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. 2023 Oct 29;16(4):e20407. doi: 10.1002/tpg2.20407

Genome‐wide identification and expression analysis of heat shock protein gene family in cassava

Changyi Wang 1,2, Fangfang Ran 1,2, Yuwei Zang 1,2, Liangwang Liu 1,2, Dayong Wang 2,3,4,✉, Yi Min 1,2,4,✉
PMCID: PMC12807021  PMID: 37899677

Abstract

Heat shock proteins are important molecular chaperones that are involved in plant growth and stress responses. However, members of the Hsp family have been poorly studied in cassava. In this study, 225 MeHsp genes were identified in the cassava genome, and their genetic structures exhibited relatively conserved features within each subfamily. The 225 MeHsp genes showed random chromosomal distribution, and at least 74 pairs of segmentally duplicated MeHsp genes. Eleven tandemly duplicated MeHsp genes were identified. Cis‐element analysis revealed the importance of MeHsps in plant adaptations to the environment. The prediction of protein interactions suggested that MeHsp70‐20 may play a critical regulatory role in the interactive network. Furthermore, the expression profiles of MeHsps in different tissues and cell subsets were analyzed using bulk transcriptomics and single‐cell transcriptomic data. Several subfamily genes exhibited unique expression patterns in the transcriptome and were selected for detailed analysis of the single‐cell transcriptome. Quantitative real‐time polymerase chain reaction (qRT‐PCR) revealed the expression patterns of these genes under temperature stress, further supporting the prediction of cis‐acting elements. This study provides valuable information for understanding the functional characteristics of MeHsp genes and the evolutionary relationships between MeHsps.

Core Ideas

  • A total of 225 Hsp genes were identified from the cassava genome.

  • The expansions of MeHsps were mostly derived from segmental duplications.

  • MeHsps display variable expression patterns across tissues.

  • Specific MeHsp subfamily genes exhibited predominant expression within distinct cell subsets.

  • Several subfamily genes were sensitive to cold and heat stress.

Abbreviations

Clp

caseinolytic protease

Cpn60

chaperonin 60

CARE

Cis‐acting regulatory element

ENA

European Nucleotide Archive

FEC

friable embryogenic callus

GFF3

general feature format version 3

Hsp

heat shock protein

NCBI

National Center for Biotechnology Information

qRT‐PCR

quantitative real‐time polymerase chain reaction

RAM

root apical meristem

1. INTRODUCTION

Cassava (Manihot esculenta Crantz) is an essential source of food and raw industrial materials (S. Zhang, Ping'An, et al., 2015). As a typical tropical crop, cassava is frequently subjected to various biotic and abiotic stressors (An et al., 2012). To acclimate to these environmental stressors, plants synthesize a wide range of proteins, notably the heat shock proteins (Hsps) (Al‐Whaibi, 2011). Hsps play crucial roles in plant responses to environmental stress (W. Wang et al., 2004). Understanding cassava Hsps holds the potential to provide practical approaches for the genetic improvement of cassava.

Heat shock proteins have been extensively characterized in many species, with 153 Hsps characterized in Arabidopsis (Arabidopsis thaliana), 72 Hsps in cucumber (Cucumis sativus), 753 Hsps in wheat (Triticum aestivum), and 283 Hsps in watermelon (Citrillus lanatus) (Altunoglu et al., 2019; X. Chen et al., 2021; A. Kumar et al., 2020; Swindell et al., 2007). Based on their molecular weight and sequence homology, Hsps can be further categorized into six subfamilies, that is, Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small heat shock proteins (Hsp20) (A. Kumar et al., 2020). Each subfamily has distinct biochemical and physiological functions in plant growth and stress responses. Hsp100 regulates the activity of protein complexes, whereas Hsp90 protects critical proteins from stress‐induced damage (Burton & Baker, 2005; G. F. Wang et al., 2011). The complex functions of Hsp70 are mainly reflected in its assistance with protein folding, disaggregation, and transportation. Simultaneously, it can form a prominent group of cellular machinery with Hsp40, thereby regulating nearly all protein‐folding processes (Caplan et al., 1993; Latijnhouwers et al., 2010). Similarly, Hsp60 is involved in protein‐folding processes specific to plastids, including the essential Rubisco enzyme (W. Wang et al., 2004). Hsp20 accumulates and forms oligomers to protect against environmental stress (Sun et al., 2016).

To date, the stress‐related functions of Hsps have been extensively documented; BIL2 (where BIL2 is brassinazole insensitive‐long hypocotyls 2), an AtHsp40 gene localized in the mitochondria, enhances plant resistance to intense light stress (Bekh‐Ochir et al., 2013). The cytoplasmic protein AtHsp70‐3 can enhance thermotolerance by inhibiting PLDδ activity (where PLD is phospholipase D) (P. Song et al., 2020). AtHsp101 and its homologous protein in rice also play crucial roles in thermotolerance (Katiyar‐Agarwal et al., 2003; Queitsch, 2000). AtHsp90.2, a cytoplasmic‐localized protein, interacts with RPM1 (where RPM1 is resistance to Pseudomonas syringae pv. maculicola 1) to modulate its stability and function and enhances plant disease tolerance (Hubert et al., 2003). Similarly, AtHsp90.1 assists in plant defense against infection by interacting with RAR1 (where RAR1 is required for Mildew Locus A12 resistance 1) and SGT1 (where SGT1 is suppressor of the G2 allele of S‐phase kinase‐associated protein 1) (Takahashi et al., 2003).

The modulation of Hsp expression has been demonstrated to enhance plant stress tolerance, rendering these genes promising targets for genetic engineering and crop improvement (Askari‐Khorasgani & Pessarakli, 2019). For example, the overexpression of AtHsp17.6B (A. thaliana) and SlDnaJ20 (Solanum lycopersicum) confers thermotolerance to transgenic plants (Tawab et al., 2019; G. Wang et al., 2019). Likewise, the overexpression of TaHsp23.9 (T. aestivum) enhances the tolerance of transgenic crops to extreme environments, whereas the overexpression of OsHsp16.9 (Oryza sativa) augments resistance to both salt and drought stress in transgenic rice (Jung et al., 2014; J. Wang et al., 2020).

Core Ideas

  • A total of 225 Hsp genes were identified from the cassava genome.

  • The expansions of MeHsps were mostly derived from segmental duplications.

  • MeHsps display variable expression patterns across tissues.

  • Specific MeHsp subfamily genes exhibited predominant expression within distinct cell subsets.

  • Several subfamily genes were sensitive to cold and heat stress.

Hsps are also involved in the regulation of chloroplast development. For instance, AtHsp21 has been identified as a necessary factor in chloroplast development (Zhong et al., 2013). In addition, three chloroplast‐targeted DnaJ proteins, AtJ8, AtJ11, and AtJ20, contribute to the stabilization of photosystem II supercomplexes (K. M. Chen et al., 2010). Hsp60 proteins with higher molecular weight, such as Cpn60α1 and Cpn60β1 (where Cpn60 is chaperonin 60), function as plastid chaperonin proteins and assist in the formation of the FtsZ ring (where FtsZ is a filamenting temperature‐sensitive mutant) (Suzuki et al., 2009). Furthermore, Cpn60β4 is involved in nicotinamide adenine dinucleotide folding, thereby influencing chloroplast development (Lalit et al., 2019; Peng et al., 2011). Hsp70 proteins localized in the cytoplasm are crucial for chloroplast development (Latijnhouwers et al., 2010). AtHsp90.5, a chloroplast‐targeted protein, interacts with the vesicle‐inducing protein in plastids 1 (VIPP1), contributes to the formation of thylakoid membranes, and plays an essential role in chloroplast biogenesis (Feng et al., 2014). Two chloroplast‐located Hsp100s, AtClpC2 and AtClpD (where Clp is caseinolytic protease), act as critical chaperones that regulate protein transport into the chloroplasts (Rosano et al., 2011). In addition to their involvement in chloroplast development, studies have highlighted the significance of AtHsp90‐1 and AtHsp90‐3 in pollen grain development (Prasinos et al., 2005). AtHsp90.2 is known to respond to jasmonates‐coronatine signaling and regulate the circadian rhythm in Arabidopsis (T. S. Kim et al., 2011; X. C. Zhang, Millet, et al., 2015). Moreover, Hsp90.9 in cassava can antagonistically regulate endogenous salicylate and auxin (Wei et al., 2021).

Members of the Hsp family have been poorly studied in cassava. Therefore, in this study, we performed a comprehensive analysis of Hsp genes in cassava, including their characteristics, chromosomal location, phylogenetic relationships, gene duplication, expression profiles in various tissues, and expression profiles in various cell subsets. Consequently, the current study establishes a foundation for future investigations of Hsp genes in cassava.

2. MATERIALS AND METHODS

2.1. Identification of the Hsp genes in cassava

The cassava genome sequence and annotation files (general feature format version 3 [GFF3], gene transfer format) were obtained from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). A full‐length representative protein sequence was obtained based on genome sequence and annotation information. The hidden Markov models for Hsp20 (PF00011), Hsp40 (PF00226), Hsp60 (PF00118), Hsp70 (PF00012), Hsp90 (PF00183), and Hsp100 (PF02861) were downloaded from the Pfam database (http://pfam.xfam.org/;Pfam35.0). The HMMer3.0 software was used to search for Hsp candidates in full‐length protein sequences based on hidden Markov models. The protein sequences with an E‐value lower than 10−2 were selected. Concurrently, BLASTp was performed with an E‐value <10−2 using the Hsp protein sequences from Arabidopsis as query sequences (X. Chen et al., 2021; Ding et al., 2017). These results were merged, and their amino acid sequences were submitted to the CDD (https://www.ncbi.nlm.nih.gov/cdd) and Pfam databases (manual verification of the complete Hsp domain). Predicted protein sequences lacking the complete Hsp domain and sHsp candidate sequences with molecular weights outside the range of 12–40 kDa were excluded (W. Wang et al., 2004). High‐quality and high‐confidence proteins (E‐value < 10−5) were assigned as cassava Hsps (MeHsps). Physicochemical properties and subcellular location predictions of MeHsps were obtained using ExPasy (http://web.expasy.org/protparam/) and WoLFPSORT (https://wolfpsort.hgc.jp/).

2.2. Phylogenetic analysis of Hsp gene families

HMMER and BLASTp were also used to identify Hsp families in the genomes of rice (O. sativa Japonica Group), yam (Dioscorea rotundata), Populus trichocarpa, and rubber tree (Hevea brasiliensis) (https://www.ncbi.nlm.nih.gov/assembly/). Protein sequences were aligned using ClustalW (Mcwilliam et al., 2013). Several protein sequences were selected to construct a maximum likelihood phylogenetic tree using MEGA7.0 (S. Kumar et al., 2016), with the following parameters: bootstrap method: 1000, passion model, partial deletion, site coverage cutoff (%): 50, ML heuristic method: nearest‐neighbor‐interchange, branch swap filter, and moderate. A phylogenetic tree of 225 MeHsp family proteins was constructed using these parameters. All phylogenetic trees were saved in the NWK format and visualized using ITOL (https://itol.embl.de/).

