Evolution and Functional Differentiation of the C-terminal Motifs of FtsZs During Plant Evolution

Abstract Filamentous temperature-sensitive Z (FtsZ) is a tubulin-like GTPase that is highly conserved in bacteria and plants. It polymerizes into a ring at the division site of bacteria and chloroplasts and serves as the scaffold protein of the division complex. While a single FtsZ is present in bacteria and cyanobacteria, there are two subfamilies, FtsZ1 and FtsZ2 in the green lineage, and FtsZA and FtsZB in red algae. In Arabidopsis thaliana, the C-terminal motifs of AtFtsZ1 (Z1C) and AtFtsZ2-1 (Z2C) display distinct functions in the regulation of chloroplast division. Z1C exhibits weak membrane-binding activity, whereas Z2C engages in the interaction with the membrane protein AtARC6. Here, we provide evidence revealing the distinct traits of the C-terminal motifs of FtsZ1 and FtsZ2 throughout the plant evolutionary process. In a range of plant species, the C-terminal motifs of FtsZ1 exhibit diverse membrane-binding properties critical for regulating chloroplast division. In chlorophytes, the C-terminal motifs of FtsZ1 and FtsZ2 exhibit both membrane-binding and protein interaction functions, which are similar to those of cyanobacterial FtsZ and red algal FtsZA. During the transition from algae to land plants, the functions of the C-terminal motifs of FtsZ1 and FtsZ2 exhibit differentiation. FtsZ1 lost the function of interacting with ARC6 in land plants, and the membrane-binding activity of FtsZ2 was lost in ferns. Our findings reveal the functional differentiation of the C-terminal motifs of FtsZs during plant evolution, which is critical for chloroplast division.


Introduction
Chloroplasts originated from endosymbiotic events that occurred ∼1 billion years ago, leading to the emergence of the Archaeplastida clade, which includes Glaucophytes, Rhodophytes (red algae), and Chlorophytes (green algae) (Osteryoung and Nunnari 2003;Zimorski et al. 2014).Chlorophytes subsequently diverged into two major lineages: Chlorophytes and Charophytes, paving the way for the evolution of various terrestrial plant groups (de Vries et al. 2016;Wang et al. 2020).During the evolution of plants, land plants have evolved into different species ranging from simple to complex, such as bryophytes, lycophytes, ferns, gymnosperms, and angiosperms (Wickett et al. 2014;Szövényi et al. 2019).
Filamentous temperature-sensitive Z (FtsZ) is a tubulinlike cytoskeletal GTPase that plays an essential role in the division of bacteria and chloroplasts (Osteryoung and Nunnari 2003;Stokes and Osteryoung 2003).FtsZ genes also exist in some non-plant eukaryotes, such as ameba, excavate, and stramenopiles, and some of those genes may be involved in mitochondrial division (Beech et al. 2000;Leger et al. 2015).In both bacteria and chloroplasts, FtsZ polymerizes to form a contractile ring-like (Z ring) complex at the division site (Vitha et al. 2001;Errington et al. 2003).The polymerization of FtsZ activates GTPase activity, catalyzing the hydrolysis of GTP to generate contractile force (Scheffers et al. 2002;Huecas et al. 2007;Olson et al. 2010).The Z ring provides a scaffold for the division apparatus and recruits other protein components (Adams and Errington 2009;Osteryoung and Pyke 2014;Chen et al. 2018).The formation of the Z ring represents the beginning of division, and Z ring localization is regulated by the Min system proteins (de Boer et al. 1989;Fujiwara et al. 2008;Zhang et al. 2013;Shaik et al. 2018;Sun et al. 2023).Incorrect localization of the Z ring causes abnormal division of bacteria and chloroplasts (Vitha et al. 2001;Adams and Errington 2009;McQuillen and Xiao 2020).
In bacteria, including cyanobacteria, the FtsZ exists as a single form, which then diverges into FtsZ1 and FtsZ2 subfamilies in plants (green algae and land plants), and FtsZA and FtsZB in red algae (Stokes and Osteryoung 2003;TerBush et al. 2013;Chen et al. 2017).In Physcomitrium patens, there is a special FtsZ, FtsZ3, besides the four FtsZs of FtsZ1 and FtsZ2 subfamilies (Martin et al. 2009a,b).The copolymerization of FtsZ1 and FtsZ2 at the division site is vital for chloroplast division in plants (McAndrew et al. 2001).Similar to FtsZ1 and FtsZ2, FtsZA and FtsZB can also copolymerize to form heteropolymers (Chen et al. 2017).
Our previous research revealed that the C-terminal motif of AtFtsZ1 in Arabidopsis thaliana comprises an amphiphilic beta-strand, exhibiting weak membrane-binding capabilities (Liu et al. 2022).The absence of this motif in Arabidopsis perturbs FtsZ assembly, leading to aberrant chloroplast division.When expressed individually in Escherichia coli, AtFtsZ2-1 formed into straight filaments.However, fusion protein expression of AtFtsZ2-1 with AtFtsZ1 C10 or co-expression with AtFtsZ1 resulted in the formation of helical structures, likely attributable to the membrane-binding property of the Z1C motif.
In this study, we found that the C-terminus of FtsZ1 proteins is composed of a simple and variable motif in different species, which is beneficial for the function of the proteins during evolution.Employing an E. coli expression system (Irieda and Shiomi 2017), we analyzed the membrane-binding activities of different FtsZ1 C-terminal motifs, including those from chlorophytes, charophytes, bryophytes, lycophytes, ferns, gymnosperms and angiosperms, alongside FtsZ in cyanobacteria and FtsZA in red algae.We also provided functional evidences of the C-terminal motifs from CreZ1 (Chlamydomonas reinhardtii FtsZ1) and PpZ1B (Physcomitrella patens FtsZ1B) with AtFZ1 (A. thaliana FtsZ1) to regulate chloroplast division in vivo.Our results illuminated the functional differentiation between FtsZ1 and FtsZ2 during plant evolution.

Evolutionary Characteristics of the FtsZ C-terminal Motif
FtsZ is conserved across cyanobacteria and plants, starting with a singular FtsZ in cyanobacteria and diverging into two distinct subfamilies: FtsZ1 and FtsZ2 in green algae and land plants, and FtsZB and FtsZA in red algae (supplementary fig.S1, Supplementary Material online).In A. thaliana, both AtFtsZ1 and AtFtsZ2-1 contain C-terminal motifs (Fig. 1a).However, their functions differ significantly (Zhang et al. 2016;Liu et al. 2022).To analyze the evolutionary trajectory of FtsZ, we aligned the C-terminal 30 amino acids (C30aa) of FtsZ in cyanobacteria and FtsZA in red algae with those of both FtsZ1 and FtsZ2 (Fig. 1b and c).The alignment revealed a core sequence, IPDFL, retained in FtsZ2 but shortened in FtsZ1 during the chlorophyta stage, eventually leading to a distinct motif in charophytes.This motif was simplified and became more conserved in angiosperms.
These findings indicate a divergence between FtsZ1 and FtsZ2, with the C-terminal motif of FtsZ1 evolving independently, resulting in distinct amino acid sequences at their C-termini and suggesting they play different roles in chloroplast division.
To further analyze the membrane-binding activity of the FtsZ1 C-terminal motifs from various species, we purified proteins GFP, GFP-PpZ1B C10, GFP-AcvZ1A C10 and GFP-PsZ1 C10 and then incubated them with liposomes.A part of the GFP-PpZ1B C10, GFP-AcvZ1A C10 and GFP-PsZ1 C10 was found to co-pellet with the liposomes, indicating a membrane-binding activity (Fig. 5a).Additionally, we investigated the relationship between membrane-binding efficacy and spiral density by expressing these proteins in bacteria, followed by lysis of equal amounts of bacteria with lysozyme.Detection of these proteins by anti-his antibodies revealed that a small fraction of GFP-PpZ1B C10 and GFP-AcvZ1A C10, and a larger fraction of GFP-PsZ1 C10, were associated with membrane (Fig. 5b).
These results indicate that the C-terminal motifs of FtsZ1 retain membrane-binding activity during the transition from algae to land plants.The unique membranebinding characteristics observed in charophytes and ferns (Figs. 2 to 4) highlight their potential role as critical evolutionary junctions due to their distinct properties.

