Abstract
Ambient temperature influences plant growth and development and minor changes can substantially impact crop yields. The underlying mechanisms for temperature perception and response are just beginning to emerge. Chromatin remodeling via the eviction of the histone variant H2A.Z in nucleosomes that alters gene expression is a critical component of thermal response in plants. However, whether chromatin-remodeling processes such as histone modifications play a global role in thermal response remains unknown. Using a combination of genetic analysis, chemical inhibition studies and RNA-seq analysis coupled with meta-analysis, here we identify POWERDRESS (PWR), a SANT-domain containing protein that is known to interact with HISTONE DEACETYLASE 9 (HDA9), as a novel key factor required for thermomorphogenesis in Arabidopsis thaliana. We identify that mutations in PWR impede thermomorphogenesis exemplified by severely attenuated temperature-induced hypocotyl/petiole elongation and early flowering. We show that inhibitors of histone deacetylases diminish temperature-induced hypocotyl elongation, which demonstrates for the first time a requirement for histone deacetylation in thermomorphogenesis. Genes that are misregulated in pwr mutants showed enrichment for GO terms associated with “response”. Our expression studies coupled with meta-analysis revealed a significant overlap between genes misregulated in pwr mutants and genes that are enriched for H2A.Z in their gene bodies. Meta-analyses reveal that genes misregulated in pwr mutants in diverse conditions also overlap with genes that are differentially expressed in the mutants of the components of the SWR1 complex that mediates H2A.Z nucleosome dynamics. Our findings thus uncover a role for PWR in facilitating thermal response and suggest a potential link between histone deacetylation and H2A.Z nucleosome dynamics in regulation of gene expression in plants.
Author summary Plant growth and development is influenced by a variety of external environmental cues. Ambient temperature affects almost all stages of plant development but the underlying molecular mechanisms remain largely unknown. In this paper, the authors show that histone deacetylation, one of the major chromatin remodeling processes, is essential for eliciting growth temperature-induced responses in plants. The authors identify POWERDRESS, a protein known to interact with HISTONE DEACETYLASE 9, as a novel key player essential for eliciting high temperature induced responses in Arabidopsis. Another chromatin remodeling mechanism that is known to play a role in thermal response is the eviction of histone variant H2A.Z from nucleosomes. Through transcriptome studies the authors demonstrate an overlap between gene regulations conferred through PWR-mediated histone H3 deacetylation and that conferred via histone H2A.Z eviction/incorporation dynamics. This study identifies a key novel gene that is essential for plants to elicit high temperature responses and reveals close links between two seemingly distinct chromatin-remodeling processes in regulating gene expression in plants.
Introduction
Plant development is highly sensitive to their growth environment. Ambient temperature, one of the major environmental factors that influences plant growth has a significant impact throughout plant development [1]. Even minor changes in temperature can modulate life history traits such as flowering time and seed set [2, 3]. Elevated temperatures modulate plant growth and development in a process termed “thermomorphogenesis” that results in a suite of phenotypes including an increase in hypocotyl elongation, petiole elongation and early flowering [1]. This is also often coupled with a dampening of defense response [4]. The molecular basis of thermal response is just beginning to emerge and appear to involve primarily changes at the level of transcription [1]. Recent work suggests that thermal cues are in part perceived through the photoreceptor phytochromes [5, 6]. For example phyB has been shown to bind to the promoters of its target genes in a temperature-dependent manner modulating transcriptional response to temperature [5].
