PLANT BIOLOGY

GRAVISENSITIVITY OF PLANT CELLS: EXPERIMENTAL DATA AND CONCEPTS

E. L. Kordyum
Institute of Botany, National Academy of Sciences of Ukraine, Tereschenkovskaya str. 2, 01004, Kiev, Ukraine

One of the fundamental achievements of modern biology is the discovery of plant cell gravisensitivity based on the data of experimental investigations in the field of space and gravitational biology. The experiments with algae, moss protonema, and higher plants in-vivo and in-vitro, which were performed in the conditions of real microgravity on board the biosatellites, space ships and orbital stations allowed to elucidate the main regularities of biological effects of microgravity and, thus, firstly to value a role of gravity in plant vital activity at the cellular and molecular levels. It was established that microgravity 1) essentially affects cell metabolism; modifications of metabolism reflected in rearrangements of organelle ultrastructure and functional load, i.e. a cell is sensitive to gravity; 2) intracellular calcium balance changes in microgravity; 3) metabolism changes in microgravity lead to acceleration of cell differentiation and aging, and 4) microgravity belongs to alteration factors which allow the adaptive reactions at the cellular and organism levels in the range of a physiological response, i.e. in the framework of genetically determinated program of ontogenesis. In connection with intensive research on biology of plant cells in microgravity, which are specialized or not specialized to gravity perception, a distinction between cell gravisensitivity and cell graviperception has to be discussed. The former is related to maintaining the cell structure and metabolism stability in the gravitational field and their changes in microgravity, and the latter is related to actively using a gravitational stimulus by cells for realization of plant normal space orientation, growth, and vital activity (gravitropism, gravitaxis). On the basis of the experimental data on the changes in cell metabolism in microgravity, it is assumed that proliferating and actively metabolizing cells are the most sensitive to the influence of altered gravity. Simultaneously, this assumption propounds the next questions: 1) what primary events underlie metabolism changes in microgravity, 2) what second messengers take part in transfer of the primary signals of microgravity, 3) whether gene expression changes in microgravity, 4) whether the parameters of a cell cycle and proliferative activity change in microgravity, and 5) how the metabolism changes in microgravity are integrated in physiological responses in the cells of different types directly connected with realization of their functions. Trying to answer these questions, a hypothesis of gravitational decompensation was proposed. According to this hypothesis, the cytoplasmic membrane is the primary site of microgravity action. The rearrangements in physical-chemical properties of the cytoplasmic membrane underlie the changes in its permeability, receptors' functions, membrane-bound enzyme activity etc., that, in its turn, leads to the next metabolism changes resulting in physiological responses of cells and organisms on the action of microgravity. In light of these ideas, it has been possible to explain the facts of cell metabolism and ultrastructure rearrangements when morphogenesis and cell differentiation are normal, as well as the complicated ways microgravity affects multicellular organisms through intercellular interaction in the tissue system. The available experimental data and theoretical ideas on cell space biology are the basis for further investigations of the structure, reproduction, differentiation and functioning of plant cells in microgravity. The main directions of future investigations of gravi-dependent and gravi-sensitive processes in plant cells, in particular the events occurring at the membrane level and providing the transduction of primary microgravity effects in the integrated intracellular processes are considered.

RESERVE UTILISATION IN ARABIDOPSIS THALIANA SEEDS GERMINATING IN MICROGRAVITY

L.G. Briarty¹and E.P. Maher²
1 9 The Cloisters, Beeston, Nottingham NG9 2FR; ex Plant Science Division, School of Biosciences, University of Nottingham, Nottingham NG7 2RD 2 The Open University in Scotland, 10 Drumsheugh Gardens, Edinburgh EH3 7QJ

It is becoming apparent that the action of microgravity conditions on plant growth can be to modulate the developmental processes, sometimes through secondary effects such as the absence of convection impeding atmospheric movement. These interactions are difficult to measure, given the complexity and constraints of in-orbit experimentation, and the real-time biochemical analyses needed to clarify such results are not yet available. Accurate, quantitative ultrastructural analysis of fixed and returned material is possible however, and the data presented are from a study of cotyledon organisation in microgravity-grown material, carried out in order to determine effects on reserve utilisation.
Samples of resin-embedded cotyledon material from 57 and 86h germinated seeds from microgravity and control conditions were sectioned for stereological analysis, and measurements made of volume density, surface density and numerical density of the main observable subcellular components.
Analysis showed that reserve lipid utilisation is significantly delayed when germination takes place in microgravity; in the 1G control material the lipid volume drops from 27% to just below 7% by 86h, while in the seedlings grown in microgravity lipid mobilisation appears slower over the same period, the volume fraction dropping from 21.5% to 15.5%. There are also differences in cotyledon cell shape.
The difference in reserve metabolism is interpreted as due to localised anoxia in the absