Results

Identification and Chromosomal Distribution of Rice HSFs

With the availability of the genomic sequences of the number of plant species, including Rice, it
is now possible to obtain the protein sequences of all the HSF members. In the present study, 10
HSFs were identified from the Rice genome. All the HSF proteins were surveyed for the
presence of DBD and OD through EMBL-EBI, employing HMM. Furthermore, SMART was
used to search the HSF-DBD to check the accuracy of the results. The characteristics of Rice
HSF genes are presented in the table below.

Gene Transcript ID Protein Chromosome Location Strand Transcrip Genomic CDS Exon Intron
ID t Count DNA Lengt
h
Os03g085450 HSFA1 3 5989254- + 1 5490 1536 2 1
0 Os03t0854500- 35992160
01
Os03g074500 Os03t0745000- HSFA2 3 0606069- _ 1 5280 1442 2 1
0 01 A 30606473
Os02g052730 Os02t0527300- HSFA3 2 19309986- - 1 4890 1287 2 1
0 01 19312780
Os01t0749300- HSFA4B 1  31370593- + 1 6270 1675 2 1
Os01g074930 02 31372497
0
HSFA5 2 17428124- + 1 5874 1599 2 1
Os02g049610 Os02t0496100- 17431026
0 01
Os09g045680 Os09t0456800- HSFB 9 17221825- + 1 5587 1546 2 1
0 01 17228613
Os04t0568700- HSFB2A 4 28574497- + 0 4567 1145 2 1
Os04g056870 00 28575516 
0
Os08t0546800- HSFB2B 8 27383114- _ 1 6754 1765 2 1
Os08g054680 01 27384387
0
Os01g062530 Os01t0625300- HSFC1A 1 24967510- + 1 6457 1689 2 1
0 01 24968608
Os01t0733200- 1 30582636- + 1 5198 1421 2 1
Os01g073320 01 30583497
0

Table 1. Detailed sequence annotation of identified Rice HSF genes

Phylogenetic Analysis and Classification of Rice HSFs

In present study, the evolutionary relationship among AtHSFs, OsHSFs, SbHSFs, BdHSFs, and
ZmHSFs was explored. A total of 33 HSFs were divided into three classes based on the
phylogenetic tree. Variation in HSF gene number was observed among different plant. HSFs of
rice were closer to wheat HSFs, which is in line with the botanical classification among
monocots. Figure 2. A neighbor-joining phylogenetic tree was constructed after aligning protein
sequences of Arabidopsis thaliana, Oryza sativa, Triticum aestivum, and Zea mays. Overall, 9
AtHSFs, 10 OsHSFs, 04 Triticum aestivum and 10 ZmHSFs were divided into three classes

Table 2. Distribution of HSF genes in different sub-classes in selected plant species.

Gene Duplication Analysis and Evolutionary Rate Calculation

In the present study, a total of 18 (18/10; 72%) Rice HSF genes were shown to be duplicated
(Table 3). Further, only one pair of a gene (ZmHSF-01/Zm-HSF-04) appeared to be tandemly
duplicated, which was recognized on chromosome number 1 (Figure 3). The rest of duplicated
genes were all segmentally duplicated, with eight different clusters containing 16 genes. These
genes were recognized on chromosomes 1–9. Moreover, the molecular evolutionary rate of
tandemly and segmentally duplicated HSF genes was calculated to gain insights into the
selective constraints on the duplicated HSF genes. The ratio of Ka and Ks substitution rate is an
effective method to investigate the selective constraint among duplicated gene pairs [64]. Hence,
in the present study, Ka, Ks, and Ka/Ks values for each paralogous gene pair were calculated
(Table 3). Here, 18 ZmHSF genes were shown to be duplicated. The Ka/Ks ratio for duplicated
ZmHSF genes ranged from 0.3415 to 0.7572. All the HSF genes in the present study have Ka/Ks
value < 1.
Protein
ID Ka/Ks Ka Ka Ks BrancKa Ka/Ka/Ks Ka BranKa Ks BraKs
Branch1 Branch1Branch1 Branch1h1 Branch2Ks Branch2ch2 Branch2nch2
Branch2

