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Black poplar (Populus nigra L.) is one of the most threatened tree species in Europe since up to 99% of its natural habitat has disappeared. Black poplar seeds are characterised by short longevity. It was recently demonstrated that black poplar seeds can be successfully stored at -- 10°C, -- 20°C and -- 196°C for at least 2 years but not at higher temperatures. In the present study, the role of oxidative stress in determining the level of viability of black poplar seeds stored at -- 196°C, -- 20°C, -- 10°C, -- 3°C and 3°C for 3 months, 1 year and 2 years was monitored. The superoxide anion radicals (O2 -- •) and hydrogen peroxide (H2O2) increased during storage and had an impact on membrane integrity as determined by changes in the content of fatty acids and phospholipids and increases in electrolyte leakage. The level of non-enzymatic and enzymatic components of the ascorbate-glutathione (AsA-GSH) cycle was also investigated. The level of O2 -- • was strongly correlated with the level of seed germination after 1 and 2 years of storage. This was accompanied by changes in the redox potential, as well as changes in the content of linoleic acid and phosphatydiloglycerol over the same period of time. In particular, the deleterious effect of H2O2 was observed after 2 years of storage when its accumulation was highly correlated with changes in the composition of fatty acids and phospholipids. Despite increased activity of AsA-GSH cycle enzymes, the level of reducing agents was insufficient and seeds exhibited large increases in the redox potential when stored at -- 3°C and still higher when stored at 3°C. Overall, the results of the study demonstrate that oxidative stress increases during seed storage, especially at the warmer temperatures and injures seed tissues; resulting in a loss of viability. [ABSTRACT FROM AUTHOR]

Key message: Adequate storability of black poplar (Populus nigraL.) seeds at temperatures below 0 °C was associated with preservation of proteins of energy and carbohydrate metabolism, protein turnover, and proteins maintaining long-term stability of dehydrated tissue. Context: Understanding seed storability is a key factor for effective seed preservation and conservation. Black poplar is an endangered tree species and its seed loses rapidly viability during storage. Aims: The aim of this study was to determine, and functionally characterise, the proteins associated with storability of black poplar seeds. Methods: Dried seeds (7.1% MC) were stored at 3 °C, − 3 °C, − 20 °C, and − 196 °C, for a period of 12 and 24 months. Proteins were extracted and separated according to their isoelectric point (pI) and mass using 2-dimensional electrophoresis. Proteins that varied in abundance for temperature and time of storage were identified by mass spectrometry. Results: A germination test showed that seeds remained viable at − 3, − 20, and − 196 °C. Storage at 3 °C caused loss of seed viability. This loss in seed vigour was related to the largest changes in protein abundance. As storage temperature decreased, a smaller number of proteins displayed changed abundance. Conclusion: Good storability of black poplar seeds under freezing conditions may be associated with the following: an inhibition of synthesis of energy and carbohydrate metabolism and protein turnover proteins, LEA proteins that maintain long-term stability of dehydrated tissue, GDSL esterases/lipases that inhibit hydrolysing activity, and by chaperonins that protect protein functionality. Storage conditions influence the preservation of protein function that governs seed viability. [ABSTRACT FROM AUTHOR]