Progress 07/01/16 to 06/30/21
Outputs
Target Audience:The target audiences are primarily scientists and students in the academic and plant biotechnology sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training in a wide array of complementary techniques, scientific writing, and presentation skills to postdocs, a graduate student and undergraduates. The postdocs and graduate students gained experience in mentoring through their interactions with the undergraduates. The postdocs and graduate student presented their findings in oral and/or poster presentations at conferences, where they also attended workshops on emerging scientific techniques and career development. Undergraduate researchers were provided with opportunities to present their findings in laboratory meetings and in posters at the Michigan State University Undergraduate Research and Arts Forum. How have the results been disseminated to communities of interest?Results have been disseminated through published papers and through oral and poster presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Plastids are essential organelles in all plant cells. The green chloroplasts in leaves carry out the life-sustaining process of photosynthesis and also synthesize many molecules critical for plant growth and development, including fatty acids, amino acids, pigments and growth regulators. The proliferation of plastids occurs by the process of division; during leaf expansion this process results in a large increase in chloroplast numbers in most leaf cells, which maximizes plant growth and productivity. Additionally, metabolic engineering of chloroplasts is being increasingly employed for the manufacture of biopharmaceuticals and other nutritionally and economically important products in plants. Our research on this project has not only elucidated fundamental new knowledge on the mechanisms underlying chloroplast division, it has also suggested new ways of manipulating chloroplast size and shape, which may have utility in downstream bioengineering and agricultural applications. Therefore, in the long run, our work has potentially significant implications for advancing agriculture and biotechnology. Our research aims to understand the molecular mechanisms of chloroplast division. Work under this project, summarized below, has advanced knowledge of this fundamentally critical process. Aim 1: We conducted experiments in vitro and in a heterologous yeast system to test whether the distinct dynamic behaviors of Arabidopsis FtsZ1 and FtsZ2 extend to FtsZA and FtsZB from the red alga Galdieria sulphuraria. We found that FtsZA and FtsZB exhibit assembly and dynamic properties similar to those of FtsZ2 and FtsZ1, respectively. Additionally, FtsZ1 reduced assembly of FtsZ2 when mixed with FtsZ2 in vitro, consistent with its effect in promoting FtsZ2 subunit exchange from coassembled filaments in yeast. FtsZB had a similar effect on FtsZA. Further studies revealed that the destabilizing effect of FtsZ1 on FtsZ2 upon their coassembly is not due to an FtsZ1-mediated stimulation of GTPase activity, unlike in bacteria where increased Z-ring dynamics are typically associated with increased GTPase activity. Extending these experiments, we tested whether the stabilizing effects of FtsZ2 and FtsZA and destabilizing effects of FtsZ1 and FtsZB are conferred by the conserved core regions common to all FtsZ proteins, or whether they are conferred by the more divergent flanking regions. We found that, for both the Arabidopsis and red algal FtsZs, the core regions behaved mostly similarly to the full-length proteins both individually and together, suggesting the core regions of distinct FtsZ pairs possess unique structural features despite their high degree of sequence conservation. Taken together, this work has shown that chloroplasts in land plants and red algae, which are separated by over a billion years of evolution, both possess one FtsZ with exceptional stability and a second that counterbalances this stability, underscoring the importance of these complementary functions in the overall regulation of chloroplast FtsZ dynamics. Aim 2: We tested the hypothesis that ARC3 functions in part by enhancing Z-ring remodeling during chloroplast division, and that this activity is activated by interaction of ARC3 with the mid-plastid division protein PARC6. Using yeast three-hybrid assays, we found that ARC3 interacts with FtsZ only in the presence of PARC6. Complementary FRAP experiments showed that ARC3 promotes exchange of subunits from Z rings reconstituted in yeast cells, also only in the presence of PARC6. Analysis of mutant proteins demonstrated that interaction of ARC3 with both PARC6 and FtsZ requires a region of ARC3 called the MORN domain; such domains occur broadly in eukaryotes but are largely of unknown function. Our findings led to a new model whereby activation of midplastid-localized ARC3 by PARC6 facilitates Z-ring remodeling during chloroplast division by promoting Z-ring dynamics, and revealed a novel function for MORN domains in regulating protein-protein interactions. Finally, in a collaboration with Dr. Li Li, we discovered that the ARC3 MORN domain also interacts with ORHIS, a protein that promotes accumulation of orange carotenoid pigments in chromoplasts, and that this interaction interferes with the interaction between ARC3 and PARC6. These and related results revealed a new function for ARC3 and suggested that ARC3-ORHIS interaction may constrain chromoplast division, leading to reduced numbers of enlarged chromoplasts and increasing the sink for carotenoid accumulation. Aim 3: For technical reasons, the proposed assays to investigate binding affinities were not feasible. To circumvent this, we carried out yeast two-hybrid and three-hybrid experiments under a range of stringencies, which yielded consistent information on the relative binding affinities between different sets of proteins. For example, these approaches provided evidence that the ARC3 MORN domain binds PARC6 with a higher affinity than ARC6, which is important for understanding the functions of these interactions in the chloroplast division complex. Aim 4: To estimate molecular ratios of plastid division proteins during plant development, we carried quantitative immunoblot analysis of FtsZ1, FtsZ2-1 (one of two FtsZ2 isoforms), ARC6 and PARC6 in Arabidopsis. Equal amounts of total protein from extracts prepared from leaf 3 of 14-, 21- and 28-day-old plants were separated by SDS-PAGE and blots were probed with the relevant antibodies. The amount of AtFtsZ1, AtFtsZ2-1, ARC6 and PARC6 protein in each lane was determined by extrapolating from standard curves constructed from immunoblots of gels loaded with different amounts of the relevant purified recombinant protein to determine the linear ranges of detection. Molecular levels of the plastid division proteins were calculated based on molecular masses of the proteins without their predicted chloroplast transit peptides. For all four proteins, the highest levels were detected in 14 d-old plants. Levels declined 36-68% by day 21, with a less significant decline between days 21 and 28. The higher levels of proteins in 14 d-old plants is consistent with the larger number of dividing chloroplasts in expanding leaves. Although protein levels decreased from 14-28 days, the data showed that the number of PARC6 molecules was approximately 20% the number of ARC6 molecules at all three time-points, suggesting this represents their stoichiometry at the chloroplast division site. Based in part on our previously published estimates of total FtsZ2 levels in isolated chloroplasts, the data also suggested that the molar ratio between FtsZ2 and ARC6, which binds FtsZ2 but not FtsZ1, is approximately 1:1 at all three stages of development. These experiments thus yielded new quantitative insights on the molecular architecture of the chloroplast division complex.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Porter K, Cao L, Chen Y, Terbush AD, Chen C, Erickson HP, Osteryoung KW (2021) The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro. J Biol Chem 296: 100627
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audiences are primarily scientists and students in the academic and plant biotechnology sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdocs involved in this work interacted closely in designing and performing synergistic cell biological and biochemical experiments, giving them valuable experience in collaborative research. All have been mentored in written communication skills, data presentation, and the responsibilities of authors and coauthors. The PI regularly discusses career development and planning with her postdocs and as indicated above one successfully moved on during this reporting period to an independent faculty position. At the suggestion of the PI one postdoc presented a virtual poster at the Plant Biology 2020 Worldwide Summit sponsored by the American Society of Plant Biologists and participated in online workshops and events at the conference. This provided new experiences in online presentation and networking skills, which are becoming increasingly important. Two undergraduate students received research training during the current reporting period, working under the guidance of postdocs. Students read and discussed original literature with the PI and their mentors as part of their work and gave updates on their research in lab meetings. Although both had planned to give poster presentations at the Michigan State University Undergraduate Research and Arts Forum this past spring semester, Covid intervened. Instead they both prepared and enthusiastically presented their posters to our lab group via Zoom. The research exposed the students to new techniques, gave them experience in data analysis, interpretation and presentation, and enriched their understanding of the scientific research process. It also provided postdocs with important mentoring skills. How have the results been disseminated to communities of interest?A manuscript on work under objective 1 was prepared, submitted, and is under revision, and a poster was presented, as described in a previos section. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Plastids are essential organelles in all plant cells. The green chloroplasts in leaves carry out the life-sustaining process of photosynthesis and also synthesize many molecules critical for plant growth and development, including lipids, amino acids and growth regulators. Other types of plastids produce important agricultural compounds such as starch and the fatty acid precursors of oils. The proliferation of plastids occurs by the process of division; during leaf expansion this process results in a large increase in chloroplast numbers in most leaf cells, which maximizes plant growth and productivity. Division of plastids is also required for their propagation during plant reproduction. Additionally, metabolic engineering of chloroplasts is being increasingly employed for the manufacture of biopharmaceuticals and other