The topics in my group concern the biosynthesis, regulation and biological role of volatiles for plants in their environment. In the last decade Petunia hybrida has emerged as the model of choice to study volatile benzenoid and phenylpropanoid synthesis, emission and regulation. These volatiles are synthesized predominantly in the corolla limb and emission is highly regulated, with a circadian rhythm, during corolla development, pollination and senescence. With all the biochemical and molecular tools available much of our understanding of volatile benzenoid/phenylpropanoid has been obtained with Petunia. The knowledge obtained with this system is being applied to tomato fruit and strawberry volatile production and thereby taste. In addition, we use tomato as a model system to study the role of terpenoids in its interaction with insects. We focus on terpene synthases expressed in trichomes and have identified several terpenoids that repel or attract whiteflies. Our aim is to engineer the production of these terpenoids for which we have identified trichome specific promoters. We use Arabidopsis for forward genetic screens to identify genes important in the response to the wound-induced C6-volatile E -2-hexenal. We also aim to identify genes that are specifically regulated by E -2-hexenal using a transcriptomics approach. Finally, a relative new topic in my group concerns effectors produced by herbivores that manipulate plant defenses. Together with the IBED at UvA we have discovered the first effectors from spider mites that manipulate the SA defense response. We are now focusing on their interactors in the plant and on effectors from whiteflies and thrips.
We study the biosynthesis of volatile benzenoids in Petunia and the regulation thereof. We have discovered, through a targeted metabolomics and transcriptomics approach, ODORANT1, the first transcription factor that regulates volatile benzenoid synthesis and provided insight in how Petunia flowers developmentally separate color and scent biosynthesis; we have recently discovered that the transcription factor EOBII (Emission of benzenoids II) acts upstream of ODO1. We are currently using Chip-Seq to identify all targets of ODO1 and EOBII. Moreover, we identified a 3-keto-acylthiolase that catalyzes an important step in benzoic acid biosynthesis, a crucial precursor for several benzenoids. Together with the lab of Natalia Dudareva (Purdue) we discovered that an ABC transporter is involved in active transport of some volatile benzenoids/phenylpropanoids.
We are particularly interested in the role of volatile terpenoids in tomato-insect interactions. We have established a role for jasmonate (JA) in the production of volatile terpenoids in tomato upon herbivory and discovered that JA induces the expression of salicylic acid (SA) methyl transferase that converts SA to the volatile methyl salicylate, an volatile crucial in attracting predatory mites as shown by silencing SAMT in tomato. We have also identified and characterized the first monoterpene synthases from tomato that are specifically expressed and induced by JA in trichomes. This was recently followed up by the identification and characterization of all terpene synthases in tomato. Recently we identified the terpenoids that are directly involved in repelling or attracting whiteflies. Through 454 and GS-flex sequencing of trichome cDNAs of wild tomatoes we identified the corresponding terpenoid synthases. We modified cultivated tomato plants to produce the relevant terpenoids using trichome specific promoters that were discovered in my lab and made them more resistant to herbivorous insects. With yeast-1-hybrid we have identified a transcription factor that specifically can upregulate certain terpene synthases in the glandular trichomes of tomato. Furthermore, we have shown that the bHLH MYC1 transcription factor is involved in both the development of type VI glandular trichomes as in regulating terpene synthases in the glandular heads.
Whitefly saliva contains many proteins and since whiteflies suppress certain defenses in plants it is thought that this occurs via effectors, similar to the situation in plant pathogenic bacteria and fungi. Together with the IBED at UvA we have discovered the first effectors from spider mites that manipulate the SA defense response and we have just submitted a manuscript describing these effectors. We are now focusing on on effectors from whiteflies and thrips and their interactors in the plant. We have discovered several plant proteins with which these effectors interact and are perturbing these interactors in planta to determine effect on insect performance.
As a fourth topic we study the role of C6 volatiles as signaling and priming molecules. These C6 volatiles are produced in most, if not all, plants upon wounding, pathogen infection and insect herbivory and can prime the defense of neighboring plants. We have identified the first Arabidopsis mutants that are resistant to E-2-hexenal (her mutants) and have mapped the her1 mutation to discover a role for GABA in this process as well as in the defense against Pseudomonas. We also mapped and cloned the her2 mutant and determined that several WRKY transcription factors may regulate the E-2-hexenal response in Arabidopsis. Moreover, in the Arabidopsis-Pseudomonas interaction, the microbe seems to hijack the C6 volatiles to enhance jasmonate biosynthesis and susceptibility. This work is continued by Silke Allmann.
I work within the Plant Physiology group headed by Prof. dr. Michel Haring. Dr. Petra Bleeker (natural variation of metabolic defense pathways) and Dr. Silke Allmann are the other staff members.
I coordinate the first year course 'Plant Biology' and the MSc course 'Biotic Interactions'. I also give lectures in various other courses. I am chair of the program committee BSc biology and are preparing the specialization Molecular Life Sciences in the third year BSc Biology.
