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

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

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

Regeneration and interspecific somatic hybridization in Allium for transfer of cytoplasmic male sterility to leek

The vast majority of the present day leek cultivars is of poor quality. The genetic constitution of leek makes it a difficult crop to breed and consequently mass, or family selection methods, both of which have a low efficiency, are mainly used. F 1 hybrid breeding seems the appropriate strategy for improvement of leek. With such a breeding system a higher uniformity of the crop, better fixation of desirable traits, such as pest and disease resistance and a better exploitation of heterosis effects can be achieved. Large-scale seed production of hybrid cultivars requires a hybridization system based on cytoplasmic male sterility (CMS), because of the considerable advantage of this system in maintaining the male sterile parent line. Since no source of CMS has yet been found in leek or related forms of A. ampeloprasum , several researchers are focused on introducing cytoplasmic male sterility into leek. In the present thesis the possibility of introducing CMS into leek via somatic hybridization with A. cepa as CMS donor was investigated. Successful application of such a technique requires an efficient system for regeneration of plants from protoplasts. At the beginning of the research project in 1990 such a protoplast-to-plant system for leek was not available. Therefore, substantial effort was directed towards the development of efficient plant regeneration methods from embryogenic callus cultures, suspension cultures and protoplasts.Initially, much attention was focused on development of regenerative callus cultures from different explants and cultivars. The highest compact callus response was obtained when mature, zygotic embryos were cultured on MS medium, containing 30 g/l sucrose and 1 mg/l 2,4-D. Significant differences were found between the cultivars and accessions for shoot formation frequency. In addition, a genotype-dependent response of leek embryo explants for the formation of shoots was evident.In contrast to plant regeneration from compact embryogenic callus of leek, which is efficient and easy to achieve, the establishment of suspension cultures from this type of callus was less successful. Although compact callus cultures in liquid medium retain their ability to form somatic embryos and shoots for a long period, they do not become a finely-dispersed suspension culture. For this purpose, a new friable embryogenic type of callus was induced on immature embryos instead of mature embryos. This friable callus comprised numerous globular embryoids, embedded in mucilage and is highly regenerative when plated on a cytokinin containing medium. It was found that the developmental stage of the immature embryo and the genotype of the donor plant significantly influenced the callus response. Characterization of the two callus types by a histological examination revealed striking differences and supported the suggestion that friable callus is more suitable for initiating suspension cultures than compact callus. A stringent selection within these friable callus cultures was necessary to obtain highly-embryogenic suspension cultures.A procedure was described for the isolation, culture and regeneration of plants from protoplasts derived from these embryogenic suspension cells. Imbedding in alginate was an important factor in increasing the plating efficiency. The regeneration frequency of the protoplast-derived calli was primarily affected by the type of callus that developed. The protoplast-to-plant system described was reproducible for at least three genotypes. Plants were regenerated within 6 months after protoplast isolation.Utilising this regeneration procedure for protoplasts, a method for symmetric hybridization between leek and onion as a CMS donor was set up. The fusion experiments yielded large numbers of hybrid calli and plants. The aneuploid status of the hybrid plants could be explained by the use of leek protoplasts, derived from an aneuploid suspension culture. This also implied that using cell suspensions as a protoplast source for fusion remains a restrictive factor in the establishment of a successful hybridization system for leek. The leaf morphology of the hybrids was intermediate between the two parents. It appeared that most of the hybrids possessed leek chloroplasts and a rearranged mitochondrial genome of both parents, but with a predominance of mtDNA fragments from leek.With the methods described in this thesis the first steps to transfer CMS to leek have been realized. The results described here show that a cytoplasm of onion can be successfully transferred to leek via somatic hybridization. Future research should focus on further improvement of the system. For this, optimization of some aspects of the regeneration process and developing an efficient selection system for the desired hybrids, containing the onion specific CMS mtDNA sequences should be accomplished. The research has shown that somatic hybridization has a high potential to obtain CMS leek plants.</p

Metabolic changes in Arabidopsis thaliana plants overexperssing chalcone synthase

