Hardware Development for Plant Cultivation Allowing In Situ Fluorescence Analysis of Calcium Fluxes in Plant Roots Under Microgravity and Ground-Control Conditions.

Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

Reactive oxygen species affect ATP hydrolysis by targeting a highly conserved amino acid cluster in the thylakoid ATP synthase γ subunit.

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

A negative effector of blue light-induced and gravitropic bending in Arabidopsis.

Although sessile, plants are able to grow toward or away from an environmental stimulus. Important examples are stem or leaf orientation of higher plants in response to the direction of the incident light. The responsible photoreceptors belong to the phototropin photoreceptor family. Although the mode of phototropin action is quite well understood, much less is known of how the light signal is transformed into a bending response. Several lines of evidence indicate that a lateral auxin gradient is responsible for asymmetric cell elongation along the light gradient within the stem. However, some of the molecular key players leading to this asymmetric auxin distribution are, as yet, unidentified. Previously, it was shown that phototropin gets autophosphorylated upon illumination and binds to a scaffold protein termed NPH3 (for nonphototropic hypocotyl 3). Using a yeast three-hybrid approach with phototropin and NPH3 as a bait complex, we isolated a protein, termed EHB1 (for enhanced bending 1), with a so far unknown function, which binds to this binary complex. This novel interacting factor negatively affects hypocotyl bending under blue light conditions in Arabidopsis (Arabidopsis thaliana) and thus seems to be an important component regulating phototropism. Interestingly, it could be shown that the gravitropic response was also affected. Thus, it cannot be ruled out that this protein might also have a more general role in auxin-mediated bending toward an environmental stimulus.

Tangent algorithm for photogravitropic balance in plants and Phycomyces blakesleeanus: Roles for EHB1 and NPH3 of Arabidopsis thaliana.

Plant organs that are exposed to continuous unilateral light reach in the steady-state a photogravitropic bending angle that results from the mutual antagonism between the photo- and gravitropic responses. To characterize the interaction between the two tropisms and their quantitative relationship we irradiated seedlings of Arabidopsis thaliana that were inclined at various angles and determined the fluence rates of unilateral blue light required to compensate the gravitropism of the inclined hypocotyls. We found the compensating fluence rates to increase with the tangent of the inclination angles (0° < γ < 90° or max. 120°) and decrease with the cotangent (90°< γ < 180° or max. 120°of the inclination angles. The tangent dependence became also evident from analysis of previous data obtained with Avena sativa and the phycomycete fungus, Phycomyces blakesleeanus. By using loss-of function mutant lines of Arabidopsis, we identified EHB1 (enhanced bending 1) as an essential element for the generation of the tangent and cotangent relationships. Because EHB1 possesses a C2-domain with two putative calcium binding sites, we propose that the ubiquitous calcium dependence of gravi- and phototropism is in part mediated by Ca2+-bound EHB1. Based on a yeast-two-hybrid analysis we found evidence that EHB1 does physically interact with the ARF-GAP protein AGD12. Both proteins were reported to affect gravi- and phototropism antagonistically. We further showed that only AGD12, but not EHB1, interacts with its corresponding ARF-protein. Evidence is provided that AGD12 is able to form homodimers as well as heterodimers with EHB1. On the basis of these data we present a model for a mechanism of early tropism events, in which Ca2+-activated EHB1 emerges as the central processor-like element that links the gravi- and phototropic transduction chains and that generates in coordination with NPH3 and AGD12 the tangent / cotangent algorithm governing photogravitropic equilibrium.

Comparative proteomic approach to study the salinity effect on the growth of two contrasting quinoa genotypes.

The aim of this study was to investigate the effect of NaCl salinity (0, 100 and 300 mM) on the individual response of the quinoa varieties Kcoito (Altiplano Ecotype) and UDEC-5 (Sea-level Ecotype) with physiological and proteomic approaches. Leaf protein profile was performed using two dimensional gel electrophoresis (2-DE). UDEC-5 showed an enhanced capacity to withstand salinity stress compared to Kcoito. In response to salinity, we detected overall the following differences between both genotypes: Toxicity symptoms, plant growth performance, photosynthesis performance and intensity of ROS-defense. We found a mirroring of these differences in the proteome of each genotype. Among the 700 protein spots reproducibly detected, 24 exhibited significant abundance variations between samples. These proteins were involved in energy and carbon metabolism, photosynthesis, ROS scavenging and detoxification, stress defense and chaperone functions, enzyme activation and ATPases. A specific set of proteins predominantly involved in photosynthesis and ROS scavenging showed significantly higher abundance under high salinity (300 mM NaCl). The adjustment was accompanied by a stimulation of various metabolic pathways to balance the supplementary demand for energy or intermediates. However, the more salt-resistant genotype UDEC-5 presented a beneficial and significantly higher expression of nearly all stress-related altered enzymes than Kcoito.

A gravitropic stimulus alters the distribution of EHB1, a negative effector of root gravitropism in Arabidopsis.

In Arabidopsis gravitropism is affected by two antagonistically interacting proteins, AGD12 (ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN) and EHB1 (ENHANCED BENDING 1). While AGD12 enhances gravitropic bending, EHB1 functions as a negative element. To further characterize their cellular function, we analyzed the location of AGD12-GFP and EHB1-GFP fusion proteins in the root apex by confocal laser-scanning microscopy after gravitropic stimulation. For this purpose, a novel method of microscopic visualization was developed with the objective and root axes aligned allowing an improved and comparable discernment of the fluorescence gradient across the columella. In vertical roots, both proteins were localized symmetrically and occurred preferentially in the outer layers of the columella. After reorienting roots horizontally, EHB1-GFP accumulated in the upper cell layers of the columella, that is, opposite to the gravity vector. The gravity-induced EHB1-GFP asymmetry disappeared after reorienting the roots back into the vertical position. No such asymmetry occurred with AGD12-GFP. Our findings reveal that after a gravitropic stimulus the cellular ratio between EHB1 and AGD12 is affected differently in the upper and lower part of the root. Its impact as a significant signaling event that ultimately affects the redirection of the lateral auxin flux toward the lower site of the root is discussed.

Short-term effects of salt exposure on the maize chloroplast protein pattern.

It is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4 h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1e and a Ca(2+)-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem II, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na(+) on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism.

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

School sport-associated injuries in adolescents: A single center experience.

BACKGROUND: Each year, on average 1.26 million students face physical injuries at German schools and universities. Implementation of systematic prevention programs against school-associated injuries in adolescents is poor. Part of the reason might be a lack of data on nature and frequency of common school-associated injuries. OBJECTIVE: The aim of this study was to obtain qualitative and quantitative data that might help to develop prevention or protection strategies against sport-associated injuries at school. METHODS: A retrospective analysis was conducted on all patients between the ages of 10 and 16 years admitted from January 1st, 2009 to December 31st, 2013 to the emergency room of an academic teaching hospital for school-associated injuries (n= 901). For injuries associated with school sports, time of injury, type of sports and injured body region were analyzed. RESULTS: A significant part of school-associated injuries occurred in school sports (55.7%). The frequency of school sport-associated injuries per school day did not differ between months or seasons of the school year. Most injuries occurred between the ages of thirteen to fifteen. Ball sports were associated with 41% of all sport-associated injuries, particularly with soccer (22%). Distal extremities like hands (38.4%) and feet/ankle (30.3%) were predominantly injured, but only 5% of cases needed surgery. CONCLUSIONS: Most school-associated injuries occur in school sport, particularly with ball sports. Mostly, distal extremities were injured. Implementation of systematic prevention strategies for a reduction in finger injuries should be subject of future research.

EHB1 and AGD12, two calcium-dependent proteins affect gravitropism antagonistically in Arabidopsis thaliana.

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca2+-activated states.

Gravitropism in Arabidopsis thaliana: Root-specific action of the EHB gene and violation of the resultant law.

Gravitropic bending of seedlings of Arabidopsis thaliana in response to centrifugal accelerations was determined in a range between 0.0025 and 4×g to revisit and validate the so-called resultant law, which claims that centrifugation causes gravitropic organs to orient parallel to the resultant stimulus vector. We show here for seedlings of A. thaliana that this empirical law holds for hypocotyls but surprisingly fails for roots. While the behavior of hypocotyls could be modeled by an arc tangent function predicted by the resultant law, roots displayed a sharp maximum at 1.8×g that substantially overshoots the predicted value and that represents a novel phenomenon, diagravitropism elicited by centrifugal acceleration. The gravitropic bending critically depended on the orientation of the seedling relative to the centrifugal acceleration. If the centrifugal vector pointed toward the cotyledons, gravitropic bending of hypocotyls and roots was substantially enhanced. The complex behavior of Arabidopsis seedlings provides strong evidence that gravitropic bending entails a cosine component (longitudinal stimulus) to which the seedlings were more sensitive than to the classical sine component. The absolute gravitropic thresholds of hypocotyls and roots were determined in a clinostat-centrifuge and found to be below 0.015×g. A tropism mutant lacking the EHB1 protein, which interacts with ARF-GAP (ARF GTPase-activating protein) and thus indirectly with a small ARF-type G protein, displayed a lower gravitropic threshold for roots and also enhanced bending, while the responses of the hypocotyls remained nearly unaffected.

Insights in small Heat Shock Protein/client interaction by combined protection analysis of two different client proteins.

sHSPs interact with clients under denaturing conditions. CPH1Δ2, a truncated version of cyanobacterial phytochrome CPH1, was introduced as a new reporter (client). Comparative analyses of At17.8 and At17.6B as cytosolic class I sHSP representatives demonstrated the advantages of a chromophore-bearing photoreversible protein as new client for analyzing sHSP holdase function in addition to malate dehydrogenase (MDH). The tested sHSPs protected both clients in similar ways but with different efficiencies. Bis-ANS binding studies with sHSPs suggested that the bis-ANS binding is dependent on interactions between different sHSPs and MDH under denaturing temperatures.

Singlet oxygen inhibits ATPase and proton translocation activity of the thylakoid ATP synthase CF1CFo.

Singlet oxygen ((1)O(2)) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of (1)O(2) generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to (1)O(2) than CF1CFo in its reduced state, a new insight on the mechanism of (1)O(2) interaction with the gamma subunit.

Singlet oxygen affects the activity of the thylakoid ATP synthase and has a strong impact on its gamma subunit.

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of (1)O2. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its gamma subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to (1)O2.

Uncapped mRNA introduced into tobacco protoplasts can be imported into the nucleus and is trapped by leptomycin B.

The mechanism of nuclear export of RNAs in yeast and animal cells is rapidly being uncovered, but RNA export in plants has received little attention. We introduced capped and uncapped fluorescent mRNAs into tobacco (Nicotiana plumbaginifolia) protoplasts and studied their cellular localization. Following insertion, capped transcripts were found in the cytoplasm, while uncapped messengers transiently appeared in the nucleus in about one-quarter to one-third of the cells. These mRNAs were trapped by the nuclear export-inhibiting drug leptomycin B, pointing to an export mechanism in plants similar to Rev-NES-mediated RNP export in other organisms.