2.3. Conserved motif, structural characterization, and chromosome localization

The conserved motifs were analyzed using the Multi Em for Motif Elicitation online program (https://meme‐suite.org/meme/) with the following parameters: classic mode; any number of repetitions; 10 motifs. The identified motifs were annotated using the InterPro dataset (http://www.ebi.ac.uk/interpro/search/sequence/). The exon/intron and localization information of MeHsps were extracted from the GFF3 file. The results were visualized using TBtools (C. Chen et al., 2020).

2.4. Synteny analysis and Ka/Ks analysis

Syntenic relationships were analyzed and visualized by TBtools using One‐step‐MCScanX and Advanced Circos, respectively (C. Chen et al., 2020; Y. Wang et al., 2012). Non‐synonymous (Ka) and synonymous (Ks) substitutions of each duplicated MeHsps were calculated using KAKS_Calculator3.0 (https://ngdc.cncb.ac.cn/biocode/tools/BT000001) (Z. Zhang, 2022).

2.5. Cis‐acting regulatory elements prediction and protein–protein interaction analysis

The upstream sequences (2000 bp) of each MeHsp coding sequence were acquired from the cassava genome. The cis‐acting regulatory elements were screened using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The protein–protein interaction among MeHsps was predicted using the STRING database (v11.50 https://string‐db.org/) with the highest confidence score (0.900). The interaction network of MeHsp proteins was analyzed using Cytoscape (v3.9.1 https://cytoscape.org/download.html).

2.6. RNA‐seq data analysis of Hsp genes

The RNA‐seq data were retrieved from the European Nucleotide Archive (ENA) database (https://www.ebi.ac.uk/ena/browser/view/PRJNA324539 (Wilson et al., 2017). RNA‐seq data were analyzed using the Trimmomatic, Hisat2, and featureCounts packages in the Galaxy database (https://galaxyproject.org/) (Gruning et al., 2017). Transcripts per million values were manually calculated and log2 transformed.

2.7. Analysis of the single‐cell transcriptome of cassava root and leaf

The expression patterns of MeHsps were examined at the single‐cell level using our single‐cell sequencing atlas of cassava tuberous roots (PRJNA895163) and leaves (PRJCA019992) (J. J. Song et al., 2023; Zang et al., 2023). The OmicSmart platform (www.omicsmart.com) was used to analyze the data, and a Uniform Manifold Approximation and Projection and gene expression bubble map were created.

2.8. Plant materials and treatments

The M. esculenta Crantz.cv.M.SC8 plants, a typical cassava cultivar, were transplanted from the Tropical Crops Genetic Resource Institute (TCGRI, Danzhou, China). All plants were cultured in Murashige and Skoog medium for 2 months. Seedlings with comparable growth status were selected for the subsequent experiments. For heat and cold stress treatments, the plantlets were exposed to temperatures of 42 and 4°C, respectively (An et al., 2017). Leaves under different treatments were collected at 0, 2, 4, 8, 12, 24, and 48 h and were immediately frozen in liquid nitrogen and stored at −80°C for subsequent experiments.

2.9. RNA extraction and quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis

Total RNA was extracted from plant samples using the RNAprep pure plant plus kits, and template cDNA was synthesized according to the instructions of the HiScript II Q RT Supermix for qPCR kit. The reaction mixture (10 µL) comprised 5 µL of 2x ChamQ universal SYBR qPCR master mix, 0.4 µL of each upstream and downstream primers, 1.2 µL of cDNA, and 3 µL of ddH2O. NCBI primer blast was employed to design gene‐specific primers targeting MeHsp genes (Table S12). β‐Tubulin (TUB) and elongation factor 1α were used as internal reference genes for qRT‐PCR (Salcedo et al., 2014; Ye et al., 2017). The following procedures were used for PCR reactions: 95°C 1 min, 95°C 5 s, 58°C 15 s, 72°C 15 s, 40 cycles, and 72°C 15 s. Each reaction was performed in triplicate for both biological and technical replicates. Relative expression levels were calculated by the 2−∆∆Ct method (Schmittgen & Livak, 2008). Statistical differences were determined using the Tukey test.

3. RESULTS

3.1. Identification of Hsp genes in cassava

After HMMER analysis and a BLASTp search, 225 Hsp genes with complete Hsp domains were identified in the cassava genome, including 8 MeHsp100s, 10 MeHsp90s, 21 MeHsp70s, 21 MeHsp60s, 109 MeHsp40s, and 56 MeHsp20s. These genes were renamed according to their screening order. Detailed information on MeHsp genes, including gene name, gene ID, chromosome location, amino acid count, coding sequence length, molecular weight, theoretical isoelectric point, instability index, aliphatic index, grand average hydropathicity, and predicted subcellular location, is listed in Table S1.