The study has shown that it is possible to introduce the heterologous CHS gene in Arabidopsis thaliana and common multicopies of transgenes containing plants were obtained. Analysis of the change in metabolome of CHS transgenic plants, high expression transgenic lines can be identified by markers such as flavonoids and phenylpropanoids. It is also clear that UV-A/blue light stress does not further increase the levels of these marker compounds in CHS transgenic Arabidopsis plants, whereas in wild type plants such a treatment results in increased levels of these compounds, in fact similar to that in the transgenic plants. There are certain physiological limitations in the accumulation of certain products. This thesis starts with a review of the function of CHS in plants and especially in plant resistance (Chapter 2). Chapter 3 deals with the work on Agrobacterium-mediated transformation of heterologous chalcone synthase in Arabidopsis thaliana Col. 0. The effect of overexpression of CHS on the transcriptional level is discribed in this chapter. The activity of the CHS enzyme in the transgenic plants is reported in Chapter 4. In Chapter 5 metabolic profiling of Arabidopsis thaliana using nuclear magnetic resonance spectroscopy (NMR) is described. In this chapter the primary and secondary metabolites of Arabidopsis thaliana Col. 0 which can be detected by NMR are reported. Chapter 6 reports the metabolic profiling of CHS transgenic Arabidopsis. Metabolomic changes upon UV-A/blue light treatment of Arabidopsis thaliana were investigated (Chapter 7). Chapter 8 deals with the study of the effect of the non-pesticide chemical, Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) on the Arabidopsis metabolome. Finally, the general summary and discussion of thesis are given in Chapter 9.LEI Universiteit LeidenVietnamese governmentMetabolemic

Genetic control of anthocyanin pigmentation in Antirrhinum flowers

The genus Antirrhinum (commonly known as snapdragons) contains more than twentyfive recognised species. The genus has been divided into three morphological subsections: Antirrhinum, Streptosepalum and Kickxiella (Rothmaler, 1956). One of the major characteristics distinguishing the three subsections is flower colour. Most species in subsection Antirrhinum have dark pink or yellow flowers, Kickxiella species are white or pale pink and Streptosepalum species have yellow or pale pink flowers. All Antirrhinum species can be crossed to produce fertile hybrids which allow the genes that underlie their differences to be identified. I used quantitative trait locus (QTL) analysis on hybrids of A. majus (dark magenta flowers) and A. charidemi (pale-pink flowers) to map genomic regions underlying differences in flower colour. This identified two major-effect loci, in Linkage Group 3 (LG3) and LG7, that explained most of the differences between these species. I used near-isogenic lines (NILs) to further test involvement of two candidate genes - Rosea (Ros) in LG3, which encodes a regulator of the anthocyanin biosynthesis pathway (ABP) and Incolorata (Inc) in LG7 which encodes a rate-limiting enzyme of the ABP. In both cases, the A. majus allele increased pigmentation. Sequence differences between Ros alleles of A. majus, A. charidemi and A. molle (a Kickxiella species with white flowers) suggest that A. molle carries a ros loss-of-function mutation and that a transposon insertion in the ROS promoter might contribute to differences in expression between A. majus and A. charidemi. Ros genotypes were found to be strongly correlated with pigmentation in the corolla tube in A. majus x A. charidemi hybrids, and to a lesser extent with corolla lobe pigmentation, although NILs suggested that ROS did not correspond to the major-effect QTL indentified in LG3. I also mapped a minor-effect QTL for tube pigmentation to a region of LG4 containing the ABP structural gene Candica. Analysis of NILs revealed that Inc was not the second major-effect QTL mapped to LG7, although sequence differences were detected between Inc alleles of A. majus and A. charidemi. I was further able to narrow down the region containing the second LG7 major-effect QTL to an interval of 11 cM, between two molecular markers, which could be used to determine the likely QTL genotypes of segregating NILs. Surprisingly, several ABP genes, particularly Nivea, Inc and Pallida, were expressed at higher levels in pale flowers that were homozygous for the A. chardemi QTL allele than in their dark flowered siblings that carried an A. majus allele. This suggests that ABP genes might be up-regulated in pale flowers as part of a negative feedback mechanism. Two potential roles of the LG7 QTL are considered 1) its requirement for anthocyanin modification or transport to the vacuole, so that a build-up of cytosolic anthocyanins or their break-down products in pale flowers increases structural gene expression but cannot compensate for the overall reduction in anthocyanin, or 2) a role in promoting production of flavonols at the expense of anthocyanins

Postharvest Treatment Effects on Health Promoting Properties and Flavonoid Genes of Grapefruit

