Genome-Wide Identiﬁcation and Characterization of Heat Shock Protein 20 Genes in Maize

: Maize is an important cereal crop worldwide and is sensitive to abiotic stresses in ﬂuctuant environments that seriously affect its growth, yield, and quality. The small heat shock protein ( HSP20 ) plays a crucial role in protecting plants from abiotic stress. However, little is known about HSP20 in maize ( ZmHSP20 ). In this study, 44 ZmHSP20s were identiﬁed, which were unequally distributed over 10 chromosomes, and 6 pairs of ZmHSP20s were tandemly presented. The gene structure of ZmHSP20s was highly conserved, with 95% (42) of the genes having no more than one intron. The analysis of the cis-element in ZmHSP20s promoter demonstrated large amounts of elements related to hormonal and abiotic stress responses, including abscisic acid (ABA), high temperature, and hypoxia. The ZmHSP20s protein had more than two conserved motifs that were predictably localized in the cytoplasm, nucleus, endoplasmic reticulum, peroxisome, mitochondria, and plasma. Phylogenetic analysis using HSP20s in Arabidopsis , rice, maize, and Solanum tuberosum indicated that ZmHSP20s were classiﬁed into 11 categories, of which each category had unique subcellular localization. Approximately 80% (35) of ZmHSP20 were upregulated under heat stress at the maize seedling stage, whereas the opposite expression proﬁling of 10 genes under 37 and 48 ◦ C was detected. A total of 20 genes were randomly selected to investigate their expression under treatments of ABA, gibberellin (GA), ethylene, low temperature, drought, and waterlogging, and the results displayed that more than half of these genes were downregulated while ZmHSP20-3 , ZmHSP20-7 , ZmHSP20-24 , and ZmHSP20-44 were upregulated under 1 h treatment of ethylene. A yeast-one-hybrid experiment was conducted to analyze the binding of four heat stress transcription factors (ZmHSFs) with eight of the ZmHSP20s promoter sequences, in which ZmHSF3, ZmHSF13, and ZmHSF17 can bind to most of these selected ZmHSP20s promoters. Our results provided a valuable resource for studying HSP20s function and offering candidates for genetic improvement under abiotic stress.


Introduction
In the changing environment, numerous adverse stress conditions such as drought, salinity, heat, cold, and chemicals, nematodes, insects, and rodents were imposed on plants, which significantly influence their growth and development [1]. These abiotic stresses can cause damage to plant cells and cause secondary damage, such as osmotic and oxidative stress [2,3]. Plants have a series of elaborate mechanisms in response to environmental changes compared to animals, including maintaining cell membrane stability [4], capturing reactive oxygen species (ROS), synthesizing antioxidants, osmotic accumulation, and osmotic regulation, inducing some enzymes in response to stress, and enhancing the transcription and signaling of partners [5], to adapt morphologically and physiologically [6]. Abiotic stresses in plants are often interrelated and lead to physiological, morphological, Life 2022, 12, 1397 3 of 16 humidity. As previously described, the treatments were imposed on seedlings at the second leaf stage [32]. For the high-temperature treatment, the seedlings were transferred to an artificial climate chamber at 37, 42, and 48 • C, and the leaves were collected after 4 h of stress. The seedling leaves under 10 • C, drought stress after 1, 2, and 4 h treatments, and waterlogged roots were also collected. For hormone treatments, 100 µM of ethylene (ET), 100 mM of ABA, and 100 mM of gibberellin (GA) were applied to treat the seedlings, and the leaves after 1, 2, and 4 h treatments were sampled. The leaves and roots of seedlings growing under 25 • C conditions were collected as the control. For each sample, more than six seedlings were mixed and immediately frozen at −80 • C.

RNA Extraction and Quantitative Reverse Transcription PCR (qRT-PCR)
Total RNA was isolated using TRIZOL reagent (Invitrogen, Gaithersburg, MD, USA) and treated with RNase-free DNase (Invitrogen). Purified RNA was used to synthesize single-stranded cDNA using recombinant M-MLV reverse transcriptase (Invitrogen). Quantitative reverse transcription PCR (qRT-PCR) was performed using gene-specific primers (Table S1) and a 2 × iTaq TM Universal SYBR Green Super Mix (Bio-Rad, Hercules, CA, USA). ZmActin1 (GRMZM2G126010) was used as an internal control for the normalization of expression data. Relative expression levels were calculated using the 2 −∆∆CT (cycle threshold) method [33]. PCR involved an initial denaturation step at 95 • C for 5 min, followed by 40 cycles of 15 s at 95 • C, 10 s at 58 • C, and 20 s at 72 • C. The primers used for qRT-PCR were designed using online software Primer3Plus (https://www.primer3plus.com/ (accessed on 1 June 2022)).

Identification of ZmHSP20s
Two approaches were applied to identify the ZmHSP20s family genes in maize. The conserved ZmHSP20 domain (PF00011) from the Pfam database [34] was used to query the maize B73 proteome (RefGen_v4) [35] with the ZmHSP20 HMM using the HMMER3.0 package [36], and the ZmHSP20 proteins were collected based on the E-value < 1 × 10 −5 . Moreover, the protein sequences of HSP20 family members in Arabidopsis and rice [37] were downloaded from TAIR [38] and the MSU Rice Genome Annotation Project [39] databases, respectively. These protein sequences from these Arabidopsis and rice HSP20 were used as queries to search against the maize proteome with an E-value < 1 × 10 −5 based on a local BLASTP program with the default parameters. The proteins from these two approaches were collected and redundant sequences were manually eliminated. The Pfam [34] and SMART [40] databases were utilized to confirm the conserved domain in the identified proteins. The molecular weight (MW) and isoelectric point (pI) of ZmHSP20s were computed with the online ExPASy tool [41]. Four online tools (Predotar [42], WOLF PSORT (https://www.genscript.com/wolf-psort.html (accessed on 6 June 2022)), TargetP [43], and CELLO [44]) were used to predict the subcellular localization. Some subcellular localizations of ZmHSP20s that cannot be predicted using software will be predicted by affinities with other species.
2.4. Analysis of Gene Structure, Chromosome Distribution, Duplication, Collinearity, and Conserved Motif DNA, coding sequences (CDSs), and protein sequences of ZmHSP20 family genes and their corresponding physical location in the maize B73 reference genome (RefGen_v4) were downloaded from the MaizeGDB database. The gene structures were drawn and displayed by Gene Structure Display Server (GSDS) [45] using DNA and CDS sequences of each gene. The online program of Multiple Em for Motif Elicitation (MEME, V5.0.3, https://meme-suite.org/meme/doc/meme.html, accessed on 6 June 2022) was applied to predict the potential motifs with default parameters. The MG2C (MapGene2Chromosome V2, http://mg2c.iask.in/mg2c_v2.0/, accessed on 8 June 2022) software was used to display the physical location of each gene in its corresponding position. According to the manual, the ZmHSP20s gene collinearity analysis within the maize genome was conducted using MCScanX software with default parameters [46].

Predicting the Cis-Regulatory Elements
The 1.5 kb sequences of the promoter of ZmHSP20 genes were obtained from the EnsemblPlants database and were then uploaded to the website of PlantCare [53] to predict the cis-regulatory DNA elements. The elements related to stress response and hormones were selected and displayed through Tbtools [54].

Prediction of the Interaction between ZmHSP20s and ZmHSFs
Protein sequences for HSF family members in maize (ZmHSFs) [55] were downloaded from maizeGDB [56], which were uploaded onto STRING database [57] to predict the interaction with ZmHSP20s. The interaction networks were drawn through Cytoscape_v3.9.1 [58]. The promoter sequences of ZmHSP20s were uploaded onto PlantRegMap [59] to predict the binding of ZmHSFs.

