LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants

Article

LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants Graphical Abstract

Authors Gerasimos Daras, Stamatis Rigas, Anastasios Alatzas, ..., Vassiliki Kostourou, George Panayotou, Polydefkis Hatzopoulos

Correspondence [email protected]

In Brief Daras et al. show that in higher land plants, both nuclear and chloroplast premRNA splicing involve a common nuclear-encoded protein, LEFKOTHEA. Starting in the early stages of embryo development, LEFKOTHEA coordinates the expression of the nuclear and chloroplast genomes, thus regulating plant growth.

Highlights d

LEFKOTHEA, a nucleus-encoded RNA-binding protein, exists only in embryophytes

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Embryo development relies on LEFKOTHEA function

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LEFKOTHEA localizes to chloroplasts and nucleus

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LEFKOTHEA catalyzes nuclear pre-mRNA and chloroplast group II intron splicing

Daras et al., 2019, Developmental Cell 50, 1–13 September 23, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.devcel.2019.07.024

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

Developmental Cell

Article LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants Gerasimos Daras,1 Stamatis Rigas,1 Anastasios Alatzas,1 Martina Samiotaki,2 Dimitris Chatzopoulos,1,3 Dikran Tsitsekian,1 Vassiliki Papadaki,2 Dimitris Templalexis,1 Georgios Banilas,1,4 Anna-Michaella Athanasiadou,1 Vassiliki Kostourou,2 George Panayotou,2 and Polydefkis Hatzopoulos1,5,* 1Department

of Biotechnology, Agricultural University of Athens, Athens, Greece for Bioinnovation, Biomedical Sciences Research Center "Alexander Fleming", Athens, Greece 3Present address: Biomedical Research Foundation of the Academy of Athens, Athens, Greece 4Present address: Department of Wine, Vine and Beverages Sciences. University of West Attica, Athens, Greece 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.07.024 2Institute

SUMMARY

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA, a nuclear encoded RNA-binding protein, participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss of function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.

INTRODUCTION The long coevolution of mitochondria and chloroplasts with their host cell has established a networking scheme highly important on developmental and cellular processes. To achieve such a progression, the host cell and the descendants of the ancestral endosymbionts, the mitochondrion and chloroplast, adopted different strategies. Besides the massive allocation of genetic material to host cell (Dyall et al., 2004; Timmis et al., 2004), the bidirectional communication strategy between the nucleus and mitochondrion or chloroplast formed a decisive platform of cellular homeostasis and differentiation. Furthermore, gene transcriptional mechanisms integrate and sustain an optimal networking level between the nucleus and the descendants of the ancestral endosymbionts necessary for cellular and developmental processes (Braunschweig et al., 2013; Jangi and Sharp, 2014).

The spliceosomal machinery in the nucleus has likely evolved from group II introns found in bacteria and organelles of fungi, algae, and higher plants (Fica et al., 2014; Lambowitz and Zimmerly, 2011). In agreement with this notion, key regions of group II introns and spliceosomal snRNAs domains share common catalytic mechanisms and structural conformations (Fica et al., 2014; Keating et al., 2010; Marcia and Pyle, 2012; Seetharaman et al., 2006). After the discovery of self-splicing group II intron RNA (Peebles et al., 1986), recent results reinforce the recruitment of a limited number of protein factors to facilitate the splicing of organellar group II introns (de Longevialle et al., 2010). On the contrary, the spliceosome exhibits exceptional compositional dynamics to splice pre-mRNA introns with diverse splicing site sequences. The spliceosome consists of five major snRNAs and hundred-plus associated proteins (Wahl et al., 2009; Yan et al., 2015), and is robust against variation in intron splice sites (Jangi and Sharp, 2014; Kitano, 2004). Despite the mechanistic parallels of the catalytic core between the spliceosomal RNA and group II introns suggesting a common evolutionary precursor (Fica et al., 2014; Keating et al., 2010; Marcia and Pyle, 2012; Seetharaman et al., 2006), it remains to be explored whether the splicing mechanisms in different cellular compartments share common regulatory components. Such a compelling strategy maintains coordinated posttranscriptional regulation, modulates functional responsiveness, and defines evolutionary process of pre-mRNAs splicing in the nucleus and splicing of group II introns in organelles. Here we identify a splicing component that controls posttranscriptional regulation and developmental progress. LEFKOTHEA, a nucleus-encoded protein with plant organelle RNA recognition (PORR) domain, promotes the splicing of both chloroplast group II introns and nuclear pre-mRNA introns, while embryogenesis is arrested in total loss of lefkothea mutant function. Our analysis reveals that transiently optimized nuclear compartmentalization of LEFKOTHEA ensures chloroplast biogenesis and advances early plant embryo development. LEFKOTHEA may represent a molecular switch for synchronization of gene expression in the nucleus and chloroplasts. In the context of nuclear-chloroplast coevolution, LEFKOTHEA has emerged as the common denominator in the RNA maturation processes of the bacterial endosymbiont and the eukaryotic lineage.

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Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

RESULTS LEFKOTHEA Controls Embryonic and Post-Embryonic Development The PORR domain proteins are nucleus-encoded RNA-binding proteins that have acquired specific roles in RNA splicing during the colonization of land by plants (Kroeger et al., 2009). Phylogenetic analysis suggests that PORR proteins are exclusively evolved in land plants and classified in two distinct groups depending on their organellar targeting pattern (Figure S1A). In model plant Arabidopsis thaliana, the PORR gene family consists of fifteen members, thirteen of which are predicted to localize in mitochondria and only two carry a transit peptide for chloroplast localization. The chloroplast isoforms At5g62990 (LEFKOTHEA) and the At4g01037 (WTF1) fall into two distinct classes emanating from a single gene duplication event and their overall predicted structure adopts a curved shape (Figure S1B), resembling the 3D structural conformation of other RNA-binding factors such as the mitochondrial transcription termination factors (mTERF) (Hammani et al., 2014). The analysis of the LEFKOTHEA (LEFKO) sequence showed that it contains a chloroplast transit peptide (TP) and unexpectedly, it also has nuclear localization signals (NLS) and a nuclear export signal (NES) (Figures 1A and S1C; Table S1). Multiple sequence alignment of the LEFKO orthologues from evolutionarily distant species shows a high degree of identity in amino acid sequence and the presence of NLS and NES motifs (Figure S1C). We screened and identified a viable chemically induced mutant allele in the At5g62990 locus. This EMS allele shows an albino cotyledon phenotype caused by a G to A transition. This mutation results in amino acid substitution of glycine (G) 373 to aspartic acid (D) within the NES motif of the LEFKO protein (Figures 1A, 1B, and S1D). Because of the viable phenotype, this allele was named Lefkothea1 (lefko1), after the name of the White Sea goddess who saved the shipwrecked Odysseus. We then examined the morphology of chloroplasts in albino lefko1 cotyledons and found that their shape is irregular and clearly have defective grana with very few and abnormal thylakoids (Figure 1C). In line with the impaired ultrastructure, components of Arabidopsis photosystems and photosynthetic pigments reveal malfunction of the photosynthetic apparatus in lefko1 cotyledons (Figures 1D and 1E). A number of Arabidopsis photosynthesis components such as D1, Cytf, Cytb6/f, and chlorophylls were also reduced in lefko1 chloroplasts of the early post-germinating tissues, i.e., first true leaves (Figures 1D, 1E, and S1E). The reduction of total chlorophyll in the first true leaves was about 70% compared to 95% reduction detected in lefko1 cotyledons (Figures 1E and S1E). In addition to the albino phenotype, the length of lefko1 primary root was shorter than the wild-type seedlings (Figures 1B and S1F). Transgenic lines expressing LEFKOTHEA in the lefko1 mutant background under control of the native or the constitutively expressed CaMV35S promoter showed normal content of the photosynthetic pigments and restored primary growth (Figures 1B, 1E, S1E, and S1F). The T-DNA recessive mutant in the At5g62990 locus, lefko2, carries an insertion at the 50 UTR and results in embryo developmental arrest (Figures 1A, 1F, 1G, and S1G). The phenotype was restored upon Agrobacterium-mediated transformation of the heterozygous mutant plants with the At5g62990 wild-type locus (Figures 1F and S1G). Together, these results indicate that 2 Developmental Cell 50, 1–13, September 23, 2019

