Gravitational effects on plant growth hormone concentration.

Numerous studies, particularly those of H. Dolk in the 1930's, established by means of bio-assay, that more growth hormone diffused from the lower, than from the upper side of a gravity-stimulated plant shoot. Now, using an isotope dilution assay, with 4,5,6,7 tetradeutero indole-3-acetic acid as internal standard, and selected ion monitoring-gas chromatography-mass spectrometry as the method of determination, we have confirmed Dolk's finding and established that the asymmetrically distributed hormone is, in fact, indole-3-acetic acid (IAA). This is the first physico-chemical demonstration that there is more free IAA on the lower sides of a geo-stimulated plant shoot. We have also shown that free IAA occurs primarily in the conductive vascular tissues of the shoot, whereas IAA esters predominate in the growing cortical cells. Now, using an especially sensitive gas chromatographic isotope dilution assay we have found that the hormone asymmetry also occurs in the non-vascular tissue. Currently, efforts are directed to developing isotope dilution assays, with picogram sensitivity, to determine how this asymmetry of IAA distribution is attained so as to better understand how the plant perceives the geo-stimulus.     

Possible effects of organelle charge and density on cell metabolism

To respond to gravity a biological system must: First, perceive the stimulus; and, second transduce the stimulus into an appropriate response. This laboratory has studied a system of perception and transduction involving the gravity-induced asymmetric distribution of a plant growth hormone. From these studies we have developed a working theory which states as its postulates that: a) The perception of the gravitational stimulus involved a perturbation of the plant's bio-electric field; and b) that the transduction of the stimulus involved voltage-gating of hormone movement from the plant's vascular tissue into the hormone responsive growing tissue. These studies may provide the simplest system for studing the mechanism whereby the gravity signal is translated into a biological response.     

Cellular responses to endogenous electrochemical gradients in morphological development.

Endogenous electric fields give vectorial direction to morphological development in Zea mays (sweet corn) in response to gravity. Endogenous electrical fields are important because of their ability to influence: 1) intercellular organization and development through their effects on the membrane potential, 2) direct effects such as electrophoresis of membrane components, and 3) both intracellular and extracellular transport of charged compounds. Their primary influence is in providing a vectorial dimension to the progression of one physiological state to another. Gravity perception and transduction in the mesocotyl of vascular plants is a complex interplay of electrical and chemical gradients which ultimately provide the driving force for the resulting growth curvature called gravitropism. Among the earliest events in gravitropism are changes in impedance, voltage, and conductance between the vascular stele and the growth tissues, the cortex, in the mesocotyl of corn shoots. In response to gravistimulation: 1) a potential develops which is vectorial and of sufficient magnitude to be a driving force for transport between the vascular stele and cortex, 2) the ionic conductance changes within seconds showing altered transport between the tissues, and 3) the impedance shows a transient biphasic response which indicates that the mobility of charges is altered following gravistimulation and is possibly the triggering event for the cascade of actions which leads to growth curvature.     

Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.     

Growth and development in higher plants under simulated microgravity conditions on a 3-dimensional clinostat.

Growth and development of etiolated pea (Pisum sativum L. cv. Alaska) and maize (Zea mays L. cv. Golden Cross Bantam) seedlings grown under simulated microgravity conditions were intensively studied using a 3-dimensional clinostat as a simulator of weightlessness. Epicotyls of etiolated pea seedlings grown on the clinostat were the most oriented toward the direction far from cotyledons. Mesocotyls of etiolated maize seedlings grew at random and coleoptiles curved slightly during clinostat rotation. Clinostat rotation promoted the emergence of the 3rd internodes in etiolated pea seedlings, while it significantly inhibited the growth of the 1st internodes. In maize seedlings, the growth of coleoptiles was little affected by clinostat rotation, but that of mesocotyls was suppressed, and therefore, the emergence of the leaf out of coleoptile was promoted. Clinostat rotation reduced the osmotic concentration in the 1st internodes of pea seedlings, although it has little effect on the 2nd and the 3rd internodes. Clinostat rotation also reduced the osmotic concentrations in both coleoptiles and mesocotyls of maize seedlings. Cell-wall extensibilities of the 1st and the 3rd internodes of pea seedlings grown on the clinostat were significantly lower and higher as compared with those on 1 g conditions, respectively. Cell-wall extensibility of mesocotyls in seedlings grown on the clinostat also decreased. Changes in cell wall properties seem to be well correlated to the growth of each organ in pea and maize seedlings. These results suggest that the growth and development of plants is controlled under gravity on earth, and that the growth responses of higher plants to microgravity conditions are regulated by both cell-wall mechanical properties and osmotic properties of stem cells.     