Yeast One-and Two-Hybrid Assays
A full-length CDS of ZmHSFs was cloned into vector pGADT7-Rec2 and the 1.5 kb promoter sequence of ZmHSP20s was cloned into vector pHIS2 using a CloneExpressII One Step Cloning Kit (Vazyme, Nanjing, China) with the corresponding primers (Table S1). Recombinant vectors were co-transfected into yeast competent AH109. Transformants were cultured on SD/-Leu-Trp and were then placed on SD/-Leu-Trp-His with a special concentration of 3-amino-1,2,4-triazole (3-AT). For the yeast-two-hybrid experiment, the full-length CDS of ZmHSFs was cloned into vector pGADT7 while the full-length CDS of ZmHSP20s was cloned into vector pGBKT7, and the transformants were screened on SD/-Leu-Trp-His-Ade.
22, ZmHSP20-28, and ZmHSP20-42. ABRE presented in 36 genes, of which ZmHSP20-1 contained 10, and ZmHSP20-10 and ZmHSP20-21 contained 8, respectively. In particula GC-motif appeared 6 times in ZmHSP20-33 but no more 2 in the other genes. Moreove 23 of ZmHSP20s contained the MBS element, 10 of ZmHSP20s contained the TGA elemen and 9 of ZmHSP20 contained the TATC_box. These results indicated that ZmHSP20s we involved in multiple hormonal and abiotic responses.   Cis-elements related to ho mone responsiveness are represented as cylindrical and cis-elements related to abiotic stress respo siveness are represented as a wedge. ABRE was the response to ABA; ARE and GC-motif were th response to anaerobic conditions; CGTCA-motif was the response to MeJA; DRE and MBS were th response to drought; G-box was light response, GARE-motif and TATC-box were the response GA; LTR was the response to low temperature; TC-rich repeat was the response to defense an stress; and TGA-element was the response to auxin.

Conserved Function of ZmHSP20s
The conserved motifs in ZmHSP20s proteins were analyzed using MEME (Figure 4 A total of five motifs were identified, of which Motif 1 was detected in all ZmHSP2 proteins, and more than two motifs in one protein were identified ( Figure 4A). Motif Motif 3, and Motif 4 were distributed on most of the proteins while Motif 5 was only foun on 8 members, including ZmHSP20-02, ZmHSP20-03, ZmHSP20-04, ZmHSP20-1 Figure 3. Characters of cis-elements in promoter regions of ZmHSP20s. Cis-elements related to hormone responsiveness are represented as cylindrical and cis-elements related to abiotic stress responsiveness are represented as a wedge. ABRE was the response to ABA; ARE and GC-motif were the response to anaerobic conditions; CGTCA-motif was the response to MeJA; DRE and MBS were the response to drought; G-box was light response, GARE-motif and TATC-box were the response to GA; LTR was the response to low temperature; TC-rich repeat was the response to defense and stress; and TGA-element was the response to auxin.

Conserved Function of ZmHSP20s
The conserved motifs in ZmHSP20s proteins were analyzed using MEME (Figure 4). A total of five motifs were identified, of which Motif 1 was detected in all ZmHSP20s proteins, and more than two motifs in one protein were identified ( Figure 4A). Motif 1, Motif 3, and Motif 4 were distributed on most of the proteins while Motif 5 was only found on 8 members, including ZmHSP20-02, ZmHSP20-03, ZmHSP20-04, ZmHSP20-16, ZmHSP20-17, ZmHSP20-18, ZmHSP20-34, and ZmHSP20-43. These ZmHSP20s were divided into two subgroups based on whether they contained the Motif 5 at the N-terminal. Interestingly, members in group 1 (containing Motif 5) were localized to the cytoplasm and had no intron except ZmHSP20-17 had 1 intron. In particular, ZmHSP20-17 in group 1 lacked Motif 3 compared with other members. The length of these conserved motifs varied from 15 to 29 amino acids ( Figure 4B). The GO enrichment analysis of 44 ZmHSP20 genes was conducted, of which 35 genes were enriched ( Figure S1). The significant GO terms mainly included the response to hydrogen peroxide, response to hydrogen peroxide, response to reactive oxygen species, response to heat, response to osmotic stress, response to stimulus, and protein oligomerization, indicating the important roles in abiotic stress. varied from 15 to 29 amino acids ( Figure 4B). The GO enrichment analysis of 44 ZmHSP20 genes was conducted, of which 35 genes were enriched ( Figure S1). The significant GO terms mainly included the response to hydrogen peroxide, response to hydrogen peroxide, response to reactive oxygen species, response to heat, response to osmotic stress, response to stimulus, and protein oligomerization, indicating the important roles in abiotic stress. To explore the evolutionary relationship of HSP20s in plants, 44 of ZmHSP20s, 18 of AtHSP20s, 18 of OsHSP20s, and 35 of StHSP20s were subjected to construction of a phylogenetic tree, which was divided into 11 categories according to a previous classification [47,60,61] (Figure 5). These proteins were predicted to localize in 6 organelles, including the cytoplasm and nucleus (C), endoplasmic reticulum (ER), peroxisome (Po), mitochondria (M), and plasma (P). The proteins in the categories of CI, CII, CIII, CV, CVI, and CVII were mainly localized in the cytoplasm and nucleus, proteins in the category of MI and MII were mainly localized in the mitochondria, while proteins in the category of ER, Po, and P were mainly localized in the endoplasmic reticulum, peroxisome, and plasma, respectively. The CI category had the largest number of members, and most of the members in category CII belonged to maize and rice. The category of CV and Po had only four members, with one member of each species. The P category had only three ZmHSP20s (ZmHSP20-5, ZmHSP20-6, and ZmHSP20-7), and the CVII category had three members To explore the evolutionary relationship of HSP20s in plants, 44 of ZmHSP20s, 18 of AtHSP20s, 18 of OsHSP20s, and 35 of StHSP20s were subjected to construction of a phylogenetic tree, which was divided into 11 categories according to a previous classification [47,60,61] (Figure 5). These proteins were predicted to localize in 6 organelles, including the cytoplasm and nucleus (C), endoplasmic reticulum (ER), peroxisome (Po), mitochondria (M), and plasma (P). The proteins in the categories of CI, CII, CIII, CV, CVI, and CVII were mainly localized in the cytoplasm and nucleus, proteins in the category of MI and MII were mainly localized in the mitochondria, while proteins in the category Life 2022, 12, 1397 9 of 16 of ER, Po, and P were mainly localized in the endoplasmic reticulum, peroxisome, and plasma, respectively. The CI category had the largest number of members, and most of the members in category CII belonged to maize and rice. The category of CV and Po had only four members, with one member of each species. The P category had only three ZmHSP20s (ZmHSP20-5, ZmHSP20-6, and ZmHSP20-7), and the CVII category had three members (AtHSP14.7, StHSP20-27, and StHSP20-28) from dicotyledonous plants, and the CVI category had four members from maize, Solanum tuberosum, and Arabidopsis. The phylogenetic relationship indicated the conservation and difference in HSP20s in plant evolution.
Life 2022, 12, x FOR PEER REVIEW 9 of Figure 5. Phylogenetic tree of HSP20 proteins of rice (Os), Arabidopsis (At), Solanum tuberosum (S and maize (Zm) using MEGA11 software based on the NJ method. Eleven subfamilies with differe colors were classified and unclassified ZmHSP20s are labeled with grey.

