The soil atmosphere composition

Partial pressure of oxygen depends on soil structure and compaction (Hadas, Reference Hadas, Benech-Arnold and Sanchez2004). In agricultural soils, oxygen content has been found to vary from 19 to 21%, thus indicating that it is not limiting if the soil is well drained (Richard and Boiffin, Reference Richard and Boiffin1990). However, the oxygen level can decrease to 10% when a crust is present at the soil surface leading to reduced exchange between the atmosphere and the soil (Richard and Guerif, Reference Richard and Guerif1988a,Reference Richard and Guerifb; Dekker, Reference Dekker, Black, Bradford and Vazquez-Ramos2000; Hadas, Reference Hadas, Benech-Arnold and Sanchez2004). In flooded soil when the pore system is filled with water (Gambrell et al., Reference Gambrell, Delaune, Patrick, Jackson, Davies and Lambers1991), the oxygen level can be less than 1% since there is a 10⁴ reduction in oxygen diffusion in water relative to air, and oxygen solubility in water decreases with increasing temperature being 14.56 mg l⁻¹ at 0°C, 11.25 mg l⁻¹ at 10°C, 9.09 mg l⁻¹ at 20°C and 7.49 mg l⁻¹ at 30°C.

The level of CO₂ does not usually exceed 0.5–1% in drained soil, but can increase up to 5–8% in flooded soils and silt loams when decomposition of organic matter occurs (Buyanovsky and Wagner, Reference Buyanovsky and Wagner1983; Nakayama and Kimball, Reference Nakayama and Kimball1988).

Seed coat structures: barriers to oxygen diffusion to the embryo

Embryo covering structures include nucellus and testa and endosperm in some species. The pericarp is fused to the seed testa in the case of caryopses, and it is not fused to the testa in the case of achenes. They are associated with various other structures among which hulls consisting of glumellae or glumes depending on the Poaceae species, and dead perianth tissues, for example in beet. They inhibit seed germination by controlling seed water uptake and oxygen supply to the embryo (Bewley and Black, Reference Bewley and Black1994; MacGregor et al., Reference MacGregor, Kendall, Florance, Fedi, Moore, Paszkiewicz, Smirnoff and Penfield2015). Surrounding structures impact oxygen diffusion and are responsible for a reduction of oxygen diffusion to the embryo that depends on their thickness and their biochemical properties. Oxygen goes through the seed coat by dissolving in the water they contain during seed imbibition, but since oxygen solubility is low, little oxygen reaches the embryo. Imbibed coats become then a barrier to oxygen diffusion since they constitute a continuous wet layer surrounding the embryo (Côme, Reference Côme1970, Reference Côme and Mazliak1982; Côme and Tissaoui, Reference Côme, Tissaoui and Heydecker1973; Côme and Corbineau, Reference Côme, Corbineau, Jiarui and Khan1992, Reference Côme, Corbineau and Mazliak1998).

