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Comparative genomics reveals conservative evolution of the
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xylem transcriptome in vascular plants
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Xinguo Li, Harry X. Wu*, and Simon G. Southerton*
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CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia
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Corresponding authors
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Email addresses:
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XL: [email protected]
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HW: [email protected]
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SS: [email protected]
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Abstract
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Background: Wood is a valuable natural resource and a major carbon sink. Wood
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formation is an important developmental process in vascular plants which played a
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crucial role in plant evolution. Although genes involved in xylem formation have been
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investigated, the molecular mechanisms of xylem evolution are not well understood.
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We use comparative genomics to examine evolution of the xylem transcriptome to
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gain insights into xylem evolution.
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Results: The xylem transcriptome is highly conserved in conifers, but considerably
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evolved in angiosperms. The functional domains of genes in the xylem transcriptome
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are moderately to highly conserved in vascular plants, suggesting the existence of a
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common ancestral xylem transcriptome. Compared to the total transcriptome derived
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from a range of tissues, the xylem transcriptome is relatively conserved in vascular
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plants. Of the xylem transcriptome, cell wall genes, ancestral xylem genes, known
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proteins and transcription factors are relatively more conserved in vascular plants. A
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total of 527 putative xylem orthologs were identified, which are unevenly distributed
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in Arabidopsis chromosomes with eight hot spots observed. Phylogenetic analysis
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revealed that evolution of the xylem transcriptome has paralleled plant evolution. We
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also identified 274 conifer-specific xylem unigenes, all of which are of unknown
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function. These xylem orthologs and conifer-specific unigenes are likely to have
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played a crucial role in xylem evolution.
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Conclusions: Conifers have highly conserved xylem transcriptomes, while
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angiosperm xylem transcriptomes are relatively diversified. Vascular plants share a
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common ancestral xylem transcriptome. In vascular plants the xylem transcriptome is
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more conserved compared to the total transcriptome.
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Background
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The evolution of xylem was a critical step that allowed vascular plants to colonize
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vast areas of the earth’s terrestrial surface. Xylem plays a crucial role in the transport
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of water and nutrients and provides mechanical support for vascular plants.
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Herbaceous vascular plants develop primary xylem, and woody plants also produce
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secondary xylem (or wood). Evolution from tracheids to vessels reflects a more
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efficient way for angiosperms to develop secondary xylem [1-3]. Primary xylem
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consists of cellulose, hemicellulose, pectin and proteins, while secondary xylem
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contains higher amounts of cellulose and lignin. From a practical point of view, wood
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represents a renewable natural resource for the timber, fibre and biofuel industries and
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it is a major carbon sink in natural ecosystems.
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In the last few decades the molecular basis of the xylem formation and evolution has
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been investigated. Syringyl (S) lignin biosynthesis was found to be an evolved lignin
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pathway in angiosperms, representing an addition to the ancient and predominant
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guaiacyl (G) lignin pathway conserved in land plants [3-6]. However, the S lignin
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pathway was also recently observed in lycophytes [7], suggesting a complex
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evolutionary history of lignin biosynthesis in vascular plants. Genes involved in wood
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formation have been identified in many plant species [8-13], allowing investigation of
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xylem evolution at the transcriptome level. A core xylem gene set was identified in
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white spruce among which 31 transcripts are highly conserved in Arabidopsis [14].
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The expanding genomic resources of model plant species [15-18] are invaluable for
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exploring many aspects of plant development and evolution, including xylem
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formation and evolution. Recent studies suggest whole-genome duplication and
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reorganization [15-17] have been a major driving force in plant evolution.
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Comparative genomics is a powerful tool for investigating plant evolution at the
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whole-genome level [19-27]. Lower collinearity across dicots and monocots [16, 21]
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and lineage-specific genes have been observed using comparative genomics [28, 29].
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Comparisons between rice and Arabidopsis showed that 50% of the rice genome was
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homologous to Arabidopsis [16], while as much as 88% of the poplar genome shares
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homology with Arabidopsis [17]. Comparative genomics of poplar with Arabidopsis
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revealed at least 11,666 protein-coding genes were present in the ancestral eurosid
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genome [17]. The recently sequenced Selaginella moellendorffii [30], moss [31] and
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Eucalyptus grandis [18] provide new opportunities for comparative genomics in
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vascular plants.
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Many commercial crops and forest trees do not have sequenced genomes; thus,
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comparative genomics in these species must rely on studies of the transcriptome
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(expressed sequence tags, ESTs). Using the transcriptome approach about half of
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loblolly pine xylem unigenes did not match Arabidopsis unigenes [32], and 39-45%
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of pine contigs had no hits in the genomes of Arabidopsis, rice and poplar [33]. Eighty
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percent of white spruce and 32-36% of sitka spruce unigenes lacked homologs in
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poplar, Arabidopsis and rice [12, 33]. Similar results were also observed in other
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comparisons between gymnosperms and angiosperms [19, 34]. In contrast,
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comparison
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transcriptomes. For example, about 84% of the white spruce transcripts had matches
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in the Pine Gene Index [12], and 60-80% of spruce contigs had homologs in loblolly
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pine and other gymnosperms [33].
of
different
gymnosperm
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species
revealed
highly
conserved
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Although comparative genomics has increased our understanding of plant evolution at
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the genome level, most studies to date have focussed on the entire genome sequence,
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and/or mixed EST resources developed from a range of tissues. In order to examine
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xylem evolution in vascular plants we focused our attention on genes expressed
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specifically in xylem. We selected ten species (see Methods) to represent different
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categories of vascular plants, including gymnosperms (pine and spruce), angiosperms
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(woody dicots, herbaceous dicots, and monocots) and lycophytes. The non-vascular
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plant moss was included as a reference for xylem evolution. Up-to-date public
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genome sequences and xylem transcriptomes of selected plant species were used for
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comparative genomic analysis, aiming to explore the evolution of the xylem
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transcriptome in vascular plants.
