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The root endosphere is colonized by soil-inhabiting bacteria, i.e., bacteria that were recruited through horizontal transmission from the soil, and by seed-inhabiting bacteria that colonize the inner tissues of the seed and might be potentially considered vertically transmitted [14, 15, 33, 34]. In view of identifying bacterial strains for application via seed coating, e.g., the inoculation of bacteria at the outside of the seed, it is important to make out whether bacteria can colonize the root endosphere from the surrounding soil or via the seeds. To this end, we set up a third experiment (experiment III; Table 1). Here, we compared bulk soil samples, root endosphere samples of 2-week-old maize seedlings grown in vitro under gnotobiotic soil-free conditions, and root endosphere samples of seedlings grown in field soil-filled pots.
Ascobic acid (AsA) is a prevailing molecule that is present in different concentrations in vacuoles, cytosols, mitochondria and chloroplasts [32,33]. AsA acts as a reducer of free radicals, mainly H2O2, forming H2O and oxidizing AsA to nontoxic dehydroascorbate (DHA) via ascorbate peroxidase [34]. Subsequently, DHA is recycled to AsA by the oxidation of reduced glutathione (GSH). In plants, AsA mainly occurs in its reduced form in nonstress conditions, and the amount of oxidized forms of AsA increases when plant cells experience oxidative stress [35]. It is believed that AsA is present in high concentrations mainly in recalcitrant seeds and is almost absent in dry orthodox seeds [36,37]. AsA also affects extension biosynthesis [38], as well as root elongation, cell vacuolization and cell wall growth [32], acting in plant growth and development and their adaptation to environmental conditions. An oxidative intercellular environment results in increased redox potential values. Another indicator used for assessing the available AsA in cells is the AsA/DHA ratio [38].
Because many forms of damage, including ROS-derived damage, serve as fundamental factors determining seed deterioration and further seed aging, the prototypical free radical theory of aging was replaced by a more accurate model that considers biological imperfectness as the true cause of aging [60]. Recently, Ren and Zhang [61] suggested that aging is encoded by genes or DNA, and pro-aging factors accelerate and promote this process in contrast to anti-aging factors that retard this process. H2O2 is considered to be a pro-aging factor [21]. As shown in this report, H2O2 levels significantly affected germination capacity and seedling emergence uniquely in P. avium (Table 1), which suggests the occurrence of oxidative stress. The impact of H2O2 levels on TBARS levels and the AsA:DHA ratio was prominent in the roots of M. sylvestris seedlings and resulted in the selection of oxidation-introducing parameters affecting seedling emergence. The controlled deterioration treatment of elm seeds resulted in doubled H2O2 levels [62]. Some treatments doubled H2O2 levels in germinated M. sylvestris seeds stored for three years emphasizing stress symptoms. However, in the majority of seeds, doubled H2O2 levels did not overlap with significantly increased TBARS levels, allowing us to hypothesize that lipid peroxidation is not the result of accumulated H2O2 but more likely lipid auto-oxidation is the origin of increased membrane permeability. Particularly, auto-oxidation is intensified in seeds dried below 6% MC [63].
The increase in TBARS levels was clearly reported in specific treatments of germinated M. sylvestris seeds. More spectacular changes in MDA levels were observed when seed viability decreased to approximately 60%. The lowest germination capacity (69% on average) occurred in M. sylvestris seeds stored for three years with 5% MC [14] notably, in these seeds, TBARS levels increased 1.6 times (Fig 2B). MDA significantly increased in orthodox-type willow seeds stored for 16 years, whereas seeds stored for up to 10 years were characterized by unaffected MDA levels [64]. In contrast, MDA levels increased by several dozen times during the accelerated aging of pepper seeds [65]. MDA, the main component of TBARS, is undoubtedly well-established marker of oxidative stress. The majority of methods enable measuring only free MDA omitting MDA-conjugates, thus not reflecting the total amount of MDA generated from lipid peroxidation [66] and rendering distinct results and their explanations.
We proved that the AsA concentrations observed in seeds stored for three years significantly affected seedling emergence in all three species, emphasizing that, AsA is a reliable seed viability marker important in maintaining a specific redox environment. The depletion of the Asc pool is likely one of the reasons for the poor seedling performance of cryostored M. sylvestris seeds previously dried to 4.9% MC. H2O2 levels significantly affected germination capacity uniquely in P. avium seeds stored for three years. Additionally, P. avium was the only species in which seedling emergence was clearly determined by the synergistic action of the three tested oxidation markers, H2O2, TBARS and AsA levels. Remarkably, P. padus seeds had a broad range of tolerance to the tested MCs and storage temperatures, resulting in balanced reduction and oxidation processes enabling a high germination capacity and successful seedling establishment. Distinct molecular responses of species, from the same category (orthodox seeds) and from the same niche after different seed storage, occurring during seed germination and establishment of healthy seedlings, emphasize differences in seed resilience concerning feasibility of their storage in ultra-low temperature and seed moisture content. 781b155fdc