Comprehensive Analysis of GH3 Gene Family in Potato and Functional Characterization of StGH3.3 under Drought Stress

Abstract

As an important hormone response gene, Gretchen Hagen 3 (GH3) maintains hormonal homeostasis by conjugating excess auxin with amino acids during plant stress-related signaling pathways. GH3 genes have been characterized in many plant species, but they are rarely reported in potato. Here, 19 StGH3 genes were isolated and characterized. Phylogenetic analysis indicated that StGH3s were divided into two categories (group I and group III). Analyses of gene structure and motif composition showed that the members of a specific StGH3 subfamily are relatively conserved. Collinearity analysis of StGH3 genes in potato and other plants laid a foundation for further exploring the evolutionary characteristics of the StGH3 genes. Promoter analysis showed that most StGH3 promoters contained hormone and abiotic stress response elements. Multiple transcriptome studies indicated that some StGH3 genes were responsive to ABA, water deficits, and salt treatments. Moreover, qRT-PCR analysis indicated that StGH3 genes could be induced by phytohormones (ABA, SA, and MeJA) and abiotic stresses (water deficit, high salt, and low temperature), although with different patterns. Furthermore, transgenic tobacco with transient overexpression of the StGH3.3 gene showed positive regulation in response to water deficits by increasing proline accumulation and reducing the leaf water loss rate. These results suggested that StGH3 genes may be involved in the response to abiotic stress through hormonal signal pathways. Overall, this study provides useful insights into the evolution and function of StGH3s and lays a foundation for further study on the molecular mechanisms of StGH3s in the regulation of potato drought resistance.

Keywords: GH3 family; expression pattern; potato; water deficit.

Figure 1

Unrooted phylogenetic tree for 19 StGH3s, 20 AtGH3s, 15 SlGH3s, and 16 OsGH3s was constructed by MEGA7.0. The neighbor connection (NJ) method was used to construct the phylogenetic tree, and the default parameter value was set to 1000.

Figure 2

The phylogenetic relationship, conserved motifs, and gene structure of StGH3. (A) A phylogenetic tree and map of the exon–intron structure were constructed. The green box represents the untranslated 5′ and 3′ regions; the orange box represents exons; the black line represents introns. (B) The conserved motifs of StGH3 proteins. (C) The conserved domain of StGH3 proteins.

Figure 3

The relationship between StGH3 gene-containing chromosomes was visualized through multiple collinear scanning toolkits (MCScanX) and TBtools. Gray lines represent collinear blocks within potato genome, while the red lines highlight GH3 gene pairs. 0-12 represents the chromosomes of potato.

Figure 4

Collinearity analysis of GH3 genes between potato and six other plant species. Gray lines represent collinear blocks in potato genome and other plant genomes, and red curves represent collinear GH3 genes.

Figure 5

Analysis of 19 cis-acting elements in the StGH3 promoter region. (A) Statistics on the number of stress-related cis-acting elements in all StGH3 gene promoters. (B) Statistics of stress-related cis-acting elements contained in the promoter of each StGH3 gene.

Figure 6

Heatmap showing expression of StGH3 genes based on RNA-seq data. Heatmap was generated based on log₂ FPKM. “QS9” and “Atl” represent the drought-tolerant potato variety “Qingshu 9” and drought-sensitive potato variety “Atlantic”, respectively. Three-week-old potato seedlings were treated in 1/2 MS medium with a final concentration of 200 mM mannitol, and the transcriptome was sequenced after 0, 2, 6, 12, and 24 h of stress.

Figure 7

The relative expression level of StGH3 genes under 200 mM mannitol stress treatments. The 0 h timepoint was taken as a reference to determine relative mRNA levels under stress conditions. Data represent the mean ± SD of three replicates. Data points marked with an asterisk (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001) indicate statistically significant differences between control and stress treatments.

Figure 8

Expression analysis of StGH3s in different tissues of flowering potato. The expression level of all genes in roots was set to 1. Error bars are the standard deviation of three measurements. The letters at the top of the bars indicate significant differences between different tissues (α = 0.05, Duncan).

Figure 9

Positive identification of StGH3.3 transgenic plants. (A) Structure of 1304 vector used for genetic transformation. (B) Tobacco used for genetic transformation. (C) GUS staining of each strain before and after water deficit treatment.

Figure 10

Effect of transient transformation of StGH3.3 gene on physiological indexes of transgenic plants. (A) Accumulation of MDA under normal and water deficit conditions. (B) Accumulation of proline under normal and water deficit conditions. The 1304 label refers to transgenic plants overexpressing empty vectors. StGH3.3 refers to transgenic plants overexpressing the target gene. Data points marked with an asterisk (*** p ≤ 0.001) indicate statistically significant differences between control and stress treatments. ns—not significant.

Figure 11

Water loss in leaves of transgenic plants in vitro. After 2 days of transient transformation, leaves of transgenic plants expressing empty vector 1304 and StGH3.3 were taken and left standing at room temperature. The leaf mass was weighed every hour, and the leaf water loss of each strain was calculated. Data points marked with an asterisk (** p ≤ 0.01, *** p ≤ 0.001) indicate statistically significant differences between control and stress treatments.

Figure 12

The relative expression level of StGH3 genes under low temperature and high salt, SA, MeJA, and ABA treatments. The expression level of all genes at 0 h was set to 1. Data represent the mean ± SD of three replicates. Data points marked with an asterisk (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001) indicate statistically significant differences between control and stress treatments.