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cite this article as doi: 10.1111/jipb.12979.

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Received May 30 2020

Accepted Jun. 7 2020

Article Type: Commentary

Edited by: Zhizhong Gong, China Agricultural University, China

Running Title: Developmental function of RdDM

RNA-directed DNA methylation has an important

developmental function in Arabidopsis that is masked by the

chromatin remodeller PICKLE

Rong Yang1†, Li He1†, Huan Huang1, Jian-Kang Zhu1,2, Rosa Lozano-Duran1, Heng Zhang1,3*

1. Shanghai Center for Plant Stress Biology, Center for Excellence for Molecular Plant Sciences, Chinese

Academy of Sciences, 3888 Chenhua Road, Shanghai 201062, China

2. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907,

USA

3. National Key Laboratory of Plant Molecular Genetics, Center for Excellence for Molecular Plant

Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China

†These authors contributed equally

*Correspondence: hengzhang@psc.ac.cn

In Arabidopsis, RNA-directed DNA methylation (RdDM) is required for the

maintenance of CHH methylation, and for de novo methylation in all (CG, CHG,

and CHH) contexts, but no obvious effect of RdDM deficiency on plant

development has been found to date. We show that the combination of

mutations in the chromatin remodeller PKL and RdDM components results in

developmental alterations, which appear in a SUPPRESSOR OF DRM1 DRM2

CMT3 (SDC)-dependent manner.

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In plants, DNA methylation in different sequence contexts (CG, CHG and CHH,

where H is A, T or C) is maintained by different DNA methyltransferases. In

Arabidopsis thaliana, the maintenance of symmetric CG methylation is

catalysed by MET1 (METHYLTRANSFERASE 1), whereas CMT3

(CHROMOMETHYLASE 3) is mainly responsible for the maintenance of CHG

methylation. The asymmetric CHH methylation is maintained by CMT2

(CHROMOMETHYLASE 2) and DRM2 (DOMAINS REARRANGED

METHYLTRANSFERASE 2). DRM2 is part of the RNA-directed DNA

methylation (RdDM) pathway, which is also responsible for establishing de

novo DNA methylation in all sequence contexts (Cao et al. 2003). The canonical

RdDM pathway requires two plant-specific RNA polymerase II-related

enzymes, Pol IV and Pol V: Pol IV generates RNA transcripts that initiate the

production of 24 nt siRNAs, which are loaded into AGO4 (Zilberman et al. 2003)

and guide this protein to scaffold RNA molecules generated by Pol V via

sequence complementarity; this leads to the recruitment of the

methyltransferase DRM2 (Gao et al. 2010; Bohmdorfer et al. 2014; Zhong et

al. 2014) and the subsequent de novo DNA methylation of adjacent DNA

sequences. Concomitant to DRM2-mediated methylation, histone modification

and chromatin remodelling occur. In Arabidopsis, methylation of the histone H3

lysine 9 (H3K9me1/2) and non-CG methylation form positive feedback loops

and facilitate the steady-state level of each other (Stroud et al. 2014). A number

of histone modifying enzymes including the histone deacetylase HDA6, histone

demethylase JMJ14 and histone deubiquitinase UBP26 are also required for

RNA-directed DNA methylation and/or RdDM-mediated transcriptional

silencing (Sridhar et al. 2007; Deleris et al. 2010; Searle et al. 2010; To et al.

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2011; Liu et al. 2012). Ultimately, RdDM generally creates a chromatin

environment refractive to gene expression.

RdDM functions in the maintenance of genome stability through suppression of

invading DNA, such as transposable elements or viruses; additionally, RdDM

participates in responses to biotic and abiotic stimuli, and is required for

genomic imprinting (recently reviewed in Zhang et al. 2018). Somewhat

surprisingly, however, despite these broad and central roles in plant biology, no

obvious immediate effect of RdDM deficiency on plant development has been

found to date in Arabidopsis.

We previously described that the chromatin remodeller PICKLE (PKL) is required to

maintain methylation patterns at RdDM target loci, unveiling a novel, unexpected

function of this protein in modulating the RdDM pathway (Yang et al. 2017). Our results

show that generation of non-coding RNAs by Pol IV and Pol V is affected in the

absence of PKL, which led us to propose that PKL promotes the generation of a

chromatin environment permissive for the activity of these RNA polymerases, and

hence conducive to transcriptional silencing, through its nucleosome remodeling

activity. In order to gain further insight into the role of PKL in the regulation of DNA

methylation and transcriptional gene silencing, we crossed the pkl mutant to other

mutants deficient in DNA methylation. We surprisingly find that plants mutated in both

PKL and RdDM genes exhibit severe developmental defects, which can be accounted

for by a strongly derepressed F-box gene SDC (SUPPRESSOR OF DRM1 DRM2

CMT3) in the double mutants. These results revealed a masked role of RdDM in

developmental regulation and suggest that PKL acts synergistically with RdDM to

repress gene transcription.

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With the aim of further exploring the role of PKL in the regulation of DNA methylation

and silencing, we crossed the pkl mutant (pkl-1) (Ogas et al. 1997) to RdDM-deficient

mutants lacking the largest subunit of Pol IV (nrpd1-3; hereafter referred to as nrpd1)

or Pol V (nrpe1-11; hereafter referred to as nrpe1). Interestingly, although the pkl

mutant exhibits several shoot phenotypes such as semi-dwarfism and reduced apical

dominance as reported (Ogas et al. 1997), the pkl nrpd1 and pkl nrpe1 double mutants

display additional developmental defects. In particular, the double mutants have

downward curled leaves (Figure 1A), a phenotype that was not observed in any pkl

mutant alleles. In addition, the double mutants displayed much smaller stature (Figure

1B), shorter siliques and reduced fertility (Figure 1C). We next crossed the pkl mutant

to mutants deficient in non-CG methylation, including cmt2 and cmt3. Both pkl cmt2

and pkl cmt3 exhibit similar developmental phenotypes as the pkl single mutant does

(Figure 1A–C). In order to test if the function of RdDM, instead of the action of Pol IV

or Pol V, is required for the developmental regulation in the pkl background, we further

crossed the pkl mutant to other RdDM-deficient mutants, including rdr2, dcl3, and drm1

drm2. We found that dysfunction in other RdDM components resulted in similar

developmental defects as those shown by pkl nrpd1 and pkl nrpe1 (Figure 1A–C). The

only exception was pkl dcl3, whose phenotype was weak and not conspicuous until

later developmental stages; this may be due to functional redundancy between the

four different DCL genes in Arabidopsis (Blevins et al. 2015; Zhai et al. 2015; Yang et

al. 2016; Ye et al. 2016).

We next performed transcriptome analyses on the double mutants pkl nrpd1 and pkl

nrpe1, and the corresponding single mutants, including pkl, nrpd1, and nrpe1.

Consistent with the extent of morphological defects, more differentially expressed

genes (DEGs) were identified in the pkl nrpd1 (N=1265) or pkl nrpe1 (N=1127) double

mutant than in any of the single mutants (Figure 2A). About 67% (752/1127) of the

DEGs identified in pkl nrpe1 were shared by pkl nrpd1 (Figure 2A). A large cluster of

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genes (cluster 2 in Figure 2B) that were derepressed in the double mutants but not in

any of the single mutants was identified. We reasoned that mis-expression of some of

these DEGs, common to both double mutants but not affected in the parental lines,

might be responsible for the observed developmental alterations in these plants.

The pkl nrpd1 and pkl nrpe1 specific developmental phenotype is reminiscent

of the one previously described for the non-CG methylation mutant drm1 drm2

cmt3 (ddc) (Cao and Jacobsen 2002; Chan et al. 2006). In the case of ddc,

altered development results from the activation of the SDC gene

(SUPPRESSOR OF drm1 drm2 cmt3) (Henderson and Jacobsen 2008), which

was shown to be necessary and sufficient for this phenotype. Strikingly, we

found SDC among the most highly up-regulated genes in the pkl nrpd1 and pkl

nrpe1 double mutants, indicating that RdDM deficiency in the pkl background

triggers a transcriptional release of this gene (Dataset S1). Quantitative RT-

PCR confirmed the results observed in the mRNA-seq experiment. The

expression of SDC is induced by at least 2100-fold in the double mutants but

less than 100 fold in any of the parental mutants (Figure 2C). In general, the

SDC expression level correlates with the severity of the developmental

abnormalities (Figures1, 2C, 2D). Derepression of SDC is observed in all

mutants, but morphological changes were clear only in mutants whose SDC

expression level is up-regulated by >1,000 fold (Figure 2D).

In order to determine whether increased expression of SDC underlies the

developmental phenotype of pkl nrpd1 and pkl nrpe1, we crossed these double

mutants to the sdc mutant (Henderson and Jacobsen 2008). As shown in Figure

1, mutation in the SDC locus is sufficient to revert morphological phenotypes of

the double mutants to that of the pkl single mutant, indicating that SDC is

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causative for the macroscopic alterations that arise in the absence of both

RdDM and PKL. The SDC promoter contains seven tandem repeats, which are

targeted by non-CG DNA methylation directed redundantly by histone

methylation and siRNA (Figure S1A); methylation at these repeats was shown

to spread beyond this region (Henderson and Jacobsen 2008). SDC silencing

has also been found to be released upon heat stress (Sanchez and Paszkowski

2014); in this case, however, only a minor decrease in CHH methylation (15%

to 10%), with no consistent change of methylation in the CG or CHG contexts,

was observed in the tandem repeats (Figure S1B). In the pkl nrpd1 and pkl

nrpe1 double mutants, changes in SDC expression do not correlate with

changes in methylation at the SDC promoter compared to nrpd1 or nrpe1

(Figure S1B). Similarly, no consistent differences in accumulation of siRNA

(Figure S1C) could be detected in this region. We further examined the level of

the repressive histone modification H3K9me2 and histone H3 at the SDC

promoter. No significant decreases in the H3K9me2 or H3 level in the pkl nrpd1

and pkl nrpe1 double mutants were observed (Figure S1D, E), indicating that

derepression of SDC in the double mutants was not due to decreases in the

level of H3K9me2 or nucleosome occupancy.

The depth and breadth of biological consequences of altering the tight genomic

control exerted by DNA methylation in general, and RdDM in particular, remains

to be explored. Here, we show that deficiency in RdDM can directly or indirectly

affect plant development, depending on the genetic context. More specifically,

the combination of mutations in the chromatin remodeller and RdDM regulator

PKL and the RdDM components results in developmental alterations, which

appear in an SDC-dependent manner.

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The molecular mechanism underlying the multi-layer epigenetic control of the

SDC gene remains to be elucidated. One possible hypothesis would be that the

synergistic effect of the pkl and nrpd1/nrpe1 mutations on the activation of SDC

relies on the promotion of a permissive chromatin state in the SDC promoter at

two different levels: the lower DNA methylation in the RdDM mutants, on one

hand, and changes in nucleosome conformation or composition that make the

promoter accessible to transcription factors, consequence of the absence of

PKL, on the other (Carter et al. 2018). Whether nucleosome organization is

indeed affected in this region remains to be determined.

It is interesting that SDC is activated in mutants affected in DNA methylation,

and the level of expression seems to correlate with the degree of methylation

deficiency. Initially, SDC was shown to be expressed in response to a lack of

non-CG methylation, in the ddc mutant; here, we report its activation in the

absence of RdDM and PKL activities. Additionally, SDC was described as up-

regulated after heat stress treatments (Sanchez and Paszkowski 2014). Taken

together, these data suggest that SDC is up-regulated in response to a release

of epigenetic silencing, raising the idea that this gene may act as a sensor of

“genomic danger”. Notably, expression of SDC had a positive effect on heat

tolerance (Sanchez and Paszkowski 2014); whether this gene promotes

resistance to other biotic or abiotic stresses remains to be determined.

Our results offer new insights into the developmental function of the RdDM

pathway, and indicate that SDC can be activated through different mechanisms

upon alteration of the genomic methylation status. The elucidation of how the

SDC gene is activated in the double mutants and what the physiological

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consequences of this transcriptional release are will require further

investigation.

ACKNOWLEDGEMENTS

This work was supported by the National Key R&D Program of China

(2016YFA0503200), National Natural Science Foundation of China

(31371312), National Key Laboratory of Plant Molecular Genetics, the Youth

Innovation Promotion Association of CAS (2014242 and Y201844), Strategic

Priority Research Program of CAS (XDB27040108), and Shanghai Municipal

Science and Technology Commission (18395801200) to H.Z.

AUTHOR CONTRIBUTIONS

H.Z. and J.K.Z designed the project; R.Y. and L.H. performed experiments;

R.Y., L.H., H.H. and H.Z. analysed data and prepared figures; R.Y., R.L.D. and

H.Z. wrote the paper. All authors read and approved the contents of this article.

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Figures

Figure 1. Simultaneous loss of RNA-directed DNA methylation and the

chromatin remodeller PICKLE alters plant development

(A) Phenotypes of four-week-old plants. One representative plant was shown

for each genotype; Col-0 is used as wild type (WT) control. (B) Phenotype of

long day-grown six-week-old plants. (C) Details of the floral stem of the same

plants from (B).

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Figure 2. Transcriptome analyses of the pkl nrpd1 and pkl nrpe1 double mutants and the parent lines

(A) Venn diagrams depicting the overlap in DEGs in single and double mutants.

(B) Heatmap showing the expression level of the combined list of differentially

expressed genes (DEGs) in single and double mutants. DEGs were identified

using WT as a control and the DEGs from all comparisons were combined to

produce the heatmap. Tiles were sorted using hierarchical clustering with

Euclidean distance as the distance measure. The scale indicates the relative

expression level of the same gene compared among different genotypes. (C)

Relative expression level of the SDC gene in pkl and non-CG methylation

mutants as measured by qRT-PCR. (D) Relative expression level of the SDC

gene in listed mutants compared to WT as measured by qRT-PCR. Values are

the mean of three biological replicates; error bars represent standard deviation.