RR/KLFF Motif is Important for the FtsZ1 Membrane Binding Activity in Various Species
To assess the role of the highly conserved C-terminal motif RR/KLFF in angiosperm FtsZ1 (Liu et al. 2022), for membrane-binding activity in other species, we modified the last five C-terminal amino acids of PpZ1B C10 from
These results not only indicate the significance of the RR/ KLFF sequence in the C-terminal of angiosperm FtsZ1 for membrane binding activity across species, but also suggest an evolutionary direction for the FtsZ1 C-terminal motifs.

The C-terminal Motif of FtsZ1 in Different Species has Similar Functions in Vivo
To assess the in vivo functionalities of FtsZ1's C-terminal motifs from various species, we engineered a fusion protein by replacing the last 10 amino acids of AtFZ1 (AtFZ1 C10) with that of CreZ1 (CreZ1 C10).AtFZ1ΔC10-CreZ1 C10 was introduced into an Atftsz1 null mutant under the control of the native AtFtsZ1 promoter.Remarkably, the chloroplast phenotype of the Atftsz1 mutant was rescued by AtFZ1ΔC10-CreZ1 C10 (Fig. 7a).Western blot analysis confirmed that the FtsZ1 protein levels in the transgenic plants were comparable to those in the wild type, and the number of chloroplasts per cell is similar to that of the wild type (Fig. 7b  and c).
Similarly, we constructed AtFZ1ΔC22-PpZ1B C22, a fusion protein in which AtFZ1's last 22 amino acids (AtFZ1 C22) were replaced with that of PpZ1B (PpZ1B C22), and introduced this construct into the Atftsz1 mutant with the native promoter of AtFtsZ1.The chloroplast phenotype in plants transformed with AtFZ1ΔC22-PpZ1B C22 was effectively rescued (Fig. 8a).Further analyzes, including immunoblotting with anti-FtsZ1 antibodies, showed that many transgenic lines had FtsZ1 protein levels and chloroplast numbers per cell close to those of the wild type (Fig. 8b and c).
Immunofluorescence staining with anti-FtsZ2-1 antibodies revealed that, unlike in the Atftsz1 mutant where long FtsZ filaments and multiple rings were observed, FtsZ in transgenic plants typically formed a single ring at the division site, similar to those observed in the wild-type (supplementary fig.S4, Supplementary Material online).
Despite the varying amino acid sequences, the C-terminal motifs of FtsZ1 from different species have similar functions in chloroplast division regulation throughout plant evolution.FtsZ1 and FtsZ2 were derived from cyanobacteria FtsZ (Stokes and Osteryoung 2003).Previous studies have reported that the C-terminus of SynFZ (Synechocystis sp.PCC6803 FtsZ), CmeFZA (Cyanidioschyzon merolae FtsZA) and AtFtsZ2 interact with the membrane protein Ftn2 in cyanobacteria or its homolog ARC6 in plants (Zhang et al. 2016;Yoshida 2018;Camargo et al. 2019).To investigate whether the C-terminal motifs of SynFtsZ and CmeFZA have membrane-binding activity, fusion proteins GFP-AtFZ2-1-SynFZ C10 and GFP-AtFZ2-1-CmeFZA C10 were expressed in E. coli cells and the fusion proteins were found to form dense helical structures, suggesting they have membrane-binding activity (supplementary fig.S5, Supplementary Material online).Thus, SynFZ C10 and CmeFZA C10 have both membrane-binding activity and protein interaction function.
FtsZ diverged into FtsZ1 and FtsZ2 during the chlorophyta stage.To explore the presence of membranebinding activity and protein interaction function in these proteins from green algae, we constructed a fusion protein, GFP-CreZ2 (C.reinhardtii FtsZ2), and expressed it in E. coli cells.Despite the significant agglomeration, the fusion protein formed a spiral structure, suggesting that CreZ2 still exhibits membrane-binding activity (supplementary fig.S5, Supplementary Material online).Further, we assessed the interactions between CreZ1, CreZ2, and CreARC6 (C.reinhardtii ARC6) using a yeast two-hybrid assay.The result revealed that both CreZ1 and CreZ2 interact with CreARC6 (Fig. 9).The essential amino acid phenylalanine (F), crucial for the interaction between FtsZ2 and ARC6 (Maple et al. 2005;Zhang et al. 2016), exists in the  1b and c).Mutating phenylalanine (F) to glycine (G) for CreZ1 F473G and CreZ2 F425G resulted in loss of interaction with CreARC6 (Fig. 9).
These findings demonstrate that the C-terminal motifs of SynFZ and CmeFZA exhibit both membrane-binding activity and protein-protein interaction functions.At the initial stage of FtsZ differentiation in chlorophytes, FtsZ1 and FtsZ2 continue to retain these crucial functions.

