Germination induction of dormant Avena fatua caryopses by KAR₁ and GA₃ involving the control of reactive oxygen species (H₂O₂ and O₂⁻) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers

Abstract

Avena fatua L. caryopses did not germinate at 20 °C in darkness because they were dormant. However, they were able to germinate in the presence of karrikinolide (KAR₁), a key bioactive compound present in smoke, and also in the presence of gibberellin A₃ (GA₃), a commonly known stimulator of seed germination. The aim of this study was to collect information on a possible relationship between the above regulators and abscisic acid (ABA), reactive oxygen species (ROS) and ROS scavenging antioxidants in the regulation of dormant caryopses germination. KAR₁ and GA₃ caused complete germination of dormant A. fatua caryopses. Hydrogen peroxide (H₂O₂), compounds generating the superoxide (O₂⁻), i.e. menadione (MN), methylviologen (MV) and an inhibitor of catalase activity, aminotriazole (AT), induced germination of dormant caryopses. KAR₁, GA₃, H₂O₂ and AT decreased ABA content in embryos. Furthermore, KAR₁, GA₃, H₂O₂, MN, MV and AT increased α-amylase activity in caryopses. The effect of KAR₁ and GA₃ on ROS (H₂O₂, O₂⁻) and activities of the superoxide dismutase (SOD) and catalase (CAT) were determined in caryopses, embryos and aleurone layers. SOD was represented by four isoforms and catalase by one. In situ localization of ROS showed that the effect of KAR₁ and GA₃ was associated with the localization of hydrogen peroxide mainly on the coleorhiza. However, the superoxide was mainly localized on the surface of the scutellum. Superoxide was also detected in the protruding radicle. Germination induction of dormant caryopses by KAR₁ and GA₃ was related to an increasing content of H₂O₂, O₂⁻and activities of SOD and CAT in embryos, thus ROS homeostasis was probably required for the germination of dormant caryopses. The above regulators increased the content of ROS in aleurone layers and decreased the activities of SOD and CAT, probably leading to the programmed cell death. The presented data provide new insights into the germination induction of A. fatua dormant caryopses by KAR₁ and also by GA₃. In A. fatua, KAR₁ or GA₃ is included in the induction germination of dormant caryopses through regulation level of ABA in embryos and ROS-antioxidant status both in embryos and aleurone layers.

Introduction

Seed dormancy can be defined as an inability of viable imbibed seeds to germinate under conditions that are favorable for the germination process (Bewley, 1997). It is commonly accepted that the balance between abscisic acid (ABA) and gibberellins (GAs) and sensitivity to these hormones are responsible for the regulation of the dormancy state and germination of dormant seeds (Cadman et al., 2006, Finch-Savage and Leubner-Metzger, 2006). ABA is the most important hormone responsible for the establishment and maintenance of dormancy in seeds. A decrease in the ABA content in seeds by physical factors, chemicals or genetic manipulation reduces dormancy (Finch-Savage and Leubner-Metzger, 2006). GAs have been considered as promotors of progression from dormancy release through germination (Finkelstein et al., 2008). Dormancy release may involve a decline in the ABA content and an increase of GAs level (Hilhorst, 2007). According to available data, reactive oxygen species (ROS), such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH), are also involved in the regulation of dormancy in seeds (El-Maarouf-Bouteau and Bailly, 2008, Whitaker et al., 2010; Diaz-Vivancos et al., 2013). Application of exogenous ROS or compounds generating ROS can break dormancy in seeds of several plant species. When a certain level of ROS is reached, it has a signaling function and consequently results in completing seed germination (Bailly et al., 2008). For example, dormancy release associated with accumulation of ROS has been noted in sunflower seeds (Oracz et al., 2007). An adequate level of ROS depends on its production and scavenging by the enzymatic system: superoxide dismutase (SOD), catalase (CAT), peroxidases, glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and non-enzymatic compounds such as the reduced glutathione and ascorbate (El-Maarouf-Bouteau and Bailly, 2008). Cross-talk between ROS and ABA or gibberellin metabolism and signaling has been discussed in the context of controlling barley caryopses dormancy (Bahin et al., 2011). It was suggested that in barley, releasing embryo dormancy by hydrogen peroxide took place through the activation of gibberellin A₃ (GA₃) signaling and synthesis rather than through the repression of ABA signaling (Bahin et al., 2011). Germination and postgermination processes of grasses i.e. wheat, barley, wild oat, depend on events in aleurone cells synthesizing and secreting hydrolytic enzymes, mainly α-amylase to starchy endosperm, in order to mobilize storage materials. Both synthesis and secretion of hydrolytic enzymes are induced by gibberellins synthesized in embryos and diffused to the starchy endosperm (Appleford and Lenton, 1997). The aleurone cells, after the completion of their secretory role, undergo programmed cell death (PCD) (Finnie et al., 2011). PCD is stimulated by gibberellins and inhibited by ABA (Bethke et al., 1999). ROS, especially H₂O₂, are considered as key inducers of PCD. Application of H₂O₂ induces cell death in cells treated with GA, but not in those treated with ABA (El-Maarouf-Bouteau and Bailly, 2008). GA₃ down-regulates the activity of antioxidant enzymes: CAT, ascorbate peroxidase and SOD, to ensure a sufficient accumulation of hydrogen peroxide prior to the onset of cell death in barley (Fath et al., 2001a, De Pinto et al., 2012). The activity of these enzymes has been maintained in ABA treated cells (Fath et al., 2001a).