High Temperature Strongly Induced the Expression of ZmHSP20s
To investigate the response of ZmHSP20s to high temperature, qRT-PCR was appli to analyze the expression level of 44 ZmHSP20s under 37, 42, and 48 °C stresses (Figu 6). Of 44 genes, 31 genes were upregulated after heat stress, while 12 genes such ZmHSP20-3, ZmHSP20-16, ZmHSP20-17, ZmHSP20-18, ZmHSP20-34, and ZmHSP20were increasingly induced under three temperature gradients ( Figure 6A). The highe upregulation of ZmHSP20s was under 42 °C stress, which was more than 1000-fold com pared with the normal condition (25 °C). Only 23 genes were upregulated under 48 stress, of which one gene, ZmHSP20-24, was only upregulated (116-fold) at this temper ture point. One gene, ZmHSP20-38, was only upregulated (32-fold) under 37 °C stress. T interaction network of ZmHSP20s showed that only 30 genes interacted with each oth (Figure 6B,C). Except for ZmHSP20-24, these 14 ZmHSP20s that were not in the netwo were not upregulated by heat stress. We further compared the expression level ZmHSP20s under 37 and 48 °C stresses, and nine genes such as ZmHSP20-20, ZmHSP2 24, ZmHSP20-28, and ZmHSP20-36 to ZmHSP20-39 displayed opposite expression prof ing under 37 and 48 °C stresses ( Figure 6B,C). Moreover, a significantly higher expressi level of ZmHSP20s under 37 and 42 °C stresses than under 48 °C stress was detected (Fi

High Temperature Strongly Induced the Expression of ZmHSP20s
To investigate the response of ZmHSP20s to high temperature, qRT-PCR was applied to analyze the expression level of 44 ZmHSP20s under 37, 42, and 48 • C stresses ( Figure 6). Of 44 genes, 31 genes were upregulated after heat stress, while 12 genes such as ZmHSP20-3, ZmHSP20-16, ZmHSP20-17, ZmHSP20-18, ZmHSP20-34, and ZmHSP20-43 were increasingly induced under three temperature gradients ( Figure 6A). The highest upregulation of ZmHSP20s was under 42 • C stress, which was more than 1000-fold compared with the normal condition (25 • C). Only 23 genes were upregulated under 48 • C stress, of which one gene, ZmHSP20-24, was only upregulated (116-fold) at this temperature point. One gene, ZmHSP20-38, was only upregulated (32-fold) under 37 • C stress. The interaction network of ZmHSP20s showed that only 30 genes interacted with each other (Figure 6B,C). Except for ZmHSP20-24, these 14 ZmHSP20s that were not in the network were not upregulated by heat stress. We further compared the expression level of ZmHSP20s under 37 and 48 • C stresses, and nine genes such as ZmHSP20-20, ZmHSP20-24, ZmHSP20-28, and ZmHSP20-36 to ZmHSP20-39 displayed opposite expression profiling under 37 and 48 • C stresses ( Figure 6B,C). Moreover, a significantly higher expression level of ZmHSP20s under 37 and 42 • C stresses than under 48 • C stress was detected ( Figure S2), implying the differential expression of ZmHSP20s under different degrees of heat stress.

Differential Expression of ZmHSP20s under Hormonal Stimuli and Abiotic Stresses
Given that a large number of cis-elements related to hormone and abiotic response occurred in the promoter region of ZmHSP20s, 20 ZmHSP20s were randomly selected to analyze their expression level under three treatments of hormone (ABA, ethylene, and GA) and three abiotic stresses (cold, drought, and waterlogging) (Figure 7). Under the ABA treatment, ZmHSP20-40 was apparently upregulated, while ZmHSP20-3, ZmHSP20-10, and ZmHSP20-30 had minor changes. All four genes (ZmHSP20-3, ZmHSP20-7, ZmHSP20-24, and ZmHSP20-44) were upregulated under 1 h of ethylene treatment, whereas ZmHSP20-3 and ZmHSP20-24 were downregulated under 2 and 4 h of treatment. ZmHSP20-4 had more than 10-fold induction after GA treatment, while ZmHSP20-27 was reduced by GA. Cold stress strongly restricted the expression of four genes (ZmHSP20-12, ZmHSP20-25, ZmHSP20-37, and ZmHSP20- 38), and the expression restriction of

