
==== Front
Plant Physiol
Plant Physiol
plphys
Plant Physiology
0032-0889
1532-2548
Oxford University Press US

38709497
10.1093/plphys/kiae256
kiae256
Research Article
Cell Biology
AcademicSubjects/SCI01270
AcademicSubjects/SCI01280
AcademicSubjects/SCI02286
AcademicSubjects/SCI02287
AcademicSubjects/SCI02288
Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas
https://orcid.org/0000-0002-2366-7153
Sun Yi Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6

https://orcid.org/0000-0002-8737-3170
Bakhtiari Shiva Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6

https://orcid.org/0000-0001-8470-3739
Valente-Paterno Melissa Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 0C7

https://orcid.org/0009-0006-2226-5757
Wu Yanxia Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6

Nishimura Yoshiki Laboratory of Plant Molecular Genetics, Department of Botany, Graduate School of Sciences, Koyoto University, Oiwake-cho, Kita-Shirakawa, Kyoto-shi 606-8502, Japan

https://orcid.org/0009-0007-3354-9023
Shen Weike School of Life Sciences, Institute of Life Sciences and Green Development, Hebei University, Baoding, Hebei 071002, China

https://orcid.org/0000-0002-6946-8029
Law Christopher Centre for Microscopy and Cell Imaging, Concordia University, Montreal, Quebec, Canada, H4B 1R6

https://orcid.org/0000-0001-8851-7562
Dhaliwal James Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6

https://orcid.org/0000-0002-9973-0446
Dai Daniel Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 0C7

https://orcid.org/0000-0003-2814-9889
Bui Khanh Huy Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 0C7

https://orcid.org/0000-0001-7227-2011
Zerges William Department of Biology, Concordia University, Montreal, Quebec, Canada, H4B 1R6

Author for correspondence: william.zerges@concordia.ca
Present address: Department of Biology, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
Yi Sun and Shiva Bakhtiari contributed equally to this work.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is William Zerges (william.zerges@concordia.ca).

Conflict of interest statement. None declared.

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Abstract

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

The expression of chloroplast proteins occurs in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

Natural Sciences and Engineering Research Council of Canada Discovery 10.13039/501100000038 217566 Japan Society for the Promotion of Science 10.13039/501100001691 22K19329 Scientific Research 21H02504 Transformative Research Areas 24H02275 Canadian Institutes of Health Research 10.13039/501100000024 PJT-156354
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pmcIntroduction

Translation can be localized in cells to direct the polypeptide product to the proper intracellular location, insert it into a membrane, or assemble it into a multisubunit complex (Schwarz and Beck 2019; Das et al. 2021). For example, cytosolic ribosomes dock on the endoplasmic reticulum (ER) and synthesize polypeptides for cotranslational import or integration into the ER membrane. Mitochondria are bound by cytosolic ribosomes synthesizing primarily inner membrane proteins, some of which undergo cotranslational import (Claros et al. 1995; Williams et al. 2014). Chloroplast protein synthesis, however, is believed to occur at random cytoplasmic locations prior to posttranslational import of the polypeptide products (Jarvis and Lopez-Juez 2013; Weis et al. 2013). This long-standing model is based on the ability of purified chloroplasts to posttranslationally import proteins and transmission electron microscopy (TEM) images of chloroplasts lacking arrays of bound cytosolic ribosomes like those on the rough ER and mitochondria (Chua and Schmidt 1978; Highfield and Ellis 1978; Carde et al. 1982; Smeekens et al. 1986). However, the possibility that at least some chloroplast proteins are synthesized at the chloroplast envelope was raised by microscopy images of a neighboring cytosolic region enriched in cytosolic ribosomes and mRNAs encoding chloroplast-localized light-harvesting complex (LHC) proteins in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii) and cytosolic ribosomal proteins in the proteome of the chloroplast envelope of Arabidopsis (Arabidopsis thaliana; Colon-Ramos et al. 2003; Uniacke and Zerges 2009; Bouchnak et al. 2019).

Chloroplasts have bacterial-type 70S ribosomes to synthesize the proteins that are encoded by the small genome in this semiautonomous organelle (Weis et al. 2013; Das et al. 2021). Some chloroplast ribosomes are bound to thylakoid membranes, into which their nascent polypeptides integrate cotranslationally and then assemble into the complexes of the photosynthetic electron transport systems (Margulies and Michaels 1974; Yamamoto et al. 1981; Zoschke and Barkan 2015; Ries et al. 2020; Trösch et al. 2022). Translation by chloroplast ribosomes for the biogenesis of photosystems I and II (PSI and PSII) is localized to the translation zone (T-zone) in the chloroplast of Chlamydomonas (Fig. 1A; Uniacke and Zerges 2007, 2009; Schottkowski et al. 2012; Sun et al. 2019). The T-zone is adjacent to the ribosome-rich cytoplasmic region (above) and envelope domains enriched in markers for the TOC and TIC translocons in the outer and inner envelope membranes of the chloroplast, which import chloroplast proteins from the cytoplasm (Schottkowski et al. 2012). These results suggest a spatial coordination of the distinct translation systems of the cytoplasm and chloroplast. Localized transcription of the chloroplast genome has been suggested by reports that nucleoids are near the pyrenoid in Chlamydomonas and bound to the chloroplast envelope in plants (Ris and Plaut 1962; Pfalz and Pfannschmidt 2013). Transcription and translation might be colocalized in chloroplasts based on evidence that they are coupled (Zoschke and Bock 2018; Zhang et al. 2023).

Figure 1. Cytosolic ribosomes are bound to the chloroplast. A) An illustration of a Chlamydomonas cell shows locations of the nucleus (N), cytoplasm (cyto), chloroplast with its lobes, pyrenoid (P), and basal region (BR). B) Immunoblot analyses of extracts of whole cells versus of purified chloroplasts revealed that cytosolic ribosomes (uL3) copurified with chloroplasts (AtpB). Controls are for contamination by organelles that bind cytosolic ribosomes: ER (BIP) and mitochondria (AOX1). Immunoblots are in Supplementary Fig. S1A (error bars = 1.0 SEM, n = 3 biological replicates from independent cultures, *P < 0.01, 2-tailed Student’s t tests). C) Heat maps show average immunoflourescence (IF) signals of uL3, AOX1, and BIP in projections of all cells or chloroplasts in representative data sets (uL3, n = 32 chloroplasts or n = 102 cells; AOX1, n = 22 chloroplasts; BIP, n = 48 chloroplasts). Size bars are not provided in C) because each projection is generated from Z stacks of individual cells or chloroplasts whose size had been scaled to a common subjective area to prevent loss of resolution. Cells and chloroplasts are oriented as illustrated in A), with anterior and posterior cell poles on the left and right, respectively. The projections of isolated chloroplasts include only the posterior basal region because the anterior lobes and central nuclear-cytoplasmic region were nonstaining and, therefore, not identified by the macro. D) IF microscopy images of purified chloroplasts show uL3 localized to a domain of their envelope (LCIA). C and D) Asterisks mark approximate position of the translation envelope domain on the longitudinal cell axis. D) “L”, anterior lobe region; “BR”, posterior basal region. The absence of LCIA signal from the lobes does not reflect a change in chloroplast morphology during isolation (Supplementary Fig. S2A; BF, bright field; size bar, 5.0 µm, applicable to all micrographs). E) The percent maximal intensities of the uL3 and LCIA IF signals are plotted along a horizontal line (not shown) drawn through the chloroplast in D).

Here, we demonstrate that cytosolic ribosomes translate on the chloroplast envelope in Chlamydomonas. Chloroplasts that had been isolated from free cytosolic ribosomes and most other cellular material retained bound both cytosolic ribosomes and mRNAs encoding chloroplast proteins on the chloroplast envelope of the basal region, primarily on a domain closest to the nucleus. Images from high-resolution electron tomography show cytosolic ribosomes on the chloroplast envelope, particularly on this domain. Translational activity of these chloroplast-bound ribosomes is supported by results of 2 assays. Roles of translation on the chloroplast envelope in directing newly synthesized proteins to discrete assembly locations within the chloroplast are suggested by differences in the distributions of mRNAs encoding subunits of photosynthesis complexes located in different chloroplast subcompartments. Finally, the localization of transcription of the chloroplast genome at the T-zone is supported by a bias in the distribution of a subpopulation of transcriptionally active chloroplast nucleoids. We propose that a spatial alignment of translation by cytosolic ribosomes on the chloroplast envelope and transcription and translation within the chloroplast facilitates the biogenesis of the photosynthetic complexes.

Results

Cytosolic ribosomes are bound to the chloroplast envelope

The possibility of localized translation on the chloroplast surface in Chlamydomonas was suggested by our finding that chloroplasts isolated from most cytoplasmic material retained more of the pool of the large ribosomal subunit protein uL3 (uL3), our marker for cytosolic ribosomes, than can be explained by contamination by known ribosome-bound organelles, ER (luminal binding protein [BIP]) and mitochondria (alternative oxidase 1 [AOX1]; Fig. 1B; Supplementary Fig. S1A). To determine whether these ribosomes were bound to the isolated chloroplasts, we looked for uL3 on their outer surface by immunoflourescence (IF) microscopy. We IF stained chloroplasts isolated from cells actively undergoing chloroplast biogenesis for uL3 and co-IF stained the chloroplast envelope for low-CO2 inducible protein LCIA (Materials and methods; Yamano et al. 2015). Although the entire envelope was IF stained for LCIA previously (Yamano et al. 2015), we did not detect LCIA on the anterior lobes of chloroplasts, possibly reflecting different CO2 concentrations of the respective culture conditions. This does not reflect a change in chloroplast morphology during the isolation procedures (Supplementary Fig. S2A).

Isolated chloroplasts showed uL3 on their surface with the highest signal intensity along a domain of the envelope of the basal region facing the nucleus (Fig. 1, D and E). The reproducibility of the enrichment of cytosolic ribosomes on this envelope domain was confirmed by the average uL3 IF signal distribution on all chloroplast images (Fig. 1C). In addition, individual chloroplasts showed weaker uL3 signal around the rest of the chloroplast, i.e. the entire basal region and the lobes (Fig. 1D). The uL3 on these chloroplasts was not of cytosolic ribosomes on contaminating ER or mitochondria because marker proteins for these organelles were not seen in this pattern (Fig. 1C; Supplementary Fig. S2, B and C). These results suggested that cytosolic ribosomes are physically associated with the chloroplast and, predominantly, on a discrete region termed here the “translation envelope domain” (Fig. 1A).

In cells, any uL3 enrichment on the chloroplast was less evident than it was on isolated chloroplasts (above), due to strong IF signal from throughout the central cytoplasmic region and our inability to distinguish bound and nonbound ribosomes (Fig. 1C; Supplementary Fig. S3A). However, in projections showing the distribution of average uL3 IF signal from all cells, one can see the ribosome-rich cytosolic regions reported previously, which likely includes ribosomes on the translation envelope domain (Colon-Ramos et al. 2003; Uniacke and Zerges 2009).

Imaging cytosolic ribosomes on the translation envelope domain by high-resolution electron tomography

The evidence against chloroplast-localized translation includes TEM images showing a ribosome-free zone around chloroplasts in spinach leaf cells (Chepko et al. 1979; Carde et al. 1982). To address this contradiction of our IF microscopy imaging of the cytosolic ribosomal marker protein (Fig. 1), we asked whether cytosolic ribosomes can be visualized on the translation envelope domain in images from 3D high-resolution electron tomography. We scored ribosomes that were within 5 nm from the chloroplast envelope (Supplementary Table S2). We did not consider others because ribosomes are abundant throughout the cytosol. The chloroplast envelope on the side of the lobe facing the cell wall is largely bare of ribosomes (Fig. 2F). By contrast, ribosomes in the highest density and in clusters can be seen on the translation envelope domain and in moderate density on the inward facing envelope of the lobe, i.e. on the side of the nucleus (Fig. 2; Supplementary Videos S1 and S2). These results validate the uL3 IF signal on isolated chloroplasts as being from cytosolic ribosomes and suggest that the translation envelope domain extends from the basal region partially along the posterior-most lobe surface, facing the nucleus (Fig. 2, E and F, illustrated in Fig. 1A). Although a moderate ribosome density was seen on the anterior end of the lobe in the TEM analyses (Fig. 2E), the uL3 IF signal was rarely enriched there on isolated chloroplasts (Fig. 1, C and D). Thus, additional work is required to address any relevance of the ribosomes there. In summary, these results detract from a ribosome-free region around the chloroplast and corroborate the evidence of chloroplast-bound ribosomes from IF microscopy.

Figure 2. Electron tomograms show cytosolic ribosomes on the outer membrane of the chloroplast envelope. A) The illustration shows the regions acquired for the tomographs in B to F). B) A tomographic slice showing the region of chloroplast envelope bound by cytosolic ribosomes as seen by IF microscopy (Fig. 1D). C) In the same image in B) dots mark the cytosolic ribosomes that are within 5 nm of the envelope. D to F) Models of chloroplast envelope and bound cytosolic ribosomes (dots) as seen from the angles shown in A). Cytosolic ribosome densities on 5 regions are indicated. D) Part of the translation envelope domain (on the right) and the posterior half of the inner surface of a lobe (on the left). E) The nuclear-cytoplasmic (inner) surface and end of the lobe are modeled. Ribosome densities indicated for the anterior and posterior halves. F) The outside surface of the lobe. Derivations of cytosolic ribosome densities are listed in Supplementary Table S2 (see also Supplementary Videos S1 and S2). Scale bars, 200 nm.

Cytosolic ribosomes on the chloroplast envelope are active

We used 2 methods to determine whether the cytosolic ribosomes on the chloroplast envelope are translationally active. The ribopuromycylation (RPM) assay epitope-tags nascent polypeptides by conjugation to puromycin when this drug induces premature translation termination (Bastide et al. 2018). The resulting tagged-nascent polypeptides are used as markers to visualize the location(s) of translation with IF microscopy, using an antibody against puromycin (Bastide et al. 2018). To prevent displacement of puromycin tagged-nascent polypeptides from the location of their synthesis (Enam et al. 2020), we puromycin-treated isolated chloroplasts under conditions that preclude translation and protein import (Ramundo et al. 2020).

On isolated chloroplasts, the IF signal of puromycin-nascent polypeptides was seen predominately from the translation envelope domain, which was strongly IF staining for uL3 (Fig. 3, A and B). This was confirmed by a heat map of the average signal from all chloroplasts in the data (Supplementary Fig. S2E). Weaker puromycin IF signal was seen on the basal region perimeter and from lobes. A minority of isolated chloroplasts showed IF signal from puromycin-nascent polypeptides from 1 or 2 lobes, in addition to the translation envelope domain (Fig. 3C). The puromycin-conjugated nascent polypeptides were on the cytoplasmic side of the chloroplast envelope, as seen in the image of an individual chloroplast and in plots of the average intensities of the IF signals from the puromycin-conjugated nascent polypeptides and the envelope (LCIA) along lines drawn across the translation envelope domains of all chloroplasts of the data set (Fig. 3, C and D). Therefore, the nascent polypeptides were from cytosolic ribosomes and not chloroplast ribosomes. These results provide evidence that the chloroplast-bound cytosolic ribosomes are translationally active.

Figure 3. The chloroplast-associated cytosolic ribosomes are translationally active and tethered by their nascent polypeptides and nonionic bonds. Results of the RPM assay show IF signal of the puromycin-conjugated nascent polypeptides (Puromycin) localized A and B) at the translation envelope domain (uL3). Arrows indicate sites of colocalization of puromycin-conjugated nascent polypeptides and cytosolic ribosomes. A and C) Asterisks indicate the approximate position of the translation envelope domain on the longitudinal axis (size bars 5.0 µm). B) Percent maximal intensities of the uL3 and puromycin IF signals were plotted along a horizontal line (not shown) drawn across the center of the chloroplast in A). C) An example chloroplast shows the IF signal of puromycin-conjugated nascent polypeptides on the cytosolic (left) side of the chloroplast envelope, LCIA. A minority of chloroplasts show strong puromycin IF signal also from their lobes, as seen in this example. D) The average IF signal intensities from puromycin and LCIA (as percent maximal value for each) were plotted along across the translation envelope domain. For each chloroplast, the peak of LCIA IF signal intensity on the x axis was set to 0 to reveal, relative to the envelope, the distribution of the average puromycin-nascent polypeptide signal across all chloroplasts analyzed. Error zone indicates 1.0 Sd from mean. Significance of the distances between the positions of the puromycin and LCIA maximal values from these 30 chloroplasts on the x axis was demonstrated with a 1-sample t test with the null hypothesis = 0. A to D) The puromycin IF signal is specific (Supplementary Fig. S2D). E) Bar heights indicate the average percent of chloroplast-bound cytosolic ribosomes (uL3) that were released by each treatment (+/−, with/without) determined by immunoblot analysis of 3 biological replicates from independent cultures performed in parallel (Supplementary Fig. S1B). *P < 0.01, 2-tailed Student’s t tests. The average amount of uL3 released by high ionic strength and puromycin is designated as 100%. High ionic strength was 750 mm KCl. Error bars indicate 1.0 SEM.

As a second assay for translational activity of the ribosomes bound to the chloroplast, we used the puromycin release assay (Redman and Sabatini 1966; Kellems et al. 1974). Puromycin releases translating ribosomes from their nascent polypeptides. Therefore, ribosomes that are released by puromycin from a membrane were translating in vivo and tethered to the membrane by their nascent polypeptides engaged in the import translocon(s) (Adelman et al. 1973; Kellems et al. 1974). Ribosome release from purified ER and mitochondria also required high ionic strength (300 to 750 mm KCl) to break noncovalent bonds with receptors on the membrane. Hence, to characterize the associations of ribosomes with the chloroplast, we assayed ribosome release from isolated chloroplasts during treatments with puromycin, high ionic strength, or both. When chloroplasts were treated with both puromycin and high ionic strength (750 mm KCl), most of their ribosomes were released (Fig. 3E; Supplementary Fig. S1B). This proportion represents ribosomes that were bound to the chloroplast. Isolated chloroplasts exposed to high ionic strength in the absence of puromycin released 64% of the chloroplast-bound ribosomes. These ribosomes were bound to the chloroplast by noncovalent bonds alone. The difference between ribosomes released with versus without puromycin revealed a significant value of 36% (P = 0.037, t test). Therefore, at least this proportion of the chloroplast-bound cytosolic ribosomes was translationally active in vivo and tethered by their nascent polypeptides. Treatment with puromycin alone had no effect (P = 0.603, t test), revealing that few or no ribosomes were associated with the chloroplast by their nascent polypeptides alone. These results provide additional evidence that the cytosolic ribosomes on the chloroplast are translationally active in vivo and are bound both by noncovalent bonds and tethered by their nascent polypeptides.

The chloroplast is bound by mRNAs encoding chloroplast-localized proteins

Our proposal that the translation envelope domain is a platform for chloroplast protein synthesis predicts that it is associated with cytoplasmic mRNAs encoding specifically chloroplast proteins. We tested this prediction using FISH (Tsanov et al. 2016). According to the rationale described above, we first asked whether chloroplasts retain nuclear-cytoplasmic mRNAs encoding light-harvesting complex II (LHCPB) proteins, which are integral membrane proteins in a complex associated with PS II (Stauber et al. 2003; Nelson and Ben-Shem 2004; Dall'Osto et al. 2015). The FISH probes were complementary to the mRNAs of LHCBM2 (Cre12.g548400) and LHCBM7 (Cre12.g548950), and thus, their signal is named here “LHCBM2/7” (Supplementary Table S1). In cells, the LHCBM2/7 mRNA FISH signal was throughout the cytoplasm and enriched adjacent to the chloroplast, as was reported previously (Fig. 4E; Supplementary Fig. S3B; Uniacke and Zerges 2009). Here, however, it was not possible to determine whether any LHCBM2/7 FISH signal was from mRNAs that were chloroplast associated versus in the neighboring cytosol. However, chloroplast association of most LHCBM2/7 mRNAs was revealed upon cell breakage and the release of nonassociated material. The resulting free chloroplasts retained 96% of the cellular LHCBM2/7 FISH signal, which was primarily near the center of the translation envelope domain (Fig. 4, A and B). This was confirmed by a heat map of the average LHCBM2/7 mRNA FISH signal (Fig. 4E). This localized LHCBM2/7 mRNA FISH signal was surrounded by the cytosolic ribosome (uL3) IF signal (Fig. 4B). These results support the synthesis of LHCPs by cytosolic ribosomes on the translation envelope domain.

Figure 4. Results of FISH reveal chloroplast-bound mRNAs encoding specifically chloroplast-localized proteins. A) Bar heights represent the percent of the average FISH signal intensity retained by chloroplasts for each of the mRNAs indicated below the graph (error bars = 1.0 SEM). P-values are from 2-tailed Student’s t tests comparing n ≥ 3 biological replicates using independent cultures. In each replicate, n ≥ 30 cells or chloroplasts. *P < 0.01. B to D) Chloroplasts IF stained for cytosolic ribosomes (uL3) and FISH probed for cytoplasmic mRNAs encoding chloroplast-localized B) LHCBM2 and LHCBM 7 and C) RBCS1 and RBCS2. D) The FISH signal from TUB2 mRNA, encoding a cytoplasmic protein, serves as a control for specificity of chloroplast localization of mRNAs encoding chloroplast proteins. Size bar, 5.0 µm, is applicable to all micrographs in B to D). E) Heat maps show average FISH signals in intensity projections of image stacks from all cells or chloroplasts in each data set (n ≥ 30 cells or chloroplasts per data set). An asterisk indicates the approximate position of the translation envelope domain on the longitudinal cell axis for each. B to E) Cells and chloroplasts are oriented as the cell illustrated in Fig. 1A. D and E) The brightness of the TUB2 control FISH signal was enhanced to show its contrasting distribution with those of the much stronger RBCS1/2 and LHCBM2/7 FISH signals.

We also imaged the mRNAs of RBCS1 and RBCS2, paralogous nuclear genes encoding the small subunit of Rubisco, an enzyme located primarily in the pyrenoid of the chloroplast (Cre02.g120100 and Cre02.g120150, respectively; Goldschmidt-Clermont and Rahire 1986). (The location of the pyrenoid is illustrated in Fig. 1A.) We refer to the RBCS1 and RBCS2 mRNAs as “RBCS1/2” because our FISH probes are complementary to both (Supplementary Table S1). In most cells, the RBCS1/2 FISH signal was in a pattern consistent with the cytoplasm but otherwise not localized, as was reported previously (Supplementary Fig. S3C; Uniacke and Zerges 2009). However, the average RBCS1/2 FISH signal from all cells showed an enrichment in the approximate location of the translation envelope domain, suggesting that some RBCS1/2 mRNAs are localized there (Fig. 4E). Indeed, chloroplasts retained 80% of the cellular RBCS1/2 mRNA FISH signal, which was strongest at the center of the translation envelope domain (Fig. 4, A and C). The average RBCS1/2 mRNA signal from all chloroplasts confirmed this pattern (Fig. 4E). Furthermore, this average signal revealed an additional feature; weaker RBCS1/2 mRNA signal distributed around the perimeter of the entire basal region of the chloroplast. Minor proportions of the signals from cytosolic ribosomes (uL3) and puromycin-conjugated nascent polypeptides were also seen in this additional pattern (Figs. 1, C and D, and 3, A to D). Thus, intensity projections revealed 2 features of RBCS1/2 FISH signal that were not evident in images of individual cells or chloroplasts (Fig. 4E): enrichment at the translation envelope domain in cells and, on chloroplasts, the weaker signal around the entire basal region. These results support RBCS1/2 translation around the entire basal region, with the highest levels on the translation envelope domain.