3.2. Phylogenetic analysis of Hsp gene families

Maximum likelihood phylogenetic trees were constructed to analyze the evolutionary relationships of Hsps between cassava and other species (Table S2). The six Hsp subfamilies were further divided based on the branch status of their phylogenetic trees (Altunoglu et al., 2019). Three main groups (Groups A–C) were identified for both MeHsp100 and MeHsp90 proteins (Figure S1a,b). The phylogenetic tree of MeHsp40 proteins exhibited the most significant number of groups (Groups A–E) due to a large number of proteins, and a subset of MeHsp40 proteins was clustered separately in Group E (Figure S1e). Four main groups (Groups A–D) were defined for MeHsp20, MeHsp60, and MeHsp70 proteins (Figure S1c,d,f). Phylogenetic analysis was also conducted to infer the relationships among the 225 identified MeHsp proteins (Figure 1). Most MeHsp proteins were grouped within their respective subfamilies, with a few exceptions; some MeHsp40 proteins were clustered with MeHsp100 subfamilies, and MeHsp70‐21 and MeHsp20‐30 were grouped with MeHsp40 subfamilies.

FIGURE 1.

FIGURE 1

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Phylogenetic relationship within 225 Hsp proteins in cassava. Different colors represent various subfamilies. Bootstrap values less than 0.5 were concealed.

3.3. Conserved motif, gene structure, and chromosome localization

Ten conserved motifs were analyzed in each MeHsp subfamily (Table S3). In general, the most closely related members of the same group had similar motif compositions (Figure S2a–f). For instance, the Hsp70 KD C‐terminal motif (motif 70‐10) is unique to group A of MeHsp70s. The chaperonin 60 motif (Motif 60–7) is unique to Group A MeHsp60s, whereas the TCP_1 (where TCP_1 is tailless complex polypeptide 1) motif (Motif 60–9) is specific to other groups. The exon–intron structure was also analyzed to acquire profound insights into the Hsp gene family in cassava (Figure S2a–f). Most MeHsp20s are intronless, whereas MeHsp100s and MeHsp90s possess at least two introns. In the MeHsp70 subfamily, MeHsp70‐20 exhibited the highest number of introns and showed significant differences in gene structure compared with the other members of the MeHsp70 subfamily.

All MeHsps were unevenly distributed on the 18 cassava chromosomes, and most were located either on the proximate or distal ends of the chromosomes (Figure 2). Most MeHsps (24) were present on chromosome 14, whereas the fewest MeHsps (3) were present on chromosome 17. Among the different MeHsp subfamilies, most MeHsp90s were located on chromosomes 6 (2), 14 (2), and 18 (2). Most MeHsp40s were present on chromosome 9 (10). Most members of the MeHsp70 subfamily were located on chromosomes 7 and 10 (10). MeHsp20s were mainly located on chromosome 14 (9), whereas MeHsp60s were mainly located on chromosomes 3 and 11 (Table S4).

FIGURE 2.

FIGURE 2

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Distribution of MeHsps on the 18 chromosomes of cassava. Lines of different colors represent subfamilies within the MeHsp family.

3.4. Synteny analysis of MeHsp genes and interspecific synteny analysis

Via synteny analysis of MeHsp genes, at least 74 pairs of segmentally duplicated genes and 11 tandemly duplicated genes were identified among the 225 MeHsp genes (Figure 3A, Table S5). These results indicate that segmental duplication is the primary driving force of MeHsp expansion. Non‐synonymous‐to‐synonymous substitution ratios were calculated (Table S6), and the Ka/Ks values of the most duplicated MeHsps were less than 1, indicating that they had experienced purifying selection during gene expansion. However, one pair of tandem‐duplicated genes may have undergone positive selection (Ka/Ks > 1). To acquire further insight into the evolutionary mechanisms of the Hsp families, we created syntenic maps of cassava with respect to other species (Table S7). As shown in Figure 3B–D, the maximum number of syntenic genes was detected in H. brasiliensis (324). A total of 86 syntenic genes were detected in O. sativs (monocotyledonous model plant), and 150 syntenic genes were detected in A. thaliana (dicotyledonous model plant). Syntenic genes have also been identified in D. rotundata (90) and P. trichocarpa (286) (Figure S3a,b), revealing the evolutionary origins of MeHsps.

FIGURE 3.

FIGURE 3

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(A) Duplication gene pairs in MeHsps. The segmentally duplicated gene pairs are connected by lines. The vertical lines on a single chromosome indicate tandem replication events. (B–D) Syntenic relationship of MeHsps with A. thaliana, O. sativa, and H. brasiliensis, respectively. The numbers in blue font are chromosomal numbers of cassava, and the numbers in purple font are chromosomal numbers of the other species.

3.5. Cis‐acting regulatory elements and potential protein–protein interactions

Twenty different types of cis‐acting regulatory elements were identified (Figure S4, Table S8), including 10 plant growth‐ and development‐related cis‐elements, five hormone‐related cis‐elements, and five stress‐related cis‐elements. The most abundant cis‐acting regulatory elements are implicated in light responses. All MeHsp subfamilies contain hormone‐responsive elements, such as MeJA‐responsive and abscisic acid‐responsive elements. Furthermore, four stress‐related components were found in all MeHsp subfamilies: anaerobic induction essential elements, defense and stress‐responsive elements, drought induction elements, and low‐temperature‐responsive elements. Among the six MeHsp subfamilies, only MeHsp40s and MeHsp60s possess phytochrome downregulation expression elements. Except for MeHsp90s and MeHsp100s, all other subfamilies contained wound‐responsive elements. These results indicate that MeHsps may respond rapidly to internal and external stimuli or regulate plant development.

We also constructed an interaction network using the STRING database to investigate potential protein–protein interactions among MeHsp proteins (Figure 4). The results showed that MeHsp70‐20 had the highest number of interactions (degree number) with other MeHsp proteins (Table S9), such as MeHsp40‐44, MeHsp60‐1, MeHsp70‐12, and MeHsp90‐4. Diverse interactions have also been discovered within distinct MeHsp subfamilies, including interactions between proteins from the same MeHsp subfamily, such as MeHsp60‐1 and MeHsp60‐13. These findings imply that members of the MeHsp family may cooperate for regulating various physiological functions in cassava.

