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 lab 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 44 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 recruits HAC1 to the ERF022 promoter to increase its expression in older leaves.
Introduction
Plants continuously produce new organs. During vegetative growth, new leaves form from the shoot apical meristem, and develop into protein-rich photosynthetic factories that export sugars. Eventually, the older leaves enter senescence by catabolizing the photosynthetic apparatus and exporting nitrogen-rich amino acids to support continuing growth (Himelblau and Amasino, 2001). Understanding the regulation of leaf senescence could maximize nitrogen recycling thus producing more nutrient-rich seeds and reducing the need for fertilizers.
The transition into leaf senescence is preceded (Kim et al., 2018a) and accompanied by changes in gene expression (Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006; Breeze et al., 2011). Lists of senescence-associated genes (SAG) have been generated from these transcriptome analyses. Enriched biological processes from Gene Ontology (GO) analyses include response to the hormones salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and ethylene. Also, enrichment of GO terms autophagy, immune response, defense response, and response to reactive oxygen species demonstrates a molecular relationship between defense and leaf senescence. Additional GO terms highly represented in SAGs from age-related developmental senescence include response to chitin and glucosinolate biosynthesis (Brusslan et al., 2015). The consistent enrichment of the phosphorylation term among SAG lists is likely a result of high expression of receptor-like kinase gene-family members, which also are known to regulate defense (Antolín-Llovera et al., 2014).
Changes in chromatin structure are hypothesized to promote and/or maintain leaf senescence (Humbeck, 2013). We have previously shown a correlation between histone 3, lysine 4, trimethylation (H3K4me3) and histone 3, lysine 9 acetylation (H3K9ac) histone modifications and increased expression of senescence up-regulated genes (SURGs). A similar correlation was seen between histone 3, lysine 27 trimethylation (H3K27me3) marks and decreased expression of senescence down-regulated genes (SDRGs) (Brusslan et al., 2012; Brusslan et al., 2015). Genetic analysis suggests histone deacetylases regulate leaf senescence. HDA19 is a negative regulator of senescence (Tian and Chen, 2001) while HDA6 is a positive regulator of leaf senescence (Wu et al., 2008). HDA9 works with POWERDRESS to reduce the expression of four putative negative regulators of leaf senescence (NPX1, TMAC2, WRKY57 and APG9), thus promoting leaf senescence (Chen et al., 2016).
Recently, two studies linked chromatin changes to leaf senescence. The Polycomb Repressive Complex 2 (PRC2) catalyzes H3K27me3 for long-term repression of ABA-induced SAGs (Liu et al., 2018). Double mutants in two PRC2 subunits (clf/swn) retain high SAG expression even after these genes are repressed in WT. H3K27me3-target genes that continue to be expressed in clf/swn mutants are significantly enriched for leaf senescence-related GO terms, indicating that long-term dampening of SAG expression is mediated by the H3K27me3 repressive mark. In the second study, the Jmj16 H3K4me3 demethylase acts to keep SAGs repressed in younger leaves (Liu et al., 2019). In jmj16 mutant alleles, both WRKY53 and SAG201 were up-regulated and associated with higher levels of the H3K4me3 mark. Non-catalytic forms of JMJ16 could bind to the promoter region, but only catalytically active forms could repress WRKY53 gene expression. This second study demonstrated that changes in H3K4me3 marks can regulate SAGs.
hac1 mutant alleles were reported to have darker green leaves (Li et al., 2014a). HAC1 encodes a histone acetyl transferase from the CREB Binding Protein family (Bordoli et al., 2001; Pandey et al., 2001), which is known to acetylate histone H3 resulting in H3K9ac (Earley et al., 2007; An et al., 2017). H3K9ac is associated with open chromatin and increased gene expression, and genes directly regulated by HAC1 are expected to be down-regulated in hac1 mutants. hac1 mutants are pleiotropic and display a protruding gynoecium (Han et al., 2007). HAC1 also regulates flowering, and hac1 mutants flower late due to increased Flowering Locus C (FLC) expression (Deng et al., 2007). FLC inhibits flowering, however decreased expression of genes that negatively regulate FLC was not observed in hac1 mutants. HAC1 may have other non-histone targets or an unknown negative regulator of FLC could be down-regulated in late-flowering hac1 mutants. In addition, hac1/hac5 double mutant seedlings are hypersensitive to ethylene (Li et al., 2014b) and display the triple response (short root, short and thick hypocotyl and exaggerated apical hook) when grown in the dark in the absence of ACC, the non-gaseous precursor to ethylene. Neither single (hac1 or hac5) mutant displayed ethylene hypersensitivity.
