Nitric oxide controls shoot meristem activity via regulation of DNA methylation.

Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.

OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module driving phloem development.

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

Coupling to Pam16 differentially controls the dual role of Pam18 in protein import and respiratory chain formation.

The presequence translocase (TIM23 complex) imports precursor proteins into the mitochondrial inner membrane and matrix. The presequence translocase-associated motor (PAM) provides a driving force for transport into the matrix. The J-protein Pam18 stimulates the ATPase activity of the mitochondrial Hsp70 (mtHsp70). Pam16 recruits Pam18 to the TIM23 complex to ensure protein import. The Pam16-Pam18 module also associates with components of the respiratory chain, but the function of the dual localization of Pam16-Pam18 is largely unknown. Here, we show that disruption of the Pam16-Pam18 heterodimer causes redistribution of Pam18 to the respiratory chain supercomplexes, where it forms a homodimer. Redistribution of Pam18 decreases protein import into mitochondria but stimulates mtHsp70-dependent assembly of respiratory chain complexes. We conclude that coupling to Pam16 differentially controls the dual function of Pam18. It recruits Pam18 to the TIM23 complex to promote protein import but attenuates the Pam18 function in the assembly of respiratory chain complexes.

Structural basis for the complex DNA binding behavior of the plant stem cell regulator WUSCHEL.

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control.

Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes.

Haemodialysis effectively removes small solutes and smaller-sized middle molecules from the blood; however, the clearance of larger middle molecules, which have been associated with negative effects, is poor. The novel medium cut-off (MCO) dialysis membrane has larger pore sizes and a more open structure than other high-flux membranes, providing improved removal of larger middle molecules while retaining albumin. However, larger pore sizes may potentially increase permeability to pyrogens, including endotoxins and other bacterial contaminants, that could be present in the dialysis fluid. In this study, we tested the capacity of low-flux, high-flux, MCO and high cut-off dialyser membranes with different pore sizes to prevent pyrogens crossing from dialysate to the blood side in a closed-loop test system, differentiating among lipopolysaccharides, peptidoglycans and bacterial DNA using a toll-like receptor assay. Even though the bacterial contamination levels in our test system exceeded the acceptable pyrogen dose for standard dialysis fluid, levels of lipopolysaccharides, peptidoglycans and bacterial DNA in the blood-side samples were too low to identify potential differences in pyrogen permeability among the membranes. Our results suggest that MCO membranes are suitable for haemodialysis using ISO standard dialysis fluid quality, and retain endotoxins at a similar level as other membranes.

Retention of beneficial molecules and coagulation factors during haemodialysis and haemodiafiltration.

Middle molecules (MMs) are associated with the pathology of uraemia, and are not effectively removed by standard extracorporeal treatments. Increased convection used in haemodiafiltration (HDF) can enhance the removal of MMs; however, high-volume HDF is not available to all patients. The new medium cut-off (MCO) membrane has been developed to allow increased removal of MMs using standard haemodialysis (HD). Improved removal of MMs has been shown with the MCO membrane compared with standard high-flux dialysers, but it is not known whether the increased pore size affects the retention of commonly used medications or that of coagulation factors in dialysis patients. Using an in vitro model, the retention of erythropoietin, heparin, insulin, vancomycin and several coagulation factors (Factors II, VII and X, protein C and antithrombin III) was investigated with the MCO membrane dialyser, compared with high-flux dialysers with polysulfone (in HDF) or polyethersulfone membranes (in HD and HDF). The retention of all molecules investigated was comparable between the MCO membrane and the high-flux dialysers. Results from the in vitro studies suggest that switching from a high-flux dialyser to the MCO membrane should not require changes to the medication dosing or anti-coagulation protocols of dialysis patients.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.

Spatial specificity of auxin responses coordinates wood formation.

Spatial organization of signalling events of the phytohormone auxin is fundamental for maintaining a dynamic transition from plant stem cells to differentiated descendants. The cambium, the stem cell niche mediating wood formation, fundamentally depends on auxin signalling but its exact role and spatial organization is obscure. Here we show that, while auxin signalling levels increase in differentiating cambium descendants, a moderate level of signalling in cambial stem cells is essential for cambium activity. We identify the auxin-dependent transcription factor ARF5/MONOPTEROS to cell-autonomously restrict the number of stem cells by directly attenuating the activity of the stem cell-promoting WOX4 gene. In contrast, ARF3 and ARF4 function as cambium activators in a redundant fashion from outside of WOX4-expressing cells. Our results reveal an influence of auxin signalling on distinct cambium features by specific signalling components and allow the conceptual integration of plant stem cell systems with distinct anatomies.

