Kinetics of DNA Repair in Vicia faba Meristem Regeneration Following Replication Stress

Abstract

The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even “chaotic” events are dealt with in a proper degree of order.

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba

“…In the first step, the nuclei were marked, and the areas of each nucleus were measured as a total number of px on the image, in the second step, only the heterochromatin areas of each nucleus were marked and their area was measured (also as the number of pixels). The overall heterochromatin percentage in the nucleus was calculated as the mean heterochromatin area against the mean area of the nucleus, in percentage terms as described in [ 8 ].…”

“…Apical root fragments (approx. 1.5 cm-long) were dissected longitudinally and blotted onto a nitrocellulose membrane in accordance with [ 8 ]. The procedure performed was similar to [ 4 ].…”

“…Former studies performed by Rybaczek et al [ 2 , 3 , 4 ] have shown that even under extremely stressful conditions, V. faba can manage stress well considering the induction of premature chromosome condensation (PCC)—an aberrant event well-known for its frequent initiation of genome chaos [ 5 , 6 ]. Under the conditions of replication stress caused by hydroxyurea (HU), which arrests replication by inhibiting the enzyme ribonucleotide reductase [ 7 ], the root meristem cells of the plant have shown incredible resistance to prolonged stressful conditions [ 4 ] and were able to recover fairly quickly once released into water [ 8 ]. Although PCC induction causes some chaotic aberrations in a fraction of cells, V. faba is mostly capable of directing the fraction of highly damaged cells to the apoptotic-like programmed cell death pathway (AL-PCD).…”

“…Although PCC induction causes some chaotic aberrations in a fraction of cells, V. faba is mostly capable of directing the fraction of highly damaged cells to the apoptotic-like programmed cell death pathway (AL-PCD). The plant cells also recovered during the next cell cycle following PCC induction [ 4 , 8 ]. We believe that the high survivability rates of the V. faba family stem from the highly effective stress response mechanisms that are activated during replication and/or mitotic division.…”

Mechanisms of Genome Maintenance in Plants: Playing It Safe With Breaks and Bumps

“…Production of ROS triggers single- and double-stranded breaks (SSBs and DSBs) in the DNA either directly through destruction of bases or modifications of bases. In some crop plants, oxidative stress imbalances ROS production and consequently promotes developmental defects and growth reduction ( Rybaczek et al, 2021 ). This results in a significant decrease in plant productivity and crop quality.…”