LEFKO is involved in embryogenesis during the globular-to-heart stage transition and modulates the growth of post-embryonic tissues. LEFKO Protein Is Mainly Expressed in Meristems and Localized to Both Nuclei and Chloroplasts To gain insights into the expression of LEFKO gene, we transformed Col-0 plants with a 1.7-kb promoter region fused to the b-glucuronidase (GUS) reporter gene. Upon embryogenesis, LEFKO is expressed early at the globular or heart and at late stages of embryo development (Figure 2A). In addition, LEFKO is expressed through the primary root and mainly at the early meristem formation of lateral roots and the primary root meristem (Figure 2A). LEFKO and WTF1 gene paralogs preserve a distinct expression profile and LEFKO is expressed at lower levels than WTF1 (Figure S2A). Using complemented lefko1;LEFKOpro::LEFKO-YFP plant lines, we examined the LEFKO localization in the embryos and the root meristems. LEFKO-YFP under the control of the endogenous promoter resulted in dual localization in nuclei and chloroplasts of the embryo cells (Figures 2B and 2C). Further, LEFKO-YFP was apparently detected in nuclei of lateral root meristem cells and localized to both nuclei and plastids of primary root meristem and leaf epidermal cells (Figure S2B). Immunoblot analysis of the YFP protein tag verified that LEFKO is actually targeted in vivo to both nuclei and chloroplasts, while the observed nuclear localization is not due to cleavage of YFP (Figures S2C and S2D).These results are in agreement with the genetic analysis, indicating that the biological role of LEFKO is important for embryo development and the growth of root apical meristems. LEFKOTHEA Exhibits a Dynamic Intracellular Distribution Pattern To shed light into the LEFKO dynamic nucleocytoplasmic partitioning and chloroplast allocation, a series of LEFKO constructs under the 35S constitutive promoter were tested using the Nicotiana benthamiana transient expression system. The LEFKO-YFP fusion protein under the CaMV35S constitutive promoter was mainly targeted to chloroplasts and accumulated in structures reminiscent to chloroplast nucleoids (Figure 3A). The N-terminal YFP fusion (YFP-LEFKO) hinders the transit peptide and restricted LEFKO localization to the nucleus but not to chloroplasts (Figures 3A and S3A). We further employed virus-induced gene silencing (VIGS) to knock down the expression of Nicotiana benthamiana receptor importin-a that mediates the nuclear import of proteins containing NLSs. Contrary to the control (pTV00) that caused YFPLEFKO localization solely to the nucleus (Figures 3A, 3B, and S3A), downregulation of importin-a NbImpa1/2 gene expression (Figure S3B) resulted in about a 4-fold increase of the cells displaying signal localization into the nucleus and the cytoplasm reaching nearly 40% of total cells (Figures 3A and 3B). In similar experiments, when the enriched in positively charged arginines monopartite NLS (Figure S1C) was substituted by glycine and alanine residues, the YFP-LEFKOD(NLS) was localized to both the cytoplasm and nucleus in approximately 65% of total cells examined (Figures 3A and 3B). Given the molecular mass (~85 kDa) of YFP-LEFKO that exceeds the size exclusion limit (~40 kDa) for passive diffusion,

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

Figure 1. Embryo and Primary Growth Phenotypes of Arabidopsis lefko Mutants (A) Schematic representation of the position of lefko1 missense mutation in NES motif and lefko2 mutant allele. The inverted triangle depicts the T-DNA insertion mutation (emb1692). mNLS and bNLS represent the monopartite and bipartite NLS motifs, respectively. (B) Albino phenotype and post-germinative growth retardation of lefko1 allele, and complemented mutant plant lines using different LEFKO constructs. This panel is composed of images taken from different petri dishes. Cropping was used in the image corresponding to lefko1;LEFKOpro::LEFKO-YFP to avoid including parts of neighboring seedlings. (C) Transmission electron micrographs of lefko1 cotyledons ultrathin sections. Chl, chloroplast; Mit, mitochondrion. (D) Western blots of protein extracts from cotyledons and first leaves of 7-day- and 25-day-old seedlings, respectively. Detection of selected proteins involved in photosynthesis. Coomassie brilliant blue (CB) staining served as loading control. (E) Effect of lefko1 allele on the photosynthetic pigments of cotyledons. Complemented mutant plants using different LEFKO constructs showed nearly wild-type accumulation of photosynthetic pigments. Error bars represent ± SD from means generated from 30 seedlings in three independent experiments. (F) LEFKO is essential for embryo development. Arrowheads depict aborted seeds. lefko2/+ complemented mutant plants were generated using LEFKOpro::LEFKO-YFP. (G) Homozygous lefko2 arrests early embryo development. Heart stage embryos (left panels) and mature seeds (right panels).

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Figure 2. The Wild-Type LEFKO Protein Is Targeted to the Nucleus and Plastids of Arabidopsis Embryos (A) Histochemical detection of GUS activity in Arabidopsis embryos and root meristems. Plants were transformed with LEFKO promoter driving the expression of GUS reporter gene. (B) Dual targeting of LEFKO protein to the nucleus (arrowheads) and chloroplasts (arrows) in the embryos of lefko1;LEFKOpro::LEFKO-YFP transgenic lines. DAPI staining depicts nuclei (blue), whereas chlorophyll autofluorescence is pseudocolored to magenta. Single confocal planes are shown as merged images for each fluorescence signal. (C) Magnification of the boxed area. Two different regions with the corresponding cross-sections in xz and yz are shown along with intensity profile plots marked with white thick lines and generated using normalized pixel intensity of YFP (green), chlorophyll (magenta), and DAPI (blue).

LEFKO enters the nucleus through the importin system, suggesting an intimate relationship between the NLS and nuclear localization. To our surprise, about half of the cells expressing the YFP variant of lefko1 mutant allele (YFP-LEFKO1) presented localization to both cytoplasm and nucleus, indicating a rapid export of LEFKO1 (Figures 3A and 3B). As LEFKO contains a NES motif of conserved hydrophobic residues (Figures S1C and S3C), the presence of LEFKO1 variant also in the cytoplasm suggests that the single amino acid of G(373) to D substitution significantly enhances the predicted export capacity of the native NES (Figure S3D). Hence, the phenotype of lefko1 mutant is potentially attributed to the disturbed equilibrium of LEFKO in the nucleus. Further, immunoblot analyses demonstrated that all the expressed polypeptides remain intact and are not processed to produce YFP protein units that could be passively introduced in the nucleus due to low molecular weight (Figure S3E). To clarify the NES-dependent nuclear export of LEFKO, we applied the nuclear export inhibitor leptomycin B (LMB) known to inhibit exportin1, a receptor facilitating the nuclear export of proteins containing the NES motif (Kudo et al., 1999). In the absence of LMB, the fluorescent signal from the full-length LEFKO protein fused with YFP was detected in chloroplasts but not easily uncovered in nuclei of N. benthamiana protoplasts (Figure 3C). However, besides chloroplast deposition, the nuclear localization of LEFKO was uncovered when cells were treated with LMB (Figure 3C), indicating the nuclear export of wild-type LEFKO protein. In agreement with fluorescent localization, western blot verified that the full-length LEFKO protein was mainly immunodetected in chloroplasts (Figure S3F). Consistent with 4 Developmental Cell 50, 1–13, September 23, 2019

the localization analysis, the protein was readily immunodetected in both nuclei and chloroplasts of LMB-treated protoplasts (Figure S3F). Notably, a gel shift of approximately 7 kDa was detected in the nuclear fraction that coincides to the size of the TP, supporting the notion that LEFKO targeting follows two routes: one to the nucleus and the second to chloroplasts. In addition, we showed that the deletion of the hydrophobic residues of NES hindering the nuclear export capacity of the LEFKO-YFPD(NES) resulted in a profound accumulation of LEFKO in the nucleus besides chloroplast localization (Figure 3D). Together, our results demonstrate that the nucleusencoded LEFKO protein is localized in chloroplasts and also presents a dynamic transitory nuclear partitioning. Nucleus-Restricted Localization of LEFKO Functionally Complements lefko1 Post-Embryonic Defects Since the substitution of G to D in lefko1 allele affects LEFKO1 protein export from the nucleus, we hypothesized that the reconstitution of LEFKO protein homeostasis in the nucleus could restore lefko1 defects. We transformed lefko1 mutant plants with a LEFKO construct missing the transit peptide. Remarkably, transgenic lefko1 lines expressing D(TP)LEFKO complemented the white cotyledon phenotype (Figure 4A) and restored to nearly normal levels the accumulation of chlorophylls in chloroplasts of cotyledons (Figure 4B) and the length of the early post-embryonic primary root (Figure 4C). In lefko1 background, the expression of the endogenous LEFKO1 gene was not affected by the D(TP)LEFKO transgene and remained at similar levels when compared to the wild-type Arabidopsis plants (Figure S4A). LEFKO1 variant exhibits an enhanced nuclear export as shown

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

Figure 3. LEFKO Is Localized to the Chloroplasts and Shows Nucleocytoplasmic Partitioning (A) Epifluorescence images of N. benthamiana epidermal cells transiently expressing LEFKO constructs fused to YFP (green). Chlorophyll autofluorescence was pseudocolored to magenta. Arrows and arrowheads depict cytoplasmic and nuclear localization, respectively. (B) N. benthamiana epidermal cells transiently expressing distinct LEFKO constructs were classified according to LEFKO subcellular localization. Error bars represent ± SD from means, n = 100 cells. The asterisk indicates a statistically significant difference (t test, p < 0.001). (C) Protoplasts isolated from transiently transformed N. benthamiana cells. The effect of LMB on protein localization in the nucleus (white arrowhead). The intensity profile below the image indicates the relative fluorescent signal of YFP, chlorophyll, and DAPI. The asterisk denotes peak fluorescence from the nucleus. Magenta and blue signals depict chloroplast autofluorescence and nuclear DAPI staining, respectively. (D) Deletion of the NES motif from LEFKO results in chloroplast and nucleus protein localization (arrowhead). Cartoons depict the targeting scenarios of the nuclear encoded LEFKO.

using N. benthamiana epidermal cells (Figure 3A), suggesting that LEFKO1 is deposited mainly in chloroplasts, whereas the transgene-derived protein was restricted solely in the nucleus (Figures S4B and S4C). Our results reveal that the temporarily depleted LEFKO activity from the nucleus results in plastid morphological defects and that LEFKO has a distinctive nuclear function as D(TP)LEFKO variant complemented lefko1 mutant. LEFKO Regulates mRNA Splicing of Chloroplast and Nuclear Genes To delineate the molecular role of LEFKO, we firstly examined the splicing events of nuclear and plastidial genes in lefko2 homozy-

gous T-DNA embryos. RNA sequencing (RNA-seq) reads from globular and heart-stage mutant embryos uncovered splicing defects sorted into the major four categories: intron retention (IR), alternative 50 or 30 splice site (50 SS or 30 SS), and exon skipping (ES) (Figure S5A; Table S2). The distribution of differential alternative splicing events showed that the alternative 50 splice site was the major splicing defect in lefko2 mutant compared with the wild type. We identified 6,796 differentially expressed (DE) genes (Table S3) and 215 differentially alternative splicing (DAS) genes in lefko2 mutants (Table S2), of which 61 genes were categorized as DE and DAS (Figure S5B). The functional enrichment analysis revealed that the DE genes from lefko2 Developmental Cell 50, 1–13, September 23, 2019 5

