Priming plant defences: metabolism and reactive oxygen species

The agricultural output must increase considerably in order to meet food security and match the increase in population and loss of arable land. Pests and diseases account for major yield losses in agriculture and negatively impact global food security. There is a surge for the development of new low-input agricultural practices that are more sustainable and include the use of environmentally friendly alternatives to agrochemicals used to control plant pathogens. Among the most common approaches to deal with plant pathogens are through breeding techniques and the use of chemical pesticides. Due to the many disadvantages of these approaches, and the negative impact of pesticides on human health and the environment, research focus is shifting towards the development of biocontrol mechanisms. One approach is to harness the full potential of the plants innate immunity through priming. Priming is a technique in which plants are subjected to a temporal mild stress, or a component that mimics a stress, to place plants in an active state to respond more effectively and rapidly upon exposure to a subsequent stress condition. Several synthetic and natural compounds have been successfully identified as priming agents against a broad range of plant pathogens. Among the best described priming agents are amino acids, and structurally derived carbohydrates like chitin and oligogalacturonides. In this thesis we aimed to identify novel priming agents against biotic stress by screening stress-related compounds that naturally accumulate in plants exposed to (a)biotic stresses. We mainly focussed on fructans, the amino acid γ-aminobutyric acid (GABA), and the polyamines (PAs) spermine (Spm) and spermidine (Spd).

Fructans are fructose-based oligo- and -polysaccharides accumulating in around 15 % of flowering plants as storage compounds typically during abiotic stresses. Mounting evidence suggest that exogenous fructans can also be effective against biotic stresses. Our data indicate that inulin-type fructans are effective against the grey mold B. cinerea in the non-fructan accumulating model plant Arabidopsis thaliana. Similarly, levan oligosaccharides (LOS) derived from the bacterium Halomonas smyrnensis significantly induced resistance against B. cinerea. We found that both inulin and LOS primed the flg22 and oligogalacturonides (OGs) induced NADPH-oxidase-mediated reactive oxygen species (ROS) burst but did not directly induce a ROS burst themselves. Inulin and LOS priming resulted in the accumulation of H2O2 after priming, and significant H2O2 accumulation and increased activities of catalase (CAT) and ascorbate peroxidase (APX) after infection. In response to B. cinerea infection, primed plants accumulated glucose (Glc) and sucrose (Suc). Through a pharmacological approach we showed that a functional NADPH-oxidase is required for fructan-induced resistance against B. cinerea. In addition, inulin priming also require a functional SNF1-related kinase 1 (SnRK1). We also showed that levan, but not inulin, is effective to control apple scab (Venturia inaequalis) in apple seedlings. Levan induced resistance in an ontogenic-dependent manner, without inflicting major sugar fluctuations. Levan also directly inhibited V. inaequalis growth. We discuss our results in the context of fructans functioning as Microbe- and/or Damage-Associated Molecular Patterns (MAMPs and DAMPs) in plants.

Secondly, we showed that GABA can be equally effective compared to its structural homolog and well-described priming agent β-aminobutyric acid (BABA) against B. cinerea in Arabidopsis at low concentrations. Priming with GABA resulted in the activation of the ROS scavenging enzymes CAT and guaiacol peroxidase (GPX) accompanied by reduced H2O2 levels after B. cinerea infection. This was correlated with lower flg22- and OGs-induced ROS burst through NADPH-oxidases in GABA primed plants. GABA priming at 1 mM, but not 100 µM, resulted in endogenous GABA accumulation after treatment and a second wave after infection. GABA priming at 100 µM inhibited the active nitrate reductase (NR), whereas 1 mM induced both the active and total NR activity. GABA priming resulted in Suc accumulation, followed by Glc and fructose (Fru) accumulation after B. cinerea infection. We discuss our findings in the context of exogenous GABA uptake resulting in metabolic fluctuations and exogenous GABA signalling.

Next, we showed that exogenous Spm is more effective than Spd to induce resistance against B. cinerea in Arabidopsis. High concentrations (> 1 mM) of PAs resulted in cell death development. Spm priming resulted in the accumulation of H2O2 after treatment, and also after B. cinerea infection. Both Spd and Spm priming enhanced the activities of CAT, APX and GPX after treatment and infection with B. cinerea. PA treatment resulted in soluble sugar accumulation after treatment, and Spm treated plants accumulated sugars after infection. Spm treatment induced GABA accumulation after treatment and infection together with the accumulation of phenylalanine and asparagine after infection. Spm treatment inhibited total and active NR activity, whereas low concentrations of Spd inhibited active NR and high concentrations enhanced active NR activity. We discuss our results in light of recent findings pointing to Spm as a candidate defence activator in plants.

Finally, in the addendum we explored UDP-Glc signalling in plants, similar to animals, through the regulator of G-protein signalling 1 (RGS1) receptor. Our data showed that exogenous UDP-Glc can prime local but not systemic plant defences against B. cinerea. Arabidopsis rgs1 mutants did not respond to UDP-Glc priming. We further showed that UDP-Glc treatment results in time-and dose-dependent regulation of the flg22-induced ROS burst, without affecting MPK3 accumulation. Arabidopsis UDP-Glc pyrophoshorylase mutants with reduced endogenous UDP-Glc were not differentially susceptible to B. cinerea infection. UDP-Glc treatment resulted in apoplastic Glc accumulation in the Wt, but not rgs1 mutant. Similarly, total Suc levels increased in Wt plants, but decreased in rgs1 mutants in response to UDP-Glc treatment. We propose that exogenous UDP-Glc functions as a stress signal in plants, similar to what has been shown in animals.