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Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel‐coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red‐fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6‐phosphate. RNA‐Seq analysis showed down‐regulation of both gene‐encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6‐phosphate synthase gene TPS1.1a was down‐regulated, whereas putative regulatory TPS7 and TPS11 were strongly up‐regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up‐regulated, whereas MYB27 was also up‐regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6‐phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits. In carbon‐starved red‐fleshed kiwifruit, both carbohydrate and anthocyanin concentrations were greatly reduced. Our model suggests that anthocyanin biosynthesis was actively repressed by MYB27 via trehalose 6‐phosphate signalling to conserve carbon. [ABSTRACT FROM AUTHOR]

Summary: MYB transcription factors (TFs) regulate diverse plant developmental processes and understanding their roles in controlling pigment accumulation in fruit is important for developing new cultivars. In this study, we characterised kiwifruit TFMYB7, which was found to activate the promoter of the kiwifruit lycopene beta‐cyclase (AdLCY‐β) gene that plays a key role in the carotenoid biosynthetic pathway.To determine the role of MYB7, we analysed gene expression and metabolite profiles in Actinidia fruit which show different pigment profiles. The impact of MYB7 on metabolic biosynthetic pathways was then evaluated by overexpression in Nicotiana benthamiana followed by metabolite and gene expression analysis of the transformants.MYB7 was expressed in fruit that accumulated carotenoid and Chl pigments with high transcript levels associated with both pigments. Constitutive over‐expression of MYB7, through transient or stable transformation of N. benthamiana, altered Chl and carotenoid pigment levels. MYB7 overexpression was associated with transcriptional activation of certain key genes involved in carotenoid biosynthesis, Chl biosynthesis, and other processes such as chloroplast and thylakoid membrane organization.Our results suggest that MYB7 plays a role in modulating carotenoid and Chl pigment accumulation in tissues through transcriptional activation of metabolic pathway genes. [ABSTRACT FROM AUTHOR]

Summary: Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild‐type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB‐absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV‐B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE‐LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8‐mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land. Significance statement: Inducible flavonoid biosynthesis regulated by the photoreceptor UVR8 is key for tolerance to UVB radiation in flowering plants, but whether this is the case for other plant groups is unclear. In this study, CRISPR/Cas9 mutagenesis is used in the model species Marchantia polymorpha to show that both the UVR8 signal transduction pathway and the importance of flavonoids for UVB tolerance are conserved in one of the most distantly related groups of plants to flowering plants, the liverworts. [ABSTRACT FROM AUTHOR]

Summary: The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light‐ and nutrient‐deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress‐induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization. [ABSTRACT FROM AUTHOR]