Higher Plant FtsZ1 C-terminal Motif Lost Protein Interaction Ability
The crucial amino acid Phenylalanine (F), essential for the interaction of FtsZ2 with the membrane protein ARC6, is absent in the C-terminus of FtsZ1 in charophytes and higher plants, as shown in sequence alignments (Fig. 1b and supplementary fig.S3, Supplementary Material online).In contrast, FtsZ2 retains the Phenylalanine throughout evolution process (Fig. 1c).To investigate whether FtsZ1 from higher plants lacks interaction with ARC6, we conducted a yeast two-hybrid assay, and the results revealed that PpZ1B does not interact with PpARC6 (P.patens ARC6), whereas PpZ2-1 (P.patens FtsZ2-1) does interact with PpARC6 (Fig. 10).
To further investigate the membrane-binding activity of FtsZ2 during plant evolution, we expressed the fusion protein GFP-PpZ2-1 in bacteria and observed a high-density spiral structure (supplementary fig.S5, Supplementary Material online), indicating that PpZ2-1 contains membrane-binding activity.We also expressed GFP-AcvZ2-1 (Adiantum capillus-veneris FtsZ2-1) in E. coli cells, which formed straight filaments (supplementary fig.S5, Supplementary Material online), suggesting a loss of membrane-binding activity in AcvZ2-1.