Macromolecular Assemblies of the Mammalian Circadian Clock.

The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ∼750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ were distributed into several complexes of ∼0.9-1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ∼20-nm and ∼25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.

Definition of a High-Confidence Mitochondrial Proteome at Quantitative Scale.

Mitochondria perform central functions in cellular bioenergetics, metabolism, and signaling, and their dysfunction has been linked to numerous diseases. The available studies cover only part of the mitochondrial proteome, and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative experimental approach to define the proteome of yeast mitochondria. We classified > 3,300 proteins of mitochondria and mitochondria-associated fractions and defined 901 high-confidence mitochondrial proteins, expanding the set of mitochondrial proteins by 82. Our analysis includes protein abundance under fermentable and nonfermentable growth, submitochondrial localization, single-protein experiments, and subcellular classification of mitochondria-associated fractions. We identified mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS), and the coenzyme Q biosynthesis cluster, as well as mitochondrial proteins with dual cellular localization. The integrative proteome provides a high-confidence source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment.

Detection of Free Advanced Glycation End Products in Vivo during Hemodialysis.

Advanced glycation end products (AGEs) are often regarded as glycotoxins, which are normally removed by the kidney. Patients with end-stage renal failure rely on hemodialysis (HD) treatment to eliminate these compounds. In the present work, a highly selective LC-MS/MS method was used for quantitation of AGE levels in plasma and in dialysis fluids of HD patients, with a focus on AGE-free adducts. A broad range of 19 amino acid modifications was identified and quantitated. It was expected that the AGE-free adducts are successfully eliminated by dialysis treatment. Indeed, with a mean elimination rate of 71%, this assumption proved to be valid for all target analytes with the exception of pyrraline, which showed an opposite behavior. Here, plasma and dialysate levels increased during the treatment by about 59%. The notions that pyrraline was formed in high amounts in the patient's bloodstream (I) after intake of the corresponding precursor compound 3-deoxyglucosone with the dialysis fluid or (II) by catalytic effects on the formation by the dialysis membrane were ruled out. In contrast, in a dietary study, the comparison of pyrraline concentrations in plasma before and after food consumption confirmed that the increase in pyrraline originates solely from digestion of glycated food proteins. Additionally, by detailed analyses of the food consumed during dialysis sessions, bread rolls with a pyrraline content of about 21.7 µmol per serving were identified as the main source.

Purification and analysis of PERIOD protein complexes of the mammalian circadian clock.

In mammals, circadian rhythms are generated at least in part by a cell-autonomous transcriptional feedback loop in which the three PERIOD (PER) and two CRYPTOCHROME (CRY) proteins inhibit the activity of the dimeric transcription factor CLOCK-BMAL1, thereby repressing their own expression. Upon nuclear entry, the PER and CRY proteins form a large protein complex (PER complex) that carries out circadian negative feedback by means of at least two basic functions: (1) it brings together multiple effector proteins that repress transcription and (2) it delivers these repressive effectors directly to CLOCK-BMAL1 bound to E-box sequences of circadian target genes. At present, the composition, mechanisms of action, and dynamics of PER complexes in circadian clock negative feedback are incompletely understood. Here, we describe several experimental approaches to the study of PER complexes obtained from mammalian tissues. We focus on the isolation of nuclei from mouse tissues, the extraction of PER complexes from the isolated nuclei, characterization of native PER complexes by gel filtration and blue native polyacrylamide gel electrophoresis, preparative immunoaffinity purification of PER complexes for mass spectrometric identification of constituent proteins, and chromatin immunoprecipitation to monitor the recruitment of PER complex proteins to CLOCK-BMAL1 at E-box sites of clock-regulated genes.

Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane.

