Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies

Abstract

Sugar transporters play important roles in plant growth and development, flowering and fruiting, as well as responses to adverse abiotic and biotic environmental conditions. Lilies (Lilium spp.) are some of the most representative ornamental bulbous flowers. Sugar metabolism is critical for bulb formation in lilies; therefore, clarifying the amount and expression pattern of sugar transporters is essential for further analyzing their roles in bulb formation. In this study, based on the transcriptome data of the Lilium Oriental hybrid 'Sorbonne' and Lilium × formolongi, a total of 69 and 41 sugar transporters were identified in 'Sorbonne' and Lilium × formolongi, respectively, by performing bioinformatics analysis. Through phylogenetic analysis, monosaccharide transporters (MSTs) can be divided into seven subfamilies, sucrose transporters (SUTs) can be divided into three subgroups, and sugars will eventually be exported transporters (SWEETs) can be divided into four clades. According to an analysis of conserved motifs, 20, 14, and 12 conserved motifs were predicted in MSTs, SUTs, and SWEETs, respectively. A conserved domain analysis showed that MSTs and SUTs contained a single domain, whereas most of the SWEETs harbored two MtN3/saliva domains, also known as a PQ-loop repeat. The LohINT1, which was predicted to have a smaller number of transmembrane structural domains, was cloned and analyzed for subcellular localization. It was found that the LohINT1 protein is mainly localized in the cell membrane. In addition, the expression analysis indicated that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated in 'Sorbonne' 1 day after scale detachment treatment, suggesting that they may regulate the initiation of the bulblet. A total of 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs were upregulated 4~6 months after sowing, which corresponds to the juvenile-to-adult transition phase of Lilium × formolongi, suggesting that they may also play a role in the accompanying bulb swelling process. Combined with quantitative real-time PCR (qRT-PCR) analysis, LohSTP8 and LohSTP12 were significantly overexpressed during the extremely early stage of bulblet initiation, and LflERD6.3 was significantly overexpressed during the growth of the underground bulblet, suggesting that they may be key sugar transporters in the formation of lily bulbs, which needs further functional verification.

Keywords: Lilium; bulblet development; bulblet initiation; expression analysis; sugar transporter.

Figure 1

Phylogenetic analysis of MSTs in lilies, Arabidopsis, and rice. The sequences of 191 MSTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method. STP, sugar transport protein; VGT, vacuolar glucose transporter; TMT, tonoplastic monosaccharide transporter; INT, inositol transporter; PLT, polyol transporter; pGlcT, plastidic glucose transporter; ERD6L, plastidic glucose transporter.

Figure 2

Phylogenetic analysis of SUTs in lilies, Arabidopsis, and rice. The sequences of 21 SUTs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.

Figure 3

Phylogenetic analysis of SWEETs in lilies, Arabidopsis, and rice. The sequences of 65 SWEETs of ‘Sorbonne’, Lilium × formolongi, Arabidopsis thaliana, and Oryza sativa were aligned using the MUSCLE Wrapper tool, and a phylogenetic tree was constructed using the FastTree maximum likelihood (MJ) method.

Figure 4

Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi MSTs. (A) Phylogenetic trees of LohMSTs and LflMSTs were constructed using the maximum likelihood method. Seven subfamilies were labeled. (B) Motif compositions of LohMSTs and LflMSTs. A total of 20 motifs are shown as rectangles with different colors. (C) Domain compositions of LohMSTs and LflMSTs. (D) Amino acid sequences of the 20 conserved motifs of LohMSTs and LflMSTs are shown.

Figure 5

Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SUTs. (A) Phylogenetic trees of LohSUTs and LflSUTs were constructed using the maximum likelihood method. Three subgroups were labeled. (B) Motif compositions of LohSUTs and LflSUTs. A total of 14 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSUTs and LflSUTs. (D) Amino acid sequences of the 14 conserved motifs of LohSUTs and LflSUTs are shown.

Figure 6

Phylogenetic relationships, conserved motifs, and conserved domain analysis of ‘Sorbonne’ and Lilium × formolongi SWEETs. (A) Phylogenetic trees of LohSWEETs and LflSWEETs were constructed using the maximum likelihood method. Four clades were labeled. (B) Motif compositions of LohSWEETs and LflSWEETs. A total of 12 motifs are shown as rectangles with different colors. (C) Domain compositions of LohSWEETs and LflSWEETs. (D) Amino acid sequences of the 12 conserved motifs of LohSWEETs and LflSWEETs are shown.

Figure 7

Subcellular localization analysis of LohINT1. (A) Subcellular localization of LohINT1-GFP and free GFP. (B) Subcellular localization of LohINT1-YFP and free YFP.

Figure 8

Expression patterns of ‘Sorbonne’ sugar transporter genes at four periods under aeroponic conditions. (A) Expression patterns of LohMSTs during four periods of bulblet initiation. (B) Expression patterns of LohSUTs during four periods of bulblet initiation. (C) Expression patterns of LohSWEETs during four periods of bulblet initiation. Color scale represents reads per kilobase per million normalized log² transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. DAT, days after treatment.

Figure 9

Expression patterns of Lilium × formolongi sugar transporter genes at three periods after sowing. (A) Expression patterns of LflMSTs at three periods during the growth of the underground bulblet. (B) Expression patterns of LflSUTs at three periods during the growth of the underground bulblet. (C) Expression patterns of LflSWEETs at three periods during the growth of the underground bulblet. Color scale represents reads per kilobase per million normalized log² transformed counts, where dark red indicates high level, dark blue indicates low level, and white indicates medium. M, months.

Figure 10

Expression profiles of 28 LohSTs genes during the extremely early stage of bulblet initiation. All data are presented as mean ± standard error of mean (SEM). Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).

Figure 11

Expression profiles of 9 LflSTs genes during the growth of the underground bulblet. All data are presented as mean ± SEM. Lowercase letters above the bars indicate significant differences between periods (p-value < 0.05, LSD, Duncan).