Abstract
Key messageInduction of biphasic interphase–mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa.Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S–M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.
Highlights
The dynamics of plant growth and development depends largely on the mitotic activity of root and shoot apical meristems (RAMs and SAMs), which provide cells for organogenesis and tissue expansion
In the present study, using Allium cepa root meristem model, we have shown that many inhibitors of DNA replication, when applied at low concentrations, may generate both premature chromosome condensation (PCC)-like and biphasic IM cells similar to those formed under the prolonged (3-day) influence of HU
Considering the recognized effects of HU and its capacity to promote IM cells in onion root meristems (Żabka et al 2010, 2012), our experiments were primarily aimed at establishing the extent to which similar outcomes may be produced by other drugs known to affect different aspects of DNA synthesis
Summary
The dynamics of plant growth and development depends largely on the mitotic activity of root and shoot apical meristems (RAMs and SAMs), which provide cells for organogenesis and tissue expansion. Exposures to all kinds of chemical or physical agents that interfere with the replication machinery may lead to faulty nuclear divisions, causing cells either to lose their meristematic identity and to initiate vascular differentiation, or eventually (as a result of excessive levels of DNA damage), to employ an in-built mechanism for cellular destruction via apoptosis-like programmed cell death (AL-PCD) (Cools et al 2011; Waterworth et al 2011) It is essential, that we have a clear understanding of how root tip cells are preserved by triggering (and by skipping) cell cycle checkpoints, which block DNA replication and mitotic activity as long as the stress endures
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