HAC1 also plays a role in the response to jasmonoyl-isoleucine (JA-ile), the active form of JA. HAC1 acetylates histones associated with MYC2 target genes to promote their expression. The Mediator Complex subunit, MED25 interacts with MYC2 and directly binds to and recruits HAC1 to target genes (An et al., 2017). Transcriptome data showed that genes induced by JA-ile were less responsive in a hac1 mutant. In addition, genes co-regulated by JA-ile and HAC1 were enriched for many defense-related biological process GO terms as well as leaf senescence.
Here we show that hac1 mutants have delayed age-related developmental leaf senescence. Potential HAC1 targets are identified by RNA-seq and ChIP-seq utilizing WT and two hac1 alleles. T-DNA insertion mutants in three potential HAC1 targets were tested for leaf senescence phenotypes, and an erf022 mutant disrupting the expression of ERF022 showed delayed senescence. These findings implicate this AP2/ERF transcription factor as a novel positive effector of leaf senescence regulated by histone acetylation co-mediated by HAC1 and MED25.
Materials and Methods
Plant Growth Conditions
Arabidopsis thaliana Col-0 ecotype plants were grow in Sunshine® Mix #1 Fafard®-1P RSi (Sungro Horticulture). The soil was treated with Gnatrol WDG (Valent Professional Products) (0.3 g/500 ml H2O) to inhibit the growth of fungus gnat larvae, and plants were sub-irrigated with Gro-Power 4-8-2 (Gro-Power, Inc.) (10 ml per gallon). Plants were grown in Percival AR66L2X growth chambers under a 20:4 light:dark diurnal cycle with a light intensity of 28 umoles photons m-2 sec-1. The low light intensity prevents light stress in older leaves, which was evident as anthocyanin accumulation at higher light intensities. To compensate for the reduced light intensity, the day length was extended. Leaves were marked by tying threads around the petioles soon after emergence from the meristem. Flowering time was determined when plants had 1 cm inflorescences (bolts). Leaf #5 from three week old plants were used for dark-induced senescence, and floated on water in the dark for the indicated number of days.
Genotype analysis
Genomic DNA was isolated from two-three leaves using Plant DNAzol Reagent (ThermoFisher) following manufacturer’s instructions. Pellets were dried at room temperature for at least two hours, and resuspended in 30 uL TE (10 mM Tris, pH 8.0, 1 mM EDTA) overmight at 4°C. One microliter of genomic DNA was used as a template in PCR reactions with primers listed in Supplemental Table 2. All standard PCR reactions were performed with a 57°C annealing temperature using Taq polymerase with Standard Taq Buffer (New England Biolabs).
Chlorophyll
One hole-punch was removed from each marked or detached leaf, and incubated in 800 μL N,N-dimethyl formamide (DMF) overnight in the dark. 200 μL of sample was placed in a quartz microplate (Molecular Devices) and readings were performed at 664 nm and 647 nm using a BioTek Synergy H1 plate reader. Absorbance readings were used to determine chlorophyll concentration (Porra et al., 1989). Chlorophyll was normalized to equal leaf area. For each genotype/condition, n =6.
Total Protein
One leaf hole-punch was ground in liquid nitrogen in a 1.5 ml microfuge tube using a blue plastic pestle. 100 μL 0.1 M NaOH was added and the sample was ground for another 30 sec (Jones et al., 1989). Samples were incubated at room temperature for 30 min, centrifuged at 14000 rpm for 5 min. The Bradford protein assay (Bio-Rad Protein Assay Dye Reagent) was used to determine protein concentration in each supernatant using a bovine serum albumin standard. For each genotype/condition, n = 6.
