*3.5. Validation of Hsf and Hsp Gene Expression Levels by qRT-PCR*

To verify the accuracy of the transcriptome sequencing, the expressions of 12 randomly selected genes were validated using quantitative real-time RT-PCR (qRT-PCR). The results showed that the expression pattern of each tested gene was similar to that of the transcriptome sequencing and the increase rate of all these *Hsf* and *Hsp* genes in the thermotolerant line 05-4 were significantly higher than those in thermosensitive line 05-1 (Figure 4).

**Figure 4.** Comparison of transcripts expression results from RNA-seq and qRT-PCR analysis. Abscissa: Sample number; the ordinate (left): the relative expression of gene validated using qRT-PCR, represented by bar chart; coordinates (right): RPKM value obtained from the transcriptome sequencing, represented by triangle scatter diagram.

## **4. Discussion**

Many studies have suggested that *Hsfs* and *Hsps* play central roles in plant developmental and defense processes [29,30]. Benefiting from genome availability, the functions of the *Hsf* and *Hsp* family genes have been characterized in many plants. Although *Hsfs* and *Hsps* exist in all living organisms, their numbers vary in different plants. There are 22 *Hsfs* in Arabidopsis, 25 *Hsfs* in rice [18], 30 *Hsfs* in maize [20], 25 *Hsfs* in pepper [31] and 52 *Hsfs* in soybean [32]. Compared to the 27 *sHsp* genes in Arabidopsis [33], there are 35, 51 and 27 *sHsp* genes in pepper [31], soybean [34] and Chinese cabbage [35], respectively. Previous studies have identified 18 *Hsp70* genes in Arabidopsis and 32 genes in rice [36]. The grapevine genome contains at least seven genes encoding members of the

*Hsp90* super family [37]. Zhang et al. (2015) reported 28 *Hsf*, 37 *sHsp*, 28 *Hsp60*, 20 *Hsp70* and 5 *Hsp100* genes in the poplar genome [19]. However, with the limited investigations into the molecular mechanism of heat tolerance, little is known about the *Hsf* family in eggplant.

In the present study, we identified 24 *Hsf* genes, 39 *sHsp* genes, 21 *Hsp60* genes, 30 *Hsp70* genes, 17 *Hsp90* genes and 10 *Hsp100* genes based on the eggplant genome (Table 1). Although the total number of *Hsf* and *Hsp* genes was similar to that of Arabidopsis [18,38–40], rice [18,41] and tomato [42], the members of some specific *Hsf* and *Hsp* subclasses in eggplant were different from the other three species. Two members were identified that belonged to subclass HsfC2 in rice, while no eggplant *Hsf* members were classified into subclass HsfC2 and the same events were also observed in *Arabidopsis thaliana* [18] and pepper [31]. Rice is the model plant use for the monocot lineage and we inferred that the gene duplications led to the unique HsfC2 subclass in monocot species [17,42], which was the most marked difference between monocots and eudicots. In contrast, similar to tomato and *Arabidopsis thaliana* [18,43], eggplant also has members that were partitioned into the HsfA6 subclass, but no rice *Hsf* members were classified into subclass HsfA6 [44]. This finding suggested that *Hsf* genes were doubled and gained new functions during the evolution of the eggplant genome. Another interesting observation was that the subclass *HsfA9* had 1 member in eggplant, compared with 4 members in pepper [31] and *Eucalyptos grandis* (Myrtaceae) contained at least 17 closely related *HsfA9*-encoding genes [17], suggesting a gene loss event during the evolutionary process of eggplant. However, there were two *HsfA4* subclass genes in eggplant, more than in pepper *CaHsfA4*, which showed that some *Hsfs* might have the similar functions, as in maize [20]. The reasons for the increase in the *HsfA9* genes need further investigation.

The phylogenetic analysis revealed that eggplant *Hsf* and *Hsp* members were more closely related to those from tomato than to those from Arabidopsis, which was consistent with the fact that both eggplant and tomato are members of the Solanaceae family [45]. Based on the previous analysis of the evolution of *Hsfs* and *Hsps* in Chinese cabbage [21,35], rice [46] and soybean [47], *Hsf* and *Hsp* genes essentially cover all the subfamilies and are relatively stable and conserved in the evolutionary process of eggplant and most of the *Hsf* and *Hsp* gene families were closely related to the evolutionary species.

