Identification of the Glyceraldehyde-3-Phosphate Dehydrogenase (GeGAPDH) Gene Family in Gastrodia elata Revealing Its Response Characteristics to Low-Temperature and Pathogen Stress

Abstract

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene plays a pivotal role in the glycolysis/gluconeogenesis process, contributing significantly to glycosyl donor synthesis, plant growth and development, and stress responses. Gastrodia elata Bl., a heterotrophic plant in the Orchidaceae family, has its dried tubers used as the traditional Chinese medicine. This study identified three GeGAPDH genes in G. elata, all encoding basic, stable, hydrophilic proteins. Phylogenetic analysis and subcellular localization predictions categorized GeGAPDH1 as a plastid subtype, while GeGAPDH2 and GeGAPDH3 were classified as cytoplasmic subtypes. Prokaryotic expression experiments demonstrated successful expression of the GeGAPDH1 protein in Escherichia coli, which exhibited significant GAPDH enzymatic activity. Subcellular localization experiments showed that GeGAPDH1 was localized in the plastid. Expression analysis indicated that the three GeGAPDH genes were predominantly expressed in tubers. Under low-temperature stress, although the total GAPDH enzyme activity in tubers did not change significantly, the expression of GeGAPDH1 was significantly up-regulated, while GeGAPDH2 and GeGAPDH3 were significantly down-regulated. This suggests that different subtypes of GeGAPDH may regulate cold resistance through different pathways. Upon pathogen infection, the GeGAPDH gene family exhibited pathogen-specific regulatory patterns. During infection by Fusarium oxysporum, both the expression levels of all three GeGAPDH genes and the total GAPDH enzyme activity in tubers increased significantly; however, F. solani infection induced a significant increase in total GAPDH enzyme activity without significant changes in gene expression. These results suggest that the GeGAPDH gene family may respond to different pathogen infections through transcriptional or translational regulation mechanisms. This study systematically identified and characterized the GeGAPDH gene family in G. elata, providing a theoretical foundation for understanding the functional differentiation of GAPDH in heterotrophic plants.

Keywords: Gastrodia elata; enzymatic activity; glyceraldehyde-3-phosphate dehydrogenase; stress response; subcellular localization.

Figure 1

Gene structure and conserved structural domains of the GeGAPDH family in G. elata. (A) Gene structure of the GeGAPDH family of G. elata. (B) Distribution of conserved structural domains of the GeGAPDH family. (C) Seven conserved structural domains in the GeGAPDH family. Conserved domains are shown in yellow boxes.

Figure 2

Phylogenetic tree of GAPDH family. Phylogenetic trees were constructed using 33 GAPDH protein sequences from different species. Phylogenetic analyses were performed using maximum likelihood and 1000 bootstrap replicates. The GAPDH family is divided into four subgroups (Group I–Group IV) and is distinguished by different colors. Different species are represented by different symbols.

Figure 3

Chromosomal localization and collinearity analysis of the GeGAPDH family in G. elata. (A) Chromosomal localization of the GeGAPDH family. (B) Collinearity analysis of the GAPDH families among A. thaliana, G. elata and O. sativa. The red triangle is the location of the GeGAPDH1 gene.

Figure 4

Cis-acting elements of the GeGAPDH family in G. elata. (A) Types of cis-acting elements of the GeGAPDH family. The intensity of the color represents the number of cis-acting elements. (B) Distribution of cis-acting elements of the GeGAPDH family.

Figure 5

The protein–protein interactions (PPI) of GAPDH proteins in G. elata. (A–C) Predicted protein interaction network of three GeGAPDH proteins. (D) No interactions were predicted among the three GeGAPDH proteins using identical parameters. Edges represent functional associations between proteins, indicating their potential involvement in shared biological processes. Edge colors indicate different prediction types.

Figure 6

The Gene cloning results of GeGAPDH1. RG is the reference gene sequence, and RP is the corresponding protein sequence of the reference gene. S is the result of sequencing, and the red dashed line box is the location of the base mutation. P is the protein sequence corresponding to the sequencing result.

