Oxidative damage and DNA repair in desiccated recalcitrant embryonic axes of Acer pseudoplatanus L.

Abstract

Background Most plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration. Results The loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (٠OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation. Conclusions The high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.

Epigenetic Marks, DNA Damage Markers, or Both? The Impact of Desiccation and Accelerated Aging on Nucleobase Modifications in Plant Genomic DNA

“…Embryonic axes were excised from the seeds no later than eight weeks after collection. The desiccation of embryonic axes was conducted as previously described [ 9 , 10 , 43 ]. The duration of the desiccation protocol ranged from 1 to 18 h. and was calculated on a fresh weight basis as previously described [ 44 ] ( Table 1 ).…”

“…A tetrazolium chloride (TTC) assay was used to assess the viability of axes subjected to accelerated ageing as previously described [ 43 , 44 , 45 ]. Four biological replicates comprising ten embryonic axes in each replicate were stained in 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution.…”

“…An in vitro regrowth assay was conducted as previously described [ 43 ] using five biological replicates comprising 10–15 embryonic axes in each replicate. After eight weeks, regrowth of embryonic axes was assessed and only counted as regenerated if the embryonic axis had developed either a shoot or a shoot with a root.…”

“…ROS levels were quantified in aged embryonic axes using the fluorogenic dye, 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCFDA; Invitrogen, Waltham, MA, USA) as previously described [ 43 ]. Embryonic axes were incubated in 1 mL of 10 µM H 2 DCFDA in darkness for 15 min.…”

“…Therefore, in order to address this issue in the present study, we examined the changes in m 5 C, hm 5 C, and 8-oxoG in embryonic axes of sycamore maple ( Acer pseudoplatanus L.) under desiccation stress conditions and after accelerated aging. We previously used sycamore seeds as a model in research on recalcitrant (desiccation-sensitive) seeds [ 10 , 43 ]. Here, we report for the first time on the changes in the level of three DNA modifications (m 5 C, hm 5 C, and 8-oxoG) while simultaneously tracking the level of ROS and antioxidants (AOX).…”