ABSTRACT
Phosphate (Pi) limitation is a constraint for plant growth in many natural and agricultural ecosystems. Plants possess adaptive mechanisms that enable them to cope with conditions of limited Pi supply, including a highly regulated genetic program controlling the expression of genes involved in different metabolic, signaling and development processes of plants. Recently, we showed that in response to phosphate limitation Arabidopsis thaliana sets specific DNA methylation patterns of genic features that often correlated with changes in gene expression. Our findings included, dynamic methylation changes in response to phosphate starvation and the observation that the expression of genes encoding DNA methyltransferases appear to be directly controlled by the key regulator PHOSPHATE RESPONSE 1 (PHR1). These results provide insight into how epigenetic marks can influence plant genomes and transcriptional programs to respond and adapt to harsh conditions. Here we present an analysis of DNA methylation in the upstream regions of low Pi responsive genes in Arabidopsis seedlings exposed to low Pi conditions. We found that hypo- and hyper-methylation in the vicinity of cognate binding sites for transcription factors known to regulate the phosphate starvation response clearly correlates with increased or decreased expression of low-Pi responsive genes.
KEYWORDS: Abiotic stress, DNA methylation, epigenetics, nutrient limitation, phosphate
Plants acquired during evolution a wide set of physiological, cellular, and molecular mechanisms to cope with adverse environmental conditions. These responses include modifications in developmental and metabolic programs, which are highly dependent on the regulation of gene expression. It is well documented that gene regulation at the transcriptional and posttranscriptional level plays a major role in plant stress responses.1-4 However, recent evidence suggests that epigenetic mechanisms also play an important role in modulating gene expression in response to environmental factors and that epigenetic marks can serve as a priming mechanism to prepare future generations to efficiently cope with biotic and abiotic stresses.5-7 An important epigenetic mark is DNA methylation, a mechanism by which cytosine DNA methylation regulates the silencing of transposable elements (TEs) and repetitive sequences, genomic imprinting, as well as gene expression. The establishment of epigenetic modifications provides a mechanism capable of controlling and stably propagating potentially reversible gene activity states.7,8 Therefore, epigenetic states are dynamic and can be effectively remodeled by environmental cues, developmental signals, and disease states to enhance genome transcriptional plasticity.7,9-11
Phosphorus (P) is an important macronutrient for all living organisms that is a critical component of nucleic acids and membrane phospholipids, as well as an essential element for energy-dependent metabolic processes.12 Plants absorb P as inorganic phosphate (Pi), a chemical form of P with low availability and mobility in most soils.12 Hence, Pi is one of the most limiting nutrients for plant growth and development in natural and agricultural soils.12,13 Therefore is critical to improve our understanding of the molecular mechanisms involved in Pi homeostasis to generate plant varieties with increased Pi uptake, acquisition and utilization efficiency to complement agricultural practices and to implement an integral and sustainable production framework.
In a recent work,14 we reported that Arabidopsis plants in Low Pi availability an extensive remodeling of global DNA methylation takes place, which in many cases correlated with changes in gene expression. Modifications in global methylation patterns within genic regions was highly correlated with transcriptional activation or repression, revealing an important role of dynamic methylation changes in modulating the expression of genes in response to Pi starvation. Also, an analysis of Arabidopsis DNA methyltransferases mutants revealed that changes in DNA methylation patterns are required for the regulation of a number of Pi-Starvation-Responsive genes and that DNA methylation is required to establish proper morphologic and physiologic phosphate starvation responses.
Differential methylation nearby Pi responsive motif sequences correlates with gene expression modulation
Methylome analysis in Arabidopsis revealed that a discrete set of differentially expressed genes in response to Pi availability also showed differential methylation (Pi-methDEGs) in response to Pi starvation.14 Methylation changes in Pi-methDEGs were mainly localized in regions within 2000 bp upstream of the transcription start site, as well as in gene body regions. From the complete set of Pi-methDEGs, 190 genes presented differential methylation either in the form of Differentially methylated regions (DMRs) or Differentially methylated cytosines (DmCs) in upstream regulatory regions. We analyzed the upstream region of these Pi-methDEGs to determine whether differential methylation was correlated with the presence of cis-regulatory elements known to be involved in the transcriptional responses to Pi-starvation or whether there was an enrichment of other cis-regulatory elements (Fig. 1A). A number of transcription factors including the master regulator PHOSPHATE RESPONSE 1 (PHR1), and several members of the MYB and WRKY families of transcription factors have been shown to regulate the transcriptional activation of low-phosphate responsive genes and their corresponding binding sites P1BS, MBS and the W-box, respectively, act as cis-acting elements that mediate Pi starvation-responsive expression.15-20 A search for these cis-acting motifs in Pi-methDEGs showed that 39 of these genes contained P1BS motifs, that present the palindromic 8 base pair sequence (GNATATNC) where PHR1 binds to DNA as a dimer,15 93 genes had W-box elements which are the cognate binding sites of WRKY transcription factors, which have been characterized as key regulators of several processes in plants, including some members of this family which are specifically involved in the response to low Pi conditions,17,19,21,22 and 80 genes contained MYB transcription factors Binding sites (MBS) (Fig. 1B). Analysis of the upstream region of Pi-methDEGs for other known cis-regulatory elements showed a significant enrichment for 7 different motifs: abscisic-acid-responsive NAC (ANAC), PDK1-interacting fragment (PIF), MYC, DREB ethylene responsive factor-associated amphiphilic repression (DEAR), ethylene response factor (ERF), TCP and Arabidopsis thaliana ERF (AtERF) motifs, which were present in the upstream region of 104, 77, 73, 45, 38, 34 and 32 Pi-methDEGs, respectively (Fig. 1B).
