Genome-Wide Survey and Expression Analyses of Hexokinase Family in Poplar (Populus trichocarpa)

Abstract

Hexokinase (HXK) family proteins exert critical roles in catalyzing hexose phosphorylation, sugar sensing, and modulation of plant growth and stress adaptation. Nevertheless, a large amount remains unknown about the molecular profile of HXK enzymes in Populus trichocarpa, a woody model tree species. A genome-wide survey of HXK-encoding genes, including phylogenies, genomic structures, exon/intron organization, chromosomal distribution, and conserved features, was conducted, identifying six putative HXK isogenes (PtHXK1-6) in the Populus genome. The evolutionary tree demonstrated that 135 homologous HXKs between 17 plant species were categorized into four major subfamilies (type A, B, C, and D), clustering one plastidic (PtHXK3) and five mitochondrial PtHXKs grouped into type A and B, respectively. The in silico deduction prompted the presence of the conserved sugar-binding core (motif 4), phosphorylation sites (motif 2 and 3), and adenosine-binding domains (motif 7). The transcriptomic sequencing (RNA-seq) and the quantitative real-time PCR (qRT-PCR) assays revealed that three isogenes (PtHXK2, 3, and 6) were abundantly expressed in leaves, stems, and roots, while others appeared to be dominantly expressed in the reproductive tissues. Under the stress exposure, PtHXK2 and 6 displayed a significant induction upon the pathogenic fungi (Fusarium solani) infection and marked promotions by glucose feeding in roots. In contrast, the PtHXK3 and 6 are ABA-responsive genes, following a dose-dependent manner. The comprehensive analyses of the genomic patterns and expression profiling provide theoretical clues and lay a foundation for unraveling the physiological and signaling roles underlying the fine-tuned PtHXKs responding to diverse stressors.

Keywords: Populus; hexokinase; stress and defense; sucrose metabolism; sugar signaling.

Figure 1

Phylogenetic relationships of clustered homologous HXKs between 17 plant species. The evolution tree was generated using the Maximum Likelihood method in MEGA X with 1000 bootstrap replicates. The four classified HXKs subgroups were represented as type A, B, C, and D.

Figure 2

The genomic features of the HXKs between P. trichocarpa and Arabidopsis. (a) The chromosomal distribution of isogenes shows the duplicated gene pair with a straight line. (b) The in silico prediction of the cis-regulatory elements in 1.5 kb gene promoters labeled in colors. (c) Genomic structures show the patterns of the exon/intron organization. The exon lengths are displayed proportionally to the scale on the bottom.

Figure 3

The conserved motif distribution in HXKs. (a) Motifs were analyzed using the MEME web server, and ten conserved motifs were boxed in colors. (b) The amino acid logos of respective motifs were detailed on the right side.

Figure 4

The conserved fragments and amino acid residues between PtHXKs and other functional homologs. Six conserved sites with different numbers of amino acids were lined in black annotated based on homologous regions to the HXK II in yeast, and two active binding sites were marked with asterisks.

Figure 5

Transcriptomic and expression profiles of PtHXKs in vegetative tissues and roots challenged by F. solani (Fs). (a) The heatmap shows the transcript in vegetative tissues of P. trichocarpa. (b) The qRT-PCR evaluation shows the tissue-specific expression of PtHXKs. (c) The heatmap shows the transcriptomic profile of PtHXK isogenes in Fs infected roots (0, 24, 48, and 72 h of post-inoculation, hpi). (d) Validation of responsive PtHXKs compared to the control (0 hpi) by qRT-PCR. The RNA-seq data were given in the Log10 of the fragments per kilobase per million reads (FPKM) expression values. At least three independent biological replicates were conducted for qRT-PCR analyses. PtβActin, PtUBIC, and PtEF-α1 were used as the internal control. Data represent mean values standard error (±SE) of at least three independent biological replicates. Asterisks indicate significant differences relative to the control using the Student’s t-test: *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 6

Expression effects of three PtHXKs in the roots feeding with various sugars and ABA. The qRT-PCR analyses show the transcript responsiveness upon the altered concentration of sugars (glucose, fructose, and sucrose) and ABA. The relative expression of the control was set as 1. Data represent mean values ±SE of at least three independent biological replicates. PtβActin, PtUBIC, and PtEF-α1 were used as reference genes. Asterisks indicate significant differences in comparison with the control using the Student’s t-test: ** p < 0.001, * p < 0.01.