*3.4. BrHSFA2 and Its Target BrHSP18.2s*

The expression of *Arabidopsis HSFA2* is dependent on HS (the expression of which is amplified by the production of its alternatively spliced form), increases the expression of target HSR genes such as *HSP18.2*, and confers acquired thermotolerance or HS memory. In the current study, *BrHSFA2* expression and splicing, and the expression of its target gene, *BrHSP18.2s* (Figure 6; Table 5), followed a similar pattern to that of *Arabidopsis* under warming conditions, implying that the warming response of *B. rapa* is similar to acquired thermotolerance in *Arabidopsis*.

Acquired thermotolerance by exposure to moderate HS confers tolerance to normally lethal HT [7]; this thermotolerance is maintained as HS memory for several days [32,49,50]. Three HS memory maintenance-related genes have been identified in *Arabidopsis*, which maintain this memory for several days after the plant returns to nonstress temperature conditions: two days for *HSFA2* [23,24,28], three days for heat stress-associated 32 kD protein gene (*Hsa32*) [49], and three days for *miR156* [22]. The maintenance of HS memory results from the induced hypermethylation of target genes (*HSR* genes), although not all target genes are hypermethylated [51].

*Arabidopsis* HSFA2 is a key regulator of responses to various types of stress including heat, high light, and ROS stress and is required for extending acquired thermotolerance by maintaining the expression of *HSP* genes [26,28,31]. HSFA2 is a regulatory component responsive to the accumulation of misfolded proteins in the cytosol [52]. HSFA2 also induces abscisic-acid-mediated heat tolerance by upregulating *HSPs* in both a monocot (fescue) and a dicot (*Arabidopsis*) [53]. HSFA2 is responsible for maintaining HS memory up to two days by maintaining histone methylation, thereby inducing HSR gene expression upon recurring HS [23,24]. Many small *HSP* genes, such as *HSP21*, *HSP22.0*, *HSP18.2*, and *ASCORBATE PEROXIDASE 2* (*APX2*), are HS memory-related genes in *Arabidopsis* [22,24], whereas *Hsp70* (AT3G12580) and *Hsp101* (AT1G74310) are non-HS memory-related genes [24]. *Arabidopsis HSFA2* produces an alternatively spliced form (truncated form) upon HS, and this truncated form in turn increases *HSFA2* transcription levels [54]. This scenario appears to operate in *B. rapa* as well, where *BrHSFA2* undergoes alternative splicing, is upregulated, and induces/maintains target gene expression/memory. Alternative splicing by a spliceosome complex is an important mechanism in the sensing of (and adaptation to) small variations in ambient temperature [14], as well as acquired thermotolerance conditions [48], in *Arabidopsis*. These findings imply that temperature changes, including HS and priming, lead to alternative splicing. Alternative splicing might contribute to long-term adaptation to HT in Chinese cabbage, in which thermotolerance does not appear to be due to morphological and architectural changes caused by high ambient temperatures, as found in other plants (thermomorphogenesis) [11].
