Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata
Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata
Luis Valledor 0 1
Jess Pascual 0 1
Mnica Meijn 0 1
Mnica Escandn 0 1
Mara Jess Caal 0 1
0 1 Plant Physiology, Faculty of Biology, University of Oviedo , Cat. Rodrigo Uria s/n, E-33071, Oviedo , Spain , 2 Department of Biology and CESAM, University of Aveiro, Campus Universitario de Santiago , P-3810-193, Aveiro , Portugal , 3 Regional Institute for Research and Agro-Food Development (SERIDA) , Finca Experimental La Mata s/n, E-33825, Grado , Spain
1 Academic Editor: Hector Candela, Universidad Miguel Hernandez de Elche , SPAIN
Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species.
Funding: This work was supported by the Ministry of
Science and Innovation of the Spanish Government,
(Projects AGL2007-62907 and AGL2011-27904). LV
and MM were funded by Juan de la Cierva
Programme, (Spanish Ministry of Economy and
Competitiveness, ref. JCI-2012-12444 and
JCI-201108958, respectively). ME and JPs fellowships were
respectively supported by Severo Ochoa, (Gobierno
del Principado de Asturias, Spain, ref. BP11117) and
Leaf development is a complex process that involves many cell and tissue differentiation and
remodelling processes . During this period, two different stages can be observed: the
Competing Interests: The authors have declared
that no competing interests exist.
proliferative stage, which covers the initial period of development and is characterised by low
cell differentiation and a high rate of cell division that will define the organ layers and shape,
and the expansion stage characterised by cell expansion and differentiation and ending with
the gain of physiological competence and maturity . Despite the gene interaction
networks and regulatory cascades which lead to leaf primordium initiation and organ shape
formation are mostly defined , the mechanisms for activating the biochemical pathways
that define organ functions are still poorly understood.
In Pinus radiata, needles reach maturity after 12 months of development . During this
period primary metabolism is directed towards photosynthesis and other biosynthetic
pathways, that will support normal tree growth and function while, at the same time,
growth-related activities such as increased protein and structural biosynthesis, cell division and
morphogenesis become down-regulated or completely silenced .
It is clear that organ induction, development, and maturation are the consequence of highly
specific regulatory mechanisms that control gene expression and protein modification and
degradation, required for successful organ development [4,6,10,13]. Among these mechanisms,
epigenetic regulation is one of the most determinant regulatory pathways . DNA methylation
and histone post-translational modifications (PTMs) have been revealed as key mechanisms for
controlling chromatin structure and function  and regulating cell growth and differentiation
. These mechanisms are dynamic and can consequently be reverted or adapted to
particular environmental situations, constituting a link between genotype and phenotype .
Despite the availability of genome-wide mapping of DNA methylation and histone PTMs in
Arabidopsis thaliana (Arabidopsis), Oryza sativa (Rice), and Populus trichocarpa (Poplar)
[22,2426] and the common consensus of the permissive (AcH4, H3K4me3) and repressive
(H3K9me3 and H3K27me3) effects of histone PTMs at the transcriptional level [17,27],
indepth studies of the gene-specific epigenetic mechanisms involved in the control of plant organ
differentiation and maturation are still limited. Focusing on leaves, previous studies have
shown that some leaf morphogenesis-related genes such as FLOWERING LOCUS C (FLC),
KNOTTED1-LIKE HOMEOBOX (KNOX) family and SHOOT MERISTEMLESS (STM)
[6,28,29] or the carbon-concentrating mechanism-related genes phosphoenolpyruvate
carboxylase (PEPC) and MALIC ENZYME (ME)  are epigenetically regulated. The transition from
proliferative growth to the expansion and differentiation stages increases global DNA
methylation and causes changes in histone PTMs in Arabidopsis ; however, the specific regulation
of enzymes related to primary and secondary metabolism at any developmental stage or
environmental situation is still poorly studied and has only been addressed in a small number of
high-throughput analyses. Charron et al.  analysed the landscape of H3K9ac, H3K9me3,
H3K27ac, and H3K27me3 during the de-etiolation process in Arabidopsis, showing a novel
insight into the epigenetic regulation of a specific physiological process. Recently, Lafos et al. 
provided an overview of H3K27me3 during Arabidopsis de-differentiation, pointing to the
importance of this repressive mark via a comparison of stem and leaf differentiated cells.