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Figure 4. Nuclear Function of Complements lefko1 Phenotypes

LEFKO

(A–C) Specific-nuclear targeting of D(TP)LEFKOYFP complemented the defects of lefko1 (A), including chlorophyll accumulation (B) and primary root length (C). The asterisks indicate a statistically significant difference (t test, p < 0.05).

background were involved in several biological processes, mainly stress response (Figure S5C). Further, the genes displaying abnormal mRNA splicing in the lefko2 mutant were subjected to gene ontology (GO) enrichment analysis and found to be enriched among others in purine ribonucleoside binding (Figure S5C). To validate the role of LEFKO in splicing, we performed RT-PCR analysis of a group of randomly selected nuclear genes that displayed splicing defects (Table S2). The chloroplast-encoded rpl2 and petB genes were also selected to be examined, as the PORR domain proteins are required for their splicing (Francs-Small et al., 2012; Kroeger et al., 2009; Hsu et al., 2019). The results of RT-PCR analysis showed that IR events were clearly evident in both nuclear and chloroplast genes of lefko2 embryos (Figure 5A). Given the albino phenotype of lefko1, we assessed the RNA defects in cotyledons. RNA-seq reads derived from lefko1 mutant cotyledons revealed splicing defects to a similar extent with those detected in lefko2 mutant, when compared to Col-0 (Figure S5B). Intron retention events were the major splicing defect in lefko1 mutant (Figure S5A). We identified 3,383 DE genes (Table S3) and 281 DAS genes in lefko1 mutants (Table S2), of which 44 genes were categorized as DE and DAS (Figure S5B). In agreement with the white cotyledon phenotype of lefko1 mutant, functional enrichment analysis demonstrated that the DE genes were involved in biological processes such as photosynthesis and stress response (Figure S5D). DAS genes of lefko1 mutant were mainly involved in RNA processing. Interestingly, RT-PCR analysis confirmed splicing defects in lefko1 similarly to lefko2, whereas the aberrant splicing of chloroplast genes was not observed despite the lefko1 white cotyledon phenotype (Figure S6A). This observation supports the notion that the G to D substitution does not interfere with the functional capacity of the LEFKO1 variant in chloroplasts. In conjunction with these results, a comparison between the 50 or 30 splice sites of the significantly affected splicing events between lefko mutants (Table S2) 6 Developmental Cell 50, 1–13, September 23, 2019

and the Arabidopsis consensus sequences (Reddy, 2007) revealed that 50 and 30 splice sites were well conserved (Figure S6B). To discriminate whether the splicing defects of nuclear genes observed in lefko1 cotyledons were caused by aberrant protein equilibrium in the nucleus or induced by a retrograde signaling pathway, we applied the pesticide norflurazon, a carotenoid inhibitor resulting in photobleaching, on wildtype seedlings to mimic the lefko1 phenotype. Despite the chlorotic phenotype, the analysis of the model genes revealed that the pharmacological application failed to cause any splicing defects of nuclear genes (Figure S6A). The importance of nuclear allocated LEFKO protein function was further proved as pre-mRNAs were correctly matured and introns were normally removed in transgenic lefko1 lines expressing D(TP)LEFKO (Figure S6A). Taking these points into consideration, lefko1 developmental and morphological defects are associated with the enhanced LEFKO1 protein export prediction from the nucleus (Figure S3D) and not attributed to a secondary chloroplast retrograde signaling pathway. LEFKOTHEA Binds Nuclear and Chloroplast mRNA Targets To clarify whether LEFKO has an RNA binding capacity, we performed in vitro gel-shift assays. The D(TP)LEFKO variant was expressed in Escherichia coli as a fusion with maltose-binding protein (MBP) (Figure S7A), and the purified protein was shown to effectively bind (Figure S7B) to nuclear pre-mRNA that showed intron retention event (Figure 5A). To demonstrate the in vivo RNA binding capacity of LEFKO, we further applied RNA immunoprecipitation (RIP) followed by reverse transcription qPCR of the chloroplast and nuclear model genes that showed intron retention events. The in vivo binding capacity of LEFKO to chloroplast and nuclear genes was examined by using the transgenic lefko1 mutant plants transformed with pLEFKO::LEFKO-YFP-FLAG. The immunoprecipitated RNA was reverse transcribed and PCR amplified with gene specific primers (Table S2). The results revealed that wild-type LEFKO protein efficiently co-immunoprecipitated the chloroplast model gene mRNAs—namely the RPL2 and PETB transcripts (Figure 5B). Likewise, LEFKO co-immunoprecipitated mRNAs derived from nuclear model genes such as At2g37510, At3g48070, At5g63260, and At4g37980 (Figure 5B). The chloroplast-encoded CLpP and nucleus-encoded GAPDH transcripts do not correspond to binding targets of LEFKO protein and served as negative controls (Figure 5B). Taken together, the results support the capacity of LEFKO to associate with nuclear and chloroplastic premature RNAs.

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Figure 5. The Role of LEFKO in the Splicing of Chloroplast Group II Introns and Nuclear Pre-mRNAs (A) Selected intron retention (IR) events in chloroplast and nuclear genes of lefko2 embryos. Wiggle plots represent the normalized read coverage in a logarithmic scale (log10) of Col-0 (green) and lefko2 (red) globular and heart-stage embryos. The intron-exon structure of annotated genes is indicated (bottom). The position of the primers is indicated by arrows. Panels on the right show the validation of IR events by RT-PCR. Spliced (S) and unspliced (U) PCR-generated products of reverse-transcribed RNAs. Splicing defects in chloroplast petB gene were detected by amplification of the indicated intron and exon regions. GAPDH (At3g04120) was used to indicate equal RNA-seq reads between the two samples and to confirm the absence of genomic (g) DNA contamination in RT-PCR analysis. (B) RNA immunoprecipitation (RIP) of RNA-LEFKO protein associations were extracted from lefko1 transgenic seedlings transformed with pLEFKO::LEFKO-YFPFLAG using anti-FLAG. LEFKO-associated nuclear and chloroplastic transcripts that showed splicing defects in lefko2 were recovered and quantitated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). ClpP and GAPDH (At3g04120) served as negative control for chloroplast and nuclear targets, respectively. RIP from wild-type Col-0 plants was used as control. qPCR results were normalized to input per sample. Data are mean ± SD from three biological replicates. Statistical significance was tested using the Student’s t test and shown as *p < 0.001. Western blot analysis of inputs and immunoprecipitated LEFKO proteins is presented.

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Figure 6. LEFKO Associates with Chloroplast Group II Intron Splicing and Spliceosomal Factors (A) Removal of the transit peptide (TP) from the protein D(TP)LEFKO restricts nuclear localization and illustrates colocalization (white) with spliceosomal factors U1-70K and SR-45. Chlorophyll autofluorescence and mCherry were pseudocolored to magenta. Blue signal depicts DAPI nuclear staining. Dotted line depicts nucleus. (B) Volcano plot of most significant chloroplast and nuclear proteins identified by LEFKO pull-down assays. Red line (significance, 0.01) separates specifically interacting proteins (top left portion of plot) from background. The plots were constructed by the t test difference versus the negative log of the p values for each protein, extracted from at least three biological experiments. Selected top hits are indicated with blue dots for nuclear proteins, dark green dots for chloroplastic proteins engaged in splicing of group II introns, light green dots for chloroplastic proteins involved in gene expression, and gray dots for miscellaneous proteins. All co-immunoprecipitated proteins are reported in Table S5. Specific interactors are listed in Table S6.

LEFKOTHEA Physically Interacts with Spliceosomal Proteins Consistent with the D(TP)LEFKO exclusive localization into the nucleus, LEFKO colocalized in nuclear speckles with the spliceosome components U1-70K and SR45 (Figure 6A). Consistent with this notion, video microscopy revealed that LEFKO speckles dynamically moved between nucleoplasm and the nucleolus (Video S1). This rapid speckle movement of LEFKO is similar to the dynamics of pre-mRNA splicing factors related to transcriptional activity (Fang et al., 2004). Given the colocalization profile of LEFKO with spliceosome components and pre-mRNA defects detected in both mutant alleles, we hypothesized that LEFKO physically interacts with spliceosomal proteins. To test this, we used quantitative interaction proteomics. Co-immunoprecipitation coupled to quantitative mass spectrometry (coIP/MS) analysis identified significant protein interactors of LEFKO protein such as the RH3 splicing component (Figure 6B; Tables S5 and S6) of chloroplast group II introns (Asakura et al., 2012), revealing that the paralogs of LEFKO and WTF1 class interact with a distinct set of plastidial splicing factors. Other factors involved in chloroplast gene expression, such as PTAC17, EF-Tu, and a Pentatricopeptide repeat protein (PPR) or few miscellaneous chloroplastic proteins, were also co-immunoprecipitated (Figure 6B; Tables S5 and S6). 8 Developmental Cell 50, 1–13, September 23, 2019

Intriguingly, this analysis also identified significant LEFKO interactors of the spliceosome machinery or rRNA processing and RNA metabolism such as NUC-L1, RS41, SR34, HDT1, HDT2, and HDT3. The NUC-L1 has been reported to precipitate with spliseosomal components (Das et al., 2013). RS41 or SR34 splicing factors and histone deacetylases (HDTs) proteins participate in the recruitment of the splicing machinery (Chen et al., 2013; Delcuve et al., 2012; Hnilicova´ et al., 2011) (Figure 6B; Tables S5 and S6). It is worth noting that most LEFKO protein interactors are components of functionally distinct pathways but tightly linked to RNA processing. Our results demonstrate that LEFKO is a splicing component of group II intron complex of chloroplasts and a factor participating in the nuclear splicing mechanism. DISCUSSION To date, cumulative evidence of the evolutionary relationship between group II intron splicing in chloroplasts and spliceosome-mediated pre-mRNA splicing in the nucleus has emerged. Nonetheless, the stereotypic exclusion of the chloroplast splicing factors from the spliceosome machinery still remains. Chloroplasts are semi-autonomous organelles responsible for energy production and synthesis of essential metabolites, including fatty acids, vitamins, tetrapyrroles, and amino acids.