nutritionally and economically important products in plants. Our research on this project is not only elucidating the fundamental mechanisms of plastid division in plants, but is also revealing new ways of manipulating chloroplast size and shape, which may have utility in downstream bioengineering and agricultural applications. Therefore, our work has potentially significant implications for advancing agriculture and biotechnology in Michigan and around the world. Objective 1: We wrote and submitted a manuscript on work described in last year's progress report on the assembly, GTPase activity and dynamic behavior of full-length Arabidopsis thaliana FtsZ1 (AtFtsZ1) and AtFtsZ2 and their respective core regions (AtFtsZ1core and AtFtsZ2core) assayed separately and together. Partly in response to reviewer feedback, we conducted the following additional experiments. We had reported assembly of AtFtsZ2 alone and in mixture with AtFtsZ1 in terms of the maximum signal in light scattering (LS) assays. To further characterize the assembly kinetics, we added data on the initial rates of assembly in all LS assays. The results showed that the extent and initial rates of assembly always followed the same trends. In mixing experiments, AtFtsZ1 reduced the rate and extent of assembly in a dose-dependent manner. Similar results were obtained with AtFtsZ1core and AtFtsZ2core. These results are consistent with our hypothesis that coassembly of AtFtsZ1 with AtFtsZ2 reduces overall protofilament stability, leading to increased dynamics. In vitro studies of bacterial FtsZs have shown that decreased protofilament assembly and stability are associated with higher GTPase activities. To determine whether mixing AtFtsZ1 with AtFtsZ2 might stimulate GTP hydrolysis, we measured specific GTPase activities at the same AtFtsZ2:AtFtsZ1 ratios used in the LS assays, but found they were only slightly higher than the specific activity of AtFtsZ2 alone, and lower than that of AtFtsZ1. Therefore, the reduced extent and initial rates of assembly in the mixtures are not explained by stimulation of specific GTPase activity. However, they could reflect reduced nucleotide availability in the presence of more total AtFtsZ, and hence faster GTP depletion. To address this possibility, we carried out additional LS experiments under conditions where GTP should remain at >38-fold excess throughout the assay. Decreases in protofilament abundance and initial assembly rates were still observed as the ratio of AtFtsZ1 was increased. These findings show that these decreases are not due to a stimulation of GTPase activity, but rather to a more direct effect of AtFtsZ1 on AtFtsZ2 assembly and protofilament dynamics. Towards defining the mechanism by which AtFtsZ1 promotes protofilament dynamics, we used LS assays to test the effect of adding AtFtsZ1 to preassembled AtFtsZ2. Experiments were performed in excess GTP to ensure that GTP was not depleted by hydrolysis during the period of monitoring. In a control assay where only buffer was added, a very slow decrease in LS was observed, likely due to the resulting dilution of AtFtsZ2. Addition of AtFtsZ1 produced a more rapid, yet gradual, decrease in LS. The gradual reduction in the LS signal suggests that as AtFtsZ2 subunits dissociate from protofilaments, their net reassembly back onto protofilaments is diminished due to interactionwith AtFtsZ1. In a previous FRAP study in yeast, we found that the ability of AtFtsZ1 to increase turnover of AtFtsZ2 from coassembled structures was dependent on AtFtsZ1 GTPase activity. Therefore, we asked if AtFtsZ1 activity is required for the reduced AtFtsZ2 assembly in vitro. To this end, we purified AtFtsZ1D275A, the same mutant used in the FRAP study, in which a highly conserved aspartate required for full GTP hydrolysis in bacterial FtsZs was altered. The activity of AtFtsZ1D275A was about 16% that of AtFtsZ1. AtFtsZ1D275A behaved similarly to AtFtsZ1 in that assembly on its own was not evident by sedimentation or LS assays. However, increasing AtFtsZ1D275A in mixture with AtFtsZ2 neither decreased the proportion of AtFtsZ2 in the pellet fraction in sedimentation assays as did AtFtsZ1, nor did it decrease assembly to the same extent as AtFtsZ1 in LS assays. These data show that AtFtsZ1 requires its GTPase activity to constrain the assembly of AtFtsZ2. The results of this work represent the first comparative in vitro analysis of the mature, soluble forms of chloroplast FtsZ1 and FtsZ2 alone and in mixture. The most important conclusions are that AtFtsZ2 assembles exceptionally stable protofilaments on its own, and that AtFtsZ1 complements this stability by reducing the rate and extent of assembly in a manner that does not entail an increase in overall GTPase activity. These findings provide new evidence that restraining the self-assembly potential of FtsZ in chloroplasts is a critical function of FtsZ1. Our results also reveal that the distinct attributes and complementary functions of AtFtsZ1 and AtFtsZ2 are conferred largely by the conserved core regions common to all FtsZ proteins. In complementary experiments building on data reported last year, we continued FRAP analysis of fluorescent fusion proteins to probe the dynamics of filaments and rings assembled by full-length Galdieria sulphuraria (Gs) FtsZA and GsFtsZB and their conserved core regions in Pichia for comparison to the Arabidopsis proteins. Previously we showed that GsFtsZA and GsFtsZAcore filaments are less dynamic than GsFtsZB and GsFtsZBcore filaments, respectively. The