A deeper knowledge of the epigenetic regulation of key pathways involved in maturation and
plant survival, such as carbon fixation and stress responses, and its potential reversion, has a
direct biotechnological application in clonal breeding, since ageing and maturation is the most
important barrier in clonal forestry programmes . To date, efforts towards the characterisation
of the epigenetic role of tree cell plasticity and in growth and development processes have been
focused on global epigenetic dynamics for defining phase change, maturation stage, bud set and
burst, and production hallmarks (see Brutigam et al.  for a review), with no record to our
knowledge, of any study dealing with gene-specific epigenetic dynamics during leaf maturation.
To fill this gap we have studied four genes that had demonstrated a differential
accumulation of its products during needle development  and have a major function in needle
development and plant growth covering photosynthesis (RUBISCO ACTIVASE, PrRBCA;
RUBISCO SMALL SUBUNIT, PrRBCS), one carbon metabolism (SERINE
HYDROXYMETHYL TRANSFERASE 4, PrSHMT4) , and developmental control and stress response
(COLD SHOCK DOMAIN PROTEIN 2, PrCSDP2) . We cloned these genes and
investigated their transcription levels in three needle developmental stages and a de-differentiated tissue
(white, non photosynthetic, calli derived from needles which is showing active growth) used as
control. Moreover, we described the epigenetic regulation mechanisms acting on the promoter
and first exon sequences either by cytosine methylation, and/or by specific histone PTMs
AcH4, H3K4me3, H3K9me3 and H3K27me3. The employ of inhibitors of histone deacetylases
(HDACs) and histone acetyltransferases (HAT) provided a deeper understanding of the role of
AcH4. These results provide new insights over how the epigenetic mechanisms regulate key
metabolic pathways during needle maturation.
Materials and Methods
Plant material and treatments
Needles that were either fully developed and mature (12-month-old; B12), those that were
developed but in transition between cell proliferation and expansion (5-month-old; B5), or
immature (three- to five-week-old; B1) (Fig 1a1c), were collected from three different adult
Pinus radiata trees grown in a test-garden of the University of Oviedo during the active growth
season (spring), washed with tap water, dried with filter paper, excised from the tree and frozen
in situ in liquid nitrogen. Samples were stored at -80C prior to DNA or RNA extraction.
Pinus radiata callus (Fig 1d) was employed as a de-differentiated control. Calli were induced
from explants taken from the same trees from which needles were sampled. After surface
sterilisation of the explants, callus was induced in EDM6 medium  supplemented with 9 M
2,4-Diclorophenoxyacetic acid and 2.66 M 6-Benzylaminopurine and grown in the dark at
25C. Once initiated, calli were subcultured every 15 days in the same medium and conditions.
One-year-old seedlings of Pinus radiata grown in greenhouse were treated in independent
blocks of 3 individuals with Suberoylanilide Hydroxamic Acid (SAHA) (SML0061-5MG,
Sigma), an inhibitor of HDACs ; Anacardic Acid (AnAc) (A7236-5MG, Sigma), an
inhibitor of HATs ; and a mock control (9 plants were considered for this assay). Drugs were
dissolved in DMSO and then diluted in ddH2O to 200 M. We performed previous dosage trials
and this concentration was previously proved to be efficient for slight modifications of the
epigenome without altering cell viability in cell cultures (data not shown). Drugs were applied in
solution to the media for the calli and by nebulization over the needles for the seedlings.
Seedlings were treated twice, with a gap of 48 h between re-treatment. Plant material was sampled
after 48 h of the last treatment, flash-frozen, and kept at -80C until use.
Fig 1. Different developmental stages analyzed in this work: B12 mature needles (a), B5 developed needles (b), B1 immature needles (c) and calli
(d) were used as a control of de-differentiated tissue with high growth capacity. Horizontal bar represents 1 cm length.
Combined isolation of DNA, RNA, and Proteins
The different biomolecules were extracted from the same samples employing a sequential
fractionation as described in Valledor et al. . In brief, samples were ground to powder in liquid
nitrogen and washed with chloroform:methanol:water (1:2.5:0.5). After centrifugation,
supernatants were discarded, and resulting pellets were resuspended in 7M Guanidine HCl, 2% (v/v)
TWEEN 20, 4% (v/v) NP-40, 50 mM Tris, pH 7.5, 1% (v/v) -mercaptoethanol. DNA was
purified from the mixture by filtering the solution through a silica mini-spin column (EconoSpin,
Epoch Life Science). 300 L of acetonitrile were then added to the flow-through containing
RNA and proteins, and RNA was isolated from the solution by filtering through a new mini
spin column. Spin columns were washed with 600 L of 2 mM Tris pH 7.5, 20 mM NaCl, 0.1
mM EDTA, 90% ethanol, and then 600 L of 2 mM Tris pH 7.5, 20 mM NaCl, 0.1 mM EDTA,
70% ethanol. Nucleic acids were eluted from the columns by adding 50 L of ddH2O. Nucleic
acids were quantified by spectrophotometry and its integrity was checked in denaturing
agarose gels. RNA samples were treated with DNase I (Fermentas) and its possible contamination
with DNA was checked by PCR.
