Research Projects in the Department of Molecular Ecology

As the coordinator of the analytical platform I am responsible for the development and optimization of analytical tools in the department of Molecular Ecology. My major research focus is the development of high throughput methods for the metabolic characterization of large populations of recombinant inbred lines (RILs). With a combination of targeted and untargeted metabolomics analysis and ecological phenotyping we are trying to identify important chemical traits mediating biotic interactions and the underlying genetic background regulating them. I am also a member of the Sonderforschungsbereich ‘ChemBioSys’ and the Cluster of Excellence ‘Balance of the Microverse’, which are funded by the German Science Foundation.

My main research focus is the identification of plant traits controlling the interactions with herbivores, their natural enemies as well as pollinators and the recruitment of the plant microbiome using the forward genetics MAGIC platform developed in the department over the last decade. The 26-parent MAGIC mapping population captures a large part of the natural variation found in Nicotiana attenuata throughout its native range. We will use a combination of natural history observations in the field, targeted as well as unbiased metabolomics analyses and microbiome sequencing to identify loci controlling traits involved in these complex ecological interactions. Functional tests using our reverse genetics platform will be conducted at our field sites in Utah and Arizona. This new approach will allow unbiased screening for novel, ecologically important traits and the underlying genetic control, which were elusive targets in reverse genetics experiments based on a priori knowledge of genes.


We are investigating the role of regulatory small RNAs (smRNAs) such as microRNAs (miRNAs) in modulating defense plasticity of plants during its interactions with environmental factors such as herbivores, pathogens and the beneficial arbuscular mycorrhizal associations (AMF). We use a genomics-guided integrative biology approach employing deep sequencing, computational biology, molecular biology, analytical chemistry and field biology approaches to understand how plants tailor responses to their environment, and how regulatory traits have been evolving. Ongoing projects include:

Composition of miRNome its reprograming during stress response: We aim to elucidate all the miRNAs that are expressed and reprogrammed during biotic interactions of herbivory, pathogenesis and AMF colonization by deep-sequencing of microRNA (miRNA) landscapes of the host plant.

Identity and evolution of effectors of smRNA machinery: The process of smRNA-mediated regulation of expression may be divided into two major events: (i) smRNA biogenesis, and (ii) their action. We have already characterized two biogenesis components, Dicer-like (DCLs) and RNA-directed RNA polymerases (RdRs) in past, yet biological functions of the Argonaute (AGO) effectors has been understudied. In N. attenuata, AGO family comprises of 11 genes. We hypothesize that the evolution of AGOs produced the signatures of functional diversification in the smRNA pathways.

Functional specificity in biological actions of N. attenuata AGOs: We are testing the hypothesis of functional specificity of AGO effectors by characterizing loss-of-function phenotypes of the N. attenuata AGOs in nature against important environmental factors, such as herbivores, infection of pathogens and AMF colonization.

Ecological functions of conserved miRNAs: Several conserved miRNAs are differentially accumulated during plant’s response to stress. These miRNAs form important hubs in the regulatory networks that modulate defense-signaling. Biological functions of such conserved miRNAs are being investigated.

Understand cross-talk of transcriptional and post-transcriptional regulation of gene expression for traits important for stress adaptation: 24-nucleotide (nt) small-interfering RNAs (siRNAs) modulate transcriptional gene silencing (TGS), whereas miRNAs are often involved in post-transcriptional gene silencing (PTGS). How do TGS and PTGS components crosstalk to fine-tune defense-signaling is an open question.


I manage both small and large scale projects, with the aim of optimizing existing procedures and protocols to gain publishable results. My position extends to the management of field season operations and logistic, fieldwork protocol management, team management, international reporting and compliance and general project management.

In 2019 I am working to optimize the processing of multiple simultaneous High-throughput (HTP) transformations of N. attenuata and N. obtusifolia using a variety of constructs. I am also working with electroporation-based pollination methods in both glasshouse and field conditions, as well as developing new methods of sowing and germinating N. attenuata to promote plant fitness in preparation for our field seasons.

Currently I am working on pollination-based mate-choice experiments with N. attenuata testing RNASe I and II function, mixed and single-parent pollination, circadian clock related pollen-loading and contribute to the management and maintenance of seedbanks for future experiments. Additionally, I am working on a project relating to natural seedbanks, which involves testing several soil treatments, cataloging developmental stages of plant growth and processing samples collected from the 2019 field season.


Plants have evolved the ability to produce a plethora of specialized metabolites. These chemicals have important ecological functions for plant survival in nature. For instance, plants respond to herbivores of pathogen attack by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. This process is regulated by a signaling cascade that involves jasmonates (JA), ubiquitous oxylipin-derived phytohormines that also play essential roles in the regulation of many developmental and growth processes. Since the functional/ecological roles of JA-regulated specialized metabolites are largely unknown, I am to disentangle JA-regulated metabolic pathways of functional significance in N. attenuata. My research has been focused on developing efficient means of manipulating biosynthesis of specialized metabolites (e.g. acyl sugar, triterpenes and phenolyic derivatives) for functional ecological studies through integrating the analysis of genomes, transcriptomes and metabolomes in the MAGIC-RIL population, wih the hope that this will lead to the eventual elucidation of functional/ecological roles of these important compounds.



I use computational metabolomics to analyze N. attenuata’s responses to multi-layered biotic interactions. My research interests are to characterize the structure of diverse small molecules using mass spectrometry and investigate the expression patterns of secondary metabolites under different conditions such as herbivory elicitation in a time scale and from different tissues and ontogeny. Furthermore, my research involves the characterization of the regulatory basis of the secondary metabolites by applying integrative analysis of transcriptome and metabolome and bioinformatics.


My research focuses on one class of abundant second metabolites in N. attenuata, 17-hydroxygernallinalool diterpene glucosides (HGL-DTGs). The major three directions of my research are: how do plants synthesize HGL-DTGs? What is the effect of HGL-DTGs on a plant’s physiology? What is the effect of HGL-DTGs on a plant’s herbivore defense.  My research will illuminate how these abundant metabolites help N. attenuata defend itself against herbivores in nature.


The physiological function of strigolactone (SL) and karrikin (KAR) signaling in plants has been well-studied, while their ecological function remains to be explored. By using well-established molecular, analytical and ecological tools, I plan to investigate the function of SL and KAR signaling in plant defense in N. attenuata and reveal their associated molecular mechanisms.


Plants partner with an incredibly diverse list of microbiomes from their native soil. Microbiomes are the “extended phenotypes” of an active ecological society and a healthy microbiome is a prerequisite for an ecologically sustainable environment. My research question explores the “Recruitment and maintenance of the Nicotiana attenuata root microbiome” funded by the Balance of the Microverse cluster, Jena School of Microbial Communication. Through my research, I intend to understand the fundamental molecular mechanisms involved in plant-microbe interactions by experimentally manipulating the abundance and composition of a synthetic community of native microbes, and further exploring the quantitative genomics and metabolomics signatures of N. attenuata. We aim to understand how host plants recruit and maintain functional consortia by utilizing the extremely resourceful in-house molecular tool-box of N. attenuata along with high-throughput screening methods such as Nanostring nCounter, and HPLC-MS/MS approaches.


Research Description:

My main project aims to identify regulatory genes that lie at the crossroads between the circadian clock and the auxin signaling pathway; two networks that are tightly interwoven and regulate multiple developmental processes. I am also exploring the function of JA-mediated epigenetic modifications in the regulation of defense responses to herbivory. Specifically, how JA-induced changes in the methylome, triggered by the RNA-directed DNA methylation pathway, influence intergenerational responses to JA and other phytohormones. I approach scientific questions with creative thinking and I have an intrinsic curiosity for all scientific fields, especially neurobiology.


Research Description:

My research explores the functional effects of a key gene that controls a water-wasting phenotype in N. attenuata plants: mitogen-activated protein kinase 4 (MPK4). I use plants stably silenced in MPK4 expression (irMPK4) and by growing these plants with EV (empty vector, Wt-like) plants in pairs, populations, and communities at different abundances, I examine the productivity of each population’s individuals as well as the population as a whole. My research focuses on the performance of these populations under drought stress, and/or while interacting with arbscular mycorrhizal fungi (AMF). I'm interested in the effect of MPK4 on how a plant responds to drought tolerance and recovery through changes in photosynthetic rates, efficiencies, and structures, as well as through metabolomic accumulation in leaves, branches and roots. Below ground interactions between plants, facilitated by a fungus such as AMF, are key to how irMPK4 plants can affect community drought response and recovery, and I seek to define some of the responsible mechanisms.


When plants are attacked by pathogens or herbivores, they reconfigure their transcriptomes to combat these stresses and maximize their fittness. How these transcriptional responses, which include the reprogramming of phytohomone signals, defense metabolites and the master transcription factors, are regulated is poorly understood. Rapid changes in gene expression may be modulated by regulatory small-RNAs (smRNAs), such as the microRNAs and small-interfering RNAs (miRNAs, siRNAs). When N. attenuata plants are attacked by herbivores, a smRNA-pathway mediated by RNA-directed RNA polymerase 1 (RdR1) and Dicer-like proteins 3 and 4 (DCL3 and 4) is recruited. The effectors of the smRNA pathways are the family of Argonaute (AGO) proteins and these are the focus of my research. Which AGO proteins are recruited during elicitation of a herbivory-specific smRNA pathway remains unknown, and I hope to identify herbivory-specific AGO effectors that would enable the construction of smRNA pathways tailored to regulate a plant’s induced-defense responses. In addition, I hope to elucidate the roles of AGOs in mediating the plant’s responses to phytopathogens.


