Conserved roles of the HSFA-mediated JA pathway in regulating anthocyanin biosynthesis under HT in Arabidopsis and tea
Numerous studies have reported that JA participates in the heat stress response; however, its specific physiological role in heat response remains ambiguous in plants (Pan et al., 2019; Wu et al., 2022). Here, we applied the same JA-related treatments described above in tea plants (MeJA, ibuprofen, or MeJA + MG132) to Arabidopsis in order to explore the conserved roles of JA in plant HSR. Following application of MeJA, there was a clear increase in the transcript abundance ofAtCHS , AtDFR , and AtANS ; moreover, treatment with ibuprofen decreased expression of these genes much as HT did, whereas MeJA partially rescued the influence of HT (Figure 7a, b). In addition, the patterns of anthocyanin content observed in Arabidopsisleaves were consistent with the patterns of catechin content in tea leaves. As expected, anthocyanin accumulation was severely reduced upon treatment with ibuprofen (67%), whose effect was similar with that of HT (75%), whereas JA could partially relieve the impact of HT (Figure 7c). These results suggest that JA also acts as a core mediator in the regulatory effect of HT on anthocyanin synthesis, another important branch of the flavonoid pathway.
Previous studies have indicated that HT decreases the expression ofArabidopsis genes required in both early and late steps of the anthocyanin biosynthetic pathway; consequently, HT represses anthocyanin biosynthesis (Kim et al., 2017; Chen et al., 2017). However, the potential roles of HSFs in regulating anthocyanin biosynthesis under HT have not been revealed. It has been reported that mutation ofAtHSFA2 , which is homologous to CsHSFA1b andCsHSFA2, causes greater sensitivity to heat stress inArabidopsis (Charng et al., 2007; Zhang et al., 2020a). Accordingly, we analyzed the expression of flavonoid biosynthesis genes in Col-0 and the athsfa2 mutant. The qRT-PCR results revealed that transcript levels of AtDFR and AtANS were significantly up-regulated in the athsfa2 mutant. Furthermore, the expression of these genes in both Col-0 and the athsfa2mutant was obviously changed by HT Figure S2). It has been reported that detached leaves of mature Arabidopsis exhibit purple-red coloration due to excessive anthocyanin accumulation (Chen et al., 2017). Observations of detached leaves of Col-0 and athsfa2plants exposed to HT (40 ℃, 48 h) revealed Col-0 leaves to have significantly lighter purple-red color than those of athsfa2following HT treatment (Figure 8a). This is consistent with the anthocyanin content in Col-0 was greatly reduced by HT, whereas that inathsfa2 was less affected (Figure 8a, Fig S4a).
To further define the conserved roles of HSFs, at least for those HSFA family members that mediate the effect of HT on anthocyanin biosynthesis, we over-expressed CsHSFA1b and CsHSFA2driven by the 35S promoter in Col-0 and the athsfa2 mutant. Two overexpression lines for each combination of construct and background with” (implies 8 lines total, 2 constructs x 2 backgrounds x 2 replicates) with relatively higher expression levels were then selected for further analysis (Figure S3). As expected, anthocyanin content was significantly reduced in these overexpression lines (Figure 8b). Correspondingly, the transcript levels of anthocyanin biosynthetic genes such as AtCHS , AtDFR, and AtANS were lower inCsHSFA1bOE3 /Col-0, CsHSFA2OE3 /Col-0,CsHSFA2OE3/athsfa2 , and CsHSFA2OE3/athsfa2 (Figure 8c, Figure S4), and HT treatment exacerbated this down-regulation (Figure 8c, Figure S4). These findings are further supported by the purple-red color of HT-treated CsHSFA1bOE3/ Col-0 and CsHSFA2OE3/ Col-0 leaves being significantly lighter upon visual inspection than in leaves of Col-0.
To verify that HSFs participate in regulating MeJA biosynthesis, we then examined MeJA in the overexpression lines by LC-MS. Compared to WT, MeJA content was significantly reduced in CsHSFA1bOE3/ Col-0,CsHSFA2OE3/ Col-0, CsHSFA1bOE3/athsfa2 , andCsHSFA2OE3/athsfa2 , and decreased even further when exposed to HT (Figure 8d). Taken together, these findings suggest that HSFA family members are activated by HT and negatively regulate JA as well as flavonoid biosynthesis, and this working model is conserved betweenArabidopsis and tea.