Percent Nitrogen
Elemental analysis for % nitrogen was done by Midwest Microlab, Indianapolis, IN. 100 dried seeds from one individual plant were in each sample (n = 8 for each genotype).
Gene Expression
Total RNA was isolated from the Indicated leaves using Trizol reagent. 1000 ng of extracted RNA was used as a template for cDNA synthesis using MMLV-reverse transcriptase (New England Biolabs) and random hexamers to prime cDNA synthesis. The cDNA was diluted 16-fold and used as a template for real-time qPCR using either ABsolute QPCR Mix, SYBR Green, ROX (Thermo Scientific) or qPCRBIO SyGreen Blue Mix Hi-Rox (PCR Biosystems), in Step One Plus or Quant Studio 6 Flex qPCR machines. All real-time qPCR reactions used a 61°C annealing temperature.
For chlorophyll, total protein, percent nitrogen and gene expression, significant differences were determined using a t-test.
RNA-seq
Indicated leaves were harvested and stored in liquid nitrogen. RNA was extracted and RNA-seq library production was performed using the breath adapter directional sequencing (BrAD-seq) method (Townsley et al., 2015). Real-time qPCR using ACT2 primers was the initial quality test. Libraries were sequenced at the Genome High-Throughput Facility (GHTF) at University of California, Irvine (UCI).
ChIP-seq
Nuclei preparation and ChIP was performed as described previously (Brusslan et al., 2012). Libraries were produced and sequenced at the GHTF at UCI.
Bioinformatics
RNA-seq raw data reads were aligned to the Arabidopsis TAIR 10 genome using Rsubread (Liao et al., 2013), and subject to quality control of count data and differential expression using NOISeq (Tarazona et al., 2015). The values were FPKM normalized using Tmisc and HTSFilter removed genes with low expression levels (Rau et al., 2013). A threshold value of q = 0.8 and a 2-fold change as the cut-off point was used to determine DEGs. ChIP-seq data were analyzed by MACS (Zhang et al., 2008) to find peaks of enrichment in comparison to input samples. MANorm (Shao et al., 2012) identified regions of differential histone modification. TopGO performed GO Biological Process enrichment and GAGE (Luo et al., 2009) performed pathway enrichment.
Results and Discussion
hac1 Mutants Show Delayed Senescence
Two Arabidopsis hac1 alleles [hac1-1 (SALK_080380) and hac1-2 (SALK_136314), Supplemental Figure 1] displayed darker green leaves when compared to WT. Age-related chlorophyll loss is shown in Figure 1A. At 28 days, total chlorophyll levels in leaf 7 were equal, but as the leaves aged, chlorophyll levels decreased faster in WT than the two hac1 alleles. A significant difference in chlorophyll levels was detected between WT and both hac1 alleles at day 48. The retention of chlorophyll was accompanied by reduced mRNA levels for genes associated with leaf senescence (Figure 1B). AtNAP encodes a positive regulator of leaf senescence associated with ABA synthesis (Liang et al., 2014; Yang et al., 2014). NIT2 encodes a nitrilase that is highly expressed in leaf senescence, and contributes to auxin synthesis (Normanly et al., 2007) and glucosinolate catabolism (Vorwerk et al., 2001). NYC1 encodes a chlorophyll b reductase required for light harvesting complex disassembly (Kusaba et al., 2007). The chlorophyll and gene expression data show that hac1 alleles display delayed leaf senescence.
The reduction of total chlorophyll was also evaluated in detached leaves floated in water in the dark (dark-induced senescence), and no difference was noted between WT and the two hac1 alleles (Figure 1C). There are molecular differences in the signaling pathways between dark-induced and developmental senescence; most prominent is the role of SA in developmental, but not in dark-induced senescence (Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006; Guo and Gan, 2012). Thus, it is possible that alterations in the signaling of developmental senescence do not necessarily accompany changes in dark-induced senescence. These results support a role for HAC1 as a promoter of age-related, developmental leaf senescence.
