Plants, Gravity, and Space
Studies of plants and their responses to gravity and the space environment have concentrated on three main themes. The first is space horticulture, the study of how to grow plants successfully in space, either for experimental purposes or for human consumption. This involves assessments of the conditions needed for optimal crop yield, the best plants to grow in space, and the problems inherent in growing plants in a gravity-free and totally enclosed system.
The second theme is the necessity for gravity, or whether there is any facet of a plant's growth, development, and metabolism that is impaired if there is no gravity. In other words, are there plant processes where the mere presence of gravity is essential, regardless of the actual direction of the gravity vector?
Finally, there is the response to the direction of the gravity vector, or how plants respond to specific directions of gravity by altering their pattern of growth and development. For example, plant stems and roots will alter their direction of growth to maintain a set angle with the gravity vector (gravitropism).
Research on each of these three themes has been supported by NASA in the past decade, but each needs additional experimentation and study.
Reasons for Studies on Space Horticulture
There are three reasons for studying space horticulture. First, if the microgravity environment of spacecraft is to be used to carry out scientific experiments on basic mechanisms of plant responses to gravity, the plants must be grown under optimal conditions. Sick plants are not suitable scientific subjects.
Second, if a lunar (or Mars) colonization base is ever to be set up, it will have to be able to grow its own food. It is doubtful that anyone has actually succeeded in growing crops in a totally enclosed system such as would be needed on the moon. The problems that have been encountered in closing the plant growth system can best be worked out on Earth or in a spacecraft and cannot be delayed until it is time to start building a lunar colony. Ground-based studies may be more relevant than spaceflight studies here, because plants on the moon will be subjected to substantial gravity (0.16 g), even if it is less than on Earth.
The third often-stated reason is to provide food for astronauts during prolonged spaceflights. An as yet unsettled question is the minimum length of the mission that would justify the weight of on-board crop-growing facilities, since for shorter flights it will be more weight efficient to carry food rather than grow it in space. Some current estimates have suggested that the spaceflight mission must exceed 2.6 years before space horticulture would be of value,1 2 and this estimate assumes that there will be sufficient power to provide optimal illumination of the plants. It is therefore unlikely that space horticulture will actually be used to provide food aboard a spacecraft in the near future, but the development of the needed technology is worth pursuing now, to prepare for later space endeavors.
Until 1997, studies in space horticulture in NASA were primarily located in the Closed Ecological Life Support System (CELSS) program. In 1997, the CELSS program was merged with the nonbiological life support studies into the Advanced Life Support (ALS) program. The CELSS program expended considerable effort to obtain basic information needed for space horticulture. One focus of activity has been to determine the maximum usable biomass per square meter that can be obtained with selected crop plants. When selected varieties of wheat were grown in dense stands on Earth under high light intensities (150 moles m-2 d-1), yields as high as 60 g m-2 d-1 were obtained. 3 These are among the highest plant yields ever recorded on Earth. High yields under similar circumstances have been obtained with potatoes as well.4 These studies, supported by the CELSS program, have indicated that maximum efficiency may best be achieved by using stands of crop plants far denser than normally used in agriculture, as long as there is sufficient light available. Experiments have reduced the estimate of the amount of growing space needed to support an astronaut in space from 25 m2 to as low as 10 m2, assuming that comparable yields can be obtained in space.5 Algae have often been suggested as a potential food source in space and they can be grown in a spacecraft, but to date the difficulty is producing palatable food from them.
A major problem has been how to close the plant growth unit completely to the outside environment. A second major problem has been developing the lighting systems to provide maximum photosynthetically active radiation with minimum power. Studies are ongoing to improve and adapt light-emitting diode (LED) and microwave lights for use in plant spacecraft facilities. By closed conditions it is meant that the plants must be grown in a system in which there is no exchange of gases with the outside and no input of water or nutrients after the start of the cultivation, and one in which all wastes are recycled or destroyed. The large environmental chamber (known as the Breadboard project) at Kennedy Space Center has come as close as any to reaching that goal,6 but there are still many problems to be overcome. The experience with the Biosphere 2 project in Arizona has shown just how difficult it is to close such a system successfully. Plant experiments conducted in space so far have not used a closed system; there is constant exchange of gases with the cabin atmosphere, and often the addition of water and nutrients from outside sources. But the system will have to be completely closed if it is to be used for farming on the moon. The technology to recycle water, nutrients, and gases must be developed first.
For a crop plant to be useful as a seed source in space, it must be able to go through a complete life cycle. A number of attempts at this have been made, generally with rather limited success. For example, wheat plants were recently grown to maturity on the Mir space station, but while many heads were produced, none contained seeds.7 The reasons for the failures are believed to be primarily hardware problems, such as low light intensities, inability to control atmospheric contaminants, and problems with nutrient and water delivery. On the other hand, plants of the rapid-cycling brassica (Brassica rapa) have been successfully raised through two generations on the Mir, although the growth and number of buds produced were significantly lower in space in the second-generation seeds (M.E. Musgrave, personal communication, 1998). Normal-appearing potato microtubers have been grown on potato stem cuttings in space, but no attempt has yet been made to grow a real potato crop in space.
Future research on space horticulture, supported by NASA, should be focused along two lines. The first and highest-priority objective is to develop a system on Earth for growing plants under completely closed conditions. This would require developing the most effective methods for removing biologically active atmospheric pollutants (such as ethylene) that might be emitted by the plant, and learning how best to maintain the O2 and CO2 levels in the plant chamber. The technology for recycling of water from both the atmosphere and the root environment as well as the nutrients from the soil solution and the unused parts of the harvested plants needs to be perfected. This technology development should be concentrated on a system that can be used for basic scientific experiments aboard the International Space Station (ISS); this will mean that the size of the growth chamber will of necessity be small. It will be necessary, then, to focus attention initially on small plants such as Arabidopsis or on plants that can thrive under low light intensities, because power will be a limiting factor on the ISS. The engineering problems associated with developing a totally closed plant growth system are not trivial, but the need is pressing.
Certain engineering problems will require tests in the microgravity environment. In particular, the delivery system for water and nutrients to the roots must be tested in microgravity; it is not possible to validate such systems in the presence of 1 g. Such tests are already under way and should be continued and expanded. However, flight experiments should be limited to those needed to develop the closed plant growth system for use in space. When a plant growth system suitable for growing small test plants in space has been perfected, it will then be time to attempt to grow crops of seed- or tuber-producing food plants in space.
A second, lower-priority focus of activity should be to study the responses of plants to specific stresses that plants in spaceflight might undergo. In particular, the response of plants to elevated CO2 needs to be studied. Plants on Earth grow in the presence of 0.035 percent CO2, but in the ISS the CO2 levels will be as high as 0.7 percent for up to 180 days.8 In general, plants can tolerate CO2 levels as high as 0.1 percent, but their ability to tolerate the levels of CO2 expected on-board ISS is unknown. One solution is to keep the atmosphere in the plant chambers completely separate from the cabin atmosphere; another is to develop strains of plants capable of handling such high CO2 levels. The CELSS program should consider supporting studies on the adaptation of plants to super-high CO2 levels.
A second stress of importance is vibration. Plants transported to and from orbit on the shuttle are subjected to rather considerable vibration for short periods. Both the immediate effects of this vibration and the long-term responses of the plants need to be understood so that they will not be mistaken for responses to the lack of gravity. NASA is already supporting research in this area (e.g., Xu et al. 9), but more experimentation is needed. These studies can best be conducted on the ground.
- The ALS program should concentrate its ground-based research on developing a completely enclosed plant growth system. This effort will require close collaboration between engineers and plant environmental scientists.
- The ALS spaceflight program should focus on testing the potentially gravity-sensitive components of the closed plant growth system, such as the nutrient delivery system.
- A lower-priority concern is a ground-based study of the problems and mechanisms associated with stresses that plants in space might encounter, including super-high CO2 levels and vibrations.
