RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula

Abstract

An intron-spliced hairpin RNA approach was used for the targeted silencing of the MtTdp1α gene encoding the αisoform of tyrosyl-DNA phosphodiesterase 1 in Medicago truncatula Gaertn. Tyrosyl-DNA phosphodiesterase 1, involved in the repair of DNA topoisomerase I-mediated DNA damage, has been poorly investigated in plants. RNASeq analysis, carried out in the MtTdp1α-depleted plants, revealed different levels of transcriptional modulation (up-and down-regulation, alternative splicing, activation of alternative promoter) in genes involved in DNA damage sensing, DNA repair, and chromatin remodelling. It is suggested that the MtTdp1α gene has new, previously undetected roles in maintaining genome integrity. Up-regulation of senescence-associated genes and telomere shortening were observed. Moreover, impaired ribosome biogenesis indicated that the MtTdp1α gene is required for the nucleolar function. In agreement with the RNA-Seq data, transmission electron microscopy detected an altered nucleolar architecture in the MtTdp1α-depleted cells. Based on the reported data, a working hypothesis related to the occurrence of a nucleolar checkpoint in plant cells is proposed.

DNA repair and telomere maintenance during seed imbibition: correlation of transcriptional patterns

“…The DNA Damage Response (DDR) was permanently activated in the Arabidopsis tert mutants, as indicated by the up-regulation of several DDR genes, among which RAD51, PARP1 and BRCA1 [9] . A link between telomere maintenance and the DNA repair enzyme Tdp1α (Tyrosyl-DNA phosphodiesterase), involved in the removal of topoisomerase I-mediated DNA damage, was evidenced since the antisense-mediated down-regulation of the MtTdp1α gene in Medicago truncatula was associated with telomere shortening [10] . RNA-Seq also revealed the differential expression of Pif1 and RecQ genes encoding helicases involved in telomere maintenance, and the HnRNPA3 (Heterogeneous RiboNuclear Protein A3) gene.…”

Depletion of tyrosyl‐DNA phosphodiesterase 1α (MtTdp1α) affects transposon expression in Medicago truncatula

“…Since it is known that in yeast the Tdp1 gene is required to ensure the accurate processing of DNA ends during non‐homologous end joining (NHEJ) (Bahmed et al ), it is possible that the impairment of DNA transposon mobility in the MtTdp1α ‐depleted cells might be caused by defective NHEJ. Although no significant differences were evidenced by RNA‐Seq analysis in the expression profiles of M. truncatula genes involved in the NHEJ pathway (Donà et al ), the occurrence of post‐transcriptional regulation of NHEJ proteins cannot be ruled out. To date, no direct evidence of the involvement of Tdp1 enzyme in the resolution of specific DNA structures that mediate TE transposition is available.…”

“…The Tdp1 gene family from Medicago truncatula Gaertn., including MtTdp1α and MtTdp1β genes, has been characterized (Macovei et al ). RNA‐Seq analysis, carried out in the MtTdp1α ‐depleted M. truncatula plants, has identified gened differentially expressed that are involved in DNA damage sensing, DNA repair and chromatin remodelling (Donà et al ).…”

“…There is still limited information on TEs mobilization profiles occurring in planta in presence of defective DNA repair. In this work, the molecular characterization of the M. truncatula MtTdp1α ‐depleted line Tdp1α‐2a (Donà et al ) has been expanded, assessing the global cytosine methylation profile, TEs expression patterns, occurrence of TEs‐associated stress‐responsive cis ‐regulatory elements and possible changes in the methylation patterns at their promoter regions. In our opinion, the reported data add novel information concerning the role of Tdp1 in relation to genome stability issues in plants, expanding the current knowledge on a crucial, still uncovered, molecular aspect of plant physiology.…”

“…In the present work, the link between the lack of MtTdp1α gene function and TEs transcriptional profiles has been investigated in the model legume M. truncatula , in order to provide novel insights into the role played by the DNA repair enzyme Tdp1α in safeguarding the plant genome. The RNA‐Seq database used in the present work has been produced in a previous study (Donà et al ). The sequencing data used in this work are deposited to the NCBI Short Read Archive and are accessible through SRA accession number SRP013555 (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi).…”