The HAC1 histone acetyltransferase promotes leaf senescence and regulates the expression of ERF022

Abstract Nutrient remobilization during leaf senescence nourishes the growing plant. Understanding the regulation of this process is essential for reducing our dependence on nitrogen fertilizers and increasing agricultural sustainability. Our laboratory is interested in chromatin changes that accompany the transition to leaf senescence. Previously, darker green leaves were reported for Arabidopsis thaliana hac1 mutants, defective in a gene encoding a histone acetyltransferase in the CREB‐binding protein family. Here, we show that two Arabidopsis hac1 alleles display delayed age‐related developmental senescence, but have normal dark‐induced senescence. Using a combination of ChIP‐seq for H3K9ac and RNA‐seq for gene expression, we identified 43 potential HAC1 targets during age‐related developmental senescence. Genetic analysis demonstrated that one of these potential targets, ERF022, is a positive regulator of leaf senescence. ERF022 is regulated additively by HAC1 and MED25, suggesting MED25 may recruit HAC1 to the ERF022 promoter to increase its expression in older leaves.

analysis. This dropped the number of decreased H3K4me3 peaks from 548 to 366. A revised Venn Diagram is now shown in Figure 3. I apologize for this oversight in the original submitted version of the manuscript.
4. The bioinformatics part in Methods described the packages used for GO enrichment and pathway analysis, but no specific result or figure was presented in the manuscript.
Line 178-185 discuss the GO terms and a new Supplemental Table 5 lists Pathway and GO Biological Process results with q-value or FDR. The Panther GO Enrichment tool, available through www.arabidopsis.org was used for GO enrichment as it has been recently updated (2/2019). This change is indicated in the Methods (line 134-135).
Minor: 1. page number need to be inserted Done 2. "MANorm " (line 130) need to be changed to "MAnorm" Done Reviewer #2: In this manuscript, the involvement of the HAC1 histone acetyltransferase in leaf senescence was investigated. It was shown that two Arabidopsis hac1 alleles display delayed age-related developmental senescence. Using a combination of ChIP-seq for H3K9ac and RNA-seq for gene expression, 44 potential HAC1 targets during age-related developmental senescence were identified. Genetic analysis demonstrated that one of these potential targets, ERF022, is a positive regulator of leaf senescence. ERF022 is regulated additively by HAC1 and MED25. It was proposed that MED25 may recruit HAC1 to the ERF022 promoter to increase its expression in older leaves. This is an interesting paper revealing a possible mechanism of epigenetic regulation in leaf senescence through regulation of ERF022 by HAC1. However, I think the following comments need to be addressed to improve the manuscript.
1. Please add a figure to show the structure of the HAC1gene, the T-DNA insertion sites of hac1 and hac2 mutants, and the positions of the primers used for PCR and RT-PCR analysis. In addition, please also show the data indicating that hac1 and hac2 mutants are indeed gene knock-out mutants. 2. Figure 1 Please indicate how many plants for each line were used for data analysis. For Figure 1B, the authors need to analyze gene expression in both hac1 and hac2 mutants.
The legend for Figure 1   Please explain why H3K4me3 modifications are affected in hac1-1 and hac1-2 mutants.
The following was added to lines 171-172, "In addition, histone acetylation and the H3K4me3 mark are shared components of one of four chromatin signatures in Arabidopsis (Roudier et al., 2011)." 4. Figure 4A-B and Figure 5A-B Please indicate how many plants for each line were used for data analysis.
We now indicate that n=6 single hole punches from 6 individual plants for chlorophyll and protein (lines 103 and 109) and for gene expression, "Each leaf sample was from one individual plant." (Line 117-118) 5. Page 7, line 234 -"These data suggest that MED25 guides HAC1 to histones at the ERF022 locus to direct histone acetylation for increased chromatin accessibility". This sentence was changed to " These data suggest that MED25 may guide HAC1 to histones at the ERF022 locus to direct histone acetylation for increased chromatin accessibility." (line 241).
There is no direct evidence to support that either MED25 or HAC1 targets directly at the ERF022 locus. To analyze whether MED25 and HAC1 target directly at the ERF022 locus, the authors need to carry our ChIP experiment using the MED25 or HAC1 antibody.
Yes, we definitely agree. These are the experiments we are planning to do. We have been trying to obtain HA-tagged or GFP-tagged HAC1 or MED25 lines from other groups who have published work, but seeds have not been sent nor are they available at the ABRC. A recent paper (doi: 10.1104/pp.19.00511) listed the peptide used to generate an anti-HAC1 antibody, and we plan on generating HA-tagged MED25 and Flag-tagged ERF022 to see if we can isolate a ternary complex. This is beyond the scope of this manuscript, but the suggestion of next steps is hugely appreciated.
6. Although the expression of ERF022 is affected in hac1 and hac2 mutants, more evidences are required to support the claim that the HAC1 histone acetyltransferase promotes leaf senescence via regulation of ERF022. I suggest the authors change the title of the manuscript to "The HAC1 histone acetyltransferase promotes leaf senescence and regulates the expression of ERF022".
The title has been changed, as suggested.