To address whether specifically mRNAs encoding chloroplast proteins are associated with the chloroplast, we visualized the FISH signal of the TUB2 mRNA encoding β2-tubulin, a protein of the cytoplasm and cilia (Cre12.g549550; Silflow and Rosenbaum 1981). In cells, the TUB2 FISH signal was dispersed and in a cytosolic pattern, as was reported previously (Fig. 4E; Supplementary Fig. S3D; Colon-Ramos et al. 2003). Chloroplasts retained only 2% of the cellular TUB2 mRNA signal, which was not from the translation envelope domain (Fig. 4, A, D, and E). Therefore, this result supports the chloroplast-localized synthesis of specifically chloroplast proteins.

A chloroplast nucleoid subpopulation is transcriptionally active and near the T-zone

Finally, we asked whether transcription of the chloroplast genome is localized within the chloroplast. In the Chlamydomonas chloroplast, 80 to 100 copies of the chloroplast genome are organized in 5 to 10 nucleoids (Nishimura 2023). We used Click-iT RNA imaging to label newly transcribed RNA in vivo with 5-ethynyl uridine (EU), which was used as an epitope-tagged marker for the location of transcription in IF microscopy images (Jao and Salic 2008). The EU IF signal was compared to the locations of chloroplast nucleoids, which were stained by DAPI (Fig. 5A). Speckle-like clusters of EU IF signal partially overlapped with 39.2% of the 51 nucleoids analyzed. These nucleoids were likely transcriptionally active, whereas the non-EU staining nucleoids were less active or inactive. Interestingly, the active nucleoids were within or near the T-zone (Fig. 5B). These results reveal that a minority of chloroplast nucleoids are transcriptionally active and located in the vicinity of translating ribosomes in the T-zone and on the translation envelope domain.

Figure 5. Transcriptionally active nucleoids are localized in and near the T-zone. Click-iT RNA imaging of newly synthesized RNAs in cells entrained to a 12-h light/12-h dark regime and taken 4 h into the light phase (at ZT4). A) Fluorescence microscopy images of 4 cells. The IF signal from EU label of newly synthesized RNA (white arrow). Chloroplast nucleoids are the DAPI-stained bodies that do not correspond to the nucleus (N; blue, black arrow). Dotted white lines outline each cell imaged. B) Illustrations of a cell show the relative intracellular locations of the 51 nucleoids in the 9 cells analyzed. For each cell, a central axis was established between the anterior and posterior cell poles. For each chloroplast nucleoid, the distance from the center of the nucleus (denoted as “r”) and the angle from the central axis (denoted as “θ”) were measured using Fiji and relative intracellular locations (r′/r cos θ, r′/r sin θ) were calculated. To display cells differing in size, axes are relative distances from the center of the nucleus normalized to the distance between the center of nucleus to anterior cell pole in each cell.

Discussion

While temporal coordination of the expression of nuclear and chloroplast genes encoding chloroplast proteins has been described, little is known of any spatial coordination (Choquet and Wollman 2009; Sun and Zerges 2015; Rochaix 2023). Our results contribute to a revelation that cytosolic ribosomes and chloroplast ribosomes translate in closely aligned subcompartments on their respective sides of a domain of the chloroplast envelope. We propose that this organization facilitates the localization of newly synthesized subunits to the T-zone for the assembly of the photosynthesis complexes. For example, LHCB2/7 mRNA translation on the translation envelope domain could facilitate the import of newly synthesized LHCPs to the T-zone for assembly with the newly synthesized chloroplast genome-encoded PSII subunits to form the PSII-LHCII supercomplex. Heat map images of the average FISH signals revealed a distinction between the distributions of the RBCS1/2 and LHCB2/7 mRNAs on the chloroplasts (Fig. 4E). While both FISH signals were strongest at the translation envelope domain, weaker RBCS1/2 mRNA signal was also seen around the perimeter of the chloroplast basal region (Fig. 4E). The latter pattern was not seen for the LHCB2/7 mRNAs. Within the chloroplast, the rbcL mRNA is translated in the pyrenoid periphery (Uniacke and Zerges 2009). Hence, we speculate that RBCS1/2 translation occurs on the entire basal region to facilitate the localization of newly synthesized RBCS to the pyrenoid for localized assembly with RBCL and the subsequent localization of newly assembled Rubisco to the pyrenoid where it functions in photosynthesis (Barrett et al. 2021). These results also point to an open question regarding the roles of the complex and stereotypic morphology of the Chlamydomonas chloroplast (Fig. 1A) in the compartmentalization of biogenic and other organellar processes.

Our results suggest a revision of the long-standing model in which chloroplast proteins are synthesized at random locations throughout the cytoplasm and imported posttranslationally. Our finding that the cytosolic ribosomes are likely tethered to the chloroplast by their nascent polypeptides raises the possibility of cotranslational chloroplast protein import (Redman and Sabatini 1966; Chua et al. 1973; Kellems et al. 1974). Chloroplast-localized translation and cotranslational import of LHCPs would be consistent with 2 general principles. First, the cotranslational role of the signal recognition particle (SRP) in all systems studied to date, to our knowledge, contrasts the proposed posttranslational roles of SRP subunit homologs in chloroplasts (Claros et al. 1995; Ott and Herrmann 2010; Williams et al. 2014; Ziehe et al. 2018; Sun et al. 2019). Second, multipass integral membrane proteins, except LHCPs, require cotranslational membrane integration to prevent their misfolding and aggregation of their hydrophobic trans-membrane domains in the aqueous cytoplasm (Ries et al. 2020; Ott and Herrmann 2010; Aviram and Schuldiner 2017). RBCS1/2 and LHCB2/7 nascent polypeptides emerging from a ribosome on the chloroplast envelope probably have sufficient length to reach the stroma and a thylakoid membrane, respectively, during cotranslational targeting (Materials and methods; Engel et al. 2015; Staehelin and Paolillo 2020). This also would hold for most chloroplast proteins because their average size (32.5 kb) is larger than those of RBCS (15 kDa) and LHCB2/7 (22 kDa; Khrebtukova and Spreitzer 1996; Ferrante et al. 2012; Mohanta et al. 2022). Our results, and the proximity of the translation envelope domain to the membranes of the photosystem biogenesis in the T-zone (Sun et al. 2019), provide a context for testing the hypothesis that LHC subunits undergo cotranslational import and thylakoid membrane insertion.

We extend the role of the T-zone to the transcription of the chloroplast genome with our finding therein of a subpopulation of transcriptionally active nucleoids (Fig. 5; Zones et al. 2015; Strenkert et al. 2019; Sun et al. 2019). Localized transcription could be a mechanism to direct newly synthesized mRNAs to the T-zone. It might reflect a coupling of transcription and translation in chloroplasts (Zoschke and Bock 2018; Zhang et al. 2023). Finally, it might result from the activation of transcription in the nucleoids that are closest to the localized import of transcription factors by translocons in envelope regions adjacent to the T-zone (Schottkowski et al. 2012).

Organelles and bacteria contain diverse compartments dedicated to specific processes, including membrane domains for protein synthesis and targeting (Hoffman et al. 2019; Iovine et al. 2021; Lithgow et al. 2023). For example, the biogenesis of the inner membrane of mitochondria involves a spatial coordination of the cytoplasmic and organellar translation systems. Like the T-zone, domains of the mitochondrial inner membrane at cristae junctions compartmentalize translation by mitochondrial ribosomes for the biogenesis of the respiratory electron transport system and ATP synthase (Watson 1972; Vogel et al. 2006; Zorkau et al. 2021). Like the translation envelope domain, domains of the outer mitochondrial membrane are bound by translating cytosolic ribosomes, enriched in the protein import translocons, and aligned with localized translation by mitochondrial ribosomes at cristae junctions (Vogel et al. 2006; Garcia et al. 2007; Gold et al. 2017; Stoldt et al. 2018). Cotranslational import of inner membrane proteins into mitochondria has been demonstrated and is hypothesized to facilitate their membrane insertion and assembly to form the respiratory complexes and ATP synthase (Garcia et al. 2007; Fox 2012; Williams et al. 2014; Formosa and Ryan 2018). The initial evidence that cytosolic ribosomes bind receptors on mitochondria included a requirement for high ionic strength to dissociate them (Kiebler et al. 1990; Szyrach 2003; Lesnik et al. 2015; Bykov et al. 2020). Our similar results raise this possibility for cytosolic ribosome association with chloroplasts (Fig. 3E). Thus, the spatial coordination of the cytoplasmic and organellar translation systems appears to be a general principle in the biogenesis of the semiautonomous organelles.

Materials and methods

Strains and culture conditions

For chloroplast purification, the Chlamydomonas strain CC-400 (CW15, cell wall defective) was cultured on high salt minimal medium (Harris 1989) containing 1.0% (w/v) sorbitol, at 24 °C, with stirring by magnetic bar and illuminated by 2 circular fluorescent bulbs 40 μE/cm2 from all lateral directions to a density of 2 to 3 × 106 cells/mL. For electron tomography and Click-iT imaging, cultures of the wild-type strain CC-125 were entrained to a 12-h light:12-h dark cycle as described previously (Sun et al. 2019). Cells were harvested at the fourth hour of the final light phase (ZT4) by centrifugation (3,000 × g, 5 min at room temperature (RT)).

Chloroplast isolation was performed as described previously with the following modifications (Mason et al. 2006; Zhan et al. 2018). Cell pellets were resuspended to 1 × 108 cells/mL in isolation buffer (IB; 300 mm sorbitol, 50 mm HEPES-KOH pH 7.5, 25 mm MgCl2, and 0.1% [w/v] BSA). Saponin (Sigma, # 47036) freshly dissolved in IB (10% w/v) was added to 0.4% (w/v), followed by incubation at 22 °C for 10 min with gentle agitation. The resuspension was passed twice through a 27-gauge needle at 0.1 mL/s. Cells and chloroplasts were collected by centrifugation at 750 × g for 2 min at 4 °C, and the pellet was resuspended in IB. This suspension was used when chloroplasts were analyzed by FISH. Percent mRNA retention in Fig. 4A was the ratio of the average of the signals for an mRNA from chloroplasts versus whole cells in the same images. Chloroplasts and unbroken cells were examined together by FISH analyses because chloroplast isolation was unnecessary, and this allowed direct comparisons of signals from chloroplasts versus cells processed together on the same slide. For the IF staining without co-FISH and the puromycin release assays, chloroplasts were purified on a discontinuous Percoll gradient as described previously (Mason et al. 2006). The material (chloroplasts) at the 45% to 65% interface was collected, diluted with 4 vol of ice-cold IB, pelleted by centrifugation (670 × g, 1 min, 4 °C), and resuspended according to the downstream use (below).

Immunoblot analyses

Proteins were transferred to a membrane of PVDF (BIO-RAD) or, for AOX1 detection, nitrocellulose (BIO-RAD) and reacted with primary and secondary antibodies as described previously (Sambrook and Russell 2001; Supplementary Fig. S1A). The primary antibodies were α-BIP (Santa Cruz sc-33757; 1:150), α-AOX1 (Agrisera AS06 152, 1:150,000), α-uL3 (1:6,000; Fleming et al. 1987), and α-AtpB (1:6,000). uL3 was called cyL4 in an outdated nomenclature. The polyclonal antibody against it was raised previously against this protein from highly purified 60S ribosome subunits from Chlamydomonas (Fleming et al. 1987; Manuell et al. 2005; Scarpin et al. 2023). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (KPL). Signals were detected using an ECL substrate (Thermo Fisher) with an Imager 600 (Amersham/GE) according to the manufacturer’s protocols. Signal quantification was conducted with Imager 600 Analysis Software (Amersham).

IF staining

IF staining was performed as described previously (Uniacke et al. 2011). The primary antibodies and the dilutions were α-uL3 (1:1,000; α-cyL4 in Fleming et al. 1987), α-AOX1 (1:1,200), α-phosphoribulokinase (PRK; 1:3,000), and α-BIP (1:100). The secondary antibody was AlexaFluor568 conjugated to goat anti-rabbit IgG (Thermo Fisher). For dual IF staining with rabbit antisera against uL3 and LCIA (Fig. 1D), chloroplasts were first reacted with α-LCIA (1:700) and then indirectly IF labeled by AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H + L) conjugated to AlexaFluor488 (Jackson ImmunoResearch Inc.). Isolated chloroplasts were reacted with α-uL3 (1:1,000) and then indirectly IF labeled by goat anti-rabbit IgG conjugated to AlexaFluor568 (Thermo Fisher). For consistency, the uL3 IF signal is presented in magenta and other signals in green.

FISH was carried out as described previously, except that hybridizations contained BSA (Bioshop) at 4.5 mg/mL and all steps (starting with cell breakage) had heparin (sodium salt, Bioshop) at 0.5 mg/mL (Tsanov et al. 2016). Chloroplasts and whole cells were processed together using the crude suspension taken immediately prior to isolation on Percoll gradient step (above). For the results in Fig. 4A, the average FISH signal intensity from the probe with random sequence was subtracted from the average intensities of the mRNA FISH probes from chloroplasts or cells in the same images. P-values are from 2-tailed Student’s t tests comparing n ≥ 3 biological replicates using independent cultures. The brightness of the TUB2 mRNA FISH signal was enhanced to reveal its distribution with respect to the distributions of the stronger RBCS1/2 and LHCBM2/7 FISH signals (Fig. 4, B to E). Specificities of the FISH signals of the RBCS1/2 and LHCBM2/7 mRNAs were demonstrated previously (Uniacke and Zerges 2009).

Fluorescence microscopy was carried out with a Leica DMI6000B inverted epifluorescence microscope with a 63× Plan Apo objective (NA 1.4) and further magnified by a 1.6× tube lens. Images were acquired on a Hamamatsu Orca R2 C10600-10B camera controlled by Volocity software (Improvision). Filters used were Texas Red (562/40 nm ex: 624/40 nm em) for AlexaFluor568 and GFP (472/30 nm ex: 520/35 nm em) for AlexaFluor488. Acquired images were taken using Z plane stacks with a spacing of 0.2 µm per section; exposure settings, gain, and excitation intensity were kept constant where comparisons between intensities were required. For deconvolution, Z stacks were taken by series capture at a thickness of 0.2 µm per section and were deconvoluted with AutoQuant X3 (Media Cybernetics Inc.). Intensity plots in Figs. 1E and 3, B and D, were generated in Fiji (Schindelin et al. 2012) by horizontal line selection spanning the center of the chloroplast in the panel immediately above whereupon the “plot profile” function was performed on each channel. The resulting data were normalized so that the maximum intensity in each channel was equal to 1.0 and then plotted in Prism (GraphPad). For Fig. 3D, from all isolated chloroplasts co-IF stained for puromycin and LCIA (n = 30), we quantified these signals along a line drawn in Fiji across the translation envelope domain of each. Readings from each of the 30 chloroplasts were aligned by setting the position with maximal LCIA signal at 0. The line plots are of the average values. Significance of the distances between the positions of the puromycin and LCIA maximal values from these 30 chloroplasts on the x axis was demonstrated with a 1-sample t test with the null hypothesis = 0. To determine the average distributions of fluorescent signals from all cells or chloroplasts in a data set, we used an in-house macro within Fiji (https://github.com/Zergeslab/cellHarvester; Sun et al. 2019). The intensity projections (Figs. 1C and 4E; Supplementary Fig. S2, A and E) did not reveal well the anterior lobes of the chloroplast probably because their positions are somewhat variable thereby blurring their appearance in the projections. This did not reflect a change in chloroplast morphology (Supplementary Fig. S2A).

High-resolution electron tomography

Sections of 300 nm thickness from the resin-embedded cells above were collected on Formvar support slot grids and stained (Elimam et al. 2016). The dual-axis tilt series were collected using the FEI Tecnai G2 F20 200 kV TEM equipped with a Gatan Ultrascan 4000 4k × 4k CCD Camera System Model 895 and a single tilt holder. Tilt series were then acquired at 2° increment from −60° to 60°, at 19,000× magnification, 5.91 Å pixel size using SerialEM (Mastronarde 2005). For the second axis tilt series acquisition, the slot grid was rotated 90° manually and the same area of interest was searched manually. The dual-axis tomograms were reconstructed from the tilt series using IMOD software package (Kremer et al. 1996). The modeling and visualization of the membrane and cytosolic ribosomes were done also by IMOD. Cytosolic ribosomes were identified as was described previously (Bourque et al. 1971). To determine the density of cytosolic ribosomes in the membrane regions in Fig. 2, D to F, IMOD was used to calculate the perimeters of each region. Then the areas were calculated by multiplying the perimeters with the height. Distances between cytosolic ribosomes and the outer envelope membrane and identification of ribosomes based on dimensions were determined on the basis of pixel length (1.182 μm).