One of the other key molecular events in thermal response involves chromatin remodeling associated with the eviction/incorporation dynamics of the histone variant H2A.Z in the nucleosomes [7]. Higher temperatures has been shown to lead to the eviction of the histone variant H2A.Z from nucleosomes [7]. H2A.Z eviction upon exposure to elevated temperature loosens the chromatin resulting in changes in gene expression [7]. H2A.Z nucleosome dynamics appears to be critical not only for temperature response, but also for general response to external stimuli [8]. H2A.Z is enriched in gene bodies of the “responsive genes”, and h2a.z mutants display mis-regulation of genes associated with response to environmental stimuli [8]. The eviction/incorporation of H2A.Z on to the nucleosomes is mediated through the SWR1 complex in Arabidopsis that consists of proteins encoded by ACTIN RELATED PROTEIN 6 (ARP6), SWC6 and PHOTOPERIOD INDEPENDENT EARLY FLOWERING 1 (PIE1). Mutations in these genes result in pleiotropic phenotypes [9-12]. In contrast to our understanding of the temperature-induced H2A.Z eviction, very little is known about the global role of other chromatin remodeling processes such as histone modifications in ambient temperature response [1, 13].
A central integrator in this transcriptional network is PHYTOCHROME INTERACTING FACTOR 4 (PIF4), which encodes a transcription factor that mediates several temperature-associated phenotypes including defense response [1, 14-18]. PIF4 is regulated at multiple levels via complex transcriptional and post-transcriptional regulatory mechanisms in response to temperature [1]. Subsequently, PIF4 regulates downstream target genes, primarily through transcription [1]. For example, PIF4 regulates temperature-induced hypocotyl elongation via stimulating auxin biosynthesis by binding to the promoters of auxin biosynthesis genes, including YUCCA8 [14-16, 19]. Thus transcriptional responses at multiple levels play critical roles in governing temperature responses in plants.
Here, we identify POWERDRESS (PWR), which is known to interact with HISTONE DEACETYLASE 9 (HDA9) [20, 21] as a novel key factor that is essential for thermomorphogenesis in Arabidopsis, and uncover a central role for histone deacetylation in mediating thermal response. We demonstrate that blocking histone deacetylation abolishes temperature-induced hypocotyl elongation. Through RNA-seq experiments, we show that PWR suppresses defense gene expression at elevated temperatures. Using our RNA-seq data with the meta analysis of published genome-wide H2A.Z ChIP-seq data we show that there is a significant overlap between genes that are mis-regulated in the pwr mutants and those that are modulated through H2A.Z eviction/incorporation dynamics. Thus, our findings reveal a global role for histone deacetylation in thermal response. In addition our findings also show a general association between two distinct chromatin remodeling mechanisms viz., histone H3 deacetylation and H2A.Z nucleosome dynamics in regulating gene expression that extends beyond thermal response.
Results
Elevated temperatures result in increased hypocotyl elongation in Arabidopsis thaliana [19]. To identify new genes that facilitate thermomorphogenesis, we carried out a forward genetic screen. T-DNA insertion lines [22] allow simultaneous screening of phenotypes at multiple conditions. We screened more than 7000 lines at 23 °C and 27 °C for attenuated response in temperature-induced hypocotyl elongation and identified 4 potential mutants with altered thermal response. One of the lines that carried an insertion at At3g52250/POWERDRESS (PWR) displayed a severely diminished thermal response in hypocotyl elongation (Fig. 1A, B, pGxE<0.0001). We examined additional independent T-DNA insertion lines at this locus and found the reduction-offunction lines to display a reduced thermal response, which suggests that PWR is essential for temperature-induced hypocotyl elongation (Fig. 1B & S1). An ems-induced mutant for PWR (pwr-1), has been previously isolated as an enhancer of agamous [23]. This pwr-1 allele in the Ler background also displayed a similar impairment in temperature-induced hypocotyl elongation (Fig 1B, pGxE<0.0001). The impaired thermal response was abolished in the pPWR::PWR-GFP line, independently confirming that PWR is the causal locus for the attenuated thermal response (Fig. 1B).
