Systematic analysis of DEMETER-like DNA glycosylase genes shows lineage-specific Smi-miR7972 involved in SmDML1 regulation in Salvia miltiorrhiza

Abstract

DEMETER-like DNA glycosylases (DMLs) initiate the base excision repair-dependent DNA demethylation to regulate a wide range of biological processes in plants. Six putative SmDML genes, termed SmDML1-SmDML6, were identified from the genome of S. miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies. Integrated analysis of gene structures, sequence features, conserved domains and motifs, phylogenetic analysis and differential expression showed the conservation and divergence of SmDMLs. SmDML1, SmDML2 and SmDML4 were significantly down-regulated by the treatment of 5Aza-dC, a general DNA methylation inhibitor, suggesting involvement of SmDMLs in genome DNA methylation change. SmDML1 was predicted and experimentally validated to be target of Smi-miR7972. Computational analysis of forty whole genome sequences and almost all of RNA-seq data from Lamiids revealed that MIR7972s were only distributed in some plants of the three orders, including Lamiales, Solanales and Boraginales, and the number of MIR7972 genes varied among species. It suggests that MIR7972 genes underwent expansion and loss during the evolution of some Lamiids species. Phylogenetic analysis of MIR7972s showed closer evolutionary relationships between MIR7972s in Boraginales and Solanales in comparison with Lamiales. These results provide a valuable resource for elucidating DNA demethylation mechanism in S. miltiorrhiza.

Figure 1

Gene structures of DMLs in S. miltiorrhiza, Arabidopsis and rice. Exons are presented by filled green boxes. Introns are presented by lines.

Figure 2

Amino acid sequence alignment of the conserved DNA glycosylase domain of DML proteins in S. miltiorrhiza, Arabidopsis and rice. Numbers show the position of amino acids. Identical amino acids are highlighted in red. Similar amino acid residues are showed in red.

Figure 3

Distribution of conserved motifs of DML proteins from S. miltiorrhiza, Arabidopsis and rice.

Figure 4

Phylogenetic analysis of DML proteins from 16 plant species. Monocot and dicot are colored green and red, respectively. The bootstrap values are shown.

Figure 5

Expression patterns of SmDMLs in roots (Rt), stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza. The expression levels were analyzed using the quantitative RT-PCR method. Fold changes of SmDML expression are shown. Expression level in leaves was arbitrarily set to 1 and the levels in other organs were given relative to this. One-way ANOVA was calculated using IBM SPSS 20 software. P < 0.05 was considered statistically significant and was represented by different letters. Error bars was indicated by the standard deviations of three biological replicates.

Figure 6

Responses of SmDML genes to 5Aza-C treatment. Fold changes of SmDMLs in leaves of S. miltiorrhiza plantlets treated with 1, 5, 10, 30 or 50 µM of 5Aza-C for 15 days are shown. The expression levels were analyzed using the quantitative RT-PCR method. Expression level in leaves without treatment (0 µM) was arbitrarily set to 1 and the levels in leaves of 5Aza-C-treated plantlets were given relative to this. One-way ANOVA was calculated using IBM SPSS 20 software. P < 0.05 was considered statistically significant and was represented by asterisks.

Figure 7

Smi-miR7972 in S. miltiorrhiza. (a) The hairpin structure of Smi-miR7972. Smi-miR7972a and Smi-miR7972b are indicated by red and green lines. (b) Expression patterns of Smi-miR7972a and Smi-miR7972b in roots (Rt), stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza. Fold changes of Smi-miR7972a and Smi-miR7972b are shown. Expression level of Smi-miR7972a in roots was arbitrarily set to 1 and the levels of Smi-miR7972a and Smi-miR7972b were given relative to this. Error bars was indicated by the standard deviations of three biological replicates. (c) Sequence alignment of miR7972s from S. miltiorrhiza, R. glutinosa and N. benthamiana. (d) Validation of Smi-miR7972a- and Smi-miR7972b-mediated cleavage using the modified 5′ RLM RACE method. Heavy black line represents open reading frame of SmDML1. The complementary sites of Smi-miR7972 in SmDML1 are represented by A and B and shown in red. The nucleotide sequences of Smi-miR7972a and Smi-miR7972b from 3′ to 5′ and the complementary sites of SmDML1 from 5′ to 3′ are shown in the expanded regions. Vertical dashes indicate Watson-Crick pairing. Circles indicate G:U wobble pairing. Vertical arrows indicate the 5′ termini of Smi-miR7972-mediated cleavage products, as obtained by 5′ RLM-RACE, with the frequency of clones shown.

Figure 8

Phylogenetic relationships of MIR7972 precursors in various Lamiids species. It includes Ruellia speciosa (rsp), Mentha longifolia (mlo), Ocimum tenuiflorum (ote), Fraxinus excelsior (fex), Dorcoceras hygrometricum (dhy), Sesamum indicum (sin), Erythranthe guttata (egu), Nicotiana obtusifolia (nob), Ipomoea nil (ini), Ipomoea trifida (itr), Nicotiana attenuata (nat), Nicotiana sylvestris (nsy), Nicotiana tomentosiformis (nto), Nicotiana tabacum (nta), Nicotiana benthamiana (nbe), Andrographis paniculata (apa), Jasminum sambac (jsa), Syringa oblata (sob), Fraxinus pennsylvanica (fpe), Olea europaea (oeu), Osmanthus fragrans (ofr), Alectra vogelii (avo), Rehmannia glutinosa (rgl), Phtheirospermum japonicum (pja), Pedicularis keiskei (pke), Conopholis americana (cam), Paulownia fortunei (pfo), Paulownia tomentosa (pto), Plantago ovata (pov), Plantago lagopus (pla), Lippia dulcis (ldu), Tectona grandis (tgr), Ocimum basilicum (oba), Perilla frutescens (pfr), Rosmarinus officinalis (rof), Mentha spicata (msp), Lithospermum erythrorhizon (ler), Arnebia euchroma (aeu). Species from Lamiales, Solanales and Boraginales are shown red, green and purple, respectively. MIR7972s could be divided into two groups, including group I and group II. The branch length is shown.