A trending increase in total leaf protein concentration accompanied the significant increase in chlorophyll levels in both hac1 alleles (Figures 2A-B). However, the delayed senescence in the hac1 alleles did not result in greater percentage of seed nitrogen (Figure 2C). Delayed senescence in wheat was reported to increase grain nitrogen concentration (Zhao et al., 2015), however the relationship between percentage of seed nitrogen and leaf senescence is complex (Chardon et al., 2014; Havé et al., 2017).
hac1 Mutants Display Altered Levels of Histone Modifications and Changes in Gene Expression During Leaf Senescence
ChIP-seq was performed on the same tissue shown in Figure 2 to identify genes associated with a loss of H3K9ac and/or H3K4me3 histone modifications in both hac1 alleles. HAC1 catalyzes H3K9 acetylation, and both H3K9ac and H3K4me3 are associated with active gene expression (Berr et al., 2011). As expected, H3K9ac significantly decreased at 968 loci and increased at only 555 loci in both hac1 alleles. H3K4me3 modifications were similarly affected, with 548 loci showing a loss and only 33 loci showing a gain of H3K4me3 marks. RNA-seq was used to identify differentially expressed genes (DEGs) between WT and both hac1 alleles. Accordingly, the number of up-regulated DEGs (12) was much smaller than the number of down-regulated DEGs (143) in both hac1 alleles. These 143 down-regulated DEGs were subject to pathway enrichment analysis, and significant enrichment of glucosinolate biosynthesis, plant-pathogen interaction, as well as glutathione and ascorbic acid metabolism were revealed. These pathways are stress-related and their down-regulation in hac1 likely slows the rate of leaf senescence. One GO term enriched in the up-regulated DEGs in both hac1 alleles is ribosome biogenesis, which occurs during rapid protein synthesis, and would be important for anabolic growth, not catabolic senescence. Cytokinin action delays dark-induced senescence, in part, by maintaining the expression of genes associated with ribosome GO terms (Kim et al., 2018b).
The Venn diagram in Figure 3 shows the overlap of genes with reductions in H3K9ac and H3K4me3 marks, as well as decreased expression in both hac1 alleles. Our analysis identified 44 genes (Supplemental Table 2) with reductions in H3K9ac marks and gene expression. These potential HAC1 targets have enriched GO terms including response to chitin and response to abiotic stimulus. These GO biological process terms have previously been associated with SAGs (Brusslan et al., 2015). Two of the potential HAC1 targets, IGMT1 and CYP81F2 (green highlight in Supplemental Table 2), encode indole glucosinolate biosynthetic enzymes, providing evidence that these secondary compounds are important during leaf senescence and potentially regulated via histone acetylation. We also observed significant reductions in H3K4me3 marks for these two genes in both hac1 alleles, further bolstering the presence of chromatin changes.
Analysis of Leaf Senescence Phenotypes in Potential HAC1 Targets
We measured leaf senescence in T-DNA insertion lines disrupting three regulatory genes from the list of 44 potential HAC1 targets (yellow highlights in Supplemental Table 2). These include ERF022, MYB15 and TMAC2. Two of these genes: ERF022 and TMAC2 also show a reduction in H3K4me3 marks. ERF022 and MYB15 encode transcription factors while TMAC2 plays a negative role in ABA response (Huang and Wu, 2007). Flowering time, NIT2 gene expression, and chlorophyll levels were quantified in these mutants (Figure 4A-C). We also showed that full-length mRNAs spanning the T-DNA insertion were not produced in each mutant allele (Figure 4D). The only line to show a consistent and strong significant alteration in leaf senescence was erf022, with slightly later flowering (by about three days), and after 44d of growth, reduced NIT2 expression (approximately 8-fold) and increased chlorophyll. These phenotypes indicate a delay in leaf senescence and implicate ERF022 as a positive regulator of leaf senescence.
