Effects of Clinorotation on Growth and Chlorophyll Content of Rice Seeds

Microgravity, as a different environment, has been shown to affect plant growth and development (Sievers et al. 1996; Sack 1997). In the present study, effects of slow clinorotation (2 rpm) on growth and chlorophyll content in rice (variety: PRH-10) seedlings were investigated. Rice seeds were clinorotated continuously for 3, 5 and 7 days under ambient conditions. Root and shoot lengths and weights of rice seedlings were measured on the third, fifth and seventh day. Chlorophyll was extracted using N, N-Dimethylformamide (DMF). Absorption and fluorescence spectra of chlorophyll were recorded. Chlorophyll a, chlorophyll b and total chlorophyll contents were calculated from absorption spectra using Arnon’s method. Results showed an increase in root and shoot lengths in clinorotated samples. Similar results were obtained for root and shoot weights. Absorption spectra of chlorophyll showed no shift in the absorption peaks. Chlorophyll content was increased in clinorotated samples as compared to the controls. Interestingly, the difference between chlorophyll content in control and clinorotated samples decreased as the number of days of clinorotation increased. Chlorophyll a/b ratio was lowered in clinorotated samples as compared to the controls. These results suggest that slow clinorotation (2 rpm) affects plant growth and chlorophyll content in rice seedlings.     

Investigation of ZnO nanoparticles on proline,anthocyanin contents and photosynthetic pigments and lipid peroxidation in the soybean

The interaction between nanoparticles and plants is inevitable. In this study, the effect of different concentrations of ZnO nanoparticles synthesised using olive extract on the soybean was studied. The soybean seeds were cultured in a Hoagland medium containing agar which was treated different concentrations (0, 200 and 400 ppm) of ZnO nanoparticles. After 21 days, the plants were harvested and the parameters of proline, anthocyanin, malondialdehyde (MDA), hydrogen peroxide (H₂ O₂), chlorophyll and carotenoid contents and phenylalanine ammonia‐lyase (PAL) and catalase (CAT) activity in soybeans (Glycine max) were measured. The results showed that the levels of chlorophyll a and b and carotenoid at concentrations of 200 and 400 ppm in comparison with control decreased, while carotenoid content at 200 ppm concentration at a concentration of 400 ppm was not significant. The level of anthocyanin and PAL activity increased with increasing concentration of nanoparticles, while proline content decreased. By increasing the concentration of ZnO nanoparticles, the content of MDA and hydrogen peroxide increased compared to control but CAT activity did not change significantly. This research suggests that ZnO nanoparticles synthesised using olive extract in soybean plants may be toxic by reactive oxygen species production.Inspec keywords: crops, pigments, enzymes, nanoparticles, zinc compounds, toxicology, agricultural safety, nanobiotechnology, hydrogen compoundsOther keywords: carotenoid content, proline content, ZnO nanoparticles, soybean plants, H₂ O₂ , ZnO, time 21.0 d, reactive oxygen species production, toxicity, catalase activity, phenylalanine ammonia‐lyase activity, chlorophyll content, Hoagland medium, olive extract, lipid peroxidation, photosynthetic pigments, hydrogen peroxide, anthocyanin contents     

The stress protein level under clinorotation in context of the seedling developmental program and the stress response

Heat-shock proteins (HSP70 and HSP90) are present in plant cells under the normal growth conditions. At the same time, a variety of environmental disruptions results in their rapid synthesis as a substantial part of adaptation. HSP amounts can be indicative of a cellular stress level. Altered gravity (clinorotation) is unnatural for plants, so it may be a kind of stress. The aim of this study was to analyze the influence of horizontal clinorotation on the HSP70 and HSP90 level during seedling development. Pea (Pisum sativum L.) seedlings grown for 3 days from seed imbibitions in stationary control and under slow clinorotation (2 rpm) are used for this investigation. Western blot analysis indicated that HSP70 and HSP90 were abundant in the embryos of dry seeds and their amount decreased significantly during seed germination. But under horizontal clinorotation, their level in seedlings remained higher compared to the control. Furthermore, a comparison of the influence of horizontal and vertical clinorotation on the HSP level was carried out. On the ELISA data, HSP70 and HSP90 amounts in the 3-day old seedlings were higher after horizontal clinorotation than after vertical. The obtained data show an increased HSP70 and HSP90 level in pea seedlings under clinorotation. Both, rotation and change in the cell position relatively to a gravity vector affect the HSP level.     