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Results
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The xylem transcriptome is highly conserved in conifer species
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Comparisons of xylem unigenes of the three conifer species (radiata pine, loblolly
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pine and white spruce) with other vascular plants and moss are shown in Figure 1A-F.
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At the nucleotide level 78-82% (E ≤ 10-5) and 63-66% (E ≤ 10-50) of radiata pine
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xylem unigenes have homologs in the xylem unigenes of loblolly pine and white
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spruce, while 93-95% (E ≤ 10-5) and 74-89% (E ≤ 10-50) have homologs in the gene
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indices of pine and spruce (Figure 1A). Thus, the radiata pine xylem transcriptome is
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highly conserved in other conifer species. Further analysis with loblolly pine and
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white spruce revealed that conifer xylem transcriptomes are highly conserved at both
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the nucleotide (Figure 1B and 1C) and deduced amino acid sequence level (Figure
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1D-F).
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Conifer xylem transcriptomes are considerably distinct from angiosperms and
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lycophytes. At the nucleotide level, only 15-32% (E ≤ 10-5) and 1-4% (E ≤ 10-50) of
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radiata pine xylem unigenes have homologs in the gene models or scaffolds of poplar,
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Eucalyptus, Arabidopsis, rice, Selaginella and moss (Figure 1A). Similarly low
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numbers of homologs were identified in analyses with loblolly pine (Figure 1B) and
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white spruce (Figure 1C). Unsurprisingly, the deduced amino acid sequences of
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conifer xylem unigenes have relatively more homologs in non-coniferous plants
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(Figure 1D-F). However, the number of homologs is still considerably lower than the
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number of matches among the three conifers (Figure 1D-F). Therefore, at both the
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nucleotide and amino acid level the conifer xylem transcriptome is relatively distinct
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from angiosperms, lycophytes and moss.
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We examined the average nucleotide identity among the homologous xylem unigenes
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shared by the different conifer species. The average nucleotide identity between
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radiata pine and loblolly pine is 97.5%, and 91% between radiata pine and the two
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spruce species (Figure 2A). Lower nucleotide identity (81-83%) was observed in the
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homologous unigenes of radiata pine and angiosperms (Populus, Arabidopsis and
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rice) or moss (Figure 2A). These results were also confirmed in analyses in loblolly
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pine (Figure 2B), providing additional evidence for xylem transcriptome conservation
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in conifers, and its divergence in non-coniferous plants.
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The xylem transcriptome has evolved considerably in angiosperms
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Poplars are woody angiosperms and are used as a model tree species for wood
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formation. Surprisingly, at the level of nucleotide sequence less than 5% of poplar
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xylem unigenes have homologs (E ≤ 10-50) in the scaffolds or gene models of the three
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angiosperms (Eucalyptus, Arabidopsis and rice) (Figure 3A). Poplar xylem unigenes
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also have few homologs in the gene indices of pine or spruce and in the gene models
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of Selaginella and moss (Figure 3A). These results suggest that the poplar xylem
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transcriptome is distinct, and that the xylem transcriptome has undergone considerable
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evolution in angiosperms. This contrasts with the highly conserved xylem
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transcriptomes of gymnosperms we observed earlier (Figure 1). Furthermore, poplar
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also has a unique genome (at the nucleotide level) among the species examined in this
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study (Figure 3C). However, the functional gene sequences of the poplar xylem
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transcriptome (Figure 3B) and the entire genome (Figure 3D) are moderately (36-50%
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at E ≤ 10-50) conserved, while their functional domains are highly conserved (77-85%
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at E ≤ 10-5) in other vascular and non-vascular plants. These results suggest that land
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plants share an ancestral genome and an ancestral xylem transcriptome .
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The xylem transcriptome is relatively more conserved in vascular plants
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We examined the pattern of the xylem transcriptome evolution by comparing it with
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the total transcriptome derived from a range of tissues. Although the xylem
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transcriptomes of pine, spruce and poplar have few homologs in the gene models of
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Arabidopsis, rice, Selaginella and moss (1.2-5% at E ≤ 10-50), there are about two to
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four times more homologs than there are for the total transcriptome (Figure 4A).
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When using a lower stringency E-value (≤ 10-5), the xylem transcriptomes of pine,
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spruce and poplar have more homologs (about 20-60%) in the above four model
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species compared to the total transcriptome (9-34%). Thus, at the nucleotide level the
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xylem transcriptome is relatively more conserved in vascular and non-vascular plants.