FIGURE 4.

FIGURE 4

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Protein–protein interactions networks among MeHsps. The shade of lines represents the interaction strength between proteins.

3.6. Expression patterns of MeHsps in different tissues

We analyzed bulk transcriptomic data obtained from the ENA database (PRJNA324539) to determine the expression patterns of MeHsp genes in different tissues (Figure S5a–f, Table S10). Different MeHsps displayed variable expression patterns across tissues. Among the MeHsp100s, MeHsp100‐1 showed high expression levels in various tissues, with the highest expression level observed in the leaves. Certain MeHsp60s, such as MeHsp60‐17, 9, had relatively higher expression levels in all tissues, and MeHsp60‐17 had a significantly higher expression level in the leaves than in other tissues. MeHsp40s, such as MeHsp40‐52, 12, and MeHsp40‐14, were also highly expressed in a variety of tissues. MeHsp70s were expressed in multiple tissues, except for MeHsp70‐3, 4, 5, and MeHsp70‐14. MeHsp70‐20, located at the center of the protein interaction network, was highly expressed in friable embryogenic callus (FEC) and root apical meristem (RAM). Similarly, MeHsp90‐9 and MeHsp90‐10, which were also found in the protein interaction network, were expressed in FEC and RAM. Notably, while MeHsp20‐35 exhibited high expression levels in different tissues, the majority of MeHsp20s had low expression. These genes, which have distinct expression patterns, may be crucial for plant development and are valuable for use in further research.

3.7. Expression patterns of MeHsps in single‐cell transcriptome

To investigate the expression patterns of MeHsps at the single‐cell level, we analyzed single‐cell transcriptomic data of cassava tuberous roots and leaves. At the subfamily level, certain MeHsps were preferentially expressed in specific cell subsets (Figures S6–S11). Most MeHsp20s exhibited predominant expression in mesophyll cells and vascular bundles. Genes in the MeHsp60 family, as well as most MeHsp90s, were also expressed in mesophyll and leaf‐proliferating cells (G1/S, G2/M). In tuberous roots, most MeHsp20s and MeHsp70s were highly expressed in phloem cells, and most of the MeHsp60s were highly expressed in meristem cells. However, the expression levels of MeHsp40s and MeHsp100s were dispersed throughout the cells and exhibited a scattered distribution among the different cell subsets.

Six subfamily genes (MeHsp100‐1, MeHsp90‐9, MeHsp70‐20, MeHsp60‐17, MeHsp40‐52, and MeHsp20‐35) with distinct expression patterns in the bulk transcriptome and that were present in the protein interaction prediction network were selected for a detailed single‐cell analysis (Figures 5, 6, Table S11). In the leaves, MeHsp20‐35 and MeHsp70‐20 had the highest expression levels in the vascular bundles, with the former being expressed in a greater number of cells. Similar to other MeHsp60s and MeHsp90s, MeHsp60‐17 and MeHsp90‐9 were highly expressed in the mesophyll and proliferating cells (G2/M). Additionally, MeHsp40‐52 and MeHsp100‐1 were highly expressed in the mesophyll cells and epidermis cells, with the latter being expressed in fewer cells. MeHsp20‐35 was found to be highly expressed in the phloem of tuberous roots, whereas MeHsp40‐52 was found to be highly expressed in the pericycle and vascular bundles. MeHsp60‐17 was also expressed in the meristem cells, exhibiting a high family‐specific expression. MeHsp70‐20 was shown to be highly expressed in the vascular bundles, as well as in the leaves. However, the expression of MeHsp90‐9 in tuberous roots was limited, with only a small fraction of meristem cells expressing this gene.

FIGURE 5.

FIGURE 5

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Single‐cell transcriptome analysis of selected MeHsps in cassava leaves. (A) Uniform Manifold Approximation and Projection (UMAP) of the six MeHsps. (B) Bubble diagram of the six MeHsps.

FIGURE 6.

FIGURE 6

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Single‐cell transcriptome analysis of selected MeHsps in cassava tuberous roots. (A) Uniform Manifold Approximation and Projection (UMAP) of the six MeHsps. (B) Bubble diagram of the six MeHsps.

3.8. Expression profiles of selected MeHsps in response to temperature stress

To further assess the expression profiles of the six MeHsps under temperature stress, qRT‐PCR was performed to examine their transcript levels. The results are presented in Figures 7, 8. Under conditions of 4°C cold stress, the expression of MeHsp20‐35 was initially decreased and then increased significantly. Similarly, MeHsp40‐52 was upregulated 8 h after cold treatment. In contrast, MeHsp90‐9 was significantly downregulated 2 h after cold stress. The expression of MeHsp100‐1 rapidly decreased after cold treatment and remained low. MeHsp70‐20 was significantly downregulated 4 h after cold treatment but promptly recovered to normal levels. In particular, MeHsp60‐17 exhibited increased expression at 4 h but significantly decreased expression at 12 h.

FIGURE 7.

FIGURE 7

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Expression of the selected MeHsps in response to cold treatment (4°C). The values represent the mean ± SE from three independent experiments. Means not sharing a letter are significantly different (p < 0.05), for example, “a” and “b,” “bc” and “d.”

FIGURE 8.

