Senescence-Associated Sugar Transporter1 affects developmental master regulators and controls senescence in Arabidopsis.

Sugar transport across membranes is critical for plant development and yield. However, an analysis of the role of intracellular sugar transporters in senescence is lacking. Here, we characterized the role of Senescence-Associated Sugar Transporter1 (SAST1) during senescence in Arabidopsis (Arabidopsis thaliana). SAST1 expression was induced in leaves during senescence and after the application of abscisic acid (ABA). SAST1 is a vacuolar protein that pumps glucose out of the cytosol. sast1 mutants exhibited a stay-green phenotype during developmental senescence, after the darkening of single leaves, and after ABA feeding. To explain the stay-green phenotype of sast1 mutants, we analyzed the activity of the glucose-induced master-regulator TOR (target of rapamycin), which is responsible for maintaining a high anabolic state. TOR activity was higher in sast1 mutants during senescence compared to wild types, whereas the activity of its antagonist, SNF1-related protein kinase 1 (SnRK1), was reduced in sast1 mutants under senescent conditions. This deregulation of TOR and SnRK1 activities correlated with high cytosolic glucose levels under senescent conditions in sast1 mutants. Although sast1 mutants displayed a functional stay-green phenotype, their seed yield was reduced. These analyses place the activity of SAST1 in the last phase of a leaf's existence in the molecular program required to complete its life cycle.

Natural variation of temperature acclimation of Arabidopsis thaliana.

Acclimation is a multigenic trait by which plants adjust photosynthesis and metabolism to cope with a changing environment. Here, natural variations of photosynthetic efficiency and acclimation of the central carbohydrate metabolism were analyzed in response to low and elevated temperatures. For this, 18 natural accessions of Arabidopsis thaliana, originating from Cape Verde Islands and Europe, were grown at 22°C before being exposed to 4°C and 34°C for cold and heat acclimation, respectively. Absolute amounts of carbohydrates were quantified together with their subcellular distribution across plastids, cytosol and vacuole. Linear electron transport rates (ETRs) were determined together with the maximum quantum efficiency of photosystem II (Fv/Fm) for all growth conditions and under temperature fluctuation. Under elevated temperature, ETR residuals under increasing photosynthetic photon flux densities significantly correlated with the degree of temperature fluctuation at the original habitat of accessions, indicating a geographical east/west gradient of photosynthetic acclimation capacities. Plastidial sucrose concentrations positively correlated with maximal ETRs under fluctuating temperature, indicating a stabilizing role within the chloroplast. Our findings revealed specific subcellular carbohydrate distributions that contribute differentially to the photosynthetic efficiency of natural Arabidopsis thaliana accessions across a longitudinal gradient. This sheds light on the relevance of subcellular metabolic regulation for photosynthetic performance in a fluctuating environment and supports the physiological interpretation of naturally occurring genetic variation of temperature tolerance and acclimation.

Impaired chloroplast positioning affects photosynthetic capacity and regulation of the central carbohydrate metabolism during cold acclimation.

Photosynthesis and carbohydrate metabolism of higher plants need to be tightly regulated to prevent tissue damage during environmental changes. The intracellular position of chloroplasts changes due to a changing light regime. Chloroplast avoidance and accumulation response under high and low light, respectively, are well known phenomena, and deficiency of chloroplast movement has been shown to result in photodamage and reduced biomass accumulation. Yet, effects of chloroplast positioning on underlying metabolic regulation are less well understood. Here, we analysed photosynthesis together with metabolites and enzyme activities of the central carbohydrate metabolism during cold acclimation of the chloroplast unusual positioning 1 (chup1) mutant of Arabidopsis thaliana. We compared cold acclimation under ambient and low light and found that maximum quantum yield of PSII was significantly lower in chup1 than in Col-0 under both conditions. Our findings indicated that net CO2 assimilation in chup1 is rather limited by biochemistry than by photochemistry. Further, cold-induced dynamics of sucrose phosphate synthase differed significantly between both genotypes. Together with a reduced rate of sucrose cycling derived from kinetic model simulations our study provides evidence for a central role of chloroplast positioning for photosynthetic and metabolic acclimation to low temperature.

Large scale, unbiased analysis of elementary calcium signaling events in cardiac myocytes.

