FER-like iron deficiency-induced transcription factor (FIT) accumulates in nuclear condensates

  1. Ksenia Trofimov
  2. Regina Gratz
  3. Rumen Ivanov
  4. Yvonne Stahl
  5. Petra Bauer
  6. Tzvetina Brumbarova

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Abstract

The functional importance of nuclear protein condensation remains often unclear. The bHLH FER-like iron deficiency-induced transcription factor (FIT) controls iron acquisition and growth in plants. Previously described C-terminal serine residues allow FIT to interact and form active transcription factor complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into FIT nuclear bodies (NBs). Using quantitative and qualitative microscopy-based approaches, we characterized FIT NBs as condensates that were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. FIT condensation may affect nuclear mobility and be relevant for integrating environmental and Fe nutrition signals.

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    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    Dear Editor and Reviewers,

    *We thank you for the thorough and detailed examination of our preprint and providing the very valuable comments that helped to even better present and interpret our data. *

    *Thank you in particular for appreciating the extensive set of microscopic techniques that we have combined to study in a unique manner the characteristics and functionalities of FIT nuclear bodies in living plant cells. *

    We prepared a revised preprint in which we address all reviewer comments. Our revision includes a NEW experiment (in four repetitions) that addresses one comment made by the reviewers with regard to the effects of the environmental FIT NB-inducing situation:

    • NEW Supplemental Figures S6 and S7: Analysis of previously reported intron retention splicing variants of Fe deficiency genes FIT, BHLH039, IRT1, FRO2 in new gene expression experiments (Four independent repetitions of the experiments with three biological replicates of each sample – white/blue light treatment, sufficient and deficient iron supply). In the following, please find our detailed response to all reviewer comments.

    With these changes, we hope that our peer-reviewed preprint can receive a positive vote,

    We are looking forward to your response,

    Sincerely

    Petra Bauer and Ksenia Trofimov on behalf of all authors

    Comments to the reviews:

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this paper entitled " FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) accumulates in homo- and heterodimeric complexes in dynamic and inducible nuclear condensates associated with speckle components", Trofimov and colleagues describe for the first time the function of FIT in nuclear bodies. By an impressive set of microscopies technics they assess FIT localization in nuclear bodies and its dynamics. Finally, they reveal their importance in controlling iron deficiency pathway. The manuscript is well written and fully understandable. Nonetheless, at it stands the manuscript present some weakness by the lack of quantification for co-localization and absence controls making hard to follow authors claim. Moreover, to substantially improve the manuscript the authors need to provide more proof of concepts in A. thaliana as all the nice molecular and cellular mechanism is only provided in N. bentamiana. Finally, some key conclusions in the paper are not fully supported by the data. Please see below:

    Main comments:

    1. For colocalization analysis, the author should provide semi-quantitative data counting the number of times by eyes they observed no, partial or full co-localization and indicate on how many nucleus they used.

    Authors:

    We have added the information in the Materials and Method section, lines 731-734:

    In total, 3-4 differently aged leaves of 2 plants were infiltrated and used for imaging. One infiltrated leaf with homogenous presence of one or two fluorescence proteins was selected, depending on the aim of the experiment, and ca. 30 cells were observed. Images are taken from 3-4 cells, one representative image is shown.

    In all analyzed cases, except in the case of colocalization of FIT and PIF4 fusion proteins, the ca. 30 cells had the same localization and/or colocalization patterns. This information has also been added in the figure legends. Each experiment was repeated at least 2-3 times, or as indicated in the figure legend.

    1. Do semi-quantitative co-localization analysis by eyes, on FIT NB with known NB makers in the A. thaliana root. For now, all the nicely described molecular mechanism is shown in N. benthamiana which makes this story a bit weak since all the iron transcriptional machinery is localized in the root to activate IRT1.

    Authors:

    The described approach has been very optimal, and we were able to screen co-localizing marker proteins in FIT NBs in N. benthamiana to better identify the nature of FIT NBs. This has been successful as we were able to associate FIT NBs with speckles. The N. benthamiana system allowed optimal microscopic observation of fluorescence proteins and quantification of FIT NB characteristics in contrast to the root hair zone of Arabidopsis where Fe uptake takes place. FIT is expressed at a low level in roots and also in leaves, whereby fluorescence protein expression levels are insufficient for the here-presented microscopic studies. The tobacco infiltration system is also well established to study FIT-bHLH039 protein interaction and nuclear body markers. We discuss this point in the discussion, see line 489-500.

    1. The authors need to provide data clearly showing that the blue light induce NB in A. thaliana and N. benthamiana.

    Authors:

    *For tobacco, see Figure 1B (t = 0, 5 min) and Supplemental Movies S1. For Arabidopsis, please see Figure 1A (t = 0, 90 and 120 min) and Supplemental Figure S1A. We provide an additional image of pFIT:cFIT-GFP Arabidopsis control plants, showing that NB formation is not detected in plants that were grown in white light and not exposed to blue light before inspection (Supplemental Figure S1B). We state, that upon blue light exposure, plants had FIT NBs in at least 3-10 nuclei of 20 examined nuclei in the root epidermis in the root hair zone (in three independent experiments with three independent plants). White-light-treated plants showed no NB formation unless an additional exposure to blue light was provided (in three independent experiments, three independent plants per experiment and with 15 examined nuclei per plant). *

    1. Direct conclusion in the manuscript:
    • Line 170: At this point of the paper the author cannot claim that the formation of FIT condensates in the nucleus is due to the light as it might be indirectly linked to cell death induced by photodamaging the cell using a 488 lasers for several minutes. This is true especially with the ELYRA PS which has strong lasers made for super resolution and that Cell death is now liked to iron homeostasis. The same experiment might be done using a spinning disc or if the authors present the data of the blue light experiment mentioned above this assumption might be discarded. Alternatively, the author can use PI staining to assess cell viability after several minutes under 488nm laser.

    Authors:

    As stated in our response to comment 3, we have included now a white light control to show that FIT NB formation is not occurring under the normal white light conditions. Since the formation of FIT NBs is a dynamic and reversible process (Figure 1A), it indicates that the cells are still viable, and that cell death is not the reason for FIT NB formation.

    • Line 273: I don't agree with the first part of the authors conclusion, saying that "wild-type FIT had better capacities to localize to NBs than mutant FITmSS271AA, presumably due its IDRSer271/272 at the C-terminus. This is not supported by the data. In order to make such a claim the author need to compare the FA of FIT WT with FITmSS271AA by statistical analysis. Nonetheless, the value seems to be identical on the graphs. The main differences that I observed here are, 1) NP value for FITmSS271AA seems to be lower compared to FIT-WT, suggesting that the Serine might be important to regulate protein homedimerization partitioning between the NP and the NB. 2) To me, something very interesting that the author did not mention is the way the FA of FITmSS271AA in the NB and NP is behaving with high variability. The FA of those is widely spread ranging from 0.30 to 0.13 compared to the FIT-WT. To me it seems that according to the results that the Serine 271/272 are required to stabilize FIT homodimerization. This would not only explain the delay to form the condensate but also the decreased number and size observed for FITmSS271AA compared to FIT-WT. As the homodimerization occurs with high variability in FITmSS271AA, there is less chance that the protein will meet therefore decreasing the time to homodimerize and form/aggregate NB.

    Authors:

    We fully agree. We meant to describe this result it in a similar way and thank you for help in formulating this point even better. Rephrasing might make it better clear that the IDRSer271/272 is important for a proper NB localization, lines 272-278:

    “Also, the FA values did not differ between NBs and NP for the mutant protein and did not show a clear separation in homodimerizing/non-dimerizing regions (Figure 3D) as seen for FIT-GFP (Figure 3C). Both NB and NP regions showed that homodimers occurred very variably in FITmSS271AA-GFP.

    In summary, wild-type FIT could be partitioned properly between NBs and NP compared to FITmSS271AA mutant and rather form homodimers, presumably due its IDRSer271/272 at the C-terminus.”

