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

Surviving Volcanic Environments — Interaction of Soil Mineral Content and Plant Element Composition

Different plant species were investigated fromtwo Aeolian Islands located in close vicinity, one with fumarolic activity (Vulcano) and one without (Lipari). On Vulcano, elevated concentrations of SO2/H2S determined in ambient air indicated the need of plants to adapt to harmful sulphur concentrations by detoxification strategies. The current study was focused on evaluating the element composition of plant leaves in relation to soil mineral contents. The soil of Volcano was characterised by a significantly lower pH on all three sampling sites as well as very high amounts of sulphur and plant available sulphate due to volcanic activities, compared to Lipari. By contrast, a general difference in the composition of trace elements in the soil was not observed between the islands, apart from arsenic, which was increased at all three sampling sites on Vulcano. Element accumulation in the leaves differed between the two islands. The tested species showed a significant higher accumulation of numerous elements (Al, B, Fe, K, Mg, Mn, Ni, and Zn) on Vulcano compared to Lipari, while excluding Ca and Mo. These differences in element accumulation in the leaves between the islands may be caused by the lower soil pH on Vulcano. Extreme sulphur accumulation was found for all tested species on Vulcano, but was lower in woody species with higher dry matter content compared to herbaceous species with lower dry matter content. This caused a significantly negative correlation between plant sulphur and dry matter content. From these results, it is concluded that species with higher dry matter contents possess a more effective protection against extreme sulphur accumulation. Strategies to cope with other potentially toxic elements in the soil ranged from exclusion to hyper-accumulation. Hierarchical cluster analyses of the leaf element content revealed a clear separation between two groups: First, herbaceous perennial plants as strong accumulators; and second, woody perennial plants such as shrubs or trees as less strong accumulators, with the primordial species Fumaria capreolata representing an outside group

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.Peer reviewe

Table_1_Surviving Volcanic Environments—Interaction of Soil Mineral Content and Plant Element Composition.DOCX

<p>Different plant species were investigated from two Aeolian Islands located in close vicinity, one with fumarolic activity (Vulcano) and one without (Lipari). On Vulcano, elevated concentrations of SO₂/H₂S determined in ambient air indicated the need of plants to adapt to harmful sulphur concentrations by detoxification strategies. The current study was focused on evaluating the element composition of plant leaves in relation to soil mineral contents. The soil of Volcano was characterised by a significantly lower pH on all three sampling sites as well as very high amounts of sulphur and plant available sulphate due to volcanic activities, compared to Lipari. By contrast, a general difference in the composition of trace elements in the soil was not observed between the islands, apart from arsenic, which was increased at all three sampling sites on Vulcano. Element accumulation in the leaves differed between the two islands. The tested species showed a significant higher accumulation of numerous elements (Al, B, Fe, K, Mg, Mn, Ni, and Zn) on Vulcano compared to Lipari, while excluding Ca and Mo. These differences in element accumulation in the leaves between the islands may be caused by the lower soil pH on Vulcano. Extreme sulphur accumulation was found for all tested species on Vulcano, but was lower in woody species with higher dry matter content compared to herbaceous species with lower dry matter content. This caused a significantly negative correlation between plant sulphur and dry matter content. From these results, it is concluded that species with higher dry matter contents possess a more effective protection against extreme sulphur accumulation. Strategies to cope with other potentially toxic elements in the soil ranged from exclusion to hyper-accumulation. Hierarchical cluster analyses of the leaf element content revealed a clear separation between two groups: First, herbaceous perennial plants as strong accumulators; and second, woody perennial plants such as shrubs or trees as less strong accumulators, with the primordial species Fumaria capreolata representing an outside group.</p

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

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

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

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