To assess whether the pwr mutation specifically affects hypocotyl development or generally impairs thermomorphogenesis, we evaluated other temperature-associated phenotypes. Elevated temperatures increase petiole length [14] (Fig. S2A) and plants, when shifted from 23 °C to 27 °C display an elongated petiole within 2 days (Fig. 1C-D). This marked response to temperature-shift was not observed in pwr-2 mutants (Fig. 1C-D). Higher temperatures result in early flowering in Arabidopsis [3]. While mutations in pwr also result in early flowering [23] (Fig. S2B), the thermo-sensitivity of floral induction was significantly reduced in pwr-2 (Fig. 1E, pGxE<0.0095). In addition pwr-2 mutants appeared smaller than wild type plants (Fig. S2B), which suggests that there is a general impairment of plant growth in pwr-2. The observed reduction in temperature-sensitivity correlated with an attenuated response in the temperature-induced expression of HSP70, PIF4 and YUCCA8 (Fig. 1F), genes known to be induced upon elevated temperatures. Taken together these results suggest that pwr mutants are generally impaired in thermomorphogenesis.
The attenuated expression of PIF4 and YUCCA8 in pwr-2 (Fig. 1F) suggests that PWR is required for the temperature-induced PIF4 expression and subsequent auxin biosynthesis, which could be the underlying mechanism for the impaired thermal response in hypocotyl elongation. In contrast, PWR expression remained mostly unaltered in the pif4-2 mutants (Fig. S3), which suggests that PIF4 does not regulate PWR at the transcriptional level. Consistent with the idea that PWR and PIF4 act in the same genetic cascade, the pif4 pwr double mutants were not significantly different from either single mutant, i.e., no additive or antagonistic interactions were observed in temperature-induced hypocotyl elongation (Fig 2A, Fig. S4A). However, pwr and pif4 mutants significantly differ in their flowering phenotype both at 23 °C and 27 °C with the pwr mutants displaying early flowering at both temperatures [23-25] (Fig. 1E,). We found pwr-2 pif4-2 or pwr-2 pif4-101 double mutants to be early flowering similar to pwr-2 mutants (Fig 2B, Fig. S4B). The early flowering at 27 °C was associated with an increase in FT expression, which suggests that the loss of PWR can overcome the requirement for the proposed activation of FT expression by PIF4[24] at high ambient temperatures (Fig. 2C).
PWR contains a SANT domain that has been suggested to play role in regulating chromatin accessibility by mediating the interaction between histone tails and the histone modifying enzymes [26]. To assess whether the acetylation status of histones modulate temperature-induced hypocotyl elongation, we grew plants in presence of histone acetylation/deacetylation inhibitors. While we did not detect any difference in hypocotyl length presence of histone acetylation inhibitor curcumin (Fig. S5), temperature-induced hypocotyl elongation was severely compromised in plants grown in presence of different histone deacetylase (HDAC) inhibitors viz., sodium butyrate, Droxinostat, CP64434 hydrate or trichostatin A (Fig. 3A, S6A & S6B). Western blots confirmed inhibition of deacetylation with an increase in acetylated proteins in presence of sodium butyrate (Fig. S6C). These findings confirmed that histone deacetylation is essential for thermomorphogenesis. Comparison of pwr-2 mutants with wild type Col-0 revealed significant drug x temperature (Fig. 3B, S6B) and drug x genotype (Fig. S6D) interactions confirming that the effect of histone deacetylation depends on the genotype and temperature. The effect of HDAC inhibitors were less pronounced in pwr-2 compared to Col-0, suggesting that PWR acts in the same pathway that is targeted by the HDAC inhibitors. Correlating with the phenotypes, there was a reduction in temperature-induced expression of YUCCA8 and PIF4 in presence of HDAC inhibitors (Fig. 3C, S6E). Moreover, hda9 mutants also displayed attenuated responses in temperature-induced hypocotyl elongation (Fig. 3D). Consistent with the recent findings [20, 21], we detected an increase in H3K9-acetylation in pwr-2 mutants (Fig. 3E). In addition, we also detected an increase in acetylated H2A.Z in pwr-2 (Fig. 3E). Taken together these data suggests that PWR-mediated histone deacetylation is essential for thermomorphogenesis in Arabidopsis.
Histone deacetylation is typically associated with down regulation of gene expression [27]. The requirement of PWR for thermomorphogenesis therefore indicates that down regulation of gene expression is also critical for proper thermal response. Thus the requirement of PWR-mediated histone deacetylation for the temperature-induced expression of YUCCA8 and PIF4 (Fig. 1F, Fig. 3C, S6E), suggests that these are indirect targets of the PWR/HDA9 module. To obtain further insights into PWR-mediated transcriptional regulation in response to temperature, we compared the pwr-2 and Col-0 transcriptomes at 23 °C and 2-hours after a shift to 27 °C, in 6-day old seedlings. Interestingly, the number of differentially expressed genes (DEGs) between Col-0 and pwr-2 was substantially higher at 27 °C (867 DEGs), than at 23 °C (36 DEGs) (Fig. 4A, B, Table S1 & S2). Analysis of the transcriptomes at 23 °C and 27 °C identified 30 genes to be differentially expressed in Col-0, while 623 genes to be differentially expressed in pwr-2 (Fig. 4A). Thus, the loss of PWR resulted in global mis-regulation of transcription at 27 °C, which suggests that PWR dampens transcriptional response to elevated temperatures. While the majority of the mis-regulated genes were up regulated in pwr-2 at 27 °C, consistent with the role of PWR in histone deacetylation, most of the genes that were induced by higher temperatures in Col-0 (Table S3) failed to do so in pwr-2 mutants (Fig. S7).
Gene Ontology (GO) analysis of the DEGs between Col-0 and pwr-2 at 27 °C showed enrichment for GO terms associated with “response” (Table S4, Fisher Test with Yekutieli correction). In addition to enrichment for genes associated with “response to temperature stimuli” (p<7.4e-6), various other response terms were also enriched (Table S4. e.g., response to: chemical stimuli (p<8.7e-59), stress (p<3.7e-42), carbohydrate (p<7.1e-25), other organism (p<9.5e-25), water (p<3.4e-21), defense (p<6.3e-21), hormone (p<1.6e-19), ethylene (p<4.1e-12)), which suggests that PWR, in addition to being involved in temperature response may be generally associated with the transcriptional regulation of “response” genes. A similar GO enrichment profile for “response” was previously reported for genes with H2A.Z enrichment in gene bodies (here after called high-H2A.Z) [8]. Therefore, we considered whether the genes that are differentially expressed in pwr-2 overlap with H2A.Z enriched “response” genes. To assess the significance of overlaps, in addition to calculating hypergeometric probabilities, we generated 100,000 random pairs of gene lists from Arabidopsis genome, analysed the overlaps between each pairs of the gene lists, calculated their hypergeometric probability and generated a simulated distribution (Fig. S8). Through meta-analysis of the published H2A.Z data [8], with the pwr transcriptome data, we detected a significant overlap between DEGs between pwr-2 and Col-0 with high-H2A.Z genes (p<3.24e-10, hypergeometric probability test), but not with low-H2A.Z genes (Fig. 4C), which suggests that the expression of a significant subset of H2A.Z enriched genes is regulated by PWR-mediated histone deacetylation. To assess whether histone acetylation is generally associated with H2A.Z enrichment, we carried out meta-analysis of publicly available data on H3K9acetylation across the genome with H2A.Z enrichment [8, 28]. While H3K9acetylation overlapped with both high-H2A.Z (850/1984, Fig. S9A, p<2e-7, hypergeometric probability test) and low-H2A.Z (1134/1984, Fig. S9A, p<1.71e-60, hypergeometric probability test) genes, we observed significant overlap of DEGs in pwr-2 only with H3K9acetylated genes that are also enriched for H2A.Z in their gene bodies (Fig. S9B-S9D), which suggests that PWR preferentially modulates high-H2A.Z genes.