    • Line 301: According to my previous comment (line 273), here it seems that the Serine 271/272 are required only for proper partitioning of the heterodimer FIT/BHLH039 between the NP and NB but not for the stability of the heterodimer formation. However, it might be great if the author would count the number of BHLH039 condensates in both version FITmSS271AA and FIT-WT. To my opinion, they would observe less BHLH039 condensate because the homodimer of FITmSS271AA is less likely to occur because of instability.

    Authors:

    bHLH039 alone localizes primarily to the cytoplasm and not the nucleus, and the presence of FIT is crucial for bHLH039 nuclear localization (Trofimov et al., 2019). Moreover, bHLH039 interaction with FIT depends on SS271AA (Gratz et al., 2019). We therefore did not consider this experiment for the manuscript and did not acquire such data, as we did not expect to achieve major new information.

    1. To wrap up the story about the requirements of NB in mediating iron acquisition under different light regimes, provide data for IRT1/FRO2 expression levels in fit background complemented with FITmSS271AA plants. I know that this experiment is particularly lengthy, but it would provide much more to this nice story.

    Authors:

    Data for expression of IRT1 and FRO2 in FITmSS271AA/fit-3 transgenic Arabidopsis plants are provided in Gratz et al. (2019). To address the comment, we did here a NEW experiment. We provide gene expression data on FIT, BHLH039, IRT1 and FRO2 splicing variants (previously reported intron retention) to explore the possibility of differential splicing alterations under blue light (NEW Supplemental Figure S6 and S7, lines 454-466). Very interestingly, this experiment confirms that blue light affects gene expression differently from white light in the short-term NB-inducing condition and that blue light can enhance the expression of Fe deficiency genes despite of the short 1.5 to 2 h treatment. Another interesting aspect was that the published intron retention was also detected. A significant difference in intron retention depending on iron supply versus deficiency and blue/white light was not observed, as the pattern of expression of transcripts with respective intron retentions sites was the same as the one of total transcripts mostly spliced.

    Minor comments

    In general, I would suggest the author to avoid abbreviation, it gets really confusing especially with small abbreviation as NB, NP, PB, FA.

    Authors:

    *We would like to keep the used abbreviations as they are utilized very often in our work and, in our eyes, facilitate the understanding. *

    Line 106: What does IDR mean?

    Authors:

    Explanation of the abbreviation was added to the text, lines 105-108:

    “Intrinsically disordered regions (IDRs) are flexible protein regions that allow conformational changes, and thus various interactions, leading to the required multivalency of a protein for condensate formation (Tarczewska and Greb-Markiewicz, 2019; Emenecker et al., 2020).”

    Line 163-164: provide data or cite a figure properly for blue light induction.

    Authors:

    We have removed this statement from the description, as we provide a white light control now, lines 157-158:

    “When whole seedlings were exposed to 488 nm laser light for several minutes, FIT became re-localized at the subnuclear level.”

    Line 188: Provide Figure ref.

    Authors:

    Figure reference was added to the text, lines 184-185:

    “As in Arabidopsis, FIT-GFP localized initially in uniform manner to the entire nucleus (t=0) of N. benthamiana leaf epidermis cells (Figure 1B).”

    Line 194: the conclusion is too strong. The authors conclude that the condensate they observed are NB based on the fact the same procedure to induce NB has been used in other study which is not convincing. Co-localization analysis with NB markers need to be done to support such a claim. At this step of the study, the author may want to talk about condensate in the nucleus which might correspond to NB. Please do so for the following paragraph in the manuscript until colocalization analysis has not been provided. Alternatively provide the co-localization analysis at this step in the paper.

    Authors:

    We agree. We changed the text in two positions.

    Lines 176-178: “Since we had previously established a reliable plant cell assay for studying FIT functionality, we adapted it to study the characteristics of the prospective FIT NBs (Gratz et al., 2019, 2020; Trofimov et al., 2019).”

    Lines 192-193:* “*We deduced that the spots of FIT-GFP signal were indeed very likely NBs (for this reason hereafter termed FIT NBs).”

    Line 214: In order to assess the photo bleaching due to the FRAP experiment the quantification of the "recovery" needs to be provided in an unbleached area. This might explain why FIT recover up to 80% in the condensate. Moreover, the author conclude that the recovery is high however it's tricky to assess since no comparison is made with a negative/positive control.

    Authors:

    In the FRAP analysis, an unbleached area is taken into account and used for normalization.

    We reformulated the description of Figure 1F, lines 212-214:

    “According to relative fluorescence intensity the fluorescence signal recovered rapidly within FIT NBs (Figure 1F), and the calculated mobile fraction of the NB protein was on average 80% (Figure 1G).”

    Line 220-227: The conclusion it's too strong as I mentioned previously the author cannot claim that the condensate are NBs at this step of the study. They observed nuclear condensates that behave like NB when looking at the way to induce them, their shape, and the recovery. And please include a control.

    Authors:

    Please see the reformulated sentences and our response above.

    Lines 176-178: “Since we had previously established a reliable plant cell assay for studying FIT functionality, we adapted it to study the characteristics of the prospective FIT NBs (Gratz et al., 2019, 2020; Trofimov et al., 2019).”

    Lines 192-193:* “*We deduced that the spots of FIT-GFP signal were indeed very likely NBs (for this reason hereafter termed FIT NBs).”

    Line 239: It's unappropriated to give the conclusion before the evidence.

    Authors:

    Thank you. We removed the conclusion.

    Line 240: Figure 2A, provide images of FIT-G at 15min in order to compare. And the quantification needs to be provided at 5 minutes and 15 minutes for both FIT-G WT and FIT-mSS271AA-G counting the number of condensates in the nucleus. Especially because the rest of the study is depending on these time points.

    Authors:

    *This information is provided in the Supplemental Movie S1C. *

    Line 241: the author say that the formation of condensate starts after 5 minutes (line 190) here (line 241) the author claim that it starts after 1 minutes. Please clarify.

    Authors:

    In line 190 we described that FIT NB formation occurs after the excitation and is fully visible after 5 min. In line 241 we stated that the formation starts in the first minutes after excitation, which describes the same time frame. We rephrased the respective sentences.

    *Lines 185-188: *“A short duration of 1 min 488 nm laser light excitation induced the formation of FIT-GFP signals in discrete spots inside the nucleus, which became fully visible after only five minutes (t=5; Figure 1B and Supplemental Movie S1A).”

    *Lines 239-242: *“While FIT-GFP NB formation started in the first minutes after excitation and was fully present after 5 min (Supplemental Movie S1A), FITmSS271AA-GFP NB formation occurred earliest 10 min after excitation and was fully visible after 15 min (Supplemental Movie S1C).”

    Line 254: Not sure what the authors claim "not only for interaction but also for FIT NB formation ". To me, the IDR is predicted to be perturbed by modeling when the serines are mutated therefore the IDR might be important to form condensates in the nucleus. Please clarify.

    Authors:

    The formation of nuclear bodies is slow for FITmSS271AA as seen in Figure 2. Previously, we showed that FITmSS271AA homodimerizes less (Gratz et al., 2019.) Therefore, the said IDR is important for both processes, NB formation and homodimerization. We have added this information to make the point clear, lines 253-255:

    “This underlined the significance of the Ser271/272 site, not only for interaction (Gratz et al., 2019) but also for FIT NB formation (Figure 2).”

    Line 255: It's not clear why the author test if the FIT homodimerization is preferentially associated with condensate in the nucleus.

    Authors:

    We test this because both homo- and heterodimerization of bHLH TFs are generally important for the activity of TFs, and we unraveled the connection between protein interaction and NB formation. We state this in lines 228-232.

    Line 269-272: It's not clear to what the authors are referring to.

    Authors:

    We are describing the homodimeric behavior of FIT and FITmSS271AA assessed by homo-FRET measurements that are introduced in the previous paragraph, lines 256-268.

    Line 309: This colocalization part should be presented before line 194.