Rocket seedling production on the international space station: Growth and nutritional properties

Producing sprouts directly during space missions may represent an interesting opportunity to offer high-quality fresh ready to eat food to the astronauts. The goal of this work was to compare, in terms of growth and nutritional quality, rocket (Eruca sativa Mill.) seedlings grown in the International Space Station during the ENEIDE mission with those grown in a ground-based experiment (in presence and absence of clinorotation). The rocket seedlings obtained from the space-experiment were thinner and more elongated than those obtained in the ground-based experiment. Cotyledons were often closed in the seedlings grown in the space experiment. Quantitative (germination, fresh and dry weight) and qualitative (glucose, fructose, sucrose and starch) traits of rocket seedling were negatively affected by micrograv-ity, especially those recorded on seedlings grown under real microgravity conditions The total chlorophyll, and carotenoids of seedlings obtained in the space experiment were strongly reduced in comparison to those obtained in the ground-based experiment (presence and absence of clinorotation). The results showed that it is possible to produce rocket seedlings in the ISS; however, further studies are needed to define the optimal environmental conditions for producing rocket seedlings with high nutritional value     

Physiological Responses of Synechocystis sp. PCC 6803 under Clinorotation

Photosystem efficiency and the characteristic on oxidative stress were examined to elucidate the metabolic responses of Synechocystis sp. PCC 6803 to short-term clinorotation. Results compiled when using clinostat to simulate microgravity for 60?h, showed that clinorotation clearly prohibited the photochemical quantum yield, but promoted the synthesis of chlorophyll and total protein. This may be a compensatory mechanism for the algal cell to maintain its normal metabolism. An increased malondialdehyde (MDA) content of algal cell upon clinorotation, together with an enhanced catalase (CAT) activity was observed during the whole period of clinorotation. One conclusion is that short-term clinorotation acts as a kind of stress, and that these physiological responses may be a special way for an algal cell to adapt itself to a different environment other than earth gravity.     

Vegetative growth of higher plants on a three-dimensional clinostat

Seedlings of rice, maize, cress, pea, and azuki bean were grown on a three-dimensional clinostat and changes in their vegetative growth processes were analyzed. A balanced relationship among the length or the weight of each organ was observed in these species even on the clinostat. Growth of pea second internodes is supported by the transport of sugars from the cotyledons, which was not influenced by the clinostat rotation. Thus, growth correlation and the translocation of sugars normally occurred even under simulated microgravity conditions. In contrast, morphogenesis was clearly changed by the clinostat rotation. The axiality along the gravity vector disappeared and so seedlings formed themselves into a sphere-like shape on the clinostat. The dorsiventrality was indistinct in growth of maize coleoptiles on the surface of the earth, but the clinostat rotation induced a clear dorsinventral bending. These changes in morphogenesis may influence the long-term growth phenomena and modify the life cycle of higher plants under a microgravity environment.     

Comparative studies on gravisensitive protists on ground (2D and 3D clinostats) and in microgravity

In order to prepare and support space experiments, 2D and 3D clinostats are widely applied to study the influence of simulated weightlessness on biological systems. In order to evaluate the results a comparison between the data obtained in simulation experiments and in real microgravity is necessary. We are currently analyzing the gravity-dependent behavior of the protists Paramecium biaurelia (ciliate) and Euglena gracilis (photosynthetic flagellate) on these different experimental platforms. So far, first results are presented concerning the behaviour of Euglena on a 2D fast rotating clinostat and a 3D clinostat as well as under real microgravity conditions (TEXUS sounding rocket flight), of Paramecium on a 2D clinostat and in microgravity. Our data show similar results during 2D and 3D clinorotation compared to real microgravity with respect to loss of orientation (gravitaxis) of Paramecium and Euglena and a decrease of linearity of the cell tracks of Euglena. However, the increase of the mean swimming velocities, especially during 3D clinorotation (Euglena) and 2D clinorotation of Paramecium might indicate a persisting mechanostimulation of the cells. Further studies including long-term 2D and 3D clinostat exposition will enable us to demonstrate the qualification of the applied simulation methods.     