Although our findings suggested a link between PWR-mediated histone deacetylation and H2A.Z enrichment, the observed overlap with H2A.Z enriched genes could be attributed to thermal transcriptome, as temperature also affects H2A.Z nucleosome dynamics [7]. Therefore, to assess whether there is a general association between H2A.Z enrichment and PWR/HDA9 mediated transcriptional regulation, we carried out a similar meta-analysis with genes that were reported to be differentially expressed in pwr-2 and hda9 mutants in published studies unrelated to temperature but associated with plant aging and flowering [20, 21]. In spite of the differences in the sampled tissue, developmental states, growth conditions as well as different research groups being involved, the pattern was same, where a significant overlap was observed with high-H2A.Z but not with the low-H2A.Z genes (Fig. S9, S10A-S10C). Of the 4081 high H2A.Z genes, we detected 1068 (26%, p<3.79e-29, hypergeometric probability test) to be differentially expressed in pwr and/or hda9 (S10D). These findings suggest that the association between H2A.Z enrichment and PWR/HDA9 mediated transcriptional regulation extends well beyond thermal response and hints at a general association between H2A.Z nucleosome dynamics and histone H3 deacetylation.
Histone H3 deacetylation and H2A.Z nucleosome dynamics are two fundamental, yet distinct chromatin-remodeling processes that modulate gene expression in response to diverse environmental stimuli [27, 29]. As our findings suggested a possible previously unexplored link between these two, we tested whether changes in gene expression conferred through H2A.Z nucleosome dynamics overlaps with those mediated through histone H3 deacetylation. To assess this, we compared the pwr-2 and hda9 transcriptomes with the published transcriptomes of H2A.Z mutants hta9/hta11 (defective for the two out of the three H2A.Z encoding genes) [30] and the mutants for the components of the SWR1 complex (arp6 (ACTIN RELATED PROTEIN 6), pie1 (PHOTOPERIOD INDEPENDENT EARLY FLOWERING 1) & swc6) that mediates H2A.Z eviction/incorporation in nucleosomes. In addition, we also compared the transcriptome of PIF4, one of the major downstream components of the H2A.Z nucleosome dynamics [18, 31]. Here as well, despite the differences in the sampled tissue, developmental states, growth conditions and research groups involved, we observed a significant overlap between the DEGs for all the transcriptomes (Fig. 4D, Fig. S12A, S13A, S14A, S15A, S15A & S16A), which further supports the potential nexus between histone deacetylation and H2A.Z nucleosome dynamics in regulating gene expression. Analysis of up regulated and down regulated genes in each of these transcriptomes revealed a significant overlap among up-regulated genes (Fig S11AB, S12B-C, S13B-C, S15B-C & S16B-C). Nevertheless, genes whose expression changed in opposite directions also displayed an overlap (Fig S11C-D, S12D-E, S13D-E, S15D-E & S16DE), which suggests that the target genes are modulated in a dynamic manner through this interaction. Taken together, these findings further hints at a link between H2A.Z nucleosome dynamics and histone deacetylation in the regulation of gene expression in plants.
Among the analysed transcriptomes, most significant and discernible overlaps were seen with pie1 followed by pif4 (Fig. 4D, S11-S17), exhibiting more significance than those of swc6, arp6 or hta9/hta11 (Fig. 4D, S11-S17). PIE1 also encodes a SANT domain containing protein[10], but its role in the SWR1 complex is well-studied [11, 32]. We found pie1 mutants to display a reduced response to temperature in hypocotyl and petiole elongation, similar to pwr-2, although the effect was less pronounced (Fig. S18). Double mutants of pwr-2 pie1-6 resembled the pwr-2 mutants suggesting that pwr is epistatic to pie1 (Fig. S16). Both PIE1 and PIF4 play critical roles in plant defense [18, 30, 33]. Defense responses are dampened at higher temperatures[4] and the up regulation of defense response genes in pwr-2 suggests that PWR mediated histone deacetylation is critical in suppressing defense gene expression at elevated temperatures (Table S5).
