Low temperature regulation of strawberry color by FvMAPK3

When it’s cold, the red coloration of strawberry fruits is delayed due to a repression of anthocyanin accumulation (Mirahmadi et al., 2012). Central roles in low-temperature mediated anthocyanin accumulation have been attributed to SUCROSE NONFERMENTING1-RELATED KINASE2.6 (FaSnRK2.6) in strawberries (Han et al., 2015) and to MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascades, such as MAPK3, in Arabidopsis and rice. Wenwen Mao and colleagues (Mao et al., 2022), now reveal that FvSnRK2.6 and FvMAPK3 exhibit similar roles in strawberries acting upstream of the rate-limiting anthocyanin biosynthesis enzyme CHALCONE SYNTHASE1 (FvCHS1) and of FvMYB10, a pivotal transcription factor regulating anthocyanin accumulation.

First, the authors discovered that FvSnRK2.6 and FvMAPK3 physically interact and that FvSnRK2.6 phosphorylates FvMAPK3. Phosphorylated FvMAPK3 protein levels as well as FvMAPK3 transcript levels were increased in white strawberries under low temperature. To confirm the role of FvMAPK3 as a downstream positive regulator of low temperature-mediated repression of anthocyanin, the authors generated and phenotyped both transient and stable overexpression (OE) and downregulated (RNAi and genome edited, cr) transgenic lines of FvMAPK3 (see Figure). A thorough expression study confirmed the repressed and enhanced expression of central anthocyanin biosynthesis-related genes (such as FvCHS1) and regulatory transcription factors involved in anthocyanin accumulation (such as FvMYB10) in OE and downregulated lines, respectively (see Figure).

Figure.

Fruit developmental ripening phenotypes of EV-OE, FvMAPK3-OE-L1 and FvMAPK3-OE-L4, FvMAPK3-cr-C15, and FvMAPK3-cr-C16 transgenic strawberry fruits (left), relative expression levels of reported regulators of anthocyanin biosynthesis in white strawberries of EV-OE, FvMAPK-OE, and FvMAPK3-cr lines (middle), and yeast-two-hybrid assay demonstrating interaction of FvMAPK3 with FvMYB10 and FvCHS1 (right). DPA, days post-anthesis. Adapted from Mao et al. (2022), Figures 4–6.

Secondly, the authors further examined whether FvCHS1 and FvMYB10 act downstream of FvMAPK3 at low temperature. Direct interaction between FvMAPK3 and FvCHS1, and FvMAPK3 and FvMYB10, as well as phosphorylation of both proteins by FvMAPK3 was shown in vitro (see Figure). Next, they explored the existence of two FvMAPK3-mediated independent pathways involving FvCHS1 and FvMYB10. Immunoblot analysis of FvCHS1 protein levels in white FvMAPK3-OE fruits revealed FvKFB1 ubiquitin-mediated degradation of FvCHS1 by FvMAPK3 at low temperature, whereas transactivation assays with FvMYB revealed a negative effect of the FvSnRK2.6–FvMAPK3 module on its transcriptional activity. Subsequently, both pathways lead to an inhibition of ripening-related anthocyanin accumulation in strawberry fruits. Interestingly, the authors end with showing that a phosphorylation-dead mutant of FvMYB10 has higher transcriptional activity under low temperature compared with the phosphorylated form of FvMYB10, suggesting it may protect strawberry fruits from delayed coloration.

This study provides a thorough molecular exploration on how two independent FvMAPK3-mediated signaling pathways regulate ripening-related coloration under low temperature in strawberries. Whether other MAPK cascade members are involved and whether these two pathways are conserved in other plant species remains to be investigated.