A Small Chloroplast-Encoded Protein as a Novel Architectural Component of the Light-Harvesting Antenna

A small conserved open reading frame in the plastid genome, ycf9, encodes a putative membrane protein of 62 amino acids. To determine the function of this reading frame we have constructed a knockout allele for targeted disruption of ycf9. This allele was introduced into the tobacco plastid genome by biolistic transformation to replace the wild-type ycf9 allele. Homoplasmic ycf9 knockout plants displayed no phenotype under normal growth conditions. However, under low light conditions, their growth rate was significantly reduced as compared with the wild-type, due to a lowered efficiency of the light reaction of photosynthesis. We show that this phenotype is caused by the deficiency in a pigment–protein complex of the light-harvesting antenna of photosystem II and hence by a reduced efficiency of photon capture when light availability is limiting. Our results indicate that, in contrast to the current view, light-harvesting complexes do not only consist of the classical pigment-binding proteins, but may contain small structural subunits in addition. These subunits appear to be crucial architectural factors for the assembly and/or maintenance of stable light-harvesting complexes

Der Einfluss von Hyperventilation auf die kognitiven Leistungen, die neurologisch-motorischen Fähigkeiten und den histologischen Schaden nach einem kontrollierten zerebralen Trauma bei der Ratte

The Effect of Hyperventilation on Cognitive Performance, Motor Functions and Lesion Volume after Controlled Cortical Impact in the Rat Introduction: We investigated the effects of short-term moderate hyperventilation on neurocognitive and motor functions as well as lesion volume in rats subjected to focal traumatic brain injury. Thereby the model of controlled cortical impact (CCI) was established associated with the evaluation of a battery of behavioral tests. Methode: 21 male Sprague-Dawley rats (369±15 g) were trained to achieve the modified Hole-Board Test (mHB-Test) for a period of 14 days and some more behavioral tests (Beam Walking, Beam Balance, neurologic score) for 3 days. After completion of specific baseline parameters of the mHB-Test rats were anesthetized with 1.0-1.5 Vol% halothane in O2/N2O (FiO2=0,33), intubated and mechanically ventilated for surgical preparation. After cranio-tomy CCI was induced using a pneumatic pistol (Ø 5 mm, 1,75 mm depth, 200 ms, 4 m/s). Animals were then randomly assigned to one of two groups for four hours post-traumatic ventilation with Halothan (0,8-1,0 Vol%): group 1=normoventilation (n=10; PaCO2=38-42 mmHg); group 2=hyperventilation (n=11; PaCO2=28-32 mmHg). During the entire study, brain temperature and mean arterial blood pressure were kept at normal physiological levels. Additionally breathing and heart rate, PaO2, pH, glucose and heamoglobin were messured. Upon recovery all behavioral tests were continued to euthanasia on the 20th day. During deep anesthesia rats were decapitated and their brains were sampled, frozen and then cut in 10 µm thick sections to evaluate lesion volume after cresyl violet staining. Results are tabu-lar shown Mean+/-SD and graphical Mean+/-SEM (statistic: ANOVA and post hoc t-test). Results: Hyperventilated rats developed a significant deficit in declarative memory (mHB-Test), with variances especially on days 1-2 after trauma associated with a decreased neuro-logical score on days 1-3 compared to normoventilated animals which had a decrease only on day 1. For motor impairments after CCI the Beam Walking and Beam Balance were most sensitive with deficits in both groups and a significant disability of hyperventilated rats. All impairments were just transient after the traumatic brain injury and adjusted to baseline pa-rameters on day 6. Bodyweight measurements, time of food intake or inactivity (mHB-Test) and all several motoric parameters show a marginal reduction of constitution in the rats after CCI. Exploration parameters of mHB-Test demonstrate that normoventilated rats are more active and explorativ following the CCI in comparison to hyperventilated rats. On day 20 after injury, lesion volume was significant larger in the hyperventilated group (69,7±13,0 mm3) versus the normoventilated group (48,3±15,6 mm3). Discussion: We evaluated a model of CCI, that is inducing a standardized and reproduce-able traumatic brain injury. Four hours of post-traumatic hyperventilation transiently impairs hippocampus-dependent declarative memory as well as neurocognitive and motor functions. Hyperventilation also enhances long-term histological damage. These data suggest, that hyperventilation without controlling intracranial pressure is able to deteriorate primary lesion and should be used with caution after acute head trauma.Einleitung: Im Rahmen dieser Studie wurde der Einfluss kurzfristiger, moderater Hyper-ventilation auf neurokognitive Funktionen, motorische Fähigkeiten und histologischen Schaden an Ratten nach kontrolliertem zerebralen Trauma untersucht. Zusätzlich wurde dabei das Modell des Controlled Cortical Impact (CCI) etabliert und in Kombination dazu verschiedene Verhaltenstest in ihrer Aussagekraft evaluiert. Methode: 21 männliche Sprague-Dawley Ratten (369±15 g) wurden 14 Tage im modifizier-ten Hole-Board Test (mHB-Test) und 3 Tage in weiteren Verhaltenstests trainiert (z.B. Beam Walking, Beam Balance, Neurologischer Score). Nach Erfüllung bestimmter Ausgangskriterien im mHB-Test wurden die Ratten mit 1,0-1,5 Vol% Halothan in O2/N2O (FiO2=0,33) anästhesiert, intubiert und mechanisch beatmet. Nach einer Kraniotomie wurde das kontrollierte zerebrale Trauma mit einem Luftdruck-betriebenen Schussapparat induziert (Ø 5 mm, 1,75 mm Eindringtiefe, 200 ms, 4 m/s). Die Tiere wurden in zwei Versuchs-gruppen für die vierstündige Beatmung unter Halothan (0,8-1,0 Vol%) nach dem Trauma randomisiert: Gruppe 1=Normoventilation (n=10; PaCO2=38-42 mmHg); Gruppe 2=Hyper-ventilation (n=11; PaCO2=28-32 mmHg). Während der gesamten Operation wurden Hirntemperatur und mittlerer arterieller Blutdruck im physiologischen Bereich gehalten. Zusätzlich wurden Atem- und Herzfrequenz, arterieller Sauerstoff-Partialdruck, pH-Wert, Blutglukose und Hämoglobin gemessen. Nach dem Eingriff wurden alle Verhaltenstests bis zur Euthanasie am 20. Tag weitergeführt. Dazu erfolgte die Gehirnentnahme nach Dekapitation in tiefer Narkose. Aus den tiefgefrorenen Gehirnen wurden 10 µm dicke Schnitte zur Bestimmung des Läsionsvolumens hergestellt und mit Kresylviolett gefärbt. Die Ergebnisse werden tabellarisch in Mittelwerten±Standardabweichung und graphisch in Mittelwerten±Standardfehler angegeben (Statistik: ANOVA und post hoc T-Test). Ergebnisse: Nach dem Trauma entwickelten hyperventilierte Ratten signifikante Defizite im deklarativen Gedächtnis (mHB-Test), die am deutlichsten am Tag 1 und 2 zu Veränderun-gen führten. Dementsprechend zeigten sich signifikante Unterschiede im neurologischen Score, der besonders am Tag 1 bis 3 im Vergleich zu den normoventilierten Tieren, die nur am Tag 1 Veränderungen zeigten, verschlechtert war. Am sensitivsten unter den neuro-logisch-motorischen Tests stellten sich der Beam Walking und der Beam Balance mit Defizi-ten in beiden Gruppen nach CCI, wobei die hyperventilierten Tiere signifikant schlechtere Leistungen zeigten. Alle Defizite blieben nur vorübergehen bestehen und 6 Tage nach dem Trauma erreichten die Tiere wieder Ausgangsleistungen. Alle Ratten wurden durch das Trauma nur wenig im Allgemeinbefinden reduziert, wie Körpergewichtsmessung, Fress- und Inaktivitätszeit (mHB-Test) und die einzelnen motorischen Verhaltenstests zeigten. Explorationsparameter des mHB-Tests ergaben, dass normoventilierte Ratten nach der OP aktiver und explorativ interessierter waren als Hyperventilierte. Am 20. Tag nach dem Trauma war das Läsionsvolumen signifikant größer in der hyperventilierten (69,7±13,0 mm3) im Vergleich zur normoventilierten (48,3±15,6 mm3) Versuchsgruppe. Diskussion: In vorliegender Studie wurde ein CCI-Modell evaluiert, das einen standardi-sierten und reproduzierbaren Gehirnschaden induziert. Hyperventilation in den ersten vier Stunden nach einem CCI beeinflusst vorübergehend sowohl das Hippokampus-abhängige deklarative Gedächtnis als auch neurokognitive und motorische Funktionen, der histolo-gische Schaden vergrößert sich langandauernd. Diese Ergebnisse weisen darauf hin, dass Hyperventilation ohne Hirndruckkontrolle die primäre Läsion verschlechtern kann und nur mit Vorsicht nach einem akutem Schädel-Hirn-Trauma einzusetzen ist

