Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis

  1. Christian Wiese
  2. Miriam Abele
  3. Benjamin Al
  4. Melina Altmann
  5. Alexander Steiner
  6. Nils Kalbfuß
  7. Alexander Strohmayr
  8. Raksha Ravikumar
  9. Chan Ho Park
  10. Barbara Brunschweiger
  11. Chen Meng
  12. Eva Facher
  13. David W. Ehrhardt
  14. Pascal Falter-Braun
  15. Zhi-Yong Wang
  16. Christina Ludwig
  17. Farhah F. Assaad

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Abstract

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK–TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

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  1. Version published to 10.1083/jcb.202311125
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

    In the manuscript "Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis" the authors investigate the TRAPPII interactome carried out by an already published IP-MS screen. They study previously identified shaggy-like kinases SK as TRAPII interactors and the phosphorylation sites by Y2H (interactions of wild type, deletion mutants and phosphomutants) and kinase assays (in vitro) and pharmacological inhibition in the subunit AtTRS120. The authors provide a deeper phenotypical analysis of trapii null mutant lines and classification as "decision mutants", based on "limited budget" and "conflict of interest" experiments (previously described) as a starting point of investigations of TGN function in comparison with hormone mutants. Cell elongation is used as a response phenotype. Authors focus on mainly TRS120 and phosphorylation by SK and partly on another TRAPP component, CLUB. Authors study the assays with differing kinases, e.g. Y2H with BIN2, phosphorylation with SK11.

    Major comments:

    • Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    A major issue is that new claims and conclusions are not supported by the new data provided here. The title says "in Arabidopsis", but only components are from Arabidopsis. Interactions, instead, are studied in this manuscript by Y2H in yeast, and phosphorylation in vitro. The abstract is very misleading and does not distinguish which aspects are studied in vitro and which in vivo. The Abstract does not mention that this study is based on previously identified interactome data.

    o The figure legends are often not sufficiently detailed to understand what exactly is represented.

    Therefore, it is not possible to judge in every case whether experiments are supported by data. E.g.

    Fig. 1: A, describe which data were used and which control for IP-MS had been taken into account. B, this is a plot, please describe what is represented. Explain better why Shaggy kinases were chosen. C, explain the principle and what is represented. How is this experiment controlled and how is it ensured that negative results are not caused by absent proteins.

    Fig. 2: Indicate the phosphorylation sites in the other subfigures. Fig. 2E: How was it generated, explain what is seen. Since this is the only figure illustrating the protein complex of TRAPP, this figure should be more thoroughly prepared and labeled. I recommend a better visualized protein complex. As before, Fig. 2F remains unclear.

    Fig. 3: Please add a figure illustrating the mutations. 3C: what has been diluted? Other examples are found in other figures.

    Fig. 4: Shouldn't the wild type be compared with all the mutants? Then statistics have to be conducted accordingly. Better explain G and H. If there are quotients, explain of what exactly.

    Fig. 5. Same as before. How do I see that there is a phenotype? There is no comparison with wild type. It is also unclear to which values the statistics refer to.

    Fig. 6: Please guide the reader through the figure and experiment.

    Fig. 8: I miss the connection with other shaggy-like kinases. This summary could be more complete. What about phosphorylation sites?

    o Line 133-134: "we focus on the TRAPPII complex as a starting point as it is required for all aspects of TGN function, including the sorting of proteins such as PINs to distinct membrane domains" I did not find an obvious connection to the PIN transporters as well as clear data to TGN functions. This sentence was for me misleading about the context of this manuscript.

    o Figure 1C: A supporting Western Blot control is needed, to fully validate the missing interaction of BIN2 with the truncated variants of TRS120 and CLUB. Additionally, swapping the constructs from DB to AD and vice versa will provide a better set-up of the interaction screen. This should be easily done in a few weeks.

    o Line 431-432: "This presents intriguing implications regarding the potential role of the AtSK-TRAPPII module in meeting the unique demands of endomembrane traffic in plants." Why do the authors come to this assumption? Further discussion is needed here.

    o Figure 2F: What serves as positive controls? What is the purpose of showing every panel between each TRS120-T2 variant with CLUB-C2, CLUB-C3, TRS120-T1 and TRS120-T3 and not only interactions between BIN2 and the TRS120-T2 variants? Why are there six negative controls as it is every time the same control?

    • Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether.

    Clearly, title, abstract and statements have to be formulated differently. The discussion should contain a limitations paragraph in which the authors detail that conclusions are based on in vitro, yeast and plant IP-MS screening data, and they should describe approaches how the study can be continued in the future. Which alternative explanations are possible. Are SKs and TRAPP expressed and present in the same locations?

    • If you have constructive further reaching suggestions that could significantly improve the study but would open new lines of investigations, please label them as "OPTIONAL".
    • Demonstrating interactions and phosphorylation by other approaches in vivo
    • demonstrating effects of TRAPP phosphomutants and lack of kinases in vivo
    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.
    • Are the data and the methods presented in such a way that they can be reproduced?

    o Figure S9: Is it not a loss of chlorophyll instead of GFP? Does not look like a fluorescent image.

    o Lacking information of pH of in vitro kinase assay solution with Mass-spectrometry.

    o What is the purpose of transferring 10 days old seedlings to fresh plates for scanning? Needs additional information for understanding, at the moment it sounds more like unnecessary extra stress for the seedlings.

    o Why are seedlings grown under constant light?

    • Are the experiments adequately replicated and statistical analysis adequate?

    o Figure 5: It will be good to use ANOVA for statistics here. I personally doubt the high significance of some parameters, e.g. for club-2 cell width and cell surface area between dark and darkW due to the high standard errors. Rechecking with the original values is necessary. Why is there no comparison between wild-type and the two mutants?

    o Figure 7A - C, statistic is probably not correct. For example: in A statistical differences with P<0.001 between wild-type (~100 %) and TRS120SαβγD (~80 %), in C statistical difference of only P<0.05 between wild-type type (90 %) and TRS120SαβγD (60 %)

    o No information on IP-MS replicate numbers mentioned.

    o Also see comments above to figures

    Minor comments:

    • Specific experimental issues that are easily addressable.
      • Specific experimental issues that are easily addressable.

    o Figure 4, S7, S8, S11 and S12: It will be helpful to support the data with images of the seedlings. - Are the text and figures clear and accurate? Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    o The introduction is quite lengthy with unnecessary information, e.g. about PIN transporters, but useful information about shaggy-like kinases and connection to brassinosteroid signaling is lacking.

    o Figure 1C: In the figure legend is no explanation of abbreviation "Co"; no explanation of BET3, TRS31, Tca17 and TRIPP; no indication that spots come from different plates (just visible by different brightness of the squares). Why are there eleven negative controls as it is every time the same control?

    o Figure 1C is specifically for BIN2, but BIN2 was not identified in the IP-MS screen represented in Figure 1B. Why does 1C not focus on SK11/12/32, identified in 1B?

    o Figure 1C shows several truncated variants of TRS120 and CLUB, a schematic overview as represented in Figure 2A will be helpful for the understanding of 1C. Order of variants should be the same (now: in 1C first TRS than CLUB in 2A first CLUB than TRS).

    o Figure 1C: Interaction of TRS120 full-length with BIN2 is missing in this figure but is presented in Figure 2F.

    o Result of Figure 2F is described after Figure 3. Better arrangement of Figures or text is needed here.

    o Figure 3A: Why was AtSK11 and not BIN2 used for the main figure? Better change Figure 3A with Figure S4 to keep the focus on BIN2. No explanation of the result in the text.

    o Figure 3A: In the figure legend is no explanation of abbreviation CBB. What are the non-phosporylated variants? Where are they shown? Description sounds that only TRS120-T2-SαβγA versus TRS120-T2 WT was tested by t-test is this correct? And if yes, why?

    o No need for Figure 3B, information was already given in Figure 2A + B.

    o Figure 3C: Why BIL2 for Clade II and not BIN2?

    o Figure 4: Why are A-E not directly compared to wild-type but trs120-4 as seen in 4F? What is the purpose of using different types of diagram?

    o Figure 4H: Why are phyAphyBcry1cry2 and pyrpyl1pyl2pyl4 depicted? No description in the text.

    o Figure 6: Confusing order of given information in the figure legend. Sentence one belongs to D and H only, second sentence describes whole figure. o Figure 6D + H, color difference between black and blue is hard to see, better change one into e.g. red.

    o Figure 7D - F wrong indication of D to F, named in the description as A) - C). Why is E different to D in F (D and F: 0-1 is attenuated, >1 enhanced; E the other way around).

    o Figure S9A: Indication of protein size on the Coomassie gel is missing and the respective position of 160 kDa is not visible on the gel.

    o Figure S12D: No explanation of the color code in the figure legend.

    o Consistent labelling and layout of all Figures and Supplemental Figures will be helpful. E.g., Figure 3A and S4; in S8A-E + S11A-C bars of different conditions have the same color. Most of the figure legends are quite shortly described and lack information about what kind of data is presented.

    o YFP parameters are described in material and methods, but no YFP construct appeared in the manuscript to my knowledge.