HSFA1
0 0 1e-10 0 0 1e-10

HSFA2A
0 0 1e-10 0 0 1e-10

HSFA3
0 0 1e-10 0 0 1e-10

HSFA4B
0 0 1e-10 0 0 1e-10

HSFA5
0 0 1e-10 0 0 1e-10

HSFB
0 0 1e-10 0 0 1e-10

HSFB2A
0 0 1e-10 0 0 1e-10

HSFB2B
0 0 1e-10 0 0 1e-10

HSFC1A
0 0 1e-10 0 0 1e-10

Table 3. Duplicated gene pairs, non-synonymous substitution rate (Ka), synonymous substitution
rate (Ks), nonsynonymous to synonymous substitution rate ratio (Ka/Ks), estimated time of
duplication event in a million years ago (MYA), and mode of gene duplication
Figure 3. Circular illustration of the Rice genome. The paralogous HSF genes are connected with
grey lines. The red lines on top of the chromosomal bar represent the position of HSFs.

Gene Structure and Protein Motif Analysis

To investigate the structural relationship among the HSF genes, the intron-exon organization of
all the targeted HSFs was analyzed using GSDS software. The intron-exon structure and number
play a key role in the evolution of gene families. The gene structure analysis was in line with the
phylogenetic relationship among Rice HSFs In general, the intron and exon numbers were shown
to be highly consistent. Particularly, 92% (23/10) HSFs contain only one intron except for HSF-
02 and HSF-24 (Figure 4). HSF-02 and HSF-24 were shown to contain three and five exons. In
contrast, the rest of the HSF genes contained two exons. Further, 17 HSFs contained 50 UTR and
30 UTR. The HSF genes belonging to the same class and sub-class showed a similar intron-exon
pattern in terms of intron number, exon length, intron phase, and overall gene length (Figure 4).
Figure 4. The intron-exon structure of Rice HSF genes. A black line represents the introns.
Exons are represented by a yellow rectangle and 5′ UTR and 3′ UTR by reddish-pink wedges.
The gene structure of Zea mays HSFs was in alignment with the phylogenetic relationship
MEME was used to identify the conserved motifs/regions responsible for DNA-binding,
oligomerization, nuclear localization, nuclear export, and biological activation of HSFs (Figure
5). In total, 20 motifs designated as motifs 1–20 were identified among Rice HSF proteins (Table
4). The highly conserved DBD is represented by motifs 1, 2, and 4. Motif 3 corresponds to OD
of class A and C HSFs. Figure 5. Conserved motifs in Rice HSFs as identified by MEME. The
motifs were identified by using full-length protein sequences of ZmHSFs. The name of HSFs
and p-values are indicated on the left side. Each motif is represented by a unique color, as
indicated at the bottom

Conserved motif sequence of Zea mays HSFs

Domain Analysis and Physio-Chemical Properties

The modular structure and the functional domains of HSFs have been studied and described
extensively. The HSF-type DBD was highly conserved and consisted of approximately 100
amino acids (Figure 6). The locations of DBD and OD were predicted using SMART and
MARCOIL (Table 5). The DBD of most Rice HSFs was located at the beginning of the N-
terminal. Few exceptions were ZmHSF-03, ZmHSF-10, ZmHSF-14, and ZmHSF-15. As
expected, the linker length between DBD and OD of HSFBs was larger than HSFAs and HSFCs.
The physicochemical properties of HSFs such as amino acid length, Mw, and pI were
 investigated using Expasy

Multiple sequence alignment of Rice HSFs

Protein ID DBD OD Amino pI Molecular Localization Localization
Acid Weight
CELLO WoLFPSORT
HSFA1 296- 332- 506 4.97 55277.22 Nucleus Nucleus
319 362

