Genome-wide analysis of the SWEET gene family in Hemerocallis citrina and functional characterization of HcSWEET4a in response to salt stress

Abstract

Sugars will be eventually effluxed transporters (SWEETs) have been confirmed to play diverse physiological roles in plant growth, development and stress response. However, the characteristics and functions of the SWEET genes in Hemerocallis citrina remain unclear and poorly elucidated. In this study, the whole genome of Hemerocallis citrina was utilized to conduct bioinformatics analysis and a total of 19 HcSWEET genes were successfully identified. Analysis of the physicochemical properties indicated dominant differences among these HcSWEETs. A phylogenetic analysis revealed that HcSWEET proteins can be divided into 4 clades ranging from Clade I to IV, where proteins within the same clade exhibited shared conserved motifs and gene structures. Five to six exons were contained in the majority of HcSWEET genes, which were unevenly distributed across 11 chromosomes. The gene duplication analysis showed the presence of 4 gene pairs. Comparative syntenic maps revealed that the HcSWEET gene family might present more closed homology in monocotyledons than dicotyledons. Cis-acting element analysis of HcSWEET genes indicated key responsiveness to various hormones, light, and stresses. Additionally, transcriptome sequencing analysis suggested that most HcSWEET genes had a relatively higher expression in roots, and HcSWEET4a was significantly up-regulated under salt stress. Overexpression further verified the possibility that HcSWEET4a was involved in response to salt stress, which provides novel insights and facilitates in-depth studies of the functional analysis of HcSWEETs in resistance to abiotic stress.

Keywords: Hemerocallis citrina; SWEETs; Abiotic stress; Functional characterization; Genome-wide.

Fig. 1

Phylogenetic tree analysis and classification of the SWEETs in night lily and some other plants. Gene members of Hemerocallis citrina (Hc), Hemerocallis fulva (Hf), Oryza sativa (Os), Zea mays (Zm), Arabidopsis thaliana (At), Vitis vinifera (Vv) were classified into 4 clades. A phylogenetic tree was constructed via the TBtools software with 5000 bootstrap replicates

Fig. 2

Constructed phylogenetic tree, conserved motif, conserved domain, and gene structure analysis of SWEETs in Hemerocallis citrina. (A) Intraspecific evolutionary tree of 19 HcSWEET members. (B) The compositions and distribution of HcSWEETs conserved motifs. (C) The conserved functional domains of HcSWEETs. (D) Gene structures of HcSWEET genes

Fig. 3

Chromosome distributions of SWEET genes on night lily (LG1-11). All HcSWEET genes named are displayed on night lily LGs, with the LG number marked at the edge of each strip. The 0-500 Mb scale represents chromosome length. The lines inside the LGs represent gene density

Fig. 4

Collinearity of HcSWEET gene pairs. (A) Chromosome location and inter-chromosomal relationships of SWEETs in Hemerocallis citrina. The identified duplication events are marked by red lines. The LG number is labeled within different colored rectangles. (B) Collinearity analysis between the SWEETs of Hemerocallis citrina (H. citrina), Arabidopsis thaliana (A. thaliana), and Oryza sativa (O. sativa)

Fig. 5

Distributions of cis-acting elements of the HcSWEET promoter

Fig. 6

Expression dynamics of the HcSWEET genes in different tissues of night lily and phenotypes under different abiotic stresses. (A) Expression heatmap of HcSWEETs in the tender root, mature root, bud, tender leaf, mature leaf, tender scope, and mature scope. (B, C) The phenotypic observations of night lily under drought stress (B) and salt stress (C)

Fig. 7

Expression heatmaps of HcSWEETs under drought (A) and salt (B) stress based on RNA-seq data

Fig. 8

Expression patterns of 19 HcSWEET genes after drought and salt treatment. The different lowercase letters indicate significant differences by t-test (p < 0.05)

Fig. 9

Subcellular localization of HcSWEET4a and HcSWEET5. pSuper: GFP represents the negative control. The second panel represents a positive marker for the plasma membrane. The rightmost panel indicates the fusion of green fluorescence, red fluorescence, and bright field. Bar = 25 μm

Fig. 10

Phenotype observations of HcSWEET4a overexpression lines under salt treatment. (A) Phenotype changes of transgenic lines (OE-1, OE-2) after treated for 0–24 h with NaCl (150 mM). Bar = 17 mm. (B) The relative expression levels of HcSWEET4a in OE-1 and OE-2 lines. The significance of difference is marked by two asterisks (p < 0.05)