Discussion
We have demonstrated that PWR is a critical gene required for thermomorphogenesis. Previous studies suggested a key role for PWR in diverse developmental processes including regulation of floral determinacy, flowering and senescence [20, 21, 23]. The underlying mechanism through which PWR acts on these processes appears to be via transcriptional regulation by histone H3 deacetylation. PWR modifies acetylation status through its physical interaction with HDA9 that results in histone deacetylation at specific loci across the genome [20, 21]. Histone deacetylation has been previously shown to be essential in both developmental processes and abiotic stress response [27]. Our results demonstrate conclusively that PWR-dependent histone deacetylation is a key chromatin-remodeling mechanism required for ambient temperature-response in plants.
Chromatin remodeling through the exchange of histone H2A.Z with H2A has been previously shown to be critical in mediating thermosensory response in plants [7]. Our findings therefore reveal another layer of chromatin remodeling that is essential in mediating transcriptional responses to temperature. PWR acts at the level of chromatin in conferring thermal response and thus an upstream factor in thermosensory response in plants. Our genetic analysis supports this hypothesis. Therefore, two distinct chromatin remodeling processes viz., H2A.Z nucleosome dynamics and histone H3 deacetylation appears to be essential for conferring thermal responses in plants. Nevertheless, it remains unclear as to how temperature information is perceived by the SWR1 or the PWR/HDA9 complex to regulate these chromatin-remodeling events.
Interestingly, mutations in SWR1 complex such as arp6 and pie1 result in contrasting temperature-induced hypocotyl phenotypes. While arp6 appears to have longer hypocotyls even at lower temperatures [7], mutations in pie1 result in relatively shorter hypocotyls even at elevated temperatures. Thus, the inability of pie1 mutants to respond to elevated temperature, similar to pwr, reveals the complexity at the level of chromatin-remodeling governing thermal responses. It is possible that PIE1 being a SANT-domain containing protein may have additional roles independent of the SWR1-complex. The striking overlap of pwr-2 transcriptome with pie1 when compared to the overlap with arp6, swc6 and hta9/hta11, the distinct phenotypes of pie1 and its genetic interaction with pwr-2 suggest a broader role of PIE1, some of which are H2A.Z-independent and associated directly or indirectly with the histone deacetylation cascade regulated by PWR.
PIE1 also plays a critical role in regulating the expression of defense genes and the role of PIE1 in defense also differs from arp6, swc6 and hta9/hta11[30, 33]. Trade-off between thermosensory growth and defense has recently been suggested to be coordinated by PIF4[18]. Analysis of the up-regulated genes in the pwr-2 transcriptome also revealed an enrichment of GO terms associated with defense (Table S5). The strong overlaps of pwr-2 transcriptome with pie1 and pif4 suggests that PWR-mediated histone H3 deacetylation is also critical in regulating gene expression changes that are associated with the trade off between growth and defense. Although PWR is required for PIF4 expression, we cannot rule out that PWR may also be required down stream of PIF4 in regulating defense gene expression. It is possible that PWR may act at multiple levels. It is also currently unknown whether pwr mutants indeed display enhanced disease resistance, which would be explored in future.
