Genome-wide identification, phylogeny and expression analysis of the SPL gene family and its important role in salt stress in Medicago sativa L

Abstract

Background: SQUAMOSA promoter-binding protein-like (SPL) transcription factors are widely present in plants and are involved in signal transduction, the stress response and development. The SPL gene family has been characterized in several model species, such as A. thaliana and G. max. However, there is no in-depth analysis of the SPL gene family in forage, especially alfalfa (Medicago sativa L.), one of the most important forage crops worldwide.

Result: In total, 76 putative MsSPL genes were identified in the alfalfa genome with an uneven distribution. Based on their identity and gene structure, these MsSPLs were divided into eight phylogenetic groups. Seventy-three MsSPL gene pairs arose from segmental duplication events, and the MsSPLs on the four subgenomes of individual chromosomes displayed high collinearity with the corresponding M. truncatula genome. The prediction of the cis-elements in the promoter regions of the MsSPLs detected two copies of ABA (abscisic acid)-responsive elements (ABREs) on average, implying their potential involvement in alfalfa adaptation to adverse environments. The transcriptome sequencing of MsSPLs in roots and leaves revealed that 54 MsSPLs were expressed in both tissues. Upon salt treatment, three MsSPLs (MsSPL17, MsSPL23 and MsSPL36) were significantly regulated, and the transcription level of MsSPL36 in leaves was repressed to 46.6% of the control level.

Conclusion: In this study, based on sequence homology, we identified 76 SPL genes in the alfalfa. The SPLs with high identity shared similar gene structures and motifs. In total, 71.1% (54 of 76) of the MsSPLs were expressed in both roots and leaves, and the majority (74.1%) preferred underground tissues to aerial tissues. MsSPL36 in leaves was significantly repressed under salt stress. These findings provide comprehensive information regarding the SPB-box gene family for improve alfalfa tolerance to high salinity.

Keywords: Legume; Medicago sativa; SPL gene family; Salt stress.

Fig. 1

Phylogenetic analysis of SPLs from M. sativa, M. truncatula and A. thaliana. Proteins with SBP domains from M. sativa (Ms), A. thaliana (At) and M. truncatula (Mt) were searched and designated as SPL. Neighbor-joining (NJ) was used for the unrooted phylogenetic tree with the maximum likelihood method (1000 bootstraps)

Fig. 2

Analysis of the gene structure and conserved motifs of MsSPL based on phylogenetic relationships. A A phylogenetic tree was constructed based on the full-length sequences of M. sativa SPL proteins. B Exon/intron structure of MsSPLs. The black lines indicate introns. C Motif composition of putative MsSPL proteins. Motifs are displayed by colored boxes numbered 1–20. CDS stands for coding

Fig. 3

Distribution of SPL genes in the M. sativa genome. A Location of genes on chr1.1 to 4.1. B Location of genes on chr4.2 to 8.4. Chromosomes are indicated in different colors and numbered on the top. The scale (Mb) represents the lengths of the chromosomes

Fig. 4

Schematic representation of the interchromosomal relationship among the MsSPLs. Syntenic blocks in the alfalfa genome are indicated by lines in orange

Fig. 5

Synteny analysis of SPL genes between M. sativa and three representative plant species. Gray lines in the background indicate collinear blocks within M. sativa and the indicated plant, whereas the red lines highlight syntenic SPL gene pairs

Fig. 6

Analysis of cis-acting elements in the putative promoter of MsSPLs. The number in color represents the copy of the cis-acting element (at the bottom) of individual MsSPLs (on the right)

Fig. 7

FPKM of MsSPLs in roots and leaves using RNA sequencing. A FPKM of MsSPLs expressed in both roots and leaves. The R/L ratio represents the ratio of FPKM in roots to that in leaves. B FPKM of the MsSPLs detected in roots but not leaves. C FPKM of the MsSPLs detected in leaves rather than roots. The color represents the FPKM normalized value. The blue and orange colors represent higher and lower expression, respectively

Fig. 8

Expression levels analyses of three MsSPL genes under salt treatment by qRT-PCR. A Expression levels of three MsSPL genes in root. B Expression levels of three MsSPL genes in leaf. “CK” represents normal growth condition, “Salt” represents salt treatment. The levels in root and leaf of the CK were arbitrarily set to 1. Error bars represent the standard deviations of three technical replicates