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In this manuscript, Feng et al. explores the roles of autophagy and programmed cell death in the lifespan of Arabidopsis thaliana root hairs. The authors found that wild type Arabidopsis root hair cells feature high levels of autophagy, measured by the presence of autophagic bodies while mutants in autophagy genes (ATG2, ATG5, and ATG7) show diminished levels of autophagic bodies. The root hairs of these autophagy mutants deteriorate at earlier time points than those of wild type seedlings, while displaying markers of developmental programmed cell death which are not found in young wild type root hair cells. They also found that, unlike similar phenotypes in other tissues, the premature degradation of root hair cells is not dependent on salicylic acid, jasmonic acid, or ethylene signaling, as double mutants in these pathways still presented early root hair collapse. Finally, they examined triple mutants in ATG2 and programmed cell death transcription factors ANAC046 and ANAC087 and found that mutations in these genes suppressed the early root hair cell death phenotype. This study reports interesting results that advance the field of developmental biology in roots with elegant single-cell resolution, we provide below some suggestions to increase the reproducibility of the work and clarity for a broader audience.

Major comments

- In the Introduction, there could be more elaboration on what the functions of the chosen autophagy genes are, as well as if there is any redundancy between them. In the text of the Results or the Methods section, an explanation of the types of mutations of each atg mutant would be helpful, as well as a justification for using a specific atg mutant in one experiment but not others (atg7 in Fig2A, and atg5 or atg2 throughout), using ATG8a as the autophagy reporter (Fig1) and using mutant atg7-2 with the H2A-GFP reporter, while the TOIM reporter was used for the other atg mutants (Fig2D, E).

- In the text of the Results or the Methods section, more detail of how the confocal images were taken would be required for reproducibility, such as contrast settings, how software (ex: Fiji) was used, camera type.

- To strengthen the conclusion that ATG5 expression in the xylem does not complement, although probably technically challenging, consider including evidence that it is expressed.

- The Discussion section could benefit from a deeper discussion of the results that were obtained e.g. are there other mutations that cause a root-hair less phenotype? If so, what is the phenotype of such mutants? What happens if you grow the plants longer, would an atg mutant eventually have more root hairs? It would also be interesting to include your thoughts on some of the mechanisms behind the antagonistic relationship of autophagy and programmed cell death, could there be a direct interaction between a given ATG and ANAC046 and/or 087?

A discussion of the possible redundancy between ANAC046 and 087 would be of interest. Would the single mutation of either in the atg2 background not rescue the premature root hair cell death? In Figure 4F, the ANAC046 line L7 has a few root hair tips remaining at the root junction, compared to line L4, which seems to have no root hairs. Could this be due to variable level of expression under the pE7 promoter?

Minor comments

- dPCD and ePCD are described in the introduction, and the meanings can be inferred, but it might be clearer to explain those abbreviations outright.

- Supplemental Figure 2A, dotted line in atg5-1 image is not explained, so it is not clear if it is the same as the dotted line in earlier figures (which outline the root profile).

- Figure 2 heading, “The loss function of autophagy...” should read “The loss of function.”

- At the end of the results section “... the premature dPCD of root hairs in atg mutants dependents on canonical” should read depends.

- Supplementary Figure 1H is missing a caption. It seems that the caption for Figure 1G may have been intended to address both figure panels from its description.

- To fully validate the new atg2-8 and atg2-9 mutants, consider including ATG2 RNA or protein expression compared to atg2-2.

- Define what the TOIM reporter is and what the acronym stands for.

- Fig1A, B, C: Each of the representative confocal images has a right-hand panel showing a merged image, but their purpose is not fully clear. An explanation in the figure legend would be helpful for clarity. The cytosolic level of fluorescence makes it difficult to distinguish the difference between autophagic bodies and background fluorescence, consider including the details of image acquisition in this case to ensure the comparison between WT and mutants is suitable.

- Figure 1E: Longitudinal is spelt wrong.

- Figure 1E: In the figure caption atg7-2 and atg5-1 are mentioned but not shown.

- Figure 3B: The figure is cut off and is written as “mCher”. Figure 2A, 3D, 3G, 3H, and 4F: The scale bars were also cut off (or are lacking).

- Could you explain the nomenclature pPASPA3>>H2A-GFP reporter name written with the “>>” symbol instead of a colon.

- In Figure 3B and 3F, the y-axis shows the number of cells expressing pPASPA3. It is not entirely clear in the text how this was counted: was this done using the H2A-GFP reporter?

- In Figure 3F, the line L1 appears to have a slightly lower number of cells expressing pPASPA3. Are there any possible reasons for this (such as differences in Cas9 targeting in different lines)?

- Statistical analyses: consider including an explanation for using t-test or ANOVA.

- The cross section of the root seen in Figure 3 could be helpful in Figure 1, since Figure 1E features a cross section confocal as part of the ConA imaging and/or include a symbol to indicate orientation on both the transverse and longitudinal section views.

Michael Kwakye and Warren Wilson (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Sally Abulaila, Kim Kissoon, Madaline McPherson, Madison McReynolds, Mandkhai Molomjamts, Habib Ogunyemi and Octavio Origel.

Competing interests

The authors declare that they have no competing interests.