Role Of Gravity In Plant Processes
Plants have evolved under conditions of constant 1 g, so there is a possibility that they have taken advantage of this influence to modulate some of their processes. For example, differences in density of cellular organelles might lead to a stratification of the cytoplasm that would influence the polarity of the cells. In the absence of gravity, then, the development of a polarity in plant cells might be adversely affected. This hypothesis has led to the repeated suggestion (e.g., in the Goldberg report10) that plants should be grown for protracted periods in microgravity to determine whether any such gravity-requiring step can be detected.
The basic experiment in this case is to grow plants in space through several generations (the "seed-to-seed experiment"). If there is any major process that is adversely affected by the lack of gravity, it should manifest itself by an obvious change in the pattern of development. However, the alteration may be minor in significance and difficult to spot unless directly targeted. Critical developmental steps may need to be examined in detail to discover this. For example, the location of the position of root hairs depends on the polarity of root epidermal cells;11 if the polarity of the cells is altered in microgravity, the location of the root hairs may be altered. This seed-to-seed experiment needs to be run for at least two generations. It is possible that in the absence of 1 g, maternal effects on the seeds would only be manifested in the second generation. If substantial effects are not found in two generations with a small number of species, it should be possible to conclude that gravity itself is not of major importance in plant growth and development.
One problem in the interpretation of seed-to-seed experiments is that there may be effects on plant growth in microgravity that are only indirectly the result of the lack of gravity. For example, in the absence of gravity, air convection around leaves may be significantly reduced, with subsequent reduction in uptake of CO2 needed in photosynthesis and in the transpiration of water vapor from leaves. As a result, the water use efficiency of plants (CO2 assimilated/H2O transpired) in microgravity may be considerably different from that in 1 g. Similarly, the lack of gravity may adversely affect root aeration, because in the absence of gravity water tends to pool in large, unbroken masses, causing an excess of water in some places with a concomitant lack of oxygen.
A second problem is that the plant growth unit in which this seed-to-seed experiment is conducted must be as "artifact-free" as possible. This means that the light intensity must be high enough so that the plants are actually carrying out net photosynthesis. This value can vary from 1 to 20 µmoles m-2 sec-2, depending on the plant used. The atmosphere in the chamber must be controlled and monitored to eliminate complications from ethylene or from unnaturally high levels of CO2.
It has been frequently assumed that the effect of weightlessness on plants can be mimicked by
placing them on a horizontal clinostat, rotating at a slow speed.12 If this were so, it would not be necessary to make use of the space environment to test the effects of a lack of gravity on plant processes. For example, the seed-to-seed experiment could be conducted on a clinostat rather than in space. However, the clinostat does not create a weightless environment; instead it produces a constant movement of the gravity vector so that the net effect cancels out the directional response to the gravity vector. A major question, then, has been the extent to which a clinostat actually simulates 0 g, and if so, at what rotation it should be used and whether it should be a horizontal or a 3-D clinostat.
Attempts to grow any plant from seed to seed in space have met with limited success and much frustration. The earlier Russian attempts were unsuccessful, largely because of technical problems. For example, it appears that the roots were not given sufficient aeration, and the plants suffered severe waterlogging.13 In a recent Mir experiment, the wheat plants' inability to form seeds may have been due to enhanced levels of atmospheric ethylene.14 A series of three flight experiments by Musgrave, however, has demonstrated that the problems caused by secondary effects of microgravity can be at least partly overcome. 15 She examined the ability of Arabidopsis plants to undergo one part of the life cycle: flower development and sexual reproduction. The technical problems that beset the first experiment, in which sexual reproduction in the plants failed, were partly corrected in a second experiment, which had partial success. A third, improved experiment resulted in apparently normal sexual reproduction in the plants. These experiments showed that the apparent inability of Arabidopsis to undergo seed formation in space was an artifact of the poor experimental conditions (for example, low air convection in the growth chamber, remediable by forced air circulation) and not a direct consequence of microgravity.
Rapid-cycling brassica (Brassica rapa L) plants have recently been grown through two complete generations on Mir (M.E. Musgrave, personal communication, 1998). Plants grown in space from the first, space-raised generation were generally smaller than comparable plants grown from seeds brought up from Earth. Some of these plants were frozen and brought back to Earth for analysis. A thorough analysis of these space-grown plants and a repeat of this experiment with both Brassica rapa and Arabidopsis thaliana plants will be needed to assess possible effects of a lack of weightlessness on plant development.
