Next Article in Journal
The Endothelial Transcription Factor ERG Mediates a Differential Role in the Aneurysmatic Ascending Aorta with Bicuspid or Tricuspid Aorta Valve: A Preliminary Study
Previous Article in Journal
Peroxisome Proliferator-Activated Receptor-α (PPARα) Expression in a Clinical Population of Pakistani Patients with Type 2 Diabetes and Dyslipidemia
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 18
10.3390/ijms231810849
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance

by
Xiaodong Lian
1,2,
Qiuping Wang
1,2,
Tianhao Li
1,2,
Hongzhu Gao
1,2,
Huannan Li
1,2,
Xianbo Zheng
1,2,
Xiaobei Wang
1,2,
Haipeng Zhang
1,2,
Jun Cheng
1,2,
Wei Wang
1,2,
Xia Ye
1,2,
Jidong Li
2,3,
Bin Tan
1,2,* and
Jiancan Feng
1,2,*
1
College of Horticulture, Henan Agricultural University, Zhengzhou 450002, China
2
Henan Engineering Center for Peach Germplasm Innovation and Utilization, Zhengzhou 450002, China
3
College of Forestry, Henan Agricultural University, Zhengzhou 450002, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(18), 10849; https://doi.org/10.3390/ijms231810849
Submission received: 4 August 2022 / Revised: 12 September 2022 / Accepted: 12 September 2022 / Published: 16 September 2022
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Review Reports Versions Notes

Abstract

:
The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42 PpHSP20 genes were detected in the peach genome and were randomly distributed onto the eight chromosomes. The primary modes of gene duplication of the PpHSP20s were dispersed gene duplication (DSD) and tandem duplication (TD). PpHSP20s in the same class shared similar motifs. Based on phylogenetic analysis of HSP20s in peach, Arabidopsis thaliana, Glycine max, and Oryza sativa, the PpHSP20s were classified into 11 subclasses, except for two unclassified PpHSP20s. cis-elements related to stress and hormone responses were detected in the promoter regions of most PpHSP20s. Gene expression analysis of 42 PpHSP20 genes revealed that the expression pattern of PpHSP20-32 was highly consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’, which is a temperature-sensitive semi-dwarf. PpHSP20-32 was selected for further functional analysis. The plant heights of three transgenic Arabidopsis lines overexpressing PpHSP20-32 were significantly higher than WT, although there was no significant difference in the number of nodes. In addition, the seeds of three over-expressing lines of PpHSP20-32 treated with high temperature showed enhanced thermotolerance. These results provide a foundation for the functional characterization of PpHSP20 genes and their potential use in the growth and development of peach.

1. Introduction

During these times of global environmental change, temperature is arguably the most important factor affecting plant growth and geographical distribution [1,2]. Plants experience fluctuations in the thermal environment characterized by average, maximum, and minimum daily temperatures, which change over the course of the seasons. Plants are very sensitive to temperature, showing responses to slight changes of just 1 °C [3]. However, it remains unknown how temperature signals are perceived. Extremely high or low temperatures lead to temperature stress, which is one of the most severe abiotic stressors and severely impacts plant growth [4,5,6]. Plants have evolved multiple pathways to adapt to temperature stress [7,8,9,10]. Nevertheless, the precise mechanism of temperature sensing, particularly ambient temperature, is still unclear.
Tremendous amounts of research have shown that multiple heat shock proteins (HSPs) emerge as central players in the temperature response, including the growth/development and stress responses [11,12,13,14]. According to their molecular weight, HSPs are divided into five major categories, including HSP100s, HSP90s, HSP70s, HSP60s, and HSP20s [4]. Among the five major categories, HSP20s are the most prevalent in plants. HSP20s are also known as small heat shock proteins and have molecular weights ranging from 15 to 42 kDa. Structurally, HSP20s are conserved, with the alpha-crystallin domain (ACD) in the C-terminus [15,16]. HSP20s have been identified in a few plant species, such as Arabidopsis (Arabidopsis thaliana) [17], rice (Oryza sativa) [18], pepper (Capsicum annuum) [19], tomato (Solanum lycopersicum) [20], watermelon (Citrullus lanatus) [21], grape (Vitis vinifera) [22], Sorghum bicolor [23], and apple (Malus domestica) [13].
HSP20s participate in a wide range of developmental processes and abiotic stresses, such as heat, salt, and drought [19,24,25,26]. In tomatoes, four SlHSP20s are constitutively expressed in almost all tissues, indicating that they might play specific housekeeping functions under normal growth conditions [20]. Under heat, drought, and salt stresses, the expression levels of SlHSP20s were up-regulated, implying their potential roles in regulating the response to stresses [20]. Similar results were obtained in peppers, where most CaHSP20s were highly induced by heat stress [19]. Overexpression of CaHSP16.4 enhanced the tolerance to heat and drought stresses by stabilizing the ROS-scavenging system [27]. Unlike heat stress, few HSP20s were induced by cold stress in soybean [28]. In grapes, most VvHSP20s responded to H2O2 treatment [22]. In addition, plenty of HSP20s were related to plant development, including seed maturation and germination, pollen development, and hypocotyl elongation [16]. AtHSP22 participated in auxin-regulated hypocotyl elongation under high temperatures in Arabidopsis thaliana [26].
Peach (Prunus persica L.), an economically important crop and a popular fruit with consumers, is widespread in both temperate and subtropical regions, although peach trees can also be found in high altitude and severe cold regions [29]. Due to its wide distribution, genetic diversity, and relatively small genome size, the peach is considered a model species for genomic research of rosaceae [30]. ‘Zhongyoutao 14’, a temperature-sensitive semi-dwarf (TSSD) peach cultivar, showed extremely shortened internodes below 30 °C and normal internode length above 30 °C [10,31]. Due to shorter internodes until mid-May and then normal length internode in the orchard of the Zhengzhou Fruit Research Institute, the tree heights of TSSD categorize them as semi-dwarf [31]. Whether PpHSP20 participated in the temperature sensitivity of ‘Zhongyoutao 14’ is unknown. In this study, the expression patterns of PpHSP20s were analyzed during shoot elongation of ‘Zhongyoutao 14’ at four critical growth stages. Furthermore, the function of PpHSP20-32 was analyzed via ectopic expression in transgenic Arabidopsis plants. The thermotolerant characteristics of transgenic Arabidopsis carrying PpHSP20-32 were also analyzed. This study provides valuable information as a step toward the further investigation of the functions and regulatory mechanisms of potentially important PpHSP20 genes that may be crucial in temperature tolerance and plant height in peach trees.

2. Results

2.1. Whole Genome Identification and Protein Structure of Peach HSP20 Genes

Forty-two HSP20 gene family members were identified from the peach genome and named PpHSP20-1 to PpHSP20-42, according to their physical location on the peach chromosomes (Table 1 and Table S1). The physicochemical properties of the predicted PpHSP20 proteins were analyzed. The predicted molecular weight ranged from 15.5 kDa to 100.0 kDa, except for PpHSP20-18 and PpHSP20-30, which were less than 15 kDa. The number of amino acids ranged from 87 aa (PpHSP20-30) to 919 aa (PpHSP20-42), and the predicted isoelectric point ranged from 4.59 (PpHSP20-42) to 9.44 (PpHSP20-36). Most PpHSP20 were predicted to be unstable proteins, while a small portion (19.0%) were stable, with instability index values less than 40 (which is a stable protein). The average hydrophobic index values of all family members were negative, indicating that the PpHSP20 protein family members were hydrophobic proteins.
The predicted protein secondary structure showed that all PpHSP20s contained four secondary structures. The proteins were mainly composed of alpha helix and random coil motifs, while beta turns were the least identified structures. Except for PpHSP20-34, PpHSP20-36, and PpHSP20-40, most of the PpHSP20s (71.4% of 42 PpHSP20s) contained second structures in the ranked quantities of random coils > alpha helix ≥ extended strand > beta turn, while the quantity ranking of the structures of the other members (21.4% of 42 PpHSP20s) was random coil > extended chain structure > alpha helix > beta turn (Table 2).

2.2. Chromosome Distribution, Gene Duplication, Gene Structure, and Conserved Motif Analysis of PpHSP20 Genes

The PpHSP20 genes were unevenly distributed among the eight chromosomes of peach (Figure 1). Among them, chromosomes 1 and 7 had five PpHSP20s, and chromosomes 2, 3, and 8 each had four PpHSP20s. Chromosome 4 carried three PpHSP20 genes, chromosome 5 had six, and Chromosome 6 had the most PpHSP20s, at eleven.
The expansion of the PpHSP20 genes family in peach was analyzed (Figure 1). The main expansion patterns were dispersed gene duplication (DSD; for 18 or 42.9% of the PpHSP20 genes) and tandem duplication (TD; for 13 PpHSP20 genes or 30.9%), (Figure 1 and Table S2). Eight genes arose through whole-genome duplication (WGD), including PpHSP20-7, PpHSP20-8, PpHSP20-17, PpHSP20-24, PpHSP20-25, PpHSP20-32, PpHSP20-38, and PpHSP20-42) (Figure 1).
The 42 PpHSP20s were grouped into five classes (Figure 2A), containing 20, 4, 2, 3, and 13 PpHSP20s in Class I–V, respectively. The structural differences of the PpHSP20 genes were also predicted. Fourteen PpHSP20s were intronless (33.3%), 17 (40.5%) had one intron, and seven (16.7%) had two introns (Figure 2B). The remaining four PpHSP20 genes contained more than two introns: PpHSP20-13 had five introns, PpHSP20-17 had 14 introns, PpHSP20-38 had 11 introns, and PpHSP20-42 had 12 introns.
MEME was used to analyze the conserved motifs of the PpHSP20 proteins, and the results showed that PpHSP20s contained ten conserved motifs. Among all PpHSP20 members, 40 (95.2%) contained Motif 1, 20 (47.6%) contained Motif 2, 34 (81.0%) contained Motif 3, and 32 (76.2%) contained Motif 6. Motif analysis showed that the PpHSP20s containing similar motifs were grouped in the same class (Figure 2A,C). For example, most PpHSP20s in class I contained motifs 1, 2, 3, 5, 6, and 10. Motifs 1, 2, 3, 6, 8, and 10 were contained in the HSP20 domain (Figure 2C).

2.3. Phylogenetic Analysis of PpHSP20 Proteins

A phylogenetic analysis was conducted using 128 HSP20 proteins, including 19 Arabidopsis thaliana sequences, 45 Glycine max sequences, 22 Oryza sativa sequences, and 42 peach sequences (Figure 3). The 128 HSP20s were divided into 13 different subfamilies, including CI, CII, CIII, CIV, CV, CVI, CVII (cytoplasm or nucleus), MI, MII (mitochondria), ER (endoplasmic reticulum), P (Plastid), Po (Peroxisome), and an unknown classification (Figure 3). There were 43 CIs, 13 CIIs, 21 CIIIs, 6 CIVs, 5 CVs, 3 CVIs, 1 CVIIs, 4 MIs, 5 MIIs, 11 Ps, 5 Pos, 9 ERs, and 2 in unknown classification. Most of the PpHSP20s (30, 71.4%) were classified as CI–CVI, followed by P (2), and ER (2); moreover, MI, MII, and Po contained only one PpHSP20 each. However, PpHSP20-32 and PpHSP20-41 were not classified. This indicated that the PpHSP20s likely function in the cytoplasm or nucleus, while a few were distributed in organelles.

2.4. Analysis of Cis-Acting Elements of PpHSP20s Promoters

The promoters in the upstream 2000 bp region of 42 PpHSP20 genes were analyzed to identify the cis-elements. Eleven types of cis-elements were detected. Most of the PpHSP20 genes possessed abscisic acid-responsive (ABRE), light-responsive, MeJA-responsive, and anaerobic-induction elements (Figure 4A,B). The elements were grouped into three categories, including phytohormone-responsive, abiotic, and biotic stress-responsive and plant development-related cis-elements. The phytohormone-responsive classification accounted for the highest proportion (49.2%, 186 of 378 elements), including abscisic acid-responsive), MeJA-responsive, salicylic acid-responsive (SA), auxin-responsive and gibberellin-responsive (GA). The abiotic and biotic stress-responsive elements included anaerobic induction and low temperature-responsive elements. In the plant development-related category, seed specific regulation, cell cycle regulation, and circadian control were identified. These results suggested that PpHSP20s were not only related to stress response, but also related to other physiological responses.

2.5. Expression of PpHSP20s during the Shoot Elongation of ‘Zhongyoutao 14’

The expression patterns of the PpHSP20s were compared at four critical stages (initial period, IP; initial elongation period, IEP; rapid growth period, RGP; stable growth period, SGP) of shoot elongation in the temperature-sensitive semi-dwarf peach cultivar ‘Zhongyoutao 14’, grown in the field under regular management with natural ambient temperature. According to their expression patterns, the 42 PpHSP20s could be classified into four groups (Figure 5A). Group I contained 19 PpHSP20s that showed the highest expression level during SGP. Group II contained 7 PpHSP20s that showed the lowest expression level in IP, including PpHSP20-6, PpHSP20-17, PpHSP20-18, PpHSP20-21, PpHSP20-33, PpHSP20-37, and PpHSP20-38. The transcript abundance of PpHSP20s in Group III was higher in the IP and the IEP (PpHSP20-32, PpHSP20-13, PpHSP20-14, and PpHSP20-35), compared to RGP and the SGP. Group IV, the second largest group containing 12 PpHSP20s, showed higher expression levels in IP or RGP, compared to IEP and RGP. The internodes length of IP (1.21 mm) and IEP (2.57 mm) were significantly less than that of RGP (11.27 mm) and SGP (12.54 mm) [10]. It showed a negative trend between the internode length and the expression levels of PpHSP20s in Group III (marked in red). Among the Group III genes, the expression level of PpHSP20-32 was most consistent with the terminal internode length, as shown in Figure 5B. We speculated that PpHSP20-32 might participate in temperature-induced shoot growth in this temperature-sensitive peach cultivar.

