Further Disruption of the TAS3 Pathway via the Addition of the AGO7 Mutation to the DRB1, DRB2 or DRB4 Mutations Severely Impairs the Reproductive Competence of Arabidopsis thaliana
, Jackson M. J. Oultram 1, Shaun J. Curtin 2, Christopher P. L. Grof 1 and Andrew L. Eamens 1,*Round 1
Reviewer 1 Report
Perler et al. report on the characterisation at macroscopic and molecular level of double mutants between DRB1-4 and AGO7. Whereas single mutants were already characterized in Arabidopsis thaliana before, there was no in depth characterisation of the ago7/drb double mutants. The main findings are that the macroscopic ago7 phenotype (in rosette & flower) is exacerbated in the ago7/drb1 and to a lesser extent in ago7/drb2 and ago7/drb4. Unsurprisingly drb1 mutants have reduced processing of the pre-miR390 precursors, DRB1 being known for long to be important for miRNA processing. The authors could not observe any clear effects of the mutations on TAS3 precursor abundance but did show synergetic effects on some of the ta-siARF targets ARFs.
Overall, the work is sound even if novelty is incremental over previous results. I would suggest the following changes to be implemented:
1. The title does not capture all the relevant aspects of this study; the mention of the test of the genetic interaction between DRB & AGO7 should be featured somehow.
2. The paper is too wordy, especially for the results part.
3. There are no scale bars for Fig 1 A, C, D.
4. Fig 1B, there is no mention of how many rosette leaves are used to do the measurements, and which stage of rosette leaves (Leaf position) is used.
5. Fig 2, they did not mention how many siliques they used to measure silique length and the seed number per silique.
6. They did statistical analysis for qPCR results (Fig 3, 4, 5). They did not mention the statistical methods they used for this analysis.
7. Results 3.2, line 372-378, “The severe retardation of drb1 reproductive development has previously been attributed to the impairment of miRNA production in the absence of DRB1 function. More specifically, impaired drb1 fertility has been attributed to the reduced abundance of the miR165/166 sRNA, a miRNA that regulates organ polarity sensing. Therefore, reduced miR165/166 abundance in drb1 floral tissues results in the mis-regulation of stamen polarity, which in turn leads to decreased seed set due to a reduced frequency of ovule fertilization, and ultimately, the formation of siliques of shorter length [52,53].” These sentences are more appropriate in the discussion part.
Author Response
Authors’ responses to Reviewer #1 comments;
On behalf of the authorship team, I (Andrew Eamens) would like to thank Reviewer #1 for their thorough and insightful review of our submitted manuscript. Please find below our responses to each of the comments that Reviewer #1 raised during their review.
The title does not capture all the relevant aspects of this study; the mention of the test of the genetic interaction between DRB & AGO7 should be featured somehow.
The authors thank Reviewer #1 for this helpful suggestion. In order to address this concern we have changed the original title of our manuscript “Disruption of the TAS3/tasiARF Pathway Severely Impairs the Reproductive Competence of Arabidopsis thaliana” to “Further disruption of the TAS3 pathway via addition of the AGO7 mutation to the DRB1, DRB2 and DRB4 mutations severely impairs the reproductive development of Arabidopsis thaliana”.
The paper is too wordy, especially for the results part.
We have attempted to reduce the wordiness of the manuscript, specifically concentrating on the Results section of the manuscript as suggested by Reviewer #1.
There are no scale bars for Fig 1 A, C, D.
The authors wish to thank Reviewer #1 for identifying this omission. Figure 1 has been modified to include scale bars in Figures 1A, 1C and 1D, and the size that each scale bar represents in now stated in the Figure 1 legend. We have also included the error bars missing from Figure 1B as well as to identifying the degree of significance for each rosette area measurement.
Fig 1B, there is no mention of how many rosette leaves are used to do the measurements, and which stage of rosette leaves (Leaf position) is used.
To address this concern raised by Reviewer #1, we have modified subsection 2.2 of the Methods to provide a more description on how this assessment was performed.
Fig 2, they did not mention how many siliques they used to measure silique length and the seed number per silique.
As stated above in point 4, to address this concern of Reviewer #1, we have provided additional information in subsection 2.2 of the revised manuscript’s Methods section to more clearly describe how this analysis was performed.