Some developmental abnormalities have been recorded with plants grown in in space. For example, a number of root cells, fixed either in space or after return to Earth, showed chromosomal abnormalities. 16 Whether these were a response to the lack of gravity or to some other stress the plants experienced while on board the spacecraft, such as elevated levels of ethylene or CO2, cannot yet be determined. Another abnormality is the apparent failure of decapped maize roots to reform caps while in space.17 The balance between starch and sugar in plant leaves has also been reported to be altered in plants grown in microgravity compared with the ground controls,18 but this may also be the result of secondary effects of low gravity.
One approach to the study of the role of gravity has been to examine plant response on a horizontal clinostat. Although the development of plants on a clinostat is generally similar to that of plants grown in space, some small differences have been detected (e.g., Brown19 ). A careful analysis of clinostats in Germany and Japan indicates that the best simulation of weightlessness is obtained using a 3-D clinostat rather than a horizontal one, and with the speed of rotation increased above 1 rpm.20 Clinostat experiments have proven useful for obtaining preliminary information about the role of gravity in plant processes, but they cannot substitute for an actual spaceflight experiment on how plants develop in the total absence of gravity.
The most pressing need in space horticulture is for the successful completion of a plant seed-to-seed experiment. Because a complete generation for either Arabidopsis thaliana or Brassica rapa requires a minimum of 60 days, this experiment is not feasible aboard the space shuttle but can only be performed on a space station. However, a definitive experiment will only be possible when the plant growth apparatus has been sufficiently perfected so that extraneous stresses do not interfere with the detection of any gravity-requiring processes. At the present time at least four competing plant growth units are being designed and supported by NASA. This seems an unnecessary duplication of effort. NASA should consider concentrating its efforts on obtaining one superior plant growth apparatus.
The results of the Brassica rapa seed-to-seed experiment need to be confirmed and extended. This experiment should first be carried out with Arabidopsis, which has an equally short generation time and the advantages of a small genome size, a wealth of available genetic mutants, and an already well-characterized pattern of development. If no major effects on growth or development are seen in these experiments, it will be apparent that, for at least two species, gravity is not a major requirement for development. In that case, it is doubtful that any additional effort should be expended in exploratory experiments, looking for effects that are not fundamental to all plant species.
If major effects on growth and development are found, it will be necessary to exclude secondary effects of a microgravity environment (such as gravity effects on convection, and thus on gas exchange and heat conduction) before concluding that gravity is important to plant growth. It may be difficult to develop a plant growth unit for use in the seed-to-seed experiment that does not expose the plants to any stress other than that of lack of gravity. This is not a fatal flaw as long as researchers know exactly what stresses the space-grown plants have encountered and how much. Physical measurements of stresses are difficult and require extensive instrumentation.
There are three possible solutions. One is to use an on-board 1-g centrifuge as a control. If small plants such as Arabidopsis are used, the centrifuge can be one with a relatively small radius rather than the large-diameter centrifuge planned for the ISS. Several small centrifuges have been produced or are currently under development. However, differences in air convection will still exist between plants grown in space which are on a centrifuge versus those not on a centrifuge.
A second approach is to use mutant plants that are insensitive to specific environmental stresses—for example, using ethylene-insensitive mutants to eliminate complications from exogenous ethylene. Another possible solution is to use indicator Arabidopsis plants that have been transformed with indicator genes coupled to specific stress-induced promoters. These specific promoters have already been or are being developed for stresses such as temperature extremes, ethylene, water stress, vibration, and anaerobiosis.21 It should be possible to detect and perhaps measure the amount and types of stresses the space-grown Arabidopsis undergoes by use of these plants.
The root development of Arabidopsis follows a definite pattern so that cell fates are predictable.22 It may be possible to determine whether the microgravity conditions cause chromosomal abnormalities sufficient to adversely affect development. If there is any significant cell death, it would be recorded in the pattern of root cell walls and could be visualized by examining the roots.
