Genome-Wide Identification and Expression Analysis of Heat Shock Protein 70 (HSP70) Gene Family in Pumpkin (Cucurbita moschata) Rootstock under Drought Stress Suggested the Potential Role of these Chaperones in Stress Tolerance

Abstract

Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has been characterized in several crops under drought stress conditions. However, there is no study on HSP70s in pumpkin (Cucurbita moschata). Therefore, we performed a comprehensive analysis of this gene family, including phylogenetic relationship, motif and gene structure analysis, gene duplication, collinearity, and promoter analysis. In this research, we found 21 HSP70s that were classified into five groups (from A to E). These genes were mostly localized in the cytoplasm, chloroplast, mitochondria, nucleus, and endoplasmic reticulum (ER). We could observe more similarity in closely linked subfamilies in terms of motifs, the number of introns/exons, and the corresponding cellular compartments. According to the collinearity analysis, gene duplication had occurred as a result of purifying selection. The results showed that the occurrence of gene duplication for all nine gene pairs was due to segmental duplication (SD). Synteny analysis revealed a closer relationship between pumpkin and cucumber than pumpkin and Arabidopsis. Promoter analysis showed the presence of various cis-regulatory elements in the up-stream region of the HSP70 genes, such as hormones and stress-responsive elements, indicating a potential role of this gene family in stress tolerance. We furtherly performed the gene expression analysis of the HSP70s in pumpkin under progressive drought stress. Pumpkin is widely used as a rootstock to improve stress tolerance, as well as fruit quality of cucumber scion. Since stress-responsive mobile molecules translocate through vascular tissue from roots to the whole plant body, we used the xylem of grafted materials to study the expression patterns of the HSP70 (potentially mobile) gene family. The results indicated that all CmoHSP70s had very low expression levels at 4 days after stress (DAS). However, the genes showed different expression patterns by progressing he drought period. For example, the expression of CmoHSP70-4 (in subgroup E) and CmoHSP70-14 (in subgroup C) sharply increased at 6 and 11 DAS, respectively. However, the expression of all genes belonging to subgroup A did not change significantly in response to drought stress. These findings indicated the diverse roles of this gene family under drought stress and provided valuable information for further investigation on the function of this gene family, especially under stressful conditions.

Keywords: chaperones; drought stress; expression pattern; heat shock proteins; phylogeny; pumpkin.

Figure 1

Phylogenetic analysis of HSP70 proteins of pumpkin, cucumber, and Arabidopsis. The HSP70s for pumpkin, Arabidopsis, and cucumber are shown in black, green, and red colors, respectively. The subgroups have been shown in different colors and indicated with the letters A to E.

Figure 2

Phylogenetic tree of CmoHSP70 proteins and their motif analysis. (A) Phylogenetic tree of CmoHSP70s in pumpkin. The CmoHSP70s were classified in five subgroups, from A to E, based on their similarities to Arabidopsis genes. (B) Ten conserved motif proteins of the CmoHSP70s, each small box indicating a motif. All 10 motif logos are shown below the figure. (C) Visualization of conserved domains of identified CmoHSP70s using TBtools. Each color represents a specific domain. The corresponding domain names have been shown below the figure.

Figure 3

Gene structure analysis of CmoHSP70s in pumpkin. The structures of intron and exon and untranslated regions (UTR) are shown in black line and yellow and green boxes, respectively. The scale is helpful for gene length estimation.

Figure 4

Chromosomal location of the identified CmoHSP70s in pumpkin. The genes with the same color indicate that they belong to the same subgroup based on the phylogenetic tree. The chromosome numbers have been shown below them.

Figure 5

Collinearity analysis of HSP70 gene family in pumpkin.

Figure 6

Synteny analysis of HSP70 family between pumpkin and two other species. The red lines show the HSP70 orthologous genes between two species, and the gray lines indicate all orthologous genes. The numbers in the figure indicate the chromosome numbers.

Figure 7

Cis-regulatory elements related to hormones and stress in promoter region of CmoHSP70 genes. (A) The regulatory elements of HSP70 related to hormones and (B) indicating the stress-related cis elements. AuxRE (auxin responsive element), ABRE (ABA responsive element), GARE (Gibberellin responsive element), MeJARE (methyl jasmonate responsive element), SARE (salicylic acid responsive element). (C) Sequence logo of HSE in the promoter region of CmoHSP70s.

Figure 8

Expression patterns of 21 identified CmoHSP70 genes in response to drought stress. The samples belonged to the xylem tissues below the graft union of pumpkin rootstock and were collected at 4, 6, and 11 DAS (days after drought stress). Each value is an average of three replications, and each replicate contained three individuals. Green and low colors show low and high relative expression levels, respectively.