Distribution of Calcium Ions in Cells of the Root Distal Elongation Zone Under Clinorotation

Currently, calcium ions are known to play a crucial role in the vital activity of plant cells and in stimulus–response coupling for many environmental signals, altered gravity included. The available data on changes in Ca^2?+? distribution and concentration in the cells of different organisms influenced by altered gravity allow to suggest that microgravity affects the calcium messenger system, and provide new insight for the understanding of calcium-and gravity-dependent cellular processes. We have studied with confocal microscopy the distribution and relative content of calcium ions in the Beta vulgaris root distal elongation zone cells grown under slow horizontal clinorotation, reproducing one of the microgravity particularities, namely the absence of an orienting action of the gravity vector, compared to control conditions. We demonstrate that Ca^2?+? relative content is 1.3 times higher in the roots of seedlings grown upwards and 1.2 times higher in the seedlings grown downwards compared to the control. Based on obtained data, taking into account the specific physiological properties of cells in the distal elongation zone, it is supposed that, under clinorotation, enhanced Ca^2?+? relative content affects Ca^2?+?-dependent cytoskeleton reorganization involved in cell gravisensing in altered gravity.     

Comparison Study of Gravity-Dependent Displacement of Amyloplasts in Statocytes of Cress Roots and Hypocotyls

In this study, the kinetics of gravity-dependent movement of amyloplasts (statoliths) along root statocytes and hypocotyls (endodermis cells) has been analyzed and compared in order to testify cytoskeleton involvement in the displacement of statoliths in cress (Lepidium sativum L.) seedling statocytes. After 32 h of growth at 1 g or under a fast clinorotation (50 rpm), the seedlings were treated for 24 min as follows: exposition to clinorotation or 180° inversion and the action of gravitational force in root tip or hypocotyl tip direction. Statolith displacement was studied by light microscopy on semi-thin longitudinal sections of hypocotyls and root caps, measuring the distance between the centre of plastids and morphological cell bottom. Considerable temporal differences have been determined between the kinetics of the longitudinal motion of amyloplasts in root and hypocotyl statocytes of 1-g seedlings upon exposition to fast clinorotation and inversion. In statocytes of both organs of seedlings grown under fast clinorotation, the gravity provoked displacement of statoliths in the direction of its action; however, the displacement was much faster in hypocotyl than in root statocytes. It has been assumed that the gravity-determined longitudinal transport of amyloplasts, both in hypocotyl endodermic cells and root statocytes of cress seedlings, is modulated by the cytoskeleton.     

Actin Microfilament Organization in the Transition Zone of Arabidopsis-ABD2-GFP Roots Under Clinorotation

Seedlings of Arabidopsis thaliana-ABD2-GFP were grown under slow clinorotation (2?rpm) and treated with actin and tubulin disrupting drugs in order to characterize the role of actin microfilaments in cell growth and gravisensing. Changes in microfilament organization and cell parameters have shown that the transition root zone (TZ) is rather sensitive to microfilament disruption in control plants. It is assumed that under clinorotation, organization of actin cytoskeleton in the TZ is coordinated in a different way than in the control. Organization of microfilaments depends upon organization of microtubules and clinorotation does not influence this interrelation significantly.     

Clinorotation Effect on Response of Cress Leaves to Red and Far-Red Light

Responses of cress (Lepidium sativum L.) seedling leaves to separate and simultaneous illumination with red (660 nm) and far-red (735 nm) light were studied under fast clinorotation (50 rpm) and usual gravity (1g) conditions. The monochromatic light emitting diodes (LEDs) have been used for illumination of seedlings from above. The growth and spatial orientation of leaves and the location of presumable gravisensors in petioles were analysed. Clinorotation in the dark promoted the radial expansion of leaf lamina and unfolding of leaves. It was shown that clinorotation in red light inhibited significantly the elongation of petioles as compared with that under the action of gravity force. Simultaneous red and far-red illumination promoted the growth of petioles under clinorotation, but did not affect the orientation of laminas, which remained the same as of the 1-g control ones. Red light, applied separately and simultaneously with far-red light, guided the bending of laminas as well as the unfolding of leaf petioles in both usual and clinorotation conditions. Histological and cytological analyses of petioles revealed the presence of movable amyloplasts in endodermic cells in proximal region of petioles. Comparison of intracellular positioning of amyloplasts in petioles of leaves grown under clinorotation and the action of gravity allows a presumption that these plastids may be identified as gravisensors of garden cress leaves. The obtained data imply that clinorotation and exposition to monochromatic red light or simultaneous illumination by red and far-red light affect the elongation of petioles of cress seedling leaves. Spectral components guide the unfolding of laminas in a gravity-independent manner.     

Cytosolic Calcium Concentration Changes in Neuronal Cells Under Clinorotation and in Parabolic Flight Missions

All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. To study the effects especially of microgravity on biological systems, a variety of platforms are available, from drop towers to the ISS. Due to the costs of these platforms and their limited availability, as an alternative, numerous simulators have been developed for so called “simulated” microgravity. A classical systems is a clinostat, basically rotating a sample around one axis, and by integration of the gravity vector for 360° arguing that thus the effects of gravity are depleted. Indeed, a variety of studies has shown that taking out the direction of gravity from a biological system often results in consequences similar to the exposure of the system to real microgravity. Nevertheless, the opposite has been shown, too, and as a consequence the relevance of clinostats in microgravity research is still under discussion. To get some more insight into this problem we have constructed a small fluorescence clinostat and have studied the effects of clinorotation on the cytosolic calcium concentration of neuroglioma cells. The results have been compared to experiments with identical cells in real microgravity, utilizing parabolic flight missions. Our results show that in case of a cell suspension used in a small florescence clinostat within a tube diameter of 2mm, the effects of clinorotation are comparable to those under real microgravity, both showing a significant increase in intracellular calcium concentration.     

Adaptation of a 2-D Clinostat for Simulated Microgravity Experiments with Adherent Cells

The fast-rotating 2-D clinostat, a ground-based facility for investigations in simulated microgravity, is mainly used for experiments with cell suspensions. Here, we describe the adaptation of a 2-D clinostat for adherent cell investigations using commercially available slide flasks. As a gradient of residual accelerations is present in the slide flasks during clinorotation, the range of maximal g-values has to be adjusted to the investigated cells and type of analysis. For gene expression analysis, a harvesting slide chamber was constructed, allowing collection of cells exposed to defined g-values. Using this slide chamber, human 1F6 melanoma cell line, exposed in the ranges of ≤0.012 g, ≤0.024 g, or ≤0.036 g for 24 h, was harvested and the respective mRNA levels of guanylyl cyclase A (GC-A), an enzyme catalyzing cyclic GMP synthesis, were determined by real-time quantitative PCR analysis. Our results show that the down-regulation of GC-A mRNA levels in 1F6 melanoma cells depends on the residual acceleration values with a maximal reduction at ≤0.012 g. We further used the slide flasks by the clinorotation of murine RAW 264.7 macrophage cell line for f-actin analysis. The laser scanning microscopy images of cells exposed to g-values of ≤0.006 g for 1 h show an increase in the cell size of clinorotated cells, but no rearrangement in the f-actin filament system compared to static 1-g controls. Thus, 2-D clinostats equipped with slide flasks can be used for adherent cell experiments, however, the maximal g-values have to be carefully considered.     

贮藏温度对采后上海青叶片衰老及活性成分的影响

目的为了探究不同贮藏温度对采后上海青叶片衰老及活性成分的影响。方法以上海青为实验材料,分别在0,5,10,15,20,25,30℃下贮藏,对上海青叶片的感官品质、色差、叶绿素含量、丙二醛(MDA)含量、类胡萝卜素含量、叶黄素含量、叶酸含量和亚硝酸盐含量的变化进行分析。结果与20,25,30℃相比,在10℃和15℃下贮藏可减缓上海青叶片L^*值、b^*值、MDA含量和亚硝酸盐含量的增加,以及|a^*|值、叶绿素含量、类胡萝卜素含量、叶黄素含量和叶酸含量的降低;与10℃和15℃贮藏条件相比,在0℃和5℃下贮藏可显著抑制上海青叶片L^*值、b^*值、MDA含量和亚硝酸盐含量的增加,以及|a^*|值、叶绿素含量、类胡萝卜素含量、叶黄素含量和叶酸含量的降低,减缓上海青叶片的黄化衰老进程,从而维持上海青良好的外观品质。在贮藏温度为20,25,30℃时,上海青的最大贮藏期不超过4 d;贮藏温度为10℃和15℃时,最大贮藏期分别不超过9 d和7 d;贮藏温度为0℃和5℃时,最大贮藏期分别不超过40 d和15 d。结论在低温条件下贮藏,尤其是在0℃下贮藏可最大限度地减缓采后上海青叶片的衰老进程,可维持其较高的活性成分,并抑制其组织中亚硝酸盐的积累,显著延长上海青的贮藏期。     

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.     

Live Imaging Technique for Studies of Growth and Development of Chinese Cabbage Under Microgravity in a Recoverable Satellite (SJ-8)

The live imaging techniques have been developed and applied to investigate for the first time the growth and development of Chinese cabbage for 18 days under microgravity conditions on board the Chinese SJ-8 recoverable satellite. These experiments offer insight into plant behaviors operating during plant development in space. Two automatic, preprogrammed CCD cameras were installed in the plant experimental chamber. The experimental objectives were: (1) seed germination; (2) seedling growth; (3) flower opening and pollination. The growth of seedlings and flowers were followed by time lapse photography at 2 h intervals. Serial real-time images of the Chinese cabbage plant growth under microgravity were successfully obtained through the remote operating system. The image data obtained from space experiment, in comparison with the results from ground control (1 g) and 3D clinostat stimulate experiments, showed that the height of plant and the number of leaves were significantly reduced under the microgravity conditions, but characters of leaf arrangement and leaf shape were not altered obviously. Flower opening and expansion were inhibited by exposed to space flight condition. The petals of flowers from both SJ-8 grown plants and clinostat rotated plants couldn’t fully expand before wilted. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

煮制时间对绿豆中淀粉性质的影响及相关性分析

目的 探究绿豆煮制过程中淀粉含量及理化性质的变化规律,从而根据不同目的 选用最优煮制时间,且为绿豆煮制加工提供科学数据.方法 以明绿豆为主要原料,采用传统煮制方法,以10 min为间隔时间采样,进行淀粉含量和淀粉性质测定及相关性分析.结果 随着煮制时间的延长,绿豆中总淀粉含量和直/支链淀粉含量均呈下降趋势.溶解度在煮制30 min后呈上升趋势,在60 min时达最大;膨胀力呈下降趋势,煮制50 min后趋于稳定;糊透明度在煮制40 min后明显增大,在60 min时达最大;凝沉稳定性在煮制40 min后逐渐增强.由相关性分析结果可知,煮制时间与溶解度呈极显著正相关,与绿豆中总淀粉含量和膨胀力呈极显著负相关,与透光率呈显著正相关,与直/支链淀粉含量和凝沉体积呈显著负相关.结论 煮制时间是影响绿豆中淀粉含量和性质的主要因素,而淀粉含量则可以直接影响绿豆的膨胀力、透明度等理化性质.在绿豆食品加工过程中,可根据加工目的 选择不同的煮制时间.     

Tubulin Cytoskeleton in Arabidopsis thaliana Root Cells Under Clinorotation

The effect of horizontal clinorotation (2 rpm) on the spatial organization of cortical microtubules (CMTs) in the living Arabidopsis root cells has been investigated. MTs were visualized by using a stably transformed line of transgenic Arabidopsis thaliana expressing GFP-MAP4 fusion protein. To monitor total changes in MT dynamics, the sensitivity to MT-inhibitor 5 μM/l oryzalin was used as an indicator. It was shown that CMTs in the distal elongation zone (DEZ) of clinorotated 5 day old seedlings exhibited a higher degree of disorder and also they were more sensitive to oryzalin. We suggest that rearrangement of CMTs in DEZ cells under clinorotation is caused by fast rate of MT dynamic turnover.