Transcription System (Promega) according to the supplier’s instructions. Quantitative Real-Time PCR (qRT-PCR) was carried out on an CFX96 Real-Time system (Bio-Rad), using the primer pairs reported in Supplementary Table S2. The SAND (Remans et al., 2008) and ubiquitin transcripts were used as internal references. Data from three biological and three technical replicates were analyzed with Bio-Rad CFX Manager software (V3.1).

For immunoblot analyses, total proteins were prepared as described by Martinez-Garcia et al. (1999). Total proteins, corresponding to 5 mg of leaf fresh-weight (100% of WT and atcrp1-1 samples) and isolated from plants at four-leaf rosette stage, were fractionated by SDS–PAGE (12% acrylamide [w/v]; (Schagger and von Jagow, 1987). Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Ihnatowicz et al., 2004) and replicate filters were immunodecorated with antibodies specific for PSI (PsaA, PsaC, and PsaD), PSII (D1, PsbO) Cyt b₆/f (PetA, PetB, and PetC), ATPase (ATPase-β) subunits, PSI (Lhca1, Lhca2) and PSII (Lhcb2, Lhcb3) antenna proteins, all obtained from Agrisera⁵. The GFP antibody was purchased from Life Technologies⁶.

Intact chloroplasts were isolated from 11 days old Arabidopsis plants, according to Kunst (1998), and Kupsch et al. (2012) with some modifications. Chloroplasts were directly resuspended in 300–400 μl of extraction buffer [2 mM DTT, 30 mM HEPES-KOH, pH 8.0, 60 mM KOAc, 10 mM MgOAc and proteinase inhibitor cocktail (Sigma–Aldrich-P9599)]. Two independent stromal preparations were carried out and one of them was performed in the presence of 2% sodium deoxycholate in order to solubilize the membrane-attached AtCRP1 protein fraction. Chloroplasts were then disrupted by pulling them through a syringe (0.55 mm × 40 mm) 30–40 times. The solution was centrifuged at 21,000 × g at 4°C to separate the stromal from the membrane fraction.

The isolated stromal fraction was diluted with one volume of coimmunoprecipitation (CoIP) buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 0.5% Nonidet P-40 and 0.5 μg/mL Aprotinin). Five microliters of mouse anti-GFP antibody (Roche, No. 11814460001) were added to the stromal fraction and incubated for 1 h at 4°C and 13 rpm on an overhead shaker. Thereafter the coimmunoprecipitation was performed as described by Kupsch et al. (2012). Successful precipitation of AtCRP1-GFP was confirmed by immunoblot analyses, using the same GFP antibody.

RNA immunoprecipitation-chip analyses were performed using a tiling microarray covering the complete Arabidopsis chloroplast genome (Kupsch et al., 2012). The coimmunoprecipitated RNA was isolated from pellet and supernatant fractions either by phenol-chloroform extraction or using the Direct-zol^TM RNA MiniPrep kit (Zymo Research). For the phenol-chloroform extraction, RNA samples were incubated in 1% SDS and 5 mM EDTA at room temperature for 5 min to dissociate RNA-protein complexes. RNA was phenol-chloroform extracted, ethanol precipitated with the addition of Glycoblue^TM Coprecipitant (Thermo Fisher Scientific), washed twice with 75% ethanol, air-dried and resuspended in 20 μl RNase-free water. For the replicate, RNA was extracted using the Direct-zol^TM RNA MiniPrep kit (Zymo Research) according to the manufacturer’s instructions. Before the extraction 2 μg yeast RNA was added to the coimmunoprecipitated RNA pellet. The entire RNA of the pellet fraction and 2 μg RNA of the supernatant fraction were used for labeling. The pellet and supernatant RNA were labeled with 0.5 μl Cy5 and 1 μl Cy3 dye, respectively (aRNA labeling kit, Kreatech Diagnostics). Labeling reaction, microarray hybridization, scanning, and evaluation were performed as described in Kupsch et al. (2012). Only PCR products for which more than half of all replicate spots (24 per PCR product spanning two experiments) passed our quality assessment (Kupsch et al., 2012) and were used in this analysis (Supplementary Table S1).

The putative AtCRP1 binding motif, i.e., the nucleotide preference for each of the amino acid pairs at the fifth and last position of PPR domains, was predicted in silico using the reported weighting schemes (Barkan et al., 2012; Barkan and Small, 2014; Harrison et al., 2016). The software FIMO⁷, which analyzes sequence databases for occurrences of known motifs (Grant et al., 2011), was employed to identify the potential binding sites of AtCRP1 within the regions enriched in our RIP-Chip experiment. Furthermore, the same regions were searched for the presence of sRNA native footprints, by consulting the JBrowse sRNA database⁸ (Ruwe et al., 2016). Numbers that delimit the native footprints refer to the chloroplast genome of Arabidopsis thaliana (NC_000932.1).

For GUS histochemical detection, plant material was fixed in 90% acetone at -20°C for 1 h. Samples were then washed three times with NaPi buffer (NaH₂PO₄ 50 mM, Na₂HPO₄ 50 mM; pH 7.0) and stained overnight at 37°C with X-gluc solution [1 mM 5-bromo-4-chloro-3-indolyl-β-D-clucoronide, 2 mM K₃/K₄Fe(CN)₆, 0,1% Triton (v/v), 10 mM EDTA, 50 mM NaPi pH 7.0]. 70% EtOH (v/v) was used as washing solution. Stained samples were then stored at 4°C and observed using a Zeiss Axiophot D1 microscope equipped with differential interference contrast (DIC) optics. Images were recorded with an Axiocam MRc5 camera (Zeiss) using the Axiovision program (v.4.1).

The Maize Genetics and Genomics Database (Lawrence et al., 2004)⁹ was used to identify the At5g42310 gene as the Arabidopsis ortholog of ZmCRP1 (see also Belcher et al., 2015). At5g42310 encodes a polypeptide of 709 amino acids with a calculated molecular mass of 80 kDa. Intron number (three) and position are conserved between the two genes, and BLASTP query of public Arabidopsis sequence database with ZmCRP1 amino acid sequence detected At5g42310 protein as the top hit with 55% sequence identity and 72% sequence similarity (Figure 1).

AtCRP1 is annotated as a PPR protein and shares with ZmCRP1 15 PPR tandem repeats, which were predicted by using the PlantPPR database (Cheng et al., 2016). All PPR motifs are of 35 aa, with the exception of P0 which consists of 30 aa and P2 of 37 aa in Arabidopsis and 38 aa in maize. The fifth and the last residue of each PPR domain form the amino acid pairs that specify the RNA target molecules (Cheng et al., 2016), and are labeled with gray and black stars in Figure 1. The ChloroP server (Emanuelsson et al., 1999)¹⁰ predicted the presence of a cTP of 54 residues (see amino acid residues in italics in Figure 1), indicating that AtCRP1, like ZmCRP1, could be imported into the chloroplast. To corroborate the in silico prediction, the AtCRP1-GFP fusion protein was expressed in transiently transformed Arabidopsis protoplasts (Figure 2). In agreement with the ChloroP prediction, the chimeric protein (GFP fluorescence) accumulated within the chloroplast in distinct fluorescent foci (CHL, autofluorescence of chloroplast chlorophylls, Figure 2A), resembling the nucleoid complexes. Indeed, AtCRP1-GFP chimera co-localized perfectly with the GUN1-RFP fusion protein, used as a nucleoid marker in this assay (RFP fluorescence, Figure 2B), (Koussevitzky et al., 2007; Colombo et al., 2016; Tadini et al., 2016), in tobacco leaf cells. To further localize AtCRP1, chloroplasts were fractionated to separate the stroma and thylakoid compartments. Immunoblot analysis, using a GFP specific antibody, allowed detection of AtCRP1-GFP specific signal in total chloroplasts, as well as in thylakoids and in the stromal fraction, indicating that the nucleoid AtCRP1 protein is both associated to membranes and soluble in the stroma (Figure 2C). These findings are in agreement with the identification of AtCRP1 as part of Megadalton complexes in the chloroplast stroma (Olinares et al., 2010), as well as in the grana of thylakoid membranes (Tomizioli et al., 2014).