    Authors:

    We find it convincing to first examine and characterize the process underlying FIT NB formation, then studying a possible function of NBs. The colocalization analysis is part of a functional analysis of NBs. We thank the reviewer for the hint that colocalization also confirms that indeed the nuclear FIT spots are NBs. We will take this point and discuss it, lines 516-522:

    “Additionally, the partial and full colocalization of FIT NBs with various previously reported NB markers confirm that FIT indeed accumulates in and forms NBs. Since several of NB body markers are also behaving in a dynamic manner, this corroborates the formation of dynamic FIT NBs affected by environmental signals.”

    “In conclusion, the properties of liquid condensation and colocalization with NB markers, along with the findings that it occurred irrespective of the fluorescence protein tag preferentially with wild-type FIT, allowed us to coin the term of ‘FIT NBs’.”

    Line 328: add the ref to figure, please.

    Authors:

    Figure reference was added to the text, lines 330-332:

    “The second type (type II) of NB markers were partially colocalized with FIT-GFP. This included the speckle components ARGININE/SERINE-RICH45-mRFP (SR45) and the serine/arginine-rich matrix protein SRm102-mRFP (Figure 5).”

    Line 334: It seems that the size of the SR45 has an anormal very large diameter between 4 and 6 µm. In general a speckle measure about 2-3µm in diameter. Can the author make sure that this structure is not due to overexpression in N. benthamiana or make sure to not oversaturate the image.

    Authors:

    Thank you for this hint. Indeed, there are reports that SR45 is a dynamic component inside cells. It can redistribute depending on environmental conditions and associate into larger speckles depending on the nuclear activity status (Ali et al., 2003). We include this reference and refer to it in the discussion, lines 557-564:

    “Interestingly, typical FIT NB formation did not occur in the presence of PB markers, indicating that they must have had a strong effect on recruiting FIT. This is interesting because the partially colocalizing SR45, PIF3 and PIF4 are also dynamic NB components. Active transcription processes and environmental stimuli affect the sizes and numbers of SR45 speckles and PB (Ali et al., 2003; Legris et al., 2016; Meyer, 2020). This may indicate that, similarly, environmental signals might have affected the colocalization with FIT and resulting NB structures in our experiments. Another factor of interference might also be the level of expression.”

    Line 335: It seems that the colocalization is partial only partial after induction of NB. The FIT NB colocalize around SR45. But it's hard to tell because the images are saturated therefore creating some false overlapping region.

    Authors:

    The localization of FIT with SR45 is partial and occurs only after FIT has undergone condensation, see lines 335-338.

    Line 344-345: It's unappropriated to give the conclusion before the evidence.

    Authors:

    We explain at an earlier paragraph that we will show three different types of colocalization and introduce the respective colocalization types within separate paragraphs accordingly, see lines 314-321.

    Line 353: increase the contrast in the image of t=5 for UAP56H2 since it's hard to assess the colocalization.

    Authors:

    This is done as noted in the figure legend of Figure 6.

    Line 381-382: "In general" does not sound scientific avoid this kind of wording and describe precisely your findings.

    Authors:

    We rephrased the sentence, line 387-388:

    Localization of single expressed PIF3-mCherry remained unchanged at t=0 and t=15 (Supplemental Figure S5A).

    Line 384-385: Provide the data and the reference to the figure.

    Authors:

    We apologize for the misunderstanding and rephrased the sentence, line 389-391:

    After 488 nm excitation, FIT-GFP accumulated and finally colocalized with the large PIF3-mCherry PB at t=15, while the typical FIT NBs did not appear (Figure 7A)

    Line 386: The structure in which FIT-G is present in the Figure 7A t=15 is not alike the once already observed along the paper. This could be explained by over-expression in N. benthamiana. Please explain.

    Authors:

    Thank you for the hint. We discuss this in the discussion part, see lines 555-568.

    Line 393: Explain and provide data why the morphology of PIF4/FIT NB do not correspond to the normal morphology.

    Authors:

    Thank you for the valuable hints. Several reasons may account for this and we provide explanations in the discussion, see lines 555-568.

    Line 396-398: It seems also from the data that co-expression of PIF4 of PIF3 will affect the portioning of FIT between the NP and the NB.

    Authors:

    We can assume that residual nucleoplasm is depleted from protein during NB formation. This is likely true for all assessed colocalization experiments. We discuss this in lines 492-494.

    The discussion is particularly lengthy it might be great to reduce the size and focus on the main findings.

    Authors:

    *We shortened the discussion. *

    **Referees cross-commenting**

    All good for me, I think that the comments/suggestions from Reviewer #2 are valid and fair. If they are addressed they will improve considerably the manuscript.

    Reviewer #1 (Significance (Required)):

    This manuscript is describing an unprecedent very precise cellular and molecular mechanism in nutrition throughout a large set of microscopies technics. Formation of nuclear bodies and their role are still largely unexplored in this context. Therefore, this study sheds light on the functional role of this membrane less compartment and will be appreciated by a large audience. However, the fine characterization is only made using transient expression in N. Bentamiana and only few proofs of concept are provided in A. thaliana stable line.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The manuscript of Trofimov et al shows that FIT undergoes light-induced, reversible condensation and localizes to nuclear bodies (NBs), likely via liquid-liquid phase separation and light conditions plays important role in activity of FIT. Overall, manuscript is well written, authors have done a great job by doing many detailed and in-depth experiments to support their findings and conclusions.

    However, I have a number of questions/comments regarding the data presented and there are still some issues that authors should take into account.

    Major points/comments:

    1. Authors only focused on blue light conditions. Is there any specific reason for selecting only blue light and not others (red light or far red)?

    Authors:

    There are two main reasons: First, in a preliminary study (not shown) blue light resulted in the formation of the highest numbers of NBs. Second, iron reductase activity assays and gene expression analysis under different light conditions showed a promoting effect under blue light, but not red light or dark red light (Figure 9). This indicated to us, that blue light might activate FIT, and that active FIT may be related to FIT NBs.

    1. Fig. 3C and D: as GFP and GFP-GFP constructs are used as a reference, why not taking the measurements for them at two different time points for example t=0 and t=5 0r t=15???

    Authors:

    Free GFP and GFP-GFP dimers are standard controls for homo-FRET that serve to delimit the range for the measurements.

    1. Line 27-271: Acc to the figure 3d, for the Fluorescence anisotropy measurement of NBs appears to be less. Please explain.

    Authors:

    FA in NBs with FITmSS271AA is variable and the value is lower than that of whole nucleus but not significantly different compared with that in nucleoplasm. We describe the results of Figure 3D in lines 272-275.

    1. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.

    Authors:

    Neither for FIT/bHLH039 nor the FITmSS271AA/bHLH039 pair, there is a significant decrease in the fluorescence lifetime values between t=0 and t=5/15. FIT-G is a control to delimit the range. The interesting experiment is to compare the protein pairs of interest between the different nuclear locations at t=5/15.

    1. Line 300-301: In Figure 4D and 4E. Fluorescence lifetime of G measurement at t=0 seems very similar for both FIT-G as well as FITmSS but if we look at the values of t=0 for FIT-G+bhlh039 it is greater than 2.5 and for FITmSS271AA-G+bhlh039 it is less which suggests more heterodimeric complexes to be formed in FITmSS271AA-G+bhlh039. Similar pattern is observed for NBs and NPs, according to the figure 4d and E.

    Therefore, heterodimeric complexes accumulated more in case of FITmSS271AA-G+bhlh039 as compared to FIT-G+bhlh039 (if we compare measurement values of Fluorescence lifetime of G of FITmSS271AA-G+bhlh039 with FIT-G+bhlh039).

    Please comment and elaborate about this further.

    Authors:

    These conclusions are not valid as the experiments cannot be conducted in parallel. Since the experiments had to be performed on different days due to the duration of measurements including new calibrations of the system, we cannot compare the absolute fluorescence lifetimes between the two sets.

    1. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.

    Authors:

    Please see our response to your comment 4).

    1. Line 439-400: As iron uptake genes (FRO2 and IRT1) are more induced in WT under blue light conditions and FRO2 is less induced in case of red-light conditions. So, what happens to Fe content of WT grown under blue light or red light as compared to WT grown under white light. Perls/PerlsDAb staining of WT roots under different light conditions will add more information to this.