Section 2.2 of the Methods now reads;
The rosette area of 28 day old seedlings was determined for each mutant background to establish the effect of loss of DRB1, DRB2, DRB4 and/or AGO7 function on vegetative development. This quantitative assessment was completed using ImageJ software to trace the rosette leaf blade margins and the petioles of each rosette leaf to determine the total area of the rosette. At the time that this assessment was performed, Col-0 plants and the 7 knockout mutant lines had developed 9 or 10 true leaves in addition to the cotyledons, and all leaves were used in the rosette area calculations. Furthermore, 4 biological replicates of 6 plants per replicate were used to determine the rosette area of each plant line. The length and seed set of siliques of 42 day old plants was assessed by firstly measuring the length of each silique before gently breaking open each silique sampled from along the entire length of the primary inflorescence stem and manually counting the number of seeds that each silique contained. As stated for the determination of the rosette area of each plant line, 4 biological replicates of 6 plants per replicate were used in the silique length and seed number assessments.
They did statistical analysis for qPCR results (Fig 3, 4, 5). They did not mention the statistical methods they used for this analysis.
For each Figure of the revised manuscript where we have stated the determined degree of significance, we used a standard Student’s t-test for these calculations. We now state this in the Methods section of the revised manuscript, in subsection 2.4. The authors kindly thank Reviewer #1 for identifying this oversight.
Section 2.4 of the Methods of the revised manuscript now states;
2.4. Statistical Analysis
A standard Student’s t-test was used to determine the degree of significance of phenotypic (Figure 1B) or molecular (Figures 3 to 5) variance for each of the seven mutant lines analyzed in this study compared to wild-type Arabidopsis.
Results 3.2, line 372-378, “The severe retardation of drb1 reproductive development has previously been attributed to the impairment of miRNA production in the absence of DRB1 function. More specifically, impaired drb1 fertility has been attributed to the reduced abundance of the miR165/166 sRNA, a miRNA that regulates organ polarity sensing. Therefore, reduced miR165/166 abundance in drb1 floral tissues results in the mis-regulation of stamen polarity, which in turn leads to decreased seed set due to a reduced frequency of ovule fertilization, and ultimately, the formation of siliques of shorter length [52,53].” These sentences are more appropriate in the discussion part.
The authors agree with Reviewer #1 that the above outlined text does not belong in the Results section of the manuscript, we have therefore removed this section of text from the revised version of the manuscript. It is also important to note here that Reviwer #2 has suggested that additional text should be included in the revised manuscript however we have not taken on this suggestion of Reviewer #2 to address this point of Reviewer #1. Further, we the authors, appreciate that our manuscript is lengthy, however we have performed multiple phenotypic and molecular analyses presented in Figures 1 through to 5 that detail extensive characterisation of the TAS3 pathway and feel that the degree of text included in this study is required to maintain the narrative of the study in order to adequately detail the findings presented here. In addition, this is the first study to report on the double mutant lines, drb1ago7, drb2ago7 and drb4ago7, and the comparison of the developmental and molecular phenotypes expressed by these three newly generated double mutants, to both wild-type Arabidopsis and the respective drb single mutants used to in the generation of the double mutants, greatly adds to the volume of text contained in this study.
Reviewer 2 Report
In this manuscript titled “Disruption of the TAS3/tasiARF Pathway Severely Impairs the Reproductive Competence of Arabidopsis thaliana”, the authors used published Arabidopsis mutants including drb1, drb2, drb4 and ago7 which are in the TAS3/tasiARF pathway, to generate double mutant material and analyzed the phenotype and downstream targets to explain the enhanced reproductive alterations comparing to the reported single mutants, and interpret the epistasis in the pathway.