My work is broadly focused on dissecting the molecular underpinnings of plant herbivore interaction in an ecological context using the model species Nicotiana attenuata. In order to dissect this interaction, I leverage the natural variation present in the species through the MAGIC population, and understand the functional consequences from the plant’s side. I use various statistical and bioinformatic tools to dissect these ecological and bio-chemical traits and map them on the genome to gain functional and evolutionary insights of the same. My current project is particularly focused on elucidating the early defense signaling components involved in jasmonic acid (JA) signaling and also the degradation of it. By integrating genomic, transcriptomic, and metabolomic data of the MAGIC population and the diverse reverse genetic toolbox in the department, I plan to disentangle this molecular network to gain a holistic functional understanding of JA signaling pathway and its role in plant defense.


The plant microbiome plays an important role in the protection of plants from various ecological hardships. Nicotiana attenuata uses its microbiome to defend itself against root rot and wilt diseases. It also improves its own phenotype through energy conservation by engaging microbes in performing tasks like nutrient assimilation. Little is known about the basic process involved in the early recruitment of a balanced plant microbiome. Our project focuses on understanding the question: Do plants sculpt their own microbiome by employing a fraction of microbes from the environmental marketplace by the provision of nutrients in the form of root exudates? To gain a functional understanding of the microbiome, we intend to use a native synthetic microbial consortium (nSynCom) in a gnotobiotic system during germination stages. We study alterations in the early germination stage recruitment by using high-throughput molecular platforms to monitor absolute variations in the nSynCom. In order to decipher the metabolite driving the community dynamics, we will exploit the transgenic line toolbox backed by targeted and untargeted metabolome platforms. Further, to study the natural variations observed in the recruitment and maintenance of the microbiome, we will explore Multi-Parent Advanced Generation Inter-Cross (MAGIC) and their microbiome associations through Quantitative Trait Loci (QTL) imputations, eventually testing the hypothesis in field.


From the timing of photosynthesis to the timing defense responses against herbivore attack, N. attenuata’s circadian clock is intimately involved in orchestrating stress responses with daily rhythms. However, the clock also functions in developmental responses to stresses such as water limitation and heat stress, which gradually become acute as N. attenuata’s growing season progresses in its natural environment, the Great Basin Desert. Despite these whole-plant functions of the circadian clock, circadian mechanisms may vary from tissue to tissue, and recent evidence suggests that the clock functions differently in roots and shoots. My research focuses on understanding the role of the circadian clock in these developmental responses, as well as in understanding the interplay between the root and shoot clock in response to abiotic stress. As the circadian clock gene, TIMING OF CAB EXPRESSION 1 (TOC1), is mechanistically linked to drought responses via abscisic acid (ABA) signaling, it provides a clear target for an exploration of the clock’s function in roots and shoots. My ultimate goal is to utilize N. attenuata’s extensive transgenic toolbox as well as various native accessions to elucidate the functional consequences for plant fitness of TOC1 in roots and shoots in response to drought stress.


Sven Heiling

 

 

17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) constitute a highly abundant compound class that occurs in concentration equivalent to starch (mg/g fresh mass) in aboveground tissues of Nicotiana attenuata. HGL-DTGs consist of an acyclic C20 17-hydroxygeranyllinalool skeleton conjugated to sugar groups (glucose and rhamnose) via bonds at C-3 and C-17 hydroxylated carbons; additional sugars are conjugated to the C’-2, C’-4, or C’-6 hydroxyl groups. Malonyl groups are typically connected to the C’-6 hydroxyl group of the glucose(s).Herbivory and jasmonate-mediated signaling induces the conjugation of malonyl groups to diterpene glycoside sugars and have been shown to reduce growth of the tobacco hornworm (Manduca sexta) in N. attenuata.

My objective is to elucidate the ecological function of HGL-DTGs and more precisely the herbivore induced malonylation of HGL-DTGs in N. attenuata. To this end, I am first developing a mass spectrometric-based method for the identification, classification and quantification of all HGL-DTG molecular species in metabolic profiles of diverse Solanaceae species (f. e.: Nicotiana sp., Capsicum sp., Lycium sp.) in order to unravel conserved and divergent tissue-specific variations in the accumulation of these defense molecules and to test the hypothesis that malonylation is a widespread phenomenon among the Solanaceous taxa. I intend to perform field and glasshouse bioassays to identify the bioactive portion of the HGL-DTGs. In parallel, I am identifying and functionally characterizing novel glycosyl-, rhamnosyl- and malonyl transferases that participate in the biosynthesis of HGL-DTGs in N. attenuata plants. This will allow engineering N. attenuata plants with reduced or increased levels of each HGL-DTG form to test the impact of each individual compound class for insect resistance and to understand the connectivity of HGL-DTG metabolism with other branches of the plant’s herbivory-regulated metabolism.