Mitogen-activated protein kinase kinase 6 negatively regulates anthocyanin induction in Arabidopsis

ABSTRACT

Arabidopsis mitogen-activated protein (MAP) kinase kinase 6 (MKK6) has previously been shown to play important roles in plant immunity and cytokinesis. Here we report that it is also involved in the negative regulation of anthocyanin biosynthesis in Arabidopsis. Elevated anthocyanin levels are observed in mkk6 knock out mutant plants. RNA-sequencing analysis revealed that the expression level of MYB75 (Myb transcription factor 75), which encodes a key positive regulator of anthocyanin biosynthesis, is up-regulated in mkk6 mutant plants, suggesting that increased MYB75 expression contributes to the elevated anthocyanin levels. In addition, suppression of high sucrose-induced anthocyanin production by pathogen associated molecular pattern-triggered immunity is also dependent on MKK6, further supporting that MKK6 is a key negative regulator of anthocyanin biosynthesis in Arabidopsis.

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Anthocyanins constitute a ubiquitous class of plant pigments which have been suggested to play important roles in abiotic stress responses ¹. The first major step in anthocyanin biosynthesis is production of tetrahydroxychalcone by chalcone synthase (CHS). Tetrahydroxychalcone is subsequently converted into flavanones by chalcone isomerases (CHIs), which then follow parallel but distinct pathways to produce the range of anthocyanins present in plants ¹. Genes encoding enzymes involved in early anthocyanin biosynthesis such as CHS and CHIs are regulated by R2R3-MYB DNA binding proteins, whereas genes encoding enzymes in late anthocyanin biosynthesis such as dihydroflavonol 4-reductase, anthocyanidin synthase and flavonoid specific glucosyltransferases are regulated by ternary complexes consisting of bHLH proteins, R2R3-MYB proteins and WD40 repeat domain proteins ².

High sucrose conditions strongly induce the expression of CHS-A and production of anthocyanins in Arabidopsis ³. This induction of anthocyanin biosynthesis by sucrose is dependent on the MYB75/PAP1 transcription factor ⁴, which works together with WD40 repeat domain proteins and bHLH proteins such as TT8, GL3 and EGL3 to regulate late anthocyanin biosynthesis genes ². Interestingly, the induction of anthocyanin biosynthesis by sucrose can be reversed by treatment with common pathogen associated molecular patterns (PAMPs), suggesting a crosstalk between PAMP signaling and abiotic stress response ⁵.

PAMPs are typically conserved components necessary for the lifestyles of plant pathogens, such as fungal chitin or bacterial flagellin ⁶. The plant innate immune system detects these PAMPs via pattern recognition receptors to activate PAMP-triggered immunity (PTI). One well characterized PAMP is flg22, a conserved 22 amino acid fragment from bacterial flagellin ⁷. It is detected by the membrane bound receptor-like kinase FLS2 ⁸. Perception of PAMPs usually leads to activation of downstream mitogen-activated protein kinase (MAPK) cascades ⁹. A MAPK cascade involves at least three components: an upstream MAP kinase kinase kinase (MAPKKK or MEKK), a downstream MAP kinase kinase (MKK) and a MAP kinase (MPK) even further downstream. Upon activation, the MAPKKK phosphorylates and activates the MKK, which subsequently phosphorylates and activates the MPK. The activated MPK further regulates the activity of downstream components by phosphorylation.

MPK4 is a multifunctional MAP kinase. It forms a kinase cascade with MEKK1 and MKK1/MKK2 which is important in basal resistance and guarded by the nucleotide-binding leucine-rich-repeat protein SUMM2 ^10–12. Loss of function mutants of mpk4 exhibit autoimmunity and the autoimmune phenotypes can be suppressed by mutations in SUMM2 ^12,13. MPK4 also forms a kinase cascade with the Arabidopsis Nucleus – and Phragmoplast-localized protein Kinase 1-related Protein kinases ANP2 and ANP3 and MKK6, which is involved in regulation of cytokinesis as well as immunity ^14–16. Moreover, MPK4 was found to be involved in promoting light-induced activation of anthocyanin accumulation through phosphorylation of MYB75 ¹⁷.

Interestingly, mkk6-2 but not anp2-2 anp3-3 mutant plants turn purple when they are about three to four weeks old (Figure 1A). Similarly, mkk6-3 plants in the Ler background also display a purple color (Figure 1B), suggesting that MKK6 plays a role in the regulation of anthocyanin accumulation. To test whether sucrose-induced anthocyanin accumulation is affected in mkk6-2, we compared anthocyanin levels in wild type (Col-0) and mkk6-2 seedlings following treatment with sucrose. As shown in Figure 2, whereas anp2-2 anp3-3 and mpk4-3 summ2-8 have similar levels of anthocyanin as wild type, mkk6-2 seedlings accumulate significantly higher levels of anthocyanin in high sucrose conditions, confirming that MKK6 negatively regulates anthocyanin accumulation.

Figure 1.

Morphology of wild type, anp2 anp3 and mkk6 mutants. (A) Morphology of Col-0 wild type, anp2-2 anp3–3 and mkk6-2 plants. (B) Morphology of Ler and mkk6-3 plants. The mkk6-2 and mkk6-3 mutants were described previously (Takahashi et al. 2010). The plants were grown on soil and the photos were taken when they were about four weeks old.

Figure 2.

Accumulation of anthocyanin in wild type, anp2-2 anp 3-3, mkk6-2, and mpk4-3 summ2-8 seedlings. Seedlings were grown on ½ MS plates. Low sucrose media contains 10 mM of sucrose and high sucrose media contains 100 mM of sucrose. Anthocyanin content was determined as previously described (http://www.bio-protocol.org/e1098). Error bars represent standard deviations from measurements of three different samples. Different letters indicate a statistically significant difference (P < 0.01) determined by two-factor ANOVA with Tukey HSD pairwise post hoc analysis.

Arabidopsis MYB75 is a key transcription factor that positively regulates the biosynthesis of anthocyanin ⁴. In RNA-sequencing data comparing transcripts between mkk6-2 and wild type plants ¹⁶, the transcript level of MYB75 is about ten-fold higher in mkk6-2 than in wild type. Further quantitative RT-PCR analysis confirmed that the transcript level of MYB75 in mkk6-2 is considerably higher than in wild type plants (Figure 3), suggesting that MKK6 negatively regulates the expression of MYB75. Since over-expression of MYB75 results in increased anthocyanin accumulation ¹⁸, the increased anthocyanin accumulation in mkk6-2 mutants is at least partially due to increased transcription of MYB75.

Figure 3.

Expression levels of MYB75 in wild type and mkk6-2. Seedlings were grown on ½ MS plates. Values were normalized relative to the expression of ACTIN1. Error bars represent standard deviations from three repeats. The samples were compared by one-tailed t-test (*P < 0.05).

As induction of anthocyanin production by high sucrose conditions can be reversed by treatment with flg22 ⁵, we tested the effect of flg22 treatment on anthocyanin accumulation in mkk6-2. As shown in Figure 4, high concentration of sucrose induces increased accumulation of anthocyanin in wild type seedlings and addition of flg22 to the media blocks the induction. The increased anthocyanin level in anp2-2 anp3-3 and mpk4-3 summ2-8 seedlings was also suppressed by addition of flg22. However, in mkk6-2 seedlings, treatment with flg22 did not lead to suppression of anthocyanin accumulation, suggesting that MKK6 is required for the flg22-induced suppression of anthocyanin accumulation. It is likely that a MAP kinase cascade involving MKK6 is activated upon perception of flg22, leading to suppression of anthocyanin production under high sucrose growth conditions.

Figure 4.

Effect of flg22 treatment on the accumulation of anthocyanin in wild type, anp2-2 anp3-3, mkk6-2, and mpk4-3 summ2-8 seedlings. Seedlings were grown on ½ MS plates. Low sucrose media contains 10 mM of sucrose and high sucrose media contains 100 mM of sucrose. PAMP treatment was carried out by including 100 nM of flg22 in the high sucrose media. Error bars represent standard deviations from measurements of three different samples. Pairs were compared by one-tailed t-test (*P < 0.05, **P < 0.001). ns: no significant difference.

MKK6 was originally shown to serve as a key player in the regulation of cytokinesis ¹⁵. Recently, we showed that MKK6 also functions in parallel with MKK1/MKK2 in a MAP kinase cascade consisting of MEKK1, MKK6 and MPK4 in plant defense ¹⁶. In addition, we found that the MAP kinase cascade consisting of ANP2/ANP3, MKK6 and MPK4 also plays important roles in plant immunity ¹⁶. The identification of MKK6 as a critical regulator of anthocyanin production further supports that MKK6 is a multifunctional protein involved in various biological processes. As anthocyanin accumulation is not affected in anp2-2 anp3-3 and mpk4-3 summ2-8 (Figures 2 and 4), ANP2/ANP3 and MPK4 either do not function together with MKK6 in regulating anthocyanin levels or there are additional functionally redundant MAPKKKs and MPKs involved in the process. Identification of the MPK(s) functioning downstream of MKK6 is critical for better understanding of how MKK6 negatively regulates anthocyanin production.

Funding Statement

This work was supported by the Natural Sciences and Engineering Research Council of Canada [2017-04690].

Acknowledgments

This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC, grant#2017-04690).