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. 2017 May 12;18(5):1041.
doi: 10.3390/ijms18051041.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava

Meng-Ting Geng  1 , Yuan Yao  2 , Yun-Lin Wang  3 , Xiao-Hui Wu  4 , Chong Sun  5 , Rui-Mei Li  6 , Shao-Ping Fu  7 , Rui-Jun Duan  8 , Jiao Liu  9 , Xin-Wen Hu  10 , Jian-Chun Guo  11
Affiliations

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava

Meng-Ting Geng et al. Int J Mol Sci. .

Abstract

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1-7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2-7) based on their N-terminal sequences. MeHXK1-5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6-7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.

Keywords: cassava; enzyme activities; gene expression; hexokinase; yeast complementation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chromosomal distribution of MeHXK genes in the cassava genome. Blue long lines represent the chromosome models, and the seven chromosomes are labeled. Pink short lines indicate the related position of each MeHXK gene in the cassava genome. Blue arrows indicate gene direction.
Figure 2
Figure 2
Amino acid alignment analysis of the identified cassava HXKs. Black lines indicate the regions with different functions. The black arrows show the eight conserved amino acids (counting the amino acid residues based on MeHXK1, the eight amino acids are Asp-103, Thr-107, Lys-197, Asp-232, Gly-254, Asp-349, Gly-441, and Ser-474), which likely correspond with the active residues of hexokinases (HXKs) proposed by Kuser et al. [29]. Dark-blue shading, pinkish shading and light blue shading reflect 100%, 75% and 50% amino acid residues conservation, respectively. The alignment was performed using DNAMAN 6.0 software (Lynnon LLC, San Ramon, CA, USA).
Figure 3
Figure 3
Subcellular localization prediction and N-terminal sequence analysis of the HXK proteins in M. esculenta, R. communis, and A. thaliana. The highest TargetP scores are written in bold font on the right side of Figure 3. cTP: chloroplast transit peptide score; mTP: mitochondrial targeting peptide score; SP: secretory pathway score; C: predicted chloroplast import; S: predicted secretory pathway. The black solid lines indicate predicted chloroplast transit peptides and the black dashed lines indicate predicted membrane anchor domains. Amino acid residues with a positive electrostatic potential located close to the predicted membrane anchor domain are shown in italics and bold.
Figure 4
Figure 4
Exon-intron structure of MeHXK1–7. Introns are shown as black lines and exons are shown as green boxes.
Figure 5
Figure 5
Phylogenetic analysis of HXK proteins from Manihot esculenta, Ricinus communis, Populus trichocarpa, Nicotiana tabacum, Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa. HXK proteins were classified into six major sub-families (named as I, II, III, IV, V, and VI). The Neighbor-Joining tree was constructed using Molecular Evolutionary Genetics Analysis Version 7.0 (MEGA7). The values shown at the branch nodes are the confidence levels from 1000 replicate bootstrap samplings. Red dot indicate HXKs from M. esculenta. Blue dot indicate HXKs from P. trichocarpa. Green dot indicate HXKs from R. communis.
Figure 6
Figure 6
Motif distribution in HXKs from M. esculenta, R. communis, and P. trichocarpa. Motifs were analyzed using the MEME web server (https://www.swissmodel.expasy.org/). The motifs are represented by different colors. Red stars indicate HXKs from M. esculenta.
Figure 7
Figure 7
The cartoon representation of the predicted 3-dimensional structural models of MeHXK1–6. (a) MeHXK1; (b) MeHXK2; (c) MeHXK3; (d) MeHXK4; (e) MeHXK5; and (f) MeHXK6. Green structures represent α-helices, purple arrows indicate β-folds, lines represent random coils, and red stick structures represent the catalytic residues. The image was generated using the PyMOL program (Schrödinger, Inc., New York, NY, USA).
Figure 8
Figure 8
Distribution of the residues in the enzyme active sites of MeHXK1–6. (a) MeHXK1; (b) MeHXK2; (c) MeHXK3; (d) MeHXK4; (e) MeHXK5; and (f) MeHXK6. Spherical structures indicate glucose molecules and the colored stick structures represent the catalytic residues. The image was generated using the PyMOL program.
Figure 9
Figure 9
The differential expression of the MeHXK genes in cassava organs or tissues. The amount of MeHXK mRNA was normalized by β-tubulin mRNA. The expression of MeHXK3 in leaves was used as a calibrator to compare with other genes for map-making. Each value is the mean ± SE of three biological replicates (n = 3). Notes: L, Leaf, S, Stem, TP, Tuber phloem, TX, Tuber xylem, MF, Male flower, FF, Female flower, F, Fruits.
Figure 10
Figure 10
The differential expression analysis of MeHXKs in cassava tuber phloem (a) and tuber xylem (b) during tuber developmental stages. The differential expressions of MeHXK genes were examined using qPCR at the tuber initial stage (90 days), the tuber expanding stage (135 and 180 days), and the tuber maturity stage (225 and 270 days). Each value was the mean ± SE of three biological replicates (n = 3). The amount of MeHXK mRNA was normalized by β-tubulin mRNA. The expression of MeHXK6 in tuber phloem at 225 days was used as a calibrator to compare with other genes for map-making.
Figure 11
Figure 11
HXK activity profiles of cassava tuber phloem and xylem during tuber root development at the initial tuber stage (90 days), tuber expanding stage (135 and 180 days), and tuber maturity stage (225 and 270 days). Each value was the mean ± standard error of three biological replicates (n = 3).
Figure 12
Figure 12
Complementation of the HXK-deficient yeast triple mutant YSH7.4-3C (hxk1, hxk2, glk1) with MeHXK2. −URA + Glc: the medium contained d-glucose as the sole carbon source and lacked uracil; −URA + Fru: the medium contained d-fructose as the sole carbon source and lacked uracil; MeHXK2: the pDR195-MeHXK2 vector was transformed into YSH7.4-3C; PRD195: the empty pDR195 vector was transformed into YSH7.4-3C; YSH7.4-3C: the mutant yeast cells without any vector transformation.
Figure 13
Figure 13
Enzyme activity analysis of MeHXK2 from yeast extracts (a) Hexose phosphorylation activity of MeHXK2 towards glucose; (b) Hexose phosphorylation activity of MeHXK2 towards fructose. Note the different scales of sugar concentrations; (c) Comparison of the pH and the hexose phosphorylation activity of MeHXK2. V: nmol of glucose 6-phosphate or fructose 6-phosphate that produce 1 mg of protein in 1 min.
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