Proteins were recovered by mixing the flow-through with 2 volumes of phenol:water (1:1).
Samples were then centrifuged at 10000 x g, and organic phase was recovered. Proteins were
precipitated by adding 2 volumes of 0.1 M ammonium acetate in methanol. After an overnight
incubation at -20C, tubes were centrifuged, and protein pellets were washed twice with
acetone. Dry pellets were dissolved in 4% SDS, 8 M Urea and quantified using BCA assay.
RT-qPCR expression analyses
One g of total DNA-free RNA and Superscript III first-strand cDNA synthesis kit
(Invitrogen) were used for first strand cDNA synthesis previous to quantitative PCR. Quantitative
PCR was performed as followed: 10 ng cDNA, 5pM of each primer and Perfecta SYBR Green
(Quanta) were mixed and amplified in ABI 7900HT system. Three measurements for each
transcript and sample were analyzed using Prism software (Applied Biosystems). Relative
quantifications were performed for all targets employing the expression levels of pine ACTIN
(AY172979.1), 18S (M82462.1) and GAPDH (KM496531.1) genes as loading controls. Primer
sequences are listed in S1 Table and were designed according to described GenBank sequences.
Amplification of 5 cDNA ends of candidate genes
The available sequences for the candidate genes PrRBCA (dbEST GO096305), PrRBCS (dbEST
TC92228), PrSHM4 (dbEST GO270978), and PrCSDP2 (dbEST AA216460) were amplified
towards 5 employing RML-RACE kit (Ambion) following the instructions provided by the
manufacturer. Amplified sequences were extended towards promoter using a chromosome walking
kit (Genome Walker Universal Kit, Clontech). The obtained sequences were included as
supplemental material (S1 File).
Analysis of global DNA methylation
Global DNA methylation was quantified by high performance capillary electrophoresis
(HPCE) as previously described . Quantification of the relative methylation of each DNA
sample was calculated as follows:
Analysis of sequence-specific DNA methylation
The methylation status of specific genomic DNA sequences was established by bisulfite
genomic sequencing. A total of 1.8 g of genomic DNA were bisulfite converted by using EpiTect
Bisulfite kit (Qiagen). Three different biological replicates per developmental stage were
processed to minimize potential artefacts and for ensuring conversion reproducibility. Once DNA
was converted, target sequences were amplified employing specific primers. PCR product was
cloned into pGEM-T easy (Promega). Eight colonies were sequenced for each gene and sample
to measure the methylation status of every cytosine. Cytosine-rich regions were detected
employing jEMBOSS 1.5 CpG plot utility (EMBOSS), and CNG and CNN sites were determined
manually. Cytosine-rich regions were only detected in the PrCSDP2 gene (S1 Fig). Primer
sequences and PCR conditions for methylation analysis are indicated in S1 Table.
Twenty-five g of protein were separated by electrophoresis in 15% acrylamide SDS gels and
then transferred by electroblotting (350mA for two hours) to Immobilon membranes
(Millipore). Membranes were blocked with skimmed milk and histone marks were detected with
anti-acetyl-Histone H4 (Upstate, ref. 06866), anti-trimethyl-Histone H3 (Lys4) (Upstate, ref.
07473), anti-trimethyl-Histone H3 (Lys9) (Upstate, ref. 07442) or anti-trimethyl-Histone
H3 (Lys 27) (Upstate, USA, ref. 07449) and polyclonal anti-delta-tubulin (Chemicon, ref.