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Interestingly, both the conifer xylem and the total transcriptomes have few homologs
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in poplar (a woody species) and in herbaceous species (Arabidopsis, rice, Selaginella
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and moss) (Figure 4A). This suggests that conifer xylem formation is distinct from
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xylem formation in woody angiosperms.
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The conservative evolution of the xylem transcriptome was more clearly observed
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when using deduced amino acid sequences (Figure 4B). For example, 37-46% of the
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conifer xylem transcriptome matched (E ≤ 10-50) the genomes of angiosperms
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(Populus, Arabidopsis and rice), lycophyte and moss; nearly two times more than the
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matches with the total conifer transcriptome (Figure 4B). Similar results were also
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obtained with the poplar xylem transcriptome in the comparisons with the total
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transcriptome (Figure 4B). Therefore, the functional gene sequences in the xylem
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transcriptome are significantly more conserved in vascular plants.
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Different functional gene groups in the xylem transcriptome have distinct
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patterns of evolution
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We divided the xylem transcriptome into different functional groups for investigating
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their diverse patterns of evolution. The xylem unigenes with matches (blastx, E ≤ 10-
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matches as vascular plant-specific xylem (VSX) genes. Among the ancestral xylem
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genes derived from radiata pine, loblolly pine and white spruce, 96-99%, 78-94% and
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48-60% (E ≤ 10-5), or 41-48%, 20-26% and 28-35% (E ≤ 10-50), respectively, have
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homologs (blastx) in the genomes of the three angiosperms and the lycophyte (Figure
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5A-C). In contrast, only 8-28% (E ≤ 10-5) and 0.4-1% (E ≤ 10-50) of the conifer VSX
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genes are homologous to the genomes of these model vascular plants (Figure 5A-C).
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These results suggest that the ancestral xylem genes are moderately to highly
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conserved in non-coniferous vascular plants, while considerable evolution has
) in the moss gene models are defined as ancestral xylem genes, and those with no
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occurred in VSX genes among conifers, angiosperms and lycophytes. Similar patterns
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were observed in comparisons of VSX and ancestral xylem genes with the poplar
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xylem transcriptome (Figure 5D).
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Cell wall genes were identified according to the CellWallNavigator [35] and
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MAIZEWALL [36] databases. We compared the level of conservation of cell wall
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genes to non-cell-wall genes (Figure 5A-D). In the four woody species (radiata pine,
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loblolly pine, white spruce and poplar) the cell wall genes have 1.5 to two times more
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homologs than the non-cell-wall genes in the genomes of the four herbaceous model
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plants (Figure 5A-D). This suggests that cell wall genes have been highly conserved
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across diverse land plants. Comparisons of transcription factors (TFs) (based on
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PlantTFDB [37]) with non-TFs and known functional genes with unknowns revealed
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significantly more conservation of transcription factors and known functional genes
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among different groups of vascular and non-vascular plants (Additional data file 1).
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Identification of putative xylem orthologs in vascular plants
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Putative xylem orthologs conserved in diverse plant groups were identified using
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deduced amino acid or protein sequences (Additional data file 2), and their numbers
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are briefly presented at two E-value cut-offs (≤ 10-50 and ≤ 10-5) in Figure 6. The
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xylem orthologs (9,164 at E ≤ 10-5 or 3,460 at E ≤ 10-50) shared by loblolly pine, white
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spruce and sitka spruce represent a common gene set expressed in conifer wood
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formation. While the xylem orthologs (6,641 at E ≤ 10-5 or 1,339 at E ≤ 10-50)
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common to the three conifers and poplar are putative woody plant xylem orthologs.
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Surprisingly, the number of putative xylem orthologs in other plant groups is only
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slightly smaller (Figure 6), suggesting that very limited numbers of xylem orthologs
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are specific to each plant group. The majority (93-96%) of the vascular plant xylem
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orthologs have matches in the gene models of a non-vascular plant (moss). This
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suggests that the xylem transcriptome of vascular plants has largely evolved from a
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subset of genes found in non-vascular plants. This is likely to be a group of genes
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predominantly involved in cell wall biosynthesis that have been recruited to
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synthesise secondary xylem in higher plants.
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Of the 1,003 loblolly pine xylem unigenes with homologs (tblastx or blastx, E ≤ 10-50)
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in the six vascular plants, 94% and 100% have hits in the uniprot and TIGR databases,
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respectively, and 94% are assigned with GO terms (Additional data file 2). Among
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these unigenes 349 (35%) were identified as cell wall genes, and the most abundant
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gene family is tubulin (16 unigenes). Cellulose synthase is also abundant (10). Several
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primary wall genes are abundant including pectate lyase (8), pectinesterase (7), XET
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(7), expansin (6) and peroxidase (5). Lignin biosynthesis-related genes are well
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represented in the cell wall gene lists, including SAMS (11), laccase (9), methionine
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synthase (cobalamin-independent) (6), 4CL (3), COMT (2), CAD (2) and CCoAMT
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(3). In addition, 49 transcription factors were found, including NAC, PHD, HB,
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MYB, LIM, CCCH, etc. The large number of transcription factors suggests that wood
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formation involves considerable transcriptional regulation. Interestingly, aquaporins
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(16 unigenes) is one of the largest gene families in the 1,003 unigenes, reflecting the
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importance of water transport in xylem formation.
