A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis

  1. Shanna Romand
  2. Hela Abdelkefi
  3. Cécile Lecampion
  4. Mohamed Belaroussi
  5. Melanie Dussenne
  6. Brigitte Ksas
  7. Sylvie Citerne
  8. Jose Caius
  9. Stefano D'Alessandro
  10. Hatem Fakhfakh
  11. Stefano Caffarri
  12. Michel Havaux
  13. Ben Field

  • Curated by eLife

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    Evaluation Summary:

    This manuscript reports that ppGpp accumulation is necessary for acclimation to nitrogen starvation in a model plant Arabidopsis. The authors also showed a ppGpp-mediated downregulation of chloroplast gene transcription and a coordinated plastid-nuclear gene expression under nitrogen deficiency. This represents a solid new step in our understanding of plant responses to nitrogen-limiting conditions as well as on the role of ppGpp in plants and possibly throughout the green lineage.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilization, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis . We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

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  1. Version published to 10.7554/elife.75041 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Romand et al investigates the role of hyperphosphorylated guanosine nucleotides (ppGpp) in acclimation of plant chloroplasts to nitrogen limitation. The signaling role of ppGpp as alarmone is well established in the stringent response of bacteria. The stringent response allows bacteria to adapt to amino acid or carbon starvation and other acute abiotic stress conditions by downregulation of resource-consuming cell processes. A series of studies, including the current one, have demonstrated the retention of the bacterial-type ppGpp-mediated signaling response in plant and algal chloroplasts. The current study convincingly demonstrates the involvement of ppGpp in remodeling of photosynthetic machinery under nitrogen limitation. Using three Arabidopsis RSH lines (two underaccumulators and one overaccumulator of ppGpp), the authors show that the ppGpp is required for preventing excess ROS accumulation, oxidative stress and death of cotyledons under nitrogen limiting condition. The authors show a transient accumulation in ppGpp upon nitrogen limitation, which is followed by a sustained increase in the ratio of ppGpp to GTP. There is a prompt decline in maximum photochemical efficiency of photosystem II (PSII) and linear electron transport under nitrogen deficiency in wild type and ppGpp overaccumulator plants. However, mutants with low amount of ppGpp have a delayed decrease in these photosynthetic parameters. PpGpp is further shown to decrease (or degrade) photosynthetic proteins, and a remodeling of PSII that involves uncoupling of LHC II from the reaction center core has been suggested to occur under nitrogen starvation. The authors also show a ppGpp-mediated downregulation of chloroplast gene transcription and a coordinated plastid-nuclear gene expression under nitrogen deficiency.

    Strengths

    1. The conclusions of this paper are mostly well supported by data. With three different RSH lines, there is a convincing demonstration of the specific involvement of ppGpp in nutrient acclimation. The line carrying conditional overexpression of Drosophila ppGpp hydrolase (MESH) nicely complements the RSH lines and strengthens many of the conclusions. This is a detailed analysis of ppGpp function in a plant species. The data supplement accompanying each main figure is extensive and helpful.
    2. The genomic analysis in nitrogen replete and deplete wild type uncovers an interesting regulation of RSH enzymes at the transcriptional level. This is likely to be part of a signaling response that works in conjunction with allosteric modulation of RSH activity under nitrogen limitation.
    3. The large-scale analysis of plastid and nuclear gene transcripts supports the involvement of ppGpp in coordinated repression of plastid and nuclear gene transcription.
    4. By the inclusion of mitochondrial genes and proteins in their analysis, the authors clearly show that the ppGpp action is limited to plastids and does not extend to mitochondria, which like chloroplasts, have a bacterial ancestry.
    5. The thorough demonstration of the involvement of ppGpp in low nitrogen acclimation of photosynthetic metabolism adds greatly to the understanding of plant abiotic stress tolerance mechanisms and ppGpp function in both plants and bacteria.

    We thank the reviewer for these observations on our work.

    Weaknesses:

    1. With two earlier reports from a different laboratory (Maekawa et al 2015 and Honoki et al 2018) showing the involvement of ppGpp in acclimation to nitrogen deficiency, the novelty of the current study is diminished. The authors mention that the double mutant (rsh2 rsh3) used by Honoki et al does not show a clear phenotype other than a delay in Rubisco degradation. It is not clear to me why the lack of two major RSH isoforms, involved in synthesis of ppGpp under light, would not produce any phenotype. This discrepancy should be discussed further in the manuscript.

    The work of Maekawa et al., 2015 and Honoki et al., 2018 was indeed important for highlighting the potential involvement of ppGpp in the acclimation to nitrogen deficiency. However, these studies were based on the constitutive overaccumulation of ppGpp. Here, we demonstrate a physiological requirement for ppGpp signalling by the plant to allow acclimation to an abiotic stress- we consider this to be a major step forwards in understanding the role of ppGpp in plants, and one of the few examples of a physiological requirement for ppGpp in plants.

