The Amsterdam petunia germplasm collection: A tool in plant science

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

Designing a novel tomato ideotype for future cultivation in space manned missions

Introduction: Methods for production of fresh, health food are needed in view of long-term, deep-space manned missions. To this end, crops tailored for better performance under non-terrestrial conditions may be obtained by the exploitation of biochemical patterns related to specialized metabolites known to confer protection against environmental challenges and to be beneficial to human health.Methods: In this work, for the first time, MicroTom plants have been engineered specifically for agrospace applications to express PhAN4, a MYB-like transcription factor able to regulate the biosynthesis of anthocyanins that influence tomato genes possibly involved in agrospace-relevant functions. Results:PhAN4 engineering underpinned the genetic background of the dwarf tomato MicroTom while maintaining yield and photosynthetic capacity. PhAN4 expression resulted in the accumulation of anthocyanins and polyphenols, a differential carotenoid profile, increased antioxidant scavenging capacities of fruits compared to the original genotype. Improved ability to counteract ROS generation and to preserve plant protein folding after ex-vivo gamma irradiation was observed.Discussion: These results highlights that the manipulation of specific metabolic pathways is a promising approach to design novel candidate varieties for agrospace applications

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is 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. axillaris N and P. inflata S6. The current 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 Petunia lineage has experienced at least two rounds of paleohexaploidization, the older gamma hexaploidy event, which is shared with other Eudicots, and the more recent Solanaceae paleohexaploidy event that is shared with tomato and other Solanaceae species. Transcription factors that were targets of selection during 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 color patterns and pollination systems. The high quality genome sequences will enhance the value of Petunia as a model system for basic and applied research on a variety of unique biological phenomena

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

The petunia loci anthocyanin1 (an1), an2, an4, and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1, an2, and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4, encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1–GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function

PH4 of Petunia Is an R2R3 MYB Protein That Activates Vacuolar Acidification through Interactions with Basic-Helix-Loop-Helix Transcription Factors of the Anthocyanin Pathway

The Petunia hybrida genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color, increased pH of petal extracts, and, in certain genetic backgrounds, the disappearance of anthocyanins and fading of the flower color. PH4 encodes a MYB domain protein that is expressed in the petal epidermis and that can interact, like AN2, with AN1 and the related BHLH protein JAF13 in yeast two-hybrid assays. Mutation of PH4 has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5, encoding a protease-like protein of unknown biological function. Constitutive expression of PH4 and AN1 in transgenic plants is sufficient to activate CAC16.5 ectopically. Together with the previous finding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated, this suggests that AN1 activates different pathways through interactions with distinct MYB proteins

The genesis of vacuolinos, a membrane compartment on the route to the vacuole

In plants that rely on animals for the transfer of pollen, the cells of the epidermis of flower petals are specialized to attract pollinators. The differentiation program of these cells includes the synthesis of anthocyanin pigments, their accumulation in the central vacuole of these cells, the display of the color via modulation of the pH in the lumen of the vacuole, the building of a “papillary” cell shape that provides a handy landing surface to the pollinator and contributes to the final color of the petal by affecting the refraction angle of the light. All these mechanisms are controlled by a group of transcription factors that activate different sets of target genes (1). A set of genes has been shown to encode for the biosynthetic enzymes of the anthocyanin pathway, while two other target genes encode for two different P-ATPases (PH1 and PH5) that together acidify the lumen of the central vacuole and some 10 other target genes still have not been assigned a function (2); (3). Localization studies for the tonoplast proteins PH1 and PH5 has brought to the discovery that in petal epidermal cells, the sorting of proteins to the vacuole includes an intermediate organelle on which proteins reside before reaching the central vacuole. As this organelle looks like a small vacuole and is marked by vacuolar proteins (including vacuolar SNAREs), we called it “vacuolino”. In transient expression assays, the membrane of vacuolinos is marked by the PH5-GFP fusion 24 hours after cell transformation, while the tonoplast of the central vacuole shows fluorescence only ~48 hrs after transformation. In different cell types (like leaf cells or unpigmented petal mesophyll cells) vacuolinos are absent and PH5-GFP appears on the tonoplast already 24 hrs after transformation. All other vacuolar proteins we have observed in their sorting pathway to the vacuole in petal epidermal cells, reach the final destination after a short permanence on the vacuolinos. Mutants for any of the above mentioned transcription factors do not show vacuolinos implying that a set of their target genes is involved in the genesis of these compartments. We have used different methods to compare the transcriptomics of petals mutant for each of the transcription factors involved in the presence of vacuolinos (AN1, PH3 and PH4) and we have isolated a number of target genes of these regulators to isolate candidate genes involved in vacuolinos biogenesis. We are now isolating mutants for each of these genes by the screening of petunia BLASTABLE collections of transposon insertions and by RNAi technology. The first three genes for which we could see disappearance of vacuolinos in the mutants are involved in different steps of the vacuolino pathway to the vacuole: - in mutants for one of this gene vacuolar proteins directly go to the vacuole without passing through the vacuolino (like in mesophyll cells), - in another they get stuck in small vesicle-like structures, - in the third the markers remain on the vacuolinos as vacuolinos seem not to be able to fuse to the central vacuole. We are at the moment also studying when the vacuolinos appear during bud development as this could give some clue of their function. Analysis of the localization of vacuolar GFPs in epidermal petal cells of buds at different developmental stages shows that: - in young buds, the petal epidermal cells are rather small (compared to open flowers) and have small vacuoles with huge folded tonoplast - during development the cells enlarge and the tonoplast unfold - -vacuolinos only show up after flower opening, when petals are approaching senescence All three these genes encoded unexpected players in the genesis, physiology and fusion of membrane compartments. 1. Koes R, Verweij CW, & Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 5:236-242. 2. Faraco M, et al. (2014) Hyperacidification of Vacuoles by the Combined Action of Two Different P-ATPases in the Tonoplast Determines Flower Color. Cell reports 6(1):32-43. 3. Verweij W, et al. (2008) An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nature cell biology 10(12):1456-1462