Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW and a maternal influence on leaf patterning in C 4 grasses

  1. Thomas E. Hughes
  2. Olga Sedelnikova
  3. Mimi Thomas
  4. Jane A. Langdale

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Abstract

Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C 3 or C 4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C 3 species veins are separated by a greater number of intervening mesophyll cells than in derived C 4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C 4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C 3 species rice. Here we show that SCR is required for inner leaf patterning in the C 4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells, whereas inner leaf tissues are patterned normally in scr;nkd mutants of rice. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C 4 but not C 3 grasses. Finally, we identified a maternal effect in maize in which maternally derived NKD can affect patterning of cells in leaf primordia that are initiated during embryogenesis. Together our results provide insight into the evolution of cell patterning in grass leaves, demonstrate a novel patterning role for IDD genes in C 4 leaves and suggest that NKD can influence embryonic leaf development non-cell autonomously from the surrounding maternal tissue.

Summary statement

Mutations in NKD IDD genes enhance loss of function scr phenotypes in the leaves of C 4 grasses maize and Setaria viridis but not in the C 3 grass rice, and reveal a maternal effect on cell-type patterning in leaves that are initiated during embryogenesis.

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  1. Review Commons

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    Reply to the reviewers

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

    All the conclusions are based on solid evidence and convincing, and the methodology are in detail to follow or repeat. The writing of the manuscript is logical and easy to follow.

    We thank the reviewer for these comments

    1. The mutation experiments indicated that nkd enhanced the phenotype of scr, but there is no leaf phenotype variation in nkd muations, this is some way difficult to understand, it would much better if the authors can give much more explanation in the discussion.

    We have added more discussion on this point. One possibility is that collectively the four genes function redundantly, however, due to the transcriptional negative feedback loop discovered here (Figure 3B), when NKD genes are mutated then SCR expression is enhanced, hence phenotypic perturbations are less likely to be observed than when SCR genes are mutated.

    2.The word green millet in the first paragraph should be changed to green foxtail. Millet means domesticated small cereal grains, such as foxtail millet, finger millet, proso millet etc.

    We thank the reviewer for this feedback and have made the suggested change.

    Reviewer #1 (Significance (Required)):

    The manuscript, which titled Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW and a maternal influence on leaf patterning in C4 grasses by Hughes et al., first reported that SCR works regulating both leaf inner pattern and epidermal stomatal patterning in the C4 model plant green foxtail. The functional difference of this gene in Setaria from that in maize and rice indicated that the inner leaf cell patterning regulation of SCR is not a characteristic of C4 Species; this gave us insight understanding of the complex of C4 leaf cell patterning. In addition to this important discover, the authors found that mutations in NKD IDD genes enhance loss of function scr phenotypes in the leaves of C4 maize and Setaria but not in the C3 rice, indicating NKD IDD was involved in the leaf cell patterning in C4 species, but no in C3. They also identified a maternal effect on cell-type patterning in maize leaves that are initiated during embryogenesis.

    We thank the reviewer for their kind comments and suggestions.

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

    The leaf anatomy that distinguishes C4 from C3 plants has been known for decades, with veins in C4 plants separated by 1 to 3 (generally 2) mesophyll cells whereas those in C3 plants are considerably farther apart. This anatomical pattern appears to be critical for the function of the C4 pathway, which under some environmental conditions is a more efficient way to fix carbon than the C3 pathway. Despite the obvious importance of close vein spacing, the genetic mechanisms that control it have been surprisingly difficult to untangle. The statement on the bottom on p. 2 ("To date, very few regulators of cell-patterning in inner leaf tissues have been identified...") is an understatement. The paper by Hughes et al. offers a major step in uncovering the basis of C4 vein spacing.

    We thank the reviewer for this feedback and agree that this work represents a major step forward in understanding C4 vein spacing.

    The authors build on their previous work in Scarecrow-like proteins in maize and rice. In maize, SCR controls patterning of the mesophyll, whereas in rice it controls development of stomata. This paper pursues the possibility that the differences in SCR roles may have to do with interacting proteins. Based on work in Arabidopsis the authors focus on proteins with an indeterminate domain (IDD) and specifically on the NAKED ENDOSPERM genes.