effect of coassembly has now been tested by expressing both sets of proteins in the same cells. FRAP showed that GsFtsZB and GsFtsZBcore increased turnover of GsFtsZA and GsFtsZAcore , respectively, from coassembled filaments and rings. These findings strongly suggest that FtsZB promotes FtsZ dynamics in red algal chloroplasts, similar to the effect of FtsZ1 in green plants, and that the distinct but complementary properties of GsFtsZA and GsFtsZB are determined largely the conserved core regions. Together, our results also lay the groundwork for defining the structural properties of the conserved core regions responsible for their distinct roles in regulating chloroplast Z-ring dynamics. Objectives 2 and 3: We confirmed preliminary data reported last year that ARC3 interacts with the chloroplast inner envelope protein ARC6, continued experiments to test the relative binding affinities between ARC3 and several other chloroplast division proteins, and began experiments to identify regions of ARC6 responsible for its interaction with ARC3. This work was interrupted by the departure of a postdoc for an independent faculty position, and by the lab shut-down due to the pandemic. Objective 4: This work was mostly completed during a previous reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Sun T, Yuan H, Chen C, Kadirjan-Kalbach DK, Mazourek M, Osteryoung KW, and Li L. 2020. ORHis, a natural variant of OR, specifically interacts with plastid division factor ARC3 to regulate chromoplast number and carotenoid accumulation. Mol. Plant 13: 864-878
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Coa L, Porter KP, Jennings EJ, Osteryoung KW. The conserved core regions of FtsZ proteins determine their distinct functions in chloroplast division. Virtual poster presentation, Plant Biology 2020, July 27-31, 2020.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audiences are primarily scientists and students in the academic and plant biotechnology sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training in a wide array of complementary techniques, scientific writing, and presentation skills to postdocs, a graduate student and undergraduates. The postdocs and graduate students gained experience in mentoring through their interactions with the undergraduates. A postdoc and graduate student presented their findings in oral and/or poster presentations at conferences, where they also attended workshops on emerging scientific techniques and career development. Undergraduate researchers were provided with opportunities to present their findings in laboratory meetings and in posters at the Michigan State University Undergraduate Research and Arts Forum. How have the results been disseminated to communities of interest?Results have been disseminated through published papers and through oral and poster presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?Described under accomplishments.
Impacts
What was accomplished under these goals?
Impact: Plastids are essential organelles in all plant cells. The green chloroplasts in leaves carry out the life-sustaining process of photosynthesis and also synthesize many molecules critical for plant growth and development, including lipids, amino acids and growth regulators. Other types of plastids produce important agricultural compounds such as starch and the fatty acid precursors of oils. The proliferation of plastids occurs by the process of division; during leaf expansion this process results in a large increase in chloroplast numbers in most leaf cells, which maximizes plant growth and productivity. Division of plastids is also required for their propagation during plant reproduction. Additionally, metabolic engineering of chloroplasts is being increasingly employed for the manufacture of biopharmaceuticals and other nutritionally and economically important products in plants. Our research on this project is not only elucidating the fundamental mechanisms of plastid division in plants, but is also revealing new ways of manipulating chloroplast size and shape, which may have utility in downstream bioengineering and agricultural applications. Therefore, our work has potentially significant implications for advancing agriculture and biotechnology in Michigan and around the world. Aim 1. Current work on chloroplast FtsZ pairs from evolutionarily divergent organisms is aimed at understanding how these copolymerizing proteins function together to control the constriction and dynamics of the contractile FtsZ ring (Z ring) during chloroplast division. Although the chloroplast FtsZ genes descended from an FtsZ homolog in the cyanobacterial ancestor of chloroplasts, the Z ring in cyanobacteria and other bacteria is composed of a single FtsZ protein whereas the chloroplast Z ring is composed of two distinct forms of FtsZ that copolymerize. Our previous work has revealed that FtsZ2 from Arabidopsis and other green-lineage organisms imparts stability to the FtsZ heteropolymers that form the Z ring, while FtsZ1 enables the Z ring to be dynamic.We have also shown a similar relationship between FtsZA and FtsZB from the red alga Galdieria sulphuraria. All FtsZ proteins possess a highly conserved core region responsible for GTP binding and hydrolysis as well as GTP-dependent polymerization. The core is flanked by more variable N- and C-terminal regions. Towards understanding the contributions of the core and flanking regions to the distinct functions of the two FtsZs in chloroplasts, we expressed and purified variants of Arabidopsis FtsZ1 and