Moonlighting Arabidopsis molybdate transporter 2 family and GSH-complex formation facilitate molybdenum homeostasis.

Molybdenum (Mo) as essential micronutrient for plants, acts as active component of molybdenum cofactor (Moco). Core metabolic processes like nitrate assimilation or abscisic-acid biosynthesis rely on Moco-dependent enzymes. Although a family of molybdate transport proteins (MOT1) is known to date in Arabidopsis, molybdate homeostasis remained unclear. Here we report a second family of molybdate transporters (MOT2) playing key roles in molybdate distribution and usage. KO phenotype-analyses, cellular and organ-specific localization, and connection to Moco-biosynthesis enzymes via protein-protein interaction suggest involvement in cellular import of molybdate in leaves and reproductive organs. Furthermore, we detected a glutathione-molybdate complex, which reveals how vacuolar storage is maintained. A putative Golgi S-adenosyl-methionine transport function was reported recently for the MOT2-family. Here, we propose a moonlighting function, since clear evidence of molybdate transport was found in a yeast-system. Our characterization of the MOT2-family and the detection of a glutathione-molybdate complex unveil the plant-wide way of molybdate.

Biosynthesis of polyprenylated xanthones in Hypericum perforatum roots involves 4-prenyltransferase.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

The Mechanisms of Molybdate Distribution and Homeostasis with Special Focus on the Model Plant Arabidopsis thaliana.

This review article deals with the pathways of cellular and global molybdate distribution in plants, especially with a full overview for the model plant Arabidopsis thaliana. In its oxidized state as bioavailable molybdate, molybdenum can be absorbed from the environment. Especially in higher plants, molybdenum is indispensable as part of the molybdenum cofactor (Moco), which is responsible for functionality as a prosthetic group in a variety of essential enzymes like nitrate reductase and sulfite oxidase. Therefore, plants need mechanisms for molybdate import and transport within the organism, which are accomplished via high-affinity molybdate transporter (MOT) localized in different cells and membranes. Two different MOT families were identified. Legumes like Glycine max or Medicago truncatula have an especially increased number of MOT1 family members for supplying their symbionts with molybdate for nitrogenase activity. In Arabidopsis thaliana especially, the complete pathway followed by molybdate through the plant is traceable. Not only the uptake from soil by MOT1.1 and its distribution to leaves, flowers, and seeds by MOT2-family members was identified, but also that inside the cell. the transport trough the cytoplasm and the vacuolar storage mechanisms depending on glutathione were described. Finally, supplying the Moco biosynthesis complex by MOT1.2 and MOT2.1 was demonstrated.

Physiological Importance of Molybdate Transporter Family 1 in Feeding the Molybdenum Cofactor Biosynthesis Pathway in Arabidopsis thaliana.

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein-protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.

Split-HaloTag imaging assay for sophisticated microscopy of protein-protein interactions in planta.

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.

Impact of wildfires on SO2 detoxification mechanisms in leaves of oak and beech trees.

Frequency and intensity of wildfire occurrences are dramatically increasing worldwide due to global climate change, having a devastating effect on the entire ecosystem including plants. Moreover, distribution of fire-smoke can influence the natural environment over very long distances, i.e. hundreds of kilometres. Dry plant matter contains 0.1-0.9% (w/w) sulphur, which is mainly released during combustion into the atmosphere as sulphur dioxide (SO2) resulting in local concentrations of up to 3000 nL L-1. SO2 is a highly hazardous gas, which enters plants mostly via the stomata. Toxic sulphite is formed inside the leaves due to conversion of SO2. Plants as sessile organisms cannot escape from threats, why they evolved an impressive diversity of molecular defence mechanisms. In the present study, two recent wildfires in Germany were evaluated to analyse the effect of SO2 released into the atmosphere on deciduous trees: the Meppen peat fire in 2018 and the forest fire close to Luebtheen in 2019. Collected leaf material from beech (Fagus sylvatica) and oak (Quercus robur) was examined with respect to detoxification of sulphur surplus due to the exposure to elevated SO2. An induced stress reaction in both species was indicated by a 1.5-fold increase in oxidized glutathione. In beech leaves, the enzymatic activities of the sulphite detoxification enzymes sulphite oxidase and apoplastic peroxidases were increased 5-fold and a trend of sulphate accumulation was observed. In contrast, oaks did not regulate these enzymes during smoke exposure, however, the constitutive activity is 10-fold and 3-fold higher than in beech. These results show for the first time sulphite detoxification strategies of trees in situ after natural smoke exposure. Beech and oak trees survived short-term SO2 fumigation due to exclusion of toxic gases and different oxidative detoxification strategies. Beeches use efficient upregulation of oxidative sulphite detoxification enzymes, while oaks hold a constitutively high enzyme-pool available.

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-ß-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-ß-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-ß-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Effects of free air carbon dioxide enrichment (FACE) on nitrogen assimilation and growth of winter wheat under nitrate and ammonium fertilization.

A 2-year Free Air CO2 Enrichment (FACE) experiment was conducted with winter wheat. It was investigated whether elevated atmospheric CO2 concentration (e[CO2 ]) inhibit nitrate assimilation and whether better growth and nitrogen acquisition under e[CO2 ] can be achieved with an ammonium-based fertilization as it was observed in hydroponic culture with wheat. Under e[CO2 ] a decrease in nitrate assimilation has been discussed as the cause for observed declines in protein concentration in C3 cereals. Wheat was grown under ambient [CO2 ] and e[CO2 ] (600 ppm) with three levels (deficiency, optimal, and excessive) of nitrate-based fertilization (calcium ammonium nitrate; CAN) or with optimal ammonium-based fertilization. Ammonium fertilization was applied via injection of an ammonium solution into the soil in the 1st year and by surface application of urea combined with nitrification inhibitors (UNI) in the 2nd year. Results showed that ammonium-based fertilization was successfully achieved in the 2nd year with respect to nitrification control, as soil ammonium concentration was considerably higher over the growing season for UNI fertilized plots compared to optimal CAN plots. Also, stem nitrate concentration, flag leaf nitrate reductase activity, and transcript levels were lower in UNI fertilized plants compared to optimal CAN. Regarding the e[CO2 ] effect on nitrate reductase activity and transcript levels, no alteration could be observed for any nitrogen fertilizer treatment. Flag leaf growth was stimulated under e[CO2 ] leading to an enhanced nitrate reductase activity referred to m2 ground area at late flowering being in line with a higher nitrogen acquisition under e[CO2 ]. Moreover, nitrogen acquisition was considerably higher in nitrate fertilized plants compared to ammonium fertilized plants under e[CO2 ]. Our results obtained under field conditions show that a change from nitrate- to ammonium-based fertilization will not lead to a better growth and nitrogen acquisition of winter wheat under future e[CO2 ].

The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture.

The ambient light environment controls many aspects of plant development throughout a plant's life cycle. Such complex control is achieved because a key repressor of light signaling, the Arabidopsis (Arabidopsis thaliana) COP1/SPA E3 ubiquitin ligase causes the degradation of multiple regulators of endogenous developmental pathways. This includes the CONSTANS (CO) transcription factor that is responsible for photoperiodic control of flowering time. There are 16 CO-like proteins whose functions are only partly understood. Here, we show that 14 CO-like (COL) proteins bind CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYTOCHROME A-105 (SPA)1 in vitro. We subsequently focused on COL12 and show that COL12 binds COP1 and SPA proteins in vivo. The COL12 protein is degraded in darkness in a COP1-dependent fashion, indicating that COL12 is a substrate of the COP1/SPA ubiquitin ligase. Overexpression of COL12 causes late flowering specifically in long day conditions by decreasing the expression of FLOWERING LOCUS T This phenotype is genetically dependent on CO. Consistent with this finding, COL12 physically interacts with CO in vivo, suggesting that COL12 represses flowering by inhibiting CO protein function. We show that COL12 overexpression did not alter CO protein stability. It is therefore likely that COL12 represses the activity of CO rather than CO levels. Overexpression of COL12 also affects plant architecture by increasing the number of rosette branches and reducing inflorescence height. These phenotypes are CO independent. Hence, we suggest that COL12 affects plant development through CO-dependent and CO-independent mechanisms.

The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex.

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnx1, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnx1 as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnx1. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation.

Identification of a protein-protein interaction network downstream of molybdenum cofactor biosynthesis in Arabidopsis thaliana.