Operations of a spaceflight experiment to investigate plant tropisms

Plants will be an important component in bioregenerative systems for long-term missions to the Moon and Mars. Since gravity is reduced both on the Moon and Mars, studies that identify the basic mechanisms of plant growth and development in altered gravity are required to ensure successful plant production on these space colonization missions. To address these issues, we have developed a project on the International Space Station (ISS) to study the interaction between gravitropism and phototropism in Arabidopsis thaliana. These experiments were termed TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS) in 2006. In this paper, we provide an operational summary of TROPI and preliminary results on studies of tropistic curvature of seedlings grown in space. Seed germination in TROPI was lower compared to previous space experiments, and this was likely due to extended storage in hardware for up to 8 months. Video downlinks provided an important quality check on the automated experimental time line that also was monitored with telemetry. Good quality images of seedlings were obtained, but the use of analog video tapes resulted in delays in image processing and analysis procedures. Seedlings that germinated exhibited robust phototropic curvature. Frozen plant samples were returned on three space shuttle missions, and improvements in cold stowage and handing procedures in the second and third missions resulted in quality RNA extracted from the seedlings that was used in subsequent microarray analyses. While the TROPI experiment had technical and logistical difficulties, most of the procedures worked well due to refinement during the project.     

Sedimentable amyloplasts in starch sheath cells of woody stems of Japanese cherry.

We examined whether sedimentable amyloplasts act as statolith in the perception of gravity in woody stems using the elongated internodes of Japanese cherry (Prunus jamasakura Sieb. ex Koidz.). In the internode of the seedlings grown on earth, amyloplasts were found sedimented at the distal end of each cell of the endodermal starch sheath tissue. In the internode grown on three-dimensional (3-D) clinostat, amyloplasts were dispersed throughout the cell matrix in the endodermal starch sheath tissue. After changing the positions of the internode from vertical to horizontal, re-sedimentation of amyloplasts toward the direction of gravity was completed in 1h, whereas the bending of the internode was observed after 12 days. We propose that sedimentable amyloplasts in the endodermal starch sheath cells may play a role in gravity perception leading to secondary xylem formation in the secondary thickening growth and eccentric growth in gravi-bending of tree stems.     

Automorphosis of higher plants on a 3-D clinostat.

On a three-dimensional (3-D) clinostat, various plant organs developed statocytes capable of responding to the gravity vector. The graviresponse of primary roots of garden cress and maize grown on the clinostat was the same as the control roots, whereas that of maize coleoptiles was reduced. When maize seedlings were grown in the presence of 10(-4) M gibberellic acid and kinetin, the graviresponse of both roots and shoots was suppressed. The corresponding suppression of amyloplast development was observed in the clinostatted and the hormone-treated seedlings. Maize roots and shoots showed spontaneous curvatures in different portions on the 3-D clinostat. The hormone treatment did not significantly influence such an automorphic curvature. When the root cap was removed, maize roots did not curve gravitropically. However, the removal suppressed the automorphic curvatures only slightly. On the other hand, the removal of coleoptile tip did not influence its graviresponse, whereas the spontaneous curvature of decapitated coleoptiles on the clinostat was strongly suppressed. Also, cytochalasin B differently affected the gravitropic and the automorphic curvatures of maize roots and shoots. From these results it is concluded that the graviperception and the early processes of signal transmission are unnecessary for automorphoses under simulated microgravity conditions. Moreover, the results support the view that the amyloplasts act as statoliths probably via an interaction with microfilaments.     

Growth of pea epicotyl in low magnetic field implication for space research

A magnetic field is an inescapable environmental factor for plants on the earth. However, its impact on plant growth is not well understood. In order to survey how magnetic fields affect plant, Alaska pea seedlings were incubated under low magnetic field (LMF) and also in the normal geo-magnetic environment. Two-day-old etiolated seedlings were incubated in a magnetic shield box and in a control box. Sedimentation of amyloplasts was examined in the epicotyls of seedlings grown under these two conditions. The elongation of epicotyls was promoted by LMF. Elongation was most prominent in the middle part of the epicotyls. Cell elongation and increased osmotic pressure of cell sap were found in the epidermal cells exposed to LMF. When the gravitational environment was 1G, the epicotyls incubated under both LMF and normal geomagnetic field grew straight upward and amyloplasts sedimented similarly. However, under simulated microgravity (clinostat), epicotyl and cell elongation was promoted. Furthermore, the epicotyls bent and amyloplasts were dispersed in the cells in simulated microgravity. The dispersion of amyloplasts may relate to the posture control in epicotyl growth under simulated microgravity generated by 3D clinorotation, since it was not observed under LMF in 1G. Since enhanced elongation of cells was commonly seen both at LMF and in simulated microgravity, all elongation on the 3D-clinostat could result from pseudo-low magnetic field, as a by-product of clinorotation. (i.e., clinostat results could be based on randomization of magnetic field together with randomization of gravity vector.) Our results point to the possible use of space for studies in magnetic biology. With space experiments, the effects of dominant environmental factors, such as gravity on plants, could be neutralized or controlled for to reveal magnetic effects more clearly.     

Modification of plant growth and development by acceleration and vibration: concerns and opportunities for plant experimentation in orbiting spacecraft.

Growth, development, and orientation of higher plants is altered by physical disturbances such as shaking, touching, or vibration. Plant growth responses to thigmic (contact rubbing) forces are almost always negative, whereas growth responses to periodic seismic (shaking) or vibric (vibrational) disturbances may be positive or negative, depending on intensity and duration of force, and prevailing environmental conditions. Seedlings are most sensitive to mechanical stress when grown in darkness or under the low-light conditions typically available in plant flight hardware. Brief exposure to physical perturbation causes immediate growth inhibition of dark-grown seedlings followed by gradual recovery of growth rate beginning 10-12 minutes later. For mild vibration, growth rate may overshoot that of undisturbed control plants within an hour of a stress episode, whereas for thigmic stress recovery may remain incomplete for 24 hours or longer. Lack of physical stimulation by gravity should make plants even more responsive to random physical perturbation. Threshold growth response of seedlings to vibrational parameters needs to be determined under real spaceflight conditions.     

A simulation of Martian gravity field recovery by using a near equatorial orbiter

An improvement to the Martian gravity field may be achieved by means of future orbiting spacecraft with small eccentricity and low altitude exemplified through a newly proposed mission design that may be tested in upcoming reconnaissance of Mars. Here, the near equatorial orbital character (with an inclination approximating 10°, eccentricity as 0.01 and semi-major axis as 4000 km) is considered, and its contribution to Martian gravity field solution is analyzed by comparing it with a hypothetical polar circular orbiter. The solution models are evaluated in terms of the following viewpoints: power spectra of gravity field coefficients, correlations of low degree zonal coefficients, precise orbit determination, and error distribution of both Mars free air gravity anomaly and areoid. At the same time, the contributions of the near equatorial orbiters in low degree zonal coefficients time variations are also considered. The present results show that the near equatorial orbiter allows us to improve the accuracy of the Martian gravity field solution, decrease correlation of low degree zonal coefficients, retrieve much better time variable information of low degree zonal coefficients, improve precise orbit determination, and provide more accurate Mars free air gravity anomaly and areoid around the equatorial region.     

Ontogeny of plants under various gravity condition.

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

Classification of gravity effects on “free” cells

When cell physiologists detect gravity related reactions of their objects it is often difficult to decide where the receptors for the observed effects are located. Answering this question is necessary for any further analysis of a detected gravity effect on cells. In previous papers we have discussed direct and indirect gravity effects in relation to the smallest functional units where the primary receptor, which interacts with gravity, is positioned inside and outside of such a unit, respectively. So, in a first approximation we can conclude that in a multicellular aquatic organism, which changes its metabolism in weightlessness, the primary receptors of gravity are located inside the cells of that organism. A special approach is necessary when free living cells, the density of which may be higher than the one of the (liquid) medium, or even cells living on a free surface are observed. In these two cases also indirect effects have to be taken into account, which will be demonstrated with the aid of the slime mold . Additionally the environment of the organisms can be changed directly and indirectly by gravity.     

Influence of nonuniform magnetic fields on orientation of plant seedlings in microgravity conditions.

Experiments on the spatial behavior of the flax (Linum usitatissimum, L.) seedlings in a nonuniform magnetic field were conducted on the orbital space stations "Salut" and "Mir". This field can displace sensory organelles (statoliths) inside receptor cells and such displacement should cause a physiological reaction of the plant-tropistic curvature. Experiments were conducted in the custom-built "Magnetogravistat" facility, where seeds were germinated and grown for 3-4 days in a magnetic field with the dynamic factor grad (H2/2) approximately equal to 10(7) Oe2/cm, then fixed on orbit and returned to Earth for analysis. It was found, that 93% of the seedlings were oriented in the field consistently with curvature in response to displacement of statoliths along the field gradient by ponderomotive magnetic forces, while control seedlings grew in the direction of the initial orientation of the seed. This suggests, that gravity receptors of plants recognized magnetic forces on statoliths as gravity, and that gravity stimulus can be substituted for plants by a force of a different physical nature.     

What is the threshold amount of starch necessary for full gravitropic sensitivity?

In preparation for microgravity experiments, we studied the kinetics of gravitropism in seedlings of wild-type (WT) Arabidopsis and three starch-deficient mutants. One of these mutants is starchless (ACG 21) while the other two are intermediate starch mutants (ACG 20 and ACG 27). In root cap cells, ACG 20 and 27 have 51% and 60% of the WT amount of starch, respectively. However, in endodermal cells of the hypocotyl, ACG 20 has a greater amount of starch than ACG 27. WT roots and hypocotyls were much more responsive to gravity than were the respective organs of the starchless mutant, and the intermediate starch mutants exhibited reduced gravitropism but had responses that were close to that of the WT. In roots, ACG 27 (more starch) was more responsive than ACG 20 (less starch), while in hypocotyls, ACG 20 (more starch) had a greater response than ACG 27 (less starch). Taken together, our data are consistent with the starch-statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell. These results also suggest that (in roots) 51-60% starch is close to the threshold amount of starch needed for full gravitropism and that the gravity sensing system is "overbuilt."     

1984年2月18日耀斑后环速度场的分析

本文研究了耀斑后环内物质在太阳重力、磁压力梯度和气体压力梯度联合作用下的下落运动,并在非等温状态及定常假定下计算了环内物质分布。计算结果表明:在环的中上部,下落运动可当作太阳重力作用下的自由下落运动;在环的底部,环内物质密度和磁场强度对物质下落运动影响较显着。      

Evaluation of experiments involving the study of plant orientation and growth under different gravitational conditions.

The manifestation of gravitropic reaction in plants has been considered from the phylogenetic point of view. A chart has been suggested according to which it is supposed that the first indications of the ability to identify the direction of the gravitational vector were inherent in the most ancient eukaryotes, which gave rise to green, brown, yellow-green, golden and diatomaceous algae as well as fungi. The experiments on the role of gravity in plant ontogenesis are being continued. The sum total of the data obtained in a number of experiments in space shows that under these conditions a structurally modified but normally functioning gravireceptive apparatus is formed. The data confirming the modification, under changed gravity, of the processes of integral and cellullar growth of the axial organs of seedlings as well as of the anatomo-morphological structure and developmental rates of plants during their prolonged growth in space are presented. It is assumed that this fact testifies to the presence of systems interacting with gravity during plant ontogenesis. At the same time the necessity for further experiments in order to differentiate an immediate biological effect of gravity from the ones conditioned by it indirectly due to the changes in the behavior of liquids and gases is pointed out. The methodological aspects of biological experiments in space as the main source of reliable information on the biological role of gravity are discussed.     

微重力下胶原蛋白纤维化及羟基磷灰石结晶的初步研究

在长期空间飞行过程中, 骨质丢失是一个严重问题. 羟基磷灰石(HAP)晶体是骨骼的主要成分, 骨骼中的胶原蛋白纤维在HAP生长结晶过程中起到关键作用. 研究了胶原蛋白纤维化过程在模拟微重力和常重力条件下的变化, 对以胶原 蛋白纤维作为模板生长出的HAP晶体形貌进行了观察. 结果表明, 不同浓度胶原蛋白溶液中形成的胶原蛋白纤维, 其内部孔隙数量和尺寸在模拟微重力条件下要明显大于常重力条件下, 胶原蛋白纤维内部孔隙的分布也不同于常重力条 件下的结果. 以模拟微重力条件下形成的胶原蛋白纤维为模板生长出的HAP 晶体主要为立方体状, 而以常重力条件下形成的胶原蛋白纤维为模板生长出的 HAP晶体形貌主要为板状. 该结果有助于未来进一步阐明空间骨质丢失的机理.      

Laser ablation of root cap cells: implications for models of graviperception.

The initial event of gravity perception by plants is generally thought to occur through sedimentation of amyloplasts in specialized sensory cells. In the root, these cells are the columella which are located toward the center of the root cap. To define more precisely the contribution of columella cells to root gravitropism, we used laser ablation to remove single columella cells or groups of these cells and observed the effect of their removal on gravity sensing and response. Complete removal of the cap or all the columella cells (leaving peripheral cap cells intact) abolishes the gravity response of the root. Removal of stories of columella revealed differences between regions of the columella with respect to gravity sensing (presentation time) versus graviresponse (final tropic growth response of the root). This fine mapping revealed that ablating the central columella located in story 2 had the greatest effect on presentation time whereas ablating columella cells in story 3 had a smaller or no effect. However, when removed by ablation the columella cells in story 3 did inhibit gravitropic bending, suggesting an effect on translocation of the gravitropic signal from the cap rather than initial gravity perception. Mapping the in vivo statolith sedimentation rates in these cells revealed that the amyloplasts of the central columella cells sedimented more rapidly than those on the flanks do. These results show that cells with the most freely mobile amyloplasts generate the largest gravisensing signal consistent with the starch statolith hypothesis of gravity sensing in roots.