Flower power and the mustard bomb : Comparative analysis of gene and genome duplications in glucosinolate biosynthetic pathway evolution in cleomaceae and brassicaceae

PREMISE OF THE STUDY: Glucosinolates (GS) are a class of plant secondary metabolites that provide defense against herbivores and may play an important role in pollinator attraction. Through coevolution with plant-interacting organisms, glucosinolates have diversifi ed into a variety of chemotypes through gene sub- and neofunctionalization. Polyploidy has been of major importance in the evolutionary history of these gene families and the development of chemically separate GS types. Here we study the eff ects of polyploidy in Tarenaya hassleriana (Cleomaceae) on the genes underlying GS biosynthesis. METHODS: We established putative orthologs of all gene families involved in GS biosynthesis through sequence comparison and their duplication method through calculation of synonymous substitution ratios, phylogenetic gene trees, and synteny comparison. We drew expression data from previously published work of the identifi ed genes and compared expression in several tissues. KEY RESULTS: We show that the majority of gene family expansion in T. hassleriana has taken place through the retention of polyploid duplicates, together with tandem and transpositional duplicates. We also show that the large majority (>75%) is actively expressed either globally or in specifi c tissues. We show that MAM and CYP83 gene families, which are crucial to GS diversifi cation in Brassicaceae, are also recruited into specifi c tissue expression pathways in Cleomaceae. CONCLUSIONS: Many GS genes have expanded through polyploidy, gene transposition duplication, and tandem duplication in Cleomaceae. Duplicate retention through these mechanisms is similar to A. thaliana, but based on the expression of GS genes, Cleomaceae-specifi c diversifi cation of GS genes has taken place.</p

The published terpenoid biosynthetic module in <i>Arabidopsis</i>.

<p>Gene abbreviations are adapted from the <i>Arabidopsis</i> Information Resource<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#t001fn002" target="_blank">^A</a>.</p><p>^A TAIR10, <a href="http://www.arabidopsis.org/" target="_blank">www.arabidopsis.org</a>, last accessed on December 13th, 2014.</p><p>^B Ohnolog pair according to Bowers et al., 2003 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#pone.0128808.ref008" target="_blank">8</a>].</p><p>^C for a comprehensive review, see Tholl and Lee, 2011 and Phillips et al., 2008.</p><p>^D assosciation of <i>AtDXS3</i> to MEP pathway is subject of scientific debate (see Phillips et al., 2008).</p><p>The published terpenoid biosynthetic module in <i>Arabidopsis</i>.</p

Transcript levels of <i>S</i>. <i>lycopersicum DXS</i>-like genes in different parts of the plant (leaves, roots, stems, stems without (W/O) trichomes, and isolated stem trichomes) relative to those of the reference gene <i>RCE1</i> (Solyc10g039370.1.1).

<p>Transcript levels were determined by real-time qPCR with four biological and three technical replicates for each biological sample. The error bars represent the standard error.</p

Comparative tissue-specific expression of all <i>Arabidopsis DXS</i>-like genes relative to the <i>bHLH</i> housekeeping gene.

<p>Values comprise averages of four independent ATH1 microarray experiments (Experiment ID: E-MEXP-2008, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#sec002" target="_blank">Materials & Methods</a> section). Notably, <i>DXS1</i> is the only member with annotation to “trichome” plant ontology (PO:0000282). The error bars represent the standard error.</p

Illustration showing the complete set of genes associated with all terpenoid biosynthetic modules identified in this study across 17 genome assemblies, based on the HMM-generated profiles of Table 5.

<p>For core-<i>TPS</i> genes, numbers of previously published full-length target genes is included if available. Asterisks indicate number of previously identified full-length <i>TPS</i> open reading frames and hence putative number of functional terpene synthase enzymes. Incomplete protein fragments are not included.</p

The extended terpenoid phenotypic module in <i>Arabidopsis</i>, including triterpene- specific (C₃₀) synthases.

<p>Three letter gene abbreviations are adapted from the <i>Arabidopsis</i> Information Resource<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#t002fn002" target="_blank">^A</a>.</p><p>^A TAIR10, <a href="http://www.arabidopsis.org/" target="_blank">www.arabidopsis.org</a>, last accessed on December 13th, 2014.</p><p>^B Ohnolog pair according to Bowers et al., 2003.</p><p>^C For a comprehensive review, see Tholl and Lee, 2011.</p><p>The extended terpenoid phenotypic module in <i>Arabidopsis</i>, including triterpene- specific (C₃₀) synthases.</p

Transcript levels of the <i>S</i>. <i>lycopersicum DXS</i>3 gene in different parts of the plant (leaves, roots, stems, stems without (W/O) trichomes, and isolated stem trichomes) relative to those of the reference gene <i>RCE1</i> (Solyc10g039370.1.1).

<p>Transcript levels were determined by real-time qPCR with four biological replicates and three technical replicates for each biological sample. The error bars represent the standard error.</p

Circos ideogram showing 5 <i>Arabidopsis</i> chromosomes with the extended set of genes associated with major terpenoid biosynthetic modules.

<p><b>A</b>. Gene inventory of the complete terpenoid biosynthetic pathway after initial expansion of published modules. Tandem duplicate supergenes are marked in red. Singletons are marked in green. Ohnolog duplicate gene pairs are marked in blue. Central pie chart shows a 68% tandem duplicate supergenes fraction. <b>B</b>. Subset of prenyltransferases and specific triterpene synthases marked in yellow. Central pie chart shows a 70% tandem duplicate supergenes fraction. <b>C</b>. Subset of core terpene synthase (<i>TPS</i>) genes marked in bright green. Central pie chart shows an 84% tandem duplicate supergenes fraction. <b>D</b>. Subset of genes associated with MEP and MVA pathways, including IPP isomerases, marked in blue. Central pie chart shows a 16% tandem duplicate supergenes fraction.</p

(B)LastZ two-way multiple alignment of 40kb-windows harboring the putative <i>Arabidopsis</i> gene transposition duplicate gene pair <i>DXS3</i> (AT5G11380) (upper lane, marked in purple) and <i>DXS1</i> (AT4G15560) (lower lane, marked in purple).

<p>Non-syntenic coding sequences are marked in green. Both duplicate copies form a highest-scoring sequence pair (marked in turquoise). Transposon-like sequences are marked in orange. Pseudogenes are marked in blue. Analysis can be regenerated online following the CoGe link <a href="https://genomevolution.org/r/eooq" target="_blank">https://genomevolution.org/r/eooq</a> (last accessed on December 13^th, 2014).</p

Tandem Duplicates fractions among terpenoid specialized biosynthetic module in 13^A genomes.

<p>Minus indicates absence of tandem duplicates. Asterisks indicate significant enrichment compared to genome-wide tandem duplicate fraction based on fisher's exact test on count data (p-value threshold: 0.01). For absolute gene numbers and p-values, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#pone.0128808.s005" target="_blank">S5 Table</a>.</p><p>^A<i>C</i>. <i>Sativa</i>, <i>L</i>. <i>sativa</i> and <i>N</i>. <i>benthamiana</i> and <i>C</i>. <i>gynandraare</i> excluded from this analysis due to technical reasons (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#sec002" target="_blank">Materials & Methods</a> section).</p><p>^B Averages based on numbers of tandem and singleton genes, not on percentage values since gene counts in subsets are not equal.</p><p>Tandem Duplicates fractions among terpenoid specialized biosynthetic module in 13<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128808#t003fn002" target="_blank">^A</a> genomes.</p