Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Cell wall DAMPs (damage-associated molecular patterns) and their oxidation in the Arabidopsis seed coat development and grafting

Plants have an innate immune system to respond to biotic stresses. The plant immunity relies on the recognition of pathogens through the pathogen/microbe-associated molecular patterns (PAMPs/MAMPs), microbial structures or compounds that activate the defense responses. Defense responses can be also triggered by endogenous elicitors, which are plant compounds that are released upon pathogen infection of mechanical injury and are indicated as DAMPs. Some DAMPs derive from the degradation of the cell wall polysaccharides, like the oligogalacturonides (OGs) from the homogalacturonan and cellodextrins (CDs) from cellulose. A possible mechanism to maintain the homeostasis of OGs and CDs has been recently identified in the laboratory where I performed my thesis. OGs and CDs are inactivated by specific oxidases, namely OGOX1-4 and CELLOX1, respectively, belonging to the Berberine-Bridge Enzyme-like (BBE-like) family. The work of my thesis has been divided into two different parts. The first part deals with the identification and characterization of a new member of the BBE-like family in Arabidopsis thaliana, encoded by the gene At5g44360/BBE23. This gene was chosen because is the member more closely related to CELLOX1, previously characterized. I investigated whether BBE23 is also a polysaccharide oxidase and possibly an oxidase of DAMPs, and determined its biochemical characteristics, its activity and its specific substrates and products. My results show that the enzymatic activity of BBE23, hereon named CELLOX2, is very similar to that of CELLOX1. Interestingly, the mixed-linked β-1,3/1,4-glucans (MLGs), also capable of acting as DAMPs, have been uncovered as substrates for both CELLOX1 and CELLOX2. Moreover, I also investigated the physiological role of both the paralogues CELLOX1 and CELLOX2 to assess if their action may somehow affect the plant cell wall structure. I used knockout mutants (CELLOX1 KO and CELLOX2 KO), and CELLOX1 (CELLOX1 OE) overexpressing plants. CELLOX2 is mainly expressed in the seed coat. The second part of the work aimed at addressing the possible role of OGs and CDs and their oxidases during the process of grafting. Plant grafting is characterized by tissue fusion and vascular reconnection and involves responses to wounding, which in part overlap with responses to pathogens. The molecular mechanisms of graft formation remain unknown. The elucidation of the grafting process includes the study of response to the wound itself, a process in which OGs have been already implicated by previous studies. It also includes the study of tissue regeneration and vascular reconnection, which involves a complex cross-talk among different signals including hormones, especially auxin and cytokinin. Transcriptional profiling has revealed that some defense-related genes, including the genes encoding OGOX1 and CELLOX1, are differentially expressed at the graft junction, suggesting a role during the grafting process. It is known that OGs antagonize the action of auxin. In my thesis work, I found that CDs, like OGs, inhibit auxin responses. However, the expression of the auxin-regulated DR5::GUS gene shows a positive effect on the response to CDs in the meristematic cells. I also found that OGs and CDs influence the grafting process, at the level of the vascular tissue formation and differentiation. Taken together, albeit preliminary, my results are very promising, as they suggest a role of OGs and CDs in the grafting process and may lead to a possible biotechnological optimization of the grafting process and vascular development.