Furthermore, seed coats of numerous seeds often contain phenolic compounds such as phenolic acids (p-hydroxybenzoic-, vanillic-, gallic-, p-coumaric-, caffeic-, ferulic-, sinapic-, chlorogenic acids), coumarins, flavonoids and tannins that are end products of flavonoid biosynthetic pathway (Glennie, Reference Glennie1981; Lenoir et al., Reference Lenoir, Corbineau and Côme1986; Winkel-Shirley, Reference Winkel-Shirley1998, Reference Winkel-Shirley2001; Croteau et al., Reference Croteau, Kutchan, Lewis, Buchanam, Gruissem and Jones2000; Tian et al., Reference Tian, Nakamura and Kayara2004; Lepiniec et al., Reference Lepiniec, Debeaujon, Routaboul, Baudry, Pourcel, Nesi and Caboche2006; Rodriguez et al., Reference Rodriguez, Barrero, Corbineau, Gubler and Benech-Arnold2015). In the presence of oxygen, the oxidation of the phenolic compounds is mediated by various enzymes: monophenol oxidase which converts monophenols into o-diphenols, catechol oxidase which oxidizes the o-diphenols into o-quinones and laccases (Macheix et al., Reference Macheix, Fleuriet and Billot1990, Reference Macheix, Fleuriet and Jay-Allemand2005; Debeaujon et al., Reference Debeaujon, Lepiniec, Pourcel, Routaboul, Bradford and Nonogaki2007). In both cases, quinones are then polymerized into brown pigment by a non-enzymatic mechanism. For example, flavan-4-ols, precursors of phlobaphenes, confer a red colour to wheat, maize and rice grains (Grotewold et al., Reference Grotewold, Drummond, Bowen and Peterson1994; Himi et al., Reference Himi, Mares, Yanagisawa and Noda2002, Reference Himi, Yamashita, Haruyama, Yanagisawa, Maekawa and Taketa2012), but condensation of flavan-3-ols leads to proanthocyanidin synthesis the oxidation of which confers a brown colour (Winkel-Shirley, Reference Winkel-Shirley1998).

Many studies have demonstrated that phenolic compounds, in particular flavonoids, secondary metabolites derived from the phenylpropanoid pathway (Lepiniec et al., Reference Lepiniec, Debeaujon, Routaboul, Baudry, Pourcel, Nesi and Caboche2006; Debeaujon et al., Reference Debeaujon, Lepiniec, Pourcel, Routaboul, Bradford and Nonogaki2007) reinforce coat-imposed dormancy. For example, Arabidopsis seeds accumulate flavonols, and proanthocyanidins during development, the brown colour being conferred by proanthocyanidin after oxidation. This pathway has been characterized using transparent testa tt(g) mutants that are affected in seed coat pigmentation (Debeaujon et al., Reference Debeaujon, Lepiniec, Pourcel, Routaboul, Bradford and Nonogaki2007; North et al., Reference North, Baud, Debeaujon, Dubos, Dubreucq, Grappin, Jullien, Lepiniec, Mario-Poll, Miquel, Rajjou, Routaboul and Caboche2010); mutants lacking PAs germinate faster than the corresponding wild-type brown seeds. Flavonoids, particularly condensed tannins, are known to regulate water uptake (Debeaujon et al., Reference Debeaujon, Léon-Kloosterziel and Koornneef2000; MacGregor et al., Reference MacGregor, Kendall, Florance, Fedi, Moore, Paszkiewicz, Smirnoff and Penfield2015), however, their impact on oxygen diffusion is unknown. In wheat and rice, red grains show a more intense dormancy than the white grain lines and thus have a higher resistance to pre-harvest sprouting (PHS) (Cohn and Hughes, Reference Cohn and Hughes1981; Flintham, Reference Flintham2000), this trait has provided breeders with an easy character to select cultivars with higher dormancy even if ‘red gene’ contribution to dormancy is small (Warner et al., Reference Warner, Kudrna, Spaeth and Jones2000; Himi et al., Reference Himi, Yamashita, Haruyama, Yanagisawa, Maekawa and Taketa2012).

