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  1. Author Response:

    Reviewer #2 (Public Review):

    Plants have an amazing diversity of pollen morphologies. This study set out to determine how apertures, the gaps in pollen exine wall, are specified during pollen development. The authors focused on the macaron (mcr) mutant that was identified in a previous forward genetic screen to have one aperture that extends around the circumference of the pollen grain, instead of the 3 equidistant apertures in normal Arabidopsis pollen. They identify the MCR gene as ELMOD B, a member of a small gene family with domain similarity to the animal Engulfment and Cell Motility domain (ELMOD) protein , which has been shown to be non-canonical GTPase Activating Proteins (GAPs). Genetic dosage experiments showed that increasing the expression levels of MCR and the closely related ELMOD_A gene in developing pollen also increases the number of apertures. Combined with epistasis analysis showing that MCR is upstream of INP1, INP2, and D6PKL3 (previously published regulators of aperture development), this data provides good evidence that MCR and ELMOD-A are major regulators of the number, positions, and size of pollen apertures.

    In the second part of the manuscript, the authors present a phylogenetic analysis of ELMOD proteins in plants. They show that the plant ELMOD genes likely have a common ancestor and that Angiosperms have four distinct ELMOD clades. They hypothesize that the A/B clade is necessary for aperture formation in Angiosperm pollen and that many angiosperms have more than one A/B ELMOD gene in order to provide redundancy for pollen development and/or other important functions. While intriguing, a weakness in this part of the study is that they did not test this hypothesis by checking to see if A/B clade ELMOD genes are expressed during pollen development in other Angiosperm lineages.

    We did not directly check the expression of A/B clade ELMOD genes during aperture formation in other angiosperms, but we have retrieved some expression data from public databases, such as the ones for rice and tomato. The microarray expression data from RiceXpro (https://ricexpro.dna.affrc.go.jp/index.html) suggested that LOC_ Os02g43590 and LOC_ Os04g46079, the A/B clade ELMODs from rice, are expressed in inflorescences and anthers at different development stages, including the young stages when apertures develop. Similarly, the expression data from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/) showed that Solyc10g062200 and Solyc01g089980, the A/B clade ELMODs from tomato, are also expressed in young flower buds.

    In the final experiments, the authors analyzed the predicted GAP domain for amino acids that are highly conserved in Angiosperm ELMODs and that are specific to different clades. They identified a conserved Arginine in the same position of the GAP domain as in animals. This arginine is necessary for GAP function in animals. The authors predicted that this Arginine would also be important for Arabidopsis ELMOD function and mutated this residue to Lys in MCR and ELMOD_A. Neither of these versions could complement the mcr aperture phenotype, confirming their hypothesis. One limitation of this experiment is that it only indicates that the domain might have a similar function to the animal ELMODs but does not directly test whether MCR and ELMOD_A actually have GAP activity.

    The most intriguing data comes in the final figures of the paper, where the authors compare the GAP domains in the different ELMOD clades. Sequence comparisons revealed that the A/B clade ELMODs tend to have a glycine at position 129 within the GAP domain, while clade E ELMODs have a cysteine at this position. They predicted that this amino acid position could be important for diversification of ELMOD functions. elmod-b, c, d, and e mutants did not have aperture phenotypes as single mutants nor in combination with mcr, indicating that they probably do not function in aperture development. However, when ELMOD_E was expressed in developing pollen with the MCR regulatory elements the shape of apertures changed to round instead of elongated furrows. A similar dosage study to the one described previously in the manuscript revealed that high levels of MCR protein could counteract the effects of ELMOD_E on aperture shape. When the ELMOD_E protein was mutated to be more like MCR in the GAP domain (Cys129 changed to Gly and Asn129 changed to Asp), aperture number in an mcr background was increased and some of the apertures were furrowed rather than round. A limitation in this study is that the furrowed and round apertures were counted together, thus missing an opportunity to quantify the effects of the mutated ELMOD_E on aperture shape. While the opposite experiment of changing these residues in MCR to ELMOD_E-like residues was not as striking (aperture number was complemented but they did not become round), these data are exciting because they reveal the power of small amino acid changes in one protein to dramatically change aperture number and phenotypes during pollen development.

    This manuscript will be of broad interest to scientists interested in cell polarity, patterning, and evolution of diverse morphologies. Diversification of clade A/B ELMOD genes could have played a role in generating the wide range of aperture numbers and shapes seen in Angiosperms. A mystery that remains and that can be addressed in future studies is how a protein that is localized throughout the cytoplasm and in the nucleus is able to regulate polarity during aperture formation.

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  2. Evaluation Summary:

    How pollen apertures are formed is not well understood. By studying Arabidopsis mutants producing pollen with aberrant aperture numbers, the authors identify proteins from the ELMOD protein family as important regulators for aperture formation. They use genetics, transgenic constructs, and site-directed mutagenesis to pinpoint important residues for protein function in this process, and show that changes in expression levels of one protein can have dramatic effects on patterning. This paper will interest scientists interested in cell polarity, patterning, and evolution of diverse morphologies. This is also the first study of the ELMOD protein family, whose potential GTPase activating activities have not yet been investigated in plants.