Near-term quantum-repeater experiments with nitrogen-vacancy centers: Overcoming the limitations of direct transmission

Quantum channels enable the implementation of communication tasks inaccessible to their classical counterparts. The most famous example is the distribution of secret key. However, in the absence of quantum repeaters, the rate at which these tasks can be performed is dictated by the losses in the quantum channel. In practice, channel losses have limited the reach of quantum protocols to short distances. Quantum repeaters have the potential to significantly increase the rates and reach beyond the limits of direct transmission. However, no experimental implementation has overcome the direct transmission threshold. Here, we propose three quantum repeater schemes and assess their ability to generate secret key when implemented on a setup using nitrogen-vacancy (NV) centers in diamond with near-term experimental parameters. We find that one of these schemes - the so-called single-photon scheme, requiring no quantum storage - has the ability to surpass the capacity - the highest secret-key rate achievable with direct transmission - by a factor of 7 for a distance of approximately 9.2 km with near-term parameters, establishing it as a prime candidate for the first experimental realization of a quantum repeater.Comment: 19+17 pages, 17 figures. v2: added "Discussion and future outlook" section and expanded introduction, published versio

Faithful transcription initiation from a mitochondrial promoter in transgenic plastids

The transcriptional machineries of plastids and mitochondria in higher plants exhibit striking similarities. All mitochondrial genes and part of the plastid genes are transcribed by related phage-type RNA polymerases. Furthermore, the majority of mitochondrial promoters and a subset of plastid promoters show a similar structural organization. We show here that the plant mitochondrial atpA promoter is recognized by plastid RNA polymerases in vitro and in vivo. The Arabidopsis phage-type RNA polymerase RpoTp, an enzyme localized exclusively to plastids, was found to recognize the mitochondrial atpA promoter in in vitro assays suggesting the possibility that mitochondrial promoters might function as well in plastids. We have, therefore, generated transplastomic tobacco plants harboring in their chloroplast genome the atpA promoter fused to the coding region of the bacterial nptII gene. The chimeric nptII gene was found to be efficiently transcribed in chloroplasts. Mapping of the 5′ ends of the nptII transcripts revealed accurate recognition of the atpA promoter by the chloroplast transcription machinery. We show further that the 5′ untranslated region (UTR) of the mitochondrial atpA transcript is capable of mediating translation in chloroplasts. The functional and evolutionary implications of these findings as well as possible applications in chloroplast genome engineering are discussed

Y3IP1, A nucleus-encoded Thylakoid Protein, Cooperates with the Plastid-Encoded YCf3 Protein in Photosystem I Assembly of Tobacco and Arabidopsis

The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C- terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35–HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35–HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes

Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

<p>Abstract</p> <p>Background</p> <p>We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.</p> <p>Methods</p> <p>Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.</p> <p>Results</p> <p>Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).</p> <p>Conclusion</p> <p>Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.</p

Improving plant drought tolerance and growth under water limitation through combinatorial engineering of signalling networks

Agriculture is by far the biggest water consumer on our planet, accounting for 70 per cent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently ('more crop per drop'). Water-use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here, we describe a combinatorial engineering approach to optimize signalling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium-dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water-limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water-limited conditions. Our work provides an efficient strategy for engineering complex signalling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function