Growth and development in Arabidopsis thaliana through an entire life cycle under simulated microgravity conditions on a clinostat.

The effects of simulated microgravity conditions produced by a horizontal clinostat on the entire life cycle of Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes.     

Plant growth processes in Arabidopsis under microgravity conditions simulated by a clinostat.

The life cycle of Arabidopsis plants was examined by growing them on a horizontal clinostat. Seeds on agar media were allowed to germinate and seedlings were grown under a simulated microgravity on a horizontal clinostat. Clinorotation (3 rpm) did not appear to interfere with germination of plant seeds and development of cotyledons and leaves. Stress relaxation parameters of the cell wall, the minimum relaxation time and the relaxation rate did not appear to be affected by clinostat rotation. On the other hand, the length of inflorescences was reduced to 61-62% by clinostat rotation. Rotation was found to inhibit the polar transport of auxin, although inflorescence growth and auxin transport were not completely inhibited. From these facts, it is possible that the life cycle in Arabidopsis plants could be accomplished in space, although growth phenomena involving auxin transport and its action may be disturbed. Plants may have a capacity to grow in space and we may be able to cultivate crops in space.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Effect of simulated microgravity on auxin polar transport in inflorescence axis of Arabidopsis thaliana.

The morphology, growth and development of higher plants are strongly influenced by environmental stimuli on the earth, which affect the changes in the dynamics of plant hormones in plants. Qualitative and quantitative changes in plant hormones are the most important internal factor to regulate plant growth and development. Among them, auxin (IAA) is of most significant. There are numerous reports concerning the physiological roles of auxin in plant growth and development (Matthysse and Scott 1984). One of the characteristics of auxin is to have the ability of polar transport along the vector of gravity on the earth (Schneider and Wightman 1978), suggesting that the activity of auxin polar transport is also important for the growth and development of plants. It has recently been reported that the normal activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana was required for flower formation (Okada et al. 1991, Ueda et al. 1992). Considering the above evidence together with the fact that gravity affects the morphology, growth and development of higher plants, gravity might affect the qualitative and quantitative changes in plant hormones including the activity of auxin polar transport. In this paper, we report the effect of microgravity condition simulated by a three-dimensional (3-D) or a horizontal clinostat on the activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana.     

Automorphosis of maize shoots under simulated microgravity on a three-dimensional clinostat

Seedlings of maize ( Zea mays L. cv. Golden Cross Bantam T-51) were grown under microgravity conditions simulated by a three-dimensional clinostat. On the clinostat, maize shoots exhibited curvatures in three different portions: (1) the basal transition zone connecting roots and mesocotyls, (2) the coleoptile node located between mesocotyls and coleoptiles, and (3) the elongating region of the coleoptiles. Even non-clinostatted control shoots showed some degree of curvature away from the caryopsis in the transition zone and bending toward the caryopsis in the coleoptile node. Clinostat rotation greatly stimulated these curvatures. Control coleoptiles elongated almost straightly, whereas coleoptiles on the clinostat bent either away from or toward the caryopsis depending on the timing of rotation. The curvature in all three portions became larger with time, both in control and clinostatted seedlings. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any portion. However, in coleoptile nodes and coleoptiles, the faster-expanding convex side exhibited a higher extensibility of the cell wall than the opposite side, and this appears to be a cause of the curvature. Thus, changes in the cell wall metabolism may be involved in automorphosis, which governs the life cycle of plants under a microgravity environment.     

Expression of PIN and AUX1 genes encoding putative carrier proteins for auxin polar transport in etiolated pea epicotyls [correction of epicotyles] under simulated microgravity conditions on a three-dimensional clinostat.

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under simulated microgravity conditions on a 3-dimensional clinostat showed automorphosis-like growth and development similar to that observed in true microgravity conditions in space. Application of inhibitors of auxin polar transport phenocopied automorphosis-like growth on 1 g conditions, suggesting that automorophosis is closely related to auxin polar transport. Strenuous efforts to know the relationships between automorphosis and auxin polar transport in pea seedlings at molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin efflux and influx carrier protein, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 and AtPINs, and between PsAUX1 and AtAUX1. Expression of PsPIN1 and PsAUX1 genes in etiolated pea seedlings grown on the clinostat were substantially affected, but that of PsPIN2 was not. Roles of these genes in auxin polar transport and automorphosis of etiolated pea seedlings are also described.     

Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment.

The principal objectives of the space experiment, BRIC-AUX on STS 95, were the integrated analysis of the growth and development of etiolated pea and maize seedlings in space and a study of the effects of microgravity conditions in space on auxin polar transport in these segments. Microgravity significantly affected the growth and development of etiolated pea and maize seedlings. Epicotyls of etiolated pea seedlings were the most oriented toward about 40 to 60 degrees from the vertical. Mesocotyls of etiolated maize seedlings were curved at random during space flight but coleoptiles were almost straight. Finally the growth inhibition of these seedlings in space was also observed. Roots of some pea seedlings grew toward to the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles, which were germinated and grown under microgravity conditions in space, were significantly low as compared with those grown on the ground of the earth. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

The senescence of oat leaf segments is promoted under simulated microgravity condition on a three-dimensional clinostat.

Plants have evolved on the earth, indicating the morphology, growth and development, and life cycle of plants are highly influenced by gravity as well as other environmental stimuli. Indeed, simulated microgravity on a clinostat or hypergravity on a centrifuge has recently been reported to change the growth and development of plants (Hoson et al. 1992, 1993, 1995, Rasmussen et al. 1994, Kasahara et al. 1995). Senescence is a final drastic phenomenon in life cycle of plants, which is characterized by the loss of total chlorophyll and protein, and/or the formation of the abscission (Osborne 1973, Thimann 1977, Addicott 1982). Many environmental stimuli as well as the qualitative and quantitative changes of plant hormones have been reported to affect plant senescence. Among those stimuli, light is the most important factor to regulate plant senescence (Leopold 1964). Dark condition promotes leaf senescence due to the decrease in endogenous level of cytokinin and/or the increase in that of abscisic acid or ethylene (Tetley and Thimann 1974, Gepstein and Thimann 1980). However, there are few reports concerning the effect of gravity on leaf senescence. Strenuous effort to learn leaf senescence under microgravity condition has been done using a three-dimensional (3-D) clinostat. In this paper, we report that simulated microgravity condition on a 3-D clinostat promoted the senescence of oat leaf segments in the dark. A possible mechanism of microgravity condition on promoting the senescence is also discussed.     

Automorphosis of etiolated pea seedlings in space is simulated by a three-dimensional clinostat and the application of inhibitors of auxin polar transport

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under microgravity conditions in space show automorphosis: bending of epicotyls, inhibition of hook formation and changes in root growth direction. In order to determine the mechanisms of microgravity conditions that induce automorphosis, we used a three-dimensional clinostat and obtained the successful induction of automorphosis-like growth of etiolated pea seedlings. Kinetic studies revealed that epicotyls bent at their basal region towards the clockwise direction far from the cotyledons from the vertical line (0 degrees) at approximately 40 degrees in seedlings grown both at 1 g and in the clinostat within 48 h after watering. Thereafter, epicotyls retained this orientation during growth in the clinostat, whereas those at 1 g changed their growth direction against the gravity vector and exhibited a negative gravitropic response. On the other hand, the plumular hook that had already formed in the embryo axis tended to open continuously by growth at the inner basal portion of the elbow; thus, the plumular hook angle initially increased; this was followed by equal growth on the convex and concave sides at 1 g, resulting in normal hook formation; in contrast, hook formation was inhibited on the clinostat. The automorphosis-like growth and development of etiolated pea seedlings was induced by auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid), but not by anti-auxin (p-chlorophenoxyisobutyric acid) at 1 g. An ethylene biosynthesis inhibitor, 1-aminooxyacetic acid, inhibited hook formation at 1 g, and ethylene production of etiolated seedlings was suppressed on the clinostat. Clinorotation on the clinostat strongly reduced the activity of auxin polar transport of epicotyls in etiolated pea seedlings, similar to that observed in space experiments (Ueda J, Miyamoto K, Yuda T, Hoshino T, Fujii S, Mukai C, Kamigaichi S, Aizawa S, Yoshizaki I, Shimazu T, Fukui K (1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J Plant Res 112: 487492). These results suggest that clinorotation on a three-dimensional clinostat is a valuable tool for simulating microgravity conditions, and that automorphosis of etiolated pea seedlings is induced by the inhibition of auxin polar transport and ethylene biosynthesis.     

Phenotypic characterization of Aspergillus niger and Candida albicans grown under simulated microgravity using a three-dimensional clinostat

The living and working environments of spacecraft become progressively contaminated by a number of microorganisms. A large number of microorganisms, including pathogenic microorganisms, some of which are fungi, have been found in the cabins of space stations. However, it is not known how the characteristics of microorganisms change in the space environment. To predict how a microgravity environment might affect fungi, and thus how their characteristics could change on board spacecraft, strains of the pathogenic fungi Aspergillus niger and Candida albicans were subjected to on-ground tests in a simulated microgravity environment produced by a three-dimensional (3D) clinostat. These fungi were incubated and cultured in a 3D clinostat in a simulated microgravity environment. No positive or negative differences in morphology, asexual reproductive capability, or susceptibility to antifungal agents were observed in cultures grown under simulated microgravity compared to those grown in normal earth gravity (1 G). These results strongly suggest that a microgravity environment, such as that on board spacecraft, allows growth of potentially pathogenic fungi that can contaminate the living environment for astronauts in spacecraft in the same way as they contaminate residential areas on earth. They also suggest that these organisms pose a similar risk of opportunistic infections or allergies in astronauts as they do in people with compromised immunity on the ground and that treatment of fungal infections in space could be the same as on earth.     

Growth and photosynthesis of Japanese flowering cherry under simulated microgravity conditions.

The photosynthetic rate, the leaf characteristics related to photosynthesis, such as the chlorophyll content, chlorophyll a/b ratio and density of the stomata, the leaf area and the dry weight in seedlings of Japanese flowering cherry grown under normal gravity and simulated microgravity conditions were examined. No significant differences were found in the photosynthetic rates between the two conditions. Moreover, leaf characteristics such as the chlorophyll content, chlorophyll a/b ratio and density of the stomata in the seedlings grown under the simulated microgravity condition were not affected. However, the photosynthetic product of the whole seedling under the simulated microgravity condition increased compared with the control due to its leaf area increase. The results suggest that dynamic gravitational stimulus controls the partitioning of the products of photosynthesis.     

Gravity-controlled asymmetrical transport of auxin regulates a gravitropic response in the early growth stage of etiolated pea (Pisum sativum) epicotyls: studies using simulated microgravity conditions on a three-dimensional clinostat and using an agravitropic mutant, ageotropum

Increased expression of the auxin-inducible gene PsIAA4/5 was observed in the elongated side of epicotyls in early growth stages of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown in a horizontal or an inclined position under 1 g conditions. Under simulated microgravity conditions on a 3D clinostat, accumulation of PsIAA4/5 mRNA was found throughout epicotyls showing automorphosis. Polar auxin transport in the proximal side of epicotyls changed when the seedlings were grown in a horizontal or an inclined position under 1 g conditions, but that under clinorotation did not, regardless of the direction of seed setting. Accumulation of PsPIN1 and PsPIN2 mRNAs in epicotyls was affected by gravistimulation, but not by clinorotation. Under 1 g conditions, auxin-transport inhibitors made epicotyls of seedlings grown in a horizontal or inclined position grow toward the proximal direction to cotyledons. These inhibitors led to epicotyl bending toward the cotyledons in seedlings grown in an inclined position under clinorotation. Polar auxin transport, as well as growth direction, of epicotyls of the agravitropic mutant ageotropum did not respond to various gravistimulation. These results suggest that alteration of polar auxin transport in the proximal side of epicotyls regulates the graviresponse of pea epicotyls.     

Growth,yield and reproduction of dwarf tomato grown under simulated microgravity conditions

Abstract

A closed hydroponic system combined with a horizontal uniaxial clinostat has been used to grow tomato plants (Solanum lycopersicum L.) under simulated microgravity conditions. The study was carried out to evaluate the quanti-qualitative traits (growth, yield and quality) of the dwarf tomato variety ‘Micro-Tom’ grown under simulated microgravity conditions and to determine if tomato plants would complete their life cycle (‘seed-to-seed’). Morphological and growth characteristics of ‘Micro-Tom’ were modified during clinorotation treatment. The ‘Micro-Tom’ plants grown under simulated microgravity exhibited a spreading growth and an increasing of the internode length. Total fruit yield, small fruit yield, leaf area, leaf dry weight, fruit dry weight, total dry weight and shoot – root ratio were lower in the clinorotated tomato plants than those grown in the control treatment. Foliar amount of carotenoids, and chlorophyll a and b were also substantially reduced under simulated microgravity conditions. Quality parameters (total soluble solids and fruit dry matter) of tomato plants were also negatively affected by clinorotation. The number of flowers per plant was increased by 32% in clinorotated plants versus controls. Fruit setting was reduced by 46% under clinorotation, while no significant difference was recorded for the pollen fertility and the seed number in small and large fruits. Clinorotation-exposed and control seeds were used in a germination trial in order to evaluate whether the seeds so formed were viable and if subsequent generations might be obtained in microgravity. Seeds formed under simulated microgravity proved to be biologically and functionally complete (germination = 78.6%) showing that ‘Micro-Tom’ plants could realize complete ontogenesis, from seed to seed in microgravity.     

Psychophysiological monitoring under conditions of real and simulated microgravity.

Dynamics of indices characterizing the process of adaptation of operators to complicated conditions of vital activity was studied with psychophysiological monitoring. This technique is based on the complex analysis of subjective and objective estimations of the current psychoemotional state and physiological and biochemical indices. We analyzed the data obtained during the examination of three healthy volunteers, who participated in the HUBES experiment (135-day isolation in the ground-based model of the orbital station "Mir"), and during a biorhythmological study conducted by six Russian cosmonauts of the main expeditions on the station "Mir." It was shown that psychophysiological monitoring is an informative method for studying different parameters of the operator's state and regulatory processes, the dynamics of biological rhythms, and individual characteristics of adaptation. By means of the analysis of a population of the variation in the characteristics of the parameters under study the experimental period of isolation was divided into three stages. The first and third stages were characterized by a relatively high variability, which was minimized at the second stage. The phasic character of the adaptation processes with a phase duration of about 60 days was revealed and a correlation was shown between the dynamics of parameters obtained by means of psychophysiological monitoring and specific features of the system regulation in different phases of the space flight and its simulation. It is suggested that the phases of the minimal variability of the studied characteristics are related to the periods of the minimal reliability of an operator's work. [Translated from Fiziologiya Cheloveka, vol. 25, no. 5, 1999 Sep-Oct, p. 86-91]     

The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Arabidopsis pinoid mutants show a strong phenotypic resemblance to the pin-formed mutant that is disrupted in polar auxin transport. The PINOID gene was recently cloned and found to encode a protein-serine/threonine kinase. Here we show that the PINOID gene is inducible by auxin and that the protein kinase is present in the primordia of cotyledons, leaves and floral organs and in vascular tissue in developing organs or proximal to meristems. Overexpression of PINOID under the control of the constitutive CaMV 35S promoter (35S::PID) resulted in phenotypes also observed in mutants with altered sensitivity to or transport of auxin. A remarkable characteristic of high expressing 35S::PID seedlings was a frequent collapse of the primary root meristem. This event triggered lateral root formation, a process that was initially inhibited in these seedlings. Both meristem organisation and growth of the primary root were rescued when seedlings were grown in the presence of polar auxin transport inhibitors, such as naphthylphtalamic acid (NPA). Moreover, ectopic expression of PINOID cDNA under control of the epidermis-specific LTP1 promoter provided further evidence for the NPA-sensitive action of PINOID. The results presented here indicate that PINOID functions as a positive regulator of polar auxin transport. We propose that PINOID is involved in the fine-tuning of polar auxin transport during organ formation in response to local auxin concentrations.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

Automorphogenesis of maize roots under simulated microgravity conditions

Plant seedlings show exaggerated growth responses on a three-dimensional clinostat. Such an automorphogenesis appears to be one of major factors which govern the life cycle of higher plants under a microgravity environment. On the three-dimensional clinostat, maize roots exhibited curvatures in three different portions; 1) the basal region just protruding from the coleorhiza, 2) the region between the mature and the elongation zone, and 3) the elongation zone, several mm from the tip. Even non-clinostatted control roots showed some degree of curvature. The curvature occurred at random without any dorsiventrality. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any region. However, the convex, rapidly expanding side exhibited a higher extensibility of the cell wall in some regions, which appears to be a cause of the curvature. In order to understand the role of gravity in regulation of plant growth and development, we should clarify a series of events by which an automorphogenesis is induced under simulated microgravity conditions.