Biochemische Charakterisierung der Fettsäure-Beta-Oxidation als Teil des Dunkelstoffwechsels von Arabidopsis thaliana

Die Blattstärke ist das primäre Endprodukt der Photosynthese und dient als zentraler Kohlenstoff- und Energiespeicher für die Pflanze. Am Ende einer normalen 8 h Nacht sind die Levels auf 5-10% abgesunken, bereits wenige Stunden später sind sie vollständig erschöpft. Da Pflanzen des Modellorganismus Arabidopsis thaliana mehrere Tage in völliger Dunkelheit überlebensfähig sind, müssen ihr alternative Energiequellen zur Verfügung stehen. Die Fettsäure-Beta-Oxidation wurde bis dato vor allem bezüglich ihrer physiologischen Relevanz während der Keimung untersucht. Daten und Kenntnisse zur Bedeutung dieses biochemischen Pathways im adulten Blattgewebe waren äußerst limitiert und beschäftigten sich vor allem mit der Produktion von Jasmonsäure als Reaktion auf Verwundungen. In der vorliegenden Arbeit wurde anhand von Beta-Oxidations-defizienten Mutanten Untersuchungen zu deren Überlebensfähigkeit in verlängerten Nachtperioden angestellt. Hierfür dienten vor allem pxa1 Mutanten als Studienobjekte. Der Genlokus PXA1 kodiert für einen peroxisomalen ABC-Transporter, dessen Verlust zu einer Inhibition der Fettsäure-Beta-Oxidation führt. Interessanteweise zeigten diese Mutanten nach bereits 36 h in Dunkelheit letale Schädigungen des Blattgewebes. Weitere Studien an Beta-Oxidations-defizienten Mutanten mit Defekten in anderen Loki bestätigten, dass die Blockade der Fettverbrennung ursächlich für das Auftreten des Dunkelphänotyps war. In detaillierten Metabolitanalysen konnte ein früher Anstau an Speicherlipiden aus Bestandteilen der Thylakoidmembranen, gefolgt von freien Fettsäuren gefunden werden. Die hierbei dominierende alpha-Linolensäure zeigte toxische Eigenschaften und bewirkte eine Organell- und Gewebeauflösung in den Mutanten. Begleitet wurde dies von einer massiven Freisetzung der Chlorophyllpigmente aus dem Photosystem II, sodass phototoxische Chlorophyll-Abbauprodukte anstauten und einen additiven, phototoxischen Phänotyp erzeugten. Durch Generierung von Doppelmutanten mit verschiedenen stärkefreien Linien konnte erstmals ein zeitlicher und physiologischer Zusammenhang zwischen Stärke- und Fettsäuremetabolismus gezeigt werden. Besagte stärkefreie, Beta-Oxidations-defiziente Doppelmutanten starben bereits nach 16 h Dunkelheit und waren bei Langtaganzucht zwergwüchsig. Aus den erhaltenen Daten wurde ein Modell entwickelt, demzufolge Pflanzen nach Ausschöpfung des Stärkepools, die notwendige Energie aus der Verbrennung von Fettsäure beziehen

Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast

Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced -proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7) and the stroma (pH 8) is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+, or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+)/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function.Funded by ERDF-co-financed grants from the Ministry of Economy and Competitiveness (BIO2012-33655) and Junta de Andalucía (CVI-7558) to KV. HHK and RH were funded by an NSF Career Grant IOS- 1553506, the School of Biological Sciences and the College of Arts and Sciences at Washington State University. AA acknowledges the receipt of a grant for his PhD studies from the Agricultural Research for Development Fund (ARDF, Egypt).Peer reviewedPeer Reviewe

Probing the in situ volumes of Arabidopsis leaf plastids using three‐dimensional confocal and scanning electron microscopy

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm3. Wild-type guard cell plastids are approximately 18 μm3. Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment

Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII

The photosynthetic machinery of plants can acclimate to changes in light conditions by balancing light-harvesting between the two photosystems (PS). This acclimation response is induced by the change in the redox state of the plastoquinone pool, which triggers state transitions through activation of the STN7 kinase and subsequent phosphorylation of light-harvesting complex II (LHCII) proteins. Phosphorylation of LHCII results in its association with PSI (state 2), whereas dephosphorylation restores energy allocation to PSII (state 1). In addition to state transition regulation by phosphorylation, we have recently discovered that plants lacking the chloroplast acetyltransferase NSI are also locked in state 1, even though they possess normal LHCII phosphorylation. This defect may result from decreased lysine acetylation of several chloroplast proteins. Here, we compared the composition of wild type (wt), stn7 and nsi thylakoid protein complexes involved in state transitions separated by Blue Native gel electrophoresis. Protein complex composition and relative protein abundances were determined by LC–MS/MS analyses using iBAQ quantification. We show that despite obvious mechanistic differences leading to defects in state transitions, no major differences were detected in the composition of PSI and LHCII between the mutants. Moreover, both stn7 and nsi plants show retarded growth and decreased PSII capacity under fluctuating light as compared to wt, while the induction of non-photochemical quenching under fluctuating light was much lower in both nsi mutants than in stn7.</p

Envelope K +

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation

Natural Variation in Small Molecule–Induced TIR-NB-LRR Signaling Induces Root Growth Arrest via EDS1- and PAD4-Complexed R Protein VICTR in Arabidopsis

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Ion Transport in Chloroplast and Mitochondria Physiology in Green Organisms

Chloroplasts and mitochondria both have a prokaryotic origin, carry essential genes on their own highly reduced genome and generate energy in the form of ATP for the plant cell. The ion composition and concentration in these bioenergetic organelles impact photosynthesis, respiration and stress responses in plants. Early electrophysiological and biochemical studies provided strong evidence for the presence of ion channels and ion transporters in chloroplast and mitochondrial membranes. However, it wasn’t until the last decade that the development of model organisms such as Arabidopsis thaliana and Chlamydomonas reinhardtii along with improved genetic tools to study cell physiolgy have led to the discovery of several genes encoding for ion transport proteins in chloroplasts and mitochondria. For the first time, these discoveries have enabled detailed studies on the essential physiological function of the organellar ion flux. This Research Topic welcomed updated overviews and comprehensive investigations on already identified and novel ion transport components involved in physiology of chloroplasts and mitochondria in green organisms

Rapid hyperosmotic-induced Ca2+ responses in Arabidopsis thaliana exhibit sensory potentiation and involvement of plastidial KEA transporters

Plants experience hyperosmotic stress when faced with saline soils and possibly with drought stress, but it is currently unclear how plant roots perceive this stress in an environment of dynamic water availabilities. Hyperosmotic stress induces a rapid rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) in plants, and this Ca(2+) response may reflect the activities of osmo-sensory components. Here, we find in the reference plant Arabidopsis thaliana that the rapid hyperosmotic-induced Ca(2+) response exhibited enhanced response magnitudes after preexposure to an intermediate hyperosmotic stress. We term this phenomenon "osmo-sensory potentiation." The initial sensing and potentiation occurred in intact plants as well as in roots. Having established a quantitative understanding of wild-type responses, we investigated effects of pharmacological inhibitors and candidate channel/transporter mutants. Quintuple mechano-sensitive channels of small conductance-like (MSL) plasma membrane-targeted channel mutants as well as double mid1-complementing activity (MCA) channel mutants did not affect the response. Interestingly, however, double mutations in the plastid K(+) exchange antiporter (KEA) transporters kea1kea2 and a single mutation that does not visibly affect chloroplast structure, kea3, impaired the rapid hyperosmotic-induced Ca(2+) responses. These mutations did not significantly affect sensory potentiation of the response. These findings suggest that plastids may play an important role in early steps mediating the response to hyperosmotic stimuli. Together, these findings demonstrate that the plant osmo-sensory components necessary to generate rapid osmotic-induced Ca(2+) responses remain responsive under varying osmolarities, endowing plants with the ability to perceive the dynamic intensities of water limitation imposed by osmotic stress