Abstract
Roots of intact olive seedlings, axenically cultured, were alternatively placed in contact with Rhizophagus irregularis (mycorrhizal) or Verticillim dahliae (pathogenic) fungi. MeJA treatments were also included. In vivo redox activities in the apoplast of the intact roots (anion superoxide generation, superoxide dismutase and peroxidase activities) were measured. All our results showed that apoplastic redox activities of intact seedling roots in contact with the compatible mycorrhizal fungus were clearly attenuated in comparison with the pathogenic fungus or treated with MeJA, even at the early stages of treatment used. Total phenolics, flavonoids and phenylpropanoid glycosides were also quantified. Roots in contact with the mycorrhizal fungus did not enhance the biosynthesis of phenolic compounds with respect to controls, while those in contact with the pathogenic one significantly enhanced the biosynthesis of all phenolic fractions measured. Reactive oxygen species and nitric oxid accumulation in roots were examined by fluorescence microscopy. All of them presented much higher accumulation in roots in contact with the pathogenic than with the mycorrhizal fungus. Altogether these results indicate that intact olive seedling roots clearly differentiated between mycorrhizal and pathogenic fungi, attenuating defense reactions against the first to facilitate its establishment, while inducing a strong and sustained defense reaction against the second. Both reactive oxygen and nitrogen species seemed to be involved in these responses from the first moments of contact. However, further investigations are required to clarify the proposed crosstalk between them and their respective roles in these responses since fluorescence images of roots revealed that reactive oxygen species were mainly accumulated in the apoplast (congruently with the measured redox activities in this compartment) while nitric oxid was mainly stored in the cytosol.
Highlights
Root defense reactions imply an oxidative burst with a rapid generation of Reactive Oxygen Species (ROS) in the apoplast, as superoxide (O22) and hydrogen peroxide (H2O2) [1],[2]
Roots in contact with arbuscular mycorrhizal fungus (AMF) hyphas showed a significant O22 generation peak after 5 min contact, but this was considerably lower (1.75-fold) than that induced by MeJA
With respect to the measurements of the oxidative burst (O22 generation) and the related antioxidant activities (ECSOD and exocellular nonspecific peroxidase (ECPOX)) we want to emphasize the use of intact seedling roots to measure such redox activities, working under in planta non invasive physiological conditions, avoiding cell or root disorganization
Summary
Root defense reactions imply an oxidative burst with a rapid generation of Reactive Oxygen Species (ROS) in the apoplast, as superoxide (O22) and hydrogen peroxide (H2O2) [1],[2]. Avirulent pathogens induced a biphasic ROS production [8], but in the case of virulent pathogens only the first phase has been detected [9] Pathogenic fungi extend their hyphae and directly penetrate into the epidermal or mesophyll layers of plant cells, developing specialized structures for the exchange of nutrients such as haustorium [10]. These structures released different effectors to the plant apoplast, as small peptides [11] that act by enhancing the infection and enzymatic activities assigned to suppress the defense response [12]. The production of ROS can be achieved by the action of the RBOH and/or apoplastic peroxidases [4], [14]
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