Identification and expression analysis of pineapple sugar transporters reveal their role in the development and environmental response

Abstract

In plants, sugars are required for several essential functions, including growth, storage, signaling, defense and reproduction. Sugar transporters carry out the controlled movement of sugars from source (leaves) to sink (fruits and roots) tissues and determine the overall development of the plant. Various types of sugar transporter families have been described in plants, including sucrose transporters (SUC/SUT), monosaccharide transporter (MST) and SWEET (from "Sugar Will Eventually be Exported Transporters"). However, the information about pineapple sugar transporters is minimal. This study systematically identified and classified 45 MST and 4 SUC/SUT genes in the pineapple genome. We found that the expression patterns of sugar transporter genes have a spatiotemporal expression in reproductive and vegetative tissues indicating their pivotal role in reproductive growth and development. Besides, different families of sugar transporters have a diel expression pattern in photosynthetic and non-photosynthetic tissues displaying circadian rhythm associated participation of sugar transporters in the CAM pathway. Moreover, regulation of the stress-related sugar transporters during cold stress indicates their contribution to cold tolerance in pineapple. Heterologous expression (yeast complementation assays) of sugar transporters in a mutant yeast strain suggested that SUT1/2 have the ability to transport sucrose, and STP13, STP26, pGlcT-L2 and TMT4 are able to transport glucose, whereas SWEET11/13 transport both sucrose and fructose. The information provided here would help researchers further explore the underlying molecular mechanism involved in the sugar metabolism of pineapple.

Keywords: circadian; fruit development; gene expression; pineapple; sugar transporter.

Figure 1

The distribution of sugar transporter genes in pineapple (Ananas comosus) genome.

Figure 2

Exon–intron structure of sugar transporter genes identified in pineapple. The gene models of sugar transporters are shown in a graphic representation using the online Gene Structure Display Server (GSDS 2.0). Introns are represented as black lines; blue boxes show coding sequences, and red boxes represent upstream or downstream regions.

Figure 3

Conserved motifs and domains in sugar transporter genes of pineapple. Distribution of conserved motifs in the sugar transporter genes. The different-colored boxes represent different motifs and their position in each sugar transporter protein sequence.

Figure 4

Phylogenetic relationship of sugar transporters among representative monocotyledons and dicotyledons. The protein sequences were used to build the phylogenetic tree of sugar transporters among representative monocots and eudicots. Sugar transporters proteins of pineapple are marked with blue circles.

Figure 5

Phylogenetic tree of sugar transporters subfamilies among representative monocots and eudicots. The trees were built with the neighbor-joining algorithm using protein sequences. (A) The phylogenetic tree of the SUC/SUT subfamily. (B) The phylogenetic tree of the PMT subfamily. Sugar transporters genes of pineapple in the phylogenetic trees are marked with blue, and the sugar transporters genes of other species are marked with different colors as labeled at the bottom.

Figure 6

Phylogenetic tree of sugar transporters subfamilies. The trees were built with the neighbor-joining algorithm using protein sequences. (A) The phylogenetic tree of the STP subfamily. (B) The phylogenetic tree of the TMT, vGT, pGlcT and ITR/INT subfamilies. Sugar transporters genes of pineapple in the phylogenetic trees are marked with blue, and the sugar transporters genes other species are marked with different colors as labeled at the bottom.

Figure 7

Expression profile of pineapple sugar transporter genes during different stages of reproductive development. The heatmap was created based on the log2 (TPM + 0.01) value of the genes and normalized by row. Red color represents a high transcript abundance, and blue represents low transcript abundance, and asterisks (*) represent the FKM values above 100. The right side of the figure shows the scale. Details of the samples are mentioned at the bottom of each lane: sepal Se1–Se4, gynoecium Gy1- Gy7, ovule Ov1–Ov7, petal Pe1–Pe3, stamen St1–St6, fruit ‘Fr_S1–Fr_S7’, where ‘S’ is the abbreviation for ‘stage’.

Figure 8

Expression pattern and pineapple sugar transporter genes across circadian rhythm in green (photosynthetic, left panel) tissue at the leaf tip and white (non-photosynthetic, right panel) tissue. The heatmap was created based on the log2 (TPM + 0.01) value of the genes and normalized by row. Red color represents a high transcript abundance, and blue represents low transcript abundance, and asterisks (*) represent the FKM values above 100.

Figure 9

Expression profile of pineapple sugar transporter genes under cold stress (4°C). High and low expression is indicated by red and blue colors, respectively, and asterisks (*) represent the FKM values above 100.

Figure 10

The relative expression levels of 6 sugar transporter genes of pineapple verified by quantitative real-time PCR (RT-qPCR). Gene expression is represented in fold change of expression calculated against pineapple EF1α by the Livak method (2^−ΔΔCT). Vertical bars represent the mean± SE of three biological replicate assays. The asterisks represent statistically significant values (*p < 0.05, **p < 0.01 and *** p < 0.001).

Figure 11

Heterologous expression of representative sugar transporters in the hexose transport-defective yeast strain EBY.VW4000. The yeast cultures harboring respective vectors were incubated until OD₆₀₀ reached 0.2 and then grown in serial dilutions on SD (-Trp) media containing either 2% maltose (positive control), 2% sucrose, 2% glucose or 2% fructose. The plates were incubated at 30°C, and the growth was documented after 3d.