Protein farnesylation is a post-translational modification involving the addition of a 15-carbon farnesyl isoprenoid to the carboxy terminus of select proteins(1-3). Although the roles of this lipid modification are clear in both fungal and animal signalling, many of the mechanistic functions of farnesylation in plant signalling are still unknown. Here, we show that CYP85A2, the cytochrome P450 enzyme that performs the last step in brassinosteroid biosynthesis (conversion of castasterone to brassinolide)(4), must be farnesylated to function in Arabidopsis. Loss of either CYP85A2 or CYP85A2 farnesylation results in reduced brassinolide accumulation and increased plant responsiveness to the hormone abscisic acid (ABA) and overall drought tolerance, explaining previous observations(5). This result not only directly links farnesylation to brassinosteroid biosynthesis but also suggests new strategies to maintain crop yield under challenging climatic conditions.

Pathogenesis‐related (PR) proteins constitute a broad class of plant proteins with analogues found throughout nature from bacteria to higher eukaryotes. PR proteins were first noted in plants as part of the hypersensitive response, but have since been assigned an array of biological roles. The PR10/Bet v1‐like proteins are a subset of PR proteins characterized by an ability to bind a wide range of lipophilic ligands, uniquely positioning them as contributors to specialized biosynthetic pathways. PR10/Bet v1‐like proteins participate in the production of plant alkaloids and phenolics including flavonoids, both as general binding proteins and in special cases as catalysts. Owing initially to the perceived allergenic properties of PR10/Bet v1‐like proteins, many were studied at the structural level to elucidate the basis for ligand binding. These studies provided a foundation for more recent efforts to understand higher‐level structural order and how PR10/Bet v1‐like proteins catalyse key reactions in plant pathways. Synthetic biology aimed at reconstituting plant‐specialized metabolism in microorganisms uses knowledge of these proteins to fine‐tune performance in new systems.

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Protein farnesylation refers to the addition of a 15-carbon farnesyl isoprenoid to the cysteine residue of the CaaX motif at the carboxy terminus of target proteins. In spite of its known roles in plant development and abiotic stress tolerance, how these processes are precisely regulated by farnesylation had remained elusive. We recently showed that CYP85A2, the cytochrome P450, which converts castasterone to brassinolide in the last step of brassinosteroid synthesis must be farnesylated in order to function in this pathway. Lack of either CYP85A2 or the farnesylation motif of CYP85A2 resulted in reduced brassinolide accumulation, hypersensitivity to ABA, and increased plant drought tolerance. In this study, we have assessed the influence of the N-terminal secretory signal and the C-terminal CaaX motif of CYP85A2 in mediating CYP85A2 function and targeting to endomembrane compartments. We show that CaaX motif could still target CYPA85A2 in the absence of an intact N-terminal secretory signal to the respective membrane compartments and partially rescue cyp85a2-2 phenotypes. However, in the absence of both the CaaX motif and the secretory signal, CYP85A2 is not targeted to the membranes and becomes unstable.

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