**8. Management of oxidative status**

Management of oxidative status is also considered as an important part of primed seeds physiology [18, 36, 161, 162] (**Figure 2**). Beginning with the seed development through maturation and germination, the seed moisture content as well as seed metabolic activity is subjected to dramatic changes. The biochemical and cellular events triggered by water uptake and subsequent loss are accompanied by a generation of reactive oxygen species (ROS) [151, 163]. During seed imbibition and early stages of germination, ROS production occurs mainly through respiratory activities of mitochondria, activities of β-oxidation pathways and enzymes such as NADPH oxidases, extracellular peroxidases, and oxalate oxidases [163]. ROS accu‐ mulation and associated oxidative damage can be regarded as a source of stress that may affect the successful completion of germination. As ROS, particularly H2O2 can act as signaling molecules, seeds must be endowed with a ROS removing system that tightly regulates their concentration. Scavenging of ROS is carried out by antioxidant system, a multifunctional machine, which includes enzymes (i.e. catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), monodehydroascorbate reductase (DHAR), and glutathione reductase (GR)) as well as nonenzymatic compounds (i.e. ascorbic acid (AsA) or reduced glutathione (GSH) [1, 151]. Metabolism of ROS, mainly H2O2, which is believed to play a central role in oxidative status signaling, is strictly associated to other reactive species and signaling molecules such as nitric oxide and hydrogen sulfide, which contribute and regulate the transition from dormant to germination phase [151, 163, 164].

tubulins (mainly β-tubulin) was stated during germination as compared to unprimed seeds

20 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

During Phase II of seed germination, when water uptake is severely limited, major metabolic processes are activated [111]. One of the most important events undergoing during Phase II is DNA repair, which precedes cell cycle activation [142, 150]. The process of DNA replication is preceded by repair of DNA damage caused mainly by reactive oxygen species, which are accumulated during seed storage and aging [151]. DNA repair covers first period of DNA synthesis, while the second period of DNA synthesis (replication) is observed before cell division. DNA synthesis in Phase II of germination and also during seed priming corresponds rather to DNA repair, mainly in organelle such as plastids and mitochondria [152]. Increased number of mitochondria in leek embryo cell of osmoprimed seeds was observed by Ashraf and Bray [153]. Mitochondria biogenesis before mitochondria division involved the transition of promitochondria to mature mitochondria. This process is accompanied by the expression of genes of nucleotide biosynthesis, transport, and organelle RNA- and DNA-related functions [154, 155]. Pre-sowing seed osmopriming induced higher expression of genes corresponding to mitochondria biogenesis such as translocases of the inner membrane (TIM) complex TIM10 and TIM23-1, mitochondrial ribosomal protein and translational elongation factor EF2, which

There are still some gaps in comprehensive understanding of pre-sowing seed priming impact on DNA repair and cell cycle regulation. Activation of different DNA repair mechanism has been observed during seed imbibition preceding germination and they are believed to be essential for successful reactivation of cell cycle [111]. They include α and β tyrosyl-DNA phosphodiesterase 1, α and β DNA topoisomerase I [156], 8-oxoguanine DNA glycosylase/ lyase and formamidopyrimidine-DNA glycosylase [157], transcription elongation factor II-S [158], DNA ligase VI and IV [159]. Varier et al. [16] have suggested that in primed seeds DNA damage is repaired before replication, primarily through DNA synthesis. However, in a study on *Cicer arietinum* primed seeds, the role of DNA repair genes in enhancing the physiological quality of seeds was postulated [160]. The authors tested the expression level of genes encoding proteins with already proved function on DNA repair mechanisms in relation to priming methods and seed size. Moreover, enhanced accumulation of transcripts was found in dry and imbibed osmoprimed *Brassica napus* seeds [36] for genes involved in DNA repair according to function description in databases, such as DUTP-pyrophosphatase-like 1, endonuclease V family protein, ribonucleoside-diphosphate reductase subunit M2 (TSO), casein kinase II, replicon protein A2, DNA glycosylase DEMETER (DME), BARD1, RECQ helicase L4B, and MUTS homolog 2. Thus, activation of DNA repair mechanisms in seeds occurs prior to their germination and contributes to enhanced germination rate and better quality of seeds under‐

Management of oxidative status is also considered as an important part of primed seeds physiology [18, 36, 161, 162] (**Figure 2**). Beginning with the seed development through

[136, 142].

is targeted into mitochondria [36].

going pre-sowing seed priming.

