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.