    Authors:

    We focused on the relatively short-term effects of blue light on signaling of nuclear events that could be related to FIT activity directly, particularly gene expression and iron reductase activity as consequence of FRO2 expression. These are both rapid changes that occur in the roots and can be measured. We suspect that iron re-localization and Fe uptake also occur, however, in our experience differences in metal contents will not be directly significant when applying the standard methods like ICP-MS or PERLs staining.

    Minor comments:

    Line 75-76: Rephrase the sentence

    Authors:

    We rephrased the sentence, lines 73-74:

    “As sessile organisms, plants adjust to an ever-changing environment and acclimate rapidly. They also control the amount of micronutrients they take up.”

    Line 119: Rephrase the sentence

    Authors:

    We rephrased the sentence, line 118-119:

    “Various NBs are found. Plants and animals share several of them, e.g. the nucleolus, Cajal bodies, and speckles.”

    Line 235-236: rephrase the sentence

    Authors:

    We rephrased the sentence, line 232-234:

    “In the work of Gratz et al. (2019), the hosphor-mimicking FITmS272E protein did not show significant changes in its behavior compared to wild-type FIT.”

    Line 444: Correct the sentence “Fe deficiency versus sufficiency”

    Authors:

    We corrected that, line 449-451:

    “In both, the far-red light and darkness situations, FIT was induced under iron deficiency versus sufficiency, while on the other side, BHLH039, FRO2 and IRT1 were not induced at all in these light conditions (Figure 9I-P).”

    **Referees cross-commenting**

    I agree with R1 suggestions/comments and i think manuscript quality will be much better if authors carry out the experiments suggested by R1. I believe this will also strengthen their conclusions.

    Reviewer #2 (Significance (Required)):

    Overall, manuscript is well written, authors have done a nice job by doing several key experiments to support their findings and conclusions. However, the results and manuscript can be improved further by addressing some question raised here. This study is interesting for basic scientists which unravels the crosstalk of light signaling in nutrient signaling pathways.

  4. Version published to 10.1083/jcb.202311048
  5. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Dear Editor,

    Herewith we submit our fully revised peer-reviewed preprint that had been reviewed by Review Commons. We thank the Review Commons team and reviewers for thoroughly commenting on our preprint and providing very useful additional points for consideration and discussion.

    You will find - the revised manuscript (third preprint version uploaded on biorxiv)
    - two reviewer letters (through Review Commons), - our rebuttal letter
    - a revised manuscript version with highlighted changes.

    Our manuscript reports that an active form of FIT, an essential transcription factor for root iron acquisition in plants, forms dynamic nuclear condensates in response to a blue light stimulus.
    A hallmark of our work is the thorough investigation of the nature of the FIT nuclear bodies in plant cells, that we were able to characterize as highly dynamic condensates in which active FIT homo- and heteromeric protein complexes can accumulate preferentially. Through co-localization with nuclear body markers, we found that these FIT condensates are related to speckles, which are a sub-type of nuclear bodies connected with splicing activities. This suggests that FIT condensates are linked with post-transcriptional regulation mechanisms.

    The reviewers highlight that an “impressive set of microscopic techniques” has been combined to study in a unique manner the characteristics and functionalities of FIT nuclear bodies in living plant cells. We show that FIT nuclear bodies can be formed in roots of Arabidopsis thaliana. The microscopic imaging techniques we used to characterize the nature and functionalities of FIT nuclear bodies in plant cells have several constraints related to sensitivity and a required strength of fluorescent protein signal. For technical reasons to be able to apply qualitative and quantitative imaging techniques, we conducted the investigation of FIT condensates in Nicotiana benthamiana, a classical and widely used plant protein expression system.

    As stated in the reviews, the connection between plant nutrition and nuclear bodies is an “unprecedented” new mode of regulation. The significance of our work is underlined by the fact that we report a “very precise cellular and molecular mechanism in nutrition” that is as yet “still largely unexplored in this context”. Therefore, our study “sheds light on the functional role of this membrane-less compartment and will be appreciated by a large audience.”

    We propose that condensate formation is a mechanism that may steer iron nutrition responses by providing a link between iron and light signaling. For sessile plants, it is absolutely essential that environmental signals are sensed and integrated with developmental and physiological programs so that plants can rapidly adjust to a changing environment and potential stress situations. Since iron is a micronutrient that may be toxic when present in excess, e.g. through catalyzing oxidative stress, plants strictly control the acquisition and allocation of iron. Hence, FIT nuclear bodies may be regulatory hubs that integrate at the sub-nuclear level environmental signaling inputs in the control of micronutrient uptake, possibly connected with splicing.

    Our work lays the ground for future studies that can address the proof of concept in more detailed manner in plants exposed to varying environmental conditions to reveal the interconnection of environmental and nutritional signaling.

    We prepared a revised preprint in which we address all reviewer comments. Please find our revision and our detailed response to all reviewer comments.

    With these changes, we hope that our peer-reviewed preprint can receive a positive vote,

    We are looking forward to your response,

    Sincerely

    Petra Bauer and Ksenia Trofimov on behalf of all authors

    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    In this paper entitled " FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) accumulates in homo- and heterodimeric complexes in dynamic and inducible nuclear condensates associated with speckle components", Trofimov and colleagues describe for the first time the function of FIT in nuclear bodies. By an impressive set of microscopies technics they assess FIT localization in nuclear bodies and its dynamics. Finally, they reveal their importance in controlling iron deficiency pathway. The manuscript is well written and fully understandable. Nonetheless, at it stands the manuscript present some weakness by the lack of quantification for co-localization and absence controls making hard to follow authors claim. Moreover, to substantially improve the manuscript the authors need to provide more proof of concepts in A. thaliana as all the nice molecular and cellular mechanism is only provided in N. bentamiana. Finally, some key conclusions in the paper are not fully supported by the data.
    Please see below:

    Main comments:

    1. For colocalization analysis, the author should provide semi-quantitative data counting the number of times by eyes they observed no, partial or full co-localization and indicate on how many nucleus they used.

    Authors:

    We have added the information in the Materials and Method section, lines 731-734:

    In total, 3-4 differently aged leaves of 2 plants were infiltrated and used for imaging. One infiltrated leaf with homogenous presence of one or two fluorescence proteins was selected, depending on the aim of the experiment, and ca. 30 cells were observed. Images are taken from 3-4 cells, one representative image is shown.

    In all analyzed cases, except in the case of colocalization of FIT and PIF4 fusion proteins, the ca. 30 cells had the same localization and/or colocalization patterns. This information has also been added in the figure legends. Each experiment was repeated at least 2-3 times, or as indicated in the figure legend.

    1. Do semi-quantitative co-localization analysis by eyes, on FIT NB with known NB makers in the A. thaliana root. For now, all the nicely described molecular mechanism is shown in N. benthamiana which makes this story a bit weak since all the iron transcriptional machinery is localized in the root to activate IRT1.

    Authors:

    The described approach has been very optimal, and we were able to screen co-localizing marker proteins in FIT NBs in N. benthamiana to better identify the nature of FIT NBs. This has been successful as we were able to associate FIT NBs with speckles. The N. benthamiana system allowed optimal microscopic observation of fluorescence proteins and quantification of FIT NB characteristics in contrast to the root hair zone of Arabidopsis where Fe uptake takes place. FIT is expressed at a low level in roots and also in leaves, whereby fluorescence protein expression levels are insufficient for the here-presented microscopic studies. The tobacco infiltration system is also well established to study FIT-bHLH039 protein interaction and nuclear body markers. We discuss this point in the discussion, see line 489-500.