Figure 7

SDS-PAGE, concentration and GAPDH activity detection. (A) The protein expression of GeGAPDH1. (B) The protein expression of empty vector. M: protein marker (11~180 kDa); CK: non-induced bacterial solution; T: total protein; S: supernatant after crushing; P: precipitation after crushing; FT: flow-through after incubation binding; W1–W3, washing solution 1–3; E1–E3, protein elution solution 1–3 The predicted molecular mass of the target protein is labeled on the right side of the gel graph. (C) The concentration of protein P was the total protein, GeGAPDH1 was the purified protein of GeGAPDH1, and the control was the purified protein of the empty vector. n = 3 technical replicates. (D) The results of GAPDH1 activity assay. n = 3 technical replicates. (E) Reactions occurring in enzyme activity assays. In this reaction, GAPDH catalyzes BPGA and NADH to G3P and NAD⁺. The decrease of NADH measured at 340 nm can reflect the level of GAPDH activity. Consumption of 1 nmol of NADH per minute per mg of protein was defined as one unit of enzyme activity.

Figure 8

Bidirectional enzyme activity and kinetic characterization of GeGAPDH1. (A) The phosphorylation reaction uses G3P as a substrate to generate NADH. (B) The dephosphorylation reaction using BPGA as a substrate consumes NADH. (C) Michaelis-Menten kinetic analysis of GeGAPDH1 with varying G3P concentrations.

Figure 9

Subcellular localization of GeGAPDH1. (A) The fusion vector pCAMBIA1300-35S-GeGAPDH1-EGFP is transiently expressed in N. benthamiana. (B) The empty vector pCAMBIA1300-35S-EGFP is transiently expressed in N. benthamiana. GFP represents the green fluorescence field, CHI represents the chloroplast spontaneous fluorescence field, DIC represents the bright field, and Merge represents the superposition field. GFP field: excitation 488 nm, emission 500–550 nm; CHI field: excitation 488 nm, emission 650–750 nm. Scale bar = 20 µm.

Figure 10

Relative expression of GeGAPDH genes in different tissues. The X-axis represents different tissues, and the Y-axis represents the relative expression levels normalized to the expression values of the tuber. Error bars represent the standard error. Significant differences are indicated as follows: ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Values represent means ± SD (n = 3).

Figure 11

Relative expression of GeGAPDH family in G. elata under low-temperature stress. (A–C) Relative expression levels of GeGAPDH genes were treated at different temperatures on day 7. (D–F) Relative expression levels of GeGAPDH genes were treated at different temperatures on day 14. The X-axis represents different temperatures, and the Y-axis represents the relative expression levels normalized to the expression values of 24 °C. Error bars represent the standard error. Significant differences are indicated as follows: ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Values represent means ± SD (n = 4).

Figure 12

Relative expression of GeGAPDH family in G. elata under Fusarium stress. (A) Healthy control: G. elata treated with PDA medium for 7 days; (B,C) G. elata were infected by F. oxysporum (B) and F. solani (C) for 7 days, respectively, showing typical symptoms of brown rot. (D–F) Relative expression levels of GeGAPDH genes under pathogen stress for 7 days. Health represents the healthy control group, Fo represents the results of F. oxysporum infection, and Fs represents the results of F. solani infection. The X-axis represents different fungal pathogen treatments; the Y-axis represents relative expression levels normalized to the expression values of healthy G. elata. Error bars represent the standard error. Significant differences are indicated as follows: ns, non-significant; * p < 0.05; ** p < 0.01. Values represent means ± SD (n = 4).

Figure 13

GAPDH enzyme activity under low temperature and Fusarium stress. (A) The results of GAPDH enzyme activity assay under low-temperature stress for 7 days. (B) The results of GAPDH enzyme activity assay under low-temperature stress for 14 days. (C) The results of GAPDH enzyme activity assay exposed to Fusarium stress for 7 days. Health represents the healthy control group, Fo represents the results of F. oxysporum infection, and Fs represents the results of F. solani infection. Error bars represent the standard error. Significant differences are indicated as follows: ns, non-significant; * p < 0.05. Values represent means ± SD (n = 4).