Exploring microRNA Signatures of DNA Damage Response Using an Innovative System of Genotoxic Stress in Medicago truncatula Seedlings

Abstract

One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (SOG1), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants.

Integrative Transcriptomics Data Mining to Explore the Functions of TDP1α and TDP1β Genes in the Arabidopsis thaliana Model Plant

“…Among the chemical treatments applied to seeds for a period of 24 h ( Figure 5 a) [ 37 ], dimethyl sulfoxide (DMSO, 1%) was used as a control and an agent to dissolve the chemical compounds, even though this compound can impair seed germination and plant development when used in high concentrations [ 77 ]. Indeed, the DMSO treatment seems to affect the expression of the TDP1 genes, in agreement with previous studies from other model systems [ 31 ]. Cycloheximide (CHX, 1 µM) strongly induces the expression of TDP1β , while 2,4-dinitrophenol (2,4-DNP, 25 μM) treatment acts oppositely on both genes, thus downregulating TDP1β and upregulating TDP1α .…”

“…Considering the mined data for co-expression networks and putative protein–protein interactions (see Section 3.1 ), the two genes’ expression data further support the involvement of these genes in DNA damage repair and the response to genotoxic stresses, in agreement with previous publications [ 14 , 25 , 31 ].…”

“…In M. truncatula (barrel medic), it was shown to be ubiquitously expressed in all plant tissues and developmental stages [ 17 , 28 ]. Moreover, the TDP1β gene was differentially expressed upon exposure to NSC120686 (inhibitor of hTdp1 acting as substrate mimetic [ 29 ]), being downregulated in calli and upregulated in young seedlings [ 30 , 31 ], possibly suggesting its implication in different developmental aspects. In both A. thaliana and barrel medic plants subjected to multiple abiotic stress conditions (cold, heat, salinity, osmotic stress, and UV-B), TDP1β was highly upregulated at the earliest timepoints (0.5–1 h) following exposure to stress [ 19 ].…”

Exploring the putative microRNAs cross-kingdom transfer in Solanum lycopersicum-Meloidogyne incognita interactions

“…In addition to the essential role of DNA repair in maintaining genome stability, recent works are discussing the involvement of DNA repair proteins in plant-pathogen interactions and SAR ( Fu and Dong, 2013 ; Camborde et al., 2019 ). Moreover, the miRNA-mediated control over DNA damage responses is starting to gain more interest from both an endogenous ( Gualtieri et al., 2021 ; Macovei et al., 2021 ) and cross-kingdom fashion ( Bellato et al., 2019 ). An interesting finding in this sense is the case of let-7, one of the most abundant miRNAs found in nematodes, putatively targeting the nuclear pore complex protein Nup98-Nup96 in tomatoes.…”

Genotype-specific germination behavior induced by sustainable priming techniques in response to water deprivation stress in rice

“…The FPG (formamidopyrimidine-DNA glycosylase) gene, encoding for a DNA repair enzyme that excises oxidized purines from damaged DNA, and the OGG1 (8-oxoguanine glycosylase) gene, involved in the removal of 8-oxoG products, were proven to be highly induced during seed imbibition in multiple species ( Macovei et al., 2011 ; Forti et al., 2021 ; Kowalik and Groszyk, 2023 ). The two TDP1 (tyrosyl-DNA phosphodiesterase 1) genes, TDP1α and TDP1β , encoding enzymes involved in hydrolyzing phosphodiester bond formed between topoisomerase I and the 3’-DNA, were proved to be highly active during seed imbibition as well as seedling growth under stress ( Macovei et al., 2010 ; Gualtieri et al., 2021 ). PARP1 (poly-(ADP-ribose) polymerase 1) gene plays an important role in mediating the response to DNA damage during gametophyte development ( Banerjee and Roy, 2021 ).…”