Seed priming, a pre-sowing technique that enhances the antioxidant/DNA repair activities during the pre-germinative metabolism, still retains empirical features. We explore for the first time the molecular dynamics of pre-germinative metabolism in primed eggplant (Solanum melongena L.) seeds in order to identify hallmarks (expression patterns of antioxidant/DNA repair genes combined with free radical profiles) useful to discriminate between high- and low-quality lots. The hydropriming protocol hereby developed anticipated (or even rescued) germination, when applied to lots with variable quality. ROS (reactive oxygen species) raised during hydropriming and dropped after dry-back. Upregulation of antioxidant/DNA repair genes was observed during hydropriming and the subsequent imbibition. Upregulation of SmOGG1 (8-oxoguanine glycosylase/lyase) gene detected in primed seeds at 2 h of imbibition appeared as a promising hallmark. On the basis of these results, the investigation was restricted within the first 2 h of imbibition, to verify whether the molecular landscape was reproducible in different lots. A complex pattern of antioxidant/DNA repair gene expression emerged, reflecting the preponderance of seed lot-specific profiles. Only the low-quality eggplant seeds subjected to hydropriming showed enhanced ROS levels, both in the dry and imbibed state, and this might be a useful signature to discriminate among lots. The plasticity of eggplant pre-germinative metabolism stimulated by priming imposes a plethora of heterogeneous molecular responses that might delay the search for quality hallmarks. However, the information hereby gained could be translated to eggplant wild relatives to speed-up their use in breeding programs or other agronomical applications.

Because high-quality seeds are essential for successful crop production in challenging environments, understanding the molecular bases of seed vigour will lead to advances in seed technology. Histone deacetylase inhibitors, promoting histone hyperacetylation, are used as tools to explore aspects still uncovered of the abiotic stress response in plants. The aim of this work was to investigate novel signatures of seed germination in Medicago truncatula, using the histone deacetylase inhibitor sodium butyrate (NaB) as stress agent. NaB-treated and untreated seeds collected at 2 and 8 hr of imbibition and at the radicle protrusion stage underwent molecular phenotyping and nontargeted metabolome profiling. Quantitative enrichment analysis revealed the influence of NaB on seed nucleotide, amino acid, lipid, and carbohydrate metabolism. Up-regulation of antioxidant and polyamine biosynthesis genes occurred in response to NaB. DNA damage evidenced in NaB-treated seeds correlated with up-regulation of base-excision repair genes. Changes in N -methyladenosine and N -methylguanine were associated with up-regulation of MtALKBH1 (alkylation repair homolog) gene. N ,N -dimethylguanosine and 5-methylcytidine, tRNA modifications involved in the post-transcriptional regulation of DNA damage response, were also accumulated in NaB-treated seeds at the radicle protrusion stage. The observed changes in seed metabolism can provide novel potential metabolic hallmarks of germination.

This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA) during Medicago truncatula 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 M. truncatula 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 MtOGG1(8-OXOGUANINE GLYCOSYLASE/LYASE) gene involved in the removal of oxidative DNA lesions, MtLIGIV(LIGASE IV) gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and TDP(TYROSYL-DNA PHOSPHODIESTERASE) 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 MtSOD(SUPEROXIDE DISMUTASE, MtAPX(ASCORBATE PEROXIDASE) and MtMT2(TYPE 2 METALLOTHIONEIN). 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 MtTRRAP(TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN) 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 MtTRRAP gene and the predicted interacting partners MtHAM2 (HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY) and MtADA2A (TRANSCRIPTIONAL ADAPTOR) 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 MtOGG1 and MtTRRAP genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers.

The pre-germinative metabolism is among the most fascinating aspects of seed biology. The early seed germination phase, or pre-germination, is characterized by rapid water uptake (imbibition), which directs a series of dynamic biochemical events. Among those are enzyme activation, DNA damage and repair, and use of reserve storage compounds, such as lipids, carbohydrates and proteins. Industrial seedling production and intensive agricultural production systems require seed stocks with high rate of synchronized germination and low dormancy. Consequently, seed dormancy, a quantitative trait related to the activation of the pre-germinative metabolism, is probably the most studied seed trait in model species and crops. Single omics, systems biology, QTLs and GWAS mapping approaches have unveiled a list of molecules and regulatory mechanisms acting at transcriptional, post-transcriptional and post-translational levels. Most of the identified candidate genes encode for regulatory proteins targeting ROS, phytohormone and primary metabolisms, corroborating the data obtained from simple molecular biology approaches. Emerging evidences show that epigenetic regulation plays a crucial role in the regulation of these mentioned processes, constituting a still unexploited strategy to modulate seed traits. The present review will provide an up-date of the current knowledge on seed pre-germinative metabolism, gathering the most relevant results from physiological, genetics, and omics studies conducted in model and crop plants. The effects exerted by the biotic and abiotic stresses and priming are also addressed. The possible implications derived from the modulation of pre-germinative metabolism will be discussed from the point of view of seed quality and technology.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is involved in DNA repair pathways as it mends the topoisomerase I—DNA covalent complexes. In plants, a small Tdp1 gene family, composed by Tdp1α and Tdp1β genes, was identified, but the roles of these genes in abiotic stress responses are not fully understood. To investigate their specific stress response patterns, the present study made use of bioinformatic and molecular tools to look into the Tdp1β gene function, so far described only in the plant kingdom, and compare it with Tdp1α gene coding for the canonical, highly conserved α isoform. The expression profiles of Tdp1α and Tdp1β genes were examined under abiotic stress conditions (cold, heat, high osmolarity, salt, and UV-B) in two model species, Arabidopsis thaliana and Medicago truncatula. The two isoforms of topoisomerase I (TOP1α and TOP1β) were also taken into consideration in view of their known roles in DNA metabolism and cell proliferation. Data relative to gene expression in Arabidopsis were retrieved from the AtGenExpress microarray dataset, while quantitative Real-Time PCR was carried out to evaluate the stress response in M. truncatula cell cultures. These analyses revealed that Tdp1β gene expression was enhanced during the first hour of treatment, whereas Tdp1α enhanced expression succeeded at subsequent timepoints. In agreement with the gene-specific responses to abiotic stress conditions, the promoter regions of Tdp1α and Tdp1β genes are well equipped with stress-related cis-elements. An in-depth bioinformatic characterization of the HIRAN motif, a distinctive feature of the Tdp1β protein, showed its wide distribution in chromatin remodeling and DNA repair proteins. The reported data suggests that Tdp1β functions in the early response to abiotic stresses.

