Assessing Anthocyanin Biosynthesis in Solanaceae as a Model Pathway for Secondary Metabolism

Solanaceae have played an important role in elucidating how flower color is specified by the flavonoid biosynthesis pathway (FBP), which produces anthocyanins and other secondary metabolites. With well-established reverse genetics tools and rich genomic resources, Solanaceae provide a robust framework to examine the diversification of this well-studied pathway over short evolutionary timescales and to evaluate the predictability of genetic perturbation on pathway flux. Genomes of eight Solanaceae species, nine related asterids, and four rosids were mined to evaluate variation in copy number of the suite of FBP enzymes involved in anthocyanin biosynthesis. Comparison of annotation sources indicated that the NCBI annotation pipeline generated more and longer FBP annotations on average than genome-specific annotation pipelines. The pattern of diversification of each enzyme among asterids was assessed by phylogenetic analysis, showing that the CHS superfamily encompasses a large paralogous family of ancient and recent duplicates, whereas other FBP enzymes have diversified via recent duplications in particular lineages. Heterologous expression of a pansy F3050H gene in tobacco changed flower color from pink to dark purple, demonstrating that anthocyanin production can be predictably modified using reverse genetics. These results suggest that the Solanaceae FBP could be an ideal system to model genotype-to-phenotype interactions for secondary metabolism

A model system for comparative research

Research today aims to analyse the development of plant processes over evolutionary time. To obtain a representative view, a range of plant species covering at least the crucial nodes in phylogeny must be selected for an in depth analysis. Here we present Petunia as one of the available systems: as a representative of the Solanaceae it has the advantages of good culture conditions and the availability of a range of materials, techniques and strategies that can be used to research an interesting and diverse set of questions. Why an array of model systems? Plants have evolved to adapt to environmental cues and to conquer and establish new ecological niches. Changes in gene functions and their controlling networks over evolutionary time have enabled the development of innovative structures, such as the reproductive organs in gymnosperms and angiosperms and the many different plant-plant, plant-animal and plant-microbe interaction strategies. Such changes in genetic make-up have ultimately led to the development of the w250 000 extant plant species. Whereas systems biology approaches focus on a single species to obtain ultimately a detailed and integrated view of the function of all genes within a single organism, comparative biology has a broader view on (dis)similarities in specific developmental and functional pathways across a variety of plant species One of the challenges ahead for plant scientists is to analyse and to understand the level of diversification that has allowed for the astonishing degree of diversity among the members of the plant kingdom. To understand these developmental differences between species, we must compare gene function development for a range of species covering the evolutionary diversity of all species. It is clear that the scope of plant research needs to be broadened; we could begin with identifying a well-spread set of taxa that would enable us to further unravel the crucial nodes of evolutionary events that have shaped the diversity we see today in plant morphology and in the modes of reproduction and survival. The most advanced model plant species is Arabidopsis but, in spite of its success, it cannot represent all extant species [1], if only for the reason that it is not representative for 'all' plant processes and interaction strategies. Moreover, analysis above the ecotype level is hampered by the difficulty in obtaining fertile progenies from crosses between Arabidopsis thaliana and related species In June 1980, the interim steering committee of the Plant Molecular Biology Association published its first PMB newsletter. In the foreword, Petunia and Lycopersicon were mentioned as outstanding model systems (but with the remark that 'The main objection to Petunia is that it will never be an important food source.'). Among other reasons, 'the availability of true haploids, its easy tissue culture and the quality of leaf tissue for biochemical studies and macromolecule purification' were mentioned, aspects that remain important to this day. The second issue, which featured Petunia and Lycopersicon as model systems on its cover, was filled almost entirely with information on Petunia and Lycopersicon model systems. Nevertheless, in the first issue, Arabidopsis was also recommended, among others, as a good alternative model system, which was a fairly accurate prediction. Here we propose Petunia as one of the available comparative eudicot systems. We will detail its historical setting, the major technological possibilities of using Petunia as a model system and the main areas of current research. We will of course have to balance the use of Petunia with models for other groups, for example, rice for the grasses, poplar and Eucalyptus for trees and Medicago and Lotus for the legumes

Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida

<p>Abstract</p> <p>Background</p> <p>Identification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis.</p> <p>Results</p> <p>In an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were <it>EF1α </it>in Mitchell and <it>CYP </it>in V30, whereas the least suitable gene was <it>GAPDH </it>in both lines.</p> <p>Conclusions</p> <p>The least adequate gene turned out to be <it>GAPDH </it>indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.</p