    We mention the use of an RSH2 RSH3 mutant by Honoki et al. 2018 while putting our results into the context of previous findings in the discussion. We bring the attention of the reviewer to our analysis of an RSH2 RSH3 mutant in this study, and that in our hands the mutant phenotype was indistinguishable from the RSH quadruple mutant (rshQM) (Figure 2- figure supplement 1 panel B). Therefore, we do indeed consider that RSH2 and RSH3 are the main RSH isoforms involved in ppGpp-mediated acclimation to nitrogen deficiency, and we state this ( see p7 l161-164 in original manuscript). As we explain in the discussion there are probably technical reasons for the discrepancy with the results reported by Honoki et al. 2018. We also note here that the RSH2 RSH3 mutants used in our study and by Honoki et al. 2018 are not identical: the same SAIL insertion SAIL_305_B12 was used for rsh2, while the rsh3 allele used by Honoki was the GABIkat insertion GABI129D02 and here SAIL_99_G05). We now add this difference in the genetic identity of the mutants as an additional potential explanation for the different findings in the two studies.

    1. The authors at times show a tendency to overinterpret their results. A ppGpp-mediated repression of chloroplast transcription and translation is sufficient to explain most of the observations in this study. However, the authors seem to go beyond this simple explanatory framework by invoking specific roles for ppGpp in remodeling of PSII antenna-core interaction and in blocking of PSII reaction center repair. There is no data in the manuscript in support of these two propositions. A coordinated decrease in synthesis of most chloroplast proteins, including the D1 reaction center protein of PSII, is sufficient to explain the decrease in Fv/Fm. There is no evidence in the manuscript for "photoinactivation gaining an upper hand via ppGpp-mediated signaling"

    The circuit breaker analogy of PSII photoinhibition that the authors discuss in support is just an interpretation. The remodeling of PSII antenna-core interaction, likewise, could be a simple consequence of the ppGpp-mediated decrease in D1 protein synthesis. The high antenna-core ratio under nitrogen starvation likely reflects the lag in the decrease of LHCB1 (which eventually decreases significantly by day 16).

    Since ppGpp-signaling primarily affects plastid transcription and translation, there is a rapid decrease in plastid psbA gene product (D1) relative to the nuclear-encoded LHCB1. The unconnected LHCII might simply be a result of the mismatch in antenna-core stoichiometry rather than an active regulation of PSII functional assembly by ppGpp.

    We have re-worked the discussion to make these points more clearly, and also to tone down certain points where we may have over-stretched our interpretation.

    We think that our interpretation is essentially the same as the reviewer’s- the ppGpp mediated inhibition of chloroplast translation and transcription is sufficient to explain the majority of our results. In the discussion we also discuss the possibility that ppGpp stimulates the active degradation of some chloroplast proteins, and put this in context of studies showing that N-starvation activates the specific proteolysis of certain photosynthetic proteins in Chlamydomonas and has an effect on the half lives of different chloroplast proteins in plants. We do not propose or present data suggesting that ppGpp has any other specific targets/effectors- for example within the PSII repair cycle or in remodelling PSII stoichiometry- although we also cannot exclude the possibility of targets in these processes.

    We think that the ppGpp dependent change in PSII stoichiometry during N-starvation is not just a side effect of a general downregulation or a temporary mismatch as suggested- but due to its size, persistence and effect on photosynthesis is likely to be part of the acclimation process. For example, the ppGpp-dependent drop in Fv/Fm is maintained at day 16 and even beyond (Fig 2D). We also see that photosynthetic proteins are still degraded in low ppGpp mutants (Fig. 3A), but that the high Fv/Fm is maintained throughout. These points and the fact that the alteration of PSII stoichiometry is not caused by the direct action of ppGpp on PSII (but via transcription/translation) does not mean that it is not important or does not play a role in acclimation. Other studies report that PSII RC inactivation can protect PSI (e.g. Tikkanen et al. 2014) and ppGpp may be working in a similar fashion here by reducing the flow of energy into the photosynthetic electron transport chain. This interpretation is consistent with our results showing that wild-type plants and high ppGpp plants (rsh1-1) accumulate less ROS and ROS-related damage than plants defective in ppGpp biosynthesis (Fig. 1).

    1. The work is mostly descriptive of the involvement of ppGpp in low nitrogen tolerance without any data on how the nitrogen deficiency is sensed by the RSH enzymes and how ppGpp orchestrates the multi-faceted acclimatory response. Perhaps, these aspects are beyond scope of the current manuscript, but they could be discussed more.