The identification of spatiotemporally restricted Ca2+ signals, Ca2+ sparks, was instrumental for our understanding of cardiac Ca2+ homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca2+ sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca2+ spark analysis for huge data sets of subcellular Ca2+ signals. iSpark was developed to be scanner and detector independent. In myocytes from hearts subjected to various degrees of hypertrophy we acquired >5.000.000 Ca2+ sparks from 14 mice. The iSpark-enabled analysis of this large Ca2+ spark data set showed that the highly organized distribution of Ca2+ sparks present in healthy cells disarrayed concomitant with the development of aberrant transverse tubules and disease severity. Thus, iSpark represents a versatile and universal tool for analyzing local Ca2+ signaling in healthy as well as diseased, aberrant local Ca2+ signal transduction. The results from the unbiased analysis of large data sets provide a deeper insight into possible mechanisms contributing to the onset and progression of cardiac diseases such as hypertrophy.

A background Ca2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling.

AIMS: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown. METHODS AND RESULTS: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn(2+)-quench microfluorimetry, we identified a background Ca(2+) entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca(2+) concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca(2+)-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. CONCLUSIONS: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca(2+) cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis.

Cold acclimation has a differential effect on leaf vascular bundle structure and carbon export rates in natural Arabidopsis accessions originating from southern and northern Europe.

Acclimation to low but non-freezing temperature represents an ecologically important process for Arabidopsis thaliana but also for many other plant species from temperate regions. Cold acclimation comprises and affects numerous molecular and physiological processes and the maintenance of sugar supply of sink tissue by photosynthetically active source tissue is essential for plant survival. Here, changes in vascular bundle (VB) structure at the leaf petiole were analysed together with sucrose exudation rates before and after cold acclimation. Six natural Arabidopsis accessions originating from southern and northern Europe were compared. Photosynthetic efficiency, that is, maximum and effective quantum yield of photosystem II, revealed a significant effect of environmental condition. Only for northern accessions was a highly significant negative correlation observed between leaf sucrose exudation rates, xylem, and petiole cross-sectional areas. Furthermore, only for northern accessions was a significant increase of VB and leaf petiole cross-sectional area observed during cold acclimation. In contrast, variance of cross-sectional areas of cold acclimated southern accessions was strongly reduced compared to control plants, while mean areas remained similar under both conditions. In summary, these findings suggest that natural Arabidopsis accessions from northern Europe significantly adjust sink strength and leaf VB structure to maintain plant growth and photosynthesis under low temperature.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology.

AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.

PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris.

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.

Identification of pexophagy genes by restriction enzyme-mediated integration.

Pichia pastoris has proven to be a valuable model for examining the molecular events of the selective degradation of peroxisomes by a process called pexophagy. We have developed a protocol to rapidly identify genes essential for glucose-induced pexophagy. This method utilizes the random integration of a Zeocin resistance cassette vector into the genomic DNA thereby disrupting gene expression. Transformed yeast are selected by growth on Zeocin and pexophagy mutants identified by their inability to degrade the peroxisomal enzyme alcohol oxidase. The Zeocin vector along with flanking genomic DNA is then isolated from the mutants and the disrupted genes identified by sequencing. We have been able to isolate 59 mutants and identify 8 unique genes. The identification of 24 genes in P. pastoris and 7 genes in H. polymorpha have been reported using this approach which has been referred to as restriction-mediated integration.

A laid-back trip through the Hennigian Forests.