    The paper presents an analysis of an impressive set of mutants in three species. A major step in this paper is the comparison among three species of grasses - maize, rice, and Setaria - rather than the more common two species, usually maize and rice. Maize and rice differ in photosynthetic pathway but they also differ in many other traits that reflect the ca. 50 million years since their last common ancestor. Setaria is, like maize, C4 and the two species are more closely related to each other than either is to rice, although they represent two independent acquisitions of C4. This paper shows that SCR orthologs control stomatal patterning in both rice and Setaria implying that the stomatal function of SCR may be ancestral in the grasses and also is not directly connected to photosynthetic pathway.

    The availability of allelic combinations of SCR and NKD in maize in particular permits the inference of possible maternal effect on the vein spacing phenotype, although exactly how this happens remains unclear.

    The discussion provides a careful and logical assessment of the state of knowledge on SCR and IDD proteins in general, and the new data on SCR and NKD in particular. Many questions remain unresolved, and many additional experiments could be suggested. However, the power of the genetics and the phenotypic analysis together provide a novel direction for research on vein spacing. I will refrain in this review from suggesting what additional information would be nice to have since I think a review should assess the quality of the paper as it stands, not as it could be with months more of work.

    My only really substantive suggestion is that the micrographs of the Setaria leaves need to be improved. Specifically, in Figure 6E it is hard to see the details of the fused veins. Either the section is too thick or the camera was not focused properly. Because this image in particular is central to the entire paper I would recommend aiming for the clarity of the images of Zea cross sections, which are fine.

    We thank the reviewer for this suggestion. Obtaining leaf cross section micrographs from the Setaria scr1;scr2;nkd mutants was extremely challenging as the growth phenotype is so severe (Figure 5), meaning that the available leaves are small and extremely fragile. Multiple attempts to fix and section leaves using a microtome failed, with leaves consistently collapsing. In our hands, Setaria is not as amenable to fresh vibratome sectioning as maize, and combined with the additional challenges of handling the tiny triple mutant leaves mean that the resultant images are not of the same quality as the maize figures. We have included a supplemental figure (Figure S8) with additional examples of fused veins identified in our screening.

    Very minor point: p. 3 - "double Zmscr1;Zmscr1h mutants" - what does the "h" in Zmscr1h refer to?

    In this context h refers to this gene being a homeologous gene duplicate, as first explained in Hughes et al. (2019). We have included an explanation in the revision.

    Reviewer #2 (Significance (Required)):

    Strengths of the paper are 1) the inclusion of three species to help determine which aspects of the gene function may be ascribed to C4; 2) thoughtful and comprehensive genetic analysis; 3) careful sections of leaves; 4) outlines of the limitations of the approach. Limitations (several of which the authors acknowledge in the Discussion) include a general lack of molecular genetic data (protein interactions, DNA binding sites, RNA-seq, etc.). While this information would be great to have, I think the strength of the genetics is such that the paper will be foundational for future work in any case. The one bit of additional data that would be ideal would be information bearing on the two mechanistic hypotheses laid out on p. 10. The model that SCR and NKD promote cell division and specify mesophyll identity is the opposite of the model that SCR and NKD inhibit vein formation. An experiment that helped point the reader toward one or the other of these models would be very valuable.

    We agree that an experiment that could distinguish these possibilities would be extremely valuable, and will undoubtedly be the subject of future experimentation.

    The paper fills a critical gap. Little to nothing is known about how the internal anatomy of leaves is patterned and the data presented provide evidence that SCR and NKD are two important players. The paper also provides a conceptual advance in offering a couple of genes and some plausible mechanisms of how they might function.

    The audience will be primarily developmental geneticists and physiologists. The paper addresses an important problem that is of broad interest to developmental biologists and is potentially important for global agriculture.

    We thank the reviewer for their kind comments and suggestions.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript of Hughes et al. aimed to demonstrate the functional interactions between Naked-Endosperm IDD genes and the transcription factor SCARECROW and a maternal effect on leaf patterning in C4 grasses. To this end, the authors conducted a greenhouse and labor experiment to create mutants of related genes and assess the expression of these genes through qRT-PCR combined with fluorescence microscopic images in Rice, Maize, and Setaria. They found an increase in the proportion of fused veins with no intervening mesophyll cells in scr;nkd mutants in C4 species (Maize and Setaria) but not in C3 species (rice). In the end, they revealed a maternal effect of derived NKD on patterning cells in leaf primordia during embryogenesis.