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The 1,003 loblolly pine xylem unigenes have between 749 and 894 close homologs
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(blastx or tblastx, E ≤ 10-50) in white spruce, sitka spruce, poplar, Arabidopsis, rice
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and Selaginella, with 527 xylem orthologs common to all the above species. These
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common xylem orthologs matched (blastx, E ≤ 10-50) 501 unique gene models in moss,
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indicating that at least 501 ancestral xylem orthologs are shared in land plants. We
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also identified all loci in Arabidopsis and rice with homology to the 1,003 loblolly
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pine xylem unigenes. All possible homologs in Arabidopsis are represented by 3,115
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and 6,563 unique loci at E ≤ 10-50 or ≤ 10-5, respectively, and in rice by 3,057 and
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8,458 unique loci. This suggests that each xylem ortholog has an average of 4-8
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paralogs in Arabidopsis and 5-14 paralogs in rice.
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The xylem orthologs and paralogs have an even distribution among chromosomes of
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Arabidopsis (Additional data file 3A) and poplar (Additional data file 3C), but may be
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unevenly distributed among rice chromosomes (Additional data file 3B). However,
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eight hot spots were observed within the five Arabidopsis chromosomes (Figure 7A,
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B), which contrasts with the even distribution of all Arabidopsis genes across
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chromosomes [38] (Figure 7C). These hot spots may be due to historical patterns of
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gene and chromosome duplication [15, 39, 40]. Within the chromosomes of rice and
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poplar the xylem orthologs are relatively evenly distributed (Additional data file 4 and
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Additional data file 5). Different distribution patterns of the xylem orthologs among
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the Arabidopsis, rice and poplar genomes may reflect their diverse history of gene
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duplication and segmental chromosomal rearrangements.
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Molecular evolution of xylem orthologs in vascular plants
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We used the 501 ancestral xylem orthologs for phylogenetic analysis because they
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largely (95%) represent the 527 unique xylem orthologs and it allowed us to include
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the non-vascular plant moss as a reference. Three different trees (Additional data file
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6A-C) were constructed using four alignment methods (see Methods), in which
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ClustalX2 and Mafft generated the same tree configuration (Additional data file 6B).
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Among these trees, morphologically related species are consistently clustered in the
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same group. The trees constructed by Kalign (Additional data file 6A) and ClustalX2
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(or Mafft) (Additional data file 6B) clearly separate angiosperms and gymnosperms
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from lycophytes and moss. Based on tree B moss and Selaginella emerged at the
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earliest time point followed by the two conifers (loblolly pine and white spruce) and
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then by the two dicots (poplar and Arabidopsis). The monocot xylem transcriptome
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appears to be the most evolutionarily distinct. In summary, the phylogenetic analysis
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suggests that molecular evolution of xylem orthologs parallels plant evolution.
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Identification of putative conifer-specific xylem genes
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A total of 274 loblolly pine xylem unigenes which matched unigenes of white spruce
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and sitka spruce (tblastx, E ≤ 10-50), but have no homologs in the gene models of
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poplar, Arabidopsis, rice, Selaginella and moss, as well as no hits in any other
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angiosperms in the uniprot known protein database (blastx, E > 10-5), were identified
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as putative conifer-specific xylem unigenes (Additional data file 7). All conifer-
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specific xylem unigenes are unknown protein sequences based on the uniprot known
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protein database, and only 16% were assigned with GO terms in the TIGR gene index
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database. This contrasts with 94% of the xylem orthologs annotated with GO terms.
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The poor annotation of the conifer-specific xylem genes is likely due to the intense
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functional characterisation of genes that has taken place in angiosperms.
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Of the conifer-specific xylem unigenes several relatively abundant transcripts have
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low homology to arabinogalactan-like proteins (AGPs), glycine/proline-rich proteins
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(GPRP), metallothionein-like class II proteins, neurofilament triplet H proteins, zinc
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finger proteins, cytochrome c1, etc (Additional data file 7). Genes with low similarity
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to other cell wall proteins are present, such as alpha tubulin, cellulose synthase like
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and extensin-like proteins. The conifer-specific xylem unigenes also include some
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genes similar to transcription factors, i.e. Aux/IAA, NAC, CCAAT-binding, WRKY,
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R2R3-MYB, BHLH and CCAAT-binding (Additional data file 7). These conifer-
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specific xylem unigenes may share the functions of related homologues, but some
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may have unique roles in gymnosperm wood formation.
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Discussion
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Gymnosperms and angiosperms have distinct patterns of xylem transcriptome
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evolution
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Our data revealed that the xylem transcriptome is highly conserved in conifers but has
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undergone considerably more diversification in angiosperms. This suggests the xylem
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transcriptome has a distinct pattern of evolution in angiosperms compared to
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gymnosperms. Pine and spruce diverged 120-140 Ma [41], slightly earlier than the
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radiation of angiosperms (100-120 Ma) [16, 17]. The rapid evolution of the
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angiosperm xylem transcriptome suggests greater sensitivity to evolutionary forces in
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flowering plants. Our results also showed that poplar has a relatively distinct genome
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and xylem transcriptome, thus poplar is unlikely to be a useful model for investigating
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wood formation and other developmental processes in gymnosperms. Mechanisms
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underlying the diverse patterns of xylem transcriptome evolution in angiosperms and
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gymnosperms are not well understood. Gene duplications and segmental chromosome
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rearrangements may be the major driving forces for this diversification; however, the
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role of genome size or gene number remains unclear.
