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. 2009 Nov 20;9:134.
doi: 10.1186/1471-2229-9-134.

Genome-wide Analysis of Major Intrinsic Proteins in the Tree Plant Populus Trichocarpa: Characterization of XIP Subfamily of Aquaporins From Evolutionary Perspective

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Genome-wide Analysis of Major Intrinsic Proteins in the Tree Plant Populus Trichocarpa: Characterization of XIP Subfamily of Aquaporins From Evolutionary Perspective

Anjali Bansal Gupta et al. BMC Plant Biol. .
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Abstract

Background: Members of major intrinsic proteins (MIPs) include water-conducting aquaporins and glycerol-transporting aquaglyceroporins. MIPs play important role in plant-water relations. The model plants Arabidopsis thaliana, rice and maize contain more than 30 MIPs and based on phylogenetic analysis they can be divided into at least four subfamilies. Populus trichocarpa is a model tree species and provides an opportunity to investigate several tree-specific traits. In this study, we have investigated Populus MIPs (PtMIPs) and compared them with their counterparts in Arabidopsis, rice and maize.

Results: Fifty five full-length MIPs have been identified in Populus genome. Phylogenetic analysis reveals that Populus has a fifth uncharacterized subfamily (XIPs). Three-dimensional models of all 55 PtMIPs were constructed using homology modeling technique. Aromatic/arginine (ar/R) selectivity filters, characteristics of loops responsible for solute selectivity (loop C) and gating (loop D) and group conservation of small and weakly polar interfacial residues have been analyzed. Majority of the non-XIP PtMIPs are similar to those in Arabidopsis, rice and maize. Additional XIPs were identified from database search and 35 XIP sequences from dicots, fungi, moss and protozoa were analyzed. Ar/R selectivity filters of dicots XIPs are more hydrophobic compared to fungi and moss XIPs and hence they are likely to transport hydrophobic solutes. Loop C is longer in one of the subgroups of dicot XIPs and most probably has a significant role in solute selectivity. Loop D in dicot XIPs has higher number of basic residues. Intron loss is observed on two occasions: once between two subfamilies of eudicots and monocot and in the second instance, when dicot and moss XIPs diverged from fungi. Expression analysis of Populus MIPs indicates that Populus XIPs don't show any tissue-specific transcript abundance.

Conclusion: Due to whole genome duplication, Populus has the largest number of MIPs identified in any single species. Non-XIP MIPs are similar in all four plant species considered in this study. Small and weakly polar residues at the helix-helix interface are group conserved presumably to maintain the hourglass fold of MIP channels. Substitutions in ar/R selectivity filter, insertion/deletion in loop C, increasing basic nature of loop D and loss of introns are some of the events occurred during the evolution of dicot XIPs.

