Visualization of Plastid Peptidoglycan in the Charophyte Alga Klebsormidium Nitens Using a Metabolic Labeling Method

Visualization of Plastid Peptidoglycan in the Charophyte Alga Klebsormidium nitens Using a Metabolic Labeling Method

Hiroyoshi Takano1,2*, Takashi Tsunefuka3, Susumu Takio1,4, Hayato Ishikawa1 and Katsuaki Takechi1

1 Faculty of Advanced Science and Technology, Kumamoto University, Kumamoto 860–8555, Japan 2 Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860–8555, Japan 3 Faculty of Science, Kumamoto University, Kumamoto 860–8555, Japan 4 Center for Water Cycle, Marine Environment and Disaster Management, Kumamoto University, Kumamoto 860–8555, Japan

Received June 3, 2018; accepted June 26, 2018

Summary It is widely accepted that a symbiosis involving a cyanobacterium with a peptidoglycan wall led to the origin of plastids of photosynthetic eukaryotes. Recently, we visualized plastid peptidoglycan in a moss species by epifluorescence microscopy using metabolic labeling with click chemistry. In the present study, we applied the same method to visualize plastid peptidoglycan in the filamentous charophyte alga Klebsormidium nitens, as its genome contains a set of genes capable of synthesizing peptidoglycan. To visualize peptidoglycan, the generation of D-alanyl-D-alanine (DA-DA) must be blocked to allow the incorporation of ethynyl-DA-DA (EDA-DA) into peptidoglycan. Because a gene-targeting technique has not been established for K. nitens, we used D-cycloserine, which is an inhibitor of D-Ala : D-Ala ligase. Treatment with 500 µM D-cycloserine arrested cell division and inhibited chloroplast division. The addition of 1 mM EDA-DA restored the rate of cell division to that observed in medium without D-cycloserine, suggesting that EDA-DA can integrate plastid peptidoglycan instead of DA-DA, thus restarting chloroplast division and therefore cell division. Subsequently, cells were subjected to click chemistry to attach an azide-modified Alexa 488 fluorophore to the EDA-DA probe. Microscopic observation indicated that both sides of chloroplasts have strong Alexa 488 fluorescence. Under conditions of no cell growth, the chloroplast peptidoglycan that existed prior to treatment with D-cycloserine might remain in the cell. Therefore, the fluorescence must be indicative of the location of newly synthesized peptidoglycan. Our observations suggest that chloroplast peptidoglycan is built along the chloroplast division plane and serves as an essential system for chloroplast division in K. nitens.

Key words Ccharophyte, Evolution, Klebsormidium nitens, Plastid peptidoglycan, Streptophyte.

It is now widely accepted that a symbiosis of a cyano- plastids are assumed to lack a peptidoglycan layer, as no bacterium led to the origin of plastids of primary photo- peptidoglycan-like structure has been observed in plas- synthetic eukaryotes, such as glaucophytes, red algae, tids using electron microscopy. and green plants (Keeling 2010). Among primary plants, Previously, we documented that the moss Physcomi- glaucophytes contain unique plastids, the cyanelles, trella patens contains a set of genes capable of gen- which harbor clearly identiﬁable peptidoglycan between erating peptidoglycan (Mur genes) and that knocking the outer and inner plastid envelopes (Iino and Hashi- out these genes causes defects in chloroplast division moto 2003, Sato et al. 2009). Peptidoglycan is a continu- (Machida et al. 2006, Takano and Takechi 2010, Sato ous covalent macromolecule comprised of a sugar-amino and Takano 2017). Recently, we were able to visualize acid polymer and is present in almost all free-living peptidoglycan in moss under epiﬂuorescence micros- bacteria, including cyanobacteria (Typas et al. 2012). copy with a novel metabolic cell wall-labeling method Bacterial peptidoglycan confers mechanical resistance of D-amino acid using click chemistry (Hirano et al. to osmotic pressure, maintains cell shape, and functions 2016). D-Ala and D-Glu are common basic components in cell division. The existence of cyanelle peptidoglycan of bacterial peptidoglycan, although L-amino acids are suggests that the endosymbiotic cyanobacterium ances- the predominant form in biological molecules. The for- tor of plastids contained peptidoglycan as part of its cell mation of D-alanyl-D-alanine (DA-DA) is catalyzed by wall. Nevertheless, with the exception of glaucophytes, D-Ala:D-Ala ligase (DDL) from two molecules of D-Ala; in common bacterial peptidoglycan, DA-DA exists at * Corresponding author, e-mail: [email protected] the C-terminus of the peptide portion of a basic com- DOI: 10.1508/cytologia.83.375 ponent of peptidoglycan, UDP-N-acetylmuramic acid 376 H. Takano et al. Cytologia 83(4)