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The common ancestor of the xylem transcriptome in vascular plants
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Our data demonstrated that the nucleotide and protein sequences of the xylem
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transcriptome are highly diversified among different categories of vascular plants, but
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that their functional domains are moderately to highly conserved in vascular plants.
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This is consistent with the diversification of all vascular plants from a common
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ancestor which is thought to have lived about 400 Ma [42]. Some of these conserved
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patterns are also consistent with comparisons using the total transcriptome in previous
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studies [32, 43]. The conserved functional domains of the xylem transcriptome
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suggest the existence of a shared ancestral xylem transcriptome of vascular plants.
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The common ancestral xylem transcriptome should be present in the earliest ancient
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vascular plants, which produced hilate/trilete spores during the Late Ordovician [44].
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The lycophyte Selaginella is among the closest living relatives of these plants and its
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xylem transcriptome is likely to resemble most closely the ancestral xylem
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transcriptome. The 5,047 (27%) xylem unigenes of loblolly pine with homologs (E
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≤10-5) in the six vascular plants including Selaginella could largely represent the
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ancestral xylem transcriptome. Because the vast majority (95%) of the ancestral
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xylem transcriptome has homologs (E ≤10-5) in the moss genome, the xylem
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transcriptome is likely to have evolved largely from the cell wall transcriptome of
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non-vascular plants, which can be traced back as far as 450 Ma [31].
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Conservative evolution of the xylem transcriptome in vascular plants
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Xylem functions in the transport of water and solutes throughout the plant and
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together with the phloem makes up the vascular transport tissues of plants. Our data
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suggests that the xylem transcriptome is relatively more conserved than the total
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transcriptome in vascular plants. Several functional gene groups within the xylem
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transcriptome are significantly more conserved among vascular plants, including cell
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wall genes, ancestral xylem genes, transcription factors and known protein genes.
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This implies that xylem has evolved more slowly compared to other organs of
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vascular plants such as leaves, flowers and seeds. On the other hand, the unknown
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function and VSX genes have been more rapidly evolving in vascular plants. The
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rapid evolution of vascular plant-specific xylem (VSX) genes contrasts with the
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generally conservative evolution of the total xylem transcriptome, suggesting VSX
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genes are particularly sensitive to evolutionary forces. These genes will be useful
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targets for functional characterisation in order to understand xylem formation and
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evolution in vascular plants.
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Our data showed that the loblolly pine xylem transcriptome has significantly more
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homologs in spruce (white spruce and sitka spruce) than in poplar and Eucalyptus,
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thus it is possible to identify genes specific to gymnosperm wood formation
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(softwoods). Surprisingly, the number of homologs of the loblolly pine xylem
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transcriptome in woody angiosperms (poplar and Eucalyptus) is similar to that in
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herbaceous angiosperms (Arabidopsis and rice), suggesting that a limited number of
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xylem genes are specific to woody plants. Furthermore, the number of homologs of
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the conifer xylem transcriptome in angiosperms is only slightly more than the number
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in lycophytes and moss. The limited number of xylem genes specific to woody plants
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suggests that differential transcriptional regulation may have played an important role
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in xylem evolution; in a similar way that transcriptional regulation gives rise to plant
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diversity [20].
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Comparison of gene expression between softwoods and hardwoods
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A previous study in Cryptomeria japonica identified 56 putative conifer-specific
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transcripts, including three specific to reproductive organs and one (unknown)
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specific to woody tissues [45]. Here we identified 274 conifer-specific xylem
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unigenes, all of which are of unknown function. Transcripts with low homology to
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some cell wall genes are abundant or present in the conifer-specific xylem unigenes
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(Additional data file 7). This suggests that some cell wall genes, such as tubulin and
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CesA, may be specific to conifer wood formation. These conifer-specific xylem genes
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could provide clues to the molecular processes that give rise to the distinct cell wall
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structures and chemical properties of softwoods compared to hardwoods. Genes
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related to lignin biosynthesis are not present in the conifer-specific xylem unigenes,
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which is consistent with the earlier conclusion that the conifer lignin pathway is
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conserved in other vascular plants [3].
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Transcripts similar to genes encoding arabinogalactan proteins (AGPs) are the most
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abundant genes in the conifer-specific xylem unigenes. AGPs are a large class of
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hydroxyproline-rich glycoproteins. The conifer-specific xylem AGPs (AGP4 and
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AGP-like) identified in this study had no homologs with any of the 47 AGPs of
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Arabidopsis [46-48]. Most AGPs are anchored to the plasma membrane [46] and
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released into the cell wall after cleavage of the GPI anchor [49]. AGPs may act as cell
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wall plasticizers, enlarging the pectin matrix, and allowing wall extension and cell
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expansion [50]. In loblolly pine six AGPs including AGP4 were identified in xylem
395
tissues [51]. Radiata pine AGPs are located in the compound middle lamella of newly
396
developed tracheids [52]. The radiata pine EST resource included 11 AGPs, in which
397
PrAGP4 is highly abundant in earlywood libraries [13].