AB3203); the latter was used as a control for protein loading . Secondary antibody was
coupled to alkaline phosphatase (Calbiochem, ref. 401312). Membranes were digitized and
analysed employing Fiji , characteristic membranes are provided in S2 Fig. Relative abundance
(RA) of each histone mark was calculated as follows:
Chromatin immunoprecipitation (ChIP)
Samples were crosslinked by incubating 2 g of fresh tissue during 16 min in vacuo with
crosslinking solution (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 2%
formaldehyde). Reactions were quenched with 2 mL 2.5M glycine after 14 min. Nuclei were isolated
according to Haring et al.  and chromatin was sonicated until fragments of an average length
of 0.30.6 kb were obtained. Five g of sonicated chromatin were diluted to a final volume of 1
ml in ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM
TrisHCl pH 8, 1mM PMSF). Chromatin was precleared by adding 25 L of Dynabeads
(Invitrogen). After 2 hours of incubation at 4C Dynabeads were removed. For each test, 5 L of
antibody (see above for references) or ddH2O (NoAb), were added to pre-cleared solutions and
incubated for 10 h at 4C. Inmunocomplexes were purified with 25 uL of Dynabeads and
washed first in low salt buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1% SDS, 1% triton
X100, 2 mM EDTA), then in high salt buffer (500 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.1%
SDS, 0.5% triton X-100, 2 mM EDTA), LiCl buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1%
triton X-100, 1% sodium deoxycholate, 1 mM EDTA) and finally in 1 mL of TE buffer (10 mM
Tris-HCl pH 8.0, 1 mM EDTA). Immunocomplexes were eluted by adding 250 L of ChIP
elution buffer (1.0% SDS, 0.1 M NaHCO3), and crosslinking was reversed by adding 10 L 5 M
NaCl and incubating for 4 hours at 65C. Input fraction was constituted by adding 5 g of
sonicated chromatin to 1 mL of ChIP elution buffer. DNAs ware purified and PCR amplification
was performed as described in S1 Table.
Data (global DNA methylation and histone marks, gene expression data) were subjected to
analysis of variance (ANOVA) using the R version 2.9.2 software . Normality and
homoscedasticity were evaluated by ShapiroWilk and Levene tests respectively. Multiple
comparisons were performed by significant Tukeys HSD test. A probability level of p< 0.05 was
considered significant for all statistical analyses. Five biological replicates were analysed.
Quantitative PCR data was processed according to Hellemans et al.  and R packages EasyqPCR
and SLqPCR before applying ANOVA.
To assess how epigenetic mechanisms might regulate needle development and maturation in
Pinus radiata, we sampled needles at different developmental stages (B1 first stages of division
from needle primordia, B5 transition between proliferative and expansive stage, B12 mature
needles) and generated de-differentiated callus tissue to serve as a non-photosynthetic
pluripotent control (See methods; Fig 1). We selected two genes closely related to leaf maturation;
PrRBCA, PrRBCS, and two genes related to immature tissues PrSHMT4 and PrCSDP2, for
which partial sequences were available. These genes showed different expression levels in the
different tissues analyzed (Fig 2): PrRBCA and PrRBCS increased expression levels with leaf
maturation, being this transcript not detected in callus and showing a low expression level in
B1 needles. As expected, in a developing tissue acquiring photosynthetic ability (B5), the
PrRBCA expression level was higher than that of PrRBCS. Alternatively, PrCSDP2 was highly
expressed in callus tissue. B1 showed the highest expression level of PrCSDP2 between the
different needles and PrSHMT4 was only expressed in calli.
Needle developmental processes are associated with changes in global
Tissue differentiation was associated with an increased level of global DNA methylation (Fig
3a). Calli showed a low degree of DNA methylation, of 10.8%, while in B1 and B5 needles it
progressively increased to 15.7% and 16.4% respectively. B1 needles were rapidly developing
and were characterised by a large increase in cell number, whereas B5 needles had fewer cell
divisions and showed an increase in differentiation. The mature B12 needles showed the highest
degree of DNA methylation at 17.7%.
The analysed histone PTMs showed different abundances, and were tissue- and
modification-specific. AcH4 showed a 2.5-fold increase in B5 needles compared to B1 needles and the
lowest levels were in the differentiated tissues of B12 needles; surprisingly, calli had the lowest
abundance of this mark, whereas the other permissive mark, H3K4me3, showed different
dynamics. This result demonstrates the complexity of epigenetic regulation mechanisms. The
abundance of H3K4me3 was highest in B1 needles and was negatively correlated with needle
differentiation; calli showed the lowest levels of this mark (Fig 3b). H3K27me3 showed no
significant differences between developmental stages, but H3K9me3 abundance progressively
increased, with a maximum in B12 needles.
After 5 de novo sequencing of target genes, only cytosine rich region in the first exon of
CSDP2 gene was identified (S1 Fig). In consequence only CSDP2 was further analysed for
changes in specific DNA methylation whereas all genes were subjected to ChIP-PCR.
Fig 2. Analysis of the Relative Quantity (RQ) of PrRBCA, PrRBCS, PrSHMT4, and PrCSDP2 expression levels in different tissues obtained by
RTqPCR. Values were normalized and expressed as fold differences compared to B1 needles. Error bars show the propagated standard error of RQ, SE(RQ).