    (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 #1 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The presented results are convincing, but how diffusely located proteins might accomplish this task is still unknown. The discussion section should be tightened up; many non-pertinent or poorly supported things are discussed, whereas, for example, the anomalous finding that T-DNA insertion alleles are hypomorphic (lines 164-167) are not. This is important given their assertion that the amount of protein influences the phenotype.

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  4. Reviewer #2 (Public Review):

    Plants have an amazing diversity of pollen morphologies. This study set out to determine how apertures, the gaps in pollen exine wall, are specified during pollen development. The authors focused on the macaron (mcr) mutant that was identified in a previous forward genetic screen to have one aperture that extends around the circumference of the pollen grain, instead of the 3 equidistant apertures in normal Arabidopsis pollen. They identify the MCR gene as ELMOD B, a member of a small gene family with domain similarity to the animal Engulfment and Cell Motility domain (ELMOD) protein , which has been shown to be non-canonical GTPase Activating Proteins (GAPs). Genetic dosage experiments showed that increasing the expression levels of MCR and the closely related ELMOD_A gene in developing pollen also increases the number of apertures. Combined with epistasis analysis showing that MCR is upstream of INP1, INP2, and D6PKL3 (previously published regulators of aperture development), this data provides good evidence that MCR and ELMOD-A are major regulators of the number, positions, and size of pollen apertures.

    In the second part of the manuscript, the authors present a phylogenetic analysis of ELMOD proteins in plants. They show that the plant ELMOD genes likely have a common ancestor and that Angiosperms have four distinct ELMOD clades. They hypothesize that the A/B clade is necessary for aperture formation in Angiosperm pollen and that many angiosperms have more than one A/B ELMOD gene in order to provide redundancy for pollen development and/or other important functions. While intriguing, a weakness in this part of the study is that they did not test this hypothesis by checking to see if A/B clade ELMOD genes are expressed during pollen development in other Angiosperm lineages.

    In the final experiments, the authors analyzed the predicted GAP domain for amino acids that are highly conserved in Angiosperm ELMODs and that are specific to different clades. They identified a conserved Arginine in the same position of the GAP domain as in animals. This arginine is necessary for GAP function in animals. The authors predicted that this Arginine would also be important for Arabidopsis ELMOD function and mutated this residue to Lys in MCR and ELMOD_A. Neither of these versions could complement the mcr aperture phenotype, confirming their hypothesis. One limitation of this experiment is that it only indicates that the domain might have a similar function to the animal ELMODs but does not directly test whether MCR and ELMOD_A actually have GAP activity.

    The most intriguing data comes in the final figures of the paper, where the authors compare the GAP domains in the different ELMOD clades. Sequence comparisons revealed that the A/B clade ELMODs tend to have a glycine at position 129 within the GAP domain, while clade E ELMODs have a cysteine at this position. They predicted that this amino acid position could be important for diversification of ELMOD functions. elmod-b, c, d, and e mutants did not have aperture phenotypes as single mutants nor in combination with mcr, indicating that they probably do not function in aperture development. However, when ELMOD_E was expressed in developing pollen with the MCR regulatory elements the shape of apertures changed to round instead of elongated furrows. A similar dosage study to the one described previously in the manuscript revealed that high levels of MCR protein could counteract the effects of ELMOD_E on aperture shape. When the ELMOD_E protein was mutated to be more like MCR in the GAP domain (Cys129 changed to Gly and Asn129 changed to Asp), aperture number in an mcr background was increased and some of the apertures were furrowed rather than round. A limitation in this study is that the furrowed and round apertures were counted together, thus missing an opportunity to quantify the effects of the mutated ELMOD_E on aperture shape. While the opposite experiment of changing these residues in MCR to ELMOD_E-like residues was not as striking (aperture number was complemented but they did not become round), these data are exciting because they reveal the power of small amino acid changes in one protein to dramatically change aperture number and phenotypes during pollen development.

    This manuscript will be of broad interest to scientists interested in cell polarity, patterning, and evolution of diverse morphologies. Diversification of clade A/B ELMOD genes could have played a role in generating the wide range of aperture numbers and shapes seen in Angiosperms. A mystery that remains and that can be addressed in future studies is how a protein that is localized throughout the cytoplasm and in the nucleus is able to regulate polarity during aperture formation.

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  5. Reviewer #3 (Public Review):

    This largely genetics, morphology, molecular phylogenetic study is thorough, systematic and its presentation clear, allowing the extensive results to be quite easily followed.

    The founding mutant is called macaron (mcr) - pollen grains have a ring-shaped aperture formed by two oppositely positioned slits merging to form a sandwiched ring in the equatorial position. The first part of the study established the relationship between MCR and two previously established aperture regulators, INP1 and D6PKL3.