Discussion
In this study, our results revealed an evolutionary route that delineates the functional characters of the FtsZ C-terminal motifs throughout plant evolution (Fig. 11).In chloroplytes, both FtsZ1 and FtsZ2 retain the ability to interact with ARC6.As evolution progressed, FtsZ1 in species beyond the chloroplyta stage lost this interaction capability with ARC6.FtsZ2 maintained the interaction capability with ARC6 up to seed plants, indicating a preservation of this function throughout the plant evolution (Miyagishima 2011).For membrane-binding activity, FtsZ1 exhibits this capability up to seed plants.In contrast, FtsZ2's membrane-binding activity is retained until During evolution, FtsZ1's C-terminal motifs show flexibility and diversity, adapting readily, while FtsZ2's C-terminal motifs remain stable (Fig. 1b and c).These evolutionary distinctions are closely linked to their functional roles (Zhang et al. 2016;Liu et al. 2022).We have verified the membrane-binding activity of FtsZ1 C-terminal motif of various species in E. coli and in vitro experiments (Figs. 2 to 5, and supplementary fig.S2, Supplementary Material online), and revealing significant variability in their membrane-binding sequences across species.FtsZ1 exhibits dynamic functionality, playing a pivotal role in exerting the contraction force necessary for chloroplast division (TerBush and Osteryoung 2012;TerBush et al. 2018).The C-terminal motif of FtsZ1 is essential for its membrane binding ability and requires shorter sequence at the C-terminal end to effectively interact with the chloroplast membrane (supplementary fig.S3, Supplementary Material online).The evolutionary pattern of the Z1C motif in plants is likely more favorable due to the fewer amino acids needed for effective membrane binding while still allowing for adequate turnover and maintaining its function in generating contraction forces for chloroplast division (TerBush and Osteryoung 2012;Liu et al. 2022).
Our findings indicate that the functions of membrane binding and protein interaction observed in FtsZ1 and FtsZ2 are inherited from cyanobacterial FtsZ C-terminal motifs (supplementary fig.S5, Supplementary Material online) (Mazouni et al. 2004).Initially, in chloroplytes, FtsZ differentiated into FtsZ1 and FtsZ2, retaining both membrane-binding and protein interaction functions (Figs. 2 and 9 and supplementary fig.S5, Supplementary Material online).Subsequently, the C-termini of FtsZ1 and FtsZ2 underwent functional differentiation.This divergence likely reflects evolution of specialized chloroplast   (Miyagishima 2011;Miyagishima et al. 2011;Liu et al. 2024).
The envelope of chloroplasts changes during the evolution of plants.Cyanobacteria have a well-defined cell wall which is mostly made up of peptidoglycan (Leganés et al. 2005).The cell wall is absent in the chloroplast of red algae, green algae and other plants (Takano and Takechi 2010;Hirano et al. 2016).However, the peptidoglycan biosynthesis pathway was found to be well-conserved in bryophytes and lycophytes (Izumi et al. 2003;Dowson et al. 2022).Mutations of Mur (murein) genes in this pathway in P. patens affected chloroplast division (Machida et al. 2006).The application of penicillin, which can inhibit the biosynthesis of peptidoglycan, also blocked chloroplast division in bryophytes and lycophytes (Izumi et al. 2003;Machida et al. 2006;Takahashi et al. 2016).The MurE gene, although exists in seed plants, is not involved in chloroplast division any more (Garcia et al. 2008;Lin et al. 2017).Moreover, penicillin doesn't inhibit the division of chloroplasts in seed plants either (Kasten and Reski 1997).In this work, the C-terminus of FtsZ1 in charophytes, bryophytes, and lycophytes required longer amino acid sequences to provide membrane-binding activity (Figs. 2 and 3 and supplementary fig.S2, Supplementary Material online).While the C-terminal membrane binding activity of FtsZ1 in seed plants was provided by a shorter motif at the very end of the C-terminus (Fig. 3 and supplementary fig.S3, Supplementary Material online).Unfortunately, the related study is lacking in ferns.The significant change of the membrane-binding sequences at the FtsZ1 C-termini from bryophytes and lycophytes to seed plants could be an adaption to the change of the peptidoglycan biosynthesis-dependent chloroplast division mechanism.
This study significantly advances the understanding of the functional differentiation of FtsZ C-terminal motifs across various species during evolution.It reveals the distinctive evolutionary paths and functional divergences of FtsZ1 and FtsZ2 C-terminal motifs throughout plant evolution (Fig. 11).The study traces the origin of the membrane-binding activity of the FtsZ1 C-terminal motif and the protein interaction function of the FtsZ2 C-terminal motif to the ancestral cyanobacterial FtsZ C-terminal motif.Initially, in chlorophytes, FtsZ differentiated into FtsZ1 and FtsZ2, with both variants maintaining dual functional capabilities (Figs. 2 and 9).The evolutionary forces behind the loss of the protein interaction function in streptophyta FtsZ1 and the loss of the membrane-binding activity in fern FtsZ2 are not fully understood.Further investigations in the future are essential to explore the evolution of these characters across species, offering insights into the adaptive mechanisms underlying chloroplast division.
To obtain complementation constructs PFtsZ1: AtFtsZ1ΔC10-CreZ1 C10 and PFtsZ1: AtFtsZ1ΔC10-PpZ1B C22, genomic DNA of A. thaliana was amplified with forward primer AtFZ1-10 and reverse primers CreZ1-11 and PpZ1B-4, and then the above PCR products were further amplified with forward primer AtFZ1-10 and reverse primers CreZ1-12 and PpZ1B-5, respectively.The PCR products were digested with BamHⅠ and NcoⅠ, and then cloned into 3302Y2 vector.These constructs were transformed into an AtFtsZ1 null mutant (Liu et al. 2022) by floral dipping method.The primer sequences are shown in supplementary table S3, Supplementary Material online.

Fluorescence Microscopy and Image Analysis
The fusion proteins expressed in E. coli BL21 (DE3) cells were observed with a fluorescence microscope (NE910, Nexcope, Ningbo, China) equipped with a camera (E3ISPM).Bacterial cells were observed with an oil immersion 100× objective.Immunofluorescence staining was performed as described previously (Li et al. 2016) with the FtsZ2-1 antibodies (Liu et al. 2022)