In cereals, the covering structures result in the inability of the grains to germinate at temperature higher than 10–15°C. In barley, the glumellae have the main role in dormancy (Lenoir et al., Reference Lenoir, Corbineau and Côme1983, Reference Lenoir, Corbineau and Côme1986; Corbineau and Côme, Reference Corbineau and Côme1996), while in oat both glumellae with pericarp, and probably testa participate to dormancy (Corbineau et al., Reference Corbineau, Lecat and Côme1986). In naked grains such as sorghum and wheat, dormancy results from the pericarp, testa and endosperm (Steinbach et al., Reference Steinbach, Benech-Arnold, Kristof, Sanchez and Marcucci Poltri1995). The surrounding structures inhibit germination by reducing oxygen supply to the embryo due to enzymatic and/or non-enzymatic oxidation of the phenolic compounds (Côme, Reference Côme1967, Reference Côme1980/81; Lenoir et al., Reference Lenoir, Corbineau and Côme1986; Fig. 1). The inhibitory effect of the covering structures increases with increasing temperature since the fixation of oxygen in the surrounding structures increases with temperatures (Lenoir et al., Reference Lenoir, Corbineau and Côme1986; Lecat et al., Reference Lecat, Corbineau and Côme1992). In barley, for example, more than 50% of the total oxygen uptake by the grain results from oxidation of phenolic compounds in the glumellae, whereas it is less than 20% at 10–15°C (Côme et al., Reference Côme, Corbineau and Lecat1988; Fig. 1). More recently using fibre-optic O₂ micro-sensor, Hoang et al. (Reference Hoang, Bailly, Corbineau and Leymarie2013) demonstrated that the oxygen content in the embryo below the glumellae is around 15.8% at 15°C, temperature that allows germination, but only 0.3% at 30°C, temperature at which grains remain dormant.

Fig. 1.

Effects of the surrounding structures on oxygen supply to the embryo. (A) Effects of temperature on the oxygen flux through the coats (pericarp + glumellae) in dormant barley grains incubated at 15°C (left) and 30°C (right). Increase in temperature results in a decrease in oxygen solubility in water during seed imbibition and in an increase in oxygen uptake through oxidation of phenolic compounds. As a result, the embryo receives less oxygen at 30°C than at 10–15°C. Adapted from Côme and Tissaoui (Reference Côme, Tissaoui and Heydecker1973), Lenoir et al. (Reference Lenoir, Corbineau and Côme1983, Reference Lenoir, Corbineau and Côme1986), Côme et al. (Reference Côme, Corbineau and Lecat1988), Côme and Corbineau (Reference Côme, Corbineau, Jiarui and Khan1992) and Corbineau and Côme (Reference Corbineau, Côme, Kigel and Galili1995). (B) Oxidation of phenolic compounds in the seed coats. The phenolic compounds are first oxidized to quinones by polyphenol oxidases (PPO) and then the quinones undergo non-enzymatic oxidative polymerization. From Côme and Corbineau (Reference Côme, Corbineau, Jiarui and Khan1992) and Corbineau and Côme (Reference Corbineau, Côme, Kigel and Galili1995).

However, afterripening that breaks dormancy has no effect on the phenolic compound composition nor on the polyphenol oxidase activity, but delays in time the oxygen uptake by the glumellae allowing a good oxygen supply to the embryo during the first hours of imbibition (Lenoir et al., Reference Lenoir, Corbineau and Côme1983, Reference Lenoir, Corbineau and Côme1986; Corbineau et al., Reference Corbineau, Lecat and Côme1986).

Relationships between seed dormancy and responsiveness to oxygen

For some species, dormant seeds do not germinate in air (21% oxygen), but their germination capacities are improved in hypoxia. Table 2 shows the sensitivity to oxygen of Oldenlandia corymbosa L. seeds as a function of oxygen in dormant seeds (at harvest) and non-dormant ones (after breaking of dormancy by a chilling treatment) (Corbineau and Côme, Reference Corbineau and Côme1980a). In dormant seeds, 91–95% of the population easily germinate in a narrow range of oxygen (5–7%) when the non-dormant seeds germinate in air and in a wide range of oxygen concentration from 5 to 21%. Similar behaviour was observed in dormant apple embryos (Côme et al., Reference Côme, Perino and Ralambosoa1985); at harvest, dormant apple embryos do not germinate in air at 15°C, but their germination is improved at lower oxygen concentration. Release of dormancy enables them to germinate in a large range of oxygenation (Côme et al., Reference Côme, Perino and Ralambosoa1985).

Table 2.

Effects of oxygen tensions on the germination of dormant and non-dormant seeds of Oldenlandia corymbosa incubated for 7 d at 40°C in continuous light

Means of 3 replicates.

Modified from Corbineau and Côme (Reference Corbineau and Côme1980a, Reference Corbineau and Côme1980/81).

However, in the majority of species, seeds are more sensitive to oxygen deprivation when they are dormant than the non-dormant ones. This influence of dormancy on oxygen sensitivity has been demonstrated with cereals like barley (Corbineau and Côme, Reference Corbineau and Côme1980b; Benech-Arnold et al., Reference Benech-Arnold, Gualano, Leymarie, Côme and Corbineau2006), wheat (Corbineau et al., Reference Corbineau, Sanchez, Côme and Chaussat1981) and oat (Lecat et al., Reference Lecat, Corbineau and Côme1992), sunflower (Gay et al., Reference Gay, Corbineau and Côme1991; Corbineau and Côme, Reference Corbineau, Côme, Jiarui and Khan1992, Reference Corbineau, Côme, Kigel and Galili1995) and Douglas fir (Pseudotsuga menziesii) (Corbineau et al., Reference Corbineau, Bianco, Garello and Côme2002). Figure 3 shows, in barley, the changes in oxygen sensitivity of the intact grains (Fig. 3A) and isolated embryos (Fig. 3B) incubated on water at 20°C at harvest (i.e. dormant grains) and during afterripening (grains dry-stored for 5 and 9.5 months at 25°C). At harvest in 2000, less than 50% of the grain population can germinate at 20°C in air (21% oxygen) and no germination occurred in atmosphere containing less than 3% oxygen (Fig. 3A). Dry storage of dormant grains for 5 and 9.5 months at 25°C results in breaking of dormancy allowing germination of all the grain population in air and reducing the oxygen concentration at which 50% of the population germinate to 4.8% and 0.4% (Fig. 3A). By contrast, isolated embryos from dormant grains at harvest germinate in atmosphere containing at least 5% oxygen (Fig. 3B, curve 1) demonstrating that the covering structures are involved in oxygen diffusion (c.f. Oxygen requirement for germination of non-dormant seeds). Afterripening also resulted in an improvement of embryo germination in hypoxia: 100% of the population germinate in 4 and 0.5% oxygen, respectively, after 5 and 9.5 months of dry storage at 25°C (Fig. 3B, curves 2 and 3).

Fig. 3.

Effects of oxygen concentration in the atmosphere on the germination percentages obtained after 5 d at 20°C with barley caryopses (A) and embryos (B) at harvest (1) and after 5 months (2) and 9.5 months (3) of afterripening at 25°C. Seeds harvested in 2000 and stored in the open air for 5 and 9.5 months at 25°C. Modified from Bradford et al. (Reference Bradford, Benech-Arnold, Côme and Corbineau2008).

Using a population-based threshold model, Bradford et al. (Reference Bradford, Benech-Arnold, Côme and Corbineau2008) have calculated a median base (or threshold) O₂ [(Ox(b)(50)]. The model predicted that 36.3% oxygen would be required for 50% germination of intact dormant grains at harvest in 2000, while the germination of isolated embryos was predicted to require only 0.123% oxygen to achieve 50% germination. For the intact grains, the Ox(b)(50) value was 2.87% and 0.30% after 5 and 9.5 months of afterripening, respectively (Bradford et al., Reference Bradford, Benech-Arnold, Côme and Corbineau2008). Environmental conditions during seed production influence their sensitivity to oxygen. Hence, the Ox(b)(50) is dependent of the year of harvest and was decreased to 15.7% for grains obtained in 2002 compared to 36.3% for those obtained in 2000 (Fig. 3A and Table 3).

Table 3.

Effects of ABA or GA₃ concentration on oxygen tensions on the median O₂ threshold Ox_b(50) calculated using a population-based threshold model in dormant seeds and isolated embryos in barley

Seeds harvested in 2002.

Modified from Bradford et al. (Reference Bradford, Benech-Arnold, Côme and Corbineau2008).