During seed imbibition, lipids are engaged in membrane reorganization while facing free radical-mediated oxidative injury. In the present work, we explored changes in lipid components at different timepoints of imbibition (0.5, 2, 4, 6, and 8 h) in the legume Medicago truncatula, by combining biochemical approaches with targeted lipidomics and untargeted metabolomics. ROS and RNS (reactive oxygen and nitrogen species) accumulation was observed throughout the tested timepoints whereas lipid peroxidation increased at 4 h of imbibition. The seed response to oxidative damage was evidenced by a significant increase in tocopherols starting from 0.5 h of imbibition as well as by the reduction in total thiol content occurring at 2 h of imbibition. Since under physiological conditions, the proper functions of the cell membranes are strongly dependent on the qualitative and quantitative balance of fatty acid residues in phospholipids, the investigation was expanded to the fatty acid cohort of M. truncatula seeds. Total saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), omega(ω)-3 and omega(ω)-6 fatty acids showed fluctuations during seed imbibition. The most remarkable finding was the profile of the ω-3 PUFA docosopentaenoic acid (DPA, 7 cis, 10 cis, 13 cis, 16 cis, and 19 cis-22:5) that showed a peak (up to 1.0% of the total fatty acid content) at 0.5 and 8 h of imbibition, concomitant with the peaks observed in tocopherol levels. It is possible that the observed changes in DPA alter the physical properties of membranes, as reported in animal cells, triggering signaling pathways relevant for the cell defense against oxidative injury. Furthermore, the content and balance between tocopherols and PUFAs is regarded as a determinant of storage stability. No enhancement in trans-fatty acids occurred throughout imbibition, suggesting for a proper antioxidant response carried by the seed. Fatty acids profiles were integrated with data from untargeted metabolomics showing changes in lipid sub-pathways, among which fatty acid amide, lyso-phospholipids, and phospholipid metabolism. The emerging lipid profiles and dynamics are discussed in view of the overall imbibition damage generated during M. truncatula seed imbibition.

In the present work, non-targeted metabolomics was used to investigate the seed response to kinetin, a phytohormone with potential roles in seed germination, still poorly explored. The aim of this study was to elucidate the metabolic signatures of germination triggered by kinetin and explore changes in metabolome to identify novel vigor/stress hallmarks in Medicago truncatula . Exposure to 0.5 mM kinetin accelerated seed germination but impaired seedling growth. Metabolite composition was investigated in seeds imbibed with water or with 0.5 mM kinetin collected at 2 h and 8 h of imbibition, and at the radicle protrusion stage. According to Principal Component Analysis, inositol pentakisphosphate, agmatine, digalactosylglycerol, inositol hexakisphosphate, and oleoylcholine were the metabolites that mostly contributed to the separation between 2 h, 8 h and radicle protrusion stage, irrespective of the treatment applied. Overall, only 27 metabolites showed significant changes in mean relative contents triggered by kinetin, exclusively at the radicle protrusion stage. The observed metabolite depletion might associate with faster germination or regarded as a stress signature. Results from alkaline comet assay, highlighting the occurrence of DNA damage at this stage of germination, are consistent with the hypothesis that prolonged exposure to kinetin induces stress conditions leading to genotoxic injury.

Seed priming can circumvent poor germination rate and uniformity, frequently reported in eggplant (Solanum melongena L.) and its crop wild relatives (CWRs). However, there is still a gap of knowledge on how these treatments impact the pre-germinative metabolism in a genotype- and/or species-dependent manner. The CWR Solanum villosum Miller (hairy nightshade) investigated in this study showed a quite unique profile of fast germination. Although this accelerated germination profile would not apparently require further improvement, we wanted to test whether priming would still be able to impact the pre-germinative metabolism, eventually disclosing the predominant contribution of specific antioxidant components. Hydropriming followed by dry-back resulted in synchronized germination, as revealed by the lowest MGR (Mean Germination Rate) and U (Uncertainty) values, compared to unprimed seeds. No significant changes in ROS (reactive oxygen species) were observed throughout the treatment. Increased tocopherols levels were detected at 2 h of hydropriming whereas, overall, a low lipid peroxidation was evidenced by the malondialdehyde (MDA) assay. Hydropriming resulted in enhanced accumulation of the naturally occurring antioxidant phenolic compounds chlorogenic acid and iso-orientin, found in the dry seeds and ex novo accumulation of rutin. The dynamic changes of the pre-germinative metabolism induced by hydropriming are discussed in view of future applications that might boost the use of eggplant CWRs for breeding, upon upgrade mediated by seed technology.