Figures

Figure 1
Figure 1
Evolutionary relationship of Populus MIPs. Phylogenetic analysis of all Populus MIPs is shown along with MIPs from Arabidopsis. Neighbor-Joining (NJ) method was used to create this unrooted tree. NJ method used the multiple sequence alignment generated by T-COFFEE to generate the tree. Populus MIP subfamilies PtPIPs, PtTIPs, PtNIPs and PtSIPs clustered with the corresponding Arabidopsis MIP subfamilies. XIPs observed only in Populus clearly form a separate group. Each MIP subfamily is shown with a specific background color to distinguish them from others. A similar result is obtained when the same analysis was carried out with rice and maize MIPs (Additional files 2 to 4).
Figure 2
Figure 2
Phylogenetic analysis of XIPs. All 35 XIPs from dicot plants, fungi, moss and protozoa have been used to construct the phylogenetic tree using NJ method. Multiple sequence alignment for creating the phylogenetic tree was generated by T-COFFEE. XIPs from dicot plants, fungi and moss cluster separately. All dicot XIPs cluster into two subgroups XIP1 and XIP2. All fungi XIPs and some dicot XIPs (Table 2) have been identified in this study. Other dicot XIPs, moss XIPs and the lone XIP from protozoa were identified by Danielson and Johanson. The names of Populus XIPs, dicot XIPs identified in this study and fungi XIPs are given in Tables 1, 2 and 3 respectively. The names of other dicot and the protozoan XIPs are given as follows with their IDs (EST/RefSeq/whole genome shotgun sequence) in brackets: SlXIP1;1 -- Solanum lycopersicum (BT014197), CcXIP1;1 -- Citrus clementina (DY275505), GrXIP1;1 -- Gossypium raimondi (CO092422), RcXIP1;1 -- Ricinus communis (EG656577), RcXIP2;1 -- Ricinus communis (EG666650), AfXIP1;1 -- Aquilegia formosa × Aquilegia pubescens (DR936893 and DT742029), DdXIP1;1 -- Dictyostelium discoideum (XM_639170) and VvXIP1;1 and VvXIP1;2 -- Vitis vinifera (AM455454).
Figure 3
Figure 3
Ar/R selectivity filters of PtPIP2;10 and PtSIP1;1. Ar/R selectivity filters of non-XIP Populus MIPs that are not found in their counterparts from Arabidopsis, rice and maize. Transmembrane regions of the MIP models from Populus were superposed on the experimentally determined structures of glycerol transporter GlpF (PDB ID: 1FX8) and the water-transporting spinach aquaporin SoPIP2;1 (PDB ID: 1Z98). Populus MIP residues are shown in stick representation with nitrogen and oxygen atoms in blue and red colors respectively. PtMIP residues are displayed in one letter code and their corresponding positions in the selectivity filter are indicated. For comparison purpose, the ar/R filters of GlpF and SoPIP2;1 are also shown in blue and pink respectively. (A) Out of 54 PIPs from four plants, PtPIP2;10 is the only PIP in which the Phe at H2 position is substituted by an Asn, making it more hydrophilic. (B) Residues forming the ar/R filter of PtSIP1;1 are very hydrophobic. Even the normally conserved Arg at LE2 position is substituted by a hydrophobic Phe. This will be one of the most hydrophobic constrictions in known MIPs.
Figure 4
Figure 4
Ar/R selectivity filters of PtXIP2;1 and F-FoXIP. Ar/R selectivity filters of two XIPs, one from a dicot plant (PtXIP2;1) and the other from fungi (F-FoXIP). XIP models were first individually superposed on the experimentally determined structures of GlpF and SoPIP2;1 as described in Figure 3. Residues of XIP models are shown in stick representation with nitrogen and oxygen atoms displayed in blue and red respectively. For other details, see the caption of Figure 3. (A) Ar/R selectivity filter of PtXIP2;1 has three hydrophobic residues and is likely to transport a more hydrophobic solute. (B) The presence of Asn and Arg along with two small residues makes the ar/R selectivity filter of F-FoXIP more hydrophilic and result in a wider constriction. Such XIPs are likely to transport bulkier hydrophilic solutes.
Figure 5
Figure 5
Alignment of loop C residues of XIPs. The sequence regions containing loop C are aligned for all XIPs. Residues forming the last turn of H3 and the first turn of H4 are shown in gray background. All Gly and Pro residues are displayed in red and pink color respectively. The conserved Cys which is part of the 'GGC' motif is shown in green.
Figure 6
Figure 6
Alignment of loop D residues of XIPs. Multiple sequence alignment of residues forming loop D is shown for all XIPs. Residues forming the last turn of H4 and the first turn of H5 are shown in gray background. All basic (Arg, Lys and His) and acidic (Asp and Glu) residues are displayed in red and blue colors respectively. ClustalW (version 1.82) was used to perform the multiple sequence alignment of both loops.
Figure 7
Figure 7
Gene structure of non-XIP MIPs from Populus, Arabidopsis and rice. Exon-intron organization of non-XIP MIP genes from Populus, Arabidopsis and rice is depicted for the PIP, TIP, NIP and SIP subfamilies. The exon-intron pattern observed in majority of MIPs within a subfamily is shown in gray background. In this case, only the number of MIPs having that pattern is indicated for each plant species. For example, "At:12/13" indicates that 12 out of 13 AtPIPs have the same gene structure. For those members with different exon-intron organization, the MIP name is explicitly given (example: Os:2.8 in PIP subfamily). The six TM regions are shown in black bars and the loops B and E are shown in oval shape. The intron positions are indicated by inverted triangle.
Figure 8
Figure 8
Exon-intron pattern observed in Populus and fungi XIPs. Exon-intron organization of XIP MIP genes from Populus and fungi is displayed. Five out of 6 PtXIPs have similar exon-intron organization and the pattern is shown in gray background. The sixth PtXIP (PtXIP2;1) has no introns and its gene structure is shown separately. The six TM regions are shown in black bars and the loops B and E are shown in oval shape. The intron positions are indicated by inverted triangle. For details about fungi XIPs, see Table 3.
Figure 9
Figure 9
Relative transcript abundance profiles of Populus MIPs. A heat map showing the transcript abundance of MIPs from all Populus subfamilies [85] is displayed using the program "Heatplus" [86]. This heat map is produced using the expression data obtained from Populus eFP browser [103]. The transcript abundance levels for the Populus MIPs were clustered using hierarchical clustering based on Pearson correlation coefficients. Each row corresponds to the normalized expression profile of a particular gene and their names are shown. Data obtained for nine different tissues for each gene are represented in columns. Symbols in the map represent as follows: ML -- mature leaf; YL -- young leaf; R -- root; DG -- dark-grown seedlings, etiolated; DL -- dark grown seedling etiolated and then exposed to light for 3 hrs; CL -- continuous light-grown seedling; FC -- female catkins; MC -- male catkins; X -- xylem. The data is normalized for each gene (row-normalized). The relative transcript accumulation is represented in a color code with green and red showing respectively the lower and higher levels of transcript accumulation.
Figure 10
Figure 10
Evolution of dicot XIPs. A likely scenario for the evolution of dicot XIPs. The evolutionary events are indicated at the point where the XIPs diverged. Dicot XIPs evolved from fungi and moss through substitutions at ar/R selectivity filter, insertion/deletion of loop C and loss of an intron. However, small and weakly polar residues occurring at the helix-helix interface are highly group-conserved.

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