Heat stress leads to reduced growth and lower final yields of plants, and the negative impact of high temperatures has been reported for
P. ternata [
18,
21]. Due to the ability of plants to activate a large number of stress-related genes and to synthesize a variety of functional proteins to counteract heat stress, it is important to understand the mechanism of
P. ternata’s response to heat stress. Suppression subtractive hybridization (SSH) was used to clone transcripts that were up-regulated during heat stress in the leaves of
P. ternata, and 44 singletons were grouped into 17 functional categories, such as responses to stress, primary metabolic processes, macromolecule metabolism and cellular processes [
22]. However, most biological functions in a cell are executed by proteins and not by mRNA, and the transcription patterns are not always consistent with the protein expression levels, as shown in a previous study [
20].
More needs to be known about the mechanism of action of proteins in response to heat stress. To the best of our knowledge, there have not been any proteomic analyses of
P. ternata subjected to heat stress. Proteomics analyses of
P. ternata have been difficult to perform due to the lack of genomic information [
23]. However, cross-species identification is a powerful option for protein identification whenever a genome is poorly characterized [
24,
25]. In this approach, proteins are identified by comparing peptides from the proteins of interest with orthologous proteins of species that are well characterized. In this study, protein database searches were performed using all available green plant proteins in the NCBI protein databases. In the present study, the physiological data showed that the leaves of plants treated with high temperature for 24 h showed stress damage. A 2-DE-MS-based proteomic approach was used to identify proteins with different expression levels in response to heat stress. A total of 24 differentially expressed proteins and protein species were detected and functionally characterized. Many protein species were identified in more than one spot, although they were excised from the same gel. This phenomenon may result from the presence of different protein isoforms, post-translational modifications or degradation [
26]. The identified proteins provide useful information on the details of the responsive mechanisms of
P. ternata exposed to high temperatures.
3.2. Small Heat Shock Proteins
sHSPs are highly conserved proteins of approximately 15–40 kD and are characterized by the possession of an á-crystalline domain at the
C-terminus. sHSPs can be classified into at least six categories according to their sequence homology and subcellular localization: cytosolic class I, cytosolic class II, mitochondrial, chloroplast localized, peroxisomal and endoplasmic [
28].
Several proteomic analyses have been performed to analyze the plant proteome in response to heat stress. In most cases, sHSPs belonging to the cytoplasmic sHSPs class as well as mitochondrial-targeted and chloroplast-targeted sHSPs have been observed. In the present study, 14 sHSPs were detectable only in the stressed sample. The protein sequences were analyzed with PSORT [
29] to predict the proteins’ subcellular localization. To determine the categories of these identified sHSPs, we searched for homologs with BLASTP [
30]. These searches combined with the sequence alignments and other data from the literature allowed these proteins to be classified.
In this study, eight spots (11–17) were identified as cytoplasmic class I family sHSPs, and spot 19 and 20 were identified as cytoplasmic class II family sHSPs. One sHSPs located in the mitochondria was present only under heat stress. Meanwhile, protein spots 3, 4, 6 and 8 were identified as homologous to chloroplast sHSPs.
The cytoplasmic sHSPs represent the most abundant sHSPs in plants [
31]. Many of them act as small heat shock proteins with important functions not only in the protection of proteins against damage due to stress but also in the folding, intracellular distribution and degradation of proteins. Transgenic plants that overexpress sHSPs have improved agronomic traits with respect to basal thermotolerance [
32–
34]. In contrast, plants with reduced expression of sHSPs have compromised acquired thermotolerance [
35].
To investigate the gene and protein expression patterns of cytoplasmic sHSPs in
P. ternata leaves, real-time quantitative PCR and Western blot analysis were performed at different time intervals during high temperature stress at 38 °C. Using 2-DE Western blot analysis, we confirmed the identities of the sHSPs-CII identified by MS. Transcriptional profile analysis clearly showed that the
sHSPs-CI and
sHSPs-CII genes are highly inducible by heat stress in leaves but they exhibited diverse expression profiles in response to heat stress. The
sHSPs-CI expression reached a peak at 2 h of heat shock but then declined at 8 h of heat stress before increasing again. The expression of
sHSPs-CII increased gradually with treatment duration, reaching a peak at the 24 h time-point but then declined at 72 h of heat stress. In addition, the protein levels of sHSPs-CI increased with the time of heat treatment, and the sHSPs-CII exhibited the same expression patterns for their mRNA and protein levels. When the CK sample was analyzed, proteins in both classes were nearly absent. A heat shock time-course experiment was designed to analyze the influence of heat shock on the expression of
sHSPs-MIT in
P. ternata leaves under stressful conditions. The results are shown in
Figure 5. The level of
sHSPs-MIT transcripts increased rapidly in
P. ternata leaves. The
sHSPs-MIT expression reached a peak at 8 h of heat shock but then declined at 12 h of heat stress before progressively increasing again.