Different letters between bars corresponding to each gene indicate significant expression differences between developmental stages (ANOVA followed by a
Tukey HSD test; p < 0.05).
Fig 3. Analysis of global DNA methylation in the different needle developmental stages. (a). Relative abundance (R.A.) of the permissive, AcH4 and
H3K4me3 (b), and repressive, H3K27me3 and H3K9me3 (c) histone marks in the indicated developmental stages. Different letters between bars
corresponding to each histone PTM indicate significant expression differences between developmental stages (ANOVA followed by a Tukey HSD test;
p < 0.05).
PrCSDP2 was regulated epigenetically by specific DNA methylation and
specific patterns of histone modifications in the first exon region but not
in the promoter
We observed differential patterns of DNA methylation after bisulfite sequencing the previously
defined cytosine rich region of PrCSDP2 (Fig 4a). Calli showed the greatest number of
methylated cytosines, with five heavily methylated residues. B1 needles showed a lower overall
number of methylated cytosines but the number of methylation loci increased to 11, indicating de
novo methylation events and targeted demethylation compared to calli. B5 showed a transitory
landscape, whereas B12 needles showed the highest number of methylated loci, with seven
from 19 being heavily methylated.
ChIP studies revealed an enrichment of AcH4 in the first exon of the PrCSPD2 gene (Fig 4b)
in calli and B1 that was not detected in B5 and B12 needles. The enrichment of the H3K4me3
fraction was also observed in less differentiated tissues and the enrichment of H3K9me3 was
positively correlated with needle development. These results correlate with the expression levels
described for this gene, which decrease with an increase in tissue differentiation, and are
supported by the fact that the application of HDAC or HAT inhibitors altered the expression
pattern of this gene (see below). The promoter region did not show significant epigenetic changes
between the different developmental stages (data not shown).
PrSHMT4 transcription was only detected in calli and was repressed in needles (Fig 2). As is
described for the other genes, the promoter and the first exon regions behaved differentially.
Focusing on the promoter, calli are characterised by a strong enrichment of the H3K4me3
marker and also by the presence of AcH4 above the control threshold (Fig 5). An absence of
the AcH4 mark, and the enrichment of the H3K9me3 fraction, correlates with the silencing of
PrSHMT4 in B1 needles. B5 and B12 needles only exhibited repressive marks in the promoter.
The first exon showed a similar behaviour to that of the promoter, with a predominance of
H3K4me3. However, we were unable to detect strong repressive marks in this region at all
PrRBCA and PrRBCS are regulated by histone post-translational
modifications at the promoter level
The interplay between AcH4 and H3K27me3 appears to play an important role in the
regulation of expression of the studied photosynthetic genes. In calli, a non-green heterotrophic tissue
in which PrRBCA and PrRBCS are repressed, strong epigenetic silencing marks were found in
the promoters (Fig 6). In the case of PrRBCS, the presence of AcH4 in green autotrophic tissues
and also one repressive mark like H3K27me3, which is lost in mature needles, appeared to
explain the progressive increase in the expression levels of this gene throughout development, and
its silencing in calli (H3K4me3 and H3K9me3 fractions did not show significant differences
between developmental stages). The PrRBCA promoter showed an enrichment of the AcH4
histone fraction in all studied material; however, an enrichment of the repressive marks H3K9me3
and H3K27me3 was only found in calli, demonstrating the importance of these marks for
defining green photosynthetic and non-photosynthetic tissues. However, we did not observe any
difference between histone marks within the first exon at different needle developmental stages,
which suggests that the epigenetic regulation of these genes occurs via promoter region.
Fig 4. (a) Bisulfite methylation analysis of PrCSDP2 in callus, growing needle and mature needles. Approximately 250 bp of the first exon were sequenced.
Cytosines in grey were present in the indicated region, but not analyzed (b) Effects of the needle developmental stage over the ChIP-based enrichment of the
first exon of PrCSDP2 using the indicated anti histone-PTMs antibodies. Image corresponds to a representative gel electrophoresis using primers for
amplifying a region corresponding to the promoter of PrCSDP2 gene.
Fig 5. Effects of the needle developmental changes upon the ChIP-based enrichment of chromatin fractions corresponding to the promoter or first
exon of PrSHMT4 using the indicated anti histone PTMs antibodies.