The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane. We report that the small hydrophobic protein Mgr2 controls the lateral release of preproteins. Mgr2 interacts with preproteins in transit through the TIM23 complex. Overexpression of Mgr2 delays preprotein release, whereas a lack of Mgr2 promotes preprotein sorting into the inner membrane. Preproteins with a defective inner membrane sorting signal are translocated into the matrix in wild-type mitochondria but are released into the inner membrane in Mgr2-deficient mitochondria. We conclude that Mgr2 functions as a lateral gatekeeper of the mitochondrial presequence translocase, providing quality control for the membrane sorting of preproteins.

The mitochondrial ADP/ATP carrier associates with the inner membrane presequence translocase in a stoichiometric manner.

The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.

Mitochondrial inner membrane protease promotes assembly of presequence translocase by removing a carboxy-terminal targeting sequence.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) is essential for importing cleavable preproteins into mitochondria. The preproteins contain amino-terminal targeting sequences that are removed by the mitochondrial processing peptidase (MPP). Some preproteins carry bipartite presequences that are cleaved twice, by MPP and the inner membrane protease (IMP). Here, we report that the TIM23 complex is altered in mitochondria lacking the IMP subunit Imp1 although none of the TIM23 components contains a bipartite presequence. We show that the TIM23 subunit Mgr2 is processed by IMP, but not by MPP. The cytosolic precursor of Mgr2 contains a carboxy-terminal sequence that promotes targeting to mitochondria, but impairs stable assembly and function of the mature TIM23 complex. IMP removes the carboxy-terminal targeting sequence and thus promotes proper assembly of the TIM23 complex. Our results reveal carboxy-terminal processing as a new mechanism in the biogenesis of the mitochondrial inner membrane.

Magnesium deficiency phenotypes upon multiple knockout of Arabidopsis thaliana MRS2 clade B genes can be ameliorated by concomitantly reduced calcium supply.

Plant MRS2 membrane protein family members have been shown to play important roles in magnesium uptake and homeostasis. Single and double knockouts for two Arabidopsis thaliana genes, AtMRS2-1 and AtMRS2-5, have previously not shown significant phenotypes even under limiting Mg(2+) supply although both are strongly expressed already in early seedlings. Together with AtMRS2-10, these genes form clade B of the AtMRS2 gene family. We now succeeded in obtaining homozygous AtMRS2-1/10 double and AtMRS2-1/5/10 triple knockout lines after selection under increased magnesium supply. Although wilting early, both new mutant lines develop fully and are also fertile under standard magnesium supply, but show severe developmental retardation under limiting Mg(2+) concentrations. To investigate nutrient dependency of germination and seedling development under various conditions, including variable supplies of Mg(2+), Ca(2+), Zn(2+), Mn(2+), Co(2+), Cd(2+) and Cu(2+), in a reproducible and economical way, we employed a small-scale liquid culturing system in 24-well plate set-ups. This allowed the growth and monitoring of individual plantlets of different mutant lines under several nutritional conditions in parallel, and the scoring and statistical evaluation of developmental stages and biomass accumulation. Detrimental effects of higher concentrations of these elements were similar in mutants and the wild type. However, growth retardation phenotypes seen upon hydroponic cultivation under low Mg(2+) could be ameliorated when Ca(2+) concentrations were concomitantly lowered, supporting indications for an important interplay of these two most abundant divalent cations in the nutrient homeostasis of plants.

The mitochondrial protein import machinery has multiple connections to the respiratory chain.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and protein translocases required for the import of mitochondrial precursor proteins. These complexes are functionally interdependent, as the import of respiratory chain precursor proteins across and into the inner membrane requires the membrane potential. Vice versa the membrane potential is generated by the proton pumping complexes of the respiratory chain. Besides this basic codependency four different systems for protein import, processing and assembly show further connections to the respiratory chain. The mitochondrial intermembrane space import and assembly machinery oxidizes cysteine residues within the imported precursor proteins and is able to donate the liberated electrons to the respiratory chain. The presequence translocase of the inner membrane physically interacts with the respiratory chain. The mitochondrial processing peptidase is homologous to respiratory chain subunits and the carrier translocase of the inner membrane even shares a subunit with the respiratory chain. In this review we will summarize the import of mitochondrial precursor proteins and highlight these special links between the mitochondrial protein import machinery and the respiratory chain. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes.

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.

Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.

Structural basis for the function of Tim50 in the mitochondrial presequence translocase.

Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding ß-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.