- The seed-to-seed experiment, using Arabidopsis thaliana and Brassica rapa plants, should be a top priority for the ISS. This experiment must be conducted on the ISS, because the plants should be grown through at least two generations in space.
- To conduct a meaningful seed-to-seed experiment, NASA needs to develop the following:
- A superior plant growth unit, with adequate lighting, gas exchange, and water and/or nutrient delivery; and
- Arabidopsis thaliana plants that are insensitive to expected environmental stresses (such as ethylene) and that contain indicator genes for all the expected environmental stresses, such as high levels of CO 2, vibration, anaerobiosis, water stress, and temperature stresses.
- In the interim, before the ISS is functional, studies on specific stages of plant development in space should be limited to small plants with short life cycles (e.g., Arabidopsis thaliana or Brassica rapa). Whenever possible, a 1-g on-board centrifuge should be available.
Responses Of Plants To A Change In The Direction Of The Gravity Vector
Plants have three major responses to changes in the gravity vector. First, plant stems and roots can alter the direction of their growth in relation to the direction of gravity (gravitropism). An organ will assume a specific direction at some defined angle to the gravity vector, and if displaced from that direction, will curve until the original direction is resumed. Gravitropism is by far the most extensively studied plant response to the gravity vector. In the second response (gravitaxis), the swimming direction of some unicellular algae is directed by the gravity vector. Finally, plant development can be influenced by the direction of the gravity vector. For example, the weight of a tree limb alters the pattern of formation of new wood to provide additional support for the limb. This extra strengthening, called reaction wood, is in response to the tissue stresses, which are directly related to the direction of the stresses within the tissues, as well as the direction of gravity itself.
The Component Parts
Gravitropism consists of a series of sequential events leading to an alteration in the growth pattern of the affected organ. In all organs studied so far, the gravity-induced curvature of the organ is a result of a differential in the rate of cell enlargement on the organ's two sides, rather than a change in the number of cells on the two sides.23 Over the years gravitropism has attracted the attention of a wide range of biologists, including Charles Darwin. 24 One reason for this is that a change in the direction of the gravity vector is one of the few methods that causes changes in plant development in a fast, reversible, but noninvasive way. The graviresponses of plants are a powerful system with which to determine the mechanisms controlling plant development, information that will be of fundamental value in agricultural sciences.
In general, gravitropism can be broken down into four steps: perception, intracellular transduction, translocation, and reaction. The perception step involves the cell's perception of the direction of the gravity vector—the displacement of some component of a cell, up or down. This component can be the whole mass of the protoplast as it presses against and interacts with the cell wall, or it may be a sedimenting heavy organelle (statolith), such as an amyloplast. In the cellular transduction step, some biochemical or structural asymmetry is set up in the perceiving cell so that the cell is polarized with
respect to the direction of gravity. The result of cellular transduction is the movement of some growth-altering factor laterally. This sets up an asymmetry of this factor across the organ, coupled with longitudinal movement of the factor from the site of perception to the site of reaction. In the reaction phase, the unequal concentration of growth factors creates an unequal rate of cell elongation on the two sides of the organ.
Gravitropism in Single Cells
The simplest gravitropic system, in theory, is one in which the whole process takes place within a single cell. There is no need for a translocation step, because the reaction occurs in the gravity-perceiving cell. Several such systems are known. For example, the curvature of the rhizoid of the green alga Chara occurs within the same cell that perceives the gravity,25 as does the gravitropism of the protonema of the moss Ceratodon.26
In both systems, it appears that the direction of gravity is perceived by the sedimentation of statoliths, tethered by the cytoskeleton. The result is a change in the location of the tip growth by which such cells elongate. The steps in between are largely a mystery. Possible involvement of localized cytoplasmic calcium and cytoskeletal elements such as actin have been suggested.27 To understand how this gravitropic response is controlled, the process of tip growth will need to be studied in greater detail to learn what controls the location of this growth. Root hairs and pollen tubes also grow by tip growth, so learning about the control of tip growth in these gravitropic systems could have major benefits to other areas of plant science. To do this, the enzymes, cell wall components, and vesicle trafficking involved need to be identified. It would help greatly if mutants altering the gravitropic process in tip-growing cells were available and if the genetics of these organisms were better understood. Nevertheless, these tip-growing gravitropic systems hold great promise for some comprehensive study of the cell biology involved and may prove to be the systems that lead to an understanding of gravitropism in more complex, multicellular systems.