RPM and puromycin release assays

The RPM assay was carried out as described previously (David et al. 2012). Isolated chloroplasts (1 × 108 mL−1 in IB) were treated with 1.0 mm puromycin (Bioshop) for 10 min at RT. Movement of puromycin-nascent polypeptides from their sites of synthesis was minimized by excluding ATP and puromycin treating isolated chloroplasts, rather than live cells. IF staining was with a mouse monoclonal antibody against puromycin (DSHB Hybridoma Product PMY-2A4, deposited by J. Yewdell) and carried out as described above. The IF signal was specific (Supplementary Fig. S2D). The puromycin release assay followed protocols that were used to show ribosome association to ER, mitochondria, and thylakoid membranes with the following modifications (Adelman et al. 1973; Chua et al. 1973; Kellems et al. 1974). Chloroplasts were resuspended with 1.0 mL IB (150 µL), pelleted at 1,000 × g for 3 min at RT, resuspended with 1.0 mL of 1 of the following 4 conditions: (i) IB + 5 mm DTT, (ii) IB + 5 mm DTT + 750 mm KCl, (iii) IB + 5 mm DTT + 1 mm puromycin + 750 mm KCl, and (iv) IB + 5 mm DTT + 1 mm puromycin. Samples were incubated at RT for 20 min. Chloroplasts were pelleted by centrifugation (1,000 × g, 3 min, RT). Total protein extracts were subjected to immunoblot analysis (Supplementary Fig. S1B). Results are from 3 concurrent biological replicate experiments using independent cultures. Averages are plotted as percent of releasable ribosomes (Fig. 3E), as was done previously and because nonreleasable ribosomes likely were trapped within unbroken cells and membranous vesicles (Redman and Sabatini 1966; Chua et al. 1973; Kellems et al. 1974). P-values are from 2-tailed Student’s t tests comparing 3 biological replicates using independent cultures.

Visualization of newly synthesized RNA

A Click-iT RNA imaging kit (Alexa 488) was used to visualize newly synthesized RNAs according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA, USA). Cells were synchronized under the 12-h light/12-h dark condition, and EU was added to the medium 4 h into the light phase. Cells were fixed with 4% formaldehyde (w/v) on ice for 10 min, permeabilized with 0.5% Triton X in Tris-buffered saline (TBS) for 30 min on ice, and washed with TBS. The cell pellet was then gently mixed with Click-iT reaction cocktail prepared following the manufacturer’s instruction. After the Click-iT reaction, cells were washed and costained with 0.5 µg/mL DAPI. Cells were observed under a fluorescent microscope (BX51, Evident Corp., Tokyo, Japan) equipped with a color CCD camera (DP71, Evident). Analyses were performed on 51 cp nucleoids in 9 cells. Images were measured using an open-source biological image analysis software, Fiji (Schindelin et al. 2012).

Calculation of the lengths of nascent polypeptides for cotranslational targeting

For RBCS1/2 and LHCB2/7, the lengths of nascent polypeptide extending from a ribosome on the chloroplast surface were estimated as follows. The polypeptide lengths in nanometers were calculated using 0.4 nm as the contour length of an amino acid residue and the lengths of RBCS1/2 (185 aa) and LHCB2/7 (249 aa; Goldschmidt-Clermont and Rahire 1986; Ainavarapu et al. 2007; Ferrante et al. 2012). From each, we subtracted the length of polypeptide in the exit tunnel of the ribosome (10 nm; Fedyukina and Cavagnero 2011). For LHCB2/7, we also subtracted the length of the transit peptide (18 nm), whose removal might be required for insertion of the mature protein into a thylakoid membrane (Ferrante et al. 2012). The resulting lengths of nascent polypeptide between the ribosome and N-terminus for RBCS1/2 (64 nm) and LHCB2/7 (71.6 nm) are sufficient to reach the stroma or a thylakoid membrane adjacent to the envelope, respectively, during their cotranslational import (Wietrzynski et al. 2020).

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers provided in Supplementary Table S1.

Supplementary Material

kiae256_Supplementary_Data

Acknowledgments

For infrastructure and technical support, we thank the Centre for Microscopy & Cell Imaging (Concordia University), the Centre for Structural & Functional Genomics (Concordia University), and Jeannie Mui and the Facility for Electron Microscopy Research (McGill University). For generous gifts of antibodies, we thank Prof. Hideya Fukuzawa (LCIA, Kyoto University), Dr. Elizabeth Harris (αAtpB and α-uL3, Duke University), Drs. Pierre Crozet and Stephane Lemaire (α-PRK), and Dr. Jonathan Yewdell (PMY-2A4).

Author contributions

Y.S., S.B., Y.N., K.H.B., and W.Z. designed the research. Y.S., S.B., M.V.-P., Y.W., Y.N., W.S., C.L., J.D., D.D., and K.H.B. performed research. C.L. contributed new analytic/computational/etc. tools. Y.S., S.B., Y.N., W.S., C.L., J.D., and W.Z. analyzed data. Y.S., S.B., and W.Z. wrote the paper.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Primary immunoblot results for results in Figs. 1B and 3E.

Supplementary Figure S2. Experimental controls and validation.

Supplementary Figure S3. Localization patterns reported previously (Colon-Ramos et al. 2003; Uniacke and Zerges 2009).

Supplementary Table S1. smiFISH probe sequences.

Supplementary Table S2. Derivation of cytosolic ribosome densities in Fig. 2, D to F.

Supplementary Video S1. Slices from a tomographic volume and different views of the corresponding 3D reconstruction. The tomograph shows the region of the chloroplast envelope that was bound by cytosolic ribosomes as seen by IF microscopy (Fig. 2, C and D). The 3D model illustrates the cytoplasmic face of the outer chloroplast envelope (light gray) and cytosolic ribosomes on the envelope (blue spheres).

Supplementary Video S2. Slices from a tomographic volume and different views of the corresponding 3D reconstruction showing the chloroplast lobe in Fig. 2, E and F. The 3D model illustrates the cytoplasmic face of the outer chloroplast envelope (light gray), the stromal face of the inner membrane (dark gray), and cytosolic ribosomes on the envelope (blue spheres).

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant (217566) to W.Z.; Japan Society for the Promotion of Science (Grant-in-Aid for Challenging Exploratory Research [22K19329], Scientific Research [21H02504], and Transformative Research Areas [24H02275]) to Y.N.; and Canadian Institutes of Health Research (PJT-156354) and Natural Sciences and Engineering Research Council of Canada (RGPIN-2022-04774) to K.H.B.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

TUB2 Gramene: Cre12.g549550

TUB2 Araport: Cre12.g549550

SAPONIN CHEBI: CHEBI:26605
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