HSFA2A 164- 231- 376 4.97 40847.53 Nucleus Nucleus

219 254
CHLOROPLAST
HSFA3 263- 356- 498 4.73 55098.54 Nucleus Nucleus
291 382

HSFA4B 133- 264- 440 5.26 49387.38 Nucleus Nucleus

183 417

HSFA5 357- 430- 475 5.38 52884.68 Nucleus Nucleus

415 475

HSFB 180- 116- 302 9.36 32798.98 Nucleus Nucleus

209 184

HSFB2A 192- 164- 305 5.19 32809.00 Nucleus Nucleus

278 193
HSFB2B 165- 215- 590 5.21 41374.05 Nucleus Nucleus
212 244

HSFC1A 227- 176- 250 8.93 27219.03 Nucleus Nucleus

248 212

HSFC1B 199- 144- 339 6.21 36862.73 Nucleus Nucleus

226 186

The physicochemical properties of Rice HSFs proteins include group, protein ID, domains,
amino acid physio-chemical properties, and subcellular localization.

Protein-Protein Interaction Network Analysis

The protein network interaction analysis can help understand protein biological functions and
mechanisms. Since both the RNA-seq data and GO annotation analysis suggested the role of
HSFs in stress conditions and normal growth, we performed network analysis to predict the
interacting partners of ZmHSFs (Table S7). The results showed that Rice HSFs interact with
themselves and a range of proteins with well-known functions in cellular growth and stress
responses (Figure 12). For example, HSFs were shown to interact with molecular chaperons
HSP101, HSP82 (belongs to HSP90 family), HSBP-2, and DnaJ-like protein (belongs to the
HSP40 family). It was reported that HSP101 and HSA32 interact with each other and promote
acquired thermotolerance in Arabidopsis. The HSP82 was reported to be induced by higher
temperatures. A higher concentration of HSP82 is required for normal cellular growth in yeast at
higher temperatures [73]. Gu et al. reported that Rice HSBP-2 and HSFA2 interact with each
other and modulate raffinose biosynthesis. HSFA2 was shown to bind to the promoter sequence
of HSBP-2 and activate its expression. Higher raffinose synthesis improved HS tolerance of
Arabidopsis thaliana. The DnaJ-like proteins are molecular co-chaperones that interact with
HSP70s and control protein homeostasis. DnaJ proteins have been reported to play a critical role
in plant growth, development, and HS tolerance. ZmHSFs also interact with two major proteins,
i.e., multi-protein bridging factor 1c (MBF1c) and DREB2A. Both these proteins have been
shown to accumulate under diverse abiotic stress conditions. DREB2A is a major protein, and its
overexpression improves plant HS, drought stress, cold stress, etc., tolerance . MBF1c is a
transcriptional co-activator that modulates the expression of DREB2A, some HSFs, and
phytohormones Interestingly, MBF1c is necessary for basal thermotolerance but not for acquired
thermotolerance In addition, MBF1c is also shown to be required for plant developmental
responses
Protein-protein network of Rice HSFs. The line connecting two proteins represents that an
interaction exists between them.
Rice HSFs are also predicted to interact with SUMO proteins. SUMOylation is a post-
translational phenomenon where SUMO proteins are covalently attached and detached to target
proteins [81]. This process affects several biological processes inside the cell, including
transcriptional regulation of gene expression, apoptosis, programmed cell death, cellular
response to stress, stability of proteins, etc. [81]. Rytz et al. reported that SIZI, a SUMO protein,
targets multiple TFs, chromatin remodelers, transcriptional co-activators/repressors connected to
abiotic and biotic stress responses [82]. This suggests Rice HSFs may also be SUMOylated
under diverse biological conditions and stress responses. To conclude, PPI analysis aligned with
the RNA-seq and GO annotation analysis, which indicated that HSFs of Zea mays play an
important role in abiotic stress conditions and Rice growth and metabolism.