Bioinformatic analysis of plant TPX2 family proteins
To identify putative plant TPX2 family proteins, the conserved TPX2 (PF06886) domains were searched from the PFAM database. The database currently holds a total of 763 sequences from 188 species. Based on the presence of other domains, these sequences can be classified into 17 different domain architectures (Additional file 1: Figure S1). We extracted all 763 sequences from PFAM database and identified that 574 of these sequences were from 45 different species of plants (Viridiplantae) (Additional file 2: Table S1). These sequences include multiple splice variants for different genes. The plant TPX2 sequences were aligned using ClustalX2.1 and a phylogenetic tree was produced (Fig. 1).
The phylogenetic tree reveals 6 main clades. Clade 1 contains MAP20, the protein first reported from wood forming tissues of Populus [12] and its homologues from different plant species [27]. Clade 2 does not contain any protein that has been previously described. Since these are closest to MAP20 clade, we name this clade as MAP20L. However, it should be considered that these proteins are much larger than MAP20 (Fig. 2). Clade 3 contains the best studied plant TPX2 protein, the AtTPX2 [22]. Clades 4–6 contain WVD2 and WDL proteins [23, 26, 28, 29]. Some of the sequences fell in between the clades. While these sequences contain the TPX2 domain and are mostly from lower plants, these are sufficiently different to produce large insertions in the global alignment of plant TPX2 proteins. Hence, for some of the analyses presented here, these sequences were not used. A full list of number of sequences in each clade is presented on Additional file 3: Table S2.
A global alignment of plant TPX2 proteins from 6 main clades (Fig. 2) reveals that the clade 1 (MAP20) proteins are the smallest of the TPX2 proteins while the clade 2 (MAP20L) and clade 3 (TPX2) are the largest. Proteins in clades 4–6 (WDLs) are of intermediate size. An analysis of sequence conservation across various clades (Fig. 2b) reveals that there is little or no sequence conservation among the proteins outside of the TPX2 domain. It is likely that members of different clades would serve different functions. They all contain the TPX2 domain which has been shown to bind the microtubules [20, 22]. It is likely these proteins will bind to microtubules and at the same time interact with other proteins to play an important role in a variety of plant developmental processes involving in a role for microtubules.
An analysis of extracted TPX2 domains from all plant TPX2 proteins reveals that there are some differences in the sequences between the different clades. The most apparent of these differences is the presence of a KLEEK motif in the clades 4–6. This motif is absent in proteins from clades 1–3 (Fig. 3).
In the PFAM database, there were 31 sequences from Arabidopsis (At) and 19 sequences from Eucalyptus (Eg). Since these sequence sets include the splice variants, the fully sequenced genomes available at Phytozome were scanned to identify 15 TPX2 loci in Arabidopsis and 12 loci in Eucalyptus (Additional file 4: Table S3). The Eucalyptus proteins were named based on known Arabidopsis homologues. A similar nomenclature approach has been used previously for CESA proteins [30]. The final list of Eucalyptus loci included EgMAP20 (Clade 1), EgMAP20L (Clade 2); EgTPX2 (Clade 3); EgWDL1, EgWDL3 and EgWDL3L (Clade 4); EgWDL4, EgWDL5 and EgWDL6 (Clade5) and EgWDL7, EgWDL8 and EgWDL8L (Clade 6).
Genetic properties of Eucalyptus TPX2 family genes
The Eucalyptus TPX2 family gene structures were compared using online software GSDS 2.0 [31]. The core TPX2 domain is usually spread across three exons in all Eucalyptus TPX2 genes, which are followed by another exon downstream (Fig. 4a). EgMAP20 is the smallest of the 12 Eucalyptus TPX2 genes and has only one more exon on the 5′ side of the TPX2 domain. EgMAP20L on the other hand has four exons upstream of TPX2 domain, making a total of 8 exons for this gene. EgTPX2 is the largest of the 12 genes and comprises of a total of 20 exons and 19 introns. The remaining genes, 9 WDL genes, have between 2 and 5 exons upstream of the TPX domain making a total of 6–9 exons in these genes (Fig. 4a).