    • Are prior studies referenced appropriately?
      • Lines 243-245: Text is nearly identical to Kalbfuß et al., 2022.
      • Lines 246-254: Text is identical to Kalbfuß et al., 2022.
    • Are the text and figures clear and accurate?

    Please see the above and below comments to figures and figure legends.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Overall, the manuscript may have very interesting data and new findings. It is very interesting that the authors study the regulation of a protein complex that may mediate environment responses and intracellular Golgi functions. However, it is very difficult to follow and understand the ideas and concept of the manuscript. This manuscript is based on a previously published interactome study by Rybek et al. 2014, Steiner et al. 2016, Kalde et al. 209. Moreover, a physiological approach is published in Kalbfuß et al. 2022. The outcomes and conclusions from these previously published manuscripts and the emanating open questions addressed here should be clearly described in the introduction. This is currently not the case. Moreover, many experimental approaches and results (e.g. figures, figure legends) are not properly described. Overall, it is therefore not possible to understand the manuscript without studying in depth all other manuscripts. Before the manuscript can be more thoroughly judged, it is necessary that the authors rewrite the manuscript, reorganize it and explain better their ideas and approaches. It is also necessary to explain and define unusual terms such as "decision mutants", "limited budget" and "conflict of interest" experiments, which are crucial for the understanding. The importance of the TRAPPII complex should be illustrated using specific physiological examples and the context in which this complex is studied here has to be explained. Before this is not corrected, the following assessment will remain rather incomplete. Another complication is that two subunits of TRAPP were studied and different types of SKs, however, authors did not systematically analyze all interactions. At least it should be thoroughly described, and a flow chart would be helpful as supplemental figure clearly describe which types of proteins were tested in the different assays. The introduction is not well written. It is very lengthy, however the important messages from previous publications are left out. Thus the open question is not understandable (see above). Instead, the results parts start with introduction again. Explanations are also lacking in every result paragraph on the approach and expected data. The Discussion is also not very well written. It is much focused on physiological and molecular actions and consequences in plants. However, there should be at first a technical discussion on the relevance since in the study is based on in vitro and heterologous expression data, and the physiological analysis was only conducted with knockouts but not phosphomutants. Therefore, the link between the protein interaction and physiological functions needs to be worked out.

    Referees cross-commenting

    My colleague and I have read thoroughly the manuscript and found a number of issues which we indicated in our review. These points can be fixed by the authors, if they formulate more carefully and remove the overstatements. They should also work on reorganizing and including more explanations.

    Significance

    Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested.

    The following aspects are important:

    One new aspect of this story is the validation of interaction of TRAPII subunits as substrate for AtSKs and their action as phosphorylation agents shown in vitro. The other new aspect is the phenotypical characterization of trapii mutants under stress-conditions (grown in darkness) and additive stress (with additional drought stress). The potential interaction with brassinosteroid signaling via BIN2 is intriguing.

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? A strength is that a new interaction is further studied. A weakness is that the studies are primarily conducted in yeast and in vitro, leaving open how relevant this process is in plants. A strength is further studies and phenotypic analysis of trapii mutant effects. A weakness is that this mutant analysis is disconnected from the action of SKs.

    Further, the writing should be improved and more clear (see comments above).

    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). The introduction gives the impression of a stronger investigation of TGN function, which is from my point of view not the case and should be reformulated and/or put into a deeper context with known literature. The authors switch several times between the different TRAPPII subunits and shaggy-like kinases in the main figures which made it for me very confusing. I believe that rearranging some data/figures will improve the understanding of the story. The text is also lacking explanations of many abbreviations and gene names which caused more difficulties in understanding the story and slowed down the reviewing process. From my point of view it seems to be necessary to read the often cited Kalbfuß et al., 2022 publication before, as many important technical aspects and scientific background, e.g. the reason to use specific control mutants, are well explained there, but are lacking in this manuscript and needs improvement.
    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? Based on the cited literature in this manuscript the direction of the story with "limited budget" and "conflict of interest" situations to classify mutants methodically seems to be a recently emerged approach. Apart from that this manuscript provides only new impact on TRAPII and AtSKs specific knowledge based on well-established and frequently used techniques that address the problem in vitro and in a heterologous system. Therefore, this story will be interesting for researchers specialized in stress responses, TGN and growth defects as well as important for basic research. Limitations in interpretation are present.
    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Our field of research is related to nutrition-regulated processes especially in Arabidopsis with a strong methodological background in interactomics, physiological, morphological and molecular responses and biochemical approaches and microscopy.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript adopted the concept that plants have cognitive ability and proposed the hypothesis that the trans-Golgi network plays a role in the decision process of cells. It investigates how this organelle function in order to reach the right decision when exposed to the combined drought stress and dark or to germination under osmotic stress. The study tests the hypothesis focusing on the on the TRAPPII complex. The authors demonstrated that defects (mutations) on TRAPPII complex cause wrong growth decisions, particularly when seedling are exposed to decisions with trade-offs.