    1. The authors need to provide data clearly showing that the blue light induce NB in A. thaliana and N. benthamiana.

    Authors:

    For tobacco, see Figure 1B (t = 0, 5 min) and Supplemental Movies S1. For Arabidopsis, please see Figure 1A (t = 0, 90 and 120 min) and Supplemental Figure S1A. We provide an additional image of pFIT:cFIT-GFP Arabidopsis control plants, showing that NB formation is not detected in plants that were grown in white light and not exposed to blue light before inspection (Supplemental Figure S1B). We state, that upon blue light exposure, plants had FIT NBs in at least 3-10 nuclei of 20 examined nuclei in the root epidermis in the root hair zone (in three independent experiments with three independent plants). White-light-treated plants showed no NB formation unless an additional exposure to blue light was provided (in three independent experiments, three independent plants per experiment and with 15 examined nuclei per plant).

    1. Direct conclusion in the manuscript:
    • Line 170: At this point of the paper the author cannot claim that the formation of FIT condensates in the nucleus is due to the light as it might be indirectly linked to cell death induced by photodamaging the cell using a 488 lasers for several minutes. This is true especially with the ELYRA PS which has strong lasers made for super resolution and that Cell death is now liked to iron homeostasis. The same experiment might be done using a spinning disc or if the authors present the data of the blue light experiment mentioned above this assumption might be discarded. Alternatively, the author can use PI staining to assess cell viability after several minutes under 488nm laser.

    Authors:

    As stated in our response to comment 3, we have included now a white light control to show that FIT NB formation is not occurring under the normal white light conditions. Since the formation of FIT NBs is a dynamic and reversible process (Figure 1A), it indicates that the cells are still viable, and that cell death is not the reason for FIT NB formation.

    • Line 273: I don't agree with the first part of the authors conclusion, saying that "wild-type FIT had better capacities to localize to NBs than mutant FITmSS271AA, presumably due its IDRSer271/272 at the C-terminus. This is not supported by the data. In order to make such a claim the author need to compare the FA of FIT WT with FITmSS271AA by statistical analysis. Nonetheless, the value seems to be identical on the graphs. The main differences that I observed here are, 1) NP value for FITmSS271AA seems to be lower compared to FIT-WT, suggesting that the Serine might be important to regulate protein homedimerization partitioning between the NP and the NB. 2) To me, something very interesting that the author did not mention is the way the FA of FITmSS271AA in the NB and NP is behaving with high variability. The FA of those is widely spread ranging from 0.30 to 0.13 compared to the FIT-WT. To me it seems that according to the results that the Serine 271/272 are required to stabilize FIT homodimerization. This would not only explain the delay to form the condensate but also the decreased number and size observed for FITmSS271AA compared to FIT-WT. As the homodimerization occurs with high variability in FITmSS271AA, there is less chance that the protein will meet therefore decreasing the time to homodimerize and form/aggregate NB.

    Authors:

    We fully agree. We meant to describe this result it in a similar way and thank you for help in formulating this point even better. Rephrasing might make it better clear that the IDRSer271/272 is important for a proper NB localization, lines 272-278:

    “Also, the FA values did not differ between NBs and NP for the mutant protein and did not show a clear separation in homodimerizing/non-dimerizing regions (Figure 3D) as seen for FIT-GFP (Figure 3C). Both NB and NP regions showed that homodimers occurred very variably in FITmSS271AA-GFP.

    In summary, wild-type FIT could be partitioned properly between NBs and NP compared to FITmSS271AA mutant and rather form homodimers, presumably due its IDRSer271/272 at the C-terminus.”

    • Line 301: According to my previous comment (line 273), here it seems that the Serine 271/272 are required only for proper partitioning of the heterodimer FIT/BHLH039 between the NP and NB but not for the stability of the heterodimer formation. However, it might be great if the author would count the number of BHLH039 condensates in both version FITmSS271AA and FIT-WT. To my opinion, they would observe less BHLH039 condensate because the homodimer of FITmSS271AA is less likely to occur because of instability.

    Authors:

    bHLH039 alone localizes primarily to the cytoplasm and not the nucleus, and the presence of FIT is crucial for bHLH039 nuclear localization (Trofimov et al., 2019). Moreover, bHLH039 interaction with FIT depends on SS271AA (Gratz et al., 2019). We therefore did not consider this experiment for the manuscript and did not acquire such data, as we did not expect to achieve major new information.

    1. To wrap up the story about the requirements of NB in mediating iron acquisition under different light regimes, provide data for IRT1/FRO2 expression levels in fit background complemented with FITmSS271AA plants. I know that this experiment is particularly lengthy, but it would provide much more to this nice story.

    Authors:

    Data for expression of IRT1 and FRO2 in FITmSS271AA/fit-3 transgenic Arabidopsis plants are provided in Gratz et al. (2019). To address the comment, we did here a NEW experiment. We provide gene expression data on FIT, BHLH039, IRT1 and FRO2 splicing variants (previously reported intron retention) to explore the possibility of differential splicing alterations under blue light (NEW Supplemental Figure S6 and S7, lines 454-466). Very interestingly, this experiment confirms that blue light affects gene expression differently from white light in the short-term NB-inducing condition and that blue light can enhance the expression of Fe deficiency genes despite of the short 1.5 to 2 h treatment. Another interesting aspect was that the published intron retention was also detected. A significant difference in intron retention depending on iron supply versus deficiency and blue/white light was not observed, as the pattern of expression of transcripts with respective intron retentions sites was the same as the one of total transcripts mostly spliced.

    Minor comments

    In general, I would suggest the author to avoid abbreviation, it gets really confusing especially with small abbreviation as NB, NP, PB, FA.

    Authors:

    We would like to keep the used abbreviations as they are utilized very often in our work and, in our eyes, facilitate the understanding.

    Line 106: What does IDR mean?

    Authors:

    Explanation of the abbreviation was added to the text, lines 105-108:

    “Intrinsically disordered regions (IDRs) are flexible protein regions that allow conformational changes, and thus various interactions, leading to the required multivalency of a protein for condensate formation (Tarczewska and Greb-Markiewicz, 2019; Emenecker et al., 2020).”

    Line 163-164: provide data or cite a figure properly for blue light induction.

    Authors:

    We have removed this statement from the description, as we provide a white light control now, lines 157-158:

    “When whole seedlings were exposed to 488 nm laser light for several minutes, FIT became re-localized at the subnuclear level.”

    Line 188: Provide Figure ref.

    Authors:

    Figure reference was added to the text, lines 184-185:

    “As in Arabidopsis, FIT-GFP localized initially in uniform manner to the entire nucleus (t=0) of N. benthamiana leaf epidermis cells (Figure 1B).”

    Line 194: the conclusion is too strong. The authors conclude that the condensate they observed are NB based on the fact the same procedure to induce NB has been used in other study which is not convincing. Co-localization analysis with NB markers need to be done to support such a claim. At this step of the study, the author may want to talk about condensate in the nucleus which might correspond to NB. Please do so for the following paragraph in the manuscript until colocalization analysis has not been provided. Alternatively provide the co-localization analysis at this step in the paper.

    Authors:

    We agree. We changed the text in two positions.

    Lines 176-178_: “_Since we had previously established a reliable plant cell assay for studying FIT functionality, we adapted it to study the characteristics of the prospective FIT NBs (Gratz et al., 2019, 2020; Trofimov et al., 2019).”

    Lines 192-193: _“_We deduced that the spots of FIT-GFP signal were indeed very likely NBs (for this reason hereafter termed FIT NBs).”

    Line 214: In order to assess the photo bleaching due to the FRAP experiment the quantification of the "recovery" needs to be provided in an unbleached area. This might explain why FIT recover up to 80% in the condensate. Moreover, the author conclude that the recovery is high however it's tricky to assess since no comparison is made with a negative/positive control.

    Authors:

    In the FRAP analysis, an unbleached area is taken into account and used for normalization.

    We reformulated the description of Figure 1F, lines 212-214:

    “According to relative fluorescence intensity the fluorescence signal recovered rapidly within FIT NBs (Figure 1F), and the calculated mobile fraction of the NB protein was on average 80% (Figure 1G).”

    Line 220-227: The conclusion it's too strong as I mentioned previously the author cannot claim that the condensate are NBs at this step of the study. They observed nuclear condensates that behave like NB when looking at the way to induce them, their shape, and the recovery. And please include a control.

    Authors:

    Please see the reformulated sentences and our response above.