The exogenous application of exogenous HATs and HDACs inhibitors
altered gene expression levels of target genes in calli and seedlings
The application of AnAc (inhibits the binding of the HAT activators p300 and
p300/CBP-associated factor, resulting in a decrease of acetylated histones ) or SAHA (chelates Zn- ions at
active centre of HDACs, resulting in the accumulation of acetylated histones ) at low
concentration resulted in an altered gene expression in calli (S3A Fig). Both AnAc and SAHA
increased the expression of PrCSDP2. On contrary, AnAc reduced PrRBCA expression, while
SAHA did not have any effect. Acetylation drugs didnt alter the expression of PrRBCS and
PrSHTM so we hypothesize a possible regulation by specific histone methylation marks. In
needles (S3B Fig) the treatment with SAHA increased the abundance of PrRBCS but reduced
the expression levels of PrRBCA and PrSHMT4 probably because SAHA has pleiotropic effects
over other regulatory network linked to these genes. On the other hand the low permeability of
needles to AnAc probably reduced the effects of this compound as it is shown in S3B Fig.
Fig 6. Effects of the needle developmental changes upon the ChIP-based enrichment corresponding
to chromatin fractions of the promoters of PrRBCS and PrRBCA genes using the indicated anti
histone PTMs antibodies.
The expression levels of PrRBCA, PrRBCS, PrCSDP2, and PrSHMT4
can be used to distinguish between needle maturation stages
Leaf development is a complex process in which different systems act co-ordinately to acquire
full physiological competence. In Arabidopsis, these processes are divided into two different
stages: proliferative, characterised by a large number of cell divisions, and expansion, in which
cells expand and reach physiological maturity [3,5]. Each stage is further characterised by the
activation or repression of different molecular mechanisms involved in the different process.
The selection of candidate genes to define these developmental stages in Pinus was partly
limited by the few studies dealing with the molecular aspects of needle maturation  and also by
the almost complete absence of full-length Pinus radiata sequences within public databases.
To firstly describe the different developmental stages and then to analyse the gene-specific
regulation of needle maturation in P. radiata, we selected four genes which expression is
characteristic of mature (PrRBCA, PrRBCS) and immature tissues (PrCSDP2, PrSHMT4), which
are differentially expressed between growing and mature needles . The study of PrRBCA
and PrRBCS expression as a marker of photosynthetic capacity allowed the differentiation of
the different stages of needle maturation. Interestingly, the expression level of these genes in B5
needles, in a transition between proliferative and expansive stages, was significantly higher
than in B1 (+6.3-fold) and heterotrophic calli, and 7-fold lower than B12. Andriankaja et al. 
established the importance of chloroplast differentiation as a regulator of the simultaneous
onset of cell expansion and photosynthesis in Arabidopsis. In contrast to photosynthetic genes,
the maximal expression of PrCSDP2 and PrSHMT4 was found in calli as these genes are
reporters of undifferentiated tissues in Arabidopsis being also related to stress protection [35,36,50].
These genes were highly expressed in calli and were drastically reduced in expression in
differentiated tissues. These results underline the suitability of this set of genes for studying the
epigenetic processes that lead to specific gene regulation during needle development.
Needle maturation correlates with changes in global DNA methylation,
AcH4, H3K4me3 and H3K9me3 levels
The evolution of the global DNA methylation level during maturation and ageing of specific
tissues, organs, and species has been previously reported [22,5153]. In Pinus, different DNA
methylation levels are associated with ageing and phase change , organ maturation ,
and stress response . In contrast to these studies, which focused almost exclusively on
DNA methylation and in the comparison of extreme situations, we analysed the progression of
DNA methylation and four histone marks along a developmental gradient allowing the specific
temporal dynamics to be described.
The degree of DNA methylation increased together with organ maturation, with the lowest
level found in calli (de-differentiated tissue) and the highest in B12 needles. Surprisingly B1
and B5 needles showed the same methylation level, probably reflecting that in B5 needles late
proliferative and early expansive stages are occurring at the same time requiring a large number
of genes to be expressed. The plasticity of DNA methylation allows the quick change in the
regulation of specific genes while maintaining the same global level, by interacting with different
epigenetic mechanisms [57,58] and does not regulate all genes [25,52,59], with different
transcriptional intensities at specific loci having the same global DNA methylation level.