    Major comments

    The experiments made for this study are enough to support the conclusions, and they were performed with adequate procedures. I just make the comment that follows.

    Lines 468-472: The authors propose that "signal integration and decision-making occur at the AtSK-TRAPPII interface". It should be considered whether the decision is made in a specific step and location or if all the cascade of responses is the decision process. It is proposed that TRAPPII makes the decision and the Rab GTPase cascades (or the downstream signals) implement the decision. The authors demonstrated that a defect on TRAPPII causes wrong-decisions, but what would happen if TRAPPII were normal but something downstream was defective and could would proceed the regular process? Would it also lead to wrong growth decisions? I am afraid that any defect may cause wrong decisions because the decision is the full metabolic process and not a single step in the route.

    Minor comments

    Lines 259-260: To make it easier to follow the reasoning, the reader should be informed what were the expected responses in case of "primary defects in cytokinesis".

    Line 413: Correct Kim et al (2023).

    Significance

    This study offers an important contribution to the discussion on how plants make decisions. The signals and the cascade of stimuli flows through an intricate network. This study demonstrates that on of the streams of information used for growth decisions passes through the Golgi complex.

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    Referee #1

    Evidence, reproducibility and clarity

    Universally present across the eukaryotic world, TRAPPII is a hetero-oligomeric complex that plays a key role in the regulation of the TGN that has often been classified as a multisubunit tethering complex, although conclusive evidence for the tethering role is still lacking. In contrast, it is well established that the complex acts as a GEF for RAB11, primarily by studies carried out with model fungi such as Saccharomyces cerevisiae and Aspergillus nidulans. Atomic structures have revealed that a few amino acids in one of the subunits of the complex suffice to "kick off" GDP from the active site of the substrate RAB. While some of the subunits are necessary to place the RAB nucleotide binding pocket at the right distance from the key TRAPPII residues on the target membrane, it seems unlikely that the sole function played by this Md complex is catalysing the exchange of GDP by GTP in the target GTPase. In this particular regard, our understanding of other potential physiological roles of the complex is utterly incomplete

    This very well written manuscript explores the regulation of TRAPPII by phosphorylation, more precisely, the role of phospho-sites in the regulation of TRS120/TRAPPC9. Using co-immunoprecipitation strategies coupled to mass spectrometry, the authors identified a member of the saggy/GSK family of kinases. Given that the interaction of protein kinases with their substrates is supposed to be transient, this is technically sound result that testifies to the impeccable methodology used by authors. They further exploit MS methodology and two hybrid analysis to identify region of TRAPPC9 interacting with the kinase, as well as the phosphorylated residues. The authors close the circle by establishing that substitutions of these residues result in modified responses to abiotic stresses. Thus, a significant merit of this work is opening up the can of regulation by phosphorylation of TRAPP functions

    Experiments/challenges for the future are determining the downstream components that govern these physiological changes in response to phosphorylation of TRAPPII, and expanding these phosphorylation studies to TRAPPIII. This latter complex is involved both in exocytosis and in autophagy, and it seems plausible that alternation between these two different fates is governed by post-translational modifications of the type studied here.

    My suggestions to the authors: there has been a burst of atomic structures of TRAPPs recently, with three papers authored by the Fromme, Munro and Sui labs. It mighty worth comparing the longer Trs120 in these structures with the prediction of the shorter protein from Arabidopsis. A very minor point is that possibly the most thorough report on the localisation of TRAPPII to the TGN is that co-signed by Mario Pinar and myself in the Journal of cell science

    Referees cross-commenting

    I have nothing to add to my review, which was made from the point of view of TRAPP researcher. if my colleagues understand that the manuscript is hyperbolic in places, over statements should be removed

    Significance

    TRAPPII is regulated by TRAPPC9 phosphorylation in cruciferae. Convincing evidence. Impeccable presentation and writing.

  10. Version published to 10.1101/2023.04.24.537966v3 on bioRxiv
  11. Version published to 10.1101/2023.04.24.537966v2 on bioRxiv
  12. Version published to 10.1101/2023.04.24.537966v1 on bioRxiv