Comparative phylogenetic and expression analysis of small GTPases families in legume and non-legume plants

Abstract

Background: Small monomeric GTPases act as molecular switches in several processes that involve polar cell growth, participating mainly in vesicle trafficking and cytoskeleton rearrangements. This gene superfamily has largely expanded in plants through evolution as compared with other Kingdoms, leading to the suggestion that members of each subfamily might have acquired new functions associated to plant-specific processes. Legume plants engage in a nitrogen-fixing symbiotic interaction with rhizobia in a process that involves polar growth processes associated with the infection throughout the root hair. To get insight into the evolution of small GTPases associated with this process, we use a comparative genomic approach to establish differences in the Ras GTPase superfamily between legume and non-legume plants.

Results: Phylogenetic analyses did not show clear differences in the organization of the different subfamilies of small GTPases between plants that engage or not in nodule symbiosis. Protein alignments revealed a strong conservation at the sequence level of small GTPases previously linked to nodulation by functional genetics. Interestingly, one Rab and three Rop proteins showed conserved amino acid substitutions in legumes, but these changes do not alter the predicted conformational structure of these proteins. Although the steady-state levels of most small GTPases do not change in response to rhizobia, we identified a subset of Rab, Rop and Arf genes whose transcript levels are modulated during the symbiotic interaction, including their spatial distribution along the indeterminate nodule.

Conclusions: This study provides a comprehensive study of the small GTPase superfamily in several plant species. The genetic program associated to root nodule symbiosis includes small GTPases to fulfill specific functions during infection and formation of the symbiosomes. These GTPases seems to have been recruited from members that were already present in common ancestors with plants as distant as monocots since we failed to detect asymmetric evolution in any of the subfamily trees. Expression analyses identified a number of legume members that can have undergone neo- or sub-functionalization associated to the spatio-temporal transcriptional control during the onset of the symbiotic interaction.

Keywords: Arf; Rab; Rop; biological nitrogen fixation; comparative genomics; symbiosis.

Figure 1.

Phylogenetic analysis of the small GTPase superfamily in legume and non-legume plants. Amino acid sequences corresponding to small GTPases from A thaliana, O. sativa, S. lycopersicum, Z. mays, L. japonicus, M. truncatula, P. vulgaris, and G. max were retrieved from genomic databases. Unrooted neighbor-joining trees were obtained using the Mega 7 software. Subfamilies were identified for each species: Rab (green), Arf (orange), Rop (blue) and Ran (pink).

Figure 2.

Multiple sequence alignment of P. vulgaris RABA2 (Phvul.011G061100) and proteins with the highest sequence identity from M. truncatula (Medtr4g064897), G. max (Glyma11g14360), L. japonicus (chr3.CM0792.300.r2.d), A. thaliana (At1g07410), S. lycopersicum (Solyc06g076450), Z. mays (GRMZM2G473906) and O. sativa (LOC_Os03g60870). Black boxes indicate identical residues and gray ones indicate conservative substitutions. Alignments were generated with Clustal Omega in MEGA7 and formatted with Boxshade. The red arrow indicates a conservative amino acid substitution in legume versus non-legume sequences. The conserved domains of Rabs are indicated by blue lines.

Figure 3.

Multiple sequence alignment of M. truncatula ROP9 (Medtr5g022600) and proteins with the highest sequence identity from, P. vulgaris (Phvul.002G106600), G. max (Glyma01g36880), L. japonicus ROP6 (chr2.CM0272.860.r2.m), A. thaliana (At2g17800), S. lycopersicum (Solyc02g083580), Z. mays (GRMZM2G375002) and O. sativa (LOC_Os02g58730). Black boxes indicate identical residues and gray ones indicate conservative substitutions. Alignments were generated with Clustal Omega in MEGA7 and formatted with Boxshade. Red arrows indicate amino acid substitutions in legumes versus non-legumes. The conserved domains of ROPs are indicated by blue lines.

Figure 4.

Multiple sequence alignment of M. truncatula ROP10 (Medtr3g078260) and proteins with the highest sequence identity from P. vulgaris (Phvul.009G180800), G. max (Glyma04g35110), L. japonicus (chr1.CM0166.830.r2.m), A. thaliana (At3g48040), S. lycopersicum (Solyc03g114070), Z. mays (GRMZM2G415327) and O. sativa (LOC_Os02g50860). Black boxes indicate identical residues and gray ones indicate conservative substitutions. Alignments were generated with Clustal Omega in MEGA7 and formatted with Boxshade. Red arrows indicate amino acid substitutions in legumes versus non-legumes. The conserved domains of ROPs are indicated by blue lines. The sequence from Z. mays is truncated at its C terminus.

Figure 5.

Three dimensional models of RABA2 from O. sativa and P. vulgaris, ROP10 from A. thaliana and M. truncatula, ROP3 from A. thaliana and ROP9 from M. truncatula. Arrows and yellow boxes indicate the position of the substitutions observed in legume versus non-legume species.

Figure 6.

Comparison of expression data obtained by RT-qPCR with microarray data of selected M. truncatula genes encoding small GTPases. Expression levels of the four transcripts in roots and nodules (Nod) at 10 or 21 days post inoculation (dpi) with S. meliloti were measured by RT-qPCR or by microarray analysis. Bars in qPCR graphs represent media and SE of two biological replicates. Expression levels were normalized with HIS3L and presented relative to the values of root tissue, which was set at 1.