Formation of plant metabolic gene clusters within dynamic chromosomal regions

Abstract

In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants.

Fig. 1.

A candidate metabolic gene cluster in Arabidopsis. Maps of (A) the thalianol gene cluster (21) and (B) a candidate metabolic gene cluster on Arabidopsis chromosome 5. The boxes represent exons. Genes from the same lineage-specific clades are colored similarly, and T-DNA insertion mutants are indicated in B. At5g42591 is a nonconserved ORF that is predicted to encode a small 35-amino acid peptide of unknown function and for which there are no large-scale expression data. (C) Microarray expression profiles of the genes within the candidate cluster shown in B and four flanking genes [image adapted from Genevestigator (46)]. The putative cluster genes shown in B are indicated in bold. They are expressed only in root tissue, in a pattern similar to the thalianol cluster genes (Fig. S6).The genes flanking the candidate cluster region are not coexpressed and do not have any obvious predicted functions in secondary metabolism. Data are displayed as a heat map (blue, expressed; white, not expressed) scaled to the expression potential of each gene. (D) Structures of thalianol and marneral.

Fig. 2.

Detection of MRN1 products in yeast and Arabidopsis. Saponified extracts from yeast and Arabidopsis were analyzed for triterpene content by GC-MS. TIC, total ion chromatogram; EIC 191, extracted ion chromatogram at m/z 191. (A) Yeast empty vector control. (B) Yeast expressing the MRN1 cDNA. (C) Root extracts from wild-type Arabidopsis. (D and E) Root extracts from CYP71A16-knockout lines mro1-1 (D) and mro1-2 (E). (F) Root extracts from mro1-2 overexpressing CYP71A16. Data are representative of at least two separate experiments. The y axis (ion count) of each chromatogram is scaled to the highest peak. Arrows show peaks representing trimethylsilylated marnerol. Unlabeled peaks are trimethylsilylated sterols.

Fig. 3.

CYP71A16 modifies the product of MRN1. Neutral extracts from Arabidopsis leaves were analyzed for triterpene content by GC-MS. TIC, total ion chromatogram; EIC 191, extracted ion chromatogram at m/z 191; EIC 586, extracted ion chromatogram at m/z 586. Leaf extracts from wild-type plants (A) or plants overexpressing MRN1 (B), CYP71A16 (C), or both MRN1 and CYP71A16 (D). Overexpression of the two enzymes resulted in the loss of the trimethylsilyl marnerol peak and the appearance of four peaks (labeled 1–4) that have ionization spectra consistent with desaturated hydroxy-marnerol. These peaks were identified using chromatogram analysis software and cannot be seen in the complex TIC. An additional three peaks of lower abundance could be detected. Ionization spectra for individual compounds are shown in Fig. S2. The plants overexpressing MRN1 and CYP71A16 presented an extreme dwarfing phenotype indicating that the products of marneral modification by CYP71A16 may inhibit plant growth and development (Fig. S3). Data are representative of at least two separate experiments. The y axis (ion count) of each column of chromatograms is to the same scale, indicated in the top left corners in A.

Fig. 4.

(A) Proposed scheme for formation of the thalianol and marneral clusters, based on the assumption that the two clusters were founded by duplication of an ancestral OSC/CYP705 gene pair. (B) Timing of cluster assembly. The evolutionary tree highlights the period over which the lineage-specific OSC and P450 clades arose and the thalianol and marneral clusters formed (pink shaded region). The α and β whole-genome duplication events are indicated as circles.

Fig. 5.

The chromosomal context of the thalianol and marnerol gene clusters. (A) Both the thalianol and marneral clusters are located in islands between chromosomal segments that were duplicated in the α whole-genome duplication event [89% of all genes are contained within α duplication segment pairs (31)]. Apart from the gene clusters, the two junction regions do not share any other duplicated genes or extensive stretches of intergenic homology. (B) Comparative maps of the thalianol and marneral cluster regions in A. thaliana and A. lyrata. Genes are indicated by filled arrowheads; cluster genes are in red, syntenic genes in blue, and nonsyntenic genes in white. Regions of DNA with homology in both species are indicated by blue bars. (C) Distribution of gene density (black line), TE density (blue dashed line), and the TE/gene density ratio (red line) across a section of chromosome 5 that contains both the marneral (M) and thalianol (T) clusters. Centromeric and pericentromeric regions are marked by horizontal black bars. Gene and TE densities were calculated for windows of 100 kb with an overlap of 5 kb. Full chromosome plots are shown in Fig. S5.