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Figure 7. Schematic Model of LEFKO Splicing Role in Plant Growth In the cytoplasm, ribosomes translate LEFKO mRNA to protein. Driven by the N-terminal transit peptide, LEFKO is targeted to chloroplasts, where it directly regulates the splicing of group II introns. The nucleocytoplasmic partitioning of LEFKO is controlled by the NLS and NES motifs. In the nucleus, LEFKO physically interacts with spliceosomal components to accomplish nuclear premRNA splicing.

They derived from endosymbiotic-free living cyanobacteria and, over evolutionary time, donated most of their genome to the nucleus of the host cell (Dyall et al., 2004; Timmis et al., 2004). Chloroplast gene expression or metabolism requires a number of proteins encoded by genes that have already been transferred to the nucleus. The mRNAs are translated in cytoplasm, and the resulting polypeptides are targeted to chloroplasts. A wide range of transcriptional and post-transcriptional processes, including DNA replication, RNA transcription, RNA processing, editing, splicing, and translation necessitate nucleus-encoded proteins (Barkan, 2011; de Longevialle et al., 2010). This nuclear process demand illustrates the fundamental role of the cell nucleus in controlling chloroplast activities. However, the compartmentalization of genes and gene products generates a barrier that has to be abolished in order to regulate coordinated nuclear and chloroplast gene expression and cellular homeostasis. Most introns in the chloroplast genome of angiosperms are classified as group II and appear to have lost their ability for self-excision (de Longevialle et al., 2010). Nucleus-encoded proteins construct a splicing machinery to remove group II introns (Belcher et al., 2015), providing evidence of a molecular crosstalk emerged during chloroplast and nuclear coevolution. PORR domain proteins are RNA-binding proteins encoded by nuclear genes and in plant organelles are engaged in group II intron splicing (Francs-Small et al., 2012; Konishi and Sugiyama, 2006; Kroeger et al., 2009). LEFKO defines a distinct class of

PORR domain proteins that exists only in land plants and shows a dynamic intracellular distribution pattern. The chloroplast and nuclear localization of LEFKO is a unique and intriguing feature. While LEFKO, like the protein paralog WTF1, directly associates with splicing components of group II introns in chloroplasts, we demonstrate that it also interacts with nuclear pre-mRNA splicing factors (Figure 7). Nuclear allocation of LEFKO at early globular or heart-stage embryos promotes splicing of pre-mRNAs. Together with the chloroplastdeposited LEFKO splicing activity, this protein constitutes a functional factor to initiate chloroplast biogenesis at early stages of plant embryo development and to coordinate gene expression in both compartments. As LEFKO contributes to the splicing of nuclear introns from genes encoding for proteins engaged in diverse functional modules, LEFKO exerts a tight control of cellular homeostasis. A consequence of the loss-of-function lefko2 homozygous mutation is an embryo developmental arrest due to splicing malfunction of group II introns and pre-mRNAs in chloroplasts and the nucleus, respectively. Consistent with this observation, LEFKO is highly conserved in higher land plants, also known as embryophytes. Specialized structures of embryophytes nurture the young embryo sporophyte during the early stages of its multicellular development (Pires and Dolan, 2012). Several loss-of-function mutations in genes encoding for splicing components of chloroplast group II introns result in lethality (Khrouchtchova et al., 2012; Schmitz-Linneweber et al., 2006; Zhang et al., 2015). Similarly, null alleles of genes encoding for spliceosome components are embryonic lethal (Huang et al., 2013; Marquardt et al., 2014; Sasaki et al., 2015). Our data confirmed that the predicted TP, NLS, and NES motifs of LEFKO control a dynamic nuclear and chloroplast compartmentalization. LEFKO localization in the nucleus and plastids was evident in cells of meristematic tissues that show high transcriptional activity. Nevertheless, using different types of tags at the C terminus, LEFKO under the CaMV35S constitutive or native promoter was detected mainly in chloroplasts, implying a transient presence in the nucleus. Strikingly, LEFKO protein was exclusively deposited in the nucleus by deleting or masking the chloroplast TP. Further, inhibition of nuclear export by LMB or NES removal also revealed nuclear localization of the LEFKO polypeptide, supporting the exceptional rapid export of LEFKO from the nucleus. Our results showed that both the two NLSs and the NES motifs are vital to sustain the necessary protein transient distribution in the nucleus, and the TP to target the polypeptide in chloroplasts. The lefko1 mutation lies within the NES, and our data analysis showed that the modified motif has a higher nuclear export prediction score. We confirmed that LEFKO1 variant has significantly higher capacity of nuclear export than the wild-type protein. This irregular protein distribution between the two subcellular compartments results in a white cotyledon phenotype due to abnormal chloroplast biogenesis. In agreement with the enhanced nuclear export of LEFKO1 variant, splicing defects were only detected in pre-mRNAs of lefko1 mutant. Given that a chloroplast retrograde signal regulates nuclear alternative splicing of factors engaged in spliceosome (Petrillo et al., 2014), we examined whether the nuclear splicing defects detected in lefko mutants were originated from the nucleusdependent function of LEFKO. Intriguingly, the LEFKO protein Developmental Cell 50, 1–13, September 23, 2019 9

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

isoform without the chloroplastic transit peptide complemented the white cotyledon phenotype of lefko1 and restored the accumulation of chlorophylls in chloroplasts of cotyledons. Furthermore, the nuclear function of LEFKO was substantiated as the splicing defects of pre-mRNAs detected in lefko1 were repaired by the exclusive nuclear D(TP)LEFKO variant, enforcing the fact that the chloroplast to nucleus retrograde route was not the primary effect of aberrant splicing of nuclear pre-mRNAs. In agreement, imitation of the lefko1 phenotype by pharmacological inhibition of photosynthetic pigments of wild-type seedlings revealed no splicing defects in the nuclear genes that were affected in lefko1 mutant background. Further, lefko1 mutant cotyledons exhibit low chlorophyll content and aberrant photosynthetic capacity. Contrary to cotyledons, the chlorophyll content and components of the photosynthetic apparatus are gradually restored in the first true leaves. It is recently widely appreciated that chloroplasts in leaves differ from the chloroplasts present in embryonic and post-embryonic tissues, including cotyledons. During embryogenesis, chloroplasts undergo a transient photosynthetic stage followed by chloroplast dedifferentiation to small non-green plastids (proplastids) at the end of seed development (Allorent et al., 2015; Bo¨rner et al., 2015). Hence, in cotyledons contrary to true leaves, chloroplasts are initially developed from proplastids, which are functionally immature organelles for photosynthesis. Our results support the role of LEFKOTHEA during this stage of plastidial cycle to synchronize RNA processing and thereby gene expression in the nucleus and chloroplasts upon embryogenesis and postembryonic seedling establishment. Maize WTF1 interacts with RNC1 and CFM4 factors (Kroeger et al., 2009), while RH3, a distinct splicing component (Asakura et al., 2012; Gu et al., 2014), is the interactor of Arabidopsis LEFKO. All represent components of the complex engaged in the splicing of group II introns (Asakura et al., 2012; Watkins et al., 2007). Additionally, while WTF1 presents a similar spatiotemporal expression pattern with LEFKOTHEA and both proteins share extensive amino acid sequence homology, LEFKO gene knockout in lefko2 allele causes embryo lethality with splicing defects in chloroplast mRNAs. This observation supports the notion that in terms of functional properties, WTF1 and LEFKO are not redundant, supporting a specialized role of LEFKOTHEA upon embryogenesis. The SR34, an orthologue of human ASF/SF2 splicing factor (Barta et al., 2010), interacted with LEFKO. ASF/SF2 promotes the recruitment of U1 snRNP to the 50 splice site to facilitate splicing reactions as it associates with U2 snRNP (Jamison et al., 1995; Zahler and Roth, 1995). Another LEFKO interactor, RS40/RS41 is a characterized serine-arginine (SR)-rich splicing factor (Chen et al., 2013). Interestingly, ASF/SF2 and SR factors interact with the nuclear export protein TAP to control the export of mature mRNAs from the nucleus (Lai and Tarn, 2004). Histone deacetylases (HDTs) known to participate in the recruitment of the splicing mechanism (Delcuve et al., 2012; Hnilicova´ et al., 2011) also interacted with LEFKO. LEFKOTHEA localizes to both the nucleoplasm and nucleolus. This localization pattern in intranuclear compartments is especially evident upon D(TP)LEFKO-YFP transient expression. LEFKOTHEA colocalizes with U1-70K and SR45 spliceosome components, which have been reported to localize in the nucleoplasm and the nucleolus together with proteins involved in pre10 Developmental Cell 50, 1–13, September 23, 2019

