Identification and Characterization of Salt- and Drought-Responsive AQP Family Genes in Medicagosativa L

Abstract

Aquaporins (AQP) are distributed ubiquitously in plants, and they play important roles in multiple aspects of plant growth and development, as well as in plant resistance to various environmental stresses. In this study, 43 MsAQP genes were identified in the forage crop Medicago sativa. All the MsAQP proteins were clustered into four subfamilies based on sequence similarity and phylogenetic relationship, including 17 TIPs, 14 NIPs, 9 PIPs and 3 SIPs. Analyses of gene structure and conserved domains indicated that the majority of the deduced MsAQP proteins contained the signature transmembrane domains and the NPA motifs. Analyses on cis-acting elements in the promoter region of MsAQP genes revealed the presence of multiple and diverse stress-responsive and hormone-responsive cis-acting elements. In addition, by analyzing the available and comprehensive gene expression data of M. truncatula, we screened ten representative MtAQP genes that were responsive to NaCl or drought stress. By analyzing the sequence similarity and phylogenetic relationship, we finally identified the corresponding ten salt- or drought-responsive AQP genes in M. sativa, including three MsTIPs, three MsPIPs and four MsNIPs. The qPCRs showed that the relative expression levels of these ten selected MsAQP genes responded differently to NaCl or drought treatment in M. sativa. Gene expression patterns showed that most MsAQP genes were preferentially expressed in roots or in leaves, which may reflect their tissue-specific functions associated with development. Our results lay an important foundation for the future characterization of the functions of MsAQP genes, and provide candidate genes for stress resistance improvement through genetic breeding in M. sativa.

Keywords: AQP genes; Medicago sativa; aquaporin; phylogenetic analysis; salt and drought stresses.

Figure 1

The phylogenetic analysis of AQP proteins from M. sativa, M. truncatula and Arabidopsis. The deduced full-length protein sequences of AQPs were constructed by using MEGA7 software based on the Maximum likelihood (ML) method, with bootstrap value of 1000 replicates. Members of four subfamilies of TIPs, PIPs, NIPs and SIPs are highlighted with purple, green, yellow and red backgrounds, respectively. The pink circles indicate AQP proteins from M. sativa; the cyan circles indicate AQP proteins from M. truncatula; and the light-blue circles indicate AQP proteins from Arabidopsis. The AQP proteins from M. truncatula are highlighted in red; while those from M. sativa are highlighted in green.

Figure 2

Chromosomal distribution of AQP family genes in M. sativa. (a) The chromosomal location and inter-chromosomal relationships of AQP genes on the eight chromosomes of M. sativa. The segmentally duplicated genes are in red and connected by red lines. The tandem duplicated genes are in red and connected by cyan lines; (b) Synteny analysis of AQP genes among M. sativa, Arabidopsis, and M. truncatula. Gray lines in the background indicate the collinear blocks within M. sativa, and M. truncatula/Arabidopsis, and the orange lines highlight the syntenic AQP gene pairs.

Figure 3

Analyses of the phylogenetic relationships, motifs and gene structure of AQP genes from M. sativa. Phylogenetic relationships (a); location of motifs (b); and gene structure of MsAQP genes (c). In (c), blue boxes indicate 5′- or 3′- untranslated regions, red boxes indicate exons, and black lines indicate introns.

Figure 4

Analyses of cis-acting elements in the upstream promoter region of AQPs in M. sativa. (a) The phylogenetic analysis of the deduced AQP proteins of M. sativa; (b) The colors and numbers on the grid indicated the numbers of different cis-acting elements in these MsAQP genes, which is presented in the form of heatmap; (c) Colored blocks represented different types of cis-acting elements and their relative location in the promoter region of each MsAQP gene.

Figure 5

Expression profiles of MtAQP genes under different treatments at different time points were retrieved from the M. truncatula gene expression atlas with multiple GeneChip^® data. The color of circles from white to red indicates the expression level from low to high after normalization. (a) Expression profiles of MtAQP genes in roots under 180 mM NaCl treatment at 0, 6, 24 and 48 h; (b) Expression profiles of MtAQP genes in roots under 200 mM NaCl treatment at 0, 1, 2, 5, 10 and 24 h; (c) Expression profiles of MtAQP genes in roots under drought treatment at 2, 3, 4, 7, 10, 14 d and rewater 1d after 14 d drought treatment; (d) Expression profiles of MtAQP genes in shoots under drought treatment at 2, 3, 4, 7, 10, 14 d and rewater 1 d after 14 d drought treatment.

Figure 6

Determination of gene expression levels for 10 MsAQP genes under stresses by using qPCR. Data are the average of three independent biological samples ± SE and vertical bars indicate standard deviation. (a) The value of X axis represents treatment period with 150 mM NaCl at 0, 4, 8, 12, 24 and 48 h, respectively; the value of Y axis represents relative expressions of each gene; (b) The value of X axis represents treatment period with 15% PEG at 0, 2, 8 and 32 h, respectively; the value of Y axis represents the relative expression of each gene. Data are presented as mean ± SD, Student’s t-test (n = 3, * p <0.05, ** p <0.01).

Figure 7

Determination of relative expression levels for 10 MsAQP genes in different tissues in M. sativa by using qPCR. Data are the average of three independent biological samples ± SE and vertical bars indicate standard deviation. The value of the X axis represents root, stem, leaf, flower tissues. The expression level in root for each sample were set as value of 1.