Genome-wide identification and expression profiling analysis of the Aux/IAA gene family in Medicago truncatula during the early phase of Sinorhizobium meliloti infection

Abstract

Background: Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal findings: Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion: The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.

Figure 1. Chromosome mapping of MtIAA family genes.

The genome visualization tool CIRCOS was employed. Medicago truncatula chromosomes are arranged in a circle, and the centromere of each chromosome is marked in black. Ribbon links represent the segmental duplication region retrieved from the SyMAP database. MtIAA family genes are mapped by locus.

Figure 2. Phylogenetic relationships, exon-intron structure and protein domain analysis of MtIAA family genes.

(A) An unrooted phylogenetic tree was constructed using ClustalW by N-J method. The sister pair genes are indicated by red boxes. (B) Exon-intron structure analysis of MtIAA genes. The untranslated regions (UTRs) are indicated by thick red lines; the exons are indicated by blue boxes; the introns are indicated by gray lines. (C) Alignment of Medicago truncatula Aux/IAA proteins obtained with the ClustalW program and manual correction. Multiple alignments of the domains I–IV of the M. truncatula Aux/IAA proteins also were showed by red lines. Colorized shading indicates identical and conversed amino acid residues, respectively. The LXLXLX motif was also marked by thin yellow box. Two NLSs were marked by black asterisks.

Figure 3. Phylogenetic relationships and motifs distribution analysis.

(A) Phylogenetic relationships among Arabidopsis and M. truncatula IAA proteins. The unrooted tree was generated by MEGA5.1 program by the N-J method. Bootstrap supports from 1000 replicates are showed at each branch. (B) The motif distribution in Arabidopsis and M. truncatula IAA proteins. Motifs of Aux/IAA proteins were analyzed by MEME web server. Four motifs representing four domains I, II, III and IV were mapped on all Aux/IAA proteins by different colors. (C) The heights of each box represent the specific amino acid conservation in each motif.

Figure 4. Tissues-specific expression patterns of MtIAA genes.

Based on the phylogenetic analysis, all MtIAA genes were grouped into five subfamilies (A: subfamily A; B: subfamily B; C: subfamily C; D: subfamily D; E: subfamily E). Expression patterns of the MtIAA genes in five indicated organs were analyzed by the data of qRT-PCR. The value of ACTIN (Cotyledon)/1000 defines as 1. C: cotyledon; L: leaf; R: root; S: shoot; F: flower. The data were analyzed by five independent repeats, and standard deviations were shown with error bars.

Figure 5. Real-time quantitative RT-PCR (qRT-PCR) analyses of MtIAA genes in plants under IAA treatment in both shoots (A) and roots (B).

Total RNA was extracted from the shoots and roots of M. truncatula seedlings for basal expression. The histogram shows the relative expression level of MtIAA genes under IAA treatment compared to the mock expression level. The relative mRNA level of individual genes was normalized with respect to the MtACTIN gene. The concentration of synthetic IAA was 0.1 µM. Samples of two different organs (shoots and roots) were used to test the changes of MtIAA genes expression level at different time points (3 hr, 6 hr, 12 hr and 24 hr). The data were analyzed by five independent repeats, and standard deviations were shown with error bars.

Figure 6. Motif analysis of specific cis-elements in promoters of MtIAA family genes in M. truncatula.

The −1000 bp promoter sequences of corresponding MtIAA genes were used to analysis of specific ZRE, AuxRE and MRE cis-elements, which are given using the presented colour code. Watson and Crick words for AUX1 is TGTCTC; AUX2 is TGTVYS, three different ZREs (GRE: BACGTV; TGA: TGACG; AC-motif: ACTCAT) and two MREs (MRE1: AMCWAMC; MRE2: GGWTW).

Figure 7. Heat map showing MtIAA genes expression pattern at the early phase of Sinorhizobium meliloti infection under different conditions.

Samples of two different organs (shoots and roots) were used to test the changes of MtIAA genes expression level at different time points (3/6/12/24/48/72 hpi) treatment. The different colors correspond to the log-transcription values of the gene change-fold ratio shown in the bar at the right of figure.

Figure 8. Heat map showing MtIAA genes expression pattern at the early phase of Sinorhizobium meliloti infection under different conditions.

Samples of two different organs (shoots and roots) were used to test the changes of MtIAA genes expression level at different time points (3/6/12/24/48/72 hpi) and conditions (−Sin/−NOA, +Sin/−NOA, −Sin/+NOA and +Sin/+NOA). The treatment –Sin/−NOA was used as mock treatment. The different colors correspond to the log-transcription values of the gene change-fold ratio shown in the bar at the right of figure.