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. 2023 Aug 30;12(17):3121.
doi: 10.3390/plants12173121.

Genome-Wide Characterization of Soybean Hexokinase Genes Reveals a Positive Role of GmHXK15 in Alkali Stress Response

Feng Jiao  1 , Yang Chen  1 , Dongdong Zhang  1 , Jinhua Wu  1
Affiliations

Genome-Wide Characterization of Soybean Hexokinase Genes Reveals a Positive Role of GmHXK15 in Alkali Stress Response

Feng Jiao et al. Plants (Basel). .

Abstract

Hexokinase (HXK) proteins catalyze hexose phosphorylation and are important for the sensing and signaling of sugar. In order to determine the roles played by HXKs in soybean growth and stress responsiveness, seventeen HXK genes (GmHXK1-17) were isolated and analyzed. The phylogenic analysis and subcellular location prediction showed that GmHXKs were clearly classified into type A (GmHXK1-4) and type B (GmHXK5-17). There were similar protein structures and conserved regions in GmHXKs to the HXKs of other plants. An expression analysis of the GmHXK genes in soybean organs or tissues demonstrated that GmHXK3 and GmHXK12, 15, and 16 were the dominant HXKs in all the examined tissues. In addition, salt, osmotic, and alkaline stress treatments dramatically increased the activity and transcripts of GmHXKs. There is the possibility that a type-B isoform (GmHXK15) plays a crucial role in soybean adaptation to alkali, as the expression levels of this isoform correlate well with the HXK enzyme activity. Based on an enzyme assay performed on recombinant plant HXK15 proteins expressed in Escherichia coli, we found that GmHXK15 had functional HXK activities. A further analysis indicated that GmHXK15 specifically targeted the mitochondria, and the overexpression of the GmHXK15 gene could significantly enhance the resistance of transgenic soybean to alkali stress. The present findings will serve as a basis for a further analysis of the function of the GmHXK gene family.

Keywords: abiotic stress; enzyme activity; expression; hexokinase; soybean (Glycine max L.).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The domain structure found in the soybean HXK proteins.
Figure 2
Figure 2
Phylogenetic and multiple alignments analysis of GmCKs. (A) Phylogenetic tree of HXK proteins from G. max, A. thaliana, O. sativa, P. vulgaris, M. truncatula, and S. bicolor. (B) Multiple alignments of protein sequences of soybean HXKs.
Figure 3
Figure 3
An analysis of the GmHXK family of genes’ synteny and intron–exon arrangement. (A) Syntenic assessment of soybean HXKs with respective genes from A. thaliana, G. max, P. vulgaris, O. sativa, S. bicolor, and M. truncatula. Chromosomes are represented by circles for the species above. HXK collinearity is indicated by colored curves. (B) The intron–exon organization of AtHXKs and GmHXKs. A pink arrow indicates an untranslated region (UTR). Alternatively, colored ellipses represent exons, while gray lines represent introns.
Figure 4
Figure 4
Upstream of the GmHXKs start codon, cis elements are predicted in the 2.0 kb promoter region. Genes encoding GmHXK are colored based on their respective positions of cis elements.
Figure 5
Figure 5
The GmHXK is expressed in a variety of tissues and developmental stages. (A) Cluster analysis of expression profiles of GmHXKs that participate in tissue development. The Phytozome database was used to examine transcripts of GmHXK genes in various tissues. Heat maps illustrate the results. A deep color denotes an elevated level of transcription, and a light color denotes low-level transcription; the color scale represents log2 expression values. An enlarged circle indicates a high level of transcription, while a smaller circle indicates low levels. (B) An assessment of transcript profiles of GmHXKs in developing seeds at early maturation (EM), mid-maturation (MM), and late maturation (LM). The transcripts of GmHXK6 were used as internal references in developing seeds at the EM stage. Each tissue was analyzed in three biological replicates. Statistically significant differences from the control group, as determined by Student’s t-test, are indicated by asterisks above the bars (* p < 0.05; ** p < 0.01).
Figure 6
Figure 6
GmHXKs expression profiles in soybean plant roots subjected to 120 mM of NaCl, 100 mM of NaHCO3, 200 mM of mannitol, or water (control) for 0, 6, 12, and 24 h, each. GmHXKs were estimated under non-stressed conditions to function as a calibrator. Each experiment was performed in triplicate. Moreover, the qRT-PCR results were examined using the 2−ΔΔct method. Asterisks above bars signify a difference regarding statistical significance, as determined by Student′s t-test (* p < 0.05, ** p < 0.01).
Figure 7
Figure 7
In soybean, HXK activity levels were determined under abiotic stress treatments. (A) HXK activity was evaluated for 0 h, 6 h, 12 h, and 24 h in roots of soybean plants treated with 120 mM of NaCl, 100 mM of NaHCO3, 200 mM of mannitol, or water (control). Biological replicates were represented by means ± SD. Statistically significant differences from the control group, as determined by Student’s t-test, are indicated by asterisks above the bars (* p < 0.05; ** p < 0.01). (B) The correlation coefficient between HXK enzymatic activity and the expression levels of GmHXKs. Correlation coefficients between any two traits are shown as ellipses on each chart. The correlation magnitude is determined by the color and slope of the ellipse. Negative correlations are represented by a red ellipse, and positive correlations by a blue ellipse.
Figure 8
Figure 8
Overexpression of GmHXK15 confers alkali tolerance in transgenic soybean hairy roots. (A) Purified GmHXK15 fusion proteins were resolved by SDS-PAGE and Western blotting (Lane S). The Eadie–Hofstee plot depicts the kinetic characteristics of GmHXK15 in response to (B) glucose and (C) fructose. (D) Subcellular localization analysis of GmHXK15 by expressing GmHXK-GFP fusion protein transiently in Arabidopsis mesophyll protoplasts. (E) Performance and (F) maximum root lengths and root fresh weights of GmHXK15 -OHR and CHR plants subjected to 100 mM NaHCO3 over 5 days. Bars = 5 cm. Asterisks denote significant differences (** p < 0.01) from CHR plants, as shown by Student’s t-test.
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