Metabolism of NHP and SA

When mobile organisms face a threat, they have the options of a fight or flight as reaction. The sessile nature of plants narrows their response option down to defend themselves against the threat. Therefore, plants developed a strong innate immune system in an evolutionary context, redundant of specialized immune cells as found in animals. Besides, they are able to prime non-infected distal tissue towards a stronger immune response after pathogen attack, a phenomenon that is termed systemic acquired resistance (SAR) (Fu and Dong, 2013). Salicylic acid (SA) and N-hydroxy pipecolic acid (NHP) are small molecules and constitute two major hormones in the plant immune response. They are key molecules in basal resistance as well as to induce SAR (Delaney et al., 1994; Wildermuth et al., 2001; Chen et al., 2018; Hartmann et al., 2018; Rekhter et al., 2019b). The biosynthesis and function of SA has been intensively studied over the last decades (Wildermuth et al., 2001; Nawrath et al., 2002; Rekhter et al., 2019b; Torrens-Spence et al., 2019). In addition, the biosynthesis of NHP was unraveled recently (Chen et al., 2018; Hartmann et al., 2018). Both compounds are known to be present in a glycosylated state likely to be inactivated or stored. This is now shifting the focus towards the enzymes catalyzing the glycosylation reactions. For SA glycosylation, three UDP-dependent glycosyltransferases (UGTs) have been described: UGT74F1, UGT74F2 and UGT76B1 (Song, 2006; Dean and Delaney, 2008; von Saint Paul et al., 2011; Noutoshi et al., 2012; George Thompson et al., 2017). In terms of NHP, the NHP-O-glycoside (NHP-OGlc) was a known metabolite, without the description of a functional UGT enzyme that was able to catalyze the synthesis, prior to this thesis (Chen et al., 2018; Hartmann and Zeier, 2018). Lately, independent research groups were able to describe one of the proposed SA UGTs, UGT76B1, to be the major enzyme in the formation of NHP OGlc in Arabidopsis thaliana (Bauer et al., 2021; Cai et al., 2021; Holmes et al., 2021; Mohnike et al., 2021). In this work, the identification and functional characterization of UGT76B1 as NHP-OGlc forming enzyme is laid-out as published earlier in The Plant Cell within Mohnike et al. 2021 (Mohnike et al., 2021). The metabolite levels of NHP, SA and their respective glucosides are therein described in response to Pseudomonas syringae pv. maculicola ES4326 (P.s.m.) infection. The metabolic fate of NHP and SA in the ugt76b1 mutant was underlined by additional UV-stress experiments. In addition, we provide data about the infection phenotype against P.s.m. and Hyaloperonospora arabidopsidis Noco 2 (H.a. Noco 2), of which we deduce an enhanced resistance phenotype of the mutant. Analyzing double mutant lines of the FLAVIN-DEPENDET MONOOXYGENASE 1 (FMO1) with ugt76b1, fmo1 ugt76b1, we show that enhanced resistance and growth deficiency are FMO1-dependent, therefore, NHP-dependent. Lastly, we argue against the need of NHP-O-glucosylation for successful mobility during SAR (Chapter I). Furthermore, we used our metabolome analysis platform to search for novel, so far undescribed metabolites of NHP. A novel metabolite, which is synthesized in an infection-dependent manner, is described to be a NHP-methyl-ester (MeNHP). Its biosynthesis is shown to be AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1)- and FMO1-dependent. In addition, its retention time and tandem-mass spectrometric properties were underlined via a chemically synthesized authentic standard of MeNHP. The novel compound is synthesized in vitro by the annotated methyl transferase At4G22530 (NHPMT1). However, T-DNA insertion lines of NHPMT1, nhpmt1-1 and nhpmt1-2 are not impaired in biosynthesis of MeNHP (Chapter II). Additionally, we present a NHP and D9-labeled NHP co-infiltration experiment to identify additional in planta NHP-derivatives. Moreover, we layout results about successful repetition of earlier published work and investigated genes that remained inconclusive towards their influence and function in plant pathogen interaction mediated defense response of A. thaliana. We were able to confirm the role of ENHANCED PSEUDOMONAS SUSCEPTIBILITY1 (EPS1) on the biosynthesis of SA by the accumulation of its substrate iscochorismate-9-glutamate (IC-9-Glu) in metabolite analysis of eps1 mutant plants (Torrens-Spence et al., 2019). Furthermore, we show that the amino acid transporter LYSINE/HISTIDINE 7 (LHT7) is not solely required for NHP biosynthesis. Similarly, ABERRANT LATERAL ROOT FORMATION 5 (ALF5), ENHANCED DISEASE SUSCEPTIBLITY 5 (EDS5) and EDS5-homolog (EDS5H) are not solely required as transporters in NHP biosynthesis (Chapter III).

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