Alleviating dormancy in Brassica oleracea seeds using NO and KAR1 with ethylene biosynthetic pathway, ROS and antioxidant enzymes modifications

BackgroundSeed dormancy is a prevailing condition in which seeds are unable to germinate, even under favorable environmental conditions. HarvestedBrassica oleracea(Chinese cabbage) seeds are dormant and normally germinate (poorly) at 21 °C. This study investigated the connections between ethylene, nitric oxide (NO), and karrikin 1 (KAR1) in the dormancy release of secondary dormantBrassica oleraceaseeds.ResultsNO and KAR1 were found to induce seed germination, and stimulated the production of ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC), and both ethylene biosynthesis enzyme ACC oxidase (ACO) [1] and ACC synthase (ACS) [2]. In the presence of NO and KAR1, ACS and ACO activity reached maximum levels after 36 and 48 h, respectively. The inhibitor of ethylene 2,5-norbornadiene (NBD) had an adverse effect onBrassica oleraceaseed germination (inhibiting nearly 50% of germination) in the presence of NO and KAR1. The benefits from NO and KAR1 in the germination of secondary dormantBrassica oleraceaseeds were also associated with a marked increase in reactive oxygen species (ROS) (H2O2and O2˙ˉ) and antioxidant enzyme activity at early germination stages. Catalase (CAT) and glutathione reductase (GR) activity increased 2 d and 4 d, respectively, after treatment, while no significant changes were observed in superoxide dismutase (SOD) activity under NO and KAR1 applications. An increase in H2O2and O2˙ˉ levels were observed during the entire incubation period, which increasing ethylene production in the presence of NO and KAR1. Abscisic acid (ABA) contents decreased and glutathione reductase (GA) contents increased in the presence of NO and KAR1. Gene expression studies were carried out with seven ethylene biosynthesis ACC synthases (ACS) genes, two ethylene receptors (ETR) genes and one ACO gene. Our results provide more evidence for the involvement of ethylene in inducing seed germination in the presence of NO and KAR1. Three out of seven ethylene biosynthesis genes (BOACS7, BOACS9andBOACS11), two ethylene receptors (BOETR1andBOETR2) and one ACO gene (BOACO1) were up-regulated in the presence of NO and KAR1.ConclusionConsequently, ACS activity, ACO activity and the expression of different ethylene related genes increased, modified the ROS level, antioxidant enzyme activity, and ethylene biosynthesis pathway and successfully removed (nearly 98%) of the seed dormancy of secondary dormantBrassica olereaceseeds after 7 days of NO and KAR1 application.