To investigate the role that AtCRP1 plays in Arabidopsis, two lines carrying T-DNA insertions into the coding sequence of At5g42310, renamed atcrp1-1 (Salk_035048) and atcrp1-2 (Sail_916A02), were obtained from the T-DNA Express Arabidopsis mutant collection (Figure 3A; see also Materials and Methods).

Both T-DNA insertions completely suppressed the accumulation of the corresponding transcripts in homozygous mutant seedlings (Figure 3B), which were characterized by a paler pigmentation of cotyledons, visible even at the fully mature embryo stage (Figure 4A), and leaves (Figures 4B,C), and found to be seedling lethal under autotrophic growth conditions on soil and MS medium without sucrose, but able to develop yellow-albinotic rosette leaves and sterile inflorescence when sucrose was provided in the medium (Figure 4C). The mutant phenotype could be rescued by Agrobacterium tumefaciens-mediated transformation of heterozygous plants with either the appropriate coding sequence fused to the 35S promoter of cauliflower mosaic virus (35S-CaMV::AtCRP1-GFP), or the genomic sequence including a 1-Kbp fragment of the promoter region (AtCRP1p::AtCRP1-GFP), corroborating a direct correspondence between genotype and phenotype, and indicating that the AtCRP1-GFP chimera was fully functional, in both cases (Figure 4D). Interestingly, complemented plants carrying the AtCRP1-GFP construct under the control of the native promoter showed a fivefold increase in AtCRP1 gene expression (Figure 4E), most probably as consequence of the T-DNA insertion in a highly expressed euchromatin region of the nuclear genome. Furthermore, a complete rescue of mutant plant phenotype could only be observed in 35S::AtCRP1-GFP transgenic lines with a limited accumulation of AtCRP1 transcripts (Figures 4D,E). Higher AtCRP1 expression levels (around 15-folds in comparison to WT) led to transgenic plants with WT-like rosette but shorter and paler stems, bleached cauline leaves, together with sterile flowers (Figures 4E,F).

Temporal and spatial expression patterns of AtCRP1, monitored by fusing the promoter region of the gene upstream of the GUS reporter gene (see also Materials and Methods), support further the key role played by AtCRP1 during early stages of seedling and leaf development (Figure 5). The GUS staining could, indeed, be detected in young cotyledons and in the upper portion of the hypocotyl (Figure 5A). Furthermore, intense GUS signals were observable in young developing leaves (Figures 5C,D), whereas the GUS coloration tended to decrease in old cotyledons and leaves (Figures 5B–D). Similar results were also obtained by monitoring the expression of AtCRP1 in cotyledons and leaves using quantitative Real-Time PCR (qRT-PCR). In general, a high level of expression of AtCRP1 was observed in green developing tissues, such as young cotyledons and leaves, whereas the expression decreased in older tissues (Figure 5E).

The albino pigmentation of atcrp1 seedlings, together with their inability to grow under autotrophic conditions, indicated a defect in the thylakoid-associated photosynthetic apparatus. To verify this assumption, immunoblot analyses with antibodies specific for single subunits of the four major thylakoid protein complexes were performed on total leaf proteins. Leaf samples were harvested from atcrp1 plants at the four-leaf rosette stage and grown on MS-medium supplemented with 1% sucrose (Figure 6; see also Materials and Methods). Under standard light conditions (50 μmol photons m^-2 s^-1), subunits of Photosystem I (PsaA, PsaC, and PsaD), Photosystem II (D1, PsbO), Light harvesting complexes (Lhca1, Lhca2, Lhcb2, and Lhcb3) and ATPase (ATPase-β) accumulated to levels lower than 10% with respect to wild type plants. Furthermore, subunits of the Cyt b₆/f (PetA, PetB, and PetC) and PSI (PsaC) were below the limits of immunoblot detection.

In summary, these results indicate a general reduction of thylakoid protein complex subunits in atcrp1 leaves, with a particularly severe effect on the accumulation of the Cyt b₆/f complex and PsaC.

ZmCRP1 has been previously demonstrated to associate with the psaC and petA mRNAs in vivo by RIP-Chip analyses (Schmitz-Linneweber et al., 2005). To investigate whether AtCRP1 shares with ZmCRP1 the RNA targets, the same RIP-Chip approach employed in maize was used here. Stroma from plants expressing AtCRP1-GFP, under the control of the native promoter (AtCRP1p::AtCRP1-GFP), was isolated and the fusion protein was immunoprecipitated using an anti-GFP serum. As a control, we performed mock precipitations with stroma extracted from WT plants, using the same GFP antibody. RNA was purified from the immunoprecipitation pellets and supernatants and was labeled with Cy5 (red) and Cy3 (green) fluorescent dyes, respectively. The two RNA fractions from AtCRP1-GFP immunoprecipitations (IPs) and from mock IPs were competitively hybridized to a chloroplast genome tiling microarray (Kupsch et al., 2012). Enrichment of RNA is reflected in the ratio of red to green fluorescence for each spot on the microarray. Two biological replicate experiments were performed with stroma from AtCRP1-GFP expressing plants and two with WT stroma. Data from the four assays were normalized and used to calculate median enrichment ratios of the red and green fluorescence signals for each PCR product among the 24 replicate spots on two arrays (Supplementary Table S1). To identify enrichment of RNA species specifically in the AtCRP1-GFP immunoprecipitation, we plotted the difference in median enrichment ratio for each DNA fragment between the AtCRP1-GFP and mock experiment against the position of the product on the plastid chromosome (Figures 7A,B).

Four prominent peaks of differential enrichment were observed. One of them corresponds to the 5′UTR of psaC transcript, a target already recognized as a ligand of ZmCRP1 in RIP-Chip assays (Schmitz-Linneweber et al., 2005). A second RNA target is represented by the petB-petD intergenic region. This RNA was not identified to interact with ZmCRP1 by RIP-Chip analysis, however, ZmCRP1 is known to aid in maturation of this particular intergenic region (Barkan et al., 1994). Interestingly, the observed enrichment of rps15 transcripts might uncover a further, novel target of AtCRP1, whereas the enrichment of psbM/trnD transcripts is often observed in RIP-Chip experiments, thus this peak was considered an artifact.

To corroborate the RIP-Chip data, the AtCRP1-associated RNAs were analyzed by slot blots (Figure 7C). RNA purified from immunoprecipitation pellets and supernatants were probed with the PCR fragments that detected the most highly enriched sequences in the RIP-Chip assay. The data confirmed that the psaC and petB-petD transcripts are highly enriched in the AtCRP1-GFP immunoprecipitates, but not the rps15 RNA. ZmCRP1 was also reported to be associated with RNAs of the petA region (Schmitz-Linneweber et al., 2005; Williams-Carrier et al., 2008), however, no enrichment of petA transcripts could be observed in the AtCRP1-GFP RIP-Chip assay (Figure 7A) and a low enrichment was detected in the slot blot assay (Figure 7C), possibly indicating that the interaction of AtCRP1 with petA transcripts is not very stable. In general, our analysis cannot exclude the possibility that CRP1 binds to additional target RNAs, for example when interactions take place at chloroplast membranes. Since we are not using cross-linked material, weak RNA-protein interactions might be lost during our assay.

To support further the RIP-Chip findings, AtCRP1 target RNAs were interrogated for the presence of native footprints at the JBrowse database¹¹. The JBrowse database provides annotations of Arabidopsis thaliana organellar short RNA (sRNA), thought to be generated from protein-mediated temporary protection of target RNAs against exonucleolytic degradation (Ruwe et al., 2016; see also Figure 8). sRNAs were found within the 5′UTR of psaC (corresponding to the 117633–117597 region of chloroplast genome) and the petB-petD intergenic region (region 76318–76358), and an sRNA was also annotated in the 5′UTR of petA (region 61615–61643). Furthermore, AtCRP1 predicted RNA binding motifs were shown to co-map with the native footprints, when the corresponding sequences were searched for the occurrence of the consensus binding motif with the FIMO program in the MEME suite¹² (Figure 8B; Takenaka et al., 2013). A short RNA has been also mapped upstream of rps15, but this region was not enriched in the RIP-Chip assay and the match with the predicted binding site of AtCRP1 is weaker than for the psaC. petB-petD, and petA sRNAs.

In summary, the RIP-Chip and slot blot data together with the colocalization of native footprints and AtCRP1 RNA binding motifs indicate that AtCRP1 likely binds directly to the 5′UTR of psaC and the petB-petD intergenic region and possibly to the 5′UTR of petA. On the contrary, the absence of an AtCRP1-specific footprint within the rps15 RNA, together with the failure of slot blot enrichment, makes any AtCRP1-rps15 interaction unlikely.

To assess whether the lack of Cyt b₆/f complex and PsaC subunit, together with the marked reduction of all protein complex subunits observed in atcrp1-1 thylakoids, was caused by deficiencies in transcript accumulation and AtCRP1-dependent transcript processing, we probed the identified AtCRP1 RNA targets and other plastid transcripts by gel blot hybridization (Figure 9).

We investigated the transcripts encoding the subunits CP47 (psbB), T (psbT), and H (psbH) of photosystem II (PSII), subunits A (psaA) and C (psaC) of PSI, Cyt f (petA), Cyt b₆ (petB) and subunit IV (petD) of cytochrome b₆/f and the alpha subunit of ATPase (ATPase-α). All these transcripts accumulated in atcrp1-1 plastids to levels lower than WT, indicating that global plastid gene expression is affected by the atcrp1-1 mutation, and explaining the marked reduction of thylakoid protein accumulation observed in atcrp1-1 leaves.

Furthermore, the plastid polycistronic transcription unit psbB-psbT-psbH-petB-petD showed some striking alteration of transcript pattern in atcrp1 samples (Figure 9). In particular, the monocistronic petB (band #4, 0.8 Kb), the dicistronic psbH-petB (band #3; 1.2 Kb) and the unspliced petB (band #2, 1.6 Kb) transcripts were barely detectable in the mutant, whereas the petB-unspliced petD-spliced dicistronic transcript (band #1, 2.2 Kb), detected with probes D, E, F, and H, accumulated to even higher levels in atcrp1 plastids, presumably due to the failure of AtCRP1-dependent processing between the petB and petD coding regions, as also shown in zmcrp1 mutant plants (Barkan et al., 1994; Fisk et al., 1999). In contrast with maize, monocistronic and spliced petD transcripts of ∼600 nucleotides do not accumulate to significant levels in Arabidopsis, and thus its absence was not observed in atcrp1 plastids (Barkan et al., 1994; Barkan, 2011).

Moreover, the lack of the PsaC and PetA subunits could be the consequence of the simultaneous decrease of transcript accumulation and a possible defect in AtCRP1-dependent activation of psaC and petA translation, as shown in Zea mays (Barkan et al., 1994; Schmitz-Linneweber et al., 2005). However, the specific regulatory role of AtCRP1 in plastid protein translation is difficult to verify, owing to the general and pleiotropic decrease of mature plastid rRNA in atcrp1-1 leaves, in spite of WT-like accumulation of rrn23 and rrn4.5 precursor forms (Figure 10). This rRNA accumulation pattern is very similar to the ones of mutants with impaired chloroplast translation and has been interpreted as a secondary consequence of reduced plastid protein synthesis (Tiller et al., 2012; Tadini et al., 2016).