Modern plant genome diversity has evolved via gene deletions, small-scale duplications, partial rearrangements and large chromosomal fragment duplications, which significantly impacted the expansion in gene family members [32]. The 12 Eucalyptus TPX2 family genes showed random distribution along Eucalyptus chromosomes on the basis of the chromosome information provided by Phytozome (http://www.phytozome.net/). The 12 TPX2 family genes are distributed on 7 of the 11 Eucalyptus chromosomes (Fig. 4b). Based on the phylogenetic tree, we linked 4 pairs of the paralogous TPX2 family genes (red and blue dotted line pairs in Fig. 4b). EgWDL5/EgWDL6 and EgWDL3/EgWDL3L are the tandem duplication pairs on chromosome 3 and 8 respectively. However the paralogous duplication pairs, EgMAP20/EgMAP20L and EgWDL8/EgWDL8L are located on chromosomes 10/9 and 10/2 respectively (Additional file 5: Table S4). The conservation and micro-colinearity of EgTPX2 family genes show symbiotic evolution and suggest a common origin of these genes. Together, the diverse duplication events contributed to the complexity of TPX2 gene family in the Eucalyptus genome.
The synonymous (Ks) and non-synonymous (Ka) substitution rates ratios (Ka/Ks ratio) were used to analyse the TPX2 gene pairs. The synonymous substitutions do not change the amino acid sequence and are subjected to a lower selection pressure. On the other hand, non-synonymous substitutions change the amino acid sequence which might lead to harmful mutations and hence are under a higher selection pressure. When Ka/Ks is close to 1, it indicates evolution under neutral selection. A Ka/Ks ratio of < 1 indicates that those genes undergo a purifying (stabilizing) selection while Ka/Ks > 1 at specific sites indicates genes that are under positive selection. In most cases, the Ka/Ks ratio is less than 1 due to the purifying selection. However, when the diversifying selection exists, the Ka/Ks of the allele will increase, even significantly higher than 1 [33]. Additional file 5: Table S4 shows that the Ka/Ks ratios of all 4 duplicated pairs were all less than 0.9, indicating purifying selection.
Functional analysis of Eucalyptus TPX2 family genes
Expression analysis of TPX2 family genes in Eucalyptus
The expression patterns of genes can provide useful clues to their function. To identify the expression patterns of TPX2 family genes in plants, 6 different tissues of Eucalyptus grandis - shoot tips (ST), young leaves (YL), mature leaves (ML), phloem (PH), xylem (XL), roots (RT)) were analysed by quantitative RT-PCR (Fig. 5a). In general, expression of EgWDL5 was the highest among the Eucalyptus TPX2 genes followed by EgWDL4 and EgTPX2. All other genes were expressed at comparatively lower levels.
To compare relative expression levels of each gene across multiple tissues, we calculated the normalised expression values for each gene (Fig. 5b). Seven out of 12 Eucalyptus genes (EgMAP20, EgWDL1, EgWDL3, EgWDL3L, EgWDL6, EgWDL7 and EgWDL8) had highest expression in roots. EgMAP20L and EgTPX2 had highest levels in young leaves while EgWDL4 and EgWDL8L had highest levels in mature leaves. While the expression of EgWDL5 was the highest in the shoot tips and young leaves (Fig. 5b). The observation that most of TPX2 genes had their highest levels in root indicated that they may be functionally important in root development.