    Lines 176-178: “Since we had previously established a reliable plant cell assay for studying FIT functionality, we adapted it to study the characteristics of the prospective FIT NBs (Gratz et al., 2019, 2020; Trofimov et al., 2019).”

    Lines 192-193: _“_We deduced that the spots of FIT-GFP signal were indeed very likely NBs (for this reason hereafter termed FIT NBs).”

    Line 239: It's unappropriated to give the conclusion before the evidence.

    Authors:

    Thank you. We removed the conclusion.

    Line 240: Figure 2A, provide images of FIT-G at 15min in order to compare. And the quantification needs to be provided at 5 minutes and 15 minutes for both FIT-G WT and FIT-mSS271AA-G counting the number of condensates in the nucleus. Especially because the rest of the study is depending on these time points.

    Authors:

    This information is provided in the Supplemental Movie S1C.

    Line 241: the author say that the formation of condensate starts after 5 minutes (line 190) here (line 241) the author claim that it starts after 1 minutes. Please clarify.

    Authors:

    In line 190 we described that FIT NB formation occurs after the excitation and is fully visible after 5 min. In line 241 we stated that the formation starts in the first minutes after excitation, which describes the same time frame. We rephrased the respective sentences.

    Lines 185-188: “A short duration of 1 min 488 nm laser light excitation induced the formation of FIT-GFP signals in discrete spots inside the nucleus, which became fully visible after only five minutes (t=5; Figure 1B and Supplemental Movie S1A).”

    Lines 239-242: “While FIT-GFP NB formation started in the first minutes after excitation and was fully present after 5 min (Supplemental Movie S1A), FITmSS271AA-GFP NB formation occurred earliest 10 min after excitation and was fully visible after 15 min (Supplemental Movie S1C).”

    Line 254: Not sure what the authors claim "not only for interaction but also for FIT NB formation ". To me, the IDR is predicted to be perturbed by modeling when the serines are mutated therefore the IDR might be important to form condensates in the nucleus. Please clarify.

    Authors:

    The formation of nuclear bodies is slow for FITmSS271AA as seen in Figure 2. Previously, we showed that FITmSS271AA homodimerizes less (Gratz et al., 2019.) Therefore, the said IDR is important for both processes, NB formation and homodimerization. We have added this information to make the point clear, lines 253-255:

    “This underlined the significance of the Ser271/272 site, not only for interaction (Gratz et al., 2019) but also for FIT NB formation (Figure 2).”

    Line 255: It's not clear why the author test if the FIT homodimerization is preferentially associated with condensate in the nucleus.

    Authors:

    We test this because both homo- and heterodimerization of bHLH TFs are generally important for the activity of TFs, and we unraveled the connection between protein interaction and NB formation. We state this in lines 228-232.

    Line 269-272: It's not clear to what the authors are referring to.

    Authors:

    We are describing the homodimeric behavior of FIT and FITmSS271AA assessed by homo-FRET measurements that are introduced in the previous paragraph, lines 256-268.

    Line 309: This colocalization part should be presented before line 194.

    Authors:

    We find it convincing to first examine and characterize the process underlying FIT NB formation, then studying a possible function of NBs. The colocalization analysis is part of a functional analysis of NBs. We thank the reviewer for the hint that colocalization also confirms that indeed the nuclear FIT spots are NBs. We will take this point and discuss it, lines 516-522:

    “Additionally, the partial and full colocalization of FIT NBs with various previously reported NB markers confirm that FIT indeed accumulates in and forms NBs. Since several of NB body markers are also behaving in a dynamic manner, this corroborates the formation of dynamic FIT NBs affected by environmental signals.”

    “In conclusion, the properties of liquid condensation and colocalization with NB markers, along with the findings that it occurred irrespective of the fluorescence protein tag preferentially with wild-type FIT, allowed us to coin the term of ‘FIT NBs’.”

    Line 328: add the ref to figure, please.

    Authors:

    Figure reference was added to the text, lines 330-332:

    “The second type (type II) of NB markers were partially colocalized with FIT-GFP. This included the speckle components ARGININE/SERINE-RICH45-mRFP (SR45) and the serine/arginine-rich matrix protein SRm102-mRFP (Figure 5).”

    Line 334: It seems that the size of the SR45 has an anormal very large diameter between 4 and 6 µm. In general a speckle measure about 2-3µm in diameter. Can the author make sure that this structure is not due to overexpression in N. benthamiana or make sure to not oversaturate the image.

    Authors:

    Thank you for this hint. Indeed, there are reports that SR45 is a dynamic component inside cells. It can redistribute depending on environmental conditions and associate into larger speckles depending on the nuclear activity status (Ali et al., 2003). We include this reference and refer to it in the discussion, lines 557-564:

    “Interestingly, typical FIT NB formation did not occur in the presence of PB markers, indicating that they must have had a strong effect on recruiting FIT. This is interesting because the partially colocalizing SR45, PIF3 and PIF4 are also dynamic NB components. Active transcription processes and environmental stimuli affect the sizes and numbers of SR45 speckles and PB (Ali et al., 2003; Legris et al., 2016; Meyer, 2020). This may indicate that, similarly, environmental signals might have affected the colocalization with FIT and resulting NB structures in our experiments. Another factor of interference might also be the level of expression.”

    Line 335: It seems that the colocalization is partial only partial after induction of NB. The FIT NB colocalize around SR45. But it's hard to tell because the images are saturated therefore creating some false overlapping region.

    Authors:

    The localization of FIT with SR45 is partial and occurs only after FIT has undergone condensation, see lines 335-338.

    Line 344-345: It's unappropriated to give the conclusion before the evidence.

    Authors:

    We explain at an earlier paragraph that we will show three different types of colocalization and introduce the respective colocalization types within separate paragraphs accordingly, see lines 314-321.

    Line 353: increase the contrast in the image of t=5 for UAP56H2 since it's hard to assess the colocalization.

    Authors:

    This is done as noted in the figure legend of Figure 6.

    Line 381-382: "In general" does not sound scientific avoid this kind of wording and describe precisely your findings.

    Authors:

    We rephrased the sentence, line 387-388:

    Localization of single expressed PIF3-mCherry remained unchanged at t=0 and t=15 (Supplemental Figure S5A).

    Line 384-385: Provide the data and the reference to the figure.

    Authors:

    We apologize for the misunderstanding and rephrased the sentence, line 389-391:

    After 488 nm excitation, FIT-GFP accumulated and finally colocalized with the large PIF3-mCherry PB at t=15, while the typical FIT NBs did not appear (Figure 7A)

    Line 386: The structure in which FIT-G is present in the Figure 7A t=15 is not alike the once already observed along the paper. This could be explained by over-expression in N. benthamiana. Please explain.

    Authors:

    Thank you for the hint. We discuss this in the discussion part, see lines 555-568.

    Line 393: Explain and provide data why the morphology of PIF4/FIT NB do not correspond to the normal morphology.

    Authors:

    Thank you for the valuable hints. Several reasons may account for this and we provide explanations in the discussion, see lines 555-568.

    Line 396-398: It seems also from the data that co-expression of PIF4 of PIF3 will affect the portioning of FIT between the NP and the NB.

    Authors:

    We can assume that residual nucleoplasm is depleted from protein during NB formation. This is likely true for all assessed colocalization experiments. We discuss this in lines 492-494.

    The discussion is particularly lengthy it might be great to reduce the size and focus on the main findings.

    Authors:

    We shortened the discussion.

    Referees cross-commenting

    All good for me, I think that the comments/suggestions from Reviewer #2 are valid and fair. If they are addressed they will improve considerably the manuscript.

    Reviewer #1 (Significance):

    This manuscript is describing an unprecedent very precise cellular and molecular mechanism in nutrition throughout a large set of microscopies technics. Formation of nuclear bodies and their role are still largely unexplored in this context. Therefore, this study sheds light on the functional role of this membrane less compartment and will be appreciated by a large audience. However, the fine characterization is only made using transient expression in N. Bentamiana and only few proofs of concept are provided in A. thaliana stable line.