Based on current knowledge [17,27,58,60,61] we selected four histone PTMs markers of
gene activation (AcH4, H3K4me3) and repression (H3K9me3, H3K27me3). Less-differentiated
tissue (B1, B5) showed a higher ratio of permissive/repressive marks compared to that of
mature needles (B12) . Similar to DNA methylation, B5 needles showed an unexpected
abundance of AcH4 and H3K4me3 marks, maybe for allowing the transition between
proliferative to expansive stages, in a similar way to bud reprogramming during flowering . In
contrast, fully mature B12 needles showed the highest abundance of the repressive mark
H3K9me3. Finally, calli presented the lowest abundance of all histone marks, a striking
phenomenon also described in Arabidopsis  and might counterbalance the low DNA
methylation level. These data highlight the complexity of epigenetic networks that are extremely
dynamic, with the relevance of each mechanism depending on the development stage and
Notably, the abundance of H3K27me3, considered one of the stronger marks for gene
repression [26,60] and recently reported to be a critical mechanism for callus de-differentiation in
Arabidopsis , was not significantly altered during needle ontology. Since H3K27me3 was shown
to be a key regulatory mark for the regulation of photosynthesis genes we hypothesize that this
mark is regulating different loci during development. Although global epigenetic levels are
considered to be good biomarkers for key developmental processes [53,54] deeper epigenetic studies
focusing on specific loci are necessary to fully understand development, stress response or
environmental adaptions on forest species as a basis for improving forest productivity.
The epigenetic marks H3K4me3 and H3K9me3 can be associated to the
transcriptional activity of PrSHMT4 and, together with specific DNA
methylation patterns, that of PrCSDP2
The PrCSDP2 and PrSHMT4 genes constitute a good model for understanding the epigenetic
changes associated with gene-silencing during needle development, since their expression is
characteristic of calli and was progressively down-regulated during differentiation. The
sitespecific analysis of PrCSDP2 observed that different developmental stages showed different
DNA methylation and histone signatures. This fact suggests that the chromatin topology of
this locus changed during development, probably contributing to the different expression levels
of PrCSDP2. Changes in chromatin topology associated with gene expression levels have been
extensively studied in animal systems [27,64].
The high reproducibility of bisulfite sequencing of first exon region of PrCSDP2 in samples
from calli or B1 needles compared to other tissues could be related to the fact that these are less
differentiated, with a small number of cell lines forming the different tissues. In spite of this, a
clear increase in the proportion of methylated cytosines was observed from calli to mature
needles. The promoter of this gene did not show any significant changes of cytosine methylation
or histone modifications, possibly because it is a clear region surrounded by epigenetically
regulated sequences. The epigenetic behaviour of this gene was similar to that reported for the
CSDP2 gene of Poplar (S4 Fig), in which the promoter had strong epigenetic signals more than
1 kb upstream and its first exon was more methylated in leaves than in buds .
Unfortunately, these high-throughput analyses do not achieve a single base resolution, which precludes
further comparative analysis of the methylation of specific cytosines.
The different patterns of histone PTMs at the first exon of PrCSDP2 also highlighted the
importance of the epigenetic regulation at this locus. In Arabidopsis, Charron et al.  provided a
dataset in which H3K9me3 and H3K27me3 are associated with AtCSDP2 (AT4G38680) in
expanded leaves, however, the dynamics of histone PTMs surrounding AtCSDP2 during leaf
development have never been specifically studied. In this work, we found that the balance between
AcH4, H3K4me3 and H3K9me3 is correlated to the expression level of this gene. Despite that,
in callus, which showed the highest expression, there was a strong enrichment of H3K4me3 and
AcH4, while B12 needles were characterized by a strong H3K9me3 signal. It seems that, in
differentiated tissues, AcH4 is not playing a key role in the regulation of this gene, since the
application of acetylant drugs (SAHA) did not increase gene expression. On contrary, in calli, both
drugs induced gene expression maybe because the direct activation of this gene by SAHA and
the downregulation of a hypothetical repressor by AnAc. This results points that there should be
another mark, not present in needles, which allows effective gene expression in calli.
The enrichment of the H3K4me3 further indicates that this gene could be expressed widely
, being in concordance to its cold protective function and its role during flowering and
fruit development . H3K27me3 was also associated with this locus in mature needles. This
mark could not be detected in Arabidopsis using high throughput approaches, showing the
importance of studying the complete epigenetic context to provide a reliable epigenetic analysis.
