Calmodulin-Like (CML) Gene Family in Medicago truncatula: Genome-Wide Identification, Characterization and Expression Analysis

Abstract

Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca²⁺ signal in plants. Medicago truncatula is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between M. truncatula, Arabidopsis thaliana and Oryza sativa shared close relationships. Gene structure analysis revealed that these MtCML genes contained one to four conserved EF-hand motifs. All MtCMLs are localized to eight chromosomes and underwent gene duplication. In addition, MtCML genes were differentially expressed in different tissues of M. truncatula. Cis-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the MtCML gene family and facilitate their potential use for applications in the genetic improvement on M. truncatula in drought, cold and salt stress environments.

Keywords: CML; Medicago truncatula; abiotic stress; expression profiling; promoter cis-acting element.

Figure 1

Phylogenetic tree of calmodulin-like (CML) proteins in Medicago truncatula, Arabidopsis thaliana and Oryza sativa. Tree constructed with 1000 bootstrap replications. CMLs from M. truncatula, A. thaliana and O. sativa distinguished using green, red and blue color, respectively.

Figure 2

Characterization of MtCML genes and MtCML proteins. (A) EF-hand motifs; (B) exon–intron structure distribution.

Figure 3

Chromosomal distribution and synteny analysis of CML genes in the genomes of Medicago truncatula and Arabidopsis thaliana. (A) Paralogous MtCML genes mapped onto M. truncatula chromosomes; (B) Orthologous CML genes mapped onto M. truncatula (chromosomes 1–8) and A. thaliana (chromosomes 1–5). Red lines indicate duplicated MtCML gene pairs.

Figure 4

Expression analysis of MtCML genes in different organs. (A) Expression patterns of MtCML genes in leaves, petioles, stems, flowers, pods, roots and seeds. The expression levels of MtCML genes are shown as the log2-based fluorescence intensity values from the microarray data (MtGEA, https://mtgea.noble.org/v3/). DAP, days after pollination; (B–G) relative expression of (B) MtCML7; (C) MtCML14; (D) MtCML17; (E) MtCML20; (F) MtCML22; (G) MtCML47 in roots, stems, leaves, flowers, seeds and pods of Medicago truncatula. Expression data obtained via qRT-PCR. MtActin used as an internal control in the qRT-PCR experiments.

Figure 5

Number of MtCML genes containing various cis-acting elements. ABRE, ABA-responsive element. LTR—cis-acting element involved in low-temperature response; CGTCA-motif—cis-acting element involved in MeJA response; TCA-element—cis-acting element involved in SA response; GARE-motif and P-box—cis-acting element involved in GA response; MBS—cis-acting element involved in drought response; circadin—cis-acting element involved in biologic rhythms; AuxRR-core and TGA-element—cis-acting elements involved in auxin response.

Figure 6

Transcript profile of MtCML genes in response to salt and drought stresses. Genome-wide microarray data of M. truncatula in different tissues at various developmental stages and the response to drought and salt were retrieved from M. truncatula Gene Expression Atlas (MtGEA, https://mtgea.noble.org/v3/). (A) Two-day-old seedlings were placed on half strength of MS medium containing 180 mM NaCl for 0, 6, 24 and 48 h, (B) Two-week-old seedlings were treated in a nutrient solution containing 200 mM NaCl for 1, 2, 5, 10 and 24 h as hydroponic treatment, while those growing in the nutrient solution as control. C and D, 24-day-old seedlings growing in soil were stopped watering for drought treatment for 14 d before rewatering, followed by sampling shoot and roots, respectively for analysis of gene expression. The expression levels of the MtCML genes are shown as the log₂-based fluorescence intensity values from the microarray data (MtGEA, https://mtgea.noble.org/v3/).

Figure 7

Relative expression of (A) MtCML2, (B) MtCML6, (C) MtCML16, (D) MtCML19, (E) MtCML33 and (F) MtCML47 in response to cold treatment. Mean values and standard errors of three independent experiments presented. * indicate significant difference between cold treatment and control at p < 0.05.