    Reviewer #2 (Evidence, reproducibility and clarity):

    The manuscript of Trofimov et al shows that FIT undergoes light-induced, reversible condensation and localizes to nuclear bodies (NBs), likely via liquid-liquid phase separation and light conditions plays important role in activity of FIT. Overall, manuscript is well written, authors have done a great job by doing many detailed and in-depth experiments to support their findings and conclusions.

    However, I have a number of questions/comments regarding the data presented and there are still some issues that authors should take into account.

    Major points/comments:

    1. Authors only focused on blue light conditions. Is there any specific reason for selecting only blue light and not others (red light or far red)?

    Authors:

    There are two main reasons: First, in a preliminary study (not shown) blue light resulted in the formation of the highest numbers of NBs. Second, iron reductase activity assays and gene expression analysis under different light conditions showed a promoting effect under blue light, but not red light or dark red light (Figure 9). This indicated to us, that blue light might activate FIT, and that active FIT may be related to FIT NBs.

    1. Fig. 3C and D: as GFP and GFP-GFP constructs are used as a reference, why not taking the measurements for them at two different time points for example t=0 and t=5 0r t=15???

    Authors:

    Free GFP and GFP-GFP dimers are standard controls for homo-FRET that serve to delimit the range for the measurements.

    1. Line 27-271: Acc to the figure 3d, for the Fluorescence anisotropy measurement of NBs appears to be less. Please explain.

    Authors:

    FA in NBs with FITmSS271AA is variable and the value is lower than that of whole nucleus but not significantly different compared with that in nucleoplasm. We describe the results of Figure 3D in lines 272-275.

    1. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.

    Authors:

    Neither for FIT/bHLH039 nor the FITmSS271AA/bHLH039 pair, there is a significant decrease in the fluorescence lifetime values between t=0 and t=5/15. FIT-G is a control to delimit the range. The interesting experiment is to compare the protein pairs of interest between the different nuclear locations at t=5/15.

    1. Line 300-301: In Figure 4D and 4E. Fluorescence lifetime of G measurement at t=0 seems very similar for both FIT-G as well as FITmSS but if we look at the values of t=0 for FIT-G+bhlh039 it is greater than 2.5 and for FITmSS271AA-G+bhlh039 it is less which suggests more heterodimeric complexes to be formed in FITmSS271AA-G+bhlh039. Similar pattern is observed for NBs and NPs, according to the figure 4d and E.

    Therefore, heterodimeric complexes accumulated more in case of FITmSS271AA-G+bhlh039 as compared to FIT-G+bhlh039 (if we compare measurement values of Fluorescence lifetime of G of FITmSS271AA-G+bhlh039 with FIT-G+bhlh039).

    Please comment and elaborate about this further.

    Authors:

    These conclusions are not valid as the experiments cannot be conducted in parallel. Since the experiments had to be performed on different days due to the duration of measurements including new calibrations of the system, we cannot compare the absolute fluorescence lifetimes between the two sets.

    1. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.

    Authors:

    Please see our response to your comment 4).

    1. Line 439-400: As iron uptake genes (FRO2 and IRT1) are more induced in WT under blue light conditions and FRO2 is less induced in case of red-light conditions. So, what happens to Fe content of WT grown under blue light or red light as compared to WT grown under white light. Perls/PerlsDAb staining of WT roots under different light conditions will add more information to this.

    Authors:

    We focused on the relatively short-term effects of blue light on signaling of nuclear events that could be related to FIT activity directly, particularly gene expression and iron reductase activity as consequence of FRO2 expression. These are both rapid changes that occur in the roots and can be measured. We suspect that iron re-localization and Fe uptake also occur, however, in our experience differences in metal contents will not be directly significant when applying the standard methods like ICP-MS or PERLs staining.

    Minor comments:

    Line 75-76: Rephrase the sentence

    Authors:

    We rephrased the sentence, lines 73-74:

    “As sessile organisms, plants adjust to an ever-changing environment and acclimate rapidly. They also control the amount of micronutrients they take up.”

    Line 119: Rephrase the sentence

    Authors:

    We rephrased the sentence, line 118-119:

    “Various NBs are found. Plants and animals share several of them, e.g. the nucleolus, Cajal bodies, and speckles.”

    Line 235-236: rephrase the sentence

    Authors:

    We rephrased the sentence, line 232-234:

    “In the work of Gratz et al. (2019), the hosphor-mimicking FITmS272E protein did not show significant changes in its behavior compared to wild-type FIT.”

    Line 444: Correct the sentence “Fe deficiency versus sufficiency”

    Authors:

    We corrected that, line 449-451:

    “In both, the far-red light and darkness situations, FIT was induced under iron deficiency versus sufficiency, while on the other side, BHLH039, FRO2 and IRT1 were not induced at all in these light conditions (Figure 9I-P).”

    Referees cross-commenting

    I agree with R1 suggestions/comments and i think manuscript quality will be much better if authors carry out the experiments suggested by R1. I believe this will also strengthen their conclusions.

    Reviewer #2 (Significance):

    Overall, manuscript is well written, authors have done a nice job by doing several key experiments to support their findings and conclusions. However, the results and manuscript can be improved further by addressing some question raised here. This study is interesting for basic scientists which unravels the crosstalk of light signaling in nutrient signaling pathways.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    _The manuscript of Trofimov et al shows that FIT undergoes light-induced, reversible condensation and localizes to nuclear bodies (NBs), likely via liquid-liquid phase separation and light conditions plays important role in activity of FIT. Overall, manuscript is well written, authors have done a great job by doing many detailed and in-depth experiments to support their findings and conclusions.
    However, I have a number of questions/comments regarding the data presented and there are still some issues that authors should take into account.

    Major points/comments:

    1. Authors only focused on blue light conditions. Is there any specific reason for selecting only blue light and not others (red light or far red)?
    2. Fig. 3C and D: as GFP and GFP-GFP constructs are used as a reference, why not taking the measurements for them at two different time points for example t=0 and t=5 0r t=15???
    3. Line 27-271: Acc to the figure 3d, for the Fluorescence anisotropy measurement of NBs appears to be less. Please explain.
    4. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.
    5. Line 300-301: In Figure 4D and 4E. Fluorescence lifetime of G measurement at t=0 seems very similar for both FIT-G as well as FITmSS but if we look at the values of t=0 for FIT-G+bhlh039 it is greater than 2.5 and for FITmSS271AA-G+bhlh039 it is less which suggests more heterodimeric complexes to be formed in FITmSS271AA-G+bhlh039. Similar pattern is observed for NBs and NPs, according to the figure 4d and E.
      Therefore, heterodimeric complexes accumulated more in case of FITmSS271AA-G+bhlh039 as compared to FIT-G+bhlh039 (if we compare measurement values of Fluorescence lifetime of G of FITmSS271AA-G+bhlh039 with FIT-G+bhlh039).
      Please comment and elaborate about this further.
    6. Figure 4: For the negative controls, data is shown at only t=0, data should be shown at t=5 also to prove that there is no decrease in fluorescence in these negative controls when they are expressed alone without bhlh39 as there is no acceptor in this case.
    7. Line 439-400: As iron uptake genes (FRO2 and IRT1) are more induced in WT under blue light conditions and FRO2 is less induced in case of red-light conditions. So, what happens to Fe content of WT grown under blue light or red light as compared to WT grown under white light. Perls/PerlsDAb staining of WT roots under different light conditions will add more information to this.

    Minor comments:

    Line 75-76: Rephrase the sentence

    Line 119: Rephrase the sentence

    Line 235-236: rephrase the sentence

    Line 444: Correct the sentence "Fe deficiency versus sufficiency"

    Referees cross-commenting

    I agree with R1 suggestions/comments and i think manuscript quality will be much better if authors carry out the experiments suggested by R1. I believe this will also strengthen their conclusions.

    Significance

    Overall, manuscript is well written, authors have done a nice job by doing several key experiments to support their findings and conclusions. However, the results and manuscript can be improved further by addressing some question raised here. This study is interesting for basic scientists which unravels the crosstalk of light signaling in nutrient signaling pathways.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this paper entitled " FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) accumulates in homo- and heterodimeric complexes in dynamic and inducible nuclear condensates associated with speckle components", Trofimov and colleagues describe for the first time the function of FIT in nuclear bodies. By an impressive set of microscopies technics they assess FIT localization in nuclear bodies and its dynamics. Finally, they reveal their importance in controlling iron deficiency pathway. The manuscript is well written and fully understandable. Nonetheless, at it stands the manuscript present some weakness by the lack of quantification for co-localization and absence controls making hard to follow authors claim. Moreover, to substantially improve the manuscript the authors need to provide more proof of concepts in A. thaliana as all the nice molecular and cellular mechanism is only provided in N. bentamiana. Finally, some key conclusions in the paper are not fully supported by the data.
    Please see below:

    Main comments:

    1. For colocalization analysis, the author should provide semi-quantitative data counting the number of times by eyes they observed no, partial or full co-localization and indicate on how many nucleus they used.
    2. Do semi-quantitative co-localization analysis by eyes, on FIT NB with known NB makers in the A. thaliana root. For now, all the nicely described molecular mechanism is shown in N. benthamiana which makes this story a bit weak since all the iron transcriptional machinery is localized in the root to activate IRT1.
    3. The authors need to provide data clearly showing that the blue light induce NB in A. thaliana and N. benthamiana.
    4. Direct conclusion in the manuscript:
    • Line 170: At this point of the paper the author cannot claim that the formation of FIT condensates in the nucleus is due to the light as it might be indirectly linked to cell death induced by photodamaging the cell using a 488 lasers for several minutes. This is true especially with the ELYRA PS which has strong lasers made for super resolution and that Cell death is now liked to iron homeostasis. The same experiment might be done using a spinning disc or if the authors present the data of the blue light experiment mentioned above this assumption might be discarded. Alternatively, the author can use PI staining to assess cell viability after several minutes under 488nm laser.
    • Line 273: I don't agree with the first part of the authors conclusion, saying that "wild-type FIT had better capacities to localize to NBs than mutant FITmSS271AA, presumably due its IDRSer271/272 at the C-terminus. This is not supported by the data. In order to make such a claim the author need to compare the FA of FIT WT with FITmSS271AA by statistical analysis. Nonetheless, the value seems to be identical on the graphs. The main differences that I observed here are, 1) NP value for FITmSS271AA seems to be lower compared to FIT-WT, suggesting that the Serine might be important to regulate protein homedimerization partitioning between the NP and the NB. 2) To me, something very interesting that the author did not mention is the way the FA of FITmSS271AA in the NB and NP is behaving with high variability. The FA of those is widely spread ranging from 0.30 to 0.13 compared to the FIT-WT. To me it seems that according to the results that the Serine 271/272 are required to stabilize FIT homodimerization. This would not only explain the delay to form the condensate but also the decreased number and size observed for FITmSS271AA compared to FIT-WT. As the homodimerization occurs with high variability in FITmSS271AA, there is less chance that the protein will meet therefore decreasing the time to homodimerize and form/aggregate NB.
    • Line 301: According to my previous comment (line 273), here it seems that the Serine 271/272 are required only for proper partitioning of the heterodimer FIT/BHLH039 between the NP and NB but not for the stability of the heterodimer formation. However, it might be great if the author would count the number of BHLH039 condensates in both version FITmSS271AA and FIT-WT. To my opinion, they would observe less BHLH039 condensate because the homodimer of FITmSS271AA is less likely to occur because of instability.
    1. To wrap up the story about the requirements of NB in mediating iron acquisition under different light regimes, provide data for IRT1/FRO2 expression levels in fit background complemented with FITmSS271AA plants. I know that this experiment is particularly lengthy, but it would provide much more to this nice story.

    Minor comments

    In general, I would suggest the author to avoid abbreviation, it gets really confusing especially with small abbreviation as NB, NP, PB, FA.

    Line 106: What does IDR mean?

    Line 163-164: provide data or cite a figure properly for blue light induction.

    Line 188: Provide Figure ref.

    Line 194: the conclusion is too strong. The authors conclude that the condensate they observed are NB based on the fact the same procedure to induce NB has been used in other study which is not convincing. Co-localization analysis with NB markers need to be done to support such a claim. At this step of the study, the author may want to talk about condensate in the nucleus which might correspond to NB. Please do so for the following paragraph in the manuscript until colocalization analysis has not been provided. Alternatively provide the co-localization analysis at this step in the paper.

    Line 214: In order to assess the photo bleaching due to the FRAP experiment the quantification of the "recovery" needs to be provided in an unbleached area. This might explain why FIT recover up to 80% in the condensate. Moreover, the author conclude that the recovery is high however it's tricky to assess since no comparison is made with a negative/positive control.

    Line 220-227: The conclusion it's too strong as I mentioned previously the author cannot claim that the condensate are NBs at this step of the study. They observed nuclear condensates that behave like NB when looking at the way to induce them, their shape, and the recovery. And please include a control.

    Line 239: It's unappropriated to give the conclusion before the evidence.

    Line 240: Figure 2A, provide images of FIT-G at 15min in order to compare. And the quantification needs to be provided at 5 minutes and 15 minutes for both FIT-G WT and FIT-mSS271AA-G counting the number of condensates in the nucleus. Especially because the rest of the study is depending on these time points.

    Line 241: the author say that the formation of condensate starts after 5 minutes (line 190) here (line 241) the author claim that it starts after 1 minutes. Please clarify.

    Line 254: Not sure what the authors claim "not only for interaction but also for FIT NB formation ". To me, the IDR is predicted to be perturbed by modeling when the serines are mutated therefore the IDR might be important to form condensates in the nucleus. Please clarify.

    Line 255: It's not clear why the author test if the FIT homodimerization is preferentially associated with condensate in the nucleus.

    Line 269-272: It's not clear to what the authors are referring to.

    Line 309: This colocalization part should be presented before line 194.

    Line 328: add the ref to figure, please.

    Line 334: It seems that the size of the SR45 has an anormal very large diameter between 4 and 6 µm. In general a speckle measure about 2-3µm in diameter. Can the author make sure that this structure is not due to overexpression in N. benthamiana or make sure to not oversaturate the image

    Line 335: It seems that the colocalization is partial only partial after induction of NB. The FIT NB colocalize around SR45. But it's hard to tell because the images are saturated therefore creating some false overlapping region.

    Line 344-345: It's unappropriated to give the conclusion before the evidence.

    Line 353: increase the contrast in the image of t=5 for UAP56H2 since it's hard to assess the colocalization.

    Line 381-382: "In general" does not sound scientific avoid this kind of wording and describe precisely your findings.

    Line 384-385: Provide the data and the reference to the figure.

    Line 386: The structure in which FIT-G is present in the Figure 7A t=15 is not alike the once already observed along the paper. This could be explained by over-expression in N. benthamiana. Please explain.

    Line 393: Explain and provide data why the morphology of PIF4/FIT NB do not correspond to the normal morphology.

    Line 396-398: It seems also from the data that co-expression of PIF4 of PIF3 will affect the portioning of FIT between the NP and the NB.

    The discussion is particularly lengthy it might be great to reduce the size and focus on the main findings.

    Referees cross-commenting

    All good for me, I think that the comments/suggestions from Reviewer #2 are valid and fair. If they are addressed they will improve considerably the manuscript.

    Significance

    This manuscript is describing an unprecedent very precise cellular and molecular mechanism in nutrition throughout a large set of microscopies technics. Formation of nuclear bodies and their role are still largely unexplored in this context. Therefore, this study sheds light on the functional role of this membrane less compartment and will be appreciated by a large audience. However, the fine characterization is only made using transient expression in N. Bentamiana and only few proofs of concept are provided in A. thaliana stable line.

  8. Version published to 10.1101/2022.12.20.520918v3 on bioRxiv
  9. Version published to 10.1101/2022.12.20.520918v2 on bioRxiv
  10. Version published to 10.1101/2022.12.20.520918v1 on bioRxiv