Gravitropism in Multicellular Plants
Gravitropic curvature in response to a change in the gravity vector can occur in most stems and roots as well as some flowers and fruits, but rarely in leaves. The resultant direction of growth can be either vertical (orthogravitropic) or at some specific and set angle to the gravity vector (plageotropic). Most research into the gravitropism of higher plants has concentrated on the response of roots, although earlier work focused more on coleoptiles. The present state of knowledge and the areas where research is needed are summarized below.
Perception. In the decade researchers have made a concerted effort to determine whether the perception of the gravity vector's direction is mediated by sedimenting statoliths interacting with some cellular component, by the tension on the cytoskeleton exerted by tethered statoliths, or by the mass of the whole protoplast acting on plasma membrane attachments to the cell wall.28 In the giant cells of the alga Chara, the direction of gravity is apparently perceived by the whole cell. This information is then used to cause different rates of protoplasmic streaming on the two sides of the cell.29 In higher plants, the evidence strongly supports the idea that amyloplasts play a major role in gravity perception. For example, removal of the root cap (the only part of the growing root that has amyloplasts) renders the root insensitive to the direction of gravity.30 But it is unclear whether the falling amyloplast must interact with some cellular component such as the endoplasmic reticulum, or whether the amyloplasts create
tensions in the cytoskeleton which are transmitted to the plasma membrane. Attempts to resolve this question by disrupting and dissolving the cytoskeleton from gravity-perceptive cells have produced equivocal results, because of the difficulty in determining whether or not the whole cytoskeleton has been removed.31
Starch-containing amyloplasts are apparently not required for graviperception in Arabidopsis roots. Starchless mutants of Arabidopsis retain some ability to undergo root gravitropism,32 as do roots in which amyloplast-containing root cap cells have been eliminated by laser ablation.33 This suggests that roots may have a second, residual method for detecting the direction of the gravity vector. The importance of the dense starch grains in graviperception can be partly assessed by comparing the g-threshold for starch-containing versus starchless Arabidopsis roots. Surprisingly, the g-threshold for a tropic response in normal starch-containing roots is still uncertain.34 35 This g-threshold should be determined in space for both starch-containing and starchless Arabidopsis roots, using a variable-g centrifuge.
The ability of starchless Arabidopsis roots to have a tropic response raises the important question of the extent to which nonstatolith-containing plant cells, in general, can perceive the direction of the gravity vector. It may be that all cells perceive gravity, but the magnitude of their perception depends on the density of the cellular components. Cells with dense objects such as amyloplasts are highly sensitive to the gravity vector and are the normal gravity-perceptive cells. But other cells may be weakly gravity perceptive or may use the information about the direction of the gravity vector to do something other than change the direction of growth. If decapped maize roots, which grow well but fail to curve in response to 1 g, are subjected to higher g forces, would they then be able to perceive the direction of the gravity vector? Any cell that perceives gravity must become polarized with respect to the gravity vector. It is the transduction steps that hold the key to an understanding of gravity responses in plants.
Intracellular Transduction of Gravity Vector Information. The perception of gravity must produce an asymmetry in the perceiving cell, either in its structure or in its biochemistry. Despite the importance of this transduction step, almost nothing is known for certain about it. A number of suggestions have been made: an asymmetry in cellular calcium,36 an activation of plasma membrane ion channels localized at one position in the cell,37 a change in the structure and distribution of cytoskeletal elements, or a change in the gating of plasmodesmata between cells.38 Any or all of these might be possible, but there is no direct evidence to support any of them. Whatever the nature of the asymmetry, it must be capable of being induced at 4°C and must be "remembered" for at least an hour at 4°C.39 None of the current theories can explain these observations.
A major emphasis of NASA-sponsored research should be on the nature of this transduction step. Techniques are available to examine intracellular localization of calcium or of cytoskeletal elements. Studies should focus on comparing cells containing amyloplasts, since these cells at least have the potential for detecting the direction of gravity, with cells with starchless amyloplasts or with cells lacking amyloplasts. There is no reason why these experiments should not be conducted on single cells or even protoplasts. Any relevant asymmetry should change when the direction of the gravity vector shifts and should be either absent or greatly reduced in cells that lack amyloplasts.
The Translocation Steps. The transduction event induces a change in the perceiving cells so that some signal is translocated differentially to the site of reaction on the two sides of an organ. The Cholodny-Went hypothesis40 proposed that the signal is auxin, and that it is first transported transversely across a horizontal stem or root, and then translocated longitudinally to the site of reaction. This is clearly a
gross oversimplification. The fact that inhibitors of polar auxin transport block gravitropism of both stems and roots indicates that auxin plays some role41 and that it must be polarly transported during gravitropism in both organs. In stems, at least, auxin is not actually translocated from one side of the stem to the other; but there is apparently a differential transport of auxin from the vascular tissue to the neighboring outer cell layers.42 In roots, the signal is translocated from the root cap through the meristem to the distal elongation zone (DEZ).43 However, it is not certain that the translocated signal is auxin or that there is just one signal. Researchers have proposed that there may be rapid signals, which might be electrical or hydraulic, as well as slow signals that are hormonal.44 Rapid gravity-induced changes in membrane potential have been detected in both root cap 45 and DEZ cells,46 but their significance is open to speculation. Asymmetric concentrations of auxin have been detected in the DEZ area of maize roots,47 but whether the auxin was translocated from the root cap or from the basal portions of the root is not known.
This area of signal translocation is ripe for additional studies. The cellular pathway for auxin transport needs to be ascertained in both stems and roots. The signal may move from cell to cell through the extracellular wall solution, or intracellularly through the plasmodesmata. The interaction between the gravity vector and auxin transport has yet to be determined. It is difficult to see how an asymmetric gradient of a chemical signal can persist if the signal is free to pass through the plasmodesmata, as auxin would be expected to do. For that reason, the effect of gravity on the conductance of plasmodesmata merits study. In roots, a more extensive study of gravity-actuated electrical signals is needed, coupled with precise measurements of the timing of the arrival of electrical signals versus the start of gravitropic curvature.
The Reaction Phase. The paradigm of the reaction phase is that an asymmetric gradient of auxin, with more on the lower side of either stem or root, causes the differential in the rate of cell elongation. In stems, the greater auxin level on the lower side leads to enhanced growth; in roots, it leads to an inhibition of growth.48 There is strong evidence for such auxin asymmetry in stems and coleoptiles 49 and some evidence for such an auxin gradient in roots.50 And though the cell layers that control elongation of stems are clearly the outer layers, the location of the comparable controlling layers in roots is uncertain, although it may be the pericyle.51
In addition to the asymmetry of auxin, there may also be a gravity-induced differential sensitivity of the cells in the reaction zone to auxin. The signal moving from the site of perception might not be auxin but some other factor that alters the sensitivity of the reacting cells to it. There is some evidence of enhanced sensitivity to auxin on the lower side of horizontal soybean hypocotyls.52 The fact that the auxin-induced small auxin up-regulated (SAUR) genes are more strongly induced on the lower side of horizontal tobacco stems53 is compatible with there being either more auxin or more sensitivity to auxin on the lower side. Further investigations into the effect of the gravity vector on the sensitivity of reacting cells to auxin are needed.
The mechanism by which auxin controls the rate of cell enlargement has been extensively studied. There is mounting evidence that auxin stimulates elongation of stem and coleoptile cells in part by promoting the export of protons from the cells, with the lowered wall pH activating wall loosening proteins such as the expansins.54 But why does auxin slow root cell elongation? There are still large gaps in knowledge here, and until these are filled in, it will not be possible to fully comprehend how gravitropic curvature occurs in roots.
Needs for Future Research on Gravitropism. During the past decade the extensive research into gravitropism has greatly enhanced knowledge about this process. But future advances could be hastened by following several principles:
- Research on gravitropism should be concentrated on only a few systems. Because systems differ, the use of large numbers of different experimental systems has created confusion and diluted effort. By concentrating efforts on a few systems, results obtained by one research group would have a synergistic effect on the research of others using the same system.
- Gravitropism in single cells might best be concentrated on the Chara rhizoid and the Ceratodon protonema systems. For roots of higher plants, there are advantages to working with either Zea mays roots, which are well studied and large, or on Arabidopsis roots, with a range of known mutants, and a whole genome that will be known within a few years. Research on gravitropism in stems might well concentrate on Arabidopsis, tomato, and peas, in which gravitropic mutants exist. Coleoptiles of Avena or Zea mays have the advantage that most of their cells contain amyloplasts and are good subjects for studies on perception, especially the transduction phase.
- Mutants should be used wherever possible. The power of genetics to dissect the steps in gravitropism is immense, and the information that could be obtained by sequencing genes directly involved in this process could lead to an understanding of how the pieces fit together. Using complementation analysis, it should be possible to locate agravitropic mutants that are blocked specifically in each of the four main steps in gravitropism. Mutants have only just started to be used effectively in such studies. Another group of useful mutants are those that alter the angle at which the organs grow in response to the direction of gravity or that change this direction in response to the presence or absence of light.55
- The research should concentrate on the basic questions and not be waylaid by less important ones. For any research supported by NASA, the PI should demonstrate that the results would enhance an understanding of the overall process. Perhaps the most important question is the nature of the asymmetry that occurs in any cell that perceives the direction of the gravity vector.
- Studies of gravitropism must make use of the most advanced techniques available, including extensive use of genetics and transgenic plants. Using advanced microscopic techniques, such as confocal microscopy to study cytoplasmic calcium distribution, could provide information not obtainable by other methods.
- A primary focus of NASA-sponsored research in plant biology should be on the mechanisms of gravitropism. In particular, modern cell and molecular techniques should be used to determine the following:
- The identity of the cells that actually perceive gravity, and the role of the cytoskeleton in the process;
- The nature of the cellular asymmetry set up in a cell that perceives the direction of the gravity vector;
- The nature and mechanism of the translocation of the signals that pass from the site of perception to the site of reaction; and
- The nature of the response to the signal(s) that leads to alterations in the rate of cell enlargement.
- Maximum use should be made of mutants and gene sequencing to identify specific proteins involved in gravitropism.
- A secondary focus should be on the mechanisms of graviperception and graviresponse in single cells, especially the algae and mosses.
Some flagellated single-celled protists can orient their swimming in response to the direction of gravity. For example, young Euglena cells, in the absence of light signals, swim upward, whereas older cells swim downward.56 The perception of gravity appears to be by the whole Euglena protoplast, because the direction of swimming can be reversed if the cells are placed in a medium whose density is greater than that of the cells. The microgravity environment of the shuttle was used effectively to show that the threshold for perception of gravity by Euglena cells is about 0.16 g.57
No research on gravitaxis is currently being supported by NASA. This area that holds great promise for understanding the response of cells to gravity and might well be one that NASA should consider supporting.
As a lower priority, NASA should consider supporting research on algal gravitaxis.
Effects of Gravity-induced Tissue Stresses on Plant Development
The above-ground organs of multicellular plants can undergo considerable stress in their tissues due to suspended weight. Cells on the top of such suspended organs are under tension, whereas those on the bottom are compressed. Horizontal tree limbs undergo changes in the pattern of deposition of new secondary xylem in response to the weight of the limb. The reaction wood that is produced supports the limb and counteracts the effects of gravity. In conifers, the reaction wood forms on the lower side of the limb and forces it upward; in hardwoods, it forms on the upper side and contracts to pull the limb upward. The result is that the branch maintains a set angle with respect to gravity, regardless of the branch's weight. The formation of reaction wood has been extensively studied by forestry scientists, because the physical properties of reaction wood are different from those of regular wood. The mechanisms involved in sensing the stresses are still largely unknown.58 However, it is not recommended that NASA support work in this area.
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