Structural and functional organization of mitochondria in soybean root statocytes under microgravitation

The effects of microgravity and ethylene on morphology and ultrastructural organization of mitochondria in root statocytes of soybean seedlings grown for 6 days on the board of the space shuttle Columbia during the STS-87 mission were investigated. The spaceflight seedlings and the ground-grown control seedlings were grown in BRIG (Biological Research in Canister) in the presence of KMnO4 to remove ethylene. It was revealed that irrespectively of KMnO4 treatment the mitochondria in the spaceflight seedlings were characterized by round or oviform and by low electron density of organelle matrix, whereas the organelles in the ground controls were polymorphic in shape and had higher electron density of matrix. The possible mechanisms of morphological and ultrastructural rearrangements of mitochondria that may be involved in adaptation processes of soybean seedlings to microgravity conditions are discussed.     

A Spaceflight Experiment for the Study of Gravimorphogenesis and Hydrotropism in Cucumber Seedlings

peg , on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity-controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water-containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction. Received 13 September 1999/ Accepted in revised form 12 October 1999     

A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings.

Seedlings of Cucurbitaceae plants form a protuberance, termed peg, on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction.     

Structural and functional features of mitochondria in statocytes of soybean root under microgravity conditions

A study of the influence of microgravity and ethylene on the morphology and ultrastructural organization of mitochondria in root apex statocytes of 6-day old soybean seedlings that traveled on the Columbia space station on the STS-87 mission is presented. The seedlings were grown in hermetically sealed canisters in the presence of KMnO₄ to neutralize ethylene. It is established that, independent of KMnO₄ treatment, most of the mitochondria in the cells of the spaceflight seedlings were characterized by a round or oval shape and electronically transparent matrix, whereas in the seedlings in the ground controls the organelles were distinguished by a characteristic polymorphism, large dimensions, and higher electronic density of the matrix. One possible mechanism that could be responsible for the morphological and ultrastructural arrangement of the organelles in the process of adaptation of the seedlings to conditions of microgravity is discussed.     

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.     

Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Root growth and statocyte polarity in lentil seedling roots grown in microgravity or on a slowly rotating clinostat

The ability of clinostats to simulate microgravity was evaluated by comparing lentil ( Lens culinnrias L. cv. Verte du Puy) seedlings grown in space (Spacelab D1 Mission) with seedlings grown on a slowly rotating elinostat. Seeds were germinated and incubated for 25.5 h at 22°C (1) in microgravity, (2) on a 1g-centrifuge in space. (3) on a slowly rotating elinostat and (4) on the ground. Morphological (root length and orientation) and ultrastructural (distribution of amyloplasts, location of the nucleus in statocytes) parameters were studied. For clinostat experiments, two different configurations were employed: the longitudinal axis of the root was parallel (horizontal elinorotation) or perpendicular (vertical elinorotation) to the axis of rotation. the same configurations were used for the lg-controls. Root length and orientation were similar for roots grown on the clinostat and in microgravity. The amyloplasts were identically distributed in statocytes of horizontally clinorolated roots and in statocytes differentiated in microgravity. However, the location of the nucleus was similar in vertically rotated roots and microgravity samples. Since the involvement of the nucleus in graviperception is not known, it can be concluded that horizontal clinorotation simulates microgravity better than vertical elinorotation.     

Graviperception of lentil seedling roots grown in space (Spacelab Dl Mission)

The growth and graviresponsiveness of roots were investigated in lentil seedlings (Lens culinaris L. cv. Verte du Puy) grown (1) in microgravity, (2) on a 1 g centrifuge in space, (3) in microgravity and then placed on the 1 g centrifuge for 3 h, (4) on the ground. Dry seeds were hydrated in space (except for the ground control) and incubated for 25 h at 22°C in darkness. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in a Biorack glove box. Root length was similar for seedlings grown in space and for the ground and the 1 g centrifuge controls. The direction of root growth in the microgravity sample deviated strongly from the initial orientation of the roots of the dry seeds. This deviation could be due to spontaneous curvatures similar to those observed on clinostats. When lentil seedlings were first grown in microgravity for 25 h and then placed on the 1 g centrifuge for 3 h, their roots bent strongly under the effect of the centrifugal acceleration. The amplitude of root curvature on the centrifuge was not significantly different from that observed on ground controls growing in the vertical position and placed in the horizontal position for 3 h. The gravisensitivity of statocytes differentiated in microgravity was similar to that of statocytes differentiated on earth. There were no qualitative differences in the ultrastructural features of the gravisensing cells in microgravity and in the 1 g centrifuge and ground controls. However, the distribution of statoliths in the gravisensing cells was different in microgravity: most of them were observed in the proximal part of these cells. Thus, these organelles were not distributed at random, which is in contradiction with results obtained with clinostats. The distal complex of endoplasmic reticulum in the statocytes was not in contact with the amyloplasts. Contact and pressure of amyloplasts on the tubules were not prerequisites for gravisensing. The results obtained are not in agreement with the hypothesis that the distal endoplasmic reticulum would be the transducer of the action of the statoliths.     

Soybean root suberin: anatomical distribution, chemical composition, and relationship to partial resistance to Phytophthora sojae

Soybean (Glycine max L. Merr.) is a versatile and important agronomic crop grown worldwide. Each year millions of dollars of potential yield revenues are lost due to a root rot disease caused by the oomycete Phytophthora sojae (Kaufmann & Gerdemann). Since the root is the primary site of infection by this organism, we undertook an examination of the physicochemical barriers in soybean root, namely, the suberized walls of the epidermis and endodermis, to establish whether or not preformed suberin (i.e. naturally present in noninfected plants) could have a role in partial resistance to P. sojae. Herein we describe the anatomical distribution and chemical composition of soybean root suberin as well as its relationship to partial resistance to P. sojae. Soybean roots contain a state I endodermis (Casparian bands only) within the first 80 mm of the root tip, and a state II endodermis (Casparian bands and some cells with suberin lamellae) in more proximal regions. A state III endodermis (with thick, cellulosic, tertiary walls) was not present within the 200-mm-long roots examined. An exodermis was also absent, but some walls of the epidermal and neighboring cortical cells were suberized. Chemically, soybean root suberin resembles a typical suberin, and consists of waxes, fatty acids, omega-hydroxy acids, alpha,omega-diacids, primary alcohols, and guaiacyl- and syringyl-substituted phenolics. Total suberin analysis of isolated soybean epidermis/outer cortex and endodermis tissues demonstrated (1) significantly higher amounts in the endodermis compared to the epidermis/outer cortex, (2) increased amounts in the endodermis as the root matured from state I to state II, (3) increased amounts in the epidermis/outer cortex along the axis of the root, and (4) significantly higher amounts in tissues isolated from a cultivar ('Conrad') with a high degree of partial resistance to P. sojae compared with a susceptible line (OX760-6). This latter correlation was extended by an analysis of nine independent and 32 recombinant inbred lines (derived from a 'Conrad' x OX760-6 cross) ranging in partial resistance to P. sojae: Strong negative correlations (-0.89 and -0.72, respectively) were observed between the amount of the aliphatic component of root suberin and plant mortality in P. sojae-infested fields.     

Effects of gravitropic stress on the development of the primary root of lentil seedlings grown in space

Root growth and cell differentiation were analysed in lentil seedlings grown (1) in microgravity (F microg), (2) on the 1 x g centrifuge (F1 x g), (3) in microgravity and placed on the 1 x g centrifuge for 4 h [F(microg + 1 x g)], (4) on the 1 x g centrifuge and placed in microgravity for 4 h [F(1 x g + microg)]. In microgravity, there were strong oscillations of the root tip, even when the seedlings were grown first on the 1 x g centrifuge [F(1 x g + microg)]. In the [F(microg + 1 x g)] sample, the roots grown in microgravity were oblique with respect to the 1 x g acceleration when the seedlings were placed on the centrifuge. They were therefore gravistimulated. However, root length was similar in the 4 samples after 29 h of growth and growth rate of the root was the same between 25 h and 29 h although it appeared to be slightly greater in the [F(microg + 1 x g)] sample. Cell elongation was analysed as a function of the distance from the root cap junction. Cell length was similar in the seedlings grown in microgravity or on the 1 x g centrifuge. The transfer from the 1 x g centrifuge to microgravity [F(1 x g + microg)] did not modify cell elongation in the roots. Cell length in the roots which were grown in microgravity and gravistimulated [F(microg + 1 x g)] was different from that observed in microgravity but this was only due to gravistimulation. Thus, gravity does not have an effect on cell elongation when the roots are strictly oriented in the vertical position but it does as soon as the root tip deviates from this orientation.     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.     

The influence of microgravity and spaceflight on columella cell ultrastructure in starch-deficient mutants of Arabidopsis.

The ultrastructure of root cap columella cells was studied by morphometric analysis in wild-type, a reduced-starch mutant, and a starchless mutant of Arabidopsis grown in microgravity (F-microgravity) and compared to ground 1g (G-1g) and flight 1g (F-1g) controls. Seedlings of the wild-type and reduced-starch mutant that developed during an experiment on the Space Shuttle (both the F-microgravity samples and the F-lg control) exhibited a decreased starch content in comparison to the G-1g control. These results suggest that some factor associated with spaceflight (and not microgravity per se) affects starch metabolism. Elevated levels of ethylene were found during the experiments on the Space Shuttle, and analysis of ground controls with added ethylene demonstrated that this gas was responsible for decreased starch levels in the columella cells. This is the first study to use an on-board centrifuge as a control when quantifying starch in spaceflight-grown plants. Furthermore, our results show that ethylene levels must be carefully considered and controlled when designing experiments with plants for the International Space Station.     

Clinorotation affects morphology and ethylene production in soybean seedlings

The microgravity environment of spaceflight influences growth, morphology and metabolism in etiolated germinating soybean. To determine if clinorotation will similarly impact these processes, we conducted ground-based studies in conjunction with two space experiment opportunities. Soybean (Glycine max [L.] Merr.) seeds were planted within BRIC (Biological Research In Canister) canisters and grown for seven days at 20 degrees C under clinorotation (1 rpm) conditions or in a stationary upright mode. Gas samples were taken daily and plants were harvested after seven days for measurement of growth and morphology. Compared to the stationary upright controls, plants exposed to clinorotation exhibited increased root length (125% greater) and fresh weight (42% greater), whereas shoot length and fresh weight decreased by 33% and 16% respectively. Plants grown under clinorotation produced twice as much ethylene as the stationary controls. Seedlings treated with triiodo benzoic acid (TIBA), an auxin transport inhibitor, under clinorotation produced 50% less ethylene than the untreated control subjected to the same gravity treatment, whereas a treatment with 2,4-D increased ethylene by five-fold in the clinorotated plants. These data suggest that slow clinorotation influences biomass partitioning and ethylene production in etiolated soybean plants.     

Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station

The GRAVI-1 experiment was brought on board the International Space Station by Discovery (December 2006) and carried out in January 2007 in the European Modular Cultivation System facility. For the first run of this experiment, lentil seedlings were hydrated and grown in microgravity for 15 h and then subjected for 13 h 40 min to centrifugal accelerations ranging from 0.29 x 10(-2) g to 0.99 x 10(-2) g. During the second run, seedlings were grown either for 30 h 30 min in microgravity (this sample was the control) or for 21 h 30 min and then subjected to centrifugal accelerations ranging from 1.2 x 10(-2) g to 2.0 x 10(-2) g for 9 h. In both cases, root orientation and root curvature were followed by time-lapse photography. Still images were downlinked in near real time to ground Norwegian User Support and Operations Center during the experiment. The position of the root tip and the root curvature were analyzed as a function of time. It has been shown that in microgravity, the embryonic root curved strongly away from the cotyledons (automorphogenesis) and then straightened out slowly from 17 to 30 h following hydration (autotropism). Because of the autotropic straightening of roots in microgravity, their tip was oriented at an angle close to the optimal angle of curvature (120 degrees -135 degrees ) for a period of 2 h during centrifugation. Moreover, it has been demonstrated that lentil roots grown in microgravity before stimulation were more sensitive than roots grown in 1 g. In these conditions, the threshold acceleration perceived by these organs was found to be between 0 and 2.0 x 10(-3) g and estimated punctually at 1.4 x 10(-5) g by using the hyperbolic model for fitting the experimental data and by assuming that autotropism had no or little impact on the gravitropic response. Gravisensing by statoliths should be possible at such a low level of acceleration because the actomyosin system could provide the necessary work to overcome the activation energy for gravisensing.     

黑龙江省主要栽培大豆品种(系)对大豆疫霉根腐病的多抗性评价

用下胚轴伤口接种方法接种鉴定黑龙江省60个栽培大豆品种和育成品系对5个具有不同毒力大豆疫霉菌菌株41-4、PMCl、USAR4、PSZJ6和USAR17的抗性.有50个品种(系)抗1个或1个以上茵株或表现中间类型,其中有5个、8个、16个和21个品种(系)分别对4个、3个、2个和1个菌株表现抗性或中间类型.60个品种(系)对5个菌株共产生12种反应模式,其中呈RRSSR反应类型的品种(系)可能含有Rpslα或Rpslc基因,品系农大3861可能含有Rps3c基因,呈SSSSS反应模式的品种(系)可能含有Rps7基因,或不含抗病基因;其它9种反应模式与含有已知单基因品种或单基因组合的反应模式不同,可能具有未知抗病基因.该研究结果表明,黑龙江省具有较丰富的抗大豆疫霉根腐病大豆品种(系),大部分品种(系)的抗性是有效的,可合理地用于大豆生产和抗疫霉根腐病育种.     

Development and growth of several strains of Arabidopsis seedlings in microgravity

Growth and development of dark-grown Arabidopsis thaliana seedlings were studied in microgravity during space shuttle mission STS-84. The major purpose of this project was to determine if there were developmental differences among the four ecotypes studied--Wassilewskija (Ws), Columbia (Col), Landsberg erecta (Ler), and C24--and to evaluate whether particular ecotypes are better suited for spaceflight experimentation compared with others. A secondary goal was to study the growth of three starch-deficient strains of Arabidopsis by extending the observations made in a previously published report. For all strains, seed germination was not affected by microgravity, but seedlings were smaller in the spaceflight samples compared with the ground controls. The starch-deficient strains continued to exhibit vigorous growth until the termination of the experiment at 121 h after imbibition of seeds. However, ethylene effects, i.e., reduced growth and exaggerated hypocotyl hooks, were observed in all strains studied. Nevertheless, the Ler and C24 ecotypes seem to be more suitable for spaceflight research, compared with the other two ecotypes, based on measurements of their relative and absolute growth. This type of information should aid in the design of plant experiments for the International Space Station.