Despite these studies little is known

Despite these studies, little is known about the regulation of defence genes during AM root colonization. In the present study, we characterized the Ptc52 gene from tomato (named here as SlPtc52), a member of the gene family of non-heme oxygenases, and analysed its molecular characteristics. This gene was previously identified as specifically up-regulated in mycorrhizal tomato plants (García-Garrido et al., 2010) as codifying Rieske non-heme iron type oxygenases (RO). Given the presence of two non-heme iron-binding motifs conserved amongst aromatic ring-hydroxylating enzymes in bacteria (Ferraro et al., 2005), these oxygenase proteins were predicted to encode a protein related to the lethal leaf spot 1 (Lls1) lesion mimic gene in plants (Gray et al., 2004). Lesion mimics are sometimes associated with enhanced disease resistance, including increased expression of pathogenesis-related (PR) genes and other markers indicative of defence system activation (Wolter et al., 1993; Dietrich et al., 1994). Some of these genes responsible for the lls1 phenotype encode enzymes involved in sbha metabolism and belong to a small five-member family of LLS1-related non-heme oxygenases defined by the presence of Rieske and mononuclear iron-binding domains (Gray et al., 2004). This family includes pheophorbide a oxygenase (PAO), chlorophyll a oxygenase (CAO), choline monooxygenase (CMO), a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic55), and a 52 kDa protein isolated from chloroplasts (Ptc52) (Gray et al., 2004). In this study, we describe the tissue expression pattern of SlPtc52. We also show that the tomato Ptc52 gene is inducible during arbuscular mycorrhiza formation and that SlPtc52 expression is associated with functional mycorrhization. SlPtc52 gene silencing led to an alteration in arbuscular abundance, suggesting that SlPtc52 plays a regulatory role during AM symbiosis.
Material and methods
Results
Discussion Many genes responsible for lesion-mimic phenotypes have been identified. The Lls1 gene, whose function loss results in a light-dependent cell death phenotype mediated by chloroplasts, was first cloned from maize (Gray et al., 1997, 2002). The A. thaliana ortholog to the maize Lls1 encodes a pheophorbide a oxygenase (PAO) which catalyzes a key step in chlorophyll degradation (Pružinská et al., 2003). AtPAO is a Rieske-type iron-sulfur protein identical to Arabidopsis accelerated cell death 1 (ACD1) (Pružinská et al., 2003). In tomato, the Lls1 homolog gene, which was previously cloned, sequenced and analyzed (Spassieva and Hille, 2002), showed a high rate of conservation at the protein and nucleotide level as compared to Lls1 genes from maize and Arabidopsis. In this study, we characterized and analyzed the Ptc52 gene from tomato, a member of the non-heme oxygenase family, whose function is associated with the Lethal leaf spot 1 (lls1) lesion mimic phenotype in plants. PTC52 belongs to a clade of the PAO (LLS1)-related family of Rieske-type proteins, and the catalytic activities of three of its members PTC52, CAO, and PAO (LLS1) in Arabidopsis are involved in chlorophyll metabolism. PTC52 catalyzes the oxidation of the 7-methyl group of the tetrapyrrole ring of protochlorohyllide a to produce the formyl group (Bartsch et al., 2008). In 2014, Berim et al. reported that PTC52-like genes from sweet basil trichomes are involved in flavonoid biosynthesis exercising flavone 8-hydroxylase activity. BLAST searches also showed that putative PTC52 homologs from different plant families have lower identity levels (50–60%) than other PAO-related genes, including PAO, CAO and TIC55. These findings suggest that the clade has promising functional diversity (Berim et al., 2014). TIC55 and PTC52 are considered to be components of the TIC and PTC import complexes, respectively, in the chloroplast (Kim and Apel, 2004).