2.6. Overexpression of PpHSP20-32 in Arabidopsis Leads to an Increase in Plant Height

In order to study the function of PpHSP20-32, we constructed a PpHSP20-32 overexpression vector and transformed it into Arabidopsis thaliana using an Agrobacterium-mediated method. The phenotypes of three transgenic lines (L1, L2, and L3) and WT were recorded (Figure 6). Two weeks after being transplanted into a substrate, their rosette leaves were longer and wider than WT (average length and width), but there was no significant difference in the number of rosette leaves (Figure 6A–C). Four weeks after transplanting, the plant morphology was observed, and the height of the flowering bolt in the three transgenic lines was higher than that of WT (Figure 6D,E). The average plant height of the three transgenic lines was 34.0 cm (L1, 34.4 cm; L2, 34.3 cm; L3, 33.4 cm), which was significantly higher than the 29.3 cm of WT. The average lengths of the internodes of the transgenic line 1–3 and WT were 1.67 cm, 1.89 cm, 1.63 cm, and 1.83 cm, respectively (Figure 6F), showing no significant difference between the transgenic lines and WT. The average numbers of internodes of the transgenic line 1–3 and WT were 23, 22, 20, and 17, respectively (Figure 6G). There was also no significant difference in the number of branches among all lines (Figure 6H).

2.7. PpHSP20-32-OE Seeds Exhibit Enhanced Thermotolerance

The seeds of three PpHSP20-32-OE lines and WT were treated at 46 °C for 30 min and transferred to 25 °C to assay thermotolerance (Figure 7). By 48 h after heat stress (HS), there was no seed germination in any of the four lines (Figure 7A,B). After 60 h at high temperature, the germination rate of the three PpHSP20-32 transgenic lines was 100%, which was significantly higher than that of WT seeds (Figure 7C,D,F). For the WT, the germination was less than 10% after 60 h of HS (Figure 7C,F), but reached about 100% germination at 96 h (Figure 7E,F). These results suggested that the overexpression of PpHSP20-32 improves the resistance of Arabidopsis seeds to high temperatures.

3. Discussion

As plants sense high temperatures or heat stress, gene expression patterns will vary, especially the up-regulation of the heat shock genes [11,32]. HSPs include the HSP100s, HSP90s, HSP70s, HSP60s, and HSP20s. HSP20s are a diverse, ancient, and important family among the HSPs [16]. The number of HSP20s has been determined in numerous plants, such as 31 in Arabidopsis thaliana [17,33], 51 in Glycine max [28], 35 in Capsicum annuum [19], 42 in Solanum lycopersicum [20], 63 in Panicum virgatum [34], 48 in Solanum tuberosum [35], 48 in Vitis vinifera [22], 47 in Sorghum bicolor [23], 41 in Malus pumila [13], 47 in Cucumis sativus [14], 45 in Cucumis melo [14], and 47 in Citrullus lanatus [14]. In peach, we identified 42 PpHSP20s, a number greater than in Arabidopsis thaliana and pepper, but lower than in switchgrass, potato, and grape. The varied numbers in different plants might be due to the difference in gene duplications during the evolution of the plants.
The 42 PpHSP20s are unevenly mapped on the eight chromosomes, with Chr6, the second longest chromosome, containing the most HSP20s. The members of other gene families, such as E3 genes, were also mainly mapped on the longer chromosomes in peach [36], while the F-box genes showed a similar phenomenon in pear [37]. The E3 and F-box genes were mapped on the longer chromosome, similar to the distribution of PpHSP20s on the chromosomes. However, the biggest cluster of HSP20s was on the shortest chromosome, chromosome 8, in apple [13]. So, any rules of distribution of gene family members may be different among different families or different plants, and need to be further validated.
The PpHSP20 duplication in peach showed inconsistent patterns with those of other plants [13,28,35]. In apple, WGD and TD were the main duplication patterns [13]. In this study, the PpHSP20 family expanded more by DSD and TD. In peach, DSD was the major expansion route for other gene families, such as the F-box, U-box, RING, BTB, SKP [36], and HSF genes [38]. This phenomenon in peach might be explained by the fact that the peach genome has not undergone a recent whole-genome duplication [39].
The 42 PpHSP20s were divided into 11 classes (CI, CII, CIII, CIV, CV, CVI, MI, MII, P, ER, and Px), except for 2 unclassified PpHSP20s, based on the phylogenetic tree which was constructed using the amino acid sequences of peach, rice, Arabidopsis thaliana and soybean. In an earlier study, the AtHSP20s were divided into seven classes (CI, CII, CIII, M, P, ER, and Px), except for five genes that did not fall into any class [17]. Afterwards, the five unclassified AtHSP20s were categorized into five new classes (CIV, CV, CVI, and CVII) and MII [40]. Most of PpHSP20s were classified into nucleocytoplasmic subfamilies (CI–CVI), which indicated that the cytoplasm may be the primary site of action for the HSP20 proteins. This was also observed in other plants, for example, apple and soybean [13,28]. In this study, the HSP20s of peach lacked any proteins in the CVII subgroup, similar to soybean [28], rice [18], switchgrass [34], apple [13], and three cucurbit species (cucumber, melon, and watermelon) [14]. In Arabidopsis thaliana, the CVII subgroup gene AtHsp14.7 was constitutively expressed, and its transcript level did not change under different stresses [40]. It was speculated that AtHsp14.7-CVII was involved in specific housekeeping functions [40].
Plant HSPs are molecular chaperones that protect the functions of their target proteins under various stress conditions to help maintain growth and development [4,16]. In this study, PpHSP20s showed different expression patterns at non-stressful but elevated temperature. The expression patterns of four PpHSP20s, namely PpHSP20-13, PpHSP20-14, PpHSP20-35, and especially PpHSP20-32, showed a correlation with the length of the terminal internodes in the shoots of temperature-sensitive semi-dwarf peach. Populus trichocarpa, a transgenic line overexpressing PtHSP17.8, showed enhanced tolerance to heat and salt stresses [24]. In pepper, CaHSP16.4 participated in heat and drought stress by enhancing the scavenging of reactive oxygen species [27]. These results mainly focused on the function of HSP20s under stress conditions. Based on this study, PpHSP20s might play important role in the regulation of shoot elongation at non-stressful temperatures. In addition, HSP20 responded to the phytohormone ABA and modulated polar auxin transport [26].
In the ambient temperature-sensing pathway, AtHSP70 is expressed at a level proportionate to the ambient temperature [41]. AtHSP90 integrates environmental temperature and auxin signaling to regulate temperature-dependent plant growth by stabilizing the auxin co-receptor F-box protein TIR1 [42,43]. A recent study showed that the heat shock protein AtHSP22 promoted hypocotyl elongation at high temperatures by regulating polar auxin transport, which required the ABI1 protein phosphatase [26]. In this study, PpHSP20-32-overexpressing transgenic lines produced larger rosette leaves and taller plants than WT. The plant height of the transgenic lines was higher than that of WT. There was no significant difference in the length of the internodes between the transgenic lines and WT, indicating that the increase in plant height of the transgenic lines may be caused by the increase in the number of internodes. The PpHSP20-32-overexpressing lines also showed enhanced heat tolerance. Similar results were observed in rice, pepper, and poplar, which together demonstrate that HSP20 genes enhance thermotolerance [24,25,27].
It remains unknown how PpHSP20-32 regulates rosette leaf size and plant height. The promoter of PpHSP20-32 contained four types of phytohormone-responsive elements, namely ABRE, MeJA-responsive, salicylic acid-responsive, and gibberellin-responsive elements (Figure 4A,B). This indicated that PpHSP20-32 might serve as a phytohormone responsive factor. In Arabidopsis thaliana, AtHSP22 is regulated by ABA and auxin, while AtHSP22 potentiates the auxin efflux PIN proteins, which promotes hypocotyl elongation [26]. These results suggested that PpHSP20-32 might serve to modulate rosette leaf and flower bolt growth by orchestrating phytohormone signaling.

