Genome-Wide Identification and Expression Analysis of the SWEET Gene Family in Annual Alfalfa (Medicago polymorpha)

Abstract

SWEET (Sugars will eventually be exported transporter) proteins are a group of sugar transporters that are involved in sugar efflux, phloem loading, reproductive development, plant senescence, and stress responses. In this study, 23 SWEET transporter members were identified in the Medicago polymorpha genome, heterogeneously distributed on seven chromosomes. These MpSWEET genes were divided into four subfamilies, which showed similar gene structure and motif composition within the same subfamily. Seventeen MpSWEET genes encode seven transmembrane helices (TMHs), and all MpSWEET proteins possess conserved membrane domains and putative serine phosphorylation sites. Four and three pairs of MpSWEET genes were predicted to be segmentally and tandemly duplicated, respectively, which may have contributed to their evolution of M. polymorpha. The results of microarray and RNA-Seq data showed that some MpSWEET genes were specifically expressed in disparate developmental stages (including seedling stage, early flowering stage, and late flowering stage) or tissues such as flower and large pod. Based on protein network interaction and expression patterns of MpSWEET genes, six MpSWEET genes were selected for further quantitative real-time PCR validation in different stress treatments. qRT-PCR results showed that MpSWEET05, MpSWEET07, MpSWEET12, MpSWEET15, and MpSWEET21 were significantly upregulated for at least two of the three abiotic stress treatments. These findings provide new insights into the complex transcriptional regulation of MpSWEET genes, which facilitates future research to elucidate the function of MpSWEET genes in M. polymorpha and other legume crops.

Keywords: Medicago polymorpha; SWEET; abiotic stress; expression analysis; phylogenetic evolution.

Figure 1

Phylogenetic tree of the SWEET family from M. polymorpha, M. truncatula, Arabidopsis thaliana, and Oryza sative. The yellow triangle, orange dot, green rectangles, and red rhombuses color represent M. polymorpha and A. thaliana, M. truncatula, and O. sative, respectively.

Figure 2

Amino acid sequence alignment results of 23 conserved domains of SWEETs in M. polymorpha. The positions of the seven transmembrane domains (TMH1 to TMH7) are represented by black line segments. The serine phosphorylation sites are represented by black triangles. The residues indicated in black were fully conserved among all proteins.

Figure 3

Conserved motifs (A), and gene exon-intron structures (B) of SWEET family members in M. polymorpha.

Figure 4

Chromosome distribution and replication event of SWEET genes in M. polymorpha. The segmentally duplicated genes are connected by blue lines, and tandem duplicated genes are connected by red lines.

Figure 5

Collinearity analysis between M. polymorpha and A. thaliana, M. truncatula, and O. sative.

Figure 6

The cis-acting elements of the promoter region form SWEET genes in M. polymorpha.

Figure 7

Protein interaction networks of 23 MpSWEET proteins based on orthologs in A. thaliana. Red font means orthologous MpSWEET proteins in A. thaliana.

Figure 8

Expression patterns and hierarchical clustering of MpSWEET genes. (A) The expression levels of the orthologs in M. truncatula of MpSWEET genes in different tissues; (B) the expression profiles of MpSWEET genes at three developmental stages.

Figure 9

Expression of MpSWEET genes in response to three stresses of drought, salt, and cold. The M. polymorpha ACTIN gene (Mpo3G42410) was used as a standard control, and the 2^−∆∆CT method was used to calculate the relative levels of gene expression. Data were statistically analyzed using Duncan’s test with SPSS25 and different letters indicate statistically significant differences (p < 0.05).