Global gene expression analyses of the alkamide-producing plant Heliopsis longipes supports a polyketide synthase-mediated biosynthesis pathway

Abstract

Background: Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed.

Methods: Transcriptomics analysis allowed to identify genes highly expressed in H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea, another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS).

Results: The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS alk) previously thought to be absent from the plant kingdom.

Keywords: Affinin; Alkamides biosynthesis; Heliopsis longipes; Plants PKS; Polyketide synthase; RNA sequencing; Transcriptome analysis.

Figure 1. General structure of alkamides (alk) and the main alkamides produced in Heliopsis longipes roots.

(A) R1: α-unsaturated acyl chain C8–C18. Other double bonds may be present isolated (E) or conjugated (E,E or E,Z). R2: amine. R3: mostly H. (B) Structure of affinin (N-isobutyl-2E,6Z,8E-decatrienamide) the most abundant, represents over 90% of the total alkamides in roots. Minor alkamides are: (C) N-2-methylbutyl-2E,6Z,8E-decatrienamide (3.3%), (D) N-isobutyl-2E-en-8,10-diyn-undecanamide (2.5%), and (E) decanoic-2E,6Z,8E-bornyl-ester (1.1%). The latter, although not an alkamide, presents the same acyl moiety as the denoted alkamides.

Figure 2. Gene Ontology (GO) classification of unigenes derived from RNA-Seq in Heliopsis species studied.

A total of 29,316 unigenes in H. longipes and 29,870 unigenes in H. annua were successfully annotated and classified into three GO categories: cellular component, biological process and molecular function.

Figure 3. Relative expression of candidate genes involved in the putative acyl-chain biosynthetic pathway identified by differential gene expression analysis of Heliopsis longipes and Heliopsis annua roots and leaves transcriptomes.

(A) Quantitative real-time PCR. (B) Expression, in fragments per kilobase of exon model per million mapped reads (FPKM) from the RNA sequencing data. ACP, acyl carrier protein; AT, acyl transferase; DH, dehydratase; KS, ketoacyl synthase; OR, oxide reductase including the K: keto reductase; ER, enoyl reductase; DH, dehydratase; TE, thioesterase. The number corresponds to the transcript set identified by differential expression analysis. Error bars represent standard error for n = 2. The relative expression pattern of the candidate transcripts in the non-alkamide producing tissue was close to zero and therefore is not easily observed in the graphs.

Figure 4. Molecular phylogenetic analysis of the ketoacyl synthase (KS) domains protein sequences.

KS domain protein sequences differentially expressed in H. longipes and H. annua roots and leaves tissue were selected for phylogenetic analysis. KS domain protein sequences non-differentially expressed but annotated in the transcriptomes that coded for conserved domains of fatty acid synthases (FAS) and polyketide synthase (PKS) were used as comparison. The different KS domains selected were classified as group A, C, D, E or F for non-differentially expressed transcripts from H. annua (roots and leaves) and H. longipes (leaves). The group B for the upregulated transcripts in H. longipesroots. KS domains from: Echinacea purpura from Plant Biology Department, Michigan State University; and other KS domains of FAS and PKS from mammals, microorganisms and other plants, obtained from NCBI, were also were selected for phylogenetic analysis based on the Maximum Likelihood method. The numbers at nodes indicate bootstrap support, for 100 bootstrap replications. The analysis was conducted in MEGA 7.

Figure 5. Proposed affinin acyl chain biosynthesis mediated by polyketide synthase (PKS).

The proposed biosynthetic pathway exhibits a multi-modular architecture and requires acyl-CoA as the starter unit and malonyl-ACP as the extender unit. The KS domain catalyzes a decarboxylative Claisen (CO₂) condensation between the growing chain and an extender unit attached to the ACP domain (gray circles), whereas the AT domain selects and loads an extender unit to the ACP. The oxide-reductase that includes the KR and DH domains sequentially reduces the β-keto group to a β-hydroxy group and α-double bond. Additional ER domains reduce the α-double bond to a saturated product, and TE cleaves the synthesized acyl chain. Enzymatic domains: ACP: acyl carrier protein, AT: acyl transferase, KS: ketoacyl synthase, KR: keto reductase, DH: dehydratase, ER: enoyl reductase, TE: thioesterase.