4. Materials and Methods

4.1. Plant Materials

The peach cultivar ‘Zhongyoutao14’, a temperature-sensitive semi-dwarf, is planted in the experimental station of the Horticulture College, Henan Agricultural University (Zhengzhou, China). The shoot internode length was temperature-dependent. Shoot tips were collected at four critical growth stages, namely the initial period (IP), initial elongation period (IEP), rapid growth period (RGP), and stable growth period (SGP) [10]. The internodes’ lengths were less than 3 mm at IP and IEP with lower environmental temperature (below 30 °C). While the internodes’ lengths at RGP and SGP with higher temperatures (above 30 °C) were more than 10 mm [10]. All samples were quickly frozen in liquid nitrogen after collection and stored in the laboratory at −80 °C. Arabidopsis thaliana (L.) Heynh Columbia 0 (Col-0) was used for transformation with PpHSP20-32.

4.2. Identification and Characteristic Analysis of Peach HSP20s

The hidden Markov model (HMM) profile (PF00011), characteristic of HSP20, was downloaded from the Pfam website (http://pfam.xfam.org, accessed on 10 May 2022) and used to identify HSP20 genes in peach. An hmmsearch was performed against the peach genome files (v2.1), downloaded from the JGI database (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 10 May 2022). The isoelectric points and other physical properties were approximated from ExPASy (http://web.expasy.org/compute_pi, accessed on 10 May 2022).

4.3. Chromosome Location and Gene Structure Analysis of the PpHSP20 Genes

According to the genome location annotation given by Phytozome V12.1, the chromosome location of each PpHSP20 was mapped using TBtools [44]. According to the DNA and CDS sequences data for the peach HSP20 gene, the gene structure map was drawn using the online tool GSDS (http://gsds.cbi.pku.edu.cn/, accessed on 10 May 2022).

4.4. Phylogeny and Motif Analysis of PpHSP20s

The amino acid sequences of the HSP20 genes of Arabidopsis thaliana, Oryza sativa, Glycine max, and peach were saved as FASTA format files. The phylogenetic tree was constructed by the maximum likelihood method using MEGA 7.0 software (v7.0, Sudhir kumar, Hachioji, Tokyo, Japan) [45]. The online software MEME5.0.4 (http://alternate.meme-suite.org/tools/meme, accessed on 12 May 2022) was used to analyze the motifs in each protein sequence.

4.5. Analysis of Cis-Acting Elements of PpHSP20s

The promoter sequence of each PpHSP20 gene (2000 bps upstream of the start codons) was downloaded from the peach genome. The cis-acting elements of the HSP20 promoters were detected using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 May 2022).

4.6. PpHSP20 Gene Expression in Different Growth Stages of Peach

The expression levels of the PpHSP20 genes were obtained from our previous transcriptome data of the four critical stages of shoot growth in the cultivar ‘Zhongyoutao 14’ (Table S3) [10]. The heatmap was generated by TBtools (v1.09876, Chengjie Chen, Guangzhou, Guangdong, China) [44]. The FPKM (fragments per kilobase of exon per million fragments mapped) values of the HSP20s and the terminal internode lengths of the stems were used for the correlation analysis.

4.7. Generation and Phenotypic Observation of PpHSP20-32-Overexpression in Arabidopsis Lines

The CDS of PpHSF20-32 was amplified using PpHSP20-32-F and PpHSP20-32-R primers (Table S4). The resulting PpHSP20-32 fragment was inserted into the pMD19T vector (Takara, Dalian, China). After sequence confirmation, the full coding sequence of PpHSP20-32 was amplified with primers including restriction sites Hind III and Xba I (Table S4), and the amplified fragment was directionally inserted into the vector pSAK277. Transgenic Arabidopsis plants were generated through the floral dip method using the Agrobacterium tumefaciens strain GV3101 [46].
After screening for kanamycin resistance and PCR verification (an initial denaturing step at 98 °C for 5 min, followed by 30 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 40 s, then 72 °C for 3 min), the transgenic plants were allowed to flower. Seeds from T2 transgenic Arabidopsis lines were used for subsequent experiments. Three seedlings from each line with five rosette leaves per seedling of each line was considered one biological replicate and used for the observation of leaf phenotype (length and width). Three biological replicates were taken, for a total of nine seedlings observed. The height, the length of internodes, and the number of internodes and branches (five plants per line) in the different transgenic lines and WT were determined.

4.8. Heat Stress Treatment

To detect the function of PpHSP20-32 in heat tolerance, heat stress treatment (46 °C for 30 min) was performed. Before heat stress treatment, seeds of WT and transgenic Arabidopsis lines were sown on MS medium and kept dark at 4 °C for 2 d, and then at 22 °C for 2 d. Then, the seeds were exposed to 46 °C for 30 min, followed by being transferred into a climate chamber (22 °C, 16 h light/8 h dark cycles). The germination of seeds was counted daily and photographed. More than 60 seeds of each line were used in each plate, with three replicates. Differences in heat stress tolerance were confirmed using Student’s t-test.

4.9. Statistical Analysis

Data were analyzed by ANOVA, Tukey HSDa, and Duncan’s multiple range tests (at p < 0.05) using IBM SPSS Statistics 20 (SPSS, Armonk, New York, NY, USA).

5. Conclusions

In this study, 42 PpHSP20 genes, distributed on eight chromosomes randomly, were identified in the peach genome. Dispersed gene duplication (DSD) and tandem duplication (TD) were the primary modes of gene duplication of PpHSP20s. Except for two unclassified PpHSP20s, the other 40 PpHSP20s were classified into 11 subclasses. The gene structures, basic classification, conserved motifs, and cis-elements were also analyzed. The expression pattern of PpHSP20-32 was highly consistent with shoot length changes during four critical growth stages of temperature-sensitive semi-dwarf peach ‘Zhongyoutao 14’ in response to increasing temperature. Transgenic Arabidopsis lines overexpressing PpHSP20-32 demonstrated that the gene can increase plant height and enhance thermotolerance. The results in this study supplied general information on the PpHSP20 gene family and further revealed the potential roles of PpHSP20-32 in plant height, in addition to the response to heat stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms231810849/s1.

Author Contributions

J.F., B.T. and X.L. conceived and designed the research. Q.W., T.L., H.G. and H.L. carried out most experiments. X.Z., X.W., J.C. and W.W. performed the bioinformatics analyses. X.Y. and J.L. analyzed the data. H.Z., B.T. and X.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Special Fund of Henan Province for Agro-Scientific Research in the Public Interest (No. 201300110500), the Henan Province Outstanding Foreign Scholar Program (GZS2020007), the Modern Agricultural Industry Technology of Henan Province (HARS-22-09-G1), the Youth Fund of Henan Province (No. 30602313), and the Innovation Fund of Henan Agriculture University (No. 30500873).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Anita K. Snyder for her critical reading of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Pauli, H.; Gottfried, M.; Dullinger, S.; Abdaladze, O.; Akhalkatsi, M.; Alonso, J.L.B.; Coldea, G.; Dick, J.; Erschbamer, B.; Calzado, R.F.; et al. Recent Plant Diversity Changes on Europe’s Mountain Summits. Science 2012, 336, 353–355. [Google Scholar] [CrossRef] [PubMed]
  2. Wolkovich, E.M.; Cook, B.I.; Allen, J.M.; Crimmins, T.M.; Betancourt, J.L.; Travers, S.E.; Pau, S.; Regetz, J.; Davies, T.J.; Kraft, N.J.B.; et al. Warming experiments underpredict plant phenological responses to climate change. Nature 2012, 485, 494–497. [Google Scholar] [CrossRef] [PubMed]
  3. Argyris, J.; Truco, M.J.; Ochoa, O.; Knapp, S.J.; Still, D.W.; Lenssen, G.M.; Schut, J.W.; Michelmore, R.W.; Bradford, K.J. Quantitative trait loci associated with seed and seedling traits in Lactuca. Appl. Genet. 2005, 111, 1365–1376. [Google Scholar] [CrossRef]
  4. Wang, W.; Vinocur, B.; Shoseyov, O.; Altman, A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004, 9, 244–252. [Google Scholar] [CrossRef]
  5. Wang, N.; Guo, T.; Sun, X.; Jia, X.; Wang, P.; Shao, Y.; Liang, B.; Gong, X.; Ma, F. Functions of two Malus hupehensis (Pamp.) Rehd. YTPs (MhYTP1 and MhYTP2) in biotic- and abiotic-stress responses. Plant Sci. Int. J. Exp. Plant Biol. 2017, 261, 18–27. [Google Scholar] [CrossRef] [PubMed]
  6. Suzuki, N. Temperature Stress and Responses in Plants. Int. J. Mol. Sci. 2019, 20, 2001. [Google Scholar] [CrossRef] [PubMed]
  7. VanWallendael, A.; Soltani, A.; Emery, N.C.; Peixoto, M.M.; Olsen, J.; Lowry, D.B. A Molecular View of Plant Local Adaptation: Incorporating Stress-Response Networks. Annu. Rev. Plant Biol. 2019, 70, 559–583. [Google Scholar] [CrossRef]
  8. Quint, M.; Delker, C.; Franklin, K.A.; Wigge, P.A.; Halliday, K.J.; van Zanten, M. Molecular and genetic control of plant thermomorphogenesis. Nat. Plants 2016, 2, 15190. [Google Scholar] [CrossRef]
  9. Zhou, Y.; Xun, Q.; Zhang, D.; Lv, M.; Ou, Y.; Li, J. TCP Transcription Factors Associate with PHYTOCHROME INTERACTING FACTOR 4 and CRYPTOCHROME 1 to Regulate Thermomorphogenesis in Arabidopsis thaliana. iScience 2019, 15, 600–610. [Google Scholar] [CrossRef]
  10. Lian, X.; Tan, B.; Yan, L.; Jiang, C.; Cheng, J.; Zheng, X.; Wang, W.; Chen, T.; Ye, X.; Li, J.; et al. Transcript profiling provides insights into molecular processes during shoot elongation in temperature-sensitive peach (Prunus persica). Sci. Rep. 2020, 10, 7801. [Google Scholar] [CrossRef]
  11. Kotak, S.; Larkindale, J.; Lee, U.; von Koskull-Doring, P.; Vierling, E.; Scharf, K.D. Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 2007, 10, 310–316. [Google Scholar] [CrossRef] [PubMed]
  12. Fragkostefanakis, S.; RÖTh, S.; Schleiff, E.; Scharf, K.-D. Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks. Plant Cell Environ. 2015, 38, 1881–1895. [Google Scholar] [CrossRef] [PubMed]
  13. Yao, F.; Song, C.; Wang, H.; Song, S.; Jiao, J.; Wang, M.; Zheng, X.; Bai, T. Genome-Wide Characterization of the HSP20 Gene Family Identifies Potential Members Involved in Temperature Stress Response in Apple. Front. Genet. 2020, 11, 609184. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, X.; Zheng, Y.; Chen, B.; Zhi, C.; Qiao, L.; Liu, C.; Pan, Y.; Cheng, Z. Genome-wide identification of small heat shock protein (HSP20) homologs in three cucurbit species and the expression profiles of CsHSP20s under several abiotic stresses. Int. J. Biol. Macromol. 2021, 190, 827–836. [Google Scholar] [CrossRef] [PubMed]
  15. Vierling, E. The Roles of Heat Shock Proteins in Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991, 42, 579–620. [Google Scholar] [CrossRef]
  16. Waters, E.R. The evolution, function, structure, and expression of the plant sHSPs. J. Exp. Bot. 2013, 64, 391–403. [Google Scholar] [CrossRef]
  17. Scharf, K.D.; Siddique, M.; Vierling, E. The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins). Cell Stress Chaperones 2001, 6, 225–237. [Google Scholar] [CrossRef]
  18. Sarkar, N.K.; Kim, Y.K.; Grover, A. Rice sHsp genes: Genomic organization and expression profiling under stress and development. BMC Genom. 2009, 10, 393. [Google Scholar] [CrossRef]
