Unravelling the biological role of novel stress-induced peptides (SIPs) in Arabidopsis thaliana.

Plant peptides accommodate a broad functional diversity ranging from development, growth, fertilization, senescence, cell death, symbiotic interactions to stress tolerance. They act as key components of cell-to-cell communication, interfere with signaling and response pathways, or display antimicrobial activity. Nevertheless, the total number of peptides is largely underestimated and different types of peptides are underexplored. Most plant peptides characterized to date act as small signaling peptides or antimicrobial peptides and are derived from non-functional precursor proteins. In contrast, peptides can also be derived from a functional protein, directly translated from small open reading frames (without the involvement of a precursor) or even encoded by primary transcripts of microRNAs.

In previous doctoral research, novel stress-induced peptides (SIPs) were identified via a transcriptome analysis on paraquat treated and Botrytis cinerea infected Arabidopsis thaliana (Arabidopsis) leaves followed by a high-throughput screening in yeast to select for bioactive peptides that enhance oxidative stress tolerance. The aim of this doctoral research was to further characterize novel stress-induced peptides and unravel their biological role in the stress response of Arabidopsis. The putative peptides studied in this work are encoded by stress-induced genes named OSIP108, SIP162/163 and OSIP116.

Overexpression of the OSIP108 peptide resulted in plants with increased tolerance to paraquat. This peptide is putatively translated from a 10 codon open reading frame (ORF) encrypted in a two-exon gene containing many short ORFs that overlaps with pseudogene At3g61185, encoding a defensin-like protein. OSIP108 was locally induced in leaves upon paraquat treatment and strongly induced by salicylic acid. We suggest that OSIP108 could be a nonprecursor-derived peptide and a glutathione look-a-like that possibly enhances paraquat tolerance via salicylic acid mediated signaling.

The SIP162/163 knock-out mutant was more susceptible to the hemibiotrophic root pathogen Fusarium oxysporum. SIP162/163 is a two-exon gene embedding a first long ORF encoding one putative cysteine-rich peptide and many short ORFs. The first exon of SIP162/163 overlaps with a pseudogene encoding a Protease inhibitor/seed storage/Lipid Transfer Protein family protein. Moreover, SIP162/163 is conserved in Brassicaceae and its genomic context harbors three other Lipid Transfer Protein-family genes of which the proteins show homology with the preprotein encoded by the first ORF in SIP162/163. This gene is induced in leaves as a direct or indirect result of F. oxysporum infection and strongly induced by ethylene and salicylic acid. We suggest that SIP162/163 could be translated in a conserved, precursor-derived peptide involved in the response of Arabidopsis against F. oxysporum via ethylene- and salicylic acid-mediated signaling.

The OSIP116 knock-out mutant showed photoperiod-dependent interveinal chlorosis, likely due to reduced cell density, and was more susceptible to F. oxysporum.OSIP116 is a short, one-exon gene encoding a putative nonprecursor-derived peptide from its first and longest ORF that was recently annotated as At4g14226 and conserved in Brassicaceae. Nevertheless, overexpression of this ORF or other OSIP116 ORFs could complement the interveinal chlorosis phenotype of the OSIP116 knock-out mutant. Additionally, OSIP116 was induced by paraquat, B. cinerea infection and ethylene. We suggest that OSIP116 could be translated in a nonprecursor-derived peptide and possibly involved in the regulation of cell density through ethylene signaling in a photoperiod-dependent manner.

We have progressed in unraveling the biological role of SIPs adding to the complexity and diversity of the plant peptidome. Nevertheless, the bottleneck in this doctoral research was the in planta detection of SIPs, since up to now no SIP was detected via western blot or mass spectrometry techniques. In the future, selected reaction monitoring will be employed as a specialized mass spectrometry technique to detect OSIP108, OSIP162/163 and OSIP116 in plants upon stress. This doctoral research has contributed to expanding the diversity of the plant peptidome and the broad functionality thereof, as nonprecursor-derived peptides and precursor-derived peptides were studied in the stress response of Arabidopsis.