398
16
399
We identified 527 xylem orthologs in vascular plants, including many primary and
400
secondary cell wall genes as well as transcription factors. Conservation of cell wall
401
genes suggests that cell wall biosynthesis is a central event in xylem formation in
402
vascular plants. These xylem orthologs maintain the stability of the basic xylem
403
machinery in vascular plants and may regulate development of xylem structures and
404
properties shared by different vascular plants, such as the common features of
405
softwoods and hardwoods. COMT was previously thought to be one of three enzymes
406
(F5H/Cald5H, COMT and SAD) specifically involved in S lignin synthesis of
407
angiosperms [3]. The occurrence of COMT genes in the xylem orthologs of vascular
408
plants suggests their involvement in diverse functions other than S lignin synthesis.
409
410
Conclusions
411
The xylem transcriptome is highly conserved in conifer species, but the nucleotide
412
sequences of the xylem transcriptome are significantly distinct among angiosperms,
413
thus considerable evolution of the xylem transcriptome has occurred in angiosperms.
414
The functional domains of the xylem transcriptome are moderately to highly
415
conserved among vascular and non-vascular plants. This suggests that vascular plants
416
share an ancestral xylem transcriptome which is likely to have evolved predominantly
417
from the cell wall transcriptome of non-vascular plants. In comparison to the total
418
transcriptome from a wide range of tissues, the xylem transcriptome has evolved
419
conservatively in vascular plants. Several functional gene groups within the xylem
420
transcriptome, including cell wall genes, ancestral xylem genes, transcription factors
421
and known function genes, are relatively more conserved in vascular plants. A total of
422
527 xylem orthologs of vascular plants and 274 conifer-specific xylem genes were
423
identified in this study. These genes provide good targets for molecular investigations
17
424
of xylem formation and evolution in softwoods (conifers) and hardwoods (woody
425
angiosperms). The xylem orthologs are unevenly distributed within Arabidopsis
426
chromosomes, with eight hot spots detected. Phylogenetic analysis of the 501
427
ancestral xylem orthologs suggests that molecular evolution of the xylem
428
transcriptome has paralleled plant evolution.
429
430
Methods
431
Plant species
432
We selected ten species to represent three major classes of vascular plants
433
(gymnosperms, angiosperms and lycophytes). Four conifer species: Pinus radiata
434
(radiata pine), Pinus taeda (loblolly pine), Picea glauca (white spruce) and Picea
435
sitchensis (sitka spruce), represent gymnosperms. Four species of dicots including
436
Populus tremula × Populus tremuloides (hybrid aspen), Populus trichocarpa,
437
Eucalyptus grandis and Arabidopsis thaliana, and one monocot (Oryza sativa, rice)
438
represent angiosperms. The recently sequenced Selaginella moellendorffii [30]
439
represents lycophytes. The non-vascular plant Physcomitrella patens (moss) was
440
included as a reference. The seven woody species (four conifer and three woody dicot
441
species) both undergo primary and secondary xylem formation. Arabidopsis also
442
develops secondary xylem and has been used as a model system for secondary xylem
443
development [10, 11, 53-56]. The monocot rice and the lycophyte S. moellendorffii
444
only produce primary xylem, while the non-vascular plant moss has no xylem at all.
445
446
Public genomic resources
447
We previously developed a radiata pine xylem transcriptome resource with 3,304
448
xylem unigenes [13]. Unigenes of loblolly pine, white spruce, sitka spruce, hybrid
18
449
aspen, P. trichocarpa, Arabidopsis, rice and moss (Additional data file 8) were
450
retrieved from the NCBI UniGene database [57]. The gene indices of pine, spruce and
451
poplar (Additional data file 8) were collected from the TIGR gene index database
452
[58]. These gene indices represent the total transcriptome from a wide range of tissues
453
(such as stem, shoots, xylem, leaves, flowers, bark, roots and seeds, etc). A sub-set of
454
xylem gene indices of pine, spruce and poplar was separately retrieved from pure
455
xylem libraries. These included genes expressed in pure xylem tissues, while mixed
456
xylem tissues (such as shoots, cambial regions with phloem, etc) were not considered.
457
After removing redundant sequences a total of 14,527, 15,262 and 9,109 xylem gene
458
indices of pine, spruce and poplar were identified. To minimize the bias in
459
comparative genomics, pure xylem gene indices were reassembled using the same
460
method and parameters as used in radiata pine [13], resulting in 18,320, 12,489 and
461
7,991 pure xylem unigenes for pine, spruce and poplar, respectively.
462
463
The public transcriptome resources (unigenes and gene indices) are unlikely to
464
include all transcripts expressed in xylem formation due to sampling limitations.
465
Therefore, selected fully sequenced plant species (P. trichocarpa, E. grandis,
466
Arabidopsis, rice, S. moellendorffii and P. patens) were added for comparative
467
genomics in this study (Additional data file 8). Gene models of P. trichocarpa (v1.1),
468
S. moellendorffii (v1.0) and P. patens (moss) (v1.1) were downloaded from the JGI
469
website [59]. E. grandis scaffolds were downloaded from EucalyptusDB [18]. A.
470
thaliana gene models (TAIR8) were downloaded from the TAIR website [60] and O.
471
sativa gene models (v6.0) were downloaded from the MSU website [61].