  19. Guo, M.; Liu, J.H.; Lu, J.P.; Zhai, Y.F.; Wang, H.; Gong, Z.H.; Wang, S.B.; Lu, M.H. Genome-wide analysis of the CaHsp20 gene family in pepper: Comprehensive sequence and expression profile analysis under heat stress. Front. Plant Sci. 2015, 6, 806. [Google Scholar] [CrossRef] [PubMed]
  20. Yu, J.; Cheng, Y.; Feng, K.; Ruan, M.; Ye, Q.; Wang, R.; Li, Z.; Zhou, G.; Yao, Z.; Yang, Y.; et al. Genome-Wide Identification and Expression Profiling of Tomato Hsp20 Gene Family in Response to Biotic and Abiotic Stresses. Front. Plant Sci. 2016, 7, 1215. [Google Scholar] [CrossRef] [Green Version]
  21. He, Y.; Fan, M.; Sun, Y.; Li, L. Genome-Wide Analysis of Watermelon HSP20s and Their Expression Profiles and Subcellular Locations under Stresses. Int. J. Mol. Sci. 2018, 20, 12. [Google Scholar] [CrossRef] [PubMed]
  22. Ji, X.R.; Yu, Y.H.; Ni, P.Y.; Zhang, G.H.; Guo, D.L. Genome-wide identification of small heat-shock protein (HSP20) gene family in grape and expression profile during berry development. BMC Plant Biol. 2019, 19, 433. [Google Scholar] [CrossRef]
  23. Nagaraju, M.; Reddy, P.S.; Kumar, S.A.; Kumar, A.; Rajasheker, G.; Rao, D.M.; Kavi Kishor, P.B. Genome-wide identification and transcriptional profiling of small heat shock protein gene family under diverse abiotic stress conditions in Sorghum bicolor (L.). Int. J. Biol. Macromol. 2020, 142, 822–834. [Google Scholar] [CrossRef] [PubMed]
  24. Li, J.; Zhang, J.; Jia, H.; Li, Y.; Xu, X.; Wang, L.; Lu, M. The Populus trichocarpa PtHSP17.8 involved in heat and salt stress tolerances. Plant Cell Rep. 2016, 35, 1587–1599. [Google Scholar] [CrossRef] [PubMed]
  25. Guo, L.M.; Li, J.; He, J.; Liu, H.; Zhang, H.M. A class I cytosolic HSP20 of rice enhances heat and salt tolerance in different organisms. Sci. Rep. 2020, 10, 1383. [Google Scholar] [CrossRef] [PubMed]
  26. Li, Y.; Li, Y.; Liu, Y.; Wu, Y.; Xie, Q. The sHSP22 Heat Shock Protein Requires the ABI1 Protein Phosphatase to Modulate Polar Auxin Transport and Downstream Responses. Plant Physiol. 2018, 176, 2406–2425. [Google Scholar] [CrossRef]
  27. Huang, L.J.; Cheng, G.X.; Khan, A.; Wei, A.M.; Yu, Q.H.; Yang, S.B.; Luo, D.X.; Gong, Z.H. CaHSP16.4, a small heat shock protein gene in pepper, is involved in heat and drought tolerance. Protoplasma 2019, 256, 39–51. [Google Scholar] [CrossRef]
  28. Lopes-Caitar, V.S.; de Carvalho, M.C.C.G.; Darben, L.M.; Kuwahara, M.K.; Nepomuceno, A.L.; Dias, W.P.; Abdelnoor, R.V.; Marcelino-Guimarães, F.C. Genome-wide analysis of the Hsp 20 gene family in soybean: Comprehensive sequence, genomic organization and expression profile analysis under abiotic and biotic stresses. BMC Genom. 2013, 14, 577. [Google Scholar] [CrossRef]
  29. Li, Y.; Cao, K.; Li, N.; Zhu, G.; Fang, W.; Chen, C.; Wang, X.; Guo, J.; Wang, Q.; Ding, T.; et al. Genomic analyses provide insights into peach local adaptation and responses to climate change. Genome Res. 2021, 31, 592–606. [Google Scholar] [CrossRef]
  30. Cao, K.; Wang, L.; Zhu, G.; Fang, W.; Chen, C.; Luo, J. Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China. Tree Genet. Genomes 2012, 8, 975–990. [Google Scholar] [CrossRef]
  31. Lu, Z.H.; Niu, L.; Chagne, D.; Cui, G.C.; Pan, L.; Foster, T.; Zhang, R.P.; Zeng, W.F.; Wang, Z.Q. Fine mapping of the temperature-sensitive semi-dwarf (Tssd) locus regulating the internode length in peach (Prunus persica). Mol. Breed. 2016, 36, 20. [Google Scholar] [CrossRef]
  32. Richter, K.; Haslbeck, M.; Buchner, J. The heat shock response: Life on the verge of death. Mol. Cell 2010, 40, 253–266. [Google Scholar] [CrossRef]
  33. Martin, H.; Elizabeth, V. A First Line of Stress Defense: Small Heat Shock Proteins and Their Function in Protein Homeostasis. J. Mol. Biol. 2015, 427, 1537–1548. [Google Scholar]
  34. Yan, H.; Zhang, A.; Chen, J.; He, X.; Xu, B.; Xie, G.; Miao, Z.; Zhang, X.; Huang, L. Genome-Wide Analysis of the PvHsp20 Family in Switchgrass: Motif, Genomic Organization, and Identification of Stress or Developmental-Related Hsp20s. Front. Plant Sci. 2017, 8, 1024. [Google Scholar] [CrossRef]
  35. Zhao, P.; Wang, D.; Wang, R.; Kong, N.; Zhang, C.; Yang, C.; Wu, W.; Ma, H.; Chen, Q. Genome-wide analysis of the potato Hsp20 gene family: Identification, genomic organization and expression profiles in response to heat stress. BMC Genom. 2018, 19, 61. [Google Scholar] [CrossRef]
  36. Tan, B.; Lian, X.; Cheng, J.; Zeng, W.; Zheng, X.; Wang, W.; Ye, X.; Li, J.; Li, Z.; Zhang, L.; et al. Genome-wide identification and transcriptome profiling reveal that E3 ubiquitin ligase genes relevant to ethylene, auxin and abscisic acid are differentially expressed in the fruits of melting flesh and stony hard peach varieties. BMC Genom. 2019, 20, 892. [Google Scholar] [CrossRef]
  37. Wang, G.M.; Yin, H.; Qiao, X.; Tan, X.; Gu, C.; Wang, B.H.; Cheng, R.; Wang, Y.Z.; Zhang, S.L. F-box genes: Genome-wide expansion, evolution and their contribution to pollen growth in pear (Pyrus bretschneideri). Plant Sci. Int. J. Exp. Plant Biol. 2016, 253, 164–175. [Google Scholar] [CrossRef]
  38. Tan, B.; Yan, L.; Li, H.; Lian, X.; Cheng, J.; Wang, W.; Zheng, X.; Wang, X.; Li, J.; Ye, X.; et al. Genome-wide identification of HSF family in peach and functional analysis of PpHSF5 involvement in root and aerial organ development. PeerJ 2021, 9, e10961. [Google Scholar] [CrossRef]
  39. International Peach Genome, I.; Verde, I.; Abbott, A.G.; Scalabrin, S.; Jung, S.; Shu, S.; Marroni, F.; Zhebentyayeva, T.; Dettori, M.T.; Grimwood, J.; et al. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat. Genet. 2013, 45, 487–494. [Google Scholar]
  40. Siddique, M.; Gernhard, S.; von Koskull-Doring, P.; Vierling, E.; Scharf, K.D. The plant sHSP superfamily: Five new members in Arabidopsis thaliana with unexpected properties. Cell Stress Chaperones 2008, 13, 183–197. [Google Scholar] [CrossRef]
  41. Kumar, S.V.; Wigge, P.A. H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 2010, 140, 136–147. [Google Scholar] [CrossRef] [PubMed]
  42. Wang, R.; Zhang, Y.; Kieffer, M.; Yu, H.; Kepinski, S.; Estelle, M. HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1. Nat. Commun. 2016, 7, 10269. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Watanabe, E.; Mano, S.; Nomoto, M.; Tada, Y.; Hara-Nishimura, I.; Nishimura, M.; Yamada, K. HSP90 Stabilizes Auxin-Responsive Phenotypes by Masking a Mutation in the Auxin Receptor TIR1. Plant Cell Physiol. 2016, 57, 2245–2254. [Google Scholar] [CrossRef] [PubMed]
  44. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant. 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  45. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef]
  46. Chung, M.H.; Chen, M.K.; Pan, S.M. Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res. 2000, 9, 471–476. [Google Scholar] [CrossRef]
Ijms 23 10849 g001 550
Figure 1. Genomic distribution and duplication of the PpHSP20 genes across the eight chromosomes of peach. Forty-two PpHSP20 genes were mapped to the eight chromosomes. Syntenic pairs are linked with lines, with colors representing different pairs.
Figure 1. Genomic distribution and duplication of the PpHSP20 genes across the eight chromosomes of peach. Forty-two PpHSP20 genes were mapped to the eight chromosomes. Syntenic pairs are linked with lines, with colors representing different pairs.
Ijms 23 10849 g001
Ijms 23 10849 g002 550
Figure 2. Phylogenetic tree of the PpHSP20 genes (A), gene structures (B), and conserved motifs of the PpHSP20s (C).
Figure 2. Phylogenetic tree of the PpHSP20 genes (A), gene structures (B), and conserved motifs of the PpHSP20s (C).
Ijms 23 10849 g002
Ijms 23 10849 g003 550
Figure 3. Phylogenetic tree of HSP20s from Prunus persica (red star), Oryza sativa (yellow star), Glycine max (green star), and Arabidopsis thaliana (blue star) constructed by the neighbor-joining method.
Figure 3. Phylogenetic tree of HSP20s from Prunus persica (red star), Oryza sativa (yellow star), Glycine max (green star), and Arabidopsis thaliana (blue star) constructed by the neighbor-joining method.
Ijms 23 10849 g003
Ijms 23 10849 g004 550
Figure 4. Analysis of the cis-elements in the PpHSP20s promoters. (A) The different types of cis-elements are shown in the promoter region of each PpHSP20 in different colors. (B) The number of each cis-acting element in the promoter regions of each PpHSP20. Coloring represents the number of elements (small: white; large: red), gray indicate zero.
Figure 4. Analysis of the cis-elements in the PpHSP20s promoters. (A) The different types of cis-elements are shown in the promoter region of each PpHSP20 in different colors. (B) The number of each cis-acting element in the promoter regions of each PpHSP20. Coloring represents the number of elements (small: white; large: red), gray indicate zero.
Ijms 23 10849 g004
Ijms 23 10849 g005 550
Figure 5. Expression levels of PpHSP20s during shoot elongation in ‘Zhongyoutao 14’ and correlation with the average terminal internode length (TIL). (A) Transcriptome data was used to measure the expression level of the PpHSP20s. The growth stages were the IP (initial period), IEP (initial elongation period), RGP (rapid growth period), and SGP (stable growth period), corresponding to four key growth stages during temperature-sensitive peach shoot development. Four groups of transcriptional patterns were classified. ‘Group III’ is highlighted with red, showing a negative trend between the internode length and the expression levels of PpHSP20s in Group III. (B) Correlation between the TIL and the expression level of genes in Group III. Black dotted line represents linear trend. Grey dots represent the internode length and the FPKM value. The average length was calculated by measuring six terminal internodes at each stage, n = 6. FPKM, fragments per kilobase of transcript per million mapped reads.
Figure 5. Expression levels of PpHSP20s during shoot elongation in ‘Zhongyoutao 14’ and correlation with the average terminal internode length (TIL). (A) Transcriptome data was used to measure the expression level of the PpHSP20s. The growth stages were the IP (initial period), IEP (initial elongation period), RGP (rapid growth period), and SGP (stable growth period), corresponding to four key growth stages during temperature-sensitive peach shoot development. Four groups of transcriptional patterns were classified. ‘Group III’ is highlighted with red, showing a negative trend between the internode length and the expression levels of PpHSP20s in Group III. (B) Correlation between the TIL and the expression level of genes in Group III. Black dotted line represents linear trend. Grey dots represent the internode length and the FPKM value. The average length was calculated by measuring six terminal internodes at each stage, n = 6. FPKM, fragments per kilobase of transcript per million mapped reads.
Ijms 23 10849 g005
Ijms 23 10849 g006 550
Figure 6. Phenotypic analysis of transgenic Arabidopsis overexpressing PpHSP20-32. (A) Phenotypes of T2 transgenic plants from three lines over-expressing PpHSP20-32 after cultivation for two weeks. The length (B) and width (C) of the rosette leaves after cultivation for two weeks. (D) Phenotypes of T2 transgenic plants after cultivation for four weeks. The plant height (E), internode length (F), number of nodes (G), and branches (H) after cultivation for four weeks. Different letters indicate significant differences within treatments by ANOVA (p < 0.05).
Figure 6. Phenotypic analysis of transgenic Arabidopsis overexpressing PpHSP20-32. (A) Phenotypes of T2 transgenic plants from three lines over-expressing PpHSP20-32 after cultivation for two weeks. The length (B) and width (C) of the rosette leaves after cultivation for two weeks. (D) Phenotypes of T2 transgenic plants after cultivation for four weeks. The plant height (E), internode length (F), number of nodes (G), and branches (H) after cultivation for four weeks. Different letters indicate significant differences within treatments by ANOVA (p < 0.05).
