Francesca Quattrocchio

The family of MYB-domain transcription factors expanded greatly during plant evolution, reaching over 200 members in some species. To assess how MYBs were co-opted into distinct processes, we studied MYB regulators of distinct branches of the flavonoid pathway. To this end, we expressed different MYBs in Petunia hybrida and assessed the capacity of each one to induce anthocyanin accumulation. Study of MYBs from different species in the same expression system did not reveal the functions of individual proteins in the plants from where they originated, but clearly pinpointed functional differences. Pigmentation MYBs from different species induced transcription of different but overlapping sets of target genes when expressed in the same cells, while yeast 2-hybrid assays revealed divergence even among MYBs from the same phylogenetic subgroup for their capacity to bind specific bHLH and WD40 protein partners. These results suggest that diversification of proteins occurred after gene duplication, resulting in specificity for different target genes. Such evolution of proteins parallels the acquisition of new branches of the flavonoid pathway in different species. The overlap in the sets of induced genes could be indicative of their ancestral function and supports stepwise evolution toward regulating new pathway branches and acquiring new products in plant tissues.

Anthocyanins are vacuolar pigments that confer red-violet colors to plant tissues. Pigmentation patterns result from spatio-temporally regulated anthocyanin synthesis and degradation. Mutational inactivation of a conserved MYB-bHLH-WDrepeat-WRKY transcriptional complex (MBWW) causes degradation of anthocyanins and ‘fading’ of flower color via a pathway that involves FADING (FA) . Here we show that FA encodes a vacuolar membrane Fe-reductase-oxidase that promotes anthocyanin degradation. In wild type petals anthocyanins in the central vacuole (CV) are stable, because FA-GFP is upheld in small vacuoles (vacuolinos) and kept away from the CV, indicating that vacuolinos act as gatekeepers in protein trafficking. In cells lacking vacuolinos, including mbww- mutant petals, FA-GFP reaches the CV and triggers anthocyanin degradation. Virus-induced gene silencing (VIGS) of an FA-homolog in pepper fruits prevented the “fading” of anthocyanins during fruit maturation. These findings provide new insights to breed ornamental and food crops with increased anthocyanin-content and enhanced nutritional value of edible parts.

Petunia hybrida is a plant model system used by many researchers to investigate a broad range of biological questions. One of the reasons for the success of this organism as a lab model is the existence of numerous mutants, involved in a wide range of processes, and the ever-increasing size of this collection owing to a highly active and efficient transposon system. We report here on the origin of petunia-based research and describe the collection of petunia lines housed in the University of Amsterdam, where many of the existing genotypes are maintained.

Flavonoids are plant pigments that provide health benefits for human and animal consumers. Understanding why domesticated crops have altered pigmentation patterns and unraveling the molecular/genetic mechanisms that underlie this will facilitate the breeding of new (healthier) varieties. We present an overview of changes in flavonoid pigmentation patterns that have occurred during crop domestication and, where possible, link them to the molecular changes that brought about the new phenotypes. We consider species that lost flavonoid pigmentation in the edible part of the plant at some point during domestication (like cereals). We also consider the converse situation, for example eggplant (aubergine), which instead gained strong anthocyanin accumulation in the skin of the fruit during domestication, and some varieties of citrus and apple that acquired anthocyanins in the fruit flesh. Interestingly, the genes responsible for such changes are sometimes closely linked to, or have pleiotropic effects on, important domestication genes, suggesting accidental and perhaps inevitable changes of anthocyanin patterning during domestication. In other cases, flavonoid pigmentation patterns in domesticated crops are the result of cultural preferences, with examples being found in varieties of citrus, barley, wheat, and maize. Finally, and more recently, in some species, anthocyanins seem to have been the direct target of selection in a second wave of domestication that followed the introduction of industrial food processing.

The sour taste ofCitrusfruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show thatCitrushomologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting “acidless” varieties. Down-regulation ofCitPH1andCitPH5is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activatingPH1andPH5in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste inCitrusand other fruits.

We identified three novel members of the R2R3‐MYB clade of anthocyanin regulators in the genome of the purple flowering Petunia inflata S6 wild accession, and we called them ANTHOCYANIN SYNTHESIS REGULATOR (ASR). Two of these genes, ASR1 and ASR2, are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species P. axillaris N and P. parodii, in the red flowering P. exserta, and in several Petunia hybrida lines, including R27 and W115. P. violacea and other P. hybrida lines (M1, V30, and W59) instead harbor functional copies of the ASR genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3‐MYB genes strongly suggest that the ASR genes cluster is a duplication of the genomic fragment containing the other three R2R3‐MYB genes with roles in pigmentation that were previously defined, the ANTHOCYANIN4‐DEEP PURPLE‐PURPLE HAZE (AN4‐DPL‐PHZ) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different Petunia accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two‐hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B‐HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in P. hybrida lines, but ASR1 appeared to be the most effective. The expression patterns of ASR1 and ASR2 cover several different petunia tissues with higher expression at early stages of bud development. In contrast, ASR3 is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel ASR MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.

Summary: Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal‐specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu‐hypertolerance of a S. vulgaris copper mine population. Cu‐hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu‐polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms.

Petunia hybridais a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents,P. axillarisN andP. inflataS6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that thePetunialineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value ofPetuniaas a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.