mRNA splicing and exon-junction complex proteins, driving mRNA export and nonsense-mediated decay (Pendle et al., 2005; Koroleva et al., 2009). Additionally, members of the SR family splicing factors that also interact with LEFKOTHEA have been detected by human nucleolar proteome analysis (Andersen et al., 2005; Sakashita and Endo, 2010). It is widely appreciated that the plant nucleolus might be actively involved in the assem
and bly and/or recycling of spliceosomal complexes (Lorkovic Barta, 2008). Furthermore, LEFKOTHEA interacts with nucleolar proteins, such as nucleolin, and HDACs, corroborating this intranuclear localization and possibly suggesting additional activities of LEFKOTHEA beyond splicing. Early development of land plants is coordinated with chloroplast biogenesis. As the seedling emerges from the soil and firstly encounters light, it accommodates an incredible reprogramming of gene expression leading to the development of a fully functional photosynthetic apparatus (Gollan et al., 2015). A transient phase of photosynthetic activity is also apparent during embryogenesis (Allorent et al., 2015). The physiological reset at these early stages, requires a molecular platform to reprogram gene expression in both the nucleus and chloroplasts. Synchronously deposited LEFKO in both compartments might coordinate posttranscriptional control of gene expression of endosymbiont-chloroplast with their host cell-nucleus to advance plant development (Figure 7). LEFKO modulates the two distinct splicing mechanisms in the nucleus and chloroplasts to accommodate gene regulation from the early stages of plant growth. During the coevolution of endosymbionts with their host cell, LEFKO provides evidence of a convergence in evolution and defines a distinct clade of PORR domain proteins that exists only in higher land plants. SUPPORTING CITATIONS The following references appear in the Supplemental Information: Chen et al., 2018; Klepikova et al., 2016; Love et al., 2014; Wang et al., 2012 STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Plant Materials and Growth Conditions METHOD DETAILS B Positional Cloning of lefko1 Allele B T-DNA Genotyping of lefko2 Allele B Preparation of LEFKO Gene Constructs B In Planta Transient Transformation B Nucleus and Chloroplast Fractionation B Preparation of Protoplasts B Extraction and Quantification of Photosynthetic Pigments B Imaging and Confocal Microscopy B Transmission Electron Microscopy B GUS Staining B Immunoblotting

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

B

RNA-Seq Gene Ontology Analysis B Analysis of Intron Retention Events B In Planta Protein Co-immunoprecipitation B RNA Immunoprecipitation B Real-Time qRT-PCR B Generation of Recombinant LEFKO Protein for In Vitro Analysis B Electrophoretic Mobility Shift Assay-EMSA B Proteomic Analysis B Bioinformatics QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY B

d d

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. devcel.2019.07.024. ACKNOWLEDGMENTS The authors thank Eva-Mari Aro, University of Turku, for D1 antisera; Roberto Barbato, Universita` del Piemonte Orientale, for CP43 antisera; Reinhold G. € t Mu €nchen, for cytb/f antisera; Harald Paulsen, Johannes Herrmann, Universita Gutenberg University, for LHCP antisera; Saskia A. Hogenhout, John Innes Centre, for VIGS pTV00:NbImpa1/2; Ligeng Ma, Capital Normal University, for SR45-mCherry and U1-70K-mCherry; Micha1 Szcze
sniak and Adam Mickiewicz University for data of the Arabidopsis U2 splice sites; and Elizabeth Haswell, Washington University in St. Louis, for RecARED seeds. This work was supported by grants from the GSRT Excellence 2012-15 Grant ‘BELiCy’ to P.H., ‘‘Synthetic Biology: From omics technologies to genomic engineering (OMIC-ENGINE)’’ (MIS 5002636) and ‘‘A Greek Research Infrastructure for Visualizing and Monitoring Fundamental Biological Processes (BIOIMAGING-GR)’’ (MIS 5002755), which are implemented under the Action ‘‘Reinforcement of the Research and Innovation Infrastructure,’’ funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). G.D., A.A., and D. Tsitsekian are indebted for funding to IKY Fellowships of Excellence. AUTHOR CONTRIBUTIONS G.D., S.R., and P.H. designed the experiments. G.D. performed most of the experiments with the contribution of G.B. and A.M.A. for map-based cloning of lefko1; D.C. for protein modeling and gene expression analysis; D. Tsitsekian for gene cloning; D. Templalexis for plant transformation; A.A. for protein analysis and coIP; M.S. and G.P. for mass spectrometry; and V.P. and V.K. for confocal microscopy. G.D., S.R., A.A., M.S., and P.H. analyzed experimental data. G.D., S.R., and P.H. prepared the manuscript with valuable contribution of all authors. P.H., funding acquisition. DECLARATION OF INTERESTS

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The authors declare no competing interests.

Dyall, S.D., Brown, M.T., and Johnson, P.J. (2004). Ancient invasions: from endosymbionts to organelles. Science 304, 253–257.

Received: October 1, 2018 Revised: April 27, 2019 Accepted: July 25, 2019 Published: August 22, 2019

Fang, Y., Hearn, S., and Spector, D.L. (2004). Tissue-specific expression and dynamic organization of SR splicing factors in Arabidopsis. Mol. Biol. Cell 15, 2664–2673.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal OctA-Probe (D-8)

Santa Cruz Biotechnology

Cat#sc-807; RRID:AB_675756

Mouse monoclonal OctA-Probe (H-5)

Santa Cruz Biotechnology

Cat#sc-166355; RRID:AB_2017593

Rabbit polyclonal anti-GFP (FL)

Santa Cruz Biotechnology

Cat#sc-8334; RRID:AB_641123

Antibodies

Rabbit polyclonal anti-GFP

Cell Signaling Technology

Cat#2555S; RRID:AB_10692764

Rabbit polyclonal ant-D1

Zhang et al., 2000

N/A

Rabbit polyclonal anti-CYTF

Alt et al., 1983

N/A

Rabbit polyclonal anti-CYTB6/F

Alt et al., 1983

N/A

Rabbit polyclonal anti-LHCP

Adamska et al., 2001

N/A

Rabbit polyclonal ant-CP43

Barbato et al., 1992

N/A

Rabbit monoclonal anti-H3 (D1H2)

Cell Signaling Technology

Cat#4499; RRID:AB_10544537

Horseradish peroxidase-conjugated goat anti-mouse

Santa Cruz Biotechnology

Cat#sc-2005; RRID:AB_631736

Horseradish peroxidase-conjugated goat anti-rabbit

Santa Cruz Biotechnology

Cat#sc-2004; RRID:AB_631746

Agrobacterium tumefaciens

P.H. lab collection

C58C1

Escherichia coli ER2523

New England Biolabs

Cat#E4131

pCp-biotin

Jena Biosciences GmbH

Cat#NU-1706-BIO

HRP-Streptavidin

Abcam

Cat#ab7403

3X FLAG peptide

Sigma Aldrich

Cat#F4799

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins

T4 RNA Ligase 1

New England Biolabs

Cat#M0204S

Leptomycin B

Cayman Chemical

Cat#10004976

SIGMAFAST Protease Inhibitor Tablets

Sigma Aldrich

Cat#S8820

Protease Inhibitor Cocktail, for plant cell and tissue extracts

Sigma Aldrich

Cat#P9599

Acetosyringone

Sigma Aldrich

Cat#D134406

Cellulase Onozuka R-10

Duchefa Biochemie

Cat#C8001

Macerozyme R-10

Duchefa Biochemie

Cat#M8002

DAPI (40 ,6-Diamidino-2-Phenylindole, Dihydrochloride)

Molecular Probes

Cat# D1306

HiScribe T7 High Yield RNA Synthesis Kit

New England Biolabs

Cat#E2040S

pMAL Protein Fusion and Purification System

New England Biolabs

Cat#E8200S

anti-FLAG M2-agarose affinity gel

Sigma Aldrich

Cat#A2220

NucleoSpin Plant RNA kit

Macherey-Nagel

Cat# 740949

TruSeq Low Input kit

Illumina

Cat#FC-134

Vivacon 500, centrifugal concentrator

Sartorius

Cat#VN01H02

Trypsin/Lys-C Mix, Mass Spec Grade

Promega

Cat#V5072

Acclaim PepMap 100, trap column

Thermo Scientific

Cat#164564

Acclaim PepMap 100, analytical column

Thermo Scientific

Cat#164540

Ultimate 3000 RSLCnano system

Thermo Fisher Scientific

N/A

LTQ Orbitrap XL mass spectrometer

Thermo Fisher Scientific

N/A

this paper

http://www.ebi.ac.uk/pride/archive/; PRIDE: PXD009965

Critical Commercial Assays

Deposited Data Mass spectrometry data of LEFKOTHEA chloroplast protein partners

(Continued on next page)

e1 Developmental Cell 50, 1–13.e1–e7, September 23, 2019

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Next Generation RNA Sequencing of lefko2 mutant embryos

this paper

https://www.ncbi.nlm.nih.gov/geo/; GEO: GSE94576

Next Generation RNA Sequencing of lefko1 mutant cotyledons

this paper

https://www.ncbi.nlm.nih.gov/geo/; GEO: GSE106459

Col-0 (Arabidopsis thaliana accession)

NASC

ID: N1093

Ler-0 (Arabidopsis thaliana accession)

NASC

ID: NW20

lefko1, T-DNA mutant line of A. thaliana (At5g62990)

SALK

ID: SALK_044699/emb1692

Experimental Models: Organisms/Strains

lefko2, EMS mutant line of A. thaliana (At5g62990)

this paper

N/A

Transgenic line of A. thaliana, LEFKOpro::LEFKO

this paper

N/A

Transgenic line of A. thaliana, LEFKOpro::LEFKO-FLAG

this paper

N/A

Transgenic line of A. thaliana, LEFKOpro::LEFKO-FLAG-YFP

this paper

N/A

Transgenic line of A. thaliana, LEFKOpro::GUS

this paper

N/A

Transgenic line of A. thaliana, 35Spro::LEFKO-FLAG-YFP

this paper

N/A

Transgenic line of A. thaliana, 35Spro::D(TP)LEFKO-FLAG-YFP

this paper

N/A

this paper

N/A

this paper

N/A

this paper

N/A

Oligonucleotides See Table S4 for primer sequences Recombinant DNA See Table S7 for plasmids information Software and Algorithms See Table S8 for software and algorithms information