Divergences in coding regions, particularly those that change the function of the gene, reflect amino acid altering substitutions and/or alterations in exon–intron structure [19]. The differences in intron and exon structure play important roles in the evolution of family genes. Structural analyses showed that the eggplant *Hsf* genes contained 0–7 introns and there were significant differences in the intron length; similar results were also obtained in cucumber [48], rice [49] and chickpea [50], but this result was different from that of pepper [31], for which all members have one intron. The number of introns of the *Hsp* gene family members in eggplant also showed differences, similar to the results of previous studies on poplar *sHsp*, *Hsp60*, *Hsp70* and *Hsp100* [19]. Qiao (2015), researching the pear *Hsf* and Guo (2015), researching the pepper *Hsp20*, showed a lack of conserved motifs among all the family genes and none of these genes contained the whole sequence, consistent with the eggplant *Hsfs* and *Hsps* in the present study [31,51]. We speculated that the deletion of introns and domains leads to structural changes during evolution, leading to functional diversity in *Hsf* and *Hsp* genes in eggplant; however, this theory needs experimental confirmation.

*Hsfs*, as transcriptional activators of *Hsps*, cooperate with *Hsps* to form a network responding to various stresses. These factors play a broad role in the tolerance to multiple environmental stress treatments apart from heat stress [52,53]. The comprehensive analysis of the expression for individual *Hsf* and *Hsp* members under HS was necessary for further functional analyses in plant thermotolerance [23,54]. The present study showed that most members of the eggplant *sHsp*, *Hsp60*, *Hsp70*, *Hsp90* and *Hsp100* families were induced by HS treatment in lines 05-1 and 05-4 and only a few members were significantly downregulated. Several studies have indicated that the expression and accumulation of heat shock proteins and heat shock transcription factors can enhance the thermostability of tomato [55], wheat [56] and rice [57]. *Hsfs* are activated under HS conditions and subsequently bind the HSE elements of the promoters of the *Hsp* genes to regulate the expression of downstream *Hsp* genes [17]. The accumulation of the *Hsps* effectively reduces the damage from HS and enhances thermotolerance by binding denaturing proteins and preventing them from irreversible aggregation [58]. Thus far, only *sHsp* has been shown to play a major role in improving plant thermotolerance in the form of molecular chaperones and cell membrane stabilizing factors [59]. However, the specific mechanisms of other *Hsp* genes are less well established. Previous studies have shown that the response of plants to high temperature was a quantitative trait controlled by multiple genes; some normal genes were closed and some stress tolerance-related genes were induced under high-temperature stress, thus altering plant morphogenesis, physiological functions and biochemical and molecular structures, which in turn influenced the growth of plants [60]. In addition, heat shock proteins are different from other stress proteins and have their own unique characteristics. In the present study, *Hsps* (*sHsp*, *Hsp60*, *Hsp70*, *Hsp90* and *Hsp100*) showed species diversity, universal distribution and instantaneous response and structural conservation. For example, the synthesis of heat shock protein was fast, beginning between the first few minutes and tens of minutes and the expression lasted for up to several hours, occasionally continuing for 12 or more hours (Figure 4). Similar results were also observed in poplar [19] and grape [61].

In Arabidopsis, there are four members of the HsfA1 family, A1a, A1b, A1c and A1d [62]. Studies have shown that HsfA1a can directly sense heat stress and become activated and the same treatments also induced the binding to *Hsp18.2* and *Hsp70* promoters, as examined by chromatin immunoprecipitation [63]. Overexpressing *HsfA1a* enhances diverse stress tolerance by promoting stress-induced *Hsp18.2* and *Hsp70* gene expression [64]. In addition, *AtHsfA1* was also related to drought stress [65] and programmed cell death [66]. Thus, in eggplant, *HsfA1* may also play a similar function to *AtHsfA1* and simultaneously communicate with *Hsps*. Increasing evidence suggests that *Hsp* is one of the most important heat stress proteins regulated by *Hsf* and is the material basis of the response of plant cells to high temperature damage [67–69]. Once exposed to high temperature, most of *Hsf* and *Hsp* genes in eggplant were induced to express rapidly and the expression level of these genes in the thermotolerant line was much higher than that in the thermosensitive line. Therefore, the Hsf–Hsp involved protein degradation pathway is also the main pathway of eggplant response to high temperature stress and may play an important role in the production of heat-tolerance in eggplant. The results provide a foundation for further functional research of these genes in eggplant, which could potentially be useful for elucidating the mechanism of thermotolerance in eggplant, even in other solanaceous plants.
