Glucosinolates are well-studied plant defense metabolites that are activated by myrosinases to generate highly toxic hydrolysis products upon attack. While glucosinolates are heavily moved around in the plant, myrosinases are sequestered and immobile. Together with Dr. Sebastian Kjeldgaard-Nintemann, I contributed to elucidate the localization of glucosinolate biosynthesis in the model plant Arabidopsis thaliana. We demonstrated that they are predominantly produced in vascular cells. Intriguingly, biosynthetic enzymes were absent from the phloem cap and the epidermis, which are major storage sites of glucosinolates. Bioimaging revealed that S-cells are coupled to surrounding glucosinolate biosynthetic cells, suggesting that glucosinolates are transported from site of synthesis to site of storage via plasmodesmata. In addition, I have contributed to the studies of Dr. Deyang Xu who revealed an indirect role of GLUCOSINOLATE TRANSPORTER1-3 (GTRs) in accumulation of glucosinolates in S-cells during early stages of development. These studies indicated that biosynthesis in young tissue might not be capable to accommodate sufficient glucosinolates and require supply from distant sites. To test whether the high basal glucosinolate levels in the young parts of the plant effectively protect from attacks, I conducted insect bioassays using mutants of glucosinolate biosynthesis and transport. These experiments showed that GTRs are required to accumulate a maximum glucosinolate concentration in young leaves, and that this distribution determines the insect feeding behavior. These findings strongly support the optimal defense theory, which states that the spatial and temporal allocation of defenses correlates with the value of diverse tissues and organs. Moreover, I tested the role of GTRs in the defense of epidermal cells against powdery mildew. In contrast to insect resistance, bioassays revealed that powdery mildew resistance is GTR-independent. Quantitative bioimaging of fluorophore-tagged glucosinolate transporters and biosynthetic enzymes revealed that de novo glucosinolate biosynthesis is cell-autonomously induced. However, the adapted powdery mildew Golovinomyces orontii appeared to manipulate de novo biosynthesis, which may contribute to compatibility of this interaction. This thesis illustrates how the complex interplay of biosynthesis, transport and storage determines the dynamic allocation of glucosinolates to accommodate the diverse requirements of the plant to defend itself against a plethora of attackers with extremely different strategies. The knowledge gained here will enable future research aiming to identify additional glucosinolate transport processes and their regulation