(MurNAc)-pentapeptide (Typas et al. 2012). An in vivo conserved function in plastid division in basal strepto- peptidoglycan-labeling method uses a DA-DA dipeptide phytes, including streptophyte algae, bryophytes, and analog probe modified with an alkyne functional group, lycophytes, but not in chlorophyte algae or angiosperms ethynyl-DA-DA (EDA-DA) (Liechti et al. 2014). To (Takano and Takechi 2010). incorporate an EDA-DA, we isolated a homolog of the Consistent with gene disruption experiments in P. bacterial ddl gene from the P. patens genome (Pp-DDL) patens, previous studies have reported that antibiotics and generated a knockout line (∆Pp-ddl) (Hirano et al. that interfere with peptidoglycan biosynthesis can inhibit 2016). Due to defects in chloroplast division, ∆Pp-ddl plastid division, in turn generating giant chloroplasts contained a macrochloroplast. The EDA-DA used in the (Kasten and Reski 1997, Katayama et al. 2003). In a click reaction was able to complement the mutant pheno- previous study, we investigated the effects of antibiotics type of ∆Pp-ddl, suggesting its incorporation into pep- in the streptophyte unicellular alga, Closterium perac- tidoglycan in moss. Click reaction chemistry can attach erosum-strigosum-littorale complex (Matsumoto et al. an azide-modified fluorophore Alexa488 to the ethynyl 2012). Although normal Closterium cells have two chlo- group. The results of click chemistry for the ∆Pp-ddl roplasts before and after cell division, cells treated with line complemented with EDA-DA indicated that moss β-lactam antibiotic, ampicillin, or D-cycloserine had chloroplasts are fully surrounded by peptidoglycan (Hi- only one chloroplast after cell division. By counting cell rano et al. 2016). Our findings strongly suggest that the types, we proposed that cells with one chloroplast ap- plastids in basal green plants have a peptidoglycan wall peared first; then, a huge chloroplast was generated that containing D-amino acids. inhibited cell division, causing the appearance of long Although cyanelle peptidoglycan in glaucophytes can cells with irregular chloroplasts followed by cell death be detected under electron microscopy (Iino and Hashi- (Matsumoto et al. 2012). Unfortunately, the genome moto 2003, Sato et al. 2009), peptidoglycan has not yet sequence of the Closterium psl complex has not yet been been visualized using epifluorescent microscopy. In a determined. Therefore, in the present study, we used the previous study, we applied the above method to visualize streptophyte alga K. nitens to visualize plastid peptido- cyanelle peptidoglycan in the glaucophyte Cyanophora glycan with the metabolic cell wall-labeling method of paradoxa (Higuchi et al. 2016). D-cycloserine, a DDL D-amino acid using click chemistry. and alanine racemase inhibitor, was used to incorporate EDA-DA into peptidoglycan. Treatment with D- Materials and methods cycloserine inhibited the growth of C. paradoxa, which was thought to be caused by interference with cyanelle Culture of the alga division, similar to what occurs during treatment with Cells of K. nitens were cultured in liquid C medium ampicillin, an inhibitor of penicillin-binding proteins (Matsumoto et al. 2012) with aeration at 22°C under (Higuchi et al. 2016). The growth of cells treated with continuous light (10 µmol m-2s-1). After two weeks, D-cycloserine recovered in medium containing EDA- cells in the 3-mL culture were collected by centrifuga- DA, and the click chemistry reaction for C. paradoxa tion and stained for 1 h using 1 mL of calcofluor white cells grown with D-cycloserine and EDA-DA showed (Sigma-Aldrich, St. Louis, MO, USA) at a concentra- peptidoglycan as Alexa 488 fluorescence surrounding tion of 10 mg mL-1. After washing twice with 1 mL of cyanelles (Higuchi et al. 2016). These results confirm C medium, cells were transferred to 50 mL of fresh C that the metabolic cell wall-labeling method using click medium and cultured. On the appropriate days, K. nitens chemistry was useful for detecting plastid peptidoglycan cells were collected and observed under fluorescent mi- via fluorescent microscopy. croscopy (BIOREVO BZ-9000; Keyence, Osaka, Japan). Streptophyta is a monophyletic plant lineage consist- The number of cells lacking calcofluor-white staining ing of streptophyte green algae and embryophytes (de on the septum (at least one or both septa) was measured. Vries et al. 2016). The genome of the streptophyte alga The antibiotics used were ampicillin sodium (Nacalai K. nitens (Division Charophyta sensu lato) contains Tesque, Inc, Kyoto, Japan) and D-cycloserine (Wako a set of genes capable of synthesizing peptidoglycan Pure Chemical Industries, Ltd., Osaka, Japan). (Mur genes) (Hori et al. 2014, Sato and Takano 2017), suggesting that this species, similar to P. patens, uses Detection of peptidoglycan peptidoglycan in plastid division. On the other hand, the EDA and DA-DA were purchased from Nagase Co., genome sequences of another algal group, the chloro- Ltd. (Tokyo, Japan) and Sigma-Aldrich, respectively. phyte green algae Chlamydomonas reinhardtii and Vol- The ‘Clickable’ Alexa Fluor 488 azide and Click-iT vox carteri, do not contain Mur genes, except for MurE, Cell Reaction Buffer Kit were purchased from Invitro- suggesting that chlorophytes lack plastid peptidoglycan gen (Life Technologies, Carlsbad, CA, USA). EDA-DA (Merchant et al. 2007, Prochnik et al. 2010). Based on was synthesized according to the methods of Liechti a dataset of plant genome sequences, suggested that et al. (2014). K. nitens cells were inoculated in C liquid the bacterial peptidoglycan synthesis pathway has a medium with 1 mM EDA-DA dipeptides and 500 µM 2018 Plastids Peptidoglycan in a Charophyte Alga 377

