Cell walls are important features of plant cells that perform a number of essential functions, both during plant development and growth and in plant-microbe interactions. Recognition of endogenous molecules released during the infection process and acting as “damage-associated molecular patterns” (DAMPs) is a key feature of immunity in plants. Oligogalacturonides (OGs), generated from disruption of homogalacturonan by pathogen pectic enzymes act as DAMPs and their perception leads to a response cascade that leads to intracellular Ca2+release, oxidative burst activation and transcriptional reprogramming. Recently transgenic Arabidopsis plants expressing a chimeric protein, named “OG-machine” (OGM), constituted by a fungal PG (FpPG) and a polygalacturonase-inhibiting protein (PGIP) from common bean (PvPGIP2) have been generated. OGM-expressing plants accumulate OGs in their tissue and exhibit enhanced resistance to a variety of pathogens, thereby providing direct evidence for the function of OGs as in vivo elicitors of the plant defense responses (DAMPs). Besides inducing immunity, OGs negatively affect plant growth and development, likely due to an auxin-antagonistic activity. This effect reflects the typical trade-off that exists between defence and growth and entails that the recognition of DAMPs poses the intrinsic risk of activating an exaggerated response that reduces or completely arrests plant growth. Thus, regulatory homeostatic mechanisms that prevent the deleterious effects of DAMP hyperaccumulation are is essential for an optimal immune response. Starting from transgenic plants that express, in a β-estradiol-inducible manner, the OGM, we looked for such a regulatory mechanism in Arabidopsis thaliana by searching for elicitor-inactive OGs that may derive from active OGs through an enzymatic modification. In the presence of β-estradiol, these plants contain increased levels of elicitor-active OGs compared to wild-type plants and a large amount of modified OG-like fragments. Mass spectrometry analyses showed that the oligomers indeed corresponded to modified OGs and are characterized by a galactaric acid residue at the reducing end, leading to the conclusion they are oxidized OGs (oxOGs). oxOGs were tested for their ability to induce defense responses and antagonize auxin responses. In all experiments, they were inactive as compared to the corresponding typical OGs. The enzyme with OG oxidizing activity was identified as Isoform 2 of Reticuline oxidase-like protein and was named OGOX1. OGOX1 produces elicitor-inactive oxidized OGs and H2O2 and is encoded by the gene At4g20830. This belongs to the superfamily of genes encoding the FADbinding berberine-bridge enzyme-like proteins (BBEls). In order to analyze the role of OGOX1, I have obtained an ogox1 null insertional mutant and transgenic plants overexpressing OGOX1 under the CaMV 35S promoter (OGOX1-OE#1.9 and #11.8). Since additional T-DNA insertional mutants of OGOX1 were not available, transgenic plants (amiR-OO1) expressing a β-estradiolinducible artificial microRNA (amiR) against the OGOX1 transcript (amiR-OO1 lines #2.5 and #3.4) were also generated. The characterization of these plants suggests that OGOX are responsible of an homeostatic mechanism involved in both development and immunity. In the last part of my work the characterization of closely related Arabidopsis BBlEs has revealed the presence of at least three additional enzymes with OG oxidizing activity. Thus, OGOX is a functional and redundant key element for regulating OG homeostasis and likely avoiding an exaggerated activation of plant defences.

Dottorato di ricerca in Scienze delle produzioni vegetali e animali