Temperature-dependent fasciation mutants connect mitochondrial RNA processing to control of lateral root morphogenesis

  1. Kurataka Otsuka
  2. Akihito Mamiya
  3. Mineko Konishi
  4. Mamoru Nozaki
  5. Atsuko Kinoshita
  6. Hiroaki Tamaki
  7. Masaki Arita
  8. Masato Saito
  9. Kayoko Yamamoto
  10. Takushi Hachiya
  11. Ko Noguchi
  12. Takashi Ueda
  13. Yusuke Yagi
  14. Takehito Kobayashi
  15. Takahiro Nakamura
  16. Yasushi Sato
  17. Takashi Hirayama
  18. Munetaka Sugiyama

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Abstract

Although mechanisms that activate organogenesis in plants are well established, much less is known about the subsequent fine-tuning of cell proliferation, which is crucial for creating properly structured and sized organs. Here we show, through analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis, root redifferentiation defective 1 ( rrd1 ), rrd2 , and root initiation defective 4 ( rid4 ), that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis. These mutants formed abnormally broadened (i.e., fasciated) LRs under high-temperature conditions due to excessive cell division. All TDF proteins localized to mitochondria, where they were found to participate in RNA processing: RRD1 in mRNA deadenylation, and RRD2 and RID4 in mRNA editing. Further analysis suggested that LR fasciation in the TDF mutants is triggered by reactive oxygen species generation caused by defective mitochondrial respiration. Our findings provide novel clues for the physiological significance of mitochondrial activities in plant organogenesis.

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  1. eLife

    ###Reviewer #3:

    Otsuka et al. report the characterisation of three temperature sensitive alleles of genes which prominently lead to overproliferation of cells in lateral root primordia. Interestingly this phenotype which is not underpinned by alteration of the auxin pattern, can be phenocopied by treatment with ROS and by interfering with the mitochondrial respiratory chain. This reveals that ROS modulate cell proliferation in the LR. The cloning and biochemical characterisation of the genes affected, reveal that all three encode enzymes involved in mt RNA processing, that perturb the production of certain components of the mitochondrial electron transport chain.

    This is an excellent manuscript that points to a new and very interesting link between primary metabolism and cell proliferation in lateral roots. It is remarkably well written and presented. The conclusions are fully supported by the data. As it is the case for exciting new discoveries, they raise a lot of questions and this manuscript is no exception. It would be very interesting for future work to uncover the nature of the molecular link between ROS and cell proliferation and why are LR so sensitive to this. It'd be eventually interesting to speculate whether the reported existence of an hypoxic environment in the centre of the LRP has to do with this.

    The one point I would like to hear some comments from the authors about relates to the growth conditions used to reveal the phenotype at restrictive temperature. They mention that they use explant culture on RIM (characterised by high glucose and high 2.5µM IBA). What's the penetrance of the phenotype in standard (1/2 MS, 1% sucrose, no additional auxin/IBA)?

  2. eLife

    ###Reviewer #2:

    The manuscript by Otsuka and coworkers, describes the mapping of the mutations in rrd1, rrd2 and rid4 causing the temperature sensitive lateral root morphogenesis defects (fascinated LR meristem). Interestingly, the respective mutated genes all map to genes involved in mitochondrial mRNA processing, mRNA deadenylation, and mRNA editing. The authors propose that defective ROS homeostasis is causal to excessive cell proliferation in the lateral root primordia, and associated fasciation phenotype. Overall the manuscript is well-written, and is overall convincing with respect to characterization and mapping of the mutants, and the importance of RNA editing in mitochondria for the mutant phenotypes. I am not yet entirely convinced about the link to ROS production and the lateral root morphogenesis defects.

    1. The fascinated LR phenotype is reminiscent of mutants defective in coordination of LR emergence, such as CASP:shy2 (Vermeer et al). Suggesting that defective signaling in LR overlaying layers, could be causal to the observed phenotype. However, the phenotyping presented in this manuscript does not allow to assess this. A detailed staging of LRPs would be required, and/or an analysis of the LRP developmental dynamics using a root bending assay.

    2. Furthermore the expression domain analysis shows clear expression in LRPs. However, I suspect expression of at least RID4-GFP in LRP overlaying layers. However, the resolution of the picture, and interference of the bright PI counterstaining in Fig2B preclude a thorough assessment of this.

    3. The colocalization analysis in Fig 2D and E is not very clear. The mitotracker signal is set a bit too weak, making it difficult to assess the distinction between the GFP signal and the overlapping (yellow) signal). This could be amended by using different LUTs (also green/reds are not great for colorblind readers). Of note is the presence of a relatively large structure labeled by RDD1-GFP, that is not colocalizing with mitotracker, suggesting it also localized to another subcellular compartment. Therefore, colocalization should be addressed more quantitatively, also using additional organellar markers. Additionally, the mitochondrial localization could be further supported by western blot on purified mitochondria.