D-cycloserine, for 7 days and then used to detect pep- fer Kit according to the manufacturer’s instructions. tidoglycan. Cells were washed three times with liquid C medium, ﬁxed, and permeabilized for 1 h using 2.5% Results and discussion formalin, 0.1 M PIPES–NaOH (pH 6.8), 2.5 mM EGTA,

1 mM MgCl2, and 0.01% NP-40. Cells were then washed Effects of D-cycloserine treatment on K. nitens cells once with 1% bovine serum albumin in 1×PBS and used To visualize plastid peptidoglycan using the metabolic for the click reaction with a Click-iT Cell Reaction Buf- labeling method, the generation of DA-DA in a K. nitens cell must be blocked for the uptake and incorporation of EDA-DA. Because a gene-targeting technique has not been established for this algal species, the antibiotic D-cycloserine was used to interfere with DDL function. K. nitens usually consists of multicellular and non- branching ﬁlaments without differentiated or specialized cells (Hori et al. 2014). Although cells of the Closterium psl complex treated with the antibiotic, ampicillin or D-cycloserine, exhibited giant chloroplasts (Matsumoto et al. 2012), preliminary experiments showed that giant cells with a huge chloroplast were not observed in the K. nitens cells treated with D-cycloserine or ampicillin. However, after cultivation, the green color of the culture medium containing antibiotics was less intense than that of the medium without antibiotics, suggesting the inhibition of K. nitens cell growth by the antibiotics. Sumiya Fig. 1. Effects of ampicillin and D-cycloserine on K. nitens cell et al. (2016) reported that cell-cycle progression in the division. Changes in the frequency (%) of cells lacking glaucophyte C. paradoxa is arrested when cyanelle divi- calcoﬂuor-white- (CF) stained septum (at least one septum sion is blocked by treatment with the β-lactam antibiotic, lacked staining) during a 7-day inoculation for untreated (triangles), ampicillin- (Amp; circles), or D-cycloserine- carbenicillin. To investigate whether a similar cell-cycle (DCS; squares) treated cells. arrest occurs in K. nitens cells treated with antibiot-

Fig. 2. Representative micrographs of antibiotic-treated cells. Light (left panels) and epifluorescence (right panels) micrographs of calcofluor white-stained K. nitens cells. For controls, cells stained with calcofluor white were observed at 0 and 7 days after transfer to fresh C medium without antibiotics. Cells treated with ampicillin (Amp) or D-cycloserine (DCS) for 7 days are also shown. Arrows indicate septa without calcofluor white staining. 378 H. Takano et al. Cytologia 83(4)

Fig. 3. Recovery of cell division of D-cycloserine-treated cells with ethynyl DA-DA. (A) Changes in the frequency (%) of cells lacking calcofluor-white- (CF) stained septum (at least one septum lacked staining) during a 7-day inoculation for untreated (triangles) or D-cycloserine- (DCS; open squares) treated cells in addition to DCS-treated cells with 1 mM ethynyl DA-DA (EDA-DA) (closed squares). (B) Representative light (upper) and epifluorescence (lower) micrographs of calcofluor white-stained K. nitens cells at 7 days after transfer to fresh C medium containing 500 µM DCS and 1 mM EDA-DA. Arrows indicate septa without calcofluor white staining.

Fig. 4. Fluorescent labeling of chloroplast peptidoglycan in K. nitens cells. K.nitens cells were grown for 7 days in liquid C medium supplemented with 1 mM EDA-DA (A) or DA-DA (B) in addition to 500 µM D-cycloserine. Collected cells were fixed and permeabilized. Using click chemistry, azide-modified Alexa Fluor 488 was bound to an alkyne group, such as the ethynyl group, that presents on EDA-DA. Micrographs showing Alexa 488 fluorescence (left) were merged with images showing chlorophyll autofluorescence (middle) in the right photographs. Low-magnification photo (upper) and two examples at higher magnification are shown. No fluorescence was detected in the K. nitens cells grown with DA-DA (B). 2018 Plastids Peptidoglycan in a Charophyte Alga 379 ics, we used a method of transiently staining cell walls although the proportion of cells without at least one cal- with the fluorescent dye calcofluor white (Ohtaka et al. cofluor fluorescent wall reached 69% (Fig. 4B). 2017) to measure the rate of cell division. Ohtaka et al. The fluorescence images of K. nitens cells differed (2017) confirmed that staining with calcofluor white from those in the visualization experiments of chloro- did not affect the growth of K. nitens. After washing plast peptidoglycan for the moss P. patens. In P. patens, excess dye from K. nitens cells stained with calcofluor each chloroplast is uniformly surrounded by Alexa 488 white for 1 h, cells were transferred to fresh liquid C fluorescence (Hirano et al. 2016). One difference be- medium and cultured. Although all cell walls showed tween the P. patens experiment and the present one is strong fluorescence just after transfer to new medium, that a knockout line for the DDL gene (∆Pp-ddl) was the fluorescence of newly synthesized cell walls by cell used for the moss experiments. Because cell growth with division was weak during culture (Fig. 2). Therefore, a multiplication of chloroplasts proceeds in the ∆Pp-ddl cell with at least one septum having low fluorescence line, peptidoglycan is unlikely to exist around each chlo- was considered to be a cell undergoing division. On the roplast. The huge chloroplasts in the ∆Pp-ddl line were appropriate days, K. nitens cells were observed under a able to begin division through the addition of EDA-DA microscope, and the number of cells (out of 100) lacking (Hirano et al. 2016). Therefore, the location of peptido- calcofluor-white staining on the septum (at least one or glycan biosynthesis in chloroplasts was unlikely to be both septa) in the middle of the filaments was measured restricted and could proceed all around the chloroplasts. to detect cell division (Figs. 1 and 2). Many cell walls On the other hand, peptidoglycan biosynthesis was ar- with low fluorescence were observed in K. nitens cells rested by the addition of D-cycloserine in K. nitens after 7 days of culture without antibiotics. The frequency cells. At the same time, cell division was also inhibited of cells with weakly fluorescent septum increased to in K. nitens cells, suggesting that the chloroplast pep- approximately 80% at day 7. We used two antibiotics, tidoglycan that existed before treatment with antibiotic ampicillin and D-cycloserine, for peptidoglycan biosyn- remained around the chloroplasts. Because the addition thesis. Although treatments of ampicillin or D-cycloser- of EDA-DA was able to restart peptidoglycan synthesis, ine at a concentration of 100 µM had little effect on cell based on the restoration of cell growth, the Alexa 488 division, the proportion of cells undergoing cell division fluorescence of K. nitens cells must be indicative of the in media containing 500 µM ampicillin or D-cycloserine location of newly synthesized peptidoglycan. Therefore, at day 7 was half of that in medium without antibiot- our observations suggest that chloroplast peptidoglycan ics, suggesting that treatment with antibiotics interfered with cell division. These phenomena may be the same as those that occurred with C. paradoxa (Sumiya et al. 2016). The inhibition of chloroplast division in medium with antibiotics for peptidoglycan biosynthesis may re- sult in the arrest of cell division, thus suggesting that no giant cells with giant chloroplasts were observed.