    Major comments

    • Optional: the authors should have conducted a whole transcriptome experiment through RNA-seq on the mutants as compared to the controls to check if these genes were significantly up-related followed by qRT-PCR for validation. By doing so, the authors should be able to get a broad overview of all key plays involved in leaf patterning.

    We agree with the reviewer that it would be useful to have this data, and such an approach will undoubtedly inform future research.

    • Optional: although the authors may evoke the statistical significance of observing fused veins in mutants sr;nkd, the presence of fused veins in one mutant Svscr1;Svscr2 and Zmscr1-m2;Zmscr1h-m1 may contradict the claim that the authors made regarding the association between scr and nkd. Moreover, the sampling size is not also large enough to draw a substantial conclusion.

    We disagree with the reviewer that our sampling size is not large enough to draw a substantial conclusion. In maize we surveyed 11 quadruple mutants and 588 veins. Although this phenotype is occasionally seen in Zmscr1;Zmscr1h mutants, it is far more penetrant in Zmscr1;Zmscr1h;Zmnkd1;Zmnkd2 quadruple mutants and easily distinguished by eye when viewing each mutant, the statistical analysis only serves to make this point. In Setaria we agree that the differences are less stark, and the sampling size is necessarily lower due to the challenges of working with the triple mutant leaves which are extremely small and fragile (far more so than the maize quadruple mutant leaves). We have already included discussion as to why the phenotype may be less penetrant in setaria. Together we think that the fact the direction of the phenotype matches that of maize is convincing evidence that the increase in fused veins is a real consequence of combining the scr and nkd mutations.

    • There are two copies of nkd in maize but only one copy in rice and Setaria. Does the presence of two copies in maize has any evolutionary or functional meaning? Does the presence and absence of one or two copies has any effect on leaf patterning? It would be interesting to discuss this in the discussion section.

    We thank the reviewer for this comment and have added discussion of this in the manuscript. This situation is common in maize, which underwent a more recent whole genome duplication since its divergence from rice and setaria. Most of these gene-pairs function redundantly, however, there is evidence of functional divergence in terms of expression in some gene-pairs. We have added a sentence in the results explaining why we think the presence of two NKD gene copies in maize is unlikely to have functional significance in this case.

    • The methods section is not easy to read for a non-specialized audience. I suggest providing an explanation of the abbreviations used to describe mutants.

    We thank the reviewer for this suggestion and have made the suggested change.

    • For the results section, you should provide a table summarizing the differences between mutants and controls regarding the leaf structure.

    We have added such a table at the end of the results section and referred to it in the discussion.

    Minor comments:

    • "Zmscr1-m2;Zmscr1h-m1 seed were" seeds instead

    We have made the suggested change.

    • "Loss of NKD gene function enhances SCR mutant phenotypes in maize and setaria" This section is confusing because several perturbations were observed in triple mutants of Setaria and quadruple mutants of Maize as compared to their double mutants (Svscr1;Svscr2 and Zmscr1;Zmscr1h). You should rewrite this subtitle for clarity.

    We have changed this sub title to read “In maize and setaria, but not in rice, nkd loss of function mutations enhance scr mutant phenotypes”

    • "The accumulation of transcripts in the ground meristem cells" How do you estimate the accumulation of transcripts? What do you mean by the accumulation of transcripts? What do you consider transcripts?

    We use this term as opposed to ‘gene expression in the ground meristem cells’ because we do not know whether the presence/absence/level of detectable RNA is regulated by transcriptional or post-transcriptional mechanisms.

    Reviewer #3 (Significance (Required)):

    The manuscript of Hughes et al. is very interesting in the context of C4 photosynthesis research because there are many transcription factor candidates involved in the development of C4 leaf anatomy but few of them have been validated. However, a whole comparative transcriptome of mutants and controls should provide a broad overview and probably new insight into key players involved in leaf patterning.

    We agree with the reviewer that this would be of great interest, but we feel it is beyond the scope of this study and will be a productive avenue of future research.

    This study goes far beyond the simple validation by outlining the potential interactions between transcription factors. The authors made a substantial effort by combining gene expression results with visual data that strengthen the quality of this manuscript. Therefore, this manuscript is very interesting for the C4 research communities and for the field of developmental biology.