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents may be directed to and will be fulfilled by the Lead Contact, Polydefkis Hatzopoulos ([email protected]). This study did not generate new unique reagents. Transgenic plant lines and plasmids are available upon request from the authors. EXPERIMENTAL MODEL AND SUBJECT DETAILS Plant Materials and Growth Conditions Arabidopsis thaliana seeds were surface-sterilized and sown on Petri dishes containing 0.5x Murashige and Skoog (MS) medium (Duchefa), pH 5.7, supplemented with 1% sucrose and solidified with 0.4% phytagel (Sigma). Seedlings were grown on solid media supplemented with 20 nM norflurazon (Fluka) and showed white cotyledon phenotype. Liquid cultures were prepared without phytagel addition. After 24 h of stratification at 4 C, seedlings were positioned to grow vertically for 2–6 days after germination at 22 C in a Fitotron growth chamber (Weiss) under a long photoperiod with 16 h of light and 8 h of darkness per day and 100 mmol m–2 s–1 light intensity. METHOD DETAILS Positional Cloning of lefko1 Allele The lefko1 mutant allele was isolated from a genetic screen of an M2 ethylmethanesulfonate (EMS)-mutagenized Arabidopsis thaliana Columbia (Col-0) background seed population. Before genetic mapping, lefko1 seeds were backcrossed four times to their Col-0 wild-type genetic background. The lefko1 mutant was crossed with Arabidopsis polymorphic ecotype Landsberg erecta. Genomic DNA was isolated from 963 F2 individuals exhibiting the lefko1 phenotype. Positional cloning was performed using combinations of simple sequence length polymorphism (SSLP) and cleaved amplified polymorphic sequence (CAPS) markers (Table S4). The molecular markers were designed based on data of Arabidopsis thaliana DNA polymorphisms available from the Monsanto Company (St. Louis, MO, USA) and the Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org). Genes with Arabidopsis Genome Initiative (AGI) annotation numbers At5g62980–At5g63030 were amplified using template DNA from BAC clone MJH22 with Phusion High-Fidelity DNA Polymerase (Finnzymes Oy). Each open reading frame (ORF) was cloned into the SmaI site of the pGPTV-HPT binary vector. The Agrobacterium tumefaciens strain C58C1 Rif R containing the non-oncogenic Ti plasmid pGV3101 was transformed with the pGPTV-HPT constructs by electroporation (Gene Pulser II; Bio-Rad). The constructs were

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introduced into lefko1 mutant allele by the vacuum infiltration method. Transgenic plants were selected on 30 mg l1 hygromycin and grown to maturity for analysis of their ability to complement the lefko1 mutant phenotype. The At5g62990 locus was amplified using as template genomic DNA from lefko1 seedlings with a set of primers resulting in overlapping polymerase chain reaction (PCR) products of average size 600 bp. Sequencing analysis of these products led to the identification of the EMS mutation. T-DNA Genotyping of lefko2 Allele The lefko2 mutant is represented by the Salk T-DNA collection Unicode SALK_044699/emb1692 (http://signal.salk.edu) and was obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). Genotypic characterization of the lefko2 (emb1692) allele confirmed the T-DNA insertion site in the 50 -UTR of LEFKO (At5g62990) gene. Hemizygous T-DNA plants were characterized by triple primer PCR-based genotyping using gene [ForT/,RevT] and T-DNA-specific [nosF] primers (Table S4). Preparation of LEFKO Gene Constructs The LEFKO gene (At5g62990) was amplified from Col-0 genomic DNA by the combination of [F1] or [F1-MLU] as forward and [R1] as reverse primers (Table S2) to generate the 1507-bp fragment. R1 reverse primer carried nucleotide sequence for the FLAG epitope and a specific MluI site at the 30 end. D(TP)LEFKO was generated using the [F2] as forward and [R1] as reverse primers. LEFKO1 mutant variant was amplified from lefko1 homozygous mutant embryos using the [F1-MLU] as forward and [R1] reverse primers. LEFKO-D(NLS) was generated using two overlapped PCR fragments. The 50 fragment was amplified using the [F1] and [R2] primers, while the 30 fragment was amplified using the [F3] and [R1] primers (Table S4). In a common double template PCR the two overlapped fragments were amplified with [F1] and [R1] primers. LEFKO-D(NES) was generated after the ligation of two PCR fragments into the SacI site. The 50 fragment was amplified using the [F1] and [R3] primers, while the 30 fragment was amplified using the [F4] and [R1] primers. Preparation of LEFKOpro::LEFKO was performed by PCR amplification of the LEFKO molecular locus using Col-0 genomic DNA as template with the combination of [F5] and [R4] or [R1] as forward and reverse primers, respectively. LEFKOpro was generated using the combination of [F5] and [R5] as forward and reverse primers, respectively (Table S4). For fluorescence imaging, Citrine–YFP reporter gene was introduced into the unique MluI restriction site of 50 or 30 . To facilitate cloning, the YFP gene was PCR amplified with [F-YFP] and [R-YFP] as forward and reverse primers, respectively (Table S4). The final PCR products were cloned into the SmaI site of pBluescript II (SK) vector. The resulting transgenes were cloned into the pGPTV– HPT binary vector with or without the CaMV35S expression cassette. LEFKOpro was cloned into the SalI site of pBI101 containing the GUS gene. The final constructs were fully sequenced to confirm that translational fusions were correct. The constructs were introduced into Col-0 ecotype of Arabidopsis and lefko mutant plants by the vacuum infiltration method. Transgenic plants were selected on 30 mg l1 hygromycin and grown to maturity to collect the T2 and T3 generations for microscopy. In Planta Transient Transformation For transient expression of the YFP fusion constructs in Nicotiana benthamiana, the Agrobacterium cells were grown at 28 C to stationary phase in LB supplemented with 50 mg l–1 kanamycin, gentamycin, and rifampicin. To obtain high level of transient expression, cultures were inoculated with Agrobacterium cells carrying the p19 protein of tomato bushy stunt virus as a viral-encoded suppressor of gene silencing. The cultures carrying different LEFKO–YFP transgenes and p19 construct were precipitated by centrifugation at 3500 rpm for 10 min and re-suspended in 10 mM MgCl2 and 150 mM acetosyringone, pH 5.5 to have an optical density (OD600) of 0.7 and 1.0, respectively. The Agrobacterium cells were incubated for 3 h in this medium and then infiltrated into the abaxial air spaces of 2–4-week-old Nicotiana benthamiana leaves. Virus-induced gene silencing (VIGS) to knock down the expression of Nicotiana benthamiana receptor importin-a was employed (Kanneganti et al., 2007). Nucleus and Chloroplast Fractionation Nuclei from Arabidopsis seedlings were isolated using sucrose gradient centrifugation. Plant tissue was homogenized in ice-cold buffer A (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM PMSF) by means of SilentCrusher M grinder (Heidolph), filtered through Miracloth and centrifuged at 1900g for 20 min at 4 C. The pellet was suspended in buffer B (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF) and centrifuged as above. The pellet was suspended in buffer B containing 0.15% Triton X-100, overlaid on top of buffer C (1.7 M sucrose, 10 mM Tris-HCl pH 8.0, 2 mM MgCl2, 0.15% Triton X-100, 1 mM PMSF) and nuclei were pelleted by centrifugation at 16,000g for 45 min at 4 C. Nuclei were isolated from protoplasts of transiently transformed Nicotiana benthamiana leaves. Protoplasts were lysed in buffer containing 20 mM Tris-HCl pH 7.0, 0.25 M sucrose, 0.7% Triton X-100, 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 1x protease inhibitor cocktail and fractionated by centrifugation at 3000g. Nuclei were washed with 20 mM Tris-HCl pH 7.0, 25% glycerol, 2.5 mM MgCl2, lysed in 2x Laemmli buffer at 90 C and used for immunoblotting. Plant tissue was homogenized in ice-cold buffer containing 0.3 M sucrose, 50 mM Tricine/NaOH pH 7.8, 10 mM NaCl, 5 mM MgCl2, and freshly added 0.2% BSA. After filtration through Miracloth, the homogenate was centrifuged at 1000g for 7 min at 4 C. The green pellet was carefully suspended in the same buffer and the centrifugation step was repeated. After centrifugation, the supernatant was removed and the chloroplast pellet was collected. Nuclear and chloroplastic proteins were extracted either in Extraction buffer (50 mM sodium phosphate buffer pH 7.0, 2% SDS, 10 mM EDTA, 3.2 mM benzamidine, 1 mM PMSF, 10 mM b-mercaptoethanol, 1x protease Inhibitor Cocktail-Sigma S8820) to use for immunoblotting or in Lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, 1x protease Inhibitor Cocktail) to use for coIP experiments.