FtsZ2 composed of only the core regions (AtFtsZ1core and AtFtsZ2core), and compared their in vitro biochemical and dynamic properties to those of the full-length mature proteins (AtFtsZ1 and AtFtsZ2), which lacked the transit peptides. Specifically, we compared the GTPase activities, critical concentrations (Ccs) for assembly based on GTPase activities because hydrolysis is assembly-dependent, and assembly behavior based on light scattering (LS), sedimentation assays and transmission electron microscopy (TEM). AtFtsZ2 and AtFtsZ2core exhibited GTP-dependent assembly in all assays. The extent of assembly depended on both protein and nucleotide concentration. TEM showed that AtFtsZ2 and AtFtsZ2core both assembled bundled protofilaments, but AtFtsZ2core protofilaments were more loosely bundled and their bundles were significantly thinner than AtFtsZ2 bundles. Neither the GTPase activity nor Cc differed significantly between AtFtsZ2 and AtFtsZ2core. In contrast, we could not detect assembly of AtFtsZ1 or AtFtsZ1core by LS, sedimentation or TEM, but both proteins hydrolyzed GTP, indicating they assembled minimally as dimers. The GTPase activity of AtFtsZ1core was about half that of AtFtsZ1. However, AtFtsZ1 GTPase did not display a Cc (i.e, the Cc was ~0), suggesting it may be a dimer in solution, while AtFtsZ1core displayed a distinct Cc. The latter results suggest a lower affinity between AtFtsZ1core than AtFtsZ1 subunits. In mixing experiments, AtFtsZ1core reduced the stability and increased the dynamics of protofilaments when coassembled with AtFtsZ2core, similar to the effects observed upon coassembly of AtFtsZ1 and AtFtsZ2. LS and TEM showed that assembly of both AtFtsZ2 and AtFtsZ2core alone or in mixture with AtFtsZ1 and AtFtsZ1core, respectively, persisted well past the predicted time of GTP depletion, indicating that disassembly is not as tightly coupled to GTP hydrolysis as it is for bacterial FtsZs, and that chloroplast FtsZ protofilaments are considerably more stable than bacterial protofilaments. To complement in vitro experiments, we used fluorescence recovery after photobleaching (FRAP) to compare the dynamics of the AtFtsZ and AtFtsZcore proteins bearing fluorescent tags when expressed individually in the heterologous yeast Pichia pastoris. We also carried out similar experiments with the G. sulphuraria (Gs) proteins. All fusion proteins assembled fluorescent ring-like structures in Pichia. FRAP experiments showed that AtFtsZ1core and GsFtsZBcore filaments were more dynamic than AtFtsZ2core and GsFtsZAcore filaments, respectively, similar to the relationships observed between the full-length proteins. Taken together, the combination of in vitro experiments and FRAP analysis in yeast has revealed that the distinctive effects of the chloroplast FtsZs on the dynamic behavior of coassembled protofilaments are determined largely by their core regions, suggesting unique structural features in these regions despite their high degree of conservation. However, the results also suggest that the flanking regions enhance the GTPase activity of AtFtsZ1 and promote the bundling of AtFtsZ2 protofilaments. Future experiments will build on these findings to identify the features responsible for the distinct roles of the two chloroplast FtsZs in regulating protofilament and Z-ring dynamics. Aim 2. We wrote, submitted and published a manuscript on the results described in last year's progress report on the recruitment of ARC3 to the chloroplast division site by PARC6, and the importance of this interaction for activating ARC3-FtsZ interaction to promote Z-ring remodeling (Chen et al 2019). While conducting these experiments it became apparent that a protein in addition to PARC6 might also recruit ARC3 to the division site; we hypothesized that this protein might be ARC6, a homolog of PARC6, even though published data reported that ARC3 and ARC6 do not interact. We began testing this hypothesis using a combination of yeast two-hybrid and three-hybrid assays and discovered that ARC3 actually does interact with ARC6, and that this interaction similarly influences binding of FtsZ to ARC3. The interaction data also suggest that PARC6 and ARC6 bind competitively to the same region of ARC3, but that a region of ARC6 that is not conserved in PARC6 influences ARC3-ARC6 interaction. Work in the next reporting period will focus on verifying and expanding on these data and testing the significance of this newly identified interaction in vivo. Aim 3. The experiments performed under Aim 2 have also yielded important new information on a variety of interactions within the chloroplast division complex. For technical reasons, the proposed surface plasmon resonance assays to investigate binding affinities were not feasible. To circumvent this, we have carried out yeast two-hybrid and three-hybrid experiments under a range of stringencies, which is providing valuable and consistent information on the relative (if not absolute) binding affinities between different sets of proteins. For example, these approaches suggest that the ARC3 binds ARC6 more weakly than PARC6, which has implications for understanding the functions of these interactions during chloroplast division. Work in the next reporting period will continue exploring relative interaction strengths towards understanding more fully the dynamic operation of the division complex.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chen C, Cao L, Yang Y, Porter K, Osteryoung KW (2019) ARC3 Activation by PARC6 Promotes FtsZ-Ring Remodeling at the Chloroplast Division Site. Plant Cell 31: 862885
