Amplified sequences have been extended towards promoter making use of a chromosome walking kit (Genome Walker Common Kit, Clontech). The obtained sequences had been provided as supplemental substance (S1 File).International DNA methylation was quantified by large functionality capillary electrophoresis (HPCE) as formerly explained [41]. DigitoxinQuantification of the relative methylation of every single DNA sample was calculated as follows: %5mdC 5mdC Peak Area one hundred 5mdC dC Peak Areas the methylation standing of specific genomic DNA sequences was proven by bisulfite genomic sequencing. A complete of one.8 g of genomic DNA have been bisulfite transformed by making use of EpiTect Bisulfite package (Qiagen). Three various organic replicates for each developmental phase had been processed to minimize prospective artefacts and for making sure conversion reproducibility. As soon as DNA was transformed, concentrate on sequences were amplified utilizing particular primers. PCR item was cloned into pGEM-T simple (Promega). Eight colonies had been sequenced for each gene and sample to measure the methylation standing of each and every cytosine. Cytosine-wealthy regions have been detected using jEMBOSS 1.5 CpG plot utility (EMBOSS), and CNG and CNN web sites had been identified manually. Cytosine-abundant areas had been only detected in the PrCSDP2 gene (S1 Fig). Primer sequences and PCR situations for methylation evaluation are indicated in S1 Table.20-5 g of protein had been separated by electrophoresis in 15% acrylamide SDS gels and then transferred by electroblotting (350mA for two hours) to Immobilon membranes (Millipore). Membranes have been blocked with skimmed milk and histone marks ended up detected with anti-acetyl-Histone H4 (Upstate, ref. 0666), anti-trimethyl-Histone H3 (Lys4) (Upstate, ref. 0773), anti-trimethyl-Histone H3 (Lys9) (Upstate, ref. 0742) or anti-trimethyl-Histone H3 (Lys 27) (Upstate, Usa, ref. 0749) and polyclonal anti-delta-tubulin (Chemicon, ref. AB3203) the latter was employed as a control for protein loading [forty two]. Secondary antibody was coupled to alkaline phosphatase (Calbiochem, ref. 401312). Membranes were digitized and analysed using Fiji [forty three], characteristic membranes are supplied in S2 Fig. Relative abundance (RA) of every histone mark was calculated as follows: RA Histone mark band depth one hundred Tubulin band intensity samples have been crosslinked by incubating two g of fresh tissue for the duration of 16 min in vacuo with crosslinking answer (.four M sucrose, ten mM Tris-HCl pH eight., 1 mM EDTA pH eight., 2% formaldehyde). Reactions ended up quenched with 2 mL two.5M glycine soon after 14 min. Nuclei had been isolated according to Haring et al. [forty four] and chromatin was sonicated until finally fragments of an common size of .three.6 kb had been acquired. Five g of sonicated chromatin have been diluted to a last quantity of one ml in ChIP dilution buffer (one.one% Triton X-100, one.2 mM EDTA, 167 mM NaCl, sixteen.seven mM TrisHCl pH eight, 1mM PMSF). Chromatin was precleared by introducing twenty five L of Dynabeads (Invitrogen). Right after two hours of incubation at four Dynabeads have been taken out. For each take a look at, 5 L of antibody (see over for references) or ddH2O (NoAb), have been additional to pre-cleared answers and incubated for 10 h at 4C. Inmunocomplexes had been purified with twenty five uL of Dynabeads and washed 1st in minimal salt buffer (one hundred fifty mM NaCl, twenty mM Tris-HCl pH eight., 1% SDS, one% triton X100, 2 mM EDTA), then in higher salt buffer (500 mM NaCl, 20 mM Tris-HCl pH eight., .one% SDS, .5% triton X-100, 2 mM EDTA), LiCl buffer (250 mM LiCl, ten mM Tris-HCl pH eight., 1% triton X-one hundred, one% sodium deoxycholate, 1 mM EDTA) and ultimately in 1 mL of TE buffer (10 mM Tris-HCl pH eight., 1 mM EDTA). Immunocomplexes were eluted by introducing 250 L of ChIP elution buffer (1.% SDS, .one M NaHCO3), and crosslinking was reversed by incorporating ten L 5 M NaCl and incubating for four hrs at sixty five. Enter fraction was constituted by introducing 5 g of sonicated chromatin to one mL of ChIP elution buffer. DNAs ware purified and PCR amplification was done as described in S1 Desk.Knowledge (international DNA methylation and histone marks, gene expression knowledge) ended up subjected to analysis of variance (ANOVA) utilizing the R variation 2.nine.two software [forty five]. Normality and homoscedasticity had been evaluated by Shapiro–Wilk and Levene tests respectively. Several comparisons were done by important Tukey’s HSD check. A chance 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. [46] 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.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.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).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).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 developmental stages.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). 25733882The 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.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.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 application of AnAc (inhibits the binding of the HAT activators p300 and p300/CBP-associated factor, resulting in a decrease of acetylated histones [47]) or SAHA (chelates Zn- ions at active centre of HDACs, resulting in the accumulation of acetylated histones [48]) 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 didn’t 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.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.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 [49] 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 [12]. 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. [5] 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.
