Natural variation is driven by local adaption and of great importance for the persistence of a species. Plants, limited in their ability to evade challenging environments, are under great pressure to survive and reproduce in their particular environment. Thus, local adaption is thought to play a key role in the evolution of novel ecotypes. However, the specific adaption to one desired environment may result in performance trade-offs in other environments. The underlying genetics and action of regulatory networks are constantly challenged and potentially reshaped. Understanding the mechanisms facilitating natural variation to come about thereby depends on investigating how regulatory networks and their underlying genetics are shaped by the surrounding environment. This thesis focussed on a specific genetic region in the Arabidopsis thaliana genome coding for enzymes responsible for the modification of the defense compound glucosinolates. The AOP locus harbors two enzyme-coding genes, AOP2 and AOP3, known to shape the glucosinolate profile. Glucosinolates display great variability across populations, and thisvariation is substantially dependent on the allelic status of the AOP locus. We investigated whether additional functional units, in the form of long-non-coding RNAs (lnc RNAs) that also originate from this locus, or the genetic region itself underwent recurrent structural diversification to obtain phenotypic diversity. Previous studies reported developmental phenotypes dependent on AOP genes, manifested in changed timing of flowering. This work investigated the role of lnc RNAs derived from the AOP2 gene in the regulation of flowering time. Phenotypic analyses across different environmental settings showed that the AOP2 lnc RNA is potentially involved in developmental phase transition dependent on the environmental conditions. More precisely, the nutritional status determined by the soil borne macronutrient nitrogen was identified as potential interacting factor. Chapter 1 reviews the current literature on the potential role of nitrogen as signal shaping flowering time. Further, a meta-analysis was conducted seeking for potential candidate genes and pathways connecting flowering time control and nitrogen response. Chapter 2 elaborates on the findings and conclusions on the second regulatory entity of AOP2 as lnc RNA by generation and phenotypic characterization of transgenic plant lines, altered in expression levels and properties of the AOP2 lnc RNA. Natural variation can be associated with diverse phenotypic variation; shape, color, composition of defense compounds and differences in development. An in silico approach across 1135 accessions identified accessions displaying phenotypic variation as well as structural variation in the genetic sequence of the AOP locus. Selected accessions were analyzed for glucosinolate profiles in different organs, flowering time and finally sequenced for the AOP locus, using long read technology. This study led to the identification of novel, highly polymorph alleles, and thereby shed light on recurrent local selection that repeatedly created glucosinolate diversity (chapter 3).In addition, work was conducted to support investigations on sugar homeostasis (chapter 4), nutrient de pendent glucosinolate biosynthesis and turnover (chapter 5), the impact of glucosinolate distribution on defense (chapter 6) and the evolution of a short linear motif critical for physical interactions between glucosinolate regulators (chapter 7).