We have demonstrated that histone deacetylation is an essential aspect of thermomorphogenesis in Arabidopsis. We have also revealed that gene regulation by histone H3 deacetylation significantly overlaps with gene regulation conferred by H2A.Z nucleosome dynamics. We present a testable model that could explain our findings (Fig. 4E). We propose that histone H3 deacetylation and H2A.Z nucleosome dynamics are highly inter-connected and the response to environmental stimuli involves both processes. In addition, we hypothesise that both these processes likely influence each other. Histone H3 deacetylation regulates gene expression by altering chromatin accessibility leading to suppression of gene expression. In addition, it also affects gene regulation by potentially modulating H2A.Z eviction/incorporation dynamics. Thus, both up/down regulation of gene expression could result from same stimuli due to intra-nucleosomal interactions at the chromatin. Similar suggestions have been made in other systems. It has been suggested that histone H3 acetylation patterns can modulate H2A.Z nucleosome dynamics in yeast [34], whereas H2A.Z has been shown to promote H3 and H4 acetylation in mammalian cells [35, 36]. Exploring the mechanistic basis of this connection would be an exciting avenue for future work.
Materials and Methods
Plant material and phenotyping
All mutants were in the Col-0 background unless otherwise specified. All T-DNA insertion lines as well as most of the mutant lines used in this study were obtained from the European Arabidopsis Stock Centre. pwr-1, pwr-2, hda9-1, hda9-2, hda6 and hda19 mutants have been described [23, 37-39]. pwr-1 and pPWR::PWR-GFP lines were gifted by Prof. Xuemei Chen and pif4-101 is from Prof. Christian Fankhauser. All double mutants were obtained by crossing and confirmed by genotyping. Hypocotyl and petiole length measurements were done as described previously[40]. Briefly, seeds were sterilised, sown on Murashige-Skoog media and then stratified for 2 days at 4°C in dark. The plates were then transferred to CU41L5-Percival growth chambers (Percival Inc, Canada) at 23° C or 27° C in short day conditions (8 hour light / 16 hour dark) and grown vertically for 10 days. For the T-DNA screening, more than 20 seedlings representing each of the 5000 T-DNA lines were grown at 23 °C and 27 °C and the seedlings were visibly inspected for attenuated response. To quantify the hypocotyl elongation, subsequently, plates with plantlets were imaged and the hypocotyl length was measured using Image J (NIH). All T-DNA lines used in subsequent analysis described in this study were confirmed by using T-DNA insertion using primers listed in the Table S6. Flowering time measurements were done as described previously and total leaf number is used as a proxy for flowering time [3]. For the HDAC inhibitor assays all the compounds (Sigma-Aldrich) were dissolved in the described concentration in DMSO and the solvent lacking the compounds was used as a mock control.
DNA/RNA Analyses
DNA and RNA extractions were done as described previously [41]. For gene expression studies DNAse I (Roche)-treated 1ug of total RNA was used for cDNA synthesis using the First strand cDNA synthesis kit (Roche) and the resulting cDNA was diluted and used for realtime PCR analysis with a Lightcycler 480 system (Roche) with SYBR green. The specific primers used for real-time PCR analysis are in Table S6. Relative expression levels were obtained using the ΔΔcT method [42] using either UBIQUITIN or TUBULIN as internal controls.
Immunoprecipitation
Approximately fifty 3-week old A.thaliana seedlings were ground in liquid nitrogen and 1mL of incubation Buffer (50mM Tris-Hcl pH7.5, 150mM NaCl, 10mM EDTA, 0.2% Triton, Roche protease inhibitor cocktail) was added. The samples were spun for 15 min at 4 °C at 13200 rpm and 50μL of supernatant kept for crude extract analysis. 250ng of anti-acetylated Lysine antibody (mouse anti-acetylated-Lysine antibody Ac-K-103, Cell Signaling Technology; dilution 12000) were added to each sample and incubated with continuous shaking for 2h at 4 °C. 50 μL of protein A agarose beads were then added for 2 more hours of incubation in the same conditions. The resin was washed 3x with 1mL of incubation buffer and denaturated for 3min at 95 °C in presence of 50 μL of Laemmli buffer [43] before immunodetection through western blots.