FIGURE 8

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Expression of the selected MeHsps in response to heat treatment (42°C). The values represent the mean ± SE from three independent experiments. Means not sharing a letter are significantly different (p < 0.05), for example, “a” and “b,” “bc” and “d.”

After 42°C heat stress, the expression of MeHsp20‐35 exhibited a significant increase, peaking at 2 h. MeHsp40‐52 also showed significant induction 2 h after heat stress and then decreased. Interestingly, this trend was similar to that observed for MeHsp70‐20. The expression of MeHsp60‐17 peaked 2 h after heat treatment and declined significantly 4 h after heat stress. MeHsp90‐9 and MeHsp100‐1 were also upregulated in response to heat stress; however, their expression decreased with extended heat treatment.

4. DISCUSSIONS

Hsp family genes play crucial roles in plant growth and stress responses (Park & Seo, 2015). In this study, we identified 225 MeHsp genes, including 8 MeHsp100s, 10 MeHsp90s, 21 MeHsp70s, 21 MeHsp60s, 109 MeHsp40s, and 56 MeHsp20s. These results show similarities with previous research conducted on cassava (Muthusamy et al., 2023; Wei et al., 2020). Besides, the number of MeHsps also differs among other species. For example, cassava exhibits a greater abundance of Hsp genes than Arabidopsis (Swindell et al., 2007). This expanded gene family suggests that cassava has a greater capacity to fine‐tune its responses and for adaptability to a wide range of stressors. However, when compared to cassava, wheat has a significantly higher number of Hsp genes (A. Kumar et al., 2020), suggesting that polyploid organisms may exhibit an elevated number of Hsp genes due to gene duplication within each chromosome set.

Phylogenetic analysis is valuable for identifying orthologous or paralogous relationships between genes in different species (Altenhoff & Dessimoz, 2009; Lian et al., 2020). In this study, Hsp sequences from cassava and five other species were used to construct phylogenetic trees. Additionally, 225 MeHsps were used to construct a dedicated phylogenetic tree. The MeHsps were categorized into six groups. However, owing to the lack of overall sequence consistency, certain MeHsp40s and MeHsp70‐21 clustered independently. Introns can influence transcription efficiency, and genes with fewer introns tend to exhibit heightened sensitivity to external stressors (Chung et al., 2006; Le Hir et al., 2003). Compared with other MeHsp subfamilies, most of the MeHsp20s were intronless. The prevalence of intronless Hsp20 genes in cassava resembles the observations in potatoes (Solanum tuberosum) and peppers (Capsicum annuum L.) (Guo et al., 2015; Zhao et al., 2018), highlighting the unique gene structure organization within this specific subfamily and its potential role in facilitating swift responses to environmental stressors.

Gene duplication events, such as segmental and tandem duplications, are important for expanding the MeHsp gene family, as evidenced by the detection of 74 pairs of segmentally duplicated genes and 11 tandem duplicated genes among 225 MeHsp family genes. The evolutionary fate of duplicated genes is mainly determined by selective pressures (Jacquemin et al., 2014), and most duplicated MeHsps have undergone purification selection. Similar findings have been confirmed in many other species (X. Chen et al., 2021; Ke et al., 2022). Synteny analysis revealed the presence of syntenic Hsp genes in Arabidopsis, rice, yam, P. trichocarpa, and rubber tree. Monocotyledonous plants, such as rice and yam, have fewer syntenic Hsp genes than cassava, whereas dicotyledonous Arabidopsis and P. trichocarpa have a greater number of syntenic Hsp genes. Cassava and rubber tree, both members of the Euphorbiaceae family, have the highest number of syntenic Hsp genes.

Cis‐acting regulatory elements (CAREs) serve as crucial molecular switches for the transcriptional regulation of complex and dynamic gene networks (Yamaguchi‐Shinozaki & Shinozaki, 2005). In the present study, we discovered a significant presence of stress‐ and hormone‐responsive elements; these types of cis‐acting elements were also found in both dove (Davidia involucrate) and barley (Hordeum vulgar L.) (Chaudhary et al., 2019; Hou et al., 2022). These CAREs within Hsps suggest that Hsps regulate various physiological processes and plant growth in response to various stimuli. When the predicted protein–protein interaction networks among MeHsps, CsHSps (C. sativus), and PtHsps (P. trichocarpa) were compared, they all exhibited a significant presence of Hsp70 and Hsp60 subfamily proteins (X. Chen et al., 2021; J. Zhang, Liu, et al., 2015). These findings emphasize the importance of these two subfamilies in coordinating the heat shock protein network.

The expression patterns of MeHsps in different tissues and organs were analyzed. Different MeHsps exhibited inconsistent expression patterns in different tissues, indicating that they may have distinct functions. Some Hsp genes exhibited unique expression patterns in the transcriptome, indicating their pivotal roles in cassava growth and development. Homologous genes in Arabidopsis are involved in the regulation of plant organ development and responses to biological and abiotic stresses (Huang et al., 2014; Leng et al., 2017; Shen et al., 2011; Sjogren et al., 2004).

Single‐cell RNA sequencing allows researchers to analyze the expression patterns of genes at the single‐cell level, paving the way for further investigations of plant cell growth, development, and stress responses (Y. Wang et al., 2021). The elevated expression levels of MeHsp20‐35 and MeHsp70‐20 in the vascular bundles highlight their significant nutrient transport and storage functions. Similarly, Hsp18.1 (Hsp20s) and Hsp70 in tea plants (Camellia sinensis) have increased expression levels within the vascular bundles (Q. Wang et al., 2022). This coordinated expression within vascular tissues across species may indicate a shared functional role in facilitating the molecular processes that are critical for vascular development. In scRNA‐seq of the Arabidopsis, AtHsp101 predominantly enriched in root guard cells (T.‐Q. Zhang et al., 2021), whereas in cassava, its homologous gene MeHsp100‐1 exhibited a scattered distribution among different root cell subsets. The widespread expression of the MeHsp100‐1 suggests a more general and diversified function within different cell subsets.