Chloroplast small heat shock proteins (sHSPs-CHL) are expressed in leaves in response to heat stress.
In vitro experiments have demonstrated that sHSPs-CHL can associate with thylakoids and protect PSII during heat and other stresses, possibly by stabilizing the O
2 evolving complex (OEC) [
36]. sHSPs-MIT exhibit high similarity to sHSPs-CHL in the
C-terminal region but differ in the
N-terminal region of the proteins [
37]. Maize sHSPs-MIT have been shown to improve mitochondrial electron transport, mainly by protecting the NADH:ubiquinone oxidoreductase activity (complex I) [
38].
sHSPs are widely distributed in plants however, it will be interesting to further isolate and analyze the function of sHSPs that are unique in P. ternata. Heat stress strongly induces the protein expression of four different classes of sHSPs in P. ternata leaves. The transcripts of three sHSPs genes were increased by heat shock treatment, although with different response patterns. These results may indicate that different classes of sHSPs were regulated with different patterns or by different signals and have different assigned functions in response to heat stress. sHSPs may function together to protect the cellular machinery and are critical for the plant to tolerate heat stress.
3.4. Protein Degradation Proteins
One newly induced protein (spot 9) was identified as speckle-type POZ protein (SPOP). The protein sequence was subjected to Pfam analysis [
40] to identify conserved domains. This protein was found to contain BTB/POZ and MATH domains. In
A. thaliana, the BTB/POZ-MATH proteins (BPM) comprise a small family of six members. They have been described previously to use their broad complex, tram track, bric-a-brac/POX virus and zinc finger (BTB/POZ) domains to assemble with CUL3a and CUL3b and potentially serve as substrate adaptors for cullin-based E3-ligases, which are involved in the ubiquitin–proteasome pathway for protein degradation [
41]. BPM has been described previously as playing a role in abiotic stress tolerance. Studies on gene expression have shown that drought, osmotic stress and salt stress can induce the expression of
BPM1,
BPM4 and
BPM5 [
42]. This result suggests that protein degradation and recycling via the ubiquitin–proteasome pathway is involved in response to heat stress in
P. ternata leaves. The up-regulation of SPOP may promote programmed cell death and leaf senescence in response to heat stress.
3.5. RNA Processing Proteins
The regulation of RNA metabolism at the post-transcriptional level, including pre-mRNA splicing, capping polyadenylation, transport, turnover and translation, has been shown to play important roles in plant responses [
43]. These post-transcriptional events are largely regulated by RNA-binding proteins (RBPs). The glycine-rich RNA-binding proteins (GRPs) are found ubiquitously in plants. These proteins contain a glycine-rich region at the
C-terminus and one or more RRMs at the
N-terminus. It has been demonstrated that the expression of GRPs can be regulated by different stresses.
ZmGRP2 is up-regulated in stressed roots of
Zea may in an ABA-independent manner. Previous studies in
Arabidopsis roots have indicated that
AtGRP7 is repressed in response to ABA, high salt and mannitol treatments [
44]. The transcript levels of all
BnGRPs were markedly up-regulated by cold stress. However, their expression levels were significantly down-regulated by dehydration and high salinity stress. It has been shown that
Arabidopsis grp7 mutants display impaired mRNA export from the nucleus to the cytoplasm, suggesting a potential cellular role of GRPs in mRNA export [
45]. Wang
et al. [
46] reported that
LbGRP1 can enhance stress resistance by mediating physiological pathways in
Limonium bicolor (Bunge) Kuntze.
Four spots (22–25) were identified as GRPs. All of these spots were down-regulated in the leaves of stressed
P. ternata plants, indicative of disordered metabolism under stress conditions. The mRNA profiles of GRPs in
P. ternata leaves after different durations of heat treatment were assessed by qPCR. The results indicate that the mRNA level is not consistent with the protein level. At the beginning of the treatment, the expression of GRPs was slightly down-regulated, but after 12 h heat stress, the expression was significantly up-regulated. This phenomenon may be caused by post-transcriptional and post-translational control mechanisms [
19].
Recent studies indicate that GRPs play an important role in heat tolerance, but the exact roles that GRPs play during heat stress and the relationship between GRPs and ABA remain to be determined.