Specific effect of hypoxia and anoxia on breaking of dormancy and induction of secondary dormancy

Oxygen is required for germination, however partial or total oxygen deprivation is not always harmful at least if it is not too prolonged. Temporary anaerobiosis applied to imbibed seeds may promote germination in dormant seeds of some species (Lonchamp and Gora, Reference Lonchamp and Gora1979; Côme et al., Reference Côme, Perino and Ralambosoa1985) and may substitute for cold stratification (Côme et al., Reference Côme, Corbineau, Soudain, Jackson, Davies and Lambers1991; Corbineau and Côme, Reference Corbineau, Côme, Kigel and Galili1995). For example, incubation in anoxia (pure nitrogen) for 2 or 3 weeks breaks dormancy in apple embryos (Tissaoui and Côme, Reference Tissaoui and Côme1973), Oldenlandia corymbosa seeds (Corbineau and Côme, Reference Corbineau and Côme1988), sunflower achenes (Corbineau and Côme, Reference Corbineau, Côme, Kigel and Galili1995) and Xanthium pennsylvanicum (Esashi et al., Reference Esashi, Kotaki and Ohhara1976). In the case of apple embryos, this beneficial effect of anoxia increases with temperature (Tissaoui and Côme, Reference Tissaoui and Côme1973).

In contrast, incubation of seeds in anoxia or hypoxia, conditions which inhibit seed germination, induces secondary dormancy in barley (Hoang et al., Reference Hoang, Bailly, Corbineau and Leymarie2013), Xanthium pennsylvanicum (Esashi et al., Reference Esashi, Okazaki, Yanai and Hishinuma1978), Viola spp., Veronica hederofolia and Veronica persica (Lonchamp and Gora, Reference Lonchamp and Gora1979), lettuce (Vidaver and Hsiao, Reference Vidaver and Hsiao1975) and Brassica napus (Pekrun et al., Reference Pekrun, Lutman and Baeumer1997). In Oldenlandia corymbosa seeds, responsiveness to light depends also on the temperature and on the oxygen tension of the atmosphere. Seeds remain responsive to white light at 10 and 20°C, but lose their responsiveness during incubation in darkness at temperature of at least 30°C; this phenomenon has been observed in many photoblastic seeds (Karssen, Reference Karssen1967; Rollin, Reference Rollin1970) and is accelerated at low oxygen concentration in Oldenlandia corymbosa (Corbineau and Côme, Reference Corbineau and Côme1985).

Effects of hypoxia on ABA and GA metabolism and sensitivity

The hormonal regulation of dormancy and germination is well established (Bewley, Reference Bewley1997; Finch-Savage and Leubner-Metzger, Reference Finch-Savage and Leubner-Metzger2006; Finkelstein et al., Reference Finkelstein, Reeves, Ariizumi and Steber2008; Holdsworth et al., Reference Holdsworth, Bentsink and Soppe2008; Graeber et al., Reference Graeber, Nakabayashi, Miatton, Leubner-Metzger and Soppe2012; Shu et al., Reference Shu, Liu, Xie and He2016; Nonogaki, Reference Nonogaki2017). Oxygen uptake by the glumellae during the first 12–14 h of imbibition appears to be related to ABA metabolism in the embryo, as a transient increase in ABA content occurs in dormant grains and not in non-dormant ones (Benech-Arnold et al., Reference Benech-Arnold, Gualano, Leymarie, Côme and Corbineau2006). The presence of glumellae alters the expression of genes involved in ABA synthesis (HvNCED1, NCED2), ABA catabolism (HvABA8’OH1) and signalling (HvABI5, HvVP1 and HvPKABA) (Mendiondo et al., Reference Mendiondo, Leymarie, Farrant, Corbineau and Benech-Arnold2010). During imbibition, the decrease in embryonic ABA level is slower in hypoxia than in air, but this effect is independent of the regulation of HvABA8’OH1 expression (Mendiondo et al., Reference Mendiondo, Leymarie, Farrant, Corbineau and Benech-Arnold2010; Hoang et al., Reference Hoang, Bailly, Corbineau and Leymarie2013). Incubation of embryos of primary dormant grains in the presence of exogenous ABA at various concentrations shows that hypoxia increased embryo sensitivity to ABA by twofold (Fig. 4), and this effect was more pronounced at 30°C than at 20°C (Benech-Arnold et al., Reference Benech-Arnold, Gualano, Leymarie, Côme and Corbineau2006), suggesting that hypoxia imposed by glumellae increases embryo sensitivity to ABA.