    1. Earlier observations showed a ploidy effect on aperture number, suggest a dosage effect. Introducing mcr into mutant plants (MiMe, Mitosis instead of Meiosis, and tes, which produce pollen grains defective in aperture numbers (respectively 50% normal, 50% more apertures in MiMe, and >10 irregularly placed in tes), the authors further established the reducing effect of mcr on aperture number.

    2. Two previously studied aperture controlling proteins, INP1 and D6PKL3, were mislocalized in mcr pollen. Instead of the normal location along the three aperture domains in WT grains, these appeared in a ring-shaped location in the mcr mutant. This and the analysis of double mcr-1 inp1/2 (apertureless), mcr-1 d6pkl3 mutants (ring-shaped and partially covered by exine-pollen outer coat), established MCR as acting upstream of D6PKL3 and INP1.

    The remainder of the manuscript reports the molecular identification of MCR (with four original alleles mcr1-4; and addition T-DNA alleles mcr5-7) and the two related ELMOD proteins involved in aperture formation [ELMOD_A and _E; A is closest to MCR, in the ELMOD_B clade). MCR has a ELMOD (engulfment and cell motility) domain; in animal cells ELMOD proteins are GTPase activating proteins, activating ADP-ribosylating factors (ARF GTPases). ELMOD proteins in plants have not been studied before. The rather extensive collection of mutations in a single gene, each conferring notable aperture defect, suggests MCR function is not only crucial, but sensibly monitored during the pollen maturation aperture formation window of time; these mutations, not the focus here, will be useful entries to future study of MCR. These are followed by analysis of potential functional interactions between proteins from the various ELMOD clades.

    3. The genetic and aperture phenotype analyses of MCR and ELMOD-A (Fig. 4) showing that they are redundant, acting synergistically but MCR being more prominently needed (Fig. 4), localization studies showing that they are not located at the aperture membrane domain, but in the cytoplasm and nucleus (Fig. 5), and that the invariant R residue these noncanonical GAPs was indeed critical for their function (ability to complement the mutants) (Fig. 6) are well done.

    These proteins are most likely involved in the secretory process or in polarity (alluded to in the Discussion), but how the location of these proteins relates to a function to select membrane regions as future aperture site (i.e. beyond being needed for INP1 and D6PKL3 but in finer biochemical or cellular details) is left unaddressed. However, these are likely too much to add to an already extensive genetic and morphology-centric study.

    4. A dosage effect of MCR and ELMOD_A and aperture number, higher levels of complementing transgene product ] correlates with higher aperture numbers (>3) in rescued mutant plants, was nicely demonstrated (Fig. 7; Supp. Data).

    5. Phylogeny analysis-guided amino acid sequence comparisons (Fig. 8 associated) led to insightful predictions, some of which, such as AA121 and 129, the authors were able to test experimentally in a later section. Genetic knockouts of other ELMODs_C,D,E,F (Fig. 9 associated) did not reveal impact on aperture formation. Cross-complementation by MCRp-driven expression of these genes in mcr mutant, and later in WT, authors were able to establish that mis-expression of ELMOD_E (from the MCR promoter) led to a new phenotype of multiple short round apertures in both mcr and WT background, showing a dominant negative effect.

    On the other hand, additive expression of ELMOD_E from its own promoter in the WT background did not influence aperture phenotype. This (though not explicitly indicated, I interpreted this as probably together with the non-phenotypic elmod_e property, and low expression level during the tetrad stage) led the authors to conclude that ELMOD E, while it has the capacity to influence, is normally not involved in the aperture formation process [L466]. They went on to show high level of MCR promoter:MCR expression in these MCR promoter:ELMOD_E expressing mcr lines can counteract the impact from ELMOD_e, restoring the apertures (from round) to elongated/furrow.

    I find the findings above interesting and the conclusion from the results insightful. However, the details related and also those in the remaining study in Fig. 9 regarding elmod_e,d,f mutants interfering with the more directly related characterization of MCR and ELMOD_E (Fig. 10).

    6. The sequence comparison led the authors to identify aa121 and 129 located in the GTP activating region as potentially important, this together with the differential activity between MCR and ELMOD_E, led them to test the functional significance of these residues. [E_clade, 100% Asn121/Cys129; other clades 0%; mcr-2 has a D substituting the conserved G129 ]. (Fig. 10) ELMOD_E(N121D/C129G) acted more like MCR in restoring furrow like apertures; with only of these amino acid residue change, the mutant ELMOD_E induced a mix of multiple round apertures (similar to normal ELMOD_E), but also three furrowed pollen and pollen with a mix of furrowed and round apertures. This is a satisfactory functional follow up from a very nice phylogeny analysis.

    7. The Discussion brought out some interesting testable hypotheses for the future, e.g. the role of ELMODs in Pedicularis pollen apertures (line 560).

    In summary, this is a strong study, with two minor weaknesses (referred to in points #3 and #5). #5 is easily remedied. #3 is most likely not possible to address. The Discussion on the #3 aspect is largely and necessarily speculative, so not quite reach the intellectual satisfaction one would like to have. But it could be the basis for future investigation.

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