Developement and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera hybrida

RNA silencing is a conserved mechanism that occurs in a broad range of eukaryotes, which is regulated by small RNAs (sRNAs). RNA silencing operates to control gene expression and maintain genome integrity. Virus-induced gene silencing (VIGS) in plants is a natural antivirus mechanism that has adapted from the general RNA silencing system. To counter the antivirus RNA silencing, plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs). Nowadays VIGS is usually referred to as the technology that uses recombinant viruses to knock down the expression of plant endogenous genes. Gerbera hybrida (gerbera) is a model species in the family of Asteraceae. As a highly heterozygous species, gerbera lacks efficient functional genetic approaches other than gene transfer. The aim of the present study was to develop a Tobacco rattle virus (TRV, genus Tobravirus) induced gene silencing system for gerbera, and use TRV VIGS to characterize functions of chalcone synthase (CHS) encoding genes in the plant. Preliminary VIGS experiments on the cultivar Terraregina, by syringe infiltration and applying previously developed TRV vectors, did not result in visible VIGS phenotypes due to the absent of TRV RNA2 in the up non-infiltrated leaves. Consequently, I first aimed to study the mechanism of TRV VIGS, and tried to develop new VIGS vectors based on TRV RNA1. I investigated the role of two important TRV proteins of the 16K VSR and the 29K movement protein (MP) on TRV infection and TRV VIGS, and developed TRV RNA1 based VIGS vectors. For accomplishing this, a series of TRV RNA1 mutants have been constructed to disrupt the 16K, or to replace its 29K with Tobacco mosaic virus (TMV, genus Tobamovirus) 30K MP. TRV RNA1 vector, carrying a fragment of the gene encoding Nicotiana benthamiana PDS to replace part of the 16K sequence, induced PDS gene silencing systemically in N. benthamiana. However, this has found to be less efficiently than the original TRV VIGS system when the wild-type RNA1 and RNA2:PDS were used. The infection experiments demonstrated that 16K was required for TRV long distance movement, and helped in maintaining the integrity of the TRV RNA2 genome. In addition, TRV 29K alone did not suppress RNA silencing in the co-infiltration assay, but it could suppress RNA silencing in the context of RNA1 replication. TRV 29K may be the first VSR whose silencing suppression functions are found to be directly linked to viral replication. The original TRV vector system was finally adopted for VIGS in gerbera. TRV VIGS was optimized for gerbera by screening for TRV sensitive cultivars and by improving its inoculation methods. Intensive gene silencing phenotypes were achieved both in green tissues and in floral tissues, demonstrated by knocking down genes involved in isoprenoid biosynthesis (phytoene desaturase: GPDS; H and I subunits of Mg-chelatase: GChl-H and GChl-I), flower pigmentation (chalcone synthase: GCHS1), and flower development (GLOBOSA-like MADS domain transcription factor: GGLO1). Unexpectedly, a gerbera polyketide synthase encoding gene, G2PS1, that has no apparent connections to the carotenoid or chlorophyll biosynthesis, was knocked down by the photo-bleaching that was induced by the silencing of GPDS, GChl-H and GChl-I, or by the herbicide norflurazon. We have demonstrated for the first time that the using of VIGS in an Asteraceaeous species. Our data also suggested that the selection and use of a marker gene for VIGS should be strictly evaluated. A new CHS encoding gene, GCHS4, was characterized in gerbera. Together with the two previously identified GCHS1 and GCHS3, gerbera CHSs are represented by a three-gene family. Each gerbera CHS shows a distinct expression pattern. GCHS3 is particularly expressed in gerbera pappus. In partnership with the concomitantly expressed GCHS1, they are involved in the biosynthesis of colorless flavonoids. GCHS4 is the only CHS that is naturally expressed in the leaf petiole and inflorescence scape, and it is responsible for cyanidin biosynthesis in those tissues. GCHS4 is also the only CHS that was induced by environmental stresses in the leaf blade. Both GCHS1 and GCHS4 are markedly expressed in gerbera petals, and GCHS4 mRNA actually takes the majority of CHS mRNAs in the later stages of petal development. Nonetheless, VIGS experiments, by target silencing GCHS1 or GCHS4 independently, demonstrated that GCHS1 is the predominant functional CHS in gerbera petals. Thus, GCHS4 in gerbera petals seems to be regulated post-transcriptionally. In conclusion, the results of this study shed new light on the mechanism of TRV VIGS. The established TRV VIGS system provides a valuable tool for functional genomics in gerbera

The influence of R2R3-MYB transcription factors in Campanula medium and Petunia hybrida

