**7. Seed priming and cell cycle regulation**

(ELP) during post-priming germination was observed in rapeseed [36]. As XTHs have the ability to cleave xyloglucans and rejoin the cut ends with new partners, they are engaged in cell wall loosening during growth and in the restructuring of the cell walls after extension.

Cytoskeleton reorganization is also necessary to achieve large rates of cells elongation that precedes radicle protrusion. The component of microtubules (β-tubulin) accumulated in tomato seeds during germination and priming and the expression preceded visible germina‐ tion [142]. Higher level of β-tubulin protein accumulation was shown in rapeseed during PEG soaking, drying, and post-priming germination. The up-regulation of genes encoding γ- and

Ultrastructural observations performed during the 6-d period of solid matrix priming (SMP) of carrot (*Daucus carrota*) seeds indicated the breakdown of storage materials, specific to the catabolic phase of germination *sensu stricto*, both in the axis and in the micropylar endosperm covering the radicle tip [143]. It was found that complete degradation of storage protein and lipid bodies and subsequent starch accumulation occurred in the radicles of carrot seeds after 8-d SMP. In the endosperm, the catabolic changes were limited to the micropylar area, where extensive breakdown of storage cell walls, partial degradation of protein bodies, and no

During seed germination, storage proteins, which provide a source of reduced nitrogen, and inorganic minerals need to be mobilized to support seedling growth. In addition, a lytic aqueous vacuolar compartment building up the turgescence necessary for cell expansion and to promote radicle protrusion and embryo elongation has to be formed (**Figure 2**). Bolte et al. [145] investigated the features and the dynamics of the vacuoles during the early stages of *Arabidopsis* seed germination and indicated the successive occurrence of two different lytic compartments in the protein storage vacuoles (PSV). The first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells [145]. Different mechanisms for the transformation of PSV into lytic vacuole in the root tip cell of germinating tobacco (*Nicotiana tabacum*) seeds were proposed by Zheng and Staehelin [146]. Ultrastructural studies demonstrated that the radicle cells of tobacco contain only one type of vacuole at particular time of development. Upon rehydration, the radicle cells only contain PSVs, but during subsequent root development, the PSVs are systematically

At present, we do not have a complete view of ultrastructural changes occurring during seed priming. One would expect that similar vacuole remodeling might occur during priming, especially in embryonic axis. The maintenance of metabolism over, for instance, several days of seed priming requires mobilization of embryo reserves. It is speculated that accumulation of endogenous osmotica, cell vacuolization, together with cell wall loosening initiated during priming, might determine the embryonic axis extension and radicle protrusion during postpriming germination. Deeper and more detailed studies should be continued in order to

β-tubulins was also noticed during post-priming germination [36].

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

transformed into lytic vacuoles via cell type specific pathways.

completely clarify this phenomenon.

storage lipid hydrolysis were observed [144].

Some of the hypotheses proposing explanation for priming-induced improvement are based on its effect on DNA in relation to activation of DNA repair mechanisms, synchronization of the cell cycle in G2 and preparation to cell division (**Figure 2**). During seed maturation, most of the embryo cells are stopped at G1 or G0 phase of cell cycle and only some species have a small proportion of cells in the G2 phase [111]. During seed imbibition, meristematic activity is limited; however, some preparation to cell division occurs. In embryos of dry tomato seeds, most cells have 2C DNA level and are in G1 phase of nuclear division [147]. The authors observed that DNA synthesis preceded germination as during imbibition in water, 4C signal was found mostly in the embryonic root tip, which suggests that cell enters S phase. They also primed seeds for 14 d in PEG-6000, which enhanced the rate and uniformity of germination. The 4C DNA signal of root tip cells increased during priming starting from 3 d incubation in PEG and was constant after re-drying the seeds to the initial moisture content. This observation suggests that priming increased the ratio of cells in G2 phase to G1 phase and indicates that the beneficial effects of priming on seedling performance are associated with the replicative DNA synthesis prior to germination [147]. This is accompanied by increase in α- and δ-like DNA polymerase activities in primed seeds and during germination.

The initiation of cell cycle and proceeding the cell to S phase may depend also on a G1 checkpoint control. Most, if not all, cell cycle proteins responsible for cell cycle control appear to be already present in dry mature seeds, although some of them should be synthesized *de novo*. However, not only protein synthesis but also their modification may play a regulatory function for cell cycle control [148]. Cell division starts just after radicle protrusion, thus, seed priming, which prolongs Phase II of seed germination and is finished just before Phase III, does not affect cell division in itself [16]. Seed priming extends Phase II, when DNA repair mechanisms and expression of genes encoding proteins needed in cell cycle control and commencement are activated and overreach the level observed in unprimed seeds. Preactivation of cell cycle by priming could be through regulation of the activity of cell cycle proteins such as cyclin-dependent protein kinases and proliferating-cell nuclear antigens [16]. It was found that osmopriming of *Brassica napus* seeds induced expression of cell division control protein 48 homolog C, cyclin P4;1, cyclin like protein and topoisomerase II in dry seeds, as well as proliferating-cell nuclear antigen 2 and cyclin dependent kinase 3;2 during imbibi‐ tion [36]. Accumulation of proliferating-cell nuclear antigen during maize seed imbibition was associated with transition of the cells from G1 to G2 [149]. Moreover, microtubules, apart from cytoskeleton formation, cytoplasmic streaming, organellar movement, and cell wall formation, function in mitotic spindle formation during mitosis. Microtubules in dry seeds are depoly‐ merized and form discrete granular bodies, which become organized into the cytoskeleton during imbibition [111, 142]. Higher expression of genes encoding microtubule-associated protein 65-1 and 70-2 as well as tubulin subunits γ-1, β-1, β-3 and microtubule motor activity proteins belonging to kinesin family was also observed during PEG soaking and in dry osmoprimed *Brassica napus* seeds [36]. Enhanced expression of tubulin genes was associated with accumulation of β-tubulin protein during osmopriming and subsequent germination [36]. Also in pre-hydrated seeds of *Arabidopsis thaliana* and *Solanum lycopersicum* accumulation of tubulins (mainly β-tubulin) was stated during germination as compared to unprimed seeds [136, 142].

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 is targeted into mitochondria [36].

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‐ going pre-sowing seed priming.