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kadirjan-Kalbach DK, Turmo A, Wang J, Smith BC, Chen C, Porter K, Childs K, DellaPenna D, Osteryoung KW (2019) Allelic Variation in the Chloroplast Division Gene FtsZ2-2 Leads to Natural Variation in Chloroplast Size. Plant Physiol pp.00841.2019. DOI: https://doi.org/10.1104/pp.19.0084
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Cao L, Schmitz AJ, Osteryoung KW. The Conserved Core Regions of FtsZ Proteins Determine their Distinct Dynamics in Chloroplast Division. Oral and poster presentation, Plant Cell Dynamics Meeting, June 18-21 , 2019, University Park, Pennsylvania.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Graham G, Jennings EJ, Osteryoung KW. Determining the Interaction Ratios of FtsZ Proteins During Chloroplast Division. Poster presentation, Michigan State University Undergraduate Research and Arts Forum, April 5, 2019, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Jennings EJ, Buell CR, Osteryoung KW. Identifying regulators of organelle size using the tropical plant genus Peperomia. Poster presentation, Plant Biology 2019, August 3-7, San Jose, California.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Jackson S, Porter KJ, Chen C, Osteryoung KW. Investigation of Interaction of Intra-Membrane Chloroplast Division Protein ARC6 With Stromal Protein ARC3 Via Yeast Two-Hybrid. Poster presentation, Michigan State University Undergraduate Research and Arts Forum, April 5, 2019, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Naeem M, Chen C, Osteryoung KW. The Effect of ARC6 on the Interaction Between ARC3 and FtsZ. Poster presentation, Michigan State University Undergraduate Research and Arts Forum, April 5, 2019, East Lansing, Michigan.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audiences are primarily scientists and students in the academic and plant biotechnology sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training in a wide array of complementary techniques, scientific writing, and presentation skills to postdocs. Undergraduate researchers who have participated were provided with opporunities to present their findings in posters at the Michigan State University Undergraduate Reserach and Arts Forum. How have the results been disseminated to communities of interest?Results have been disseminated through published papers and conference presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Chloroplasts arose from a free-living cyanobacterium and carry out the life-sustaining process of photosynthesis. Plastids also synthesize many compounds critical for plant growth and development, including lipids, amino acids and growth regulators, and specialized plastid types manufacture products of major agricultural importance, such as oil, starch and vitamins. The propagation of plastids occurs by division of pre-existing organelles. During leaf expansion this process results in a developmentally programmed increase in chloroplast numbers and compartment size, which maximizes photosynthetic productivity. Chloroplasts are also being increasingly exploited as factories for the production of biopharmaceuticals and other economically important products in plants, and our work is leading to ways of manipulating chloroplast size and shape, which may have utility in downstream bioengineering and agricultural applications. Therefore, our research on the fundamentally critical process of chloroplast division in plants has potentially significant implications for agriculture and biotechnology in Michigan and around the world. We made significant progress during the current reporting period primarily on aims 1, 2 and 4. Aim 1. We are using the yeast Pichia pastoris as a heterologous system in which to study chloroplast Z-ring dynamics. Fluorescent fusions for expression in Pichia were constructed for FtsZ2 and FtsZ1 from the monocot Oryza sativa (Os) and FtsZA and FtsZB from the red alga Galdieria sulphuraria (Gs). Fusions were constructed either with or without a membrane tethering sequence (MTS) under control of a promoter that permits protein expression at either moderate or high levels. We also made an FtsZ1 construct without an MTS for the green alga Ostreococcus tauri (OtFtsZ1); several other constructs are also being generated. For evolutionary comparison, similar constructs were produced for the single FtsZs from the glaucophyte alga Cyanophora paradoxa (Cp) and the cyanobacterium Synechococcus elongatus (Se). Following induction at moderate levels, epifluorescence microscopy showed that the majority of the fusion proteins, including OtFtsZ1, GsFtsZB and SeFtsZ without the MTS, formed closed rings. In our previously published results for the individual Arabidopsis thaliana (At) proteins, only AtFtsZ2-MTS formed rings in Pichia (Yoshida et al. 2016, Nat Plants 2:16095). Our new results indicate variability in the capacity for different FtsZs to assemble rings in Pichia, but suggest a general tendency for individual FtsZs to form ring-like structures whether or not they are membrane-tethered. To begin addressing our hypothesis that Z-ring size in Pichia reflects the size of the organelle or cell, we carried out initial measurements of Z-ring circumference under moderate-expression conditions in which rings do not constrict. The circumferences of CpFtsZ, GsFtsZA and GsFtsZ-MTS rings were somewhat similar those of the plastids in C. paradoxa (~2 μm) and G. sulphuraria (~6 μm), respectively. However, SeFtsZ and