Protein Expression and Purification
To express GFP, GFP-PpZ1B C10, GFP-AvcZ1A C10, GFP-PsZ1 C10 proteins with 6×His tag, PCR-amplifications were performed with the primers GFPEcoRⅠ and AtFZ2-1-46, and plasmids pET-28a-GFP-AtFZ2-1, pET-28a-GFP-AtFZ2-1-PpZ1B C10, pET-28a-GFP-AtFZ2-1-AcvZ1A C10 and pET-28a-GFP-AtFZ2-1-PsZ1 C10 as the templates, respectively.The primers sequences are shown in supplementary table S3, Supplementary Material online.The PCR products were digested with EcoRⅠ and ligated with T4 ligases (NEB), and then amplified with primers NcoⅠGFP and T7ter, the PCR products were digested with NcoⅠ and XhoⅠ and then cloned into pET30a expression vectors.The fusion proteins were expressed in E. coli BL21 strains with a His tag.Protein purification was performed using the same protocol as described previously (Liu et al. 2022).Liposome preparation was performed as described previously (Liu et al. 2022).Proteins GFP, GFP-PpZ1B C10, GFP-AvcZ1A C10, GFP-PsZ1 C10 were incubated with liposomes or PBS (135 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) buffer for 1 h at room temperature.The mixtures were separated into supernatant and pellet with a centrifugation at 15,000 g for 10 min at room temperature, and then the pellet was washed with PBS for one time.The supernatant and pellet were probed by immunoblot with anti-GFP antibodies (Biodragon Beijing).
To analyze the relationship between membrane-binding ability and spiral density, proteins GFP, GFP-PpZ1B C10, GFP-AvcZ1A C10, GFP-PsZ1 C10 were expressed in E. coli BL21 strains in 20 mL LB medium with 50 mg•L −1 kanamycin, grown to an OD 600 value of ∼0.45 at 37 °C, and then induced with 0.1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 2 h at 20 °C to OD 600 value of ∼0.65.Bacterial cells of equal quantity were collected by centrifugation at 12,000 g for 1 min at room temperature, and resuspended with 500 μL TBS (20 mM Tris, 150 mM NaCl, pH 7.5), and then lysed for 2 min at room temperature after the addition of 50 μL lysozyme (ACE Biotechnology).Supernatants and pellets were separated with a centrifugation at 12,000 g for 5 min at room temperature, and then pellets were washed with TBS for one time.The supernatants and pellets were probed by immunoblot with anti-his antibodies (Jiaxuan biotech).

Chloroplast Phenotype Analysis
To observe the chloroplast phenotype, Arabidopsis leaves from 4-week-old plants were fixed with 3.5% glutaraldehyde in darkness for 1 h at room temperature.Then, the glutaraldehyde was replaced with 0.1 M Na 2 EDTA (pH = 9.0) and the samples were incubated in a 55 °C water bath for 2 h.The images were captured with an Olympus CX21 (Olympus, Tokyo) microscope coupled with a USB 2.0 digital camera (Changheng, Beijing).The number of chloroplasts per cell was counted and analyzed with Excel (Microsoft).

Immunoblot Analysis of Proteins in Transgenic Plants
Proteins were extracted from leaves of 4-week-old plants, and separated by SDS-PAGE gels.The total proteins were transferred to PVDF membrane (Bio-Rad), and then blocked with 5% (w/v) fat-free milk in TBST buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 2 h at room temperature.The membrane blot was probed with purified FtsZ1 antibodies with a dilution of 1:2500 for 1 h at room temperature, and then the secondary antibodies (Jiaxuan biotech) with a dilution of 1:10,000 for 1 h at room temperature.The signals were generated with an eECL Western Blot Kit (Beijing ComWin Biotech Company) and developed with a film.

MBEFig. 1 .
Fig. 1.Different sequence motifs were evolved at the C-terminus of FtsZ1 and FtsZ2 during plant evolution.a) Diagrams of AtFtsZ1 and AtFtsZ2 protein domains.The predicted transit peptide (TP) is shown in green, the core domain (CD) is shown in blue, the C-terminal motif of AtFtsZ1 is shown (Z1C) in red, and the C-terminal motif of AtFtsZ2 (Z2C) is shown in yellow.(b and c) Multiple sequence alignments of the C-terminal 30 amino acids (C30aa) from Cyanobacterial FtsZ and Rhodophyta FtsZA, alongside FtsZ1 b) and FtsZ2 c) across various species, including Chlorophytes, Charophytes, Bryophytes, Lycophytes, Pteridophytes, Gymnosperms, and Angiosperms, arranged from top to bottom.Red box b) and red triangle c) indicate the key amino acid phenylalanine f) important for the interaction with ARC6 (Maple et al. 2005; Zhang et al. 2016).