Discussion
Abiotic stress hurts crop development and yield and is a major barrier to meeting food demand worldwide. Plants have different strategies for coping with different types of stress. HSPs were induced in almost all stresses [2], and each member of the HSPs group has a unique roles [62]. HSP20s is a subfamily of HSPs groups, which is also called small HSPs. The expression levels of HSP20s were regulated by heat, salt, and powdery mildew in barley (Hordeum vulgare L.) [63], and the expression of Lilium davidii HSP16.45 in Arabidopsis thaliana enhanced the latter cell activity in heat, salt, and oxidative stress [64], indicating that HSP20s play essential roles in biotic and abiotic stresses. In the present study, a total of 44 ZmHSP20s were identified (Table S2), and four clusters in three chromosomes were detected (Figure 1). The gene structure and amino acid sequence were conserved among 44 members (Figures 2 and 4), and six pairs of genes were collinear, of which these characters were also detected in tomatoes and apples [65,66]. The analysis of the phylogenetic relationship in maize, rice, Arabidopsis, and potato demonstrated that the specific subcellular localization of each category was presented, indicating the specific function of HSP20s in each category. Some evolution-related categories such as P and CVI were also identified, which may play vital roles in maize and dicotyledonous plants, respectively. Moreover, the member of OsHSP20s was not detected in the CVI category, implying the possible association with the aquatic environment.
Gene expression was strongly affected by environmental stimuli, which was regulated through multiple factors such as cis-elements and trans-factors. The protein of transfactors can bind to the cis-elements in the promoter to activate or inhibit the expression of targets. The cis-elements in the promoter of one gene can reflect its potential expression profiling. The HSP20s participate in diverse biotic and abiotic stresses [49], which implied that some cis-elements related to stresses may be located in the promoter of HSP20s. Using the online tool PlantCare [53], the cis-elements in the promoter of 44 ZmHSP20s were identified (Figure 3), and large amounts of elements associated with hormone and abiotic stress were detected in all genes, indicating that ZmHSP20s are also tightly associated with abiotic stresses. To verify these results, qRT-PCR was conducted to analyze the response of hormone and abiotic stresses (Figure 7). All selected genes responded to hormone stimuli (ABA, GA, and ethylene) and abiotic stresses (hypoxia, low temperature, and drought),