The promoter of PrSHMT4 does not possess cytosine-rich regions and it is not methylated
in Poplar (POPTR_0017s08600) or Arabidopsis (AT4G13930), suggesting that its epigenetic
regulation is based only in histone modifications. The expression of this gene was correlated
to high levels of H3K4me3 and the presence of AcH4 in calli. Needles were characterized by a
very low expression of this gene and the presence of H3K9me3 in its promoter, supporting
the view of this modification as a strong repressive mark that is related to differentiation and
maturity . The unexpected presence of the repressive mark H3K27me3 in calli,
underlines the complexity of the epigenetic network and suggests the need for site-specific studies
to complement the available high-throughput models to provide an accurate analysis of
Photosynthetic carbon fixation regulation is associated to a crosstalk
between histone H4 acetylation and H3K9me3 at the promoter level
ChIP analyses revealed that the epigenetic regulation of PrRBCA and PrRBCS occurs via their
promoters and not via their first exons, and could be mediated by interplay between AcH4 and
H3K27me3. Despite the absence of specific studies of epigenetic regulation of photosynthesis
and its chemical unmasking, these results are in agreement with the epigenetic changes
observed during de-etiolation of Arabidopsis leaves  in which histone acetylation played an
essential role for the activation of photosynthetic machinery. The reversibility of epigenetics
could be closely related to the capacity for rapid adaptation or a return to the previous
functional status of photosynthetic activities.
Furthermore, despite the maintenance of global H3K27me3 during development, this was a
crucial mark for the repression of PrRBCA and PrRBCS in callus and during maturation in the
case of RBCS. Genome-wide analysis of H3K27me3 in fully expanded leaves of Arabidopsis
 supports our observations, since this modification was not found in AtRBCA
(AT2G39730) and in AtRBCS (AT1G67090, AT5G38430, AT5G38420, AT5G38410). The
developmental and tissue-specific transcriptional regulation of these genes justifies the absence of
differential H3K4me3 patterns for genes with a low tissue specificity, proposed by .
As the promoters of PrRBCA and PrRBCS showed a low density of cytosine residues and
consequently we suggested that DNA methylation does not have a direct regulatory function
for these genes. This hypothesis is reinforced by the analysis of the Poplar  and Arabidopsis
 leaf methylomes. In conclusion, we propose that histone acetylation and H3K27me3
specifically regulate the loci RBCA and RBCS in P. radiata and the promoter region is more
responsive to epigenetic regulation than the first exon.
The similarities in the specific epigenetic regulation of RBCA and RBCS in different plant
species suggest that this system has been conserved throughout evolution. However, further
research is needed to increase our knowledge concerning the molecular biology of these
mechanisms and their functional, ecological, and evolutionary significance.
S1 Fig. Analysis of cytosine rich regions of the indicated genes. Observed vs Expected plot
shows the ratio based on the frequency of C's and G's in that window. Percentage plot
represents the rate of Cs and Gs within the studied sequence; putative islands plot indicates the
region where cytosine rich region is predicted.
S2 Fig. Representative blots showing the identification and quantification of (a) Acetylated
Histone H4, (b) H3K27me3, (c) H3K9me3, and (d) H3K4me3 by immunobloting on
protein extracts from calli (C), mature (B12), growing (B5), and immature (B1) needles. Band
intensities were normalized against Tubulin (Tub).
S3 Fig. Analysis of the Relative Quantity (RQ) of PrCSDP2, PrRBCA, PrRBCS, and
PrSHMT4 expression levels in (a) P. radiata calli and (b) needles after co cultivation with
AnAc and SAHA. Expression levels were normalized to values in controls (untreated calli
or needles). Different letters indicate significant differences between developmental stages
(Tukey HSD test; p < 0.05).
S4 Fig. Differential DNA methylation found in the promoter of the locus
POPTR_0009s13460, ortholog of PrCSDP2, when comparing leaf and apical buds. Poplar
methylome  was visualized using gbrowse available at the Oregon State University (http://
scaffold_9:10697662..10702661). The specific locus corresponding to POPTR_0009s13460 was
defined after BLASTing the PrCSDP2 partial sequence to Populus trichocarpa genome v3
available at Phytozome (http://www.phytozome.net).
The authors wish to thank Dr. Mario F. Fraga and Dr. Agustn Fernandez for their helpful
comments. This work was supported by the Ministry of Science and Innovation of the Spanish
Government (AGL2011-27904). LV and MM were funded by Juan de la Cierva Programme
(Spanish Ministry of Economy and Competitiveness, ref. JCI-2012-12444 and JCI-2011-08958,
respectively). ME and JP fellowships were respectively supported by Severo Ochoa (Gobierno
del Principado de Asturias, Spain, ref. BP11117) and FPU (Ministry of Education, Spain, ref.
Conceived and designed the experiments: LV MM MJC. Performed the experiments: LV JP
ME MM. Analyzed the data: LV MM MJC. Wrote the paper: LV MM MJC.
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