Overall, this is a well written nice work potentially extending our current knowledge of plant small RNA. The interpretation of the results is novel and inspiring. Here are my major comments:
Multiple aspects of phenotype has been studies and discussed in this work. One thing needs to be careful is, those genes are likely involved in different small RNA functions, in different tissue/cell types and resulting these phenotypes. While quantitative PCR results in this study looks like in the flower, which is a mixture of different cell type in the pistil and petal, etc. The general trend of ARF and KAN genes may not be the same in all the tissue/cell types. It will be good to adjust the introduction, to highlight the major microRNA/tasiRNA pathways and mutants that impact the flower development and reproductive competence. Is there any other microRNA could play roles via DRB1/2/4? Also, the authors mainly discuss about the flower defects of the mutants, and they are mainly compared to arf and kan mutants. But how about tas3 mutants? Previous work pointed out supernumerary megaspore mother cell phenotype in the tas3a/b mutants. Could it be possible to provide more genetic evidence of double mutants? How the authors identify the double mutants? Was that possible to distinguish double mutants just by visible defects? Do you think there’s a redundancy between drb2 and drb4. If yes, maybe it’s worthy to make drb2drb4ago7 triple mutant? Maybe draw a cartoon to illustrate the roles of small RNA, DRBs, AGO, ARF, KAN and roles in flower development? Here are the minor comments: How many biological replicates for each genotype were used? If there were multiple, please indicate the numbers for each genotype and error bars. Is that possible to quantify the pistil phenotype?
Author Response
Authors’ responses to Reviewer #2 comments;
On behalf of the authorship team, I (Andrew Eamens) would like to thank Reviewer #2 for their insightful review of our submitted manuscript. The authors would especially like to thank Reviewer #2 that our “interpretation of the results is novel and inspiring”. Please find below our responses to each of the comments that Reviewer #2 raised during their review of our submitted manuscript.
Multiple aspects of phenotype has been studies and discussed in this work. One thing needs to be careful is, those genes are likely involved in different small RNA functions, in different tissue/cell types and resulting these phenotypes. While quantitative PCR results in this study looks like in the flower, which is a mixture of different cell type in the pistil and petal, etc. The general trend of ARF and KAN genes may not be the same in all the tissue/cell types.
Yes the authors totally agree with Reviewer #2’s insightful suggestion that ARF/KAN gene expression would likely differ between individual tissues that make up the Arabidopsis flower. However, due to the severity of the developmental phenotypes expressed by the drb1ago7, drb2ago7 and drb4ago7 double mutants, the authorship team decided to concentrate our molecular analyses on terminal floral buds to reveal molecular phenotypic differences between the seven mutant lines analysed in this study. Considering the functional roles demonstrated for DRB1, DRB2, DRB4 and AGO7 in the TAS3 pathway, the authors are of the opinion that the molecular assessment of the TAS3 pathway in its entirety (PRI-MIR390 expression; miR390 accumulation; TAS3 expression; tasiARF expression; ARF2, ARF3 and ARF4 expression, and; KAN1, KAN2, KAN3 and KAN4 expression) would be more insightful than the analysis of only parts of the TAS3 pathway in a specific tissue type and/or organ structure of an Arabidopsis flowers.
It will be good to adjust the introduction, to highlight the major microRNA/tasiRNA pathways and mutants that impact the flower development and reproductive competence. Is there any other microRNA could play roles via DRB1/2/4?
Yes, Reviewer #2 is correct in their suggestion that additional miRNA/target gene expression modules are likely involved in flower development in Arabidopsis. This has been well described elsewhere. Further, we and others have been shown the requirement of DRB1, DRB2 and/or DRB4 for the production of individual miRNAs or the regulation of miRNA target expression. However, AGO7 has only previously been demonstrated to solely function as an effector protein of miR390-directed target gene expression regulation. In addition to miR390, AGO7 has been shown to load other miRNA sRNAs, however AGO7 has only been demonstrated to use the miR390 sRNA as a guide to direct RNA silencing. We, the authors, therefore feel that due to these previous experimentally validated demonstrations, the physiological and molecular phenotypes reported here are specific to the miR390/TAS3/tasiARF pathway.
Also, the authors mainly discuss about the flower defects of the mutants, and they are mainly compared to arf and kan mutants. But how about tas3 mutants? Previous work pointed out supernumerary megaspore mother cell phenotype in the tas3a/b mutants.
