Evolution of serine carboxypeptidase-like acyltransferases in the monocots

Abstract

The serine carboxypeptidases are a large family of proteases. in higher plants some members of this family have diversified and adopted new functions as acyltransferases required for the synthesis of natural products. we recently reported the first serine carboxypeptidase-like (scpl) acyltransferase enzyme to be characterized from monocotyledonous plants.¹ This enzyme, AsSCPL1, is required for acylation of antimicrobial terpenes (avenacins) that are produced in the roots of oat (Avena spp.) and that provide protection against soil-borne pathogens. The SCPL acyltransferase enzyme family has undergone substantial expansion following the divergence of monocots and dicots. Here we discuss the evolution of this SCPL enzyme family in monocots, their contribution to metabolic diversity, and the roles of these enzymes in biotic and abiotic stress tolerance.

Acylation contributes to the considerable diversity in structure and function of plant natural products. Most plant natural product acyltransferases characterized to date belong to the BAHD family. Members of the BAHD family have roles in the acylation of a wide range of plant natural products and utilise CoA-thioester acyl-donor substrates.² SCPL acyltransferases have recently emerged as a new and distinct family of plant acyltransferases that utilise O-glucose ester acyl-donor substrates.^1,3–16 Previously characterized SCPL acyltransferases from dicots include enzymes required for synthesis of glucose polyesters implicated in insect defence in tomato³ and for synthesis of UV-protectant sinapate esters in Arabidopsis.⁴ Our finding that the oat AsSCPL1 enzyme acylates antifungal triterpenes that are required for disease resistance extends the known range of functions of this family, and also suggests that the SCPL acyltransferase family originated before the divergence of the monocots and dicots.¹ Phylogenetic analysis indicates that there are likely to be numerous SCPL acyltransferases in both dicot and monocot species and that the corresponding genes have multiplied and diversified independently in the two lineages (Fig. 1). The AsSCPL1 enzyme has diverged from related sequences in other grasses and cereals under the influence of strong diversifying selection.¹ Avenacins are produced only by oat and not by other grasses or cereals. The rapid evolution of the AsSCPL1 enzyme along with other previously characterized avenacin biosynthetic pathway enzymes^17–19 is consistent with neo-functionalization associated with the emergence of the avenacin pathway within the genus Avena. These observations raise some intriguing questions about the functions of SCPL proteins in other cereals and grasses—might they also have a role in the synthesis of compounds that confer resistance to biotic/abiotic stresses?

Figure 1.

Evolutionary relationships across the plant SCPL protein family. SCPL protein sequences from P. patens (20 sequences) and C. reinhardtii (9 sequences) were identified using www.phytozome.net and aligned with sequences from A. thaliana (50 sequences), O. sativa (31 sequences) and selected sequences for characterized SCPL acyltransferases from other plant species using Muscle 3.6. Phylogeny was reconstructed using Mega 4.0, regions corresponding to the predicted signal peptide and internal spacer were removed prior to tree construction (sequence alignments are included as Suppl. materials online) as described in ref. 1. Characterized SCPL acyltransferase enzymes included in the tree are from Arabidopsis: (SCT: sinapoyl:choline transferase; SMT : sinapoyl:malate transferase; SST: sinapoyl:sinapoyl transferase; SAT : sinapoyl:anthocyanin transferase); oat: (AsSCPL1 acyltransferase; FJ475130 circled in red); Solanum pennellii glucose acyltransferase (GAC; AAF64227); and Brassica napus sinapoyl:choline transferase (SCT; AAQ91191). Characterized serine carboxypeptidase sequences included are from barley (Hordeum vulgare): SCPI (P07519), CPDWII (P55748) and SCPIII (P21529); wheat (Triticum aestivum): CPDWII (P08819); yeast (Saccharomyces cerevisiae): CPY (NP_014026). Also included were the sorghum (Sorghum bicolour) HNL1 nitrile-lyase enzyme (CAD12888), and the Brachypodium distachyon BdSCPL1 sequence (encoded by Bd3g21550). The rice homologue of AsSCPL1, encoded by Os10g01134, is indicated by an arrow. The scale bar indicates 0.1 substitutions per site. Bootstrap support (%) is shown for selected branches.