In addition to single‐gene analysis, a comprehensive analysis of the single‐cell expression profiles of gene families can help reveal their enrichment levels and functional roles within specific cell subsets (J. Y. Kim et al., 2021). In this study, we observed that particular MeHsp subfamily genes were predominantly expressed in different cell subsets. For instance, the majority of MeHsp60s were found to be enriched in meristem‐related cell subsets, suggesting that these genes may play coordinated roles in cellular proliferation and contribute to the specific functions of these cell subsets. In contrast to MeHsp60s, most MeHsp20s were enriched in cell subsets related to substance transport. The expression preference of MeHsp subfamily genes across cell subsets suggests shared regulatory mechanisms, functional roles, or evolutionary relationships among these genes, providing additional evidence for their inclusion in the same gene family.

Industrialization and urbanization pose immense challenges to crop quality and yield (Lesk et al., 2016). Heat shock proteins were initially characterized for their essential roles in crop responses to temperature stress (ul Haq, Khan, Ali, Khattak, et al., 2019). In this study, we selected six MeHsp subfamily genes to examine their expression patterns under cold and heat stress conditions. In general, these six MeHsp genes were more sensitive to heat stress than cold stress, although some genes showed substantial downregulation after cold stress, which may be related to the heat resistance and non‐hardy properties of cassava. MeHsp40‐52, a gene encoding the J protein, is crucial for maintaining protein homeostasis in crop cells under stress conditions (G. Wang et al., 2014). The J protein plays distinct roles in the activation of the ATP hydrolysis activity of Hsp70 during cell metabolism and stress responses (Caplan et al., 1993). Under heat stress, the expression pattern of MeHsp70‐20 was similar to MeHsp40‐52, suggesting a potential synergistic response to heat stress. MeHsp60‐17 encodes a member of the chaperonin Cpn60α1, and its expression level exhibited a substantial increase under cold stress. A similar phenomenon was observed in CaHsp60s (C. annuum L.) (ul Haq, Khan, Ali, Gai, et al., 2019). Hsp90 is also a typical stress response chaperone (Xu et al., 2012), with MeHsp90‐9 exhibiting a significant increase in its expression under heat stress.

5. CONCLUSION

In this study, we identified 225 Hsp genes in the cassava genome. MeHsps within the same evolutionary branch had similar gene structures. The expansion of MeHsps is mainly derived from segmental duplications under purifying selection pressures. In addition, the MeHsp gene CAREs have multiple stress response components, and there are many protein–protein interactions among MeHsps, with MeHsp70‐20 playing a crucial role in the predicted protein interaction network. Different MeHsps display diverse expression patterns across tissues, suggesting distinctive functional roles for individual MeHsps. Certain subfamily genes were preferentially expressed in different cell subsets. Six subfamily genes were selected for a detailed single‐cell transcriptomic investigation. These genes are expressed in different cell subsets and may participate in various physiological regulatory functions. Finally, we examined the expression of the selected genes in response to temperature stress. These findings indicate that MeHsp genes may participate in regulating the temperature stress response in cassava. This study lays a solid foundation for future research by providing a thorough understanding of the cassava MeHsp genes.

AUTHOR CONTRIBUTIONS

Changyi Wang: Formal analysis; investigation; visualization; writing—original draft. Fangfang Ran: Data curation; formal analysis. Yuwei Zang: Resources; software. Liangwang Liu: Data curation; resources. Dayong Wang: Writing—review and editing. Yi Min: Conceptualization; writing—review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Supplemental Figure S1. Phylogenetic tree among six Hsp subfamilies (a). Hsp100s; (b). Hsp90s; (c). Hsp70s; (d). Hsp60s; (e). Hsp40s; (f). Hsp20s. The bootstrap value less than 50% were concealed.

Supplemental Figure S2. Conserved motif and gene structure of MeHsp subfamilies (a). Hsp100s; (b). Hsp90s; (c). Hsp70s; (d). Hsp60s; (e). Hsp40s; (f). Hsp20s. The bootstrap value less than 0.5 were concealed.

Supplemental Figure S3. Syntenic relationship of MeHsps with (a) D. rotundata (b) P. trichocarpa.

Supplemental Figure S4. Distribution of cis‐acting elements of MeHsps.

Supplemental Figure S5. Expression profile of MeHsps in different tissues. (a) MeHsp100s. (b) MeHsp90s. (c) MeHsp70s. (d) MeHsp60s. (e) MeHsp40s. (f) MeHsp20s. TPM values are converted by log2. The heat map was clustered by row.

Supplemental Figure S6. Single Cell Transcriptome analysis of MeHsp100s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S7. Single Cell Transcriptome analysis of MeHsp90s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S8. Single Cell Transcriptome analysis of MeHsp70s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S9. Single Cell Transcriptome analysis of MeHsp60s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S10. Single Cell Transcriptome analysis of MeHsp40s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S11. Single Cell Transcriptome analysis of MeHsp20s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps. MeHsp40‐12, MeHsp20‐2,21,28 were not annotated in the current Single Cell Transcriptome. They are not displayed in the figure.