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Preparation of Protoplasts Two g of 3-week-old Nicotiana benthamiana leaves were sliced with a razor into 1-mm2 and transferred to a 24-well dish containing 0.5 M mannitol for 1 h. Then the sliced material was floated in the enzyme solution 1% cellulase Onozuka R-10, 0.25% Macerozyme R-10 (Duchefa), 8 mM CaCl2, 0.4 M mannitol, pH 5.5. Digestion took place overnight in the dark at 22 C. Protoplasts were harvested from the bottom of the wells and washed twice with 0.5 M mannitol and 0.2 M CaCl2, then resuspended in 1 ml 0.5 M mannitol and counted in a hemacytometer. The nuclear export inhibitor Leptomycin B (Cayman Chemical) was dissolved in ethanol and applied at a final concentration of 2 mM. Extraction and Quantification of Photosynthetic Pigments Photosynthetic pigments were extracted from 6-day-old Arabidopsis seedlings incubated with DMSO for 30 min at 65 C. The O.D. of each extract was determined and pigments concentrations (mg/gr fresh weight) were calculated. Three independent experiments were used for each sample. Imaging and Confocal Microscopy The plant specimens of T3 Arabidopsis thaliana transgenic lines and Nicotiana benthamiana leaf epidermal cells were mounted between a slide and cover-slip in distilled water and examined using an Olympus BX-50. For staining, protoplasts were submerged in 0.53 MS liquid medium supplemented with 1 mg/ml DAPI for 10 min and washed several times with 0.53 MS liquid medium. YFP fluorescence was visualized using the fluorescein filter #41017, Endow GFP Bandpass Emission Filter (Chroma Technology Corp), chloroplast autofluorescence and mCherry fluorescence was visualized with the U-MSWG rhodamine filter set, while DAPI fluorescence was visualized with the U-MWU filter set (Olympus). Images were taken with the Olympus DP71 camera, using Cell^A (Olympus Soft Imaging Solutions). Experiments were performed in triplicates. Visualization of endogenous YFP-LEFKO fluorescence in embryos was achieved with a Leica SP8X confocal microscope using the 63x lens. The different fluorescent signals were obtained using the following set up, DAPI excitation laser UV/405, detection 415-448nm, YFP excitation laser 514nm, detection 521-555nm, chlorophyll autofluorescence, excitation 583nm, detection 595-680nm. Image cross-sections were analyzed with the Leica LAS X software. Intensity plot profiles of the fluorescent images were generated using ImageJ software package. An 8-pixel wide line was drawn across the cell nuclei and the neighboring plastids and pixel gray values for YFP/Chlorophyll/DAPI signals were calculated with the plot-profile function of ImageJ. Values were normalized after background subtraction, using the ImageJ algorithm (20px). Final merging of images was performed using Adobe Photoshop CS6 (version 13.0) software. Morphometric analysis of primary root elongation was performed by taking digital photographs with a Sony DSC-F707 camera. Images obtained were further analyzed using the ImageJ software package (https://imagej.nih.gov/ij/) and statistically processed. Transmission Electron Microscopy For transmission electron microscopy (TEM), cotyledons of 6-d-old Col-0 and lefko1 seedlings were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, at 4 C for 2–3 h, post-fixed in 1% OsO4 for 2 h, washed in buffer, dehydrated in a series of ethanol, embedded in Spurr epoxy resin and polymerized at 70 C for 36 h. Ultrathin sections were cut with a Reichert OMU-3 ultramicrotome (C. Reichert), stained with uranyl acetate and lead citrate, and examined and photographed with a Zeiss 9S-2 transmission electron microscope (Carl Zeiss). Experiments were performed in triplicates. GUS Staining Seeds and dissected embryos were collected in staining solution (50 mM sodium phosphate buffer pH 7.2, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 2 mM X-gluc), vacuum infiltrated for 5 min and incubated for 2 h at 37 C. Samples were washed twice with 70% ethanol for 30 min and covered with chloral hydrate (2.5 g to 1 ml of 30% glycerol) for several hours. Cleared tissues were mounted in chloral hydrate/glycerol solution under coverslips and photographed using an Olympus BX-50. Experiments were done in triplicates. Immunoblotting Sample preparation for SDS–PAGE was performed by adding Laemmli Sample Buffer. Protein samples were heated at 70 C for 7 min, subjected to 8% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) Immobilon-P membrane (Millipore) by the semi-dry transfer system (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in TBST for 60 min and then incubated with rabbit polyclonal primary antibodies diluted as follows, 1:100 OctA-Probe (D-8), 1:2000 anti-GFP (Santa Cruz Biotechnology), 1:1000 anti-GFP (Cell Signaling Technology) used for immunoblots of Figure S2, 1:3000 anti-D1 (Zhang et al., 2000), 1:3000 anti-CYTF (Alt et al., 1983), 1:10000 anti-CYTB6/F (Alt et al., 1983), 1:3000 anti-LHCP (Adamska et al., 2001) and 1:700 anti-CP (Barbato et al., 1992). Immunoblotting of co-immunoprecipitation experiments performed with mouse monoclonal OctA-Probe (H-5) (Santa Cruz Biotechnology) in 1:500 dilution. After three washes with TBST, the blots were incubated with 1:10000 diluted goat anti-rabbit IgG-HRP or 1:3000 diluted goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnology). Western blot detection was performed with chemiluminescence luminol reagent (Santa Cruz Biotechnology) by exposure to UltraCruz blue autoradiography film. Western blots were performed in triplicates.

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RNA-Seq Total RNA was extracted using the NucleoSpin Plant RNA kit (Macherey-Nagel) with an on-column DNase treatment according to the manufacturer’s instructions from lefko1 and wild-type cotyledons. In addition, total RNA was isolated from globular/heart-stage lefko2 and wild-type embryos. The quantity and quality of the RNA were assessed using a NanoDrop 1000 spectrophotometer and agarose gel electrophoresis. RNA-seq libraries were generated using the TruSeq Low Input kit according to the manufacturer’s instructions (Illumina). Sequencing was performed on BGISEQ-500 platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample. Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (TAIR10). RNA-seq data were analyzed using the SOAPnuke (version v.1.5.2) with parameters ‘‘-l 15 -q 0.2 -n 0.05’’ and the HISAT2 pipeline (version 2.0.4) with parameters ‘‘–phred64 –sensitive –no-discordant –no-mixed -I 1 -X 1000’’. We generated approximately 130 million reads for lefko1 and Col-0 cotyledon samples and 60 million reads for lefko2 and Col-0 embryo samples. On average, about 97% of the clean reads could be unambiguously aligned to the TAIR10 reference genome sequence. Clean reads were mapped to reference Bowtie, and then gene expression level was calculated with RSEM with default parameters. Analysis of differential gene expression was performed with DEseq2 with parameters ‘‘Fold Change R 2.00 and Adjusted Pvalue % 0.05’’. Alternative splicing events were detected for lefko mutants and wild-type using rMATS tool with parameters ‘‘-analysis U -t paired -a 8’’ (Shen et al., 2014) and ARAPORT database (Zhang et al., 2017; Cheng et al., 2017) (http://rnaseq-mats.sourceforge.net/) (Table S2), and visualized using the Integrative Genomics Viewer (IGV) tool (http://software.broadinstitute.org/software/igv/). Alternative splicing events with FDR % 0.05 were defined as significant differentially splicing events. Gene Ontology Analysis Gene ontology (GO) enrichment for the differentially expressed genes (DEG) and the differentially alternative splicing (DAS) genes were performed using the PlantRegMap database (Jin et al., 2017) (http://plantregmap.cbi.pku.edu.cn/index.php). GO categories were classified in terms of Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) GO-terms. Analysis of Intron Retention Events Total RNA was isolated from globular and heart staged embryos and cotyledons using the phenol-sodium dodecyl sulfate (SDS) extraction method. RNA concentrations were determined spectrophotometrically and verified by ethidium bromide staining on agarose gels. DNA was eliminated with RQ1 RNase-free DNase (Promega). Reverse transcription (RT) was performed on 100 ng of total DNA-free RNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA synthesis and RT–PCR analysis for each transcript were performed using gene-specific primers (Table S4). Two biological and at least three technical replicates were performed. The products were analyzed by agarose gel electrophoresis and viewed with ethidium bromide staining. For comparative analysis and normalization, glyceraldehyde 3-phosphate dehydrogenase (GAPDH-At3g04120) was chosen as an endogenous control. In Planta Protein Co-immunoprecipitation Total proteins from Arabidopsis lefko1 seedlings transformed with LEFKOpro::LEFKO-YFP bearing the FLAG epitope, were incubated with 30 ml anti-FLAG M2-agarose affinity gel (Sigma-Aldrich, Darmstadt, Germany) for 2 h at 4 C in IP buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA and Protease Inhibitor Cocktail (Sigma-Aldrich). After four washes with TBS, the co-immunoprecipitated proteins were eluted with 200 ml 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1% (v/v) Triton X-100 and 150 ng/ml 3X FLAG peptide (F4799, Sigma-Aldrich). Ten ml of the elution fractions were used for immunoblotting and the rest were subjected to proteomic analysis. RNA Immunoprecipitation lefko1 seedlings transformed with LEFKOpro::LEFKO-YFP-FLAG plants were washed four times in cold sterile water and fixed with 1% formaldehyde for 15 min under vacuum. Fixation was stopped with 125 mM glycine under vacuum. Fixed seedlings were washed four times in cold water, grounded in IP buffer and incubated with 30 ml anti-FLAG M2-agarose affinity gel (Sigma-Aldrich) for 2 h at 4 C supplemented with RNase Inhibitor (New England Biolabs). Immunoprecipitated samples were washed with 50 mM Tris-HCl pH 7.4, 150 mM NaCl supplemented with RNase Inhibitor. One milliliter buffer per sample was used for all washes; each wash requires 5 min rotation at 4 C. Immune complexes were eluted in 200 mL elution buffer (1% SDS and 0.1 M NaHCO3, RNase Inhibitor). The total 200-mL eluted sample was incubated with 5 mL 0.5 M EDTA, 10 mL 1 M Tris$HCl pH 6.5, and 1 mL 2 mg/mL proteinase K (Sigma) at 45 C for 1 h. Then equal volume of phenol:chloroform was added and the RNA was precipitated in 2.53 volume 100% ethanol at 20 C overnight. Real-Time qRT-PCR Quantitative RT-PCR was performed to determine transcripts enrichment in RNA immunoprecipitations. Purified RNA from RIP experiments was incubated with PrimeScript Reverse Transcriptase (Takara Bio, Shiga, Japan) for 60 min at 42 C to synthesize cDNA using gene specific primers (Table S4). Quantitative PCR reactions were performed in the PikoReal Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green I as the DNA-binding dye provided in SYBR Select Master Mix (Applied Biosystems) and applying the following cycler conditions: 2 min at 50 C, 2 min at 95 C, followed by 40 cycles of 15 s at 95 C, 1 min at 60 C. All quantitative PCR reactions were performed as triplicates of three biological repeats. At the end of each reaction, the cycle threshold (Ct) e5 Developmental Cell 50, 1–13.e1–e7, September 23, 2019

