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Front Plant Sci
2016 Jan 01;7:1564. doi: 10.3389/fpls.2016.01564.
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Soybean TIP Gene Family Analysis and Characterization of GmTIP1;5 and GmTIP2;5 Water Transport Activity.
Song L
,
Nguyen N
,
Deshmukh RK
,
Patil GB
,
Prince SJ
,
Valliyodan B
,
Mutava R
,
Pike SM
,
Gassmann W
,
Nguyen HT
.
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Soybean, one of the most important crops worldwide, is severely affected by abiotic stress. Drought and flooding are the major abiotic stresses impacting soybean yield. In this regard, understanding water uptake by plants, its utilization and transport has great importance. In plants, water transport is mainly governed by channel forming aquaporin proteins (AQPs). Tonoplast intrinsic proteins (TIPs) belong to the plant-specific AQP subfamily and are known to have a role in abiotic stress tolerance. In this study, 23 soybean TIP genes were identified based on the latest soybean genome annotation. TIPs were characterized based on conserved structural features and phylogenetic distribution. Expression analysis of soybean TIP genes in various tissues and under abiotic stress conditions demonstrated tissue/stress-response specific differential expression. The natural variations for TIP genes were analyzed using whole genome re-sequencing data available for a set of 106 diverse soybean genotypes including wild types, landraces and elite lines. Results revealed 81 single-nucleotide polymorphisms (SNPs) and several large insertions/deletions in the coding region of TIPs. Among these, non-synonymous SNPs are most likely to have a greater impact on protein function and are candidates for molecular studies as well as for the development of functional markers to assist breeding. The solute transport function of two TIPs was further validated by expression in Xenopus laevis oocytes. GmTIP1;5 was shown to facilitate the rapid movement of water across the oocyte membrane, while GmTIP2;5 facilitated the movement of water and boric acid. The present study provides an initial insight into the possible roles of soybean TIP genes under abiotic stress conditions. Our results will facilitate elucidation of their precise functions during abiotic stress responses and plant development, and will provide potential breeding targets for modifying water movement in soybean.
FIGURE 1. Phylogenetic relationship and exon-intron structure of soybean Tonoplast intrinsic proteins (TIPs). (A) The unrooted tree was constructed via alignment of full-length amino acid sequences from soybean using MEGA6 software by the neighbor-joining method. (B) Lengths of the exons and introns of each TIP gene are displayed proportionally. Exons and introns are indicated by yellow rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicated by blue rectangles.
FIGURE 2. Chromosomal locations and gene duplication events of soybean TIP gene family members. The segmentally duplicated gene pairs are linked by black dotted lines. The left scale represents physical distance along the chromosomes in megabases (Mb). Chromosome numbers are shown at the top of each vertical gray bar.
FIGURE 3. Protein tertiary structure showing pore morphology of GmTIP1;5 and GmTIP2;5. (A) Tertiary structures comprised of six transmembrane domains and water molecules (red) passing through the pores of GmTIP1;5 (left) and GmTIP2;5 (right) visualized with CLC genomic workbench. (B) Cross sections of the proteins showing pore. (C) Pore diameter profile of GmTIP1;5 (left) and GmTIP2;5 (right) at 3 Å steps corresponding to the pore shape in (B). Pore axis (X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): pore diameter value in Å.
FIGURE 4. Identification and distribution of conserved motifs in soybean TIP protein sequences. Distribution of conserved motifs in soybean TIP members. All motifs were identified by Multiple EM for Motif Elicitation (MEME) using the complete amino acid sequences of GmTIP proteins. Different motifs are indicated by different colored boxes numbered 1–10. The annotation of each motif is listed at the bottom. Motif 1 and motif 2 contain the NPA domain.
FIGURE 5. Heatmap of expression profiles of the soybean GmTIP gene family in nine tissues. Relative tissue expression levels of GmTIPs based on RNA-seq data were used to construct the expression patterns of soybean genes. The expression data (Reads Per Kilobase Million) values were median-centered and normalized for each gene in different tissue before transforming to color scale. The color bar at the bottom shows the range of expression values from highest expression level (red) to lowest expression level (green), 0 is the median expression level (Black).
FIGURE 6. Expression profiles of soybean TIP genes under different abiotic stress conditions or in different germplasms. (A) Heatmap representation of expression patterns of soybean TIP genes across the root, leaf, and shoot tissue in Williams 82 under varying water-deficit stress conditions (VMS: very mild stress; MS: mild stress; SS: severe stress; SR: water recovery after severe stress). (B) Expression profiles of the soybean TIP genes in leaves of Pana (fast wilting, drought vs. control), PI 567690 (slow wilting, drought vs. control), PI 408105A (flooding tolerant, flooding vs. control), S99-2281(flooding sensitive, flooding vs. control). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The color bar at the bottom shows the range of expression values from increased expression (red) to decreased expression (green), 0 means no gene expression pattern changed (Black).
FIGURE 7. Variation in pore morphology of GmTIP5;2 and GmTIP5;2m. GmTIP5;2m contains two amino acid changed (148: A<>G; 159: T<>R) due to two SNP (Gm12_895269 and Gm_895302). (A) and (B) Cross sections of the proteins showing pore of GmTIP5;2 (A) and GmTIP5;2m (B). (C) and (D) Pore diameter profile of GmTIP5;2 (C) and GmTIP5;2m (D) at 3 Å steps. Pore axis (X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): pore diameter value in Å.
FIGURE 8. Oocyte Swelling Assays with GmTIP1;5 and GmTIP2;5. The relative diameter of GmTIP-expressing oocytes following exposure to hypoosmotic or boron-containing isosmotic solutions was measured to characterize the osmotic permeability of GmTIP1;5 and GmTIP2;5-containing membranes. (A) Water transport assay with Xenopus leaevis oocytes in hypoosmotic solution showed that GmTIP2;5-mediated water uptake leads to rapid swelling and bursting (top) compared to the control (bottom). Scale bars = 1 mm. (B) Rate of oocyte swelling in hypoosmotic solution in mock-injected controls (yellow line) vs. GmTIP2;5-expressing oocytes (blue line). Oocytes were observed to burst after 10 min. (C) Rate of oocyte swelling in hypoosmotic solution in mock injected controls (yellow line) vs. GmTIP1;5-expressing oocytes (green line). Results are reported as means ± SEM (N = 5 oocytes). (D) Rate of oocyte swelling in isosmotic boric acid solution in mock injected controls (yellow line) vs. GmTIP2;5-expressing oocytes (blue line) and GmTIP1;5 (green line). The volume of each oocyte was measured for 20 min or until it burst.
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