    4. The accumulation of polyadenylated transcripts in Fig3D, seems to also display a temperature sensitivity in the WT. Why was this assay not done using a quantitativePCR, that will allow for better appreciation of temperature component.

    5. In contrast to the LR phenotyping as displayed in Fig 1, the LR phenotyping in Fig4 is done in a completely different way. Why not use a uniform way to quantify. As it was done now, the suppression of rdd1 by ags1 mutation, is not very convincing, as the rrd1 phenotype is nearly abolished in the Col-0 introgressed line (Fig 4 B), suggesting that the rrd1 phenotype is sensitized in the Ler background.

    6. While the authors focus on the LR morphology phenotype in the mutants, there is also a prominent effect on primary root growth that is not described. However, this phenotype does not seem to be very ecotype-specific, and is rescued in the ags1 background. A small phenotypic characterization of the primary root phenotype could thus be beneficial for the manuscript, and it’s wider relevance for development.

    7. Fig5. -> explain arrowheads in B, in the legend. Bar charts using mean + and - SD should be avoided when you do not have many data points, as in D and F (N=3 and 2). Better to show the raw data. Loading controls are missing for Fig5 C and E.

    8. The section about ROS is all based on ROS related pharmacology. However, ROS levels in the mutants were not assessed, making it difficult to use the pharmacological treatments to interpret the origin of the mutant phenotypes.

    9. What is the link to the temperature sensitivity. Are these mutants hypersensitive to ROS inducing treatments?

    10. While the role of ROS in LR development is key to the proposed model, the authors did not introduce what is the state of the art about ROS in lateral and primary root development.

    11. In their model the authors might need to discuss whether or not ROS from the LRP could act as an intercellular coordinative developmental signal.

  3. eLife

    ###Reviewer #1:

    This study continues research started by Professor Munetaka Sugiyama and his laboratory who identified about 20 years ago, or so, very interesting temperature-dependent fasciation (TDF) mutants affected in lateral root primordium (LRP) morphogenesis. The authors identified and reported in this study genes responsible for the mutant phenotype of the root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4). Intriguingly, all the genes are involved in RNA processing. Detailed analysis of the role of RRD2 and RID4 in mitochondrial mRNA editing and RRD1 in poly(A) degradation of mitochondrial mRNA make this work a solid and substantial study. The fact that pharmacological treatments of wild type seedlings by mitochondrial electron transport inhibitors can phenocopy the fasciated LRP phenotype is really fine. Similarly, the experiments with paraquat and ascorbate are very interesting. The main conclusion of the work (that LRP morphogenesis is linked to mitochondrial RNA processing and mitochondrion-mediated ROS generation) is novel and significant. I think this is an important step forward in our understanding of LRP morphogenesis.

    I see only one main conceptual or interpretation problem.

    The authors conclude that "that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis" (line, L, 51). A similar statement appears on L101-103 where the authors postulate that TDF encode "negative regulators of proliferation that are important for the size restriction of the central zone during the formation of early stage LR primordia". Again, similar statements appear on L151-152, 344, and in the section of discussion "Mitochondrial RNA processing is linked to the control of cell proliferation", especially where the authors say about "the control of cell proliferation at the early stage".

    To my opinion, the above conclusions are arguable and cannot be accepted. To conclude about excessive cell division, the number of anticlinal divisions must be estimated per founder cell. This analysis has not been performed. The fact that at early stages LRPs are wider in the TDF mutants suggests that a greater number of FCs in the longitudinal plane participate in LRP formation. So, if this is correct, the mutations apparently affect control of lateral inhibition, and TDF genes are negative regulators of lateral inhibition. This question should be further investigated, but currently a more careful interpretation of the results is required. Also, if TDF genes encode "negative regulators of proliferation" then more frequent divisions would occur in the mutant. This question was not addressed either. If more frequent cell division is expected in early stage LRPs, this should result in formation of smaller cells. In accordance with Fig. 1D of this study and Figs. 1b and 3a of Otsuka and Sugiyama (2012), this is not the case. Contrary, it seems that at the same developmental stage there are lower numbers of cells per unit of volume in the mutants compared to wild type. Another, possible explanation of the TDF mutant phenotype, in addition to lateral inhibition, is abnormal establishment of stem cell identity or affected stem cell function. Therefore, the mechanistic explanation of the link between TDF gene action and the respective mutant phenotype is not satisfactory. The interpretation given can be corrected and carefully rephrased throughout the text.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.

    ###Summary:

    The reviewers were very enthusiastic about your work. They identified some shortcomings, but most of it could be addressed by text edits. The reviewers were less convinced about the envisioned link to reactive oxygen species (ROS). Ideally, you should consolidate this aspect by depicting the mis-regulated ROS in the mutant, and its restoration in the suppressor double mutants (e.g. by staining).

  5. Version published to 10.1101/2020.06.09.141382v3 on bioRxiv
  6. Version published to 10.1101/2020.06.09.141382v2 on bioRxiv
  7. Version published to 10.1101/2020.06.09.141382v1 on bioRxiv