Fig. 2 .
Fig. 2. Green alga FtsZ1 C-terminal sequences have membrane-binding activity.a) GFP-AtFZ2-1 with a fusion of the C-terminal 10 amino acids of CreZ1, KniZ1 and CbrZ1 expressed in E. coli.Bars = 5 μm.b) GFP-AtFZ2-1 with fusion of longer sequences of the C-termini of CreZ1, KniZ1 and CbrZ1 formed helical structures in E. coli.Bars = 5 μm.c) Statistical analysis of the helical density of the FtsZ fusion proteins in a) and (b).t test, **P < 0.01.Error bars represent the mean ± SD.

Fig. 4 .
Fig. 4. Pattern diagram of helical structures formed by FtsZ fusion proteins expressed in E. coli.The morphology of FtsZ2-1 is altered into spiral structures due to the membrane-binding activity of FtsZ1 C-terminal motifs in various species.The characteristic of FtsZ spiral density was significantly changed in Charophytes and Pteridophyta during plant evolution (Figs. 2 and 3).

Fig. 5 .
Fig. 5.The C-terminal motifs of FtsZ1 in different species exhibit membrane-binding activity.a) Liposome co-precipitation with GFP, GFP-PpZ1B C10, GFP-AcvZ1A C10 and GFP-PsZ1 C10 in vitro.To analyze the membrane-binding activity of GFP, GFP-PpZ1B C10, GFP-AcvZ1A C10 and GFP-PsZ1 C10, the proteins were incubated with (+) liposomes or without (−) liposomes.Supernatant (S) and pellet (P) were separated by centrifugation.Immunoblots were analyzed by anti-GFP antibodies.b) Fractionation analysis of GFP fused with FtsZ C-termini from different plants.Proteins GFP, GFP-PpZ1B C10, GFP-AcvZ1A C10 and GFP-PsZ1 C10 were expressed in E. coli, and bacterial cells of equal quantity were lysed by lysozyme.Supernatant (S) and pellet (P) were separated by centrifugation.Immunoblots were analyzed by anti-his antibodies.Coomassie Brilliant Blue (CBB) staining was used as a loading control.

Fig. 7 .
Fig. 7. CreZ1 C10 has a function similar to AtFZ1 C10 in vivo.a) Chloroplast phenotype of wild type (WT), Atftsz1 and transgenic plants.Scale bar = 10 μm.All the images have the same magnification.b) Immunoblot analysis of FtsZ1 protein levels in WT, Atftsz1 and transgenic plants with anti-FtsZ1 antibodies.Coomassie Brilliant Blue (CBB) staining served as a loading control.c) Correlation between chloroplast number and cell area in WT, Atftsz1, and transgenic plants shown in a).The best-fit lines had slopes of 0.0166 (R 2 = 0.8633), 0.0017 (R 2 = 0.2697), 0.0137 (R 2 = 0.9486), and 0.012 (R 2 = 0.879) for the wild type, Atftsz1, and two transgenic lines, respectively.n > 30 cells for each sample.

Fig. 8 .
Fig. 8. PpZ1B C22 has a function similar to AtFZ1 C22 in vivo.a) Chloroplast phenotype in wild type (WT), Atftsz1 and transgenic plants.Scale bar = 10 μm.All the images have the same magnification.b) Immunoblot analysis of FtsZ1 level in WT, Atftsz1 and transgenic plants with anti-FtsZ1 antibodies.Coomassie Brilliant Blue (CBB) staining served as a loading control.c) Correlation between chloroplast number and cell area in WT, Atftsz1, and transgenic plants shown in a).The best-fit lines had slopes of 0.0139 (R 2 = 0.8836), 0.001 (R 2 = 0.1408), 0.0153 (R 2 = 0.9399), and 0.0133 (R 2 = 0.9423) for the wild type, Atftsz1, and two transgenic plant lines, respectively.n > 30 cells for each sample.