The genomes of moss (Physcomitrella patens) and of the alga Chlamydomonas reinhardtii encode 20 and 7 predicted SCPL proteins respectively. Phylogenetic analysis of these sequences, together with predicted SCPL proteins from higher plants, reveals that none of the sequences from P. patens or C. reinhardtii belong to Clade 1A (Fig. 1), which includes all of the previously characterized SCPL-acyltransferases.¹¹ Furthermore, the pentapeptide motif surrounding the catalytic serine residue, which is conserved across all members of Clade IA (the acyltransferase clade), is not found in P. patens or C. reinhardtii SCPL proteins. This motif is likely to be important for formation of a hydrogen bonding network that allows binding of the glucose ester substrates.¹⁶ The other members of the serine carboxypeptidase family have a different (but conserved) motif at this position which is integral to the hydrogen bond network of the proteases and is present in all P. patens and C. reinhardtii SCPL proteins. These phylogenetic analyses suggest that Clade 1A emerged after the separation of mosses and algae from higher plants.

Members of Clade 1A are established as acyltransferases with roles in plant secondary metabolism.^1,3–16 To investigate the possible functions of other monocot members of this clade we analysed the expression pattern of the gene encoding the closest relative to AsSCPL1 in rice (Os10g01134) (indicated by an arrow in Fig. 1) using publicly available transcriptome datasets.²⁰ The available in silico data indicate that this gene is expressed principally in the leaves, developing inflorescence and seed. Expression of Os10g01134 increases in response to biotic and abiotic stresses, including exposure to salt and arsenate and challenge with the rice blast fungus Magnaporthe oryzae. This is a markedly different expression pattern to that of the AsSCPL1 gene, which is expressed exclusively in the epidermal cells of oat root tips.¹ We carried out correlation analysis to identify genes that are co-expressed with Os10g01134 using the Rice Array Database.²¹ Of the genes that were significantly co-expressed with Os10g01134, ten had predicted functions in secondary metabolism. These included two genes with predicted roles in flavonoid biosynthesis, Os06g41810 (encoding a putative dihydroflavonol-4-reductase; correlation coefficient, r, 0.66) and Os06g27770 (encoding a putative isoflavone reductase; r = 0.61). This suggests a possible role for the rice SCPL protein encoded by Os10g01134 in the acylation of flavonoids. Clearly this hypothesis will need to be tested experimentally. Anthocyanins are common secondary metabolites that have been reported in a number of monocot species and that may be acylated with aromatic or aliphatic side chains.²² An SCPL acyltransferase from Arabidopsis has previously been implicated in the sinapoylation of anthocyanins.¹²

The identification and characterisation of AsSCPL1 as the founder member of a monocot-specific subgroup of SCPL acyltransferases has added a new dimension to the unravelling story of the evolution of the SCPL acyltransferase family in plants. Having diverged from ancestral proteases early in the evolution of flowering plants, the SCPL acyltransferase family subsequently radiated by duplication and mutation in higher plants, concomitant with the emergence and diversification of different plant natural product pathways.

Intriguingly, AsSCPL1 forms part of an operon-like gene cluster for avenacin synthesis.¹ Gene order in eukaryotes has until recently been regarded as essentially random. However metabolic gene clusters for synthesis of plant defence compounds are emerging as a new theme in plant biology. Examples have been reported in maize and rice^23–27 and we recently reported the first operon-like gene cluster to be found in Arabidopsis.²⁸ The formation of such gene clusters has most probably been driven by intense epistatic selection that favours coinheritance and co-ordinate regulation of the cognate pathway genes. The AsSCPL1 gene is likely to have been recruited into the avenacin gene cluster by gene duplication and relocation with associated acquisition of a new function, by analogy with proposed models of formation of metabolic gene clusters in yeast.²⁹

Addendum to: Mugford ST, Qi X, Bakht S, Hill L, Wegel E, Hughes RK, et al. A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats. Plant Cell. 2009;21:2473–2484. doi: 10.1105/tpc.109.065870.