Western blots
Western blots were done as described previously [44]. Equal amounts of protein were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoresis and transfer to Protran BA85 nitrocellulose membranes (Whatman, Germany). Transferred proteins were visualized by Ponceau S red staining. Plant protein samples obtained from A. thaliana (20 seedlings), were homogenized in 250 μL of Laemmli loading buffer [43]. Antibodies used for Western blotting were anti-H2A.Z (dilution 1/5000), anti-SUMO1 (dilution 1/5000), anti-H3K9ac (dilution 1/5000) goat anti-mouse and anti-rabbit IgG-HRP antibodies (Santa Cruz, dilution 110000).
Transcriptome studies
RNA-seq analysis was done as described previously [45]. About one hundred 6-day-old seedlings of Col-0 and pwr-2 each were grown at 23 °C in short days (SD) in growth chambers (GR-36, Percival Scientific, Canada). Half of the samples were moved to 27 °C. Tissue from whole seedlings were collected for RNA extraction from both 23 °C and 27 °C after 2-hours. Two biological replicates were used. Total RNA was extracted from two biological replicates using Isolate II RNA plant kit (Bioline Pty Ltd, Australia). The libraries were prepared on an Illumina HiSeqTM 2000 platform using paired-end sequencing of 90 bp in length at BGI-Shenzen (Beijing Genomics Institute). FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to perform the initial quality control check of the transcriptome data. SortmeRNA was used to filter the rRNA sequences from the datasets, using its default rRNA databases comprising of 16S, 18S 23S and 28S rRNAs[46]. The reads for each sample were aligned to Arabidopsis thaliana TAIR 10 genome using Tophat2 (v2.1.0) [47] and bowtie2 (v2.1.0) [48]. Raw abundance counts were obtained from the Bio conductor-R-subread package using featureCounts (v1.4.5) [49] from the output produced by Tophat2. Only fragments with both reads successfully aligned (specified through ‐p and ‐B parameters in featureCounts) were considered for summarization. The resulting lists of abundance counts were used as an input data for DESeq2 (v1.14.1) [50] differential expression analysis pipeline. For differential expression analysis and estimation of dispersions across libraries in DESeq2, batch effect between replicates was accounted for through a negative binomial GLM as described previously [45]. Genes with a padj<0.05 (Benjamini-Hochberg corrected p-values) were termed as differentially expressed genes (DEGs). The gene lists generated through the analysis of differential expression were used in the online program AgriGO to identify enriched GO terms [51]. Additional gene lists for overlap analysis were either obtained from published data [8, 18, 20, 30, 33]. Overlaps between gene lists were tested through hypergeometric probabilities as well as through simulation studies performed in R. 100,000 random gene lists of comparable sizes to the gene lists that were analysed (500-2000 genes) were generated from Arabidopsis. The hypergeometric probabilities for the overlaps observed in the simulated dataset were calculated using hypergeometric probability function in R.
Author contributions
CT, MvZ and SB designed and conceived the study. CT, SS, RS, LvdW and MN performed experiments. CT, ASY, DT and SB analysed the data. CT, ASY and SB wrote the manuscript with inputs from all authors.
Supplementary Tables
Table S1. List of DEGs between Col-0 and pwr-2 at 23 °C
Table S2. List of DEGs between Col-0 and pwr-2 at 27 °C.
Table S3. List of DEGs between 23 °C and 27 °C in Col-0
Table S4. GO-terms that were significantly enriched in DEGs in pwr-2 mutants. The GO terms with “response” are given in bold.
Table S5. GO-terms that were significantly enriched in genes that were up-regulated in pwr-2 mutants. The GO terms with “response” are given in bold. The GO terms associated with “defense” are highlighted.
Table S6. List of primers in used in this study.
Acknowledgments
We thank the European and North American Arabidopsis stock Centres, and Xuemei Chen for the seeds. We thank Iain Searle, John Alvarez, David Smyth and members of the SKB lab for critical comments on the manuscript. The RNA-seq data presented in this paper is available in GEO repository with the accession number GSE101782.