Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones

Highlights

• The Norway spruce 10-member SABATH family include one IAMT, one SAMT and three JAMTs.

• IAMTs are conserved in seed plants.

• JAMT evolved independently in gymnosperms and angiosperms.

Abstract

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms.

Introduction

Phytohormones are chemicals produced by plants and function at extremely low concentrations. They regulate many biological processes ranging from plant growth and development to interactions with the environment. Some phytohormones are carboxylic acids and include indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA). For each of these five types of phytohormones, their respective methyl ester forms have been identified in plants (Westfall et al., 2013). Methyl salicylate (MeSA) and methyl jasmonate (MeJA) occur widely in nature (Knudsen et al., 1993). While methyl abscisate can occur as an artifact (Milborrow and Mallaby, 1975), the methyl ester of IAA has been consistently detected from the model plant Arabidopsis (Arabidopsis thaliana) (Pan et al., 2010). The methyl esters of GAs have been found in fern during antheridium formation (Yamauchi et al., 1996). Carboxyl methylation of phytohormones can have two biological outcomes. The first is deactivation of a phytohormone, and the methyl esters serve as a storage form (Varbanova et al., 2007, Yang et al., 2008). Under certain conditions, the methyl esters can be de-methylated to produce biologically active free acid forms (Yang et al., 2008). This presents a mechanism for rapid regulation of phytohormone homeostasis. The second outcome is to produce a metabolite with new functions. For SA and JA, which are defense signaling molecules (Thaler et al., 2012), their respective methyl esters MeSA (Chen et al., 2003) and MeJA (Seo et al., 2001) function in defense response and as floral scent compounds for attracting insect pollinators (Knudsen et al., 1993).

The enzymes that catalyze the methylation of carboxylic acid phytohormones except ABA have been identified from a number of plants and designated as IAA methyltransferase (IAMT), GA methyltransferase (GAMT), SA methyltransferase (SAMT) and JA methyltransferse (JAMT) (Chen et al., 2003, Qin et al., 2005, Ross et al., 1999, Seo et al., 2001s, Varbanova et al., 2007, Zhao et al., 2008, Zhao et al., 2013, Zhao, 2010). In contrast, it remains elusive whether or not the methyl ester of ABA identified in plants are products of enzymatic reactions. IAMT, GAMT, SAMT and JAMT are S-Adenosyl-L-methionine (SAM)-dependent methyltransferases, catalyzing the transfer of a methyl group from SAM to the carboxyl group of IAA, GA, SA and JA to form their corresponding methyl esters. Interestingly, IAMT, GAMT, SAMT and JAMT are homologous enzymes, belonging to the same protein family, SABATH (D'Auria et al., 2003). The SABATH genes form a mid-sized gene family in several plants that have been studied. Arabidopsis, rice (Oryza sativa) and poplar (Populus trichocarpa) genomes contain 24, 41 and 28 SABATH genes respectively (Chen et al., 2003, Zhao et al., 2008, Zhao et al., 2013). In addition to the above enzymes, the SABATH family also contains enzymes that methylate benzoic acid (Murfitt et al., 2000), farnesoic acid (Yang et al., 2006), cinnamic/p-coumaric acid (Kapteyn et al., 2007), anthranilic acid (Köllner et al., 2010), nicotinic acid (Hippauf et al., 2010) and nitrogen-methyltransferases involved in caffeine biosynthesis (Kato et al., 2000, McCarthy and McCarthy, 2007). The SABATH family renders a useful system for understanding the structural basis underlying substrate specificity discrimination of a protein family because of a wide-range of substrates of varying structures (Pott et al., 2004).

For the SABATH proteins that methylate phytohormones, we have been interested in understanding their functions and evolution. In the pursuit of these questions, we have made two important findings. The first is that IAMT appears to be conserved in seed plants (Zhao et al., 2008). IAMTs from Arabidopsis, rice, black cottonwood and white spruce (Picea glauca) form a monophyletic clade (Zhao et al., 2009). The second finding is the moss Physcomitrella patens does not contain phytohormone-methylating SABATH proteins. The P. patens genome contains four SABATH genes, one of which encodes a protein with a thiol methylating property. None of the four SABATHs from P. patens have activity with phytohormones tested as substrates (Zhao et al., 2012). This second finding suggests that phytohormone-methylating enzymes may have evolved at a later stage of land plant evolution.

To gain further insight on the evolution of the SABATH proteins that methylate phytohormones, we elected to characterize the SABATH family in Norway spruce (Picea abies). Three SABATH genes have been previously analyzed in white spruce, which is also a gymnosperm. That study was based on expressed sequence tags and only one of the three genes was assigned with a biochemical function (Zhao et al., 2009). Norway spruce is the first gymnosperm to have its nuclear genome sequenced (Nystedt et al., 2013). The availability of the whole genome sequence enables the identification and analysis of the complete SABATH gene family in this model gymnosperm plant.