472
473
Comparative genomic analysis
19
474
Blast programs (blastn, tblastx, blastx, blastp and tblastn) were used for sequence
475
comparisons. All blast searches were performed locally using BlastStation2 (TM
476
software, Inc., CA). Expected-value (E-value or E), sequence identity and bit scores
477
were collected for evaluating single sequence similarity. Percentage of hits, average
478
sequence identity and average bit score at different E-value cut-offs were calculated
479
for the transcriptome comparisons between different plant species.
480
481
The E-value cut-offs for inferring sequence similarity vary from 10-3 [17, 24] to 10-50
482
[62, 63], but 10-10 to 10-30 have been widely used [19, 32, 43, 64-67]. To ensure high
483
confidence as previously suggested [62] and to increase the likelihood of inferring
484
biological significance, we used the following thresholds of E-values: (a) E = 0, the
485
two sequences are identical and considered to derive from the same gene; (b) 10-50 ≤ E
486
< 0, the two sequences are highly similar and likely to be from the same gene family;
487
(c) 10-5 ≤ E < 10-50, the two sequences are similar in one or more regions along the
488
whole sequence and likely to contain the same functional domain(s) or motif(s); (d) E
489
< 10-5, the two sequences are likely to be unrelated genes. As a general rule, E ≤ 10-50
490
was interpreted in this study to indicate whole sequence similarity of two given
491
sequences (gene conservation or apparent homologs); and 10-5-10-50 was interpreted to
492
indicate partial sequence similarity (domain or motif conservation). However, the
493
possible influences of different blast programs and database size were not considered
494
in setting the E-value thresholds.
495
496
Phylogenetic analysis
497
The predicted amino acid sequences of ancestral xylem orthologs from loblolly pine
498
and white spruce, and protein sequences from the five model species (poplar,
20
499
Arabidopsis, rice, Selaginella and moss) were used for phylogenetic analysis. For
500
each species all sequences of ancestral xylem orthologs were combined into one
501
sequence prior to phylogenetic analysis. Sequence alignments were compared using
502
four methods: ClustalX2 [68], Kalign [68], Mafft [68] and Mega4 [69]. Phylogenetic
503
trees were built using the neighbour-joining algorithm with a bootstrap of 1000.
504
505
Abbreviations
506
Mb, million base pairs; Ma, million years ago; EST, expressed sequence tag; E, E-
507
value or expected-value; GO, gene ontology; PGI, pine gene index; SGI, spruce gene
508
index; PplGI, Populus gene index; PGI xylem, gene index from pure xylem tissues of
509
pines; SGI xylem, gene index from pure xylem tissues of spruces; PplGI xylem, gene
510
index from pure xylem tissues of Populus; VSX genes, vascular plant-specific xylem
511
genes; TF, transcription factor; CesA, cellulose synthase; AGP, arabinogalactan
512
protein; COMT, caffeic acid 3-O-methyltransferase.
513
514
Authors’ contributions
515
XL contributed the project idea, collected relevant sequence data, conducted the
516
comparative analysis and prepared the manuscript. SS and HW proposed and guided
517
the research project. All the authors have approved the final manuscript.
518
519
Additional data files
520
Additional data file 1 is a figure showing that known protein genes and transcription
521
factors in the xylem transcriptome are relatively more conserved in diverse plants.
522
Additional data file 2 is a table listing 527 xylem orthologs in vascular plants based on
21
523
the loblolly pine xylem unigenes with homologs (tblastx or blastx, E ≤ 10-50) in the
524
unigenes or gene models of white spruce, sitka spruce, poplar, Arabidopsis, rice and
525
Selaginella. Additional data file 3 is a figure showing the distribution of xylem
526
orthologs on each chromosome of Arabidopsis, rice and poplar, and its significance
527
(P-value) compared to all loci in each chromosome. Additional data file 4 is a figure
528
showing locations of xylem orthologs on rice chromosomes (A: 3,057 homologs
529
based on 1E-50; B: 8,458 homologs based on 1E-5; C: 3,000 randomly selected loci).
530
Additional data file 5 is a figure showing locations of xylem orthologs on poplar
531
chromosomes (A: 3,523 homologs based on 1E-50; B: 7,436 homologs based on 1E-
532
5; C: 3,200 randomly selected loci). Additional data file 6 is a figure showing
533
phylogenetic trees for 501 ancestral xylem orthologs using four alignment methods (A:
534
Kalign; B: ClustalX2 or Mafft; C: Mega 4). Additional data file 7 is a table listing 274
535
putative conifer-specific xylem unigenes. Additional data file 8 is a table summarising
536
the public genomic resources of 11 plant species used in this study.
537
538
Acknowledgements
539
This work was funded by Forest and Wood Products Australia (FWPA), ArborGen
540
LLC, the Southern Tree Breeding Association (STBA), Queensland Department of
541
Primary Industry (QDPI) and the Commonwealth Scientific and Industrial Research
542
Organization (CSIRO). We thank Iain Wilson, Shannon Dillon, Colleen MacMillan,
543
Bryan Clarke and Jason Bragg for their critical comments on the manuscript.
544
545
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Figure legends
778
Figure 1. Comparisons of conifer xylem unigenes with genes in other species.
779
Xylem unigenes of radiata pine, loblolly pine and white spruce were blasted against
780
xylem unigenes, gene indices, gene models or scaffolds of other plant species.
