# Abscisic Acid--Defensive Player in Flax Response to _Fusarium culmorum_ Infection
## 4 Discussion
For instance, _Arabidopsis_ mutants with impaired NOX function (_ArthohD, AtrbU_F) were more susceptible to pathogens compared with the wild type [47]. It was evidenced that respiratory burst oxidase homolog protein D (GhRbohD) positively regulates cotton resistance to _Verticillium dahlia_[48]. Numerous studies provided evidence that the NOXs' role in plant immunity comes from their interactions with other immunity signal transducers such as Rac/Rop small GTPases [49] or ABA [50]. Thus, ABA may be perceived as ROS inducer, and in fact such observations were made for abiotically stressed plants where it is connected with stomatal closure [51] and during pathogen infection [52]. Our results show increased levels of NOX gene expression (_RbohD, RbohF_). Moreover, a positive correlation (0.79) between _RbohF_ and ABA level can be noted. In addition, we noticed an increase in _RbohD_ and _RbohF_ transcript levels in flax treated with (+)ABA (in different concentrations ranging from 2.5 uM to 1 mM). No H\({}_{2}\)O\({}_{2}\) level change was detected though, which may be due to the activation of genes encoding the enzymes processing the reactive oxygen forms (catalase and peroxidase) (Supplementary File S6). A similar observation of H\({}_{2}\)O\({}_{2}\) level and those enzymes was made for the _F. culmorum_ treated flax. Increased activity of ascorbate peroxidase (_APx_) gene (growing in each subsequent time-point) was determined. Additionally, transcript level of catalase was increased, though later compared with that of _APx_. Peroxidases are involved in a broad range of physiological processes throughout the plant life cycle and are important players in plant defense reaction. Among others, they are involved in lignin and suberin formation, cross-linking of cell wall components, and the metabolism of ROS and RNS (reactive nitrogen species). Moreover, peroxididases may play a role in the ABA-dependent signaling pathway, activated both under abiotic and biotic stress conditions [53]. Although the H\({}_{2}\)O\({}_{2}\) assay showed no statistically significant differences between _F. culmorum_ infected and untreated control flax seedlings it is known that not only hydrogen peroxide is involved in signaling but also NO and Ca\({}^{2+}\) can take part in plant responses to stresses [54]. Especially Ca\({}^{2+}\) was recently discovered as an initial factor in plant adaptation to difficult environmental conditions [55]. Worth noting is the existence of a cross talk between free radicals and this should be investigated in flax response to Fc infection in a wider perspective because NO may cause inhibition of hydrogen peroxide accumulation as it was proven for lettuce [56]. Changes detected in the transcript levels of callose synthesis genes correspond with elevated amounts of callose in 48 hpi. Callose deposits together with other possible reinforcement of cell wall through peroxidase activity may greatly contribute to the formation of physical barrier, which facilitates stopping further infection as well as spreading it to the neighboring tissues [57]. Induced callose deposition triggered by holaphylamine (steroid alkaloid) has been previously detected in flax after inoculation with _F. oxysporum_[58]. Furthermore, ABA-dependent triggering of callose synthesis and deposition was proved as a mechanism in necrotic pathogens within \(\beta\)-amino-butyric acid-induced response [33]. It can be assumed that in flax containing increased level of ABA the amount of callose also rises. Based on literature evidence, we believe that it is an effect directly corresponding with the infection [3; 59; 60]. Alternative way, presented for _Arabidopsis thaliana_, involves decreased level of callose synthase leading to complete loss of callose and initiation of disease resistance based on salicylic acid [61]. This work is the first to show the possible mechanism of the effect of hemibiotrophic _Fusarium culmorum_ on the immune response in flax driven via ROS production and ABA biosynthesis.