BACKGROUND: This paper is a comment on the idea of matrix-free Cladistics. Demonstration of this idea's efficiency is a major goal of the study. Within the proposed framework, the ordinary (phenetic) matrix is necessary only as "source" of Hennigian trees, not as a primary subject of the analysis. Switching from the matrix-based thinking to the matrix-free Cladistic approach clearly reveals that optimizations of the character-state changes are related not to the real processes, but to the form of the data representation. METHODS: We focused our study on the binary data. We wrote the simple ruby-based script FORESTER version 1.0 that helps represent a binary matrix as an array of the rooted trees (as a "Hennigian forest"). The binary representations of the genomic (DNA) data have been made by script 1001. The Average Consensus method as well as the standard Maximum Parsimony (MP) approach has been used to analyze the data. PRINCIPLE FINDINGS: The binary matrix may be easily re-written as a set of rooted trees (maximal relationships). The latter might be analyzed by the Average Consensus method. Paradoxically, this method, if applied to the Hennigian forests, in principle can help to identify clades despite the absence of the direct evidence from the primary data. Our approach may handle the clock- or non clock-like matrices, as well as the hypothetical, molecular or morphological data. DISCUSSION: Our proposal clearly differs from the numerous phenetic alignment-free techniques of the construction of the phylogenetic trees. Dealing with the relations, not with the actual "data" also distinguishes our approach from all optimization-based methods, if the optimization is defined as a way to reconstruct the sequences of the character-state changes on a tree, either the standard alignment-based techniques or the "direct" alignment-free procedure. We are not viewing our recent framework as an alternative to the three-taxon statement analysis (3TA), but there are two major differences between our recent proposal and the 3TA, as originally designed and implemented: (1) the 3TA deals with the three-taxon statements or minimal relationships. According to the logic of 3TA, the set of the minimal trees must be established as a binary matrix and used as an input for the parsimony program. In this paper, we operate directly with maximal relationships written just as trees, not as binary matrices, while also using the Average Consensus method instead of the MP analysis. The solely 'reversal'-based groups can always be found by our method without the separate scoring of the putative reversals before analyses.

An adaptation of astronomical image processing enables characterization and functional 3D mapping of individual sites of excitation-contraction coupling in rat cardiac muscle.

In beating cardiomyocytes, synchronized localized Ca2+ transients from thousands of active excitation-contraction coupling sites (ECC couplons) comprising plasma and sarcoplasmic reticulum membrane calcium channels are important determinants of the heart's performance. Nevertheless, our knowledge about the properties of ECC couplons is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle the fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion. CaCLEAN empowers the investigation of fundamental properties of ECC couplons in beating cardiomyocytes without pharmacological interventions. Upon examining individual ECC couplons at the nanoscopic level, we reveal their roles in the negative amplitude-frequency relationship and in ß-adrenergic stimulation, including decreasing and increasing firing reliability, respectively. CaCLEAN combined with 3D confocal imaging of beating cardiomyocytes provides a functional 3D map of active ECC couplons (on average, 17,000 per myocyte). CaCLEAN will further enlighten the ECC-couplon-remodelling processes that underlie cardiac diseases.

Cardiac remodeling in Gαq and Gα11 knockout mice.

BACKGROUND: Although both Gαq- and Gα11-protein signaling are believed to be involved in the regulation of cardiac hypertrophy, their detailed contribution to myocardial function remains elusive. METHODS AND RESULTS: We studied remodeling processes in healthy transgenic mice with genetically altered Gαq/Gα11-expression, in particular a global Gα11-knockout and a novel inducible cardiac specific Gαq-knockout, as well as a combined double knockout (dKO) mouse line. Echocardiography and telemetric ECG recordings revealed that compared with wild type mice, hearts of dKO mice showed an increased ejection fraction and a decreased heart rate, irrespective of age resulting in a maintained cardiac output. We attributed these findings to the lack of Gα11, which the absence was associated with a decreased afterload. Histological analysis of the extracellular matrix in the heart depicted a diminished presence of collagen in aging hearts of dKO mice compared to wild-type mice. The results of a transcriptome analysis on isolated ventricular cardiac myocytes revealed alterations of the activity of genes involved in the Gαq/Gα11-dependent regulation of the extracellular matrix, such as the matricellular protein Cyr61. CONCLUSIONS: From our data we conclude that Gαq/Gα11 signaling pathways play a pivotal role in maintaining gene activity patterns. For the heart we revealed their importance in modulating the properties of the extracellular matrix, a mechanism that might be an important contributor and mechanistic basis for the development of pressure-overload induced cardiac hypertrophy.

Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine alpha-amidating monooxygenase.

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris.

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

PpAtg9 trafficking during micropexophagy in Pichia pastoris.

PpAtg9 is essential for the selective degradation of peroxisomes (e.g., pexophagy) in Pichia pastoris. This integral membrane protein is synthesized in the endoplasmic reticulum (ER) and transported to a unique peripheral compartment (Atg9-PC). A putative ER exit motif has been identified and when deleted results in the accumulation of PpAtg9 within the ER. Upon the onset of micropexophagy, PpAtg9 transits from the Atg9-PC to perivacuolar structures (PVS) and sequestering membranes (SM) that arise from the vacuole to engulf the peroxisomes. In this article, we will discuss the transport pathways of PpAtg9 and those factors responsible for its trafficking.