SeFtsZ-MTS rings were considerably larger than the circumference of S. elongatus cells (~5 μm). These data hint that there may not be a strict correlation between Z-ring size in Pichia and cell or organelle size. Additional measurements will be performed. To investigate whether the Z rings formed in Pichia constrict, FtsZ proteins were induced to high levels under conditions that resulted in constriction of AtFtsZ2-MTS rings (Yoshida et al. 2016, Nat Plants 2:16095). None of the ring-forming constructs lacking the MTS constricted. Further, while OsFtsZ1-MTS and GsFtsZB-MTS also formed rings that were presumably membrane-tethered, neither constricted. Constriction was only observed for OsFtsZ2-MTS, GsFtsZA-MTS, and SeFtsZ-MTS rings. These results preliminarily suggest that chloroplast Z-ring constriction requires both a more ancestral form of FtsZ (FtsZA or FtsZ2) and tethering to a membrane. Additionally, we are currently generating Pichia constructs for coexpression of plant and algal FtsZ pairs at different ratios to examine the effect of FtsZ1 and FtsZB on the rate of Z-ring constriction and on the dynamic remodeling of Z rings based on fluorescence recovery after photobleaching (FRAP) measurements. Aim 2. We hypothesized that the FtsZ-assembly inhibitor ARC3 functions in part by enhancing Z-ring remodeling during chloroplast division, and that this activity is activated by interaction of ARC3 with the mid-plastid division protein PARC6. We carried out a battery of experiments towards testing this hypothesis. We completed yeast three-hybrid assays begun last year that enabled us to test interactions between three proteins, and found that ARC3 could interact with FtsZ only in the presence of PARC6. Further, we established some of the structural requirements for ARC3-PARC6 interaction. We also generated a series of constructs to test the effects of ARC3 and PARC6 on remodeling of Z rings reconstituted from Arabidopsis FtsZs in Pichia cells. Consistent with our hypothesis, FRAP analysis showed that ARC3 promotes exchange of subunits from reconstituted Z rings, but only when coexpressed with PARC6. We are preparing a manuscript describing these and related findings, which will be completed and submitted in the next reporting period. Aim 4. To begin estimating molecular ratios of plastid division proteins during plant development, we carried quantitative immunoblot analysis of FtsZ1, FtsZ2-1 (one of two FtsZ2 isoforms), ARC6 and PARC6 in Arabidopsis. Extracts were prepared from leaf 3 of 14-, 21- and 28-day-old plants and protein concentrations were determined. Equal amounts of total protein were run on SDS gels (two replicates per gel) and blots were probed with the relevant antibodies. The amount (ng) of AtFtsZ1, AtFtsZ2-1, ARC6 and PARC6 protein in each lane was determined by extrapolating from standard curves constructed from immunoblots of gels loaded with different amounts of the relevant purified recombinant protein to determine the linear ranges of detection by chemiluminescence. Preliminary analyses were performed to determine amounts of leaf extract that yielded signals within the linear range for each antibody. Molecular levels of the plastid division proteins were calculated based on molecular masses of the proteins without their predicted transit peptides. For all four proteins, the highest levels were detected in 14 d-old plants. Levels declined 36-68% by day 21, with a less significant decline between days 21 and 28. The higher levels of proteins in 14 d-old plants correlates with the larger number of dividing chloroplasts in expanding leaves. Although protein levels decreased from 14-28 days, the data show that the number of PARC6 molecules is approximately 20% the number of ARC6 molecules at all three time-points, suggesting this represents their stoichiometry at the chloroplast division site. Based on the current measurements of FtsZ2-1 levels and our previous estimates of both FtsZ2-1 and FtsZ2-2 levels in isolated chloroplasts from leaves of whole rosettes (McAndrew et al 2008, Biochem J 412: 367-378), the data also suggest that the molar ratio between FtsZ2 and ARC6, which specifically binds FtsZ2, is approximately 1:1 at all three stages of development. These experiments are thus beginning to add quantitative information on the molecular architecture of the chloroplast division complex. These experiments will be repeated in the next reporting period with added measurements of levels of FtsZ2-2 as well as other chloroplast division proteins.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Chen C, MacCready JS, Ducat DC, Osteryoung KW. 2017. The molecular machinery of chloroplast division. Plant Physiol. 176: 138151.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Sung MW, Shaik R, Terbush AD, Osteryoung KW, Vitha S, Holzenburg A (2018) The chloroplast division protein ARC6 acts to inhibit disassembly of GDP-bound FtsZ2. J Biol Chem 293: 1069210706
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audiences are primarily scientists and students in the academic and plant biotechnology sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results have been disseminated through published papers, conference presentationsand seminars. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Chloroplasts arosefrom a free-living cyanobacterium and carry out the life-sustaining process of photosynthesis. Plastids also synthesize many compounds critical for plant growth and development, including lipids, amino acids and growth regulators. Specialized plastid types manufacture products of major agricultural importance, such as oil and starch. The propagation of plastids occurs by division of pre-existing organelles. During leaf expansionthis process results in a developmentally programmed increase in chloroplast numbers and compartment size, maximizing photosynthetic productivity. Chloroplasts are also being increasingly exploited as factories for the production of biopharmaceuticals and other economically important products in plants, and our work is leading to ways of manipulating chloroplast size and shape, which may have utility in downstream bioengineering and agricultural applications. Therefore, our research on the fundamentally critical process of chloroplast division in plants has potentially significant implications for agriculture and biotechnology in Michigan and around the world. During the reporting period, we demonstrated through in vitro biochemical assays and ex vivo experiments in yeast that the FtsZA and FtsZB from the red alga Galdieria sulphurariahave properties similar to those of FtsZ2 and FtsZ1, respectively, in higher plants, i.e., that FtsZA provides the structural framework for the FtsZ ring while FtsZB promotes FtsZ ring remodeling and constuction by making FtsZ filaments more dynamic. We prepared and published two papers on these findings. We showedthere are two pools of ARC3 that function distinctly during chloroplast division in Arabidopsis. One is distributed evenly across the chloroplast stroma and probablyprevents FtsZring formation at non-division sites. A second pool colocalizes with the FtsZ ring at the division site, where its assembly-inhibitory activity likely accelerates Z-ring remodeling during chloroplast division. Recrutiment of the latter pool to the division site requiresthe FtsZ-interacting membrane proteinPARC6.ARC3 bears a C-terminal domain called the MORN domain that inibits its interaction with FtsZ but is required for its interaction with PARC6. During the reporting period, weshowed that PARC6 allows full-length ARC3 bearing the MORN domain to interact with FtsZ proteins in a yeast three-hybrid system, suggesting that PARC6 sequesters the MORN domain, enabling ARC3-FtsZ interaction. We further showed that full-length ARC3 inhibits assembly of FtsZ filaments in a heterologous yeast system only in the presence of PARC6.Our results suggest that PARC6 has at least two functions at the division site: it acts as a scaffold to bring ARC3 and FtsZ into close proximity, andit activates the inhibitory activity of ARC3 on Z-ring assembly by sequestering the MORN domain to accelerate Z-ring remodeling.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Terbush AD, MacCready JS, Chen C, Ducat DC, Osteryoung KW (2017) Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages. Plant Physiol pp.00558.2017. doi: 10.1104/pp.17.00558
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Chen Y, Porter K, Osawa M, Augustus AM, Milam SL, Joshi C, Osteryoung KW, Erickson HP (2017) The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments. J Biol Chem 292:52075215. doi: 10.1074/jbc.M116.767715
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Chen C, MacCready JS, Ducat DC, Osteryoung KW (2018) The molecular machinery of chloroplast division. Plant Physiol (under review)
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Dutta S, Cruz JA, Imran SM, Chen J, Kramer DM, Osteryoung KW (2017) Variations in chloroplast movement and chlorophyll fluorescence among chloroplast division mutants under light stress. J Exp Bot. doi: 10.1093/jxb/erx203
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Progress 07/01/16 to 09/30/16
Outputs
Target Audience:Scientists interested in plants, photosynthesis and organelles Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The work has provided training in scientific writing skills, imaging technology and phylogentic analysis to a postdoc. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Fluorescent fusions of FtsZ protein from diverse photosynthetic organisms (FtsZ2 and FtsZ1 from land plants and green algae, FtsZA and FtsZB from a red alga, and the single FtsZs from a glaucophyte alga and a cyanobacterium) were expressed in the yeast Schizosaccharomyces pombe to compare their intrinsic assembly and dynamic properties. Filament morphologies for FtsZ1 and FtsZB, and for FtsZ2 and FtsZA, were conserved across broad evolutionary distances. FtsZ pairs from plants and algae colocalized, suggesting their coassembly. Fluorescnce recovery after photobleaching experiments demonstrated that subunit exchange is greater from FtsZ1 and FtsZB filaments than from FtsZ2 and FtsZA filaments, that FtsZ2 and FtsZA turnover is greatly increased when they are coassembled with FtsZ1 and FtsZB, respectively, and that GTPase activity is critical for promoting turnover of FtsZ2 and FtsZA filaments, but not FtsZ1 or FtsZB. Glaucophyte and cyanobacterial FtsZs displayed filament assembly and turnover properties more similar to those of FtsZ2 and FtsZA. The results suggest that the distinct properties previously described for Arabidopsis FtsZ1 and FtsZ2 extend to diverse FtsZ pairs and demonstrate that the more ancestral FtsZ2 and FtsZA isoforms have retained functional attributes of their common FtsZ ancestor while FtsZ1 and FtsZB have acquired new but similar dynamic properties in the green and red lineages, respectively, through convergent evolution. A manuscript on these results is in preparation.
Publications
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