Figure 1.
Analysis of differential methylation in the boundary of cis-regulatory elements. (A) Schematic representation of a gene upstream region. Pi-methDEGs where differential methylation (DMRs and DmCs) overlapped with upstream regions were analyzed for presence of cis-regulatory elements, then the presence of DNA methylation in the boundary of these motifs was analyzed (100 bp in each direction from the start of the motif sequence). (B) Number of Pi-methDEGs with at least one cis-regulatory element in upstream regions (2000 bp) (white bars) and Number of Pi-methDEGs where differential methylation was consistently present nearby cis-regulatory elements (gray bars).
To elucidate whether differential methylation is present in close proximity or within the cis-regulatory elements previously identified in the upstream sequences of Pi-methDEGs, we decided to search for DNA methylation marks in a window of 200 bp centered at the start point of each type of the cis-regulatory sequence present in Pi-methDEGs (Fig. 1A). Cis-regulatory elements overlapping DMRs or containing at least 4 DmCs in the 200 bp window where denominated as Pi-methDEGs with motif-adjacent methylation. Interestingly only the MBS, P1BS and W-box cis-regulatory elements showed high overlap with DNA methylation (MBS 73.7%, P1BS 77% and W-box 81.7%), whereas other cis-regulatory elements only showed DNA methylation overlap between 14.7% for ERF to 35.5% for DEAR motifs (Fig. 1B). This suggests that in response to low Pi conditions specific cis-regulatory elements within Pi-methDEGs upstream regions are selectively methylated.
Since MBS, P1BS and W-box showed a significantly higher incidence of nearby differential methylation in response to Pi-deprivation, we decided to further analyze Pi-methDEGs where differential methylation was present in these particular motifs boundary. First, we examined whether MBS, P1BS or W-box are present alone or in combinations in the upstream regulatory region of Pi-methDEGs. From a total of 88 Pi-methDEGs analyzed only 31.8% (28 genes) harbored only one type of these motifs which where distributed from higher to lower in W-box (20 genes), MBS (7 genes) and P1BS (1 gene) (Fig. 2A). Genes with a combination of these cis-regulatory elements accounted for 68.2% of Pi-methDEGs (60 genes) and 17 Pi-methDEGs contained all 3 motifs. 31 Pi-methDEGs contained both W-box and MBS elements, 8 contained W-box and P1BS elements and 4 genes contained MBS and P1BS motifs (Fig. 2A). The distribution of these cis-regulatory elements in Pi-methDEGs suggests that the presence of more that one of the cis-acting elements involved in the transcriptional responses to Pi-starvation is more prone to differential methylation and probably necessary to fine-tune gene expression by affecting the binding of multiple transcription factors.
Figure 2.
Analysis of genes with cis-regulatory elements associated with differential methylation. (A) Venn Diagram of Genes with unique and shared Cis-regulatory elements (MBS, P1BS and W-box). (B) Number of Cis-regulatory element (MBS, P1BS and W-box) containing Hyper-methylated (Upmeth) or Hypo-methylated (Downmeth) Pi-methDEGs that were differentially expressed (Upregulated and Downregulated) in response to Pi starvation. (C) Gene Ontology (GO) enrichment analysis of the complete set of 88 genes where the presence of DNA methylation in the vicinity of cis-regulatory elements correlated with modulation of gene expression, values are the normalized frequency (Relative frequency of Inquiry Set/Relative frequency of reference set, RFi/RFr), Enrichment Cut-Off was 2-fold.
The effect of motif-adjacent differential methylation on the expression of Pi-methDEGs in response to Pi starvation was assessed (Fig. 2B). Hypomethylation in the boundary of cis-regulatory elements in 45 of the analyzed Pi-methDEGs was preferentially correlated with upregulation (91%, 41 genes), whereas downregulation was correlated with hypomethylation in only 4 cases (9%). Hypermethylation at cis-regulatory elements boundary was correlated with downregulation in 24 Pi-methDEGs (68.5%) and with upregulation in 11 genes (31.5%). Only in 3 cases (3.7 % of total cases) motif-adjacent differential methylation overlapped with transposable elements (TEs); interestingly, in these 3 cases (AT1G47960, CIF1; AT2G16430 and PAP10; AT3G23450) hypermethylation was correlated with upregulation. Methylation and gene expression relationships suggest that there is a strong correlation of methylation adjacent to MBS, P1BS and W-box motifs with changes in gene expression, and that, at least in Arabidopsis, differential methylation in response to low Pi availability in upstream sequences rarely overlaps with TEs.
Finally Gene Ontology (GO) enrichment analysis was performed to determine whether in the Pi-methDEGs set with differential methylation adjacent to Pi-responsive cis-acting elements presented enrichment on specific GO categories. Enriched GO categories in our Pi-methDEGs set include peptide and sugar transport as the highest enriched categories (17.5 and 11.75-Fold respectively), with other enriched categories such as detoxification (5.9-Fold), phosphate metabolism (3.8-Fold) and sensing and response to external stimulus (2.87-Fold) (Fig. 2C, Table 1).
Table 1.
Differentially expressed genes targeted by differential methylation (Pi-methDEGs) in the boundary of MBS, P1BS and W-box cis-regulatory elements. Genes are representative of GO enriched categories. Arabidopsis Locus ID and Gene Descriptions of Pi-methDEGs are shown.
| Locus ID | Gene Description |
|---|---|
| AT1G20840 | TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) Hyper, Down |
| AT1G32450 | NITRATE TRANSPORTER 1.5 (NRT1.5) Hyper, Down |
| AT2G13540 | ABA HYPERSENSITIVE 1 (ABH1) Hyper, Down |
| AT4G35090 | CATALASE 2 (CAT2) Hyper, Down |
| AT5G01180 | PEPTIDE TRANSPORTER 5 (PTR5) Hyper, Down |
| AT1G47960 | CELL WALL/VACUOLAR INHIBITOR OF FRUCTOSIDASE 1 (C/VIF1) Hyper, Up |
| AT1G74590 | GLUTATHIONE S-TRANSFERASE TAU 10 (GSTU10) Hyper, Up |
| AT2G16430 | PURPLE ACID PHOSPHATASE 10 (PAP10) Hyper, Up, TE |
| AT4G37670 | N-ACETYL-L-GLUTAMATE SYNTHASE 2 (NAGS2) Hyper, Up |
| AT5G22300 | NITRILASE 4 (NIT4) Hyper, Up |
| AT2G26760 | CYCLIN B1;4 (CYCB1;4) Hypo, Down |
| AT3G02850 | STELAR K+ OUTWARD RECTIFIER (SKOR) Hypo, Down |
| AT3G21870 | CYCLIN P2;1 (CYCP2;1) Hypo, Down |
| AT3G60840 | MICROTUBULE-ASSOCIATED PROTEIN 65-4 (MAP65-4) Hypo, Down |
| AT4G01820 | ATP-BINDING CASSETTE B3 (ABCB3) Hypo, Down |
| AT1G66570 | SUCROSE-PROTON SYMPORTER 7 (SUC7) Hypo, Up |
| AT3G05690 | NUCLEAR FACTOR Y, SUBUNIT A2 (NF-YA2) Hypo, Up |
| AT3G47420 | GLYCEROL-3-PHOSPHATE PERMEASE 1 (G3Pp1) Hypo, Up |
| AT3G59884 | MICRORNA827A (MIR827A) Hypo, Up |
| AT4G21390 | (B120) Hypo, Up |
UpUpregulated Down Downregulated
Hypo Hypo-methylated Hyper Hyper-methylated
TEOverlaped with transposable element
Conclusions
In the referenced study,14 we proposed a model where in response to phosphate starvation a specific DNA methylation program is triggered in which differential methylation at gene features correlate with changes of expression of phosphate starvation responsive genes. Here we report that in the case of several Pi-starvation responsive genes, differential methylation occurs in the vicinity of binding sites motifs of TFs known to be directly involved in the low Pi response of Arabidopsis. We propose that methylation at gene upstream sequences may affect the binding capacity of transcription factors to cis-regulatory elements and then promoting changes in the expression state of low-Pi-responsive genes. Such mechanism would relay on an overlap of DNA methylation with cis-regulatory elements that are preferentially prone to receive methylation marks in order to modulate the expression of a discrete set of genes in response to phosphate starvation. This regulation framework is an attractive model to explain how DNA methylation at upstream regions of genes modulates adaptive responses to environmental signals during growth and developmental processes in plants.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
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