781
Percentage of hits is presented in the Y-axis at E-value cut-offs ranging from 1E-5 to
782
0 in the X-axis. A: Radiata pine vs. other species (blastn); B: Loblolly pine vs. other
783
species (blastn); C: White spruce vs. other species (blastn); D: Radiata pine vs. other
784
species (tblastx or blastx); E: Loblolly pine vs. other species (tblastx or blastx); F:
785
White spruce vs. other species (tblastx or blastx).
786
787
Figure 2. Average nucleotide identity between pine unigenes and respective
788
homologs in other species. Xylem unigenes of radiata pine and loblolly pine were
789
blasted using blastn against unigenes of other species, including loblolly pine, white
790
spruce, sitka spruce, hybrid aspen, P. trichocarpa, Arabidopsis, rice and moss.
791
Homologs at E-value cut-offs ranging from 0 to 1E-5 were collected and their average
792
nucleotide identities were calculated and presented. A: Radiata pine xylem unigenes
793
vs. homologous unigenes of other species; B: Loblolly pine xylem unigenes vs.
794
homologous unigenes of other species.
795
796
Figure 3. Comparisons of genes between poplar and other species. Poplar
797
xylem unigenes and gene models were blasted against gene indices, gene models or
798
scaffolds of other plant species. Percentage of hits is presented in the Y-axis at E-
799
value cut-offs ranging from 1E-5 to 0 in the X-axis. A: Poplar xylem unigenes vs.
800
other species (blastn); B: Poplar xylem unigenes vs. other species (tblastx or blastx);
28
801
C: Poplar gene models vs. other species (blastn); D: Poplar gene models vs. other
802
species (tblastn or blastp).
803
804
Figure 4. The xylem transcriptome is relatively more conserved in vascular and
805
non-vascular plants. Xylem gene indices and total gene indices of pine, spruce and
806
poplar were blasted against the gene models of Populus, Arabidopsis, rice, Selaginella
807
and moss using blastn and blastx. Percentage of hits is presented in the Y-axis at E-
808
value cut-offs of 1E-50 and 1E-5 in the X-axis. Statistical tests for the different
809
number of hits observed with xylem and total transcriptome were performed assuming
810
a binomial distribution. Almost all comparisons showed statistical significances
811
(P<0.001) (data not show), with only two exceptions from the blastn comparisons
812
(1E-50) between the two conifer species and poplar. A: Blastn; B: Blastx.
813
814
Figure 5. Cell wall genes and ancestral xylem genes are relatively more
815
conserved in diverse plants. Putative cell wall genes, non-cell-wall genes, vascular
816
plant-specific xylem (VSX) genes and ancestral xylem genes from xylem unigenes of
817
radiata pine, loblolly pine, white spruce and poplar were blasted using tblastx with
818
pine and spruce gene indices as well as blastx with gene models of poplar,
819
Arabidopsis, rice, Selaginella and moss. Percentage of hits is presented in the Y-axis
820
at E-value cut-offs of 0, 1E-50 and 1E-5 in the X-axis. Different numbers of hits
821
observed with cell wall and non-cell-wall genes, as well as with VSX and ancestral
822
xylem genes were statistically tested assuming a binomial distribution. Almost all
823
comparisons showed statistical significances (P<0.001) (data not show), except for a
824
few comparisons among conifers at 1E-5. A: Radiata pine; B: Loblolly pine; C: White
825
spruce; D: Poplar.
29
826
827
Figure 6. Number of putative xylem orthologs in different groups of land plants.
828
Loblolly pine xylem unigenes (18,320) were blasted using tblastx against the unigenes
829
of white spruce and sitka spruce, and the gene models of poplar, Arabidopsis, rice,
830
Selaginella and moss. The number of putative xylem orthologs common to different
831
plant groups is presented at two E-value cut-offs (1E-50 and 1E-5). Conifers 1:
832
loblolly pine plus white spruce; Conifers 2: conifers 1 plus sitka spruce; Woody
833
plants: conifers 2 plus poplar; Seed plants 1: woody plants plus Arabidopsis; Seed
834
plants 2: seed plants 1 plus rice; Vascular plants: seed plants 2 plus Selaginella; Land
835
plants: vascular plants plus moss. The xylem orthologs shared in conifers 2, woody
836
plants, seed plant 1, seed plants 2 and vascular plants represent genes specific to
837
conifers, woody plants, seed plants, lower seed plants (lacking secondary xylem) and
838
vascular plants, respectively. The high proportion of orthologs common to land plants
839
suggests that the ancestral xylem genome was largely present in non-vascular plants.
840
841
Figure 7. Hot spots of putative xylem orthologs on the chromosomes of
842
Arabidopsis. The 1,003 loblolly pine xylem unigenes highly conserved in other
843
vascular plants (xylem orthologs) have strong homology (1E-50) with 3,115 loci and
844
moderate homology (1E-5) with 6,563 loci in the Arabidopsis genome. Positioning of
845
these homologs on the five chromosomes of Arabidopsis revealed eight hot spots
846
(showed as numbers 1 to 8 with red colour) of putative Arabidopsis xylem orthologs.
847
A: 3,115 highly homologous loci; B: 6,563 moderately homologous loci; C: 6,000
848
randomly selected loci.
849
850
30