The Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula

Abstract

This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA) during <i>Medicago truncatula</i> seed germination, with emphasis on the seed repair response. Seeds treated with H₂O and TSA (10 and 20 μM) were collected during imbibition (8 h) and at the radicle protrusion phase. Biometric data showed delayed germination and impaired seedling growth in TSA-treated samples. Comet assay, performed on radicles at the protrusion phase and 4-days old <i>M. truncatula</i> seedlings, revealed accumulation of DNA strand breaks upon exposure to TSA. Activation of DNA repair toward TSA-mediated genotoxic damage was evidenced by the up-regulation of <i>MtOGG1</i>(<i>8-OXOGUANINE GLYCOSYLASE/LYASE</i>) gene involved in the removal of oxidative DNA lesions, <i>MtLIGIV</i>(<i>LIGASE IV</i>) gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and <i>TDP(TYROSYL-DNA PHOSPHODIESTERASE)</i> genes encoding the multipurpose DNA repair enzymes tyrosyl-DNA phosphodiesterases. Since radical scavenging can prevent DNA damage, the specific antioxidant activity (SAA) was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu reagent assays. Fluctuations of SAA were observed in TSA-treated seeds/seedlings concomitant with the up-regulation of antioxidant genes <i>MtSOD(SUPEROXIDE DISMUTASE, MtAPX</i>(<i>ASCORBATE PEROXIDASE</i>) and <i>MtMT2</i>(<i>TYPE 2 METALLOTHIONEIN</i>). Chromatin remodeling, required to facilitate the access of DNA repair enzymes at the damaged sites, is also part of the multifaceted seed repair response. To address this aspect, still poorly explored in plants, the <i>MtTRRAP</i>(<i>TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN</i>) gene was analyzed. TRRAP is a transcriptional adaptor, so far characterized only in human cells where it is needed for the recruitment of histone acetyltransferase complexes to chromatin during DNA repair. The <i>MtTRRAP</i> gene and the predicted interacting partners <i>MtHAM2</i> (<i>HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY</i>) and <i>MtADA2A</i> (<i>TRANSCRIPTIONAL ADAPTOR</i>) showed tissue- and dose-dependent fluctuations in transcript levels. PCA (Principal Component Analysis) and correlation analyses suggest for a new putative link between DNA repair and chromatin remodeling that involves <i>MtOGG1</i> and <i>MtTRRAP</i> genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers.