SWEET Gene Family in Medicago truncatula: Genome-Wide Identification, Expression and Substrate Specificity Analysis

Abstract

SWEET (Sugars Will Eventually be Exported Transporter) proteins mediate the translocation of sugars across cell membranes and play crucial roles in plant growth and development as well as stress responses. In this study, a total of 25 SWEET genes were identified from the Medicago truncatula genome and were divided into four clades based on the phylogenetic analysis. The MtSWEET genes are distributed unevenly on the M. truncatula chromosomes, and eight and 12 MtSWEET genes are segmentally and tandemly duplicated, respectively. Most MtSWEET genes contain five introns and encode proteins with seven transmembrane helices (TMHs). Besides, nearly all MtSWEET proteins have relatively conserved membrane domains, and contain conserved active sites. Analysis of microarray data showed that some MtSWEET genes are specifically expressed in disparate developmental stages or tissues, such as flowers, developing seeds and nodules. RNA-seq and qRT-PCR expression analysis indicated that many MtSWEET genes are responsive to various abiotic stresses such as cold, drought, and salt treatments. Functional analysis of six selected MtSWEETs in yeast revealed that they possess diverse transport activities for sucrose, fructose, glucose, galactose, and mannose. These results provide new insights into the characteristics of the MtSWEET genes, which lay a solid foundation for further investigating their functional roles in the developmental processes and stress responses of M. truncatula.

Keywords: Medicago truncatula; SWEET; abiotic stress; evolution; expression analysis; sugar transport.

Figure 1

Phylogenetic relationships of the SWEET family genes in Arabidopsis, sorghum, cucumber, and M. truncatula. The sequences of the 82 SWEET proteins from the above four plant species were aligned by Clustal Omega, and the phylogenetic tree was constructed by the MEGA 7.0 using the NJ method with 1000 bootstrap replicates. The proteins from Arabidopsis, sorghum, cucumber, and M. truncatula are indicated with the prefixes of At, Sb, Cs, and Mt, respectively.

Figure 2

Locations and duplications of MtSWEET genes on M. truncatula chromosomes. The black lines indicate segmentally duplicated genes, and the tandemly duplicated genes are boxed. The scale is provided in megabase (Mb).

Figure 3

Multiple sequence alignment of MtSWEET proteins. The positions of the TMHs are underlined. The positions of the active sites of tyrosine (Y) and aspartic acid (D) are indicated by triangles. The conserved serine (S) phosphorylation sites are indicated by arrows. The sequence of MtSWEET2b was truncated.

Figure 4

Gene structures of the MtSWEET genes according to their phylogenetic relationships. The blue boxes, green boxes, and black lines indicate UTRs (untranslated regions), CDSs, and introns, respectively.

Figure 5

Expression analysis of the MtSWEET genes in different developmental tissues (A) and during nodule development (B) of M. truncatula using microarray data. The expression levels of the MtSWEET genes are shown as the log₂-based fluorescence intensity values from the microarray data (MtGEA, https://mtgea.noble.org/v3/). DAP, days after pollination. dpi, days post inoculation.

Figure 6

Expression patterns of MtSWEET genes in response to cold, drought, and salt stresses. The expression levels of the MtSWEET genes are shown as the FPKM values based on the RNA-seq data in our previous study [50].

Figure 7

Expression levels of four selected MtSWEET genes in response to cold (A), drought (B), and salt (C) stress conditions. Expression of the genes at 0 h was set as 1, and different letters indicate statistically significant differences (Duncan’s test, p < 0.05).

Figure 8

Substrate specificity analysis of six selected MtSWEET proteins in yeast mutant strains EBY.VW4000 (A) and SUSY7/ura3 (B). Cells were serially 10-fold diluted (10-, 100- and 1000-fold) and spotted on solid SD media supplemented with 2% concentration of different sugar substrates. Maltose and glucose were the sole carbon sources for the positive controls of EBY.VW4000 and SUSY7/ura3 cells, respectively.