Flower color is one of the most important traits for ornamental plants. Anthocyanin is the main pigment in flowers and the vegetative tissue in most higher plants. Both wild campanulas and petunias have blue/purple flowers because of the delphinidin-based anthocyanins. However, most of the flower color in the genus Campanula is mostly blue, rarely white, and a very few species are pink, while modern commercial petunias are known for their wide range of flower color in different patterns. Many kinds of research have proven that the R2R3-MYB transcription factors are responsible for regulating anthocyanin biosynthesis in different tissues. In Petunia spec., ANTHOCYANIN-2 (AN2) is considered to be the major contributor to coloration in the petal limb, while several other paralogs are involved in other tissues or stress responses. Unlike petunias, which have a convenient system for regeneration and a stable genetic plant transformation, there are no related reports for C. medium. Therefore, an efficient protocol for regeneration and Agrobacterium‑mediated transformation based on leaf disk by using GFP as the reporter gene for C. medium was established in the first part. In the second chapter, two strategies were designed to change the anthocyanin pattern in C. medium. The first is the redirection of delphinidin-based anthocyanins to pelargonidin-based anthocyanins in a blue cultivar. The second strategy was to improve the pigmentation in a pink-flowering variety of C. medium by overexpression of an R2R3-MYB transcription factor (PhAN2). In the third chapter, the same AN2 overexpression vectors used for Campanula and another flowerspecific guided AN2 constructs were also transferred into an an2- genetic petunia cultivar. The results indicated that overexpression of AN2 leads to various physiological changes in addition to increased pigmentation. The mechanism of the flower limb coloration of phenotypically "darkly-veined"petunias, and the roles of different R2R3-MYB genes were characterized in the last part. Overall, several conclusions obtained in this dissertation: (i) genetic transformation of C. medium is available; (ii) PhAN2 and PhAN4 can activate anthocyanin biosynthesis in several tissues of Campanula and Petunia and the role of PhAN2 in plant growth physiology may be diverse; (iii) the restoration of anthocyanins in the petals of commercial "dark-veined" petunias from a whiteflowered ancestor is achieved by up-regulation of AN4 as part of a long-term breeding process by humans rather than by natural selection

Identification of novel genes involved in petunia flower development using transcript profiling and reverse genetics

[ENG] Petals are a key element on plant life cycle as, in many species, they attract pollinators, thus aiding to reproduction. Furthermore, they have economic importance in ornamental crops. In the present study, petal transcriptional patterns were compared within the ower organs in Arabidopsis thaliana. It was found that catalytic molecular functions were overrepresented in petals. A shortlist comprising the top ten di_erentially expressed genes in petals were mapped to the model species with industrial value Petunia hybrida, and further downregulated by RNAi. The silencing phenotypes found permitted to assign functions in petal development to seven novel genes: when silenced, they triggered alterations on ower size and shape (PhCYP76, PhNPH3, PhFeSOD, PhXTH, PhCYP96 and PhWAK), petal smoothness (PhPRA), color (PhNPH3 and PhWAK) and symmetry (PhCYP76). Pleiotropic phenotypes were found, such as changes in root morphology and leaf color (PhCYP76), ower number, capsule and seed morphology (PhCYP96) and plant height (PhCYP76 and PhCYP96). To accomplish the experimental design, three methods were developed. First, the \pESTle" management system that assembles, annotates, stores and serves expressed sequence tag data. Second, a reference gene selection for real time PCR experiments that includes a new method for stability estimation based on rank aggregation of published algorithms, and concludes that a normalization factor with two members of EF1_, SAND, CYP or RAN1 is stable enough under most conditions. And third, a PCR e_ciency estimator based on amplicon characteristics which allows e_ciency-driven primer design in a Web tool. [SPA] La corola es un elemento básico en el ciclo de vida vegetal puesto que, en muchos casos, atrae a polinizadores que intervienen en su propagación. Además, posee un valor económico en especies ornamentales. En el presente trabajo se realizó un análisis comparativo contrastando el transcriptoma de pétalos frente al resto de órganos orales de Arabidopsis thaliana. Como resultado se observó la preponderancia de genes involucrados en funciones catalíticas. Aún de dilucidar el rol de estos genes, se escogieron aquellos nueve con mayor expresión diferencial y se silenciaron de forma estable mediante ARN de interferencia. De esta forma, el análisis fenotípico ha permitido asignar un papel a siete nuevos genes: al ser silenciados, provocan alteraciones en la forma y tamaño (PhCYP76, PhNPH3, PhFeSOD, PhXTH, PhCYP96 y PhWAK), textura (PhPRA), color (PhNPH3 y Ph- WAK) y simetría (PhCYP76) de los pétalos; así como el color de las hojas y la morfología radicular (PhCYP76), el número de ores y la altura de la planta (PhCYP76 y PhCYP96). Aún de respaldar el diseño experimental se desarrollaron tres métodos. En primer lugar, el gestor _pESTle_, que aúna el ensamblaje, anotación, almacenamiento y difusión de ESTs. En segundo lugar, la selección de genes de referencia para PCR en tiempo real, que reúne un nuevo método para su evaluación basado en la agregación de rangos; y la descripción de la pertinencia de emplear un factor de normalización con dos genes de entre EF1_, SAND, CYP y RAN1 en la mayor parte de los casos. Finalmente, se produjo el estimador _pcrE_ciency_, que proporciona un entorno de diseño de cebadores que simultáneamente predice su eficiencia.Universidad Politécnica de Cartagen