Visualization of plastid peptidoglycan using click chemistry To visualize plastid peptidoglycan, we examined whether the addition of EDA-DA could restore the rate of cell division of antibiotic-treated cells (Fig. 3). Indeed, the addition of 1 mM EDA-DA in medium containing 500 µM D-cycloserine resulted in the restoration of cell division to the rate observed in medium without anti- Fig. 5. Detection of newly synthesized plastid peptidoglycan in K. biotics, suggesting that EDA-DA can integrate plastid nitens cells. In this scheme, only a middle cell is proceed- peptidoglycan instead of DA-DA, thus restarting chloro- ing to cell division. Cells and chloroplasts are represented by boxes and red rounded rectangles, respectively. Because plast and cell division. Therefore, cells grown in medium D-cycloserine is the inhibitor of D-alanine: D-alanine ligase, containing EDA-DA and D-cycloserine for 7 days were the synthesis of plastid peptidoglycan was thought to inter- fixed, permeabilized, and then subjected to click chem- fere with this antibiotic in C medium (Phase I). The addition istry to attach an azide-modified Alexa 488 fluorophore of EDA-DA may restore plastid peptidoglycan synthesis by its integration into peptidoglycan, instead of DA-DA (Phase to the EDA-DA probe. Microscopic observation showed II). Green indicates newly synthesized peptidoglycan that that both sides of the chloroplasts exhibited strong Al- was detected using click chemistry with Alexa 488 azide. exa 488 fluorescence (Fig. 4A). No fluorescence was During chloroplast division, a large amount of peptidoglycan is synthesized at the plastid division plane to divide observed in cells grown with 1 mM DA-DA and D-cyclo- the plastid (Phase III). Therefore, divided cells must have a serine for 7 days, because no alkyne group was bound to chloroplast with Alexa 488 fluorescence at the end (Phase the azide-modified Alexa Fluor 488 by click chemistry, IV). 380 H. Takano et al. Cytologia 83(4) is built along the chloroplast division plane and serves as Takano, H. 2016. Moss chloroplasts are surrounded by a peptido- an essential system for chloroplast division in K. nitens. glycan wall containing D-amino acids. Plant Cell 28: 1521–1532. Hori et al. 2014. Klebsormidium flaccidum genome reveals primary Weak fluorescence at the center of chloroplasts (Fig. 4A) factors for plant terrestrial adaptation. Nat. Commun. 5: 1–9. may depend on the incorporation of EDA-DA for repair- Iino, M. and Hashimoto, H. 2003. Intermediate features of cyanelle ing peptidoglycan. 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