*3.3. Putative HT Adaptation-Related Genes in Kenshin*

To identify and characterize putative HT adaptation-related genes in Kenshin, we applied new cutoff criteria (Table 3) compared with known genes from *Arabidopsis* (Table 4) and confirmed their expression by RT-PCR (Figure 5). Unexpectedly, *B. rapa* warming genes appeared to differ from

those of *Arabidopsis*, suggesting that different warming adaptation mechanisms or different sets of genes might function in *B. rapa* upon warming conditions. *Arabidopsis* warming genes such as *PHYB*, *HSP70*, and *PIF4* were highly expressed in all *B. rapa* samples, with no notable increase upon warming treatment. Only the expression pattern of *BrPIF4* (Bra000283) appeared to be somewhat related to HT adaptation, and two homologs of acquired thermotolerance-related genes in *Arabidopsis*, *BrROF2* and *BrHSFA2*, appeared to be responsive to warming in *B. rapa*. PIF4, a basic helix-loop-helix (bHLH) transcription factor, is a central regulator of ambient temperature signaling in *Arabidopsis* [9]. PIF4-mediated thermomorphogenesis is associated with the circadian clock [39,40], auxin [9,41,42], other phytohormones [43,44], and epigenetic modification [6]. Quint et al. (2016) [11] indicated that PIF controls thermomorphogenesis via three molecular circuitries: (1) transcriptional regulation of circadian clock genes; (2) post-translation regulation by phosphorylation and degradation; and (3) phytohormonal control through interactions at various levels. These findings and our expression data suggest that BrPIF4 plays a role in the adaptation of Kenshin to HT.

Most warming-responsive genes in Kenshin are orthologs of *Arabidopsis* genes involved in acquired thermotolerance: HSR genes, s*HSP*s, peroxidase family genes, and disease-resistance genes (Table 3; Figure 5). The expression of other *HSP* genes and *BrMPSR1* also increased in response to warming conditions, suggesting their possible involvement in HT adaptation (Figure 5). The roles of a few s*HSP*s in heat tolerance have been examined, including genes encoding HSP 21 (*HSP21*; Bra026317, AT4G27670) [45] and 17.6 kDa class II HSP (*HSP17.6II*; AT5G12020; Bra006137, Bra008920) [29]. However, many s*HSP*s, such as *HSP21*, *HSP22.0*, *HSP18.2*, and *ASCORBATE PEROXIDASE 2* (*APX2*), are HS memory-related genes [22] and are targeted by HSFA2 to help maintain HS memory [31,32]. Class III peroxidases (PRXs) are plant-specific enzymes encoded by multigene families that are involved in lignification, cell elongation, stress responses, and seed germination [46]. Ascorbate peroxidase (APX), a key antioxidant enzyme, participates in various abiotic stress responses and in maintaining cellular homeostasis [47]. In *Arabidopsis*, MPSR1 (Misfolded Protein Sensing RING E3 Ligase 1) is involved in the rapid degradation of misfolded proteins due to protein-damaging stress, thereby controlling proteotoxic stress in the cytoplasm [48]. In *B. rapa*, two *MPSR1* genes (*BrMPSR1-1* (*Bra012441*) and *BrMPSR1-2*(*Bra016290*)) appear to be regulated at the transcriptional level or regulated in evolutionarily divergent ways. These genes might also participate in HT tolerance in Kenshin. Together, these findings support the notion that these genes play a role in long-term adaptation to HT in Kenshin.