TPG2-16-e20407-s001.docx (33.5MB, docx)

Supplemental Table S1. List of the 225 MeHsp genes identified in this study.

Supplemental Table S2. List of the Hsp genes in the phylogenetic tree.

Supplemental Table S3. Analysis of conserved motifs in cassava Hsp proteins.

Supplemental Table S4. Number of MeHsp genes on each chromosome.

Supplemental Table S5. Duplicated MeHsp gene pairs.

Supplemental Table S6. Ka/Ks value of duplicated MeHsp genes.

Supplemental Table S7. One‐to‐one orthologous relationships between MeHsp and other five plant species.

Supplemental Table S8. Cis‐acting regulatory elements in the promoter regions of 225 MeHsp genes.

Supplemental Table S9. Degree statistics of protein interaction nodes.

Supplemental Table S10. RNA‐seq data of MeHsp genes that were used in this study.

Supplemental Table S11. Single Cell RNA‐seq data of six selected genes that were used in this study.

Supplemental Table S12. Primers for qRT‐PCR.

TPG2-16-e20407-s002.xlsx (144.8KB, xlsx)

ACKNOWLEDGMENTS

The study was financially supported by a grant (31960039) to Yi Min from the National Natural Science Foundation of China, and a grant (822RC651) to Yi Min from the Natural Science Foundation of Hainan Province, a grant (XTCX2022JKB07) to Dayong Wang from the Cooperative Innovation Center of Hainan University, a grant (821RC1053) to D.W. from the Natural Science Foundation of Hainan Province, and a grant (31760246) to Dayong Wang from the National Science Foundation of China.

Wang, C. , Ran, F. , Zang, Y. , Liu, L. , Wang, D. , & Min, Y. (2023). Genome‐wide identification and expression analysis of heat shock protein gene family in cassava. The Plant Genome, 16, e20407. 10.1002/tpg2.20407

Assigned to Associate Editor Rupesh Deshmukh.

Contributor Information

Dayong Wang, Email: wangdy@hainanu.edu.cn.

Yi Min, Email: 992601@hainanu.edu.cn.

DATA AVAILABILITY STATEMENT

The original data presented in the study are publicly available, which can be found at the NCBI database and NGDC database: https://www.ncbi.nlm.nih.gov/bioproject/324539; https://www.ncbi.nlm.nih.gov/bioproject/895163; https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA019992.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure S1. Phylogenetic tree among six Hsp subfamilies (a). Hsp100s; (b). Hsp90s; (c). Hsp70s; (d). Hsp60s; (e). Hsp40s; (f). Hsp20s. The bootstrap value less than 50% were concealed.

Supplemental Figure S2. Conserved motif and gene structure of MeHsp subfamilies (a). Hsp100s; (b). Hsp90s; (c). Hsp70s; (d). Hsp60s; (e). Hsp40s; (f). Hsp20s. The bootstrap value less than 0.5 were concealed.

Supplemental Figure S3. Syntenic relationship of MeHsps with (a) D. rotundata (b) P. trichocarpa.

Supplemental Figure S4. Distribution of cis‐acting elements of MeHsps.

Supplemental Figure S5. Expression profile of MeHsps in different tissues. (a) MeHsp100s. (b) MeHsp90s. (c) MeHsp70s. (d) MeHsp60s. (e) MeHsp40s. (f) MeHsp20s. TPM values are converted by log2. The heat map was clustered by row.

Supplemental Figure S6. Single Cell Transcriptome analysis of MeHsp100s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S7. Single Cell Transcriptome analysis of MeHsp90s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S8. Single Cell Transcriptome analysis of MeHsp70s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S9. Single Cell Transcriptome analysis of MeHsp60s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S10. Single Cell Transcriptome analysis of MeHsp40s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps.

Supplemental Figure S11. Single Cell Transcriptome analysis of MeHsp20s in cassava tuberous root and leaves. (a) UMAP of MeHsps. (b) Bubble diagram of MeHsps. MeHsp40‐12, MeHsp20‐2,21,28 were not annotated in the current Single Cell Transcriptome. They are not displayed in the figure.

TPG2-16-e20407-s001.docx (33.5MB, docx)

Supplemental Table S1. List of the 225 MeHsp genes identified in this study.

Supplemental Table S2. List of the Hsp genes in the phylogenetic tree.

Supplemental Table S3. Analysis of conserved motifs in cassava Hsp proteins.

Supplemental Table S4. Number of MeHsp genes on each chromosome.

Supplemental Table S5. Duplicated MeHsp gene pairs.

Supplemental Table S6. Ka/Ks value of duplicated MeHsp genes.

Supplemental Table S7. One‐to‐one orthologous relationships between MeHsp and other five plant species.

Supplemental Table S8. Cis‐acting regulatory elements in the promoter regions of 225 MeHsp genes.

Supplemental Table S9. Degree statistics of protein interaction nodes.

Supplemental Table S10. RNA‐seq data of MeHsp genes that were used in this study.

Supplemental Table S11. Single Cell RNA‐seq data of six selected genes that were used in this study.

Supplemental Table S12. Primers for qRT‐PCR.

TPG2-16-e20407-s002.xlsx (144.8KB, xlsx)

Data Availability Statement

The original data presented in the study are publicly available, which can be found at the NCBI database and NGDC database: https://www.ncbi.nlm.nih.gov/bioproject/324539; https://www.ncbi.nlm.nih.gov/bioproject/895163; https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA019992.

Articles from The Plant Genome are provided here courtesy of Wiley Periodicals LLC on behalf of Crop Science Society of America

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