Fig. 4.

Effects of ABA concentration on the germination percentages obtained after 7 d with embryos isolated from dormant barley grains placed at 30°C in 21% (filled circles), 15% (open circles), 10% (filled triangles) and 5% (open triangles) oxygen. Seeds harvested in 2000. Means of 2 measurements ± arithmetical spread. From Benech-Arnold et al. (Reference Benech-Arnold, Gualano, Leymarie, Côme and Corbineau2006).

ABA delayed and reduced seed and embryo germination; using a population-based threshold model, Bradford et al. (Reference Bradford, Benech-Arnold, Côme and Corbineau2008) show that ABA concentration up to 10 μM had relatively little effect on O₂ sensitivity of seeds. However, at higher concentration, the value of Ox_b50 increased from 4.8% in the absence of ABA to 12 and 14.5% in the presence of 100 and 1000 μM ABA, respectively (Table 3). Incubation of embryos in the presence of GA₃ results in a decrease in Ox_b50 (Table 3) from 18.4% on water down to 8.2% in the presence of 1000 μM GA.

Hypoxia at 15°C which induced secondary dormancy in barley was related to an increased expression of ABA synthesis genes (HvNCED2) only after 3 d and a strong regulation of gibberellin (GA) metabolism genes with promotion in HvGA2ox3 (64-fold compared with grains in air) and inhibition of HvGA3ox2 and HvGA20ox1 after 1 d (Hoang et al., Reference Hoang, Bailly, Corbineau and Leymarie2013). In addition, embryo sensitivity to ABA increases in hypoxia and seeds became insensitive to GA at low oxygen (Benech-Arnold et al., Reference Benech-Arnold, Gualano, Leymarie, Côme and Corbineau2006). However, hypoxia did not alter the embryo sensitivity to ABA and GA which was similar in both primary and hypoxia-induced secondary dormant grains (Hoang et al., Reference Hoang, Bailly, Corbineau and Leymarie2013).

Effect on fermentation

Although EC has been cited as being essential in the regulation of germination (Raymond and Pradet, Reference Raymond and Pradet1980; Raymond et al., Reference Raymond, Al-Ani and Pradet1985), dormancy in oat does not result from an inability to synthesize ATP from ADP and AMP (Côme and Corbineau, Reference Côme, Corbineau and Taylorson1989). During seed incubation at 30°C, a temperature at which dormant grains cannot germinate, embryos from dormant and non-dormant grains exhibited a similar increase in the ATP/ADP ratio and in the EC [(ATP + 0.5 ADP)/(ATP + ADP + AMP)] as long as seeds had not germinated (Côme et al., Reference Côme, Corbineau and Lecat1988). In anoxia (nitrogen) or hypoxia (1% oxygen), the immediate response at the level of mitochondria corresponded to a decrease in ATP synthesis and in the EC within 10 min (Lecat, Reference Lecat1987; Table 4), the secondary responses resulted in an increase of EC that was stabilized after 1 to 3 h due to fermentation and accumulation of ethanol in starchy seeds (Tables 4 and 5) (Pradet and Raymond, Reference Pradet and Raymond1983; Raymond et al., Reference Raymond, Al-Ani and Pradet1985; Lecat, Reference Lecat1987). Ethanol is the major product of fermentation in higher plants whether they are tolerant to anoxia or not (Raymond et al., Reference Raymond, Al-Ani and Pradet1983, Reference Raymond, Al-Ani and Pradet1985; Ricard et al., Reference Ricard, Couée, Raymond, Saglio, Saint-Ges and Pradet1994). Late responses leading to tolerance to oxygen deprivation were associated with a decrease in ADH activity and a change in synthesis of malate, succinate, lactate and shikimate resulting from amino-acid metabolism instead of ethanol (Raymond et al., Reference Raymond, Al-Ani and Pradet1985; Bui et al., Reference Bui, Novi, Lombardi, Lannuzzi, Rossi, Santaniello, Mensuali, Corbineau, Giuntoli, Perata, Zaffagnini and Licausi2019). In higher plants, l-lactate is often produced prior to ethanol in the first min after the transfer to anoxia, and alanine is the third major fermentation product in plants. The adenylate EC under anoxia depends on the seed reserves; in group I (lipid seeds including lettuce, sunflower, radish, turnip, cabbage, flax and soybean), germination does not occur in oxygen pressure below 1 kPa and the EC is below 0.4 while it remains at a higher value (0.70–0.82) in seeds from group II (i.e. starch seeds as rice, wheat, maize, sorghum and pea) for which germination still occurs at 0.1 kPa (Table 5; Al-Ani et al., Reference Al-Ani, Bruzau, Raymond, Saint-Ges, Leblanc and Pradet1985).

