Identification of DNA methyltransferases and demethylases in Solanum melongena L., and their transcription dynamics during fruit development and after salt and drought stresses

Abstract

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species.

Fig 1

Gene and protein structure of the eggplant set of (A) C5-MTases, (B) demethylases. On the left hand panel the exon/intron structures are showed, with exons displayed as black boxes and introns as lines (generated using the tool provided at http://wormweb.org/exonintron). Name of the motifs/domains are shown inside the figure. Acronym explanations: BAH = Bromo Adjacent Homology domain; CHROMO = CHRromatin Organisation MOdifier domain; DNMT1-RFD = Cytosine specific DNA methyltransferase Replication Foci Domain; Perm-CXXC = Permuted single zf-CXXC unit; RRM_DME = RNA-recognition motif in Demeter.

Fig 2. Chromosomal location of genes encoding C5-MTase and demethylase.

The 12 pseudomolecules (chromosomes) are depicted by the set of red bars in the center, and gene density (1 Mbp windows) by the outer blue track. Location of each gene is showed.

Fig 3

Phylogenetic analysis of the genes encoding (A) C5-MTase and (B) DNA demethylases. The two unrooted, neighbour-joining trees were constructed by aligning the C5-MTase and DNA demethylases protein sequences contained in S1 and S2 Files, respectively. Colours (yellow, green and violet) in the tree A indicate the main three clades obtained for the C5-MTases corresponding to the three subclasses: MET1, CMT and DRM, respectively. Colours (pale blue, grey and orange) in the tree B indicate the main three clades obtained for the DNA demethylase corresponding to the three subclasses present in Arabidopsis: ROS, DML and DME, respectively. The number at each node represents the bootstrap percentage value from 1,000 replicates. Smel = Solanum melongena, At = Arabidopsis thaliana, Cc = Cynara cardunculus, Gm = Glycine max, Os = Oryza sativa, Sl = Solanum lycopersicum, Zm = Zea mays, Fa = Fragaria x ananassa, St = Solanum tuberosum, Sm = Salvia miltiorrhiza, Sb = Sorghum bicolor, Bd = Brachypodium distachyon, Rc = Ricinus communis, Pt = Populus trichocarpa, Br = Brassica rapa, Cr = Capsella rubella, Cs = Cucumis sativus, Cm = Cucumis melo, Fv = Fragaria vesca, Md = Malus domestica.

Fig 4. qRT-PCR based transcription profiling of eggplant C5-MTase during fruit development in the three eggplant F1 hybrids.

On “X” axis, the three eggplant F1 hybrids (CL = Clara, BR = Bella Roma and NL = Nite Lady) are represented at three fruit stages (5, 14 and 25 days after anthesis) indicated with the numbers as 1, 2 and 3, respectively. The actin eggplant gene was used as the reference sequence. Error bars represent SD (n = 3). Different letters associated with the set of means indicate a significant difference based on Tukey's HSD test (P≤0.05).

Fig 5. qRT-PCR based transcription profiling of eggplant DNA demethylase during fruit development in the three eggplant F1 hybrids.

On “X” axis, the three eggplant F1 hybrids (CL = Clara, BR = Bella Roma and NL = Nite Lady) are represented at three fruit stages (5, 14 and 25 days after anthesis) indicated with the numbers as 1, 2 and 3, respectively. The actin eggplant gene was used as the reference sequence. Error bars represent SD (n = 3). Different letters associated with the set of means indicate a significant difference based on Tukey's HSD test (P≤0.05).

Fig 6. The patterns of expression of C5-MTase and DNA demethylase in leaf tissues of the F1 hybrid ‘Nite Lady’ in response to salt stress.

The actin eggplant gene was used as the reference sequence. Error bars represent SD (n = 3). Asterisk indicates a significant difference based on Tukey's HSD (P≤0.05).

Fig 7. The patterns of expression of C5-MTase and DNA demethylase in leaf tissues of the F1 hybrid ‘Nite Lady’ in response to drought stress.

The actin eggplant gene was used as the reference sequence. Error bars represent SD (n = 3). Asterisk indicates a significant difference based on Tukey's HSD (P≤0.05).