Paula van Kleeff Postdoc
Diana Naalden Postdoc
Sarmina Dangol Postdoc
Pulu Sun Lab manager
Marieke Mastop technician
Milan Plasmeier PhD student
Pietro Zocca PhD student
Andreia Rodrigues technician
Jurre Bleeker, technician
Fariza Shaipulah (Malaysia)
Maaike Boersma (In Holland)
Ahmed Abd-el-Haliem (RijkZwaan)
Suzanne Alves Aflitos-Hoogstrate (UvA)
Sonia Jillings (In Holland)
Silke Allmann (UvA)
Michel de Vries (UvA)
Jiesen Xu (China)
Aldana Ramirez (Gent)
Chun Sui (China)
Carlos Alberto Villarroel (Chile)
Eleni Spyropoulou (UvA)
Alessandra Scala, Frontiers
Arjen van Doorn (Munich))
Petra Bleeker (UvA)
Julian Verdonk (WUR)
Merijn Kant (UvA, IBED)
Chris van Schie (ENZA Seeds)
Doerte Klaus (Toulouse)
Chunlin Liu (China)
Kai Ament (Bejo Seeds)
Alex Van Moerkercke (Sweden)
Rossana Mirabella, Fontiers
Plants are organisms that cannot move so they have evolved several mechanisms to face environmental stress. When a leaf is wounded or is infected by pathogenic bacteria many signals are triggered into the whole plant and the combination of these informations are fundamental for life and death struggle. In our lab we are interested in shedding light in this complex field, plant signal transduction, to reach a basal knowledge on which applied research is established ( develop resistance to stress ). In particular we pointed our attention on E-2-hexenal, a volatile compound, emitted under mechanical or biotic stress, that we have found is related with GABA, another molecule that is induced during several kinds of stress. Our aim is to discover the connection between these two signals and also with the others plant signals that trigger defences, such as jasmonic acid and salicylicacid.
Pseudomonas syringae and Arabidopsis thaliana
are the two components of our biological system. We have several lines of Arabidopsis, for example one which overproduce GABA, another one that has a lower synthesis of E-2-hexenal, and different strains of Pseudomonas that we would like to use to trigger a response in these plants.
To analyse the plant-pathogen system we use a wide range of experimental approaches like molecular biology techniques (DNA/RNA isolation, cDNA synthesis, PCR, quantitative PCR, plant transformation), biochemistry techniques (quantification of plant hormones an signalling molecules with LC/MS and GC/MS), biological assays (Pseudomonas infection of plants and score symptoms on leaves) and microbiology techniques (collect leaves infiltrated with bacteria and check their growth after it).
On commercial tomato three arthropod pests constitute enormous problems for agriculture; aphids, whiteflies and spider mites. In our lab we have been working on tomato-whitefly and tomato-spider mite interactions for many years. The long-term aim of this research is to develop resistance to infestation.
Whitefly ( Bemisia tabaci ) is the vector of viruses in tomato and control of virus infection will be based on prevention of infection. It appears that the host plant is an important factor in selection for resistance; some tomato cultivars appear to exhibit resistance to whitefly infestation and repel whitefly through the production of specific compounds (terpenoids). Previously we established which compounds can decrease whitefly visitation. Forthis internship we will try to elucidate partof thepathway ofterpenoid production and its regulation at the level of terpene biosynthesis which finally will result in application in breeding programmes.
We have cloned several key enzymes in the terpenoid biosynthesis pathway and have created transgenic tomato lines. These lines will be characterised at gene-expression level (Q-PCR) and production of volatile terpenes (Gas Chromatography-Mass Spectrometry). The effect of altered terpene biosynthesis on whitefly (or spider mite) visitation can then be assessed in bioassays.
New terpene synthases will be cloned in expression vectors for in-vivo production of terpenes (by expression in E. coli ), in order to find out which products they actually make. This can also be done by transient expression in a tobacco plant via A grobacterium infiltration. We check whether the gene is expressed and if so, which volatiles are produced.
Experimental techniques include molecular, biochemical and ecological assays (cloning, E. coli expression, GG-MS, DNA/RNA isolation, cDNA synthesis, PCR and quantitative PCR, bioassays)
The reproductive success of many plants is dependent on pollination by animals like hummingbirds and bees. Attractive floral traits like color and scent can enhance the reproductive success of a plant. Petunia is a wonderful plant to study the biosynthesis of these scent molecules, so called floral volatiles. A mix of floral volatiles is synthesized and emitted by Petunia flowers in a highly regulated manner. These volatiles are synthesized when the pollinators are active and the flower ready to be pollinated. The biosynthesis pathway of floral volatiles is described, but still some enzymes in this pathway are missing. Moreover, the regulation of this pathway is poorly understood. Four transcription factors are known to regulate this pathway, but how they regulate volatile biosynthesis remains largely unknown. Recently we have done Next Generation Sequencing on RNA of several transgenic petunia’s. From this sequencing data we can pick candidate genes that either regulate biosynthesis (transcription factors) or synthesize specific volatiles (biosynthetic genes).
Hypothesis and objectives
The project aims to characterize a new biosynthetic gene or transcription factor and determine its role in floral volatile biosynthesis. For this purpose three objectives will be addressed:
1. Determine the expression pattern of the candidate gene
Floral volatile genes are expressed mainly in the flower and expression cycles in time. To determine if the candidate displays the same tissue specific and rhythmic expression as other floral volatile genes expression analysis will be performed. For this purpose RNA will be isolated, cDNA synthesized and the expression level will be determined with quantitative RT-PCR.
2. To elucidate the subcellular localization of the candidate
To determine if the candidate localizes to the same subcellular compartments as related floral volatile enzymes Agrobacterium Transient Transformatiom Assays (ATTA) will be performed. The candidate will be labeled with a fluorescent tag to visualize its subcellular localization in Nicotiana benthamiana leaves and Petunia flowers.
3a. To determine the function of the biosynthetic gene
To test if the candidate has the predicted enzymatic activity it will be expressed in vitro in E.coli. The produced protein will be analyzed by Western blotting and enzymatic assays.
3b. To determine the function of the transcription factor
To elucidate the role of the transcription factor in floral volatile biosynthesis the gene will be silenced in petunia plants. For this purpose a RNAi construct will be cloned and Petunia petals will be transient silenced. Volatile emission will be measured by GC-MS.
Cloning, RNA isolation, cDNA synthesis, qPCR, ATTA, microscopy,
In vitro E. coli expression, Western blotting and enzymatic assays or GC-MS