Certain naturally occurring compounds present in grapefruit have shown several health promoting properties such as anticancer, antioxidant, antimicrobial and cardioprotective. However, the levels of these naturally occurring compounds are influenced by several preharvest and postharvest factors. The primary objective of the current research is to determine the effects of different postharvest treatments on naturally occurring compounds present in grapefruit. In the first and second studies, effect of ethylene degreening on ‘Star Ruby’ and ‘Rio Red’ grapefruit natural compounds was investigated. Degreening helped to improve grapefruit color while maintaining the health promoting compounds. Significant influence of ethylene treatment was observed on flavonoids and furocoumarins. The third and fourth study examined the changes in ‘Star Ruby’ and ‘Rio Red’ grapefruit under cold storage and with low temperature conditioning treatment. Conditioning the fruits prior to cold storage reduced the incidence of chilling injury and conditioned fruits had similar or higher levels of health promoting compounds compared to fruits stored under cold storage without conditioning. However, results suggest that for short term storage of few weeks, storing fruits at 11⁰C was better for retention of most naturally occurring compounds. The fifth study focused on use of modified atmosphere packaging (MAP) to maintain grapefruit quality and nutritional properties. Two MAP films, micro-perforated (modified oxygen, carbon dioxide and humidity levels) and macro-perforated (modified humidity only), were investigated for their influence on naturally occurring compounds and fruit quality under a prolonged storage period. MAP treatments did not have significant effect on ascorbic acid, limonoids and fruit quality parameters such as total soluble solids, acidity, fruit taste, decay and disorders. Based on our research, use of MAP films is recommended to maintain fruit quality and health promoting compounds. The sixth and seventh studies focused on variation in health promoting compounds, specifically, flavonoid pathway gene expression and volatile compounds present in ‘Rio Red’ grapefruit during fruit development and maturity. Overall expression of flavonoid pathway genes and related flavonoid content decreased as the fruits developed and matured, with the levels being highest in immature fruits harvested in June. Levels of limonene decreased as the fruits developed from June to April; while, nootkatone levels increased with fruit development and maturity. The eighth study investigated the effect of different ethylene concentrations on flavonoid pathway gene expression and related flavonoids in grapefruit. Significant effect of ethylene concentration was observed on flavonoids and furocoumarins as well as the genes involved in the flavonoid biosynthesis. Overall, significant influence of different postharvest treatments was observed on grapefruit health promoting compounds. We believe that this research will be helpful to the citrus industry to optimize the postharvest treatments in order to maximize their benefits in regards to fruit quality and nutritional value

Differential responses of bean (Phaseolus vulgaris L.) to elicitor fractions from Colletotrichum lindemuthianum

Polysaccharide elicitor preparations from culture filtrate and cell walls of Colletotrichum lindemuthianum had broadly similar monosaccharide compositions. Treatment with culture filtrate or cell wall elicitors had no effect on the electrolyte leakage from bean leaf slices or mesophyll cells and resulted in similarly reduced viability of suspension cultured bean cells. Both elicitor preparations had similar effects on the induction of extractable activities, synthesis and mRRA activities of phenylalanine ammonia-lyase, chalcone synthase and chalcone isomerase, on the induction of synthesis and mRRA activities of chalcone synthase multiple subunit isoforms in bean cell suspension cultures and on the patterns of active phenylalanine ammonia-lyase and chalcone synthase multiple forms separated by chromatofocussing. Both qualitative and quantitative differences were observed in the effects of the two elicitor preparations on the accumulation of 5-deoxy and 5-hydroxy isoflavonoids, deposition of wall-bound phenolics and on the levels of free and esterified hydroxycinnamic acids. The two elicitor preparations induced prolyl hydroxylase activity although only the cell wall elicitor induced accumulation of hydroxyproline in the cell walls of suspension cultured bean cells. In addition, although the overall patterns of polysomal mRRA activities in bean cell cultures following treatment with cell wall or culture filtrate elicitors were broadly similar, distinct differences were observed in the effects of the two elicitor preparations on the induction/reduction of the activities of mRNAs encoding a number of polypeptides as shown by two-dimensonal gel electrophoresis.An unaltered monosaccharide composition and ability to induce phenylpropanoid biosynthetic pathway enzymes were associated with all fractions obtained after chromatography of culture filtrate elicitor on the basis of size and charge. Fractions obtained after affinity chromatography on Concanavalin A-Sepharose had different monosaccharide compositions but exhibited similar effects on the viability of cultured cells and on the induction of synthesis of phenylalanine ammonia-lyase, chalcone synthase and chalcone isomerase as did the crude culture filtrate elicitor. However, although the three Concanavalin A-Sepharose-purified fractions induced chalcone synthase activity to higher levels than the crude culture filtrate elicitor preparation, little or no induction of phenylalanine ammonia-lyase and chalcone isomerase activities was observed following treatment with the Concanavalin A-unbound and a-methyl mannoside-eluted fractions. In addition, the Concanavalin A-Sepharose-purified fractions induced the activities of mRNAs encoding phenylalanine ammonia-lyase and the most basic chalcone synthase subunit isoform to higher levels than the culture filtrate elicitor. The three Concanavalin A-Sepharose-purified fractions had broadly similar effects on the overall patterns of protein synthesis in vitro although their effects were clearly different from those obtained with crude elicitor preparations. Possible physiological importance of putative multielicitar components with non-identical biological activities and their value in elucidating biochemical control mechanisms underlying regulation and co-ordination of host gene expression are discussed.<p