Subcellular localization of Eucalyptus TPX2 proteins
Despite their variable domain structures and expression patterns, one common feature of TPX2 family proteins is that they are all likely to be MAPs and have MT binding activity. To investigate their MT binding ability in vivo, we examined the subcellular localization of a selection of Eucalyptus TPX2 proteins. Coding sequences (CDS) of EgMAP20, EgWDL3 and EgWDL3L were fused to C-terminal Yellow Florescence Protein (YFP) tags (EgMAP20-YFP, EgWDL3-YFP and EgWDL3L-YFP) and the constructs were transiently expressed in tobacco leaf epidermal cells. Confocal microscopy observations of the EgMAP20-YFP, EgWDL3-YFP and EgWDL3L-YFP florescence signals form net-like structures throughout the cell (Fig. 6) indicating that these proteins are all distributed along the MT cytoskeleton. Previously, poplar PttMAP20-YFP has been shown to distribute along microtubules [12]. In addition, PttMAP20 was strongly up-regulated during secondary cell wall synthesis in hybrid aspen and tightly co-regulated with CESA genes [12]. WVD2, WDL1 and WDL3 have also been localized to cortical microtubules [23, 28]. TPX2 has been shown to localize to the cortical microtubules in interphase, and it may decorate other MT arrays during other stages of plant cell division (preprophase band, spindle, phragmoplast) [22]. TPX2 family proteins contain a conserved MT binding domain, the TPX2 domain, which plays an important role in the organization of the MT arrays, cell growth and the regulation of cell division [34]. Taken together, previous studies and our own observations suggest that EgMAP20, EgWDL3 and EgWDL3L are MT binding proteins like their homologues from Populus and Arabidopsis.
Phenotypic observation of transgenic plants
EgMAP20 overexpression in Arabidopsis leads to organ twisting
To understand the effect of the TPX2 family proteins on plant growth and development, p35S:EgMAP20 overexpressing transgenic Arabidopsis plants were analysed. The cotyledons of 12-day-old seedlings overexpressing EgMAP20 developed left/right handed twisting of epidermal cells while etiolated hypocotyls of 3-days-old seedlings showed right/left-handed twist (Fig. 7a-f).
Similar twisting phenotypes with overexpression of TPX2 proteins from other species have been reported previously. Overexpressing PttMAP20 in Arabidopsis caused cotyledon petioles with left-handed helical twisting and hypocotyl epidermal cells with a right-handed helical twist [12]. Overexpression of MT bundling proteins, WVD2 and WDL1 in Arabidopsis led to a right hand deviation of roots and left hand skewing of cotyledon petioles [26] but left-handed twisting of the rosette leaves. During organ twisting, the anisotropy of cell elongation is lost, leading to generation of short and robust cells. In addition, cortical MT arrays of root epidermal cells are also changed [28]. Overexpression of TPX2 caused the random organization of cortical microtubules and root right handed shift [25]. However, overexpression of the MT depolymerising protein MAP18 produced a left handed twist of hypocotyl epidermal cells [11].
The growth of plant cells involves an increase in cell volume, which can be achieved by cell expansion or elongation. Both of these two processes occur in a specific area of the cell surface causing the changes in cell morphology. Three kinds of MT arrays participating in cell morphogenesis exist in plants, including cortical MTs, preprophase bands and phragmoplast MTs. The cortical MTs during interphase are involved in controlling the arrangement of cellulose microfibrils in the cell wall and thereby determining the direction of cell elongation [35]. It is plausible that MAP20 plays a role in cell elongation leading to directional skewing. To understand why cotyledon and hypocotyl cells of EgMAP20 overexpression plants are sometimes left/right spiral, further experiments are needed.
EgWDL3L overexpression in Arabidopsis affects growth
The over-expression (OE) of EgWDL3L in Arabidopsis on the other hand did not lead to any helical twisting. There was no difference between the wild type and EgWDL3L OE lines grown in dark. However, the hypocotyls were shorter for the light grown seedlings (Fig. 8). Previous studies have shown that AtWDL3 is a negative regulator of the hypocotyl cell elongation and acts in a light dependent manner. In light, AtWDL3 is relatively stable and promotes MTs to form a longitudinal arrangement, thereby inhibiting hypocotyl cell elongation. However, in the dark, AtWDL3 is degraded via the 26S proteasome pathway, thereby removing inhibition of hypocotyl cell elongation [23]. Since EgWDL3L and AtWDL3 are closely related and are both part of Clade 4, EgWDL3L like AtWDL3 may also be a negative regulator of cell elongation.