Here we failed to detect significant alteration to either TAS3 transcript abundance or tasiARF sRNA abundance. However, in the double mutant lines, drb1ago7 and drb2ago7, that expressed the most severe reproductive phenotypes, we detected altered tasiARF target gene expression, namely; reduced ARF2 expression and elevated ARF3 and ARF4 expression. Furthermore, we detected altered KAN gene expression in flowers of the double mutants with KAN gene expression previously demonstrated to be regulated by ARF transcription factors. Therefore, we therefore decided to focus our attention on the phenotypes previously reported to be expressed by plant lines with altered ARF or KAN gene expression in an attempt to identify the underlying molecular alterations that were potentially responsible for the reproductive phenotypes expressed by the mutant lines assessed in this study.
Could it be possible to provide more genetic evidence of double mutants? How the authors identify the double mutants? Was that possible to distinguish double mutants just by visible defects?
The authors thank Reviewer #2 for identifying this oversight. To address this concern of Reviewer #2, we modified subsection 2.1 of the Methods of the revised manuscript to provide a more accurate detailed description on how we identified the double mutants generated for use in this study. Specifically we have added the text “Homozygosity of each of the two mutant alleles harbored by the drb1ago7, drb2ago7 and drb4ago7 double mutants was confirmed via a standard PCR-based genotyping approach prior to any experimental analyses being performed on these newly generated Arabidopsis lines.” to subsection 2.1 of the Methods. In brief, the double mutants were identified via a standard PCR-based genotyping approach and due to their generation from two existing mutant backgrounds, homozygous double mutants expressed a consistent, non-varied developmental and reproductive phenotype.
Do you think there’s a redundancy between drb2 and drb4. If yes, maybe it’s worthy to make drb2drb4ago7 triple mutant?
Yes, Reviewer #2 is correct; there is redundancy between DRB2 and DRB4 function which we and others have reported previously. These studies are cited throughout the text of the manuscript for reference. Furthermore, we have attempted a number of times to generate a triple mutant however we have been unsuccessful on each occasion, a finding that potentially indicates that the drb2drb4ago7 triple mutant is embryo lethal. In addition, currently the authorship team are not in the position to conduct such experiments, experiments that would take up to 6 months to successfully complete.
Maybe draw a cartoon to illustrate the roles of small RNA, DRBs, AGO, ARF, KAN and roles in flower development?
The authors do not believe that the addition of such a cartoon would add further value to the manuscript. The molecular interactions revealed by the experimental analyses performed in this study are well described in the text of both the Results and Discussion sections of the revised manuscript.
Here are the minor comments: How many biological replicates for each genotype were used? If there were multiple, please indicate the numbers for each genotype and error bars. Is that possible to quantify the pistil phenotype?
We thank Reviewer #2 for identifying this oversight. We have now added error bars to the phenotypic analysis detailed in Figure 1B as well as modifying subsection 2.2 of the Methods section of the revised manuscript to provide the reader with more detail on how these experiments were performed. Specifically, we have modified Methods subsection 2.2 of the revised manuscript to read as below;
The rosette area of 28 day old seedlings was determined for each mutant background to establish the effect of loss of DRB1, DRB2, DRB4 and/or AGO7 function on vegetative development. This quantitative assessment was completed using ImageJ software to trace the rosette leaf blade margins and the petioles of each rosette leaf to determine the total area of the rosette. At the time that this assessment was performed, Col-0 plants and the 7 knockout mutant lines had developed 9 or 10 true leaves in addition to the cotyledons, and all leaves were used in the rosette area calculations. Furthermore, 4 biological replicates of 6 plants per replicate were used to determine the rosette area of each plant line. The length and seed set of siliques of 42 day old plants was assessed by firstly measuring the length of each silique before gently breaking open each silique sampled from along the entire length of the primary inflorescence stem and manually counting the number of seeds that each silique contained. As stated for the determination of the rosette area of each plant line, 4 biological replicates of 6 plants per replicate were used in the silique length and seed number assessments.
We agree with Reviewer #2’s suggestion that quantification of pistil length would have been a worthy inclusion in our Figure 1 experimentation. However, unfortunately, we did not perform this measurement as part of our experimental characterisation of the drb, ago7 and drb1/2/4ago7 mutants. In addition, we are no longer in the position to perform this suggested additional phenotypic characterisation of our mutant lines: experimentation that would take many months to complete.