The molybdenum cofactor (Moco) is ubiquitously present in all kingdoms of life and vitally important for survival. Among animals, loss of the Moco-containing enzyme (Mo-enzyme) sulphite oxidase is lethal, while for plants the loss of nitrate reductase prohibits nitrogen assimilation. Moco is highly oxygen-sensitive, which obviates a freely diffusible pool and necessitates protein-mediated distribution. During the highly conserved Moco biosynthesis pathway, intermediates are channelled through a multi-protein complex facilitating protected transport. However, the mechanism by which Moco is subsequently transferred to apo-enzymes is still unclear. Moco user enzymes can be divided into two families: the sulphite oxidase (SO) and the xanthine oxidoreductase (XOR) family. The latter requires a final sulphurisation of Moco catalysed via ABA3. To examine Moco transfer towards apo-Mo-enzymes, two different and independent protein-protein interaction assays were performed in vivo: bimolecular fluorescence complementation and split luciferase. The results revealed a direct contact between Moco producer molybdenum insertase CNX1, which represents the last biosynthesis step, and members of the SO family. However, no protein contact was observed between Moco producer CNX1 and apo-enzymes of the XOR family or between CNX1 and the Moco sulphurase ABA3. Instead, the Moco-binding protein MOBP2 was identified as a mediator between CNX1 and ABA3. This interaction was followed by contact between ABA3 and enzymes of the XOR family. These results allow to describe an interaction matrix of proteins beyond Moco biosynthesis and to demonstrate the complexity of transferring a prosthetic group after biosynthesis.

The molybdenum cofactor biosynthesis complex interacts with actin filaments via molybdenum insertase Cnx1 as anchor protein in Arabidopsis thaliana.

The pterin based molybdenum cofactor (Moco) plays an essential role in almost all organisms. Its biosynthesis is catalysed by six enzymes in a conserved four step reaction pathway. The last three steps are located in the cytoplasm, where a multimeric protein complex is formed to protect the intermediates from degradation. Bimolecular fluorescence complementation was used to test for cytoskeleton association of the Moco biosynthesis enzymes with actin filaments and microtubules using known cytoskeleton associated proteins, thus permitting non-invasive in vivo studies. Coding sequences of binding proteins were cloned via the GATEWAY system. No Moco biosynthesis enzyme showed any interaction with microtubules. However, alone the two domain protein Cnx1 exhibited interaction with actin filaments mediated by both domains with the Cnx1G domain displaying a stronger interaction. Cnx6 showed actin association only if unlabelled Cnx1 was co-expressed in comparable amounts. So Cnx1 is likely to be the anchor protein for the whole biosynthesis complex on actin filaments. A stabilization of the whole Moco biosynthesis complex on the cytoskeleton might be crucial. In addition a micro-compartmentation might either allow a localisation near the mitochondrial ATM3 exporter providing the first Moco intermediate or near one of the three molybdate transporters enabling efficient molybdate incorporation.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins.

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

Visualization and quantification of protein interactions in the biosynthetic pathway of molybdenum cofactor in Arabidopsis thaliana.

The molybdenum cofactor (Moco) is the active compound at the catalytic site of molybdenum enzymes. Moco is synthesized by a conserved four-step pathway involving six proteins in Arabidopsis thaliana. Bimolecular fluorescence complementation was used to study the subcellular localization and interaction of those proteins catalysing Moco biosynthesis. In addition, the independent split-luciferase approach permitted quantification of the strength of these protein-protein interactions in vivo. Moco biosynthesis starts in mitochondria where two proteins undergo tight interaction. All subsequent steps were found to proceed in the cytosol. Here, the heterotetrameric enzyme molybdopterin synthase (catalysing step two of Moco biosynthesis) and the enzyme molybdenum insertase, which finalizes Moco formation, were found to undergo tight protein interaction as well. This cytosolic multimeric protein complex is dynamic as the small subunits of molybdopterin synthase are known to go on and off in order to become recharged with sulphur. These small subunits undergo a tighter protein contact within the enzyme molybdopterin synthase as compared with their interaction with the sulphurating enzyme. The forces of each of these protein contacts were quantified and provided interaction factors. To confirm the results, in vitro experiments using a technique combining cross-linking and label transfer were conducted. The data presented allowed the outline of the first draft of an interaction matrix for proteins within the pathway of Moco biosynthesis where product-substrate flow is facilitated through micro-compartmentalization in a cytosolic protein complex. The protected sequestering of fragile intermediates and formation of the final product are achieved through a series of direct protein interactions of variable strength.

Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors.

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.