    We agree that these are very important questions, and also that they are out of the scope of the current work. We think that our work goes beyond the descriptive by demonstrating the physiological functions of ppGpp-signalling during nitrogen deficiency and a framework for how it occurs (i.e downregulation of chloroplast function and avoidance of excess oxidative stress).

    Reviewer #3 (Public Review):

    The manuscript by Romand et al. explores the role of guanosine penta- and tetraphosphate, ppGpp, in the acclimation of plants to nitrogen limitation. It shows that an early and transient ppGpp accumulation - and a controlled ppGpp/GTP ratio - is necessary for a proper acclimation of plants to such stress. The pathway is shown to act on remodeling the photosynthetic machinery and downregulating photosynthesis during stress, thus limiting ROS damage to the plants. This regulation most likely takes place by affecting chloroplast transcription, maintaining the balance between nucleus- and chloroplast-encoded proteins.

    The manuscript proposes a thorough analysis of the ppGpp-induced response including extensive wild type and mutant analyses at the gene and protein expression level as well as at the physiological level under nitrogen limitation together with heterologous expression of ppGpp hydrolase from Drosophila. The conclusions are carefully backed by the data (but for the lack of gene expression analysis in the high ppGpp line, rsh1-1), the figures and text clear, well-written and easy to follow. Altogether it represents a solid new step in improving the comprehension of plant response to nitrogen limitation, as well as on the role of ppGpp in plants and possibly throughout the green lineage. An alternative hypothesis to ppGpp photoprotective role could be discussed in that photoprotection may be an indirect effect due to photosynthetic protein degradation enabled by ppGpp, possibly through modulation of ppGpp/GTP ratio affecting chloroplast protease activity.

    On this last point we agree with the reviewer- our data indicates that the photoprotective role of ppGpp is via the ppGpp-dependent control of the abundance of photosynthetic proteins. This is indirect in the sense that we have no evidence that ppGpp itself interacts with components of the photosynthetic machinery. However, as discussed below we do not think that photoprotection is just a side-effect of ppGpp’s action- we show that the capacity to synthetise ppGpp is required for avoiding the generation of ROS and tissue death.

  3. eLife

    Evaluation Summary:

    This manuscript reports that ppGpp accumulation is necessary for acclimation to nitrogen starvation in a model plant Arabidopsis. The authors also showed a ppGpp-mediated downregulation of chloroplast gene transcription and a coordinated plastid-nuclear gene expression under nitrogen deficiency. This represents a solid new step in our understanding of plant responses to nitrogen-limiting conditions as well as on the role of ppGpp in plants and possibly throughout the green lineage.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This manuscript entitled "A ppGpp-mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis " by Romand et al. described ppGpp accumulation is necessary for acclimation to nitrogen starvation in a model plant Arabidopsis. The authors revealed that ppGpp accumulation leads to remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity. It seems to be reasonable that the ppGpp signal works for protecting plant cells from oxidative stress. Further, the authors also clarified that ppGpp accumulation affects patterns of chloroplast gene expression during nitrogen starvation. The findings will be highly appreciated.

  5. eLife

    Reviewer #2 (Public Review):

    Romand et al investigates the role of hyperphosphorylated guanosine nucleotides (ppGpp) in acclimation of plant chloroplasts to nitrogen limitation. The signaling role of ppGpp as alarmone is well established in the stringent response of bacteria. The stringent response allows bacteria to adapt to amino acid or carbon starvation and other acute abiotic stress conditions by downregulation of resource-consuming cell processes. A series of studies, including the current one, have demonstrated the retention of the bacterial-type ppGpp-mediated signaling response in plant and algal chloroplasts. The current study convincingly demonstrates the involvement of ppGpp in remodeling of photosynthetic machinery under nitrogen limitation. Using three Arabidopsis RSH lines (two underaccumulators and one overaccumulator of ppGpp), the authors show that the ppGpp is required for preventing excess ROS accumulation, oxidative stress and death of cotyledons under nitrogen limiting condition. The authors show a transient accumulation in ppGpp upon nitrogen limitation, which is followed by a sustained increase in the ratio of ppGpp to GTP. There is a prompt decline in maximum photochemical efficiency of photosystem II (PSII) and linear electron transport under nitrogen deficiency in wild type and ppGpp overaccumulator plants. However, mutants with low amount of ppGpp have a delayed decrease in these photosynthetic parameters. PpGpp is further shown to decrease (or degrade) photosynthetic proteins, and a remodeling of PSII that involves uncoupling of LHC II from the reaction center core has been suggested to occur under nitrogen starvation. The authors also show a ppGpp-mediated downregulation of chloroplast gene transcription and a coordinated plastid-nuclear gene expression under nitrogen deficiency.

