Catch You on the Flip Side: A Critical Review of Flippase Mutant Phenotypes.

Lipid flippases are integral membrane proteins that use ATP hydrolysis to power the generation of phospholipid asymmetry between the two leaflets of biological membranes, a process essential for cell survival. Although the first report of a plant lipid flippase was published in 2000, progress in the field has been slow, partially due to the high level of redundancy in this gene family. However, recently an increasing number of reports have examined the physiological function of lipid flippases, mainly in Arabidopsis thaliana. In this review we aim to summarize recent findings on the physiological relevance of lipid flippases in plant adaptation to a changing environment and caution against misinterpretation of pleiotropic effects in genetic studies of flippases.

Evolution and a revised nomenclature of P4 ATPases, a eukaryotic family of lipid flippases.

In all eukaryotic cells, P4 ATPases, also named phospholipid flippases, generate phospholipid asymmetry across biological membranes. This process is essential for cell survival, as it is required for vesicle budding and fusion in the secretory pathway. Several P4 ATPase isoforms can be identified in all sequenced eukaryotic genomes, but their evolution and interrelationships are poorly described. In this study, we conducted a thorough phylogenetic analysis of P4 ATPases in all major eukaryotic super-groups and found that they can be divided into three distinct families, P4A, P4B and P4C ATPases, all of which have an ancient origin. While P4B ATPases have been lost in plants, P4A ATPases are present in all eukaryotic super-groups. P4C ATPases form an intermediate group between the other two but appear to share a common origin with P4A ATPases. Sequence motifs unique to P4 ATPases are situated in the basal ATP hydrolyzing machinery. In addition, no clear signature motifs within P4 ATPase subgroups were found that could be related to lipid specificity, likely pointing to an elaborate transport mechanism in which different amino acid residue combinations in these pumps can result in recognition of the same substrate.

Localization of the glucosinolate biosynthetic enzymes reveals distinct spatial patterns for the biosynthesis of indole and aliphatic glucosinolates.

Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore-tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense-related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis.

Within the cell, biosynthetic pathways are embedded in protein-protein interaction networks. In Arabidopsis, the biosynthetic pathways of aliphatic and indole glucosinolate defense compounds are well-characterized. However, little is known about the spatial orchestration of these enzymes and their interplay with the cellular environment. To address these aspects, we applied two complementary, untargeted approaches-split-ubiquitin yeast 2-hybrid and co-immunoprecipitation screens-to identify proteins interacting with CYP83A1 and CYP83B1, two homologous enzymes specific for aliphatic and indole glucosinolate biosynthesis, respectively. Our analyses reveal distinct functional networks with substantial interconnection among the identified interactors for both pathway-specific markers, and add to our knowledge about how biochemical pathways are connected to cellular processes. Specifically, a group of protein interactors involved in cell death and the hypersensitive response provides a potential link between the glucosinolate defense compounds and defense against biotrophic pathogens, mediated by protein-protein interactions.

An evolutionarily young defense metabolite influences the root growth of plants via the ancient TOR signaling pathway.

To optimize fitness a plant should monitor its metabolism to appropriately control growth and defense. Primary metabolism can be measured by the universally conserved TOR (Target of Rapamycin) pathway to balance growth and development with the available energy and nutrients. Recent work suggests that plants may measure defense metabolites to potentially provide a strategy ensuring fast reallocation of resources to coordinate plant growth and defense. There is little understanding of mechanisms enabling defense metabolite signaling. To identify mechanisms of defense metabolite signaling, we used glucosinolates, an important class of plant defense metabolites. We report novel signaling properties specific to one distinct glucosinolate, 3-hydroxypropylglucosinolate across plants and fungi. This defense metabolite, or derived compounds, reversibly inhibits root growth and development. 3-hydroxypropylglucosinolate signaling functions via genes in the ancient TOR pathway. If this event is not unique, this raises the possibility that other evolutionarily new plant metabolites may link to ancient signaling pathways.

Rhizosecretion of stele-synthesized glucosinolates and their catabolites requires GTR-mediated import in Arabidopsis.

Casparian strip-generated apoplastic barriers not only control the radial flow of both water and ions but may also constitute a hindrance for the rhizosecretion of stele-synthesized phytochemicals. Here, we establish root-synthesized glucosinolates (GLS) are in Arabidopsis as a model to study the transport routes of plant-derived metabolites from the site of synthesis to the rhizosphere. Analysing the expression of GLS synthetic genes in the root indicate that the stele is the major site for the synthesis of aliphatic GLS, whereas indole GLS can be synthesized in both the stele and the cortex. Sampling root exudates from the wild type and the double mutant of the GLS importers GTR1 and GTR2 show that GTR-mediated retention of stele-synthesized GLS is a prerequisite for the exudation of both intact GLS and their catabolites into the rhizosphere. The expression of the GTRs inside the stele, combined with the previous observation that GLS are exported from biosynthetic cells, suggest three possible routes of stele-synthesized aliphatic GLS after their synthesis: (i) GTR-dependent import to cells symplastically connected to the cortical cells and the rhizosphere; (ii) GTR-independent transport via the xylem to the shoot; and (iii) GTR-dependent import to GLS-degrading myrosin cells at the cortex. The study suggests a previously undiscovered role of the import process in the rhizosecretion of root-synthesized phytochemicals.

Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization.

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality.