3.1 Identification and Characterization of the CssHSPGene Family in Tea Plant
A total of 54 CssHSP genes were screened from the tea plant
genome through an investigation using the ACD, a conserved
carboxyl-terminal domain of approximately 90 amino acids. The results of
the analysis of physiological and biochemical properties revealed that
the lengths of most CssHSP s ranged from 129 (TEA033542) to 288
(TEA003981) amino acids, the molecular weights were between 14.96 kDa
(TEA033542) and 32 kDa (TEA003981), and pI ranged from 4.65 (TEA010603)
to 10.18 (TEA030325). These data indicate that 18 CssHSP proteins
were basic with pI >7.0, whereas the remaining 29 proteins
were acidic. In addition, the proteins with more than 300 amino acids
were TEA028261, TEA024355, TEA019050, TEA029627, TEA031251, TEA006315,
and TEA004116. More detailed data, including instability index,
aliphatic index, and subcellular localization, were also collected
(Table S1 ).
To explore the molecular evolution of and phylogenetic relationships
among sHSP genes in plants, a phylogenetic tree was constructed
from 117 sHSP domain sequences of five different species; the tree
included 14 sequences from a monocotyledonous angiosperm (rice) and 103
sequences from dicotyledonous angiosperms (A. thaliana , tomato,
and tea plant). All sHSP genes were categorized into 12 groups
(CI–CVII, P, MI–MII, ER, and Px). At least one tea plant sHSP was
determined to belong to each group except for Px. Group CI was the
largest clade, including 17 (almost 31%) of the CssHSP genes
identified (Figure 1 ). Proteins belonging to the M groups were
primarily in groups MI and MII and were closely related to those in P
subfamilies, suggesting that they have similar origins and evolutionary
history.
Then the protein sequence features of the CssHSP families were
revealed, that the 10 most conserved motifs were identified and
designated motif 1 to motif 10 by using the MEME tool (Figure2, Figure S1 ). Members of the same group usually had
similar motif compositions, and some motifs were specifically present in
one or more groups. For example, most group CI members contained motifs
1, 2, 3, 4, 5, 6, 8, and 9. The locations of the motifs were similar.
Motif 9 was unique to group CI. Motifs 7 and 10 were observed only in
groups CIII and P. In addition, protein motifs 1, 2, and 3 in theCssHSPs were determined to be highly conserved and distributed
across almost all members.
3.2 Expression Patterns of CssHSP Genes Based on
Genome Data
The expression patterns of CssHSP genes were presented, eight tea
plant tissue genomes that are available online were downloaded (Figure3A ). The transcripts per million (TPM) values of theCssHSP genes in different tissues are listed in Table S2. AllCssHSP genes had diverse expression in the
eight
tissues(Figure S2) . We found that 10 CssHSP genes had
higher expression levels [defined as log2(TPM)
> 2] in these eight tissues. Several CssHSP genes
exhibited some tissue specificity. For instance, TEA030313 and TEA025531
were only highly expressed in flowers. TEA015431 and TEA003981 were only
highly expressed in roots and fruit, respectively. In general, almost
all CssHSP genes were specifically expressed in one or more
tissue (Figure 3A and Table S3 ), implying that these
genes play different roles in the growth and development of the tea
plant.
To further demonstrate functions of CssHSP genes in responses to
abiotic and biotic stresses, the expression values of CssHSP s
after cold, drought, NaCl, and MeJA treatment were analyzed in the
genome data (Table S4 ). Heat maps were created on the basis of
expression levels, and upregulated genes are displayed in a Venn diagram
(Figure 3 ). Under cold, drought, and NaCl treatments,CssHSP s with normalized expression values ≥1 was considered to be
upregulated genes; in MeJA treatment, CssHSP s with normalized
expression values ≥3 was considered to be upregulated. Among the 54CssHSP genes, 47 genes were upregulated in response to stress,
and some were upregulated for more than one stressor. For the abiotic
stresses, 40, 8, and 17 CssHSP genes response respectively to
cold, drought, and salt. Furthermore, 25 genes were observed to be
upregulated in the presence of MeJA. For these treatments, the most
upregulated genes were TEA033542 and TEA017741 (Figure 3E and
Table S4 ).