Ijms 23 10849 g006
Ijms 23 10849 g007 550
Figure 7. Thermotolerance of the PpHSP20-32-OE lines. (A) Seeds of wild type (WT) and the PpHSP20-32-OE lines (L1, L2, and L3) were treated at 46 °C for 30 min. Photographs were taken before HS treatment. (B–E) Photographs were taken after 48 h, 60 h, 80 h, and 96 h at 25 °C. (F) Germination rates among wild-type and PpHSP20-32-OE lines transgenic plants after HS treatment. The number of germinated plants was counted at different times after HS treatment. For three replications, more than 50 seedlings were used for each line (t-test significant at p < 0.05).
Figure 7. Thermotolerance of the PpHSP20-32-OE lines. (A) Seeds of wild type (WT) and the PpHSP20-32-OE lines (L1, L2, and L3) were treated at 46 °C for 30 min. Photographs were taken before HS treatment. (B–E) Photographs were taken after 48 h, 60 h, 80 h, and 96 h at 25 °C. (F) Germination rates among wild-type and PpHSP20-32-OE lines transgenic plants after HS treatment. The number of germinated plants was counted at different times after HS treatment. For three replications, more than 50 seedlings were used for each line (t-test significant at p < 0.05).
Ijms 23 10849 g007
Table
Table 1. Predicted protein characteristics of the PpHSP family members.
Table 1. Predicted protein characteristics of the PpHSP family members.
Gene IDGene NameAmino Acid NumberMolecular Weight/DaIsoelectric PointInstability IndexAliphatic IndexAverage of
Hydropathicity
Prupe.1G237800PpHSP20-123726,812.579.1353.7157.59−0.802
Prupe.1G407100PpHSP20-216218,223.846.6453.285.99−0.54
Prupe.1G407200PpHSP20-321422,898.395.7845.8565.09−0.86
Prupe.1G407300PpHSP20-415617,514.175.5839.3880.51−0.439
Prupe.1G586200PpHSP20-520222,714.259.1234.6790.4−0.356
Prupe.2G065600PpHSP20-643847,399.439.1841.966.28−0.828
Prupe.2G243400PpHSP20-716518,345.76.2148.8876.12−0.621
Prupe.2G243800PpHSP20-815417,381.685.8155.4581.04−0.671
Prupe.2G317700PpHSP20-922324,801.187.7547.9585.61−0.533
Prupe.3G017400PpHSP20-1014316,084.376.8442.0185.17−0.45
Prupe.3G034800PpHSP20-1116117,783.216.1642.9582.92−0.463
Prupe.3G085200PpHSP20-1223426,468.866.8755.0386.2−0.672
Prupe.3G108500PpHSP20-1324527,393.149.2658.6875.27−0.469
Prupe.4G023100PpHSP20-1413315,541.75.5249.1889.4−0.353
Prupe.4G125800PpHSP20-1516018,417.877.1337.873.06−0.832
Prupe.4G201100PpHSP20-1621925,002.236.9150.1873.42−0.624
Prupe.5G004500PpHSP20-1739944,140.856.3531.6175.21−0.619
Prupe.5G006000PpHSP20-1813814,813.278.5441.03100.140.082
Prupe.5G071200PpHSP20-1920923,640.615.4670.1174.07−0.626
Prupe.5G186700PpHSP20-2032737,390.035.1355.8581.07−0.488
Prupe.5G186800PpHSP20-2123325,847.965.6144.5678.58−0.609
Prupe.5G242900PpHSP20-2221423,777.019.2628.1175.09−0.575
Prupe.6G008800PpHSP20-2314316,155.264.6967.6283.85−0.298
Prupe.6G065900PpHSP20-2415818,076.557.9150.5874.62−0.666
Prupe.6G066100PpHSP20-2515818,105.55.8349.7273.99−0.683
Prupe.6G066200PpHSP20-2615817,961.496.7648.2180.13−0.555
Prupe.6G066300PpHSP20-2716118,427.825.5749.5973.23−0.671
Prupe.6G066400PpHSP20-2815818,222.616.3451.4171.52−0.697
Prupe.6G066500PpHSP20-2915818,109.535.5755.9877.09−0.613
Prupe.6G066600PpHSP20-30879408.799.157.8374.02−0.299
Prupe.6G151500PpHSP20-3133136,970.149.3243.3763.08−0.871
Prupe.6G266500PpHSP20-3215317,931.36.4549.8965.56−0.746
Prupe.6G328400PpHSP20-3320422,150.25.129.9779.75−0.317
Prupe.7G008300PpHSP20-3417419,246.369.3339.0386.32−0.243
Prupe.7G008400PpHSP20-3516018,391.299.2843.5685.81−0.296
Prupe.7G008500PpHSP20-3614716,918.689.4432.9588.78−0.276
Prupe.7G053900PpHSP20-3742848,041.145.8843.9377.15−0.42
Prupe.7G187900PpHSP20-3857262,944.24.9348.9860.28−0.957
Prupe.8G000400PpHSP20-3920022,775.125.9146.9697.4−0.442
Prupe.8G031300PpHSP20-4048554,400.115.3650.4572.76−0.911
Prupe.8G046200PpHSP20-4116318,241.315.6248.2167.48−0.799
Prupe.8G182600PpHSP20-42919100,089.874.5943.8274.11−0.633
Table
Table 2. The secondary structure analysis and subcellular localization prediction of PpHSP proteins.
Table 2. The secondary structure analysis and subcellular localization prediction of PpHSP proteins.
Protein IDAlpha HelixExtended StrandBeta TurnRandom Coil
PpHSP20-151346146
PpHSP20-256341260
PpHSP20-3603517102
PpHSP20-449361061
PpHSP20-578291283
PpHSP20-6154368240
PpHSP20-73429993
PpHSP20-83232783
PpHSP20-9324610135
PpHSP20-1032311070
PpHSP20-115033969
PpHSP20-12843313104
PpHSP20-1351388148
PpHSP20-142831767
PpHSP20-153338683
PpHSP20-1662461893
PpHSP20-171034818230
PpHSP20-181444872
PpHSP20-19493011119
PpHSP20-20995328147
PpHSP20-21573614126
PpHSP20-2256491297
PpHSP20-2315381080
PpHSP20-2424241091
PpHSP20-252634890
PpHSP20-262931890
PpHSP20-272828897
PpHSP20-282927795
PpHSP20-293030989
PpHSP20-301314357
PpHSP20-31143208160
PpHSP20-3231351077
PpHSP20-33215313117
PpHSP20-3460303030
PpHSP20-3548341266
PpHSP20-3656291250
PpHSP20-37858427232
PpHSP20-381446723338
PpHSP20-397631984
PpHSP20-402026130192
PpHSP20-414229884
PpHSP20-4231213051426
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Lian, X.; Wang, Q.; Li, T.; Gao, H.; Li, H.; Zheng, X.; Wang, X.; Zhang, H.; Cheng, J.; Wang, W.; et al. Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance. Int. J. Mol. Sci. 2022, 23, 10849. https://doi.org/10.3390/ijms231810849

AMA Style

Lian X, Wang Q, Li T, Gao H, Li H, Zheng X, Wang X, Zhang H, Cheng J, Wang W, et al. Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance. International Journal of Molecular Sciences. 2022; 23(18):10849. https://doi.org/10.3390/ijms231810849

Chicago/Turabian Style

Lian, Xiaodong, Qiuping Wang, Tianhao Li, Hongzhu Gao, Huannan Li, Xianbo Zheng, Xiaobei Wang, Haipeng Zhang, Jun Cheng, Wei Wang, and et al. 2022. "Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance" International Journal of Molecular Sciences 23, no. 18: 10849. https://doi.org/10.3390/ijms231810849

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop