Elicitor-induced Oxidative Burst and Phenylpropanoid Metabolism in Pinus radiata Cell Suspension Cultures

Abstract

A cell wall elicitor preparation from the needle pathogen Dothistroma pini was used to induce defence responses in Pinus radiata cell suspension cultures. Addition of elicitor to cell suspensions induced a rapid, transient burst in the accumulation of H2O2, with maximal response between 20 and 40 min post-elicitation. The protein kinase inhibitors staurosporine and K252a inhibited H2O2 accumulation showing that protein phosphorylation is required in the signal transduction pathway leading to the oxidative burst. Over a more extended time period elicitation of suspension cells lead to the activation of phenylpropanoid biosynthesis. The activity of phenylalanine ammonia-lyase (EC 4.3.1.5), the first enzyme in the phenylpropanoid biosynthetic pathway, increased 8-fold following elicitation with maximal activity 36 h post-elicitation. The activity of cinnamyl alcohol dehydrogenase (EC 1.1.1.195), an enzyme involved in lignin biosynthesis, increased 2.5-fold with maximal response 48–72 h post-elicitation. Thioglycolic acid extractable material increased 2-fold with maximal response 48–72 h post-elicitation, and phloroglucinol–HCl-positive material increased over the same time course. These data show that P. radiata suspension cells are an excellent model system for investigating the biochemistry and enzymology of pathogen defence responses in P. radiata.

A review of Pinaceae resistance mechanisms against needle and shoot pathogens with a focus on the Dothistroma–Pinus interaction

“…Dothistromin injection can also generate a hypersensitive-like response in P. radiata needles, the plant producing benzoic acid as a phytoalexin and highly lignified lesiondelineating bands (Franich et al 1986). Dothistroma septosporum cell wall elicitors also induce a hypersensitive-like response in suspension-cultured P. radiata cells (Hotter 1997). However, dothistromin production is not required for infection of P. radiata, as genetically modified D. septosporum strains, unable to produce the toxin, are still able to complete their life cycle on this host (Schwelm et al 2009).…”

“…These include cell wall-bound phenolic and polyphenolic compounds, such as lignin, that can restrict pathogen growth (Vance et al 1980;Miedes et al 2014). During in vitro experiments, lignin and other cell wall-bound phenolics accumulated in P. radiata cell suspension cultures after exposure to D. septosporum cell wall elicitors (Hotter 1997). The activity of both phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase, two enzymes key to the biosynthesis of phenolic compounds, increased dramatically in these cell suspension cultures (Hotter 1997).…”

“…Another similar method is to compare the chemical and morphological response of 'resistant' and 'susceptible' plants to inoculation with a given pathogen (Jurgens et al 2003;Luchi et al 2005;Jacobs et al 2009). Host tissue cultures have also been used to determine responses to pathogen recognition (Hotter 1997). Finally, in vitro experiments have been used to investigate the impact of plant secondary metabolites on pathogen growth and their potential role in plant defence and resistance (Blodgett and Stanosz 1997).…”

Formation of plant tracheary elements in vitro – a review

“…No free single TEs were found but TEs were formed in~2.5% of all clusters. Möller et al (2003) found that Pinus radiata xylem strips-derived callus cells maintained on P6-SHv medium (Hotter 1997) supplemented with 4.5 μM 2,4-D and 4.4 μM BAP differentiated into TEs when transferred to P6-SHv medium supplemented with 2 g L −1 activated charcoal. The differentiation rate varied from 2% to 45% depending on growth conditions.…”

“…The rates varied not only between genotypes but also between explant types (root, hypocotyl, cotyledon and needle) isolated from the same plant (Devillard, unpublished). Xylogenic P. radiata cell lines can be maintained on P6-SHv medium (Hotter 1997) supplemented with 4.5 μM 2,4-D and 4.4 μM BAP. However, in vitro P. radiata xylogenic cell cultures lose their capacity to proliferate and form TEs with prolonged in vitro culture periods (Devillard, unpublished).…”