**8. Management of oxidative status**

Kubala et al. [36] have considered the priming as a process, which consists of two main phases: controlled hydration of the seeds and drying back to the initial moisture content. Seed prehydration followed by re-drying during priming treatment, similarly to seed maturation and germination, exerts changes in moisture content, which leads to ROS production and activation of the antioxidant system. The activation of APX and the accumulation of AsA and GSH during osmopriming of spinach seeds (*Spinacia oleracea*) were accompanied by the repression of SOD and CAT activity [1]. These results indicated that activation of AsA-GSH cycle during osmopriming of spinach seeds can decrease the level of lipid peroxidation products in primed seeds. Moreover, the same authors have suggested that the renewal of antioxidant defense system, possibly required by seedling establishment, occurred during the late stages of germination as a result of up-regulation of CAT activity after initial reduction and overall antioxidant activation [1]. Kubala et al. [36] have indicated through an integrated transcrip‐ tomic and proteomic approach that the priming-induced germination can be linked with the activation of antioxidant system. The authors showed that during osmopriming of *Brassica napus* seeds *CAT2* and *PER21* encoding peroxidase 21 were up-regulated and GR protein was accumulated. Moreover, the same authors have observed up-regulation of *PER13* gene and accumulation of peroxidase 12, DHAR and peroxiredoxin proteins during post-priming germination. Furthermore, Kubala et al. [162] have postulated a correlation between activation of antioxidant metabolism in osmoprimed *Brassica napus* seeds and increased tolerance to salt stress during germination. The enhanced activity of APX, SOD, and CAT corresponded with increased expression rate of *APX, SOD*, and *CAT* genes. Similar result has been obtained by Nouman et al. [165], who showed that priming of *Moringa oleifera* seeds with *Moringa* leaf extract (MLE) improves growth under saline condition mainly by activation of antioxidant system (SOD, CAT, and POD). Priming of mung bean seeds (*Vigna radiata* L. Wilczek) with βamino butyric acid (BABA) enhanced the activities of SOD and POX leading to improved tolerance to NaCl and PEG 6000 stresses [65]. Enhanced chilling stress tolerance in two tobacco varieties (MSk326 and HHDJY) was due to increased activity of antioxidant enzymes (SOD, POD, CAT, and APX) as a result of priming seeds with putrescine [165]. Results obtained by Islam et al. [167] showed that in haloprimed wheat (*Triticum aestivum*) seeds, increased activity of CAT, POD, and APX enhanced tolerance to salinity stress. Osmopriming with PEG has improved sorghum (*Sorghum bicolor*) seed germination and seedling establishment under adverse soil moisture conditions and has been correlated with antioxidant system activation (APX, CAT, POD, and SOD) [3]. Rice (*Oryza sativa*) seeds primed with polyethylene glycol (PEG) showed increased activity of APX in parallel with decreased activity of SOD, POD, and CAT under ZnO nanoparticles stress [38]. The same authors have also observed downregulation of genes encoding the antioxidant enzymes (*APXa, APXb, CATa, CATb, CATc, SOD2*, and *SOD3*) in PEG primed seeds under nano-ZnO stress. They have concluded that priming with PEG significantly alleviates the toxic effects of nano-ZnO through improved cell structures of leaf and roots.

Seed aging during storage is associated with ROS production. Appearance of oxidative stress results in a decrease of seed quality. Kibinza et al. [161] showed that priming plays an important role in seed recovery from aging through CAT activation. Their results revealed accumulation of hydrogen peroxide (H2O2) and reduction of CAT at the gene expression level and protein content during sunflower (*Helianthus annuus* L) seed aging. Interestingly, the adverse results of aging were recovered by seed osmopriming, which led to induction of CAT synthesis by activating gene expression and translation of the enzyme.

Summing up, the management of oxidative status in primed seeds plays a very important role as a machinery, which leads to protection against oxidative stress, recovery from aging, and regulation of ROS production/accumulation. Alleviation in ROS level exerts a signal, which could be perceived, transduced, and crosstalk with other signaling pathways, thus executing physiological response by activation or repression of molecular processes.