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

was automatically set up at the level that reflected the best kinetic PCR parameters by the PikoReal Software 2.1 and melting curve analysis was performed to monitor primer specificity. The percent input method was applied to estimate the transcript enrichment in the samples. The primers are listed in Table S4. Generation of Recombinant LEFKO Protein for In Vitro Analysis LEFKO was expressed in Escherichia coli ER2523 strain using the pMAL Protein Fusion and Purification System E8200S (NEB) according to manufacturer’s instructions. Briefly, the D(TP)LEFKO was generated by PCR amplification as previously described and cloned into NdeI/SalI of pMAL-c5X expression vector, down-stream from the malE gene which encodes maltose-binding protein (MBP). The DNA sequence of the insert was verified by the appropriate digestions and the MBP-DðTPÞLEFKO fusion protein was expressed by introducing the plasmid into ER2523 cells. Cultures initiated from a single colony were grown overnight at 37 C on LBR medium (1% peptone, 0.5% NaCl, 0.5% yeast extract, 0.2% glucose) with ampicillin and then were transferred in 1:100 dilution to LBR medium without ampicillin. These cultures were grown for another 1-2 h at 37 C to OD600 of 0.5. Protein expression was induced by the addition of 0.3 mM IPTG and incubation was continued for 4 h at 30 C. Cells were harvested, suspended in icecold buffer A (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol, 1X protease Inhibitor Cocktail) and lysed by sonication. The lysate was cleared by centrifugation at 20,000g for 15 min and the MBP-DðTPÞLEFKO protein was purified from the supernatant by affinity chromatography on amylose-coupled agarose resin. The lysate was diluted 1:6 and applied to a 2-ml column equilibrated in buffer A. The column was washed with 5 volumes of buffer A and bound protein was eluted in buffer A containing 10 mM maltose. The eluted protein was concentrated by centrifugation at 10000g for 15 min at 4 C in a Vivaspin-4 column, (Sartorius Stedim Biotech GmbH,), dialyzed in buffer containing 100 mM HEPES pH 7.3, 200 mM KCl, 10 mM MgCl2, 10 mM DTT, 10% glycerol, and used immediately for in vitro RNA-protein binding assay or stored at -20 C supplemented with 50% glycerol. Electrophoretic Mobility Shift Assay-EMSA Intron fragment of approximately 100bp from nuclear At3g48070 was generated by PCR amplification (Table S4) and in vitro transcribed using HiScribe T7 High Yield RNA Synthesis Kit E2040S (NEB). The RNA fragments were 30 labelled with pCp-biotin (NU-1706-BIO, Jena Biosciences GmbH) by the T4 RNA ligase 1 (M0204S, NEB). The labeled RNA fragment was diluted 1:1000 to 100 pM, heated at 95 C for 2 min and incubated with increasing concentrations of in vitro expressed LEFKO protein or MBP in 100 mM HEPES pH 7.3, 200 mM KCl, 10 mM MgCl2, 10 mM DTT, 10% glycerol for 30 min at RT. The reactions were analyzed in 5% non denaturing polyacrylamide gels prepared in 1X TBE buffer and transferred to Parablot Plus nylon membrane (Macherey-Nagel) by the semi-dry transfer system. Membranes were blocked overnight with casein at 4 C and then incubated with streptavidin-HRP (ab7403, Abcam; diluted 1:40000) for 1 h. Detection was performed by enhanced chemiluminescence. Experiments were done in triplicates. Proteomic Analysis The eluted proteins from at least three independent coIP experiments were reduced with 10 mM DTT for 30 min at 56 C and processed according to the filter aided sample preparation (FASP) protocol (Wi
sniewski et al., 2009) using spin filter devices with 10-kDa cutoff VN01H02 (Sartorius,). Specifically, the protein solutions were diluted with 1 ml 8 M urea/100 mM Tris-HCl pH 8.5 and applied serially on top of the filter unit through centrifugation at 14000g. The proteins on the filters were extensively washed (three times) with 200 ml urea solution and centrifugations. Alkylation of protein cysteines was performed using a 10 mg/ml iodoacetamide solution in urea for 30 min in the dark, again on top of the filter. The proteins on the filters were then washed three times with a solution of 50 mM NH4HCO3. Finally, the proteins were digested by adding 1 mg trypsin/LysC mix (mass spec grade, Promega) in 80 ml of 50 mM NH4HCO3 solution and incubated overnight at 37 C. The next day the peptides were eluted by adding 100-ml water and centrifugation at 14000g. The peptides were dried down by speed-vac-assisted solvent removal and reconstituted in a solution of 2% (v/v) ACN and 0.1% (v/v) formic acid. The peptide solution was incubated for 3 min in a sonication water bath. Peptide concentration was determined by NanoDrop absorbance measurement at 280 nm. Then, 2.5 mg peptides were pre-concentrated with a flow of 3 ml/min for 10 min using a C18 trap column (Acclaim PepMap100, 100 mm x 2cm, Thermo Scientific) and then loaded onto a 50 cm long C18 column (75 mm ID, particle size 2 mm, 100A˚, Acclaim PepMap RSLC, Thermo Scientific). The binary pumps of the HPLC (RSLCnano, Thermo Scientific) consisted of Solution A (2% (v/v) ACN in 0.1% (v/v) formic acid) and Solution B (80% (v/v) ACN in 0.1% (v/v) formic acid). The peptides were separated using a linear gradient of 4% B up to 40% B in 210 min with a flow rate of 300 nl/min. The column was placed in an oven operating at 35 C. The eluted peptides were ionized by a nanospray source and detected by an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) operating in a data dependent mode (DDA). Full scan MS spectra were acquired in the Orbitrap (m/z 300–1600) in profile mode with the resolution set to 60,000 at m/z 400 and automatic gain control target at 106 ions. The six most intense ions were sequentially isolated for collision-induced (CID) MS/MS fragmentation and detection in the linear ion trap. Dynamic exclusion was set to 1 min and activated for 90 s. Ions with single charge states were excluded. Lock-mass of m/z 445,120025 was used for continuous internal calibration. Xcalibur (Thermo Scientific) was used to control the system and acquire the raw files. Bioinformatics Phylogenetic tree was created using EnsemblPlants (http://plants.ensembl.org). Multiple sequence alignment of LEFKO homologues was performed using the ClustalX 1.83 software (http://bips.u-strasbg.fr/en/Products/Software). Protein structure prediction was Developmental Cell 50, 1–13.e1–e7, September 23, 2019 e6

Please cite this article in press as: Daras et al., LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants, Developmental Cell (2019), https://doi.org/10.1016/j.devcel.2019.07.024

generated by the I-TASSER application. The resulting PDB files were analyzed by PyMOL (http://www.pymol.org). Subcellular localization predictions were performed using PSORT, Predotar, TargetP, ChloroP, SLP-Local, BaCelLo, SuBloc, CELLO, NUCLEO, Nuc-PLoc and YLoc. Nuclear Localization Signals were predicted using NucPred, cNLS Mapper, NLStradamus and Nuclear Export Signals were predicted using LocNES and NetNES server. Expression profiles of LEFKOTHEA and WTF1 genes in Arabidopsis were retrieved from TRAVA applying default parameters (http://travadb.org). Sequence logos were created using WebLogo (http:// weblogo.berkeley.edu/). QUANTIFICATION AND STATISTICAL ANALYSIS The raw files of protein mass spectrometry were analyzed using MaxQuant software (version 1.5.3.30) (Tyanova et al., 2016a) against the complete UniProt proteome of Arabidopsis thaliana (version of 19/1/2016) and a common contaminants database by the Andromeda search engine. Search parameters were strict trypsin specificity, allowing up to two missed cleavages. Oxidation of methionines, deamidation of glutamine and asparagine and N-terminal acetylation were set as variable modifications. Protein abundance was calculated on the basis of the normalized spectral protein intensity as label free quantitation (LFQ intensity) enabling the ‘‘match between runs’’ option. LFQ was performed with a minimum ratio count of 1. The statistical analysis was performed using Perseus (Tyanova et al., 2016b) (version 1.5.3.2). Proteins identified as contaminants, ‘‘reverse’’ and ‘‘only identified by site’’ were filtered out and positive/negative replicates were grouped. The LFQ intensities were transformed to logarithmic (log2(x)) and the zero intensities were imputed - replaced by normal distribution, assuming that the corresponding protein is low abundant in the sample (imputation criteria: width 0.1 and down shift 1.8). Volcano plots were generated representing the statistical test results based on the two-sided Student’s t tests that were performed comparing the LEFKO-specific pull-down versus the nonspecific pull-down, using p value for truncation. Statistical analysis of photosynthetic pigments and primary root length was assessed by using three biological replicates of at least 30 and 50 seedlings each, respectively. Statistical significance of the nucleus/cytoplasm YFP-LEFKO localization (n = 100 cells) was assessed using t test p < 0.001. No sample-size calculation was performed because most of the plant samples are from mixed multiple seedlings. The sample sizes are sufficient for conclusions because p values are under threshold in statistical tests and effect sizes with >1.5 fold changes. Wild-type and mutant plants were randomly selected from Petri-dishes. DATA AND CODE AVAILABILITY The accession numbers for the Next-generation RNA sequencing raw and processed data reported in this paper are GEO: GSE94576 (lefko2) and GEO: GSE106459 (lefko1). The accession number for mass spectrometry data reported in this paper is PRIDE: PXD009965.

e7 Developmental Cell 50, 1–13.e1–e7, September 23, 2019