We detected AtCRP1 in the stroma and associated with thylakoid membranes (see Figure 2; Table 1), whereas ZmCRP1 is a stromal protein with no detectable association with chloroplast membranes (Fisk et al., 1999). The dual localization of AtCRP1 within the chloroplast is supported by proteomic studies that detected AtCRP1 in the grana-fraction of Arabidopsis thylakoids (Tomizioli et al., 2014) and in the stroma proteome, as part of Megadalton complexes (Olinares et al., 2010). In particular, AtCRP1 appeared to be highly enriched in fractions that contained ribosomal proteins, translation factors, RNA helicases and other PPR proteins, suggesting a major role of AtCRP1 in chloroplast gene expression. These data, together with the co-localization with GUN1 protein (see Figure 2), indicate that AtCRP1 is integral to chloroplast nucleoids (Koussevitzky et al., 2007; Colombo et al., 2016; Tadini et al., 2016), i.e., the DNA-containing structures without defined boundaries that harbor the plastid gene expression machinery (Pfalz and Pfannschmidt, 2013; Melonek et al., 2016). Similarly, ZmCRP1 was found to be highly enriched in the nucleoid fractions of maize plastids, together with proteins involved in DNA replication, organization and repair as well as transcription, mRNA processing, splicing and editing (Majeran et al., 2012), further supporting the involvement of CRP1 proteins in plastid gene expression.

The yellow-albinotic and seedling lethal phenotype exhibited by atcrp1 is very similar to the chlorophyll deficient and lethal phenotype of zmcrp1 plants (Barkan et al., 1994; Fisk et al., 1999). Arabidopsis mutants die at the two-cotyledon stage after germination on soil, but can overcome seedling lethality on sucrose-containing media, where they develop mature leaves and sterile flowers (see Figure 4; Table 1). Similarly, non-photosynthetic zmcrp1 plants die at about 3 weeks after germination when seed reserves are exhausted. Furthermore, the atcrp1 phenotype appears to be typical of Arabidopsis mutants lacking components of the photosynthetic apparatus and not of the gene expression machinery or of the protein import apparatus, since the latter usually result in the premature arrest at the globular-to-heart stage of embryo development, when chloroplast biogenesis begins (Ruppel and Hangarter, 2007; Romani et al., 2012; Beeler et al., 2014). Nevertheless, the pale-green pigmentation of the mutant embryo at bent-cotyledon stage (see Figure 4) and the β-glucuronidase (GUS) activity observed in young developing cotyledons and rosette leaves, but not in older tissues (see Figure 5), indicate that AtCRP1 gene expression and protein accumulation is required during the very early stages of the photosynthetic apparatus assembly. Immunoblot data indicate, indeed, that AtCRP1, like ZmCRP1, might act as a nuclear-encoded anterograde regulatory component responsible for coordination of the accumulation of Cyt b₆/f and PSI protein complexes (see Figure 6). Besides their role in linear electron transport (LET), Cyt b₆/f and PSI indeed play a key role in Cyclic Electron Transport (CET), which has been reported to be enhanced in Arabidopsis green seeds and to be required for optimal seed vigor and seed germination rate (Allorent et al., 2015).

In contrast to zmcrp1 plants (Barkan et al., 1994), the absence of AtCRP1 destabilized the entire photosynthetic apparatus, as shown by the marked reduction of PSII core, ATPase and LHC protein levels. The general down-regulation of thylakoid complexes owing to defects in the intersystem electron transport chain appears to be a common feature of Arabidopsis photosynthetic mutants and provides clear evidence of a different adaptive response between monocot and dicot plants (Meurer et al., 1996; Varotto et al., 2000, 2002; Maiwald et al., 2003; Weigel et al., 2003; Ihnatowicz et al., 2004, 2007; Belcher et al., 2015). Furthermore, the atcrp1-1 phenotype, both in terms of plastid transcript and plastid protein accumulation, appears to be much more drastic than the one of other ppr mutants required for the processing and expression of psbB-psbT-psbH-petB-petD operon, such as hcf152 (Meierhoff et al., 2003), suggesting that the absence of AtCRP1 protein might affect the activity of other factors essential for plastid gene expression. As a matter of fact, rRNA abundance is markedly reduced in atcrp1-1 plastids, indicating a general reduction of protein synthesis, as consequence of pleiotropic effects.