Table 4.

Change in energy charge (EC) and metabolic responses in oat embryos placed in anoxia (0% oxygen) or hypoxia (1% oxygen) after 8 h incubation of oat grains on water at 30°C and in air

Grains harvested in 1984. Mean of 3 measurements ± SD.

ADH, alcohol dehydrogenase.

Modified from Lecat (Reference Lecat1987).

Table 5.

Effects of a 3-h incubation in anoxia at 20°C on the energy charge (EC) and ethanol accumulated in seeds

Modified from Pradet and Raymond (Reference Pradet and Raymond1983) and Raymond et al. (Reference Raymond, Al-Ani and Pradet1985).

In contrast, change in non-adenylic triphosphate nucleotides (NTP) content within the embryo during seed incubation at 30°C depended on the dormancy state (Corbineau et al., Reference Corbineau, Vinel, Leymarie, Côme, Adkins, Ashmore and Navie2007). Thermo-dormant grains lost their ability to synthesize NTP from adenine after transfer from 30 to 20°C; the capacity of synthesis of NTPs seems then to be a good marker of dormancy in oat seeds, as in buds of Helianthus tuberosus L. tubers (Le Floc'h and Lafleuriel, Reference Le Floc'h and Lafleuriel1981), of Fraxinus excelsior L. (Lavarenne et al., Reference Lavarenne, Champciaux, Barnola and Gendraud1982), Quercus spp. (Barnola et al., Reference Barnola, Crochet, Payan, Gendraud and Lavarenne1986) and Prunus persica L. (Balandier et al., Reference Balandier, Gendraud, Rageau, Bonhomme, Richard and Parisot1993).

In addition, modulation of EC using NaN₃ and polyethylene-glycol (PEG) has demonstrated that the induction of thermodormancy in oat seeds is an active phenomenon requiring an EC higher than 0.5 (Corbineau et al., Reference Corbineau, Vinel, Leymarie, Côme, Adkins, Ashmore and Navie2007). Exogenous GA strongly stimulated germination of oat seeds at high temperature and in hypoxia (Lecat et al., Reference Lecat, Corbineau and Côme1992) but has no effect on EC.

Involvement of the N-end rule pathway

The ubiquitin-proteasome system (UPS) degrades the proteins by targeting specific signals known as degrons. Among the degradation signals, N-terminus is the one targeted at the N-terminal residues rather than the sequence elements of the protein (Bachmair et al., Reference Bachmair, Finley and Varshavsky1986). The N-end rule pathway has three branches: the Arg/N-end rule pathway, the Ac/N-end rule pathway and the Pro/N-end rule pathway (Lee et al., Reference Lee, Heo, Kim and Hwang2016; Nguyen et al., Reference Nguyen, Mun, Lee and Hwang2018, Reference Nguyen, Lee, Mun, Truong, Park and Hwang2019; Dong et al., Reference Dong, Zhang, Li, Tempel, Loppnau and Min2018, Zhang et al., Reference Zhang, Gannon, Hassall, Deery, Gibbs, Holdsworth, van der Hoorn, Lilley and Theodoulou2018a,Reference Zhang, Gannon, Jones, Rundle, Hassall, Gibbs, Holdsworth and Theodouloub). The Arg/N-end rule pathway identifies specific N-degrons such as aromatic residues (Phe, Trp, Tyr) by PROTEOLYSIS 1 (PRT1) and basic residues (Arg, His, Lys) by PRT6 in plant. Tertiary destabilizing residues (Asn, Gln and Cys) are first modified, either enzymatically (by deamidation of Asn or Gln) or chemically (by oxidation of Cys), to generate a secondary destabilizing residue (Asp, Glu and oxidized Cys). Studies reviewed by Licausi (Reference Licausi2011, Reference Licausi2013), Voesenek and Bailey-Serres (Reference Voesenek and Bailey-Serres2015) and Loreti and Perata (Reference Loreti and Perata2020) indicate that the sensory system to hypoxia relies on an oxygen-mediated branch of the N-end rule pathway for protein degradation acting on a specific clade of ethylene-responsive transcription factor (ERF-VII). Both the N-end rule pathway and ERF-VII are components of the oxygen signalling pathway, in particular involved in responses to low oxygen stress (Gibbs et al., Reference Gibbs, Lee, Isa, Gramuglia, Fukao, Bassel, Correia, Corbineau, Theodoulou, Bailey-Serres and Holdsworth2011, Reference Gibbs, Conde, Berckhan, Prasad, Mendiondo and Holdsworth2015; Licausi, Reference Licausi2011, Reference Licausi2013; Licausi et al., Reference Licausi, Kosmacz, Weits, Giuntoli, Giorgi, Voesenek, Perata and Van Dongen2011, Reference Licausi, Pucciariello and Perata2013; Hartman et al., Reference Hartman, Sasidharan and Voesenek2021). Group VII ERF transcription factors are either constitutively expressed and/or differentially transcriptionally regulated in response to different signals including low oxygen (Bailey-Serres et al., Reference Bailey-Serres, Fukao, Gibbs, Holdsworth, Lee, Licausi, Perata, Voesenek and Van Dongen2012; Gibbs et al., Reference Gibbs, Bacardit, Bachmair and Holdsworth2014a,Reference Gibbs, Isa, Movahedi, Lozano-Juste, Mendiondo and Berckhanb, Reference Gibbs, Conde, Berckhan, Prasad, Mendiondo and Holdsworth2015; Voesenek and Bailey-Serres, Reference Voesenek and Bailey-Serres2015; Loreti and Perata, Reference Loreti and Perata2020; Hartman et al., Reference Hartman, Sasidharan and Voesenek2021). Four of the five ERFs, HYPOXIA RESPONSIVE1 (HRE1 and HRE2) and RELATED TO APETALA 2.2 (RAP 2.2 and RAP 2.12) are implicated in the regulation of hypoxia-responsive genes. Under normoxia, ERFs are degraded via the N-end rule pathway of proteolysis. When oxygen becomes limiting, the degradation of the ERFs by the N-end rule pathway is inhibited because of the reduction of oxygen-mediated Cys oxidation (Bailey-Serres et al., Reference Bailey-Serres, Fukao, Gibbs, Holdsworth, Lee, Licausi, Perata, Voesenek and Van Dongen2012; Gibbs et al., Reference Gibbs, Conde, Berckhan, Prasad, Mendiondo and Holdsworth2015; Loreti and Perata, Reference Loreti and Perata2020; Hartman et al., Reference Hartman, Sasidharan and Voesenek2021).