    We thank the reviewer for their kind comments and suggestions.

    A plant biologist working on the evolution and regulation of morphological characters using transcriptomics and genomics.

  2. 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 #3

    Evidence, reproducibility and clarity

    The manuscript of Hughes et al. aimed to demonstrate the functional interactions between Naked-Endosperm IDD genes and the transcription factor SCARECROW and a maternal effect on leaf patterning in C4 grasses. To this end, the authors conducted a greenhouse and labor experiment to create mutants of related genes and assess the expression of these genes through qRT-PCR combined with fluorescence microscopic images in Rice, Maize, and Setaria. They found an increase in the proportion of fused veins with no intervening mesophyll cells in scr;nkd mutants in C4 species (Maize and Setaria) but not in C3 species (rice). In the end, they revealed a maternal effect of derived NKD on patterning cells in leaf primordia during embryogenesis.

    Major comments

    • Optional: the authors should have conducted a whole transcriptome experiment through RNA-seq on the mutants as compared to the controls to check if these genes were significantly up-related followed by qRT-PCR for validation. By doing so, the authors should be able to get a broad overview of all key plays involved in leaf patterning.
    • Optional: although the authors may evoke the statistical significance of observing fused veins in mutants sr;nkd, the presence of fused veins in one mutant Svscr1;Svscr2 and Zmscr1-m2;Zmscr1h-m1 may contradict the claim that the authors made regarding the association between scr and nkd. Moreover, the sampling size is not also large enough to draw a substantial conclusion.
    • There are two copies of nkd in maize but only one copy in rice and Setaria. Does the presence of two copies in maize has any evolutionary or functional meaning? Does the presence and absence of one or two copies has any effect on leaf patterning? It would be interesting to discuss this in the discussion section.
    • The methods section is not easy to read for a non-specialized audience. I suggest providing an explanation of the abbreviations used to describe mutants.
    • For the results section, you should provide a table summarizing the differences between mutants and controls regarding the leaf structure.

    Minor comments:

    • "Zmscr1-m2;Zmscr1h-m1 seed were" seeds instead
    • "Loss of NKD gene function enhances SCR mutant phenotypes in maize and setaria" This section is confusing because several perturbations were observed in triple mutants of Setaria and quadruple mutants of Maize as compared to their double mutants (Svscr1;Svscr2 and Zmscr1;Zmscr1h). You should rewrite this subtitle for clarity.
    • "The accumulation of transcripts in the ground meristem cells" How do you estimate the accumulation of transcripts? What do you mean by the accumulation of transcripts? What do you consider transcripts?

    Significance

    The manuscript of Hughes et al. is very interesting in the context of C4 photosynthesis research because there are many transcription factor candidates involved in the development of C4 leaf anatomy but few of them have been validated. However, a whole comparative transcriptome of mutants and controls should provide a broad overview and probably new insight into key players involved in leaf patterning.

    This study goes far beyond the simple validation by outlining the potential interactions between transcription factors. The authors made a substantial effort by combining gene expression results with visual data that strengthen the quality of this manuscript. Therefore, this manuscript is very interesting for the C4 research communities and for the field of developmental biology.

    A plant biologist working on the evolution and regulation of morphological characters using transcriptomics and genomics.

  3. 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 leaf anatomy that distinguishes C4 from C3 plants has been known for decades, with veins in C4 plants separated by 1 to 3 (generally 2) mesophyll cells whereas those in C3 plants are considerably farther apart. This anatomical pattern appears to be critical for the function of the C4 pathway, which under some environmental conditions is a more efficient way to fix carbon than the C3 pathway. Despite the obvious importance of close vein spacing, the genetic mechanisms that control it have been surprisingly difficult to untangle. The statement on the bottom on p. 2 ("To date, very few regulators of cell-patterning in inner leaf tissues have been identified...") is an understatement. The paper by Hughes et al. offers a major step in uncovering the basis of C4 vein spacing.

    The authors build on their previous work in Scarecrow-like proteins in maize and rice. In maize, SCR controls patterning of the mesophyll, whereas in rice it controls development of stomata. This paper pursues the possibility that the differences in SCR roles may have to do with interacting proteins. Based on work in Arabidopsis the authors focus on proteins with an indeterminate domain (IDD) and specifically on the NAKED ENDOSPERM genes.