RNA immunoprecipitation-Chip and slot blot data suggest a physical interaction between AtCRP1 and the transcripts of psaC. petB-petD and possibly petA, even though it is not known whether these interactions are direct or mediated by other factors (see Figure 7). However, all of these RNAs harbor a region where a native footprint is annotated, raising the tempting hypothesis that AtCRP1 is in fact the RNA-binding factor responsible for that footprint (see Figure 8). Furthermore, when these enriched fragments were searched for occurrences of the predicted binding motif of AtCRP1, each of them proved to contain a hit inside the footprint region, strongly suggesting that AtCRP1 could be the factor leaving those footprints. Nevertheless, the observation that the footprints identified in Arabidopsis psaC, petA, and petB-petD transcripts are larger than the 14 nucleotide size of the predicted AtCRP1 footprint (37, 29, and 41 nucleotides in psaC, petA, and petB-petD, respectively) supports the view that the binding of AtCRP1 to its targets in vivo could be stabilized by other protein partners. For instance, the peptide chain release factor B3 (PrfB3) has been also shown to be required for Arabidopsis autotrophic growth and for the stability of 3′ processed petB transcripts to adjust cytochrome b₆ levels (Stoppel et al., 2011), thus possibly being an AtCRP1 specific protein partner. Similarly, PPR proteins involved in RNA stabilization and editing have been shown to interact with RNA Recognition Motif (RRM) proteins and other factors, indicating that larger protein complexes assembled around a PPR protein are likely to occur (Kupsch et al., 2012; Takenaka et al., 2014; Shi et al., 2015).

The interactions with the 5′UTR of psaC and petA have also been reported in the case of ZmCRP1 (Schmitz-Linneweber et al., 2005; Williams-Carrier et al., 2008), indicating that this feature of CRP1 function is conserved between Arabidopsis and maize. ZmCRP1 was also shown to bind directly to the 5′-UTR of petA transcripts by electrophoresis mobility shift assay (Williams-Carrier et al., 2008), favoring the possibility of a direct binding of CRP1 proteins to the corresponding RNA targets (see also Figure 8). Furthermore, ZmCRP1 has been proposed to directly control the translation of petA and psaC transcripts (Barkan et al., 1994), as shown through pulse labeling and polysome loading (in the case of petA), or deduced from the reduced association of psaC RNAs with ribosomes. Interestingly, the PsaC subunit of PSI and the PetA subunit of Cyt b₆/f could not be detected in atcrp1 thylakoids, despite the accumulation of the corresponding transcripts with no processing defects (see also Figure 9), suggesting that AtCRP1 plays a major role in translation regulation also in Arabidopsis. Unfortunately, the specific requirement of AtCRP1 in plastid protein translation cannot be verified by comparing Col-0 and atcrp1-1 leaves, due to the marked reduction of rRNA accumulation in atcrp1-1 plastids.

In addition to the defects in petA translation, the complete absence of Cyt b₆/f protein complex observed in atcrp1 thylakoids can also be attributed to processing alterations of the psbB-psbT-psbH-petB-petD polycistronic transcription unit. The lack of the monocistronic petB, the dicistronic psbH-petB, and the unspliced petB transcripts, together with the direct binding of AtCRP1 to the petB-petD intergenic region, strongly support the role of AtCRP1 in the metabolism of petB and petD transcripts. PPR protein-derived RNA-footprints are considered to arise due to exonucleolytic activity (Ruwe et al., 2016). Since sRNAs corresponding to predicted binding sites of AtCRP1 are identified here, the most likely role for AtCRP1 is to block exonucleases from degrading the petB and petD transcripts. A similar defect in petB-petD maturation has been reported in zmcrp1 mutant plants (Barkan et al., 1994; Fisk et al., 1999; Schmitz-Linneweber et al., 2005), although no association was detected between ZmCRP1 and the petB-petD intergenic region (Schmitz-Linneweber et al., 2005), so it is still uncertain whether the role of ZmCRP1 is direct or indirect.