Our results suggest that H3K9 acetylation mediated by HAC1 occurs at ERF022 during leaf aging, and is accompanied by changes in H3K4me3 marks. Together, these two marks likely promote the expression of ERF022, a positive regulator of leaf senescence. ERF022 is a member of the drought-responsive element-binding (DREB) subfamily of the AP2/ERF family (Nakano et al., 2006). Protoplast transfection experiments show ERF022 to be a positive regulator of the RD29A promoter (Wehner et al., 2011), suggesting ERF022 may mediate abiotic stress. Etiolated erf022 mutant seedlings produce significantly more ethylene, suggesting that ERF022 attenuates ethylene synthesis early in development (Nowak et al., 2015). EIN2 encodes an essential component of the ethylene signaling pathway, and ein2 mutants delay leaf senescence (Oh et al., 1997), thus increased ethylene production would be expected to accelerate senescence. If ERF022 is acting similarly in seedlings and older leaves, increased ethylene would be expected to promote senescence, however a delay was observed in erf022. It is possible that ERF022 plays different roles at different times in development. JA and a necrotrophic pathogen stimulated ERF022 expression (Mcgrath et al., 2005), indicating ERF022 plays a role in defense. Defense and senescence share many genes, as noted previously. Of interest, the ethylene hypersensitivity previously observed in hac1/hac5 double mutant seedlings may be due to reduced expression of ERF022. erf022 mutants overproduce ethylene, and mutations in HAC1 and HAC5 additively displayed a constitutive triple response.
MEDIATOR25 works additively with HAC1 to regulate ERF022 expression
The MED25 subunit of the Mediator Complex can interact with HAC1. We obtained med25 mutants and produced hac1-1/med25 double mutants to evaluate genetic interaction. The longest delay in flowering was observed for med25 and hac1-1/med25 (Figure 5A), but an additive effect in flowering phenotype was not present. Chlorophyll levels were measured in leaf 7 in 45 day old plants, and higher chlorophyll levels were observed in hac1-1, med25 and the hac1-1/med25 double mutants, and although all lines were significantly greater than WT, none were significantly different from each other (Figure 5B). These data suggest that HAC1 and MED25 do not have an additive effect, as loss of one or both show similar delays in flowering and chlorophyll loss. The erf022 mutant was also included in this experiment; it bolted later and had more chlorophyll than WT, but it did not differ from the hac1-1, med25 or hac1-1/med25 mutant lines.
Gene expression was also evaluated in these mutant lines. As expected, ERF022 expression was minimally detected in the erf022 mutant. A strong additive effect was seen between hac1-1 and med25 with much lower ERF022 expression in the hac1-1/med25 double mutant than in either single mutant (Figure 5C). These data suggest that MED25 guides HAC1 to histones at the ERF022 locus to direct histone acetylation for increased chromatin accessibility. With respect to two other SAGs, NIT2 and Lhcb2.4, the erf022 mutant showed the largest effect: minimal up-regulation of NIT2 (Figure 5D) and minimal down-regulation of Lhcb2.4 (Figure 5E) as compared to hac1-1, med25 and hac1-1/med25. These data suggest that loss of ERF022 has a more profound effect on the leaf senescence phenotype than its down-regulation through loss of both HAC1 and MED25. Although the ERF022 transcript levels were similar to the hac1-1/med25 double mutant (Figure 5C), it is probable that the mRNA produced in the erf022 mutant is inefficiently translated due to the T-DNA insertion in the 3’-UTR and led to a stronger phenotype in erf022. In addition, there are likely more genes mis-regulated in hac1-1/med25 and these may have compensating effects on leaf senescence.
Conclusion
hac1 mutant alleles display a delay in leaf senescence implicating histone acetylation as a contributor to the regulation of leaf senescence. A combined approach using ChIP-seq, RNA-seq and genetic analysis, identified ERF022 as a novel positive effector of leaf senescence regulated by H3K9ac and H3K4me3 marks. ERF022 is possibly a direct target of HAC1, which operates in concert with MED25 to allow full expression of ERF022 in older leaves.
Author Contributions
WEH and KK designed and performed the research and analyzed data. JAC performed the research. JAB designed and performed the research, analyzed data and wrote the paper. All authors greatly contributed to editing.
Acknowledgements
The authors thank Soumi Barman and Glenn Nurwano for technical help in genotype analysis. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers R25GM071638 and SC3GM113810. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Footnotes
Author email addresses: willehinckley{at}gmail.com, k.kevmanesh{at}hotmail.com, cordova.a.jaime{at}gmail.com