Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble 13 phenylpropanoid biosynthesis in Arabidopsis 14

Phenylpropanoids are phenylalanine-derived specialized metabolites and include 35 important structural components of plant cell walls such as lignin and hydroxycinnamic acids 36 (HCAs), as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate 37 esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity 38 in HCE biosynthesis, such that most Arabidopsis mutants with blockages in the pathway simply 39 redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase 1 ( fah1 ) mutant 40 accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis 41 may be repressed in response to loss of ferulate 5-hydroxylase (F5H) activity. Here we show 42 that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE 43 accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or 44 flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 45 mutants, and that this repression is specific to tissues in which F5H is normally expressed. 46 Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is 47 dependent on the MED5a/5b subunits of the transcriptional co-regulatory complex Mediator, 48 which are similarly required for the repression of lignin biosynthesis and stunted growth of the 49 phenylpropanoid pathway mutant reduced epidermal fluorescence 8 ( ref8 ). Taken together, 50 these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a 51 Mediator-dependent manner in Arabidopsis fah1 mutants, and support an emerging model in 52 which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid 53 metabolism. anthocyanin and indicate of C3′H prevents the repression of anthocyanin accumulation in the fah1 mutant background, suggesting that the accumulation of a biosynthetic intermediate between C3′H and F5H may contribute to the inhibition of anthocyanin accumulation in fah1 mutants. Our experiments with soil-grown and sucrose-stressed fah1 plants demonstrate that the repression of phenylpropanoid metabolism in fah1 mutants extends to the anthocyanin branch of the pathway, and that this repression is also dependent on Mediator. In contrast with HCEs, anthocyanin accumulation is only affected by disruption of MED5a/5b in the absence of FAH1, strengthening the conclusion that phenylpropanoids are derepressed and not simply globally 311 increased in med5a/5b fah1 mutants. These observations are consistent with those of Maruta et 312 al., (2014) who recently showed that fah1 seedlings fail to accumulate wild-type levels of 313 anthocyanins in response to methylviologen-induced oxidative stress. In contrast with the 314 conclusions of Maruta et al. (2014), however, we have found evidence that the anthocyanin 315 deficiency of fah1 plants is not a result of expression changes at the transcriptional level of 316 anthocyanin biosynthetic genes. Not only do fah1 mutants show wild-type transcriptional 317 induction of anthocyanin biosynthesis genes in response to sucrose stress, but also the rescue 318 of anthocyanin accumulation in med5a/5b fah1 mutants is not associated with any changes in 319 the expression of these genes. Moreover, we have shown that overexpression of the 320 MYB75/PAP1 transcription factor leads to similar induction of anthocyanin biosynthetic genes in 321 leaf blades of pap1-D and fah1 pap1-D mutants, despite the striking anthocyanin deficiency of 322 the latter. Although we cannot formally exclude the possibility that Mediator regulates 323 anthocyanin biosynthetic genes in fah1 mutants in a limited spatial or temporal window, the most straightforward interpretation of our data that the repression of anthocyanin biosynthesis 325 in fah1 is independent of changes in the transcription of the anthocyanin biosynthetic genes. these results are similar we