    The paper presents an analysis of an impressive set of mutants in three species. A major step in this paper is the comparison among three species of grasses - maize, rice, and Setaria - rather than the more common two species, usually maize and rice. Maize and rice differ in photosynthetic pathway but they also differ in many other traits that reflect the ca. 50 million years since their last common ancestor. Setaria is, like maize, C4 and the two species are more closely related to each other than either is to rice, although they represent two independent acquisitions of C4. This paper shows that SCR orthologs control stomatal patterning in both rice and Setaria implying that the stomatal function of SCR may be ancestral in the grasses and also is not directly connected to photosynthetic pathway.

    The availability of allelic combinations of SCR and NKD in maize in particular permits the inference of possible maternal effect on the vein spacing phenotype, although exactly how this happens remains unclear.

    The discussion provides a careful and logical assessment of the state of knowledge on SCR and IDD proteins in general, and the new data on SCR and NKD in particular. Many questions remain unresolved, and many additional experiments could be suggested. However, the power of the genetics and the phenotypic analysis together provide a novel direction for research on vein spacing. I will refrain in this review from suggesting what additional information would be nice to have since I think a review should assess the quality of the paper as it stands, not as it could be with months more of work.

    My only really substantive suggestion is that the micrographs of the Setaria leaves need to be improved. Specifically, in Figure 6E it is hard to see the details of the fused veins. Either the section is too thick or the camera was not focused properly. Because this image in particular is central to the entire paper I would recommend aiming for the clarity of the images of Zea cross sections, which are fine.

    Very minor point:

    p. 3 - "double Zmscr1;Zmscr1h mutants" - what does the "h" in Zmscr1h refer to?

    Significance

    Strengths of the paper are 1) the inclusion of three species to help determine which aspects of the gene function may be ascribed to C4; 2) thoughtful and comprehensive genetic analysis; 3) careful sections of leaves; 4) outlines of the limitations of the approach. Limitations (several of which the authors acknowledge in the Discussion) include a general lack of molecular genetic data (protein interactions, DNA binding sites, RNA-seq, etc.). While this information would be great to have, I think the strength of the genetics is such that the paper will be foundational for future work in any case. The one bit of additional data that would be ideal would be information bearing on the two mechanistic hypotheses laid out on p. 10. The model that SCR and NKD promote cell division and specify mesophyll identity is the opposite of the model that SCR and NKD inhibit vein formation. An experiment that helped point the reader toward one or the other of these models would be very valuable.

    The paper fills a critical gap. Little to nothing is known about how the internal anatomy of leaves is patterned and the data presented provide evidence that SCR and NKD are two important players. The paper also provides a conceptual advance in offering a couple of genes and some plausible mechanisms of how they might function.

    The audience will be primarily developmental geneticists and physiologists. The paper addresses an important problem that is of broad interest to developmental biologists and is potentially important for global agriculture.

  4. 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

    All the conclusions are based on solid evidence and convincing, and the methodology are in detail to follow or repeat. The writing of the manuscript is logical and easy to follow.

    1. The mutation experiments indicated that nkd enhanced the phenotype of scr, but there is no leaf phenotype variation in nkd muations, this is some way difficult to understand, it would much better if the authors can give much more explanation in the discussion. 2.The word green millet in the first paragraph should be changed to green foxtail. Millet means domesticated small cereal grains, such as foxtail millet, finger millet, proso millet etc.

    Significance

    The manuscript, which titled Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW and a maternal influence on leaf patterning in C4 grasses by Hughes et al., first reported that SCR works regulating both leaf inner pattern and epidermal stomatal patterning in the C4 model plant green foxtail. The functional difference of this gene in Setaria from that in maize and rice indicated that the inner leaf cell patterning regulation of SCR is not a characteristic of C4 Species; this gave us insight understanding of the complex of C4 leaf cell patterning. In addition to this important discover, the authors found that mutations in NKD IDD genes enhance loss of function scr phenotypes in the leaves of C4 maize and Setaria but not in the C3 rice, indicating NKD IDD was involved in the leaf cell patterning in C4 species, but no in C3. They also identified a maternal effect on cell-type patterning in maize leaves that are initiated during embryogenesis.

  5. Version published to 10.1101/2022.10.28.514209v2 on bioRxiv
  6. Version published to 10.1101/2022.10.28.514209v1 on bioRxiv