Discussion
Abiotic stress hurts crop development and yield and is a major barrier to meeting food demand worldwide. Plants have different strategies for coping with different types of stress. HSPs were induced in almost all stresses [2], and each member of the HSPs group has a unique roles [62]. HSP20s is a subfamily of HSPs groups, which is also called small HSPs. The expression levels of HSP20s were regulated by heat, salt, and powdery mildew in barley (Hordeum vulgare L.) [63], and the expression of Lilium davidii HSP16.45 in Arabidopsis thaliana enhanced the latter cell activity in heat, salt, and oxidative stress [64], indicating that HSP20s play essential roles in biotic and abiotic stresses. In the present study, a total of 44 ZmHSP20s were identified (Table S2), and four clusters in three chromosomes were detected ( Figure 1). The gene structure and amino acid sequence were conserved among 44 members (Figures 2 and 4), and six pairs of genes were collinear, of which these characters were also detected in tomatoes and apples [65,66]. The analysis of the phylogenetic relationship in maize, rice, Arabidopsis, and potato demonstrated that the specific subcellular localization of each category was presented, indicating the specific function of HSP20s in each category. Some evolution-related categories such as P and CVI were also identified, which may play vital roles in maize and dicotyledonous plants, respectively. Moreover, the member of OsHSP20s was not detected in the CVI category, implying the possible association with the aquatic environment.
Gene expression was strongly affected by environmental stimuli, which was regulated through multiple factors such as cis-elements and trans-factors. The protein of trans-factors can bind to the cis-elements in the promoter to activate or inhibit the expression of targets. The cis-elements in the promoter of one gene can reflect its potential expression profiling. The HSP20s participate in diverse biotic and abiotic stresses [49], which implied that some cis-elements related to stresses may be located in the promoter of HSP20s. Using the online tool PlantCare [53], the cis-elements in the promoter of 44 ZmHSP20s were identified (Figure 3), and large amounts of elements associated with hormone and abiotic stress were detected in all genes, indicating that ZmHSP20s are also tightly associated with abiotic stresses. To verify these results, qRT-PCR was conducted to analyze the response of hormone and abiotic stresses (Figure 7). All selected genes responded to hormone stimuli (ABA, GA, and ethylene) and abiotic stresses (hypoxia, low temperature, and drought), of which all four genes increased their expression after 1 h of ethylene, suggesting their possible roles in ethylene-mediated signals. The expression of four ZmHSP20s were restricted under cold stress, similar to previous transcriptome analysis [67]. Under given conditions, some ZmHSP20s were upregulated while some ZmHSP20s were inhibited, demonstrating their differential function in response to stresses.
Heat stress seriously affects growth, development, and yield, which frequently occurs with the increasing global climate. The expression of HSP20s was activated, and yielded proteins can avoid protein degradation [13,18], which usually play roles in molecular chaperone, retaining suitable conformations [19,20]. The GO analysis of 44 ZmHSP20s displayed that these genes are mainly involved in stresses such as high temperature, osmosis, and salt stress. They also involved in protein assembly, folding, and membrane composition ( Figure S1), which were also discovered in rice [68], indicating the conserved characters of HSP20s in plants under heat stress. Transcriptome analysis of maize seedling leaves revealed that ZmHSP20 were obviously upregulated under heat stress [67], and qRT-PCR analysis of ZmHSP20 under 37, 42, and 48 • C stresses showed that approximately 80% of ZmHSP20 were upregulated ( Figure 6), implying the essential roles of ZmHSP20s under heat stress. Moreover, the differential expression profiling of ZmHSP20s under 37 and 48 • C conditions indicated their diverse roles. Specifically, the genes in cluster 3 such as ZmHSP20-16, ZmHSP20-17, and ZmHSP20-20 were significantly upregulated (more than 1000-fold) under heat stress, which would be a potential target for genetic improvement of heat stress. Moreover, the induced expression of ZmHSP20s under heat stress depended on the binding of ZmHSFs proteins in their promoter regions (Figure 8), but not on proteinprotein interaction between ZmHSFs and ZmHSP20s ( Figure S4), suggesting the molecular mechanism of ZmHSP20s in response to heat stress.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12091397/s1, Figure S1. Functional analysis of 44 ZmHSP20s based on Gene Ontology. Figure S2. Boxplots showing the expression levels of ZmHSP20s under heat stress. Expression levels of ZmHSP20s increased under 37 • C, 42 • C, and 48 • C conditions, comparing with 25 • C. Figure S3. Predicted Interaction between ZmHSP20s and ZmHSFs protein using STRING database. Figure S4. The interaction between ZmHSFs and ZmHSP20s based on yeast-two-hybrid experiments. SD, synthetic dropout medium; Leu, leucine; Trp, tryptophan; His, histidine; Ade, adenine. Table S1. The primer using in this study. Table S2. Information of 44 ZmHSP20s, including Gene, Gene ID, position start, position end, chromosome, isoelectric point (PI), molecular weight (MW), and predicted subcellular localization. Table S3. Predicted gene fragments for ZmHSP20 and HSF interaction.
Author Contributions: Conceptualization, methodology, formal analysis, and writing-original draft preparation, H.Q.; formal analysis, software, visualization, and writing-original draft preparation, X.C.; Investigation and validation, S.L., J.G. and Y.K.; formal analysis and software, H.F. and X.Z.; conceptualization, supervision, and funding acquisition, F.Y. and P.Y. All authors have read and agreed to the published version of the manuscript.