    Strengths:

    1. The conclusions of this paper are mostly well supported by data. With three different RSH lines, there is a convincing demonstration of the specific involvement of ppGpp in nutrient acclimation. The line carrying conditional overexpression of Drosophila ppGpp hydrolase (MESH) nicely complements the RSH lines and strengthens many of the conclusions. This is a detailed analysis of ppGpp function in a plant species. The data supplement accompanying each main figure is extensive and helpful.

    2. The genomic analysis in nitrogen replete and deplete wild type uncovers an interesting regulation of RSH enzymes at the transcriptional level. This is likely to be part of a signaling response that works in conjunction with allosteric modulation of RSH activity under nitrogen limitation.

    3. The large-scale analysis of plastid and nuclear gene transcripts supports the involvement of ppGpp in coordinated repression of plastid and nuclear gene transcription.

    4. By the inclusion of mitochondrial genes and proteins in their analysis, the authors clearly show that the ppGpp action is limited to plastids and does not extend to mitochondria, which like chloroplasts, have a bacterial ancestry.

    5. The thorough demonstration of the involvement of ppGpp in low nitrogen acclimation of photosynthetic metabolism adds greatly to the understanding of plant abiotic stress tolerance mechanisms and ppGpp function in both plants and bacteria.

    Weaknesses:

    1. With two earlier reports from a different laboratory (Maekawa et al 2015 and Honoki et al 2018) showing the involvement of ppGpp in acclimation to nitrogen deficiency, the novelty of the current study is diminished. The authors mention that the double mutant (rsh2 rsh3) used by Honoki et al does not show a clear phenotype other than a delay in Rubisco degradation. It is not clear to me why the lack of two major RSH isoforms, involved in synthesis of ppGpp under light, would not produce any phenotype. This discrepancy should be discussed further in the manuscript.

    2. The authors at times show a tendency to overinterpret their results. A ppGpp-mediated repression of chloroplast transcription and translation is sufficient to explain most of the observations in this study. However, the authors seem to go beyond this simple explanatory framework by invoking specific roles for ppGpp in remodeling of PSII antenna-core interaction and in blocking of PSII reaction center repair. There is no data in the manuscript in support of these two propositions. A coordinated decrease in synthesis of most chloroplast proteins, including the D1 reaction center protein of PSII, is sufficient to explain the decrease in Fv/Fm. There is no evidence in the manuscript for "photoinactivation gaining an upper hand via ppGpp-mediated signaling". The circuit breaker analogy of PSII photoinhibition that the authors discuss in support is just an interpretation. The remodeling of PSII antenna-core interaction, likewise, could be a simple consequence of the ppGpp-mediated decrease in D1 protein synthesis. The high antenna-core ratio under nitrogen starvation likely reflects the lag in the decrease of LHCB1 (which eventually decreases significantly by day 16). Since ppGpp-signaling primarily affects plastid transcription and translation, there is a rapid decrease in plastid psbA gene product (D1) relative to the nuclear-encoded LHCB1. The unconnected LHCII might simply be a result of the mismatch in antenna-core stoichiometry rather than an active regulation of PSII functional assembly by ppGpp.

    3. The work is mostly descriptive of the involvement of ppGpp in low nitrogen tolerance without any data on how the nitrogen deficiency is sensed by the RSH enzymes and how ppGpp orchestrates the multi-faceted acclimatory response. Perhaps, these aspects are beyond scope of the current manuscript, but they could be discussed more.

  6. eLife

    Reviewer #3 (Public Review):

    The manuscript by Romand et al. explores the role of guanosine penta- and tetraphosphate, ppGpp, in the acclimation of plants to nitrogen limitation. It shows that an early and transient ppGpp accumulation - and a controlled ppGpp/GTP ratio - is necessary for a proper acclimation of plants to such stress. The pathway is shown to act on remodeling the photosynthetic machinery and downregulating photosynthesis during stress, thus limiting ROS damage to the plants. This regulation most likely takes place by affecting chloroplast transcription, maintaining the balance between nucleus- and chloroplast-encoded proteins.

    The manuscript proposes a thorough analysis of the ppGpp-induced response including extensive wild type and mutant analyses at the gene and protein expression level as well as at the physiological level under nitrogen limitation together with heterologous expression of ppGpp hydrolase from Drosophila. The conclusions are carefully backed by the data (but for the lack of gene expression analysis in the high ppGpp line, rsh1-1), the figures and text clear, well-written and easy to follow. Altogether it represents a solid new step in improving the comprehension of plant response to nitrogen limitation, as well as on the role of ppGpp in plants and possibly throughout the green lineage. An alternative hypothesis to ppGpp photoprotective role could be discussed in that photoprotection may be an indirect effect due to photosynthetic protein degradation enabled by ppGpp, possibly through modulation of ppGpp/GTP ratio affecting chloroplast protease activity.

  7. Version published to 10.1101/2021.09.18.460674v1 on bioRxiv