Germination of dormant and non-dormant seeds can be also induced by ecological factors such as smoke released from fire. It has been proved that smoke can enhance the seed germination of 1200 species representing more than 80 genera worldwide (Dixon et al., 2009). A primary germination stimulant has been discovered in plant-derived smoke (Van Staden et al., 2004) and burned cellulose (Flematti et al., 2004). It was named butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one, and now is known as karrikinolide (KAR₁) (Dixon et al., 2009). KAR₁, similarly to smoke can stimulate seed germination of fire-prone and non-fire-prone plant species. Both smoke and KAR₁ can stimulate germination or seedling growth of weeds and crops (Light et al., 2009). Recently, five karrikins, namely KAR₂–KAR₆ have been found in smoke (Flematti et al., 2009).

Avena fatua (wild oat) is a persistent weed in cereal production systems in many regions of the world, including Poland. After the harvest, dormancy of caryopses of this grass can be caused by the tissue surrounding the embryo, the embryo itself, or both (Adkins and Peters, 2001). Germination of dormant florets and/or caryopses can be induced by various factors, e.g. dry storage (Foley, 1994, Kępczyński et al., 2013), gibberellin (Adkins et al., 1986, Kępczyński et al., 2006, Kępczyński et al., 2013), smoke (Adkins and Peters, 2001, Kępczyński et al., 2006, Kępczyński et al., 2010) and KAR₁ (Daws et al., 2007, Stevens et al., 2007, Kępczyński et al., 2010, Kępczyński et al., 2013). Earlier studies have shown that the response of dormant A. fatua caryopses to KAR₁ requires ethylene action (Kępczyński and Van Staden, 2012) and gibberellin biosynthesis (Kępczyński et al., 2013). A stimulatory effect of KAR₁ and GA₃ on germination of dormant A. fatua caryopses is associated with increasing dehydrogenases and α, β-amylases before radicle protrusion (Kępczyński et al., 2013).

Little information can be found in the literature on the interaction between KAR₁, which is considered as a representative of a novel class of plant growth regulators (Nelson et al., 2009), and plant hormones in seeds. There is no data on the participation of ROS in the germination of dormant A. fatua caryopses. Likewise, no information is available on the interaction between KAR₁ and ROS with respect to dormancy and germination of seeds. Similarly, it is unknown whether KAR₁ and GA₃ can control ROS levels in aleurone layers of dormant A. fatua caryopses. So far, the effect of the above regulators on ROS content and antioxidants in embryo and aleurone layers from the same A. fatua caryopsis has not been determined. Until now the effect of GAs on ROS level and antioxidants was not analyzed in cereal embryo and aleurone layers in the same experiment. Likewise, no one has compared the effect of KAR₁, GA₃ and ROS on content of ABA in dormant embryos of grasses. In addition, localization of hydrogen peroxide and superoxide in embryos from caryopses incubated in the presence of KAR₁ or GA₃ is unknown. Moreover, the effect of KAR₁ and GA₃ on isoforms activities of SOD and CAT was not analyzed in caryopses.

Therefore the aim of the present study was to examine whether germination induction of dormant A. fatua caryopses by KAR₁ and GA₃ is associated with the control of ROS level, H₂O₂ and O₂⁻, and the enzymatic antioxidants that scavenge them, CAT and SOD, in two components, embryo and aleurone layers. In addition, the effect of KAR₁ and GA₃ on in situ localization of H₂O₂ and O₂⁻ in embryo and isoenzymes activity of CAT and SOD were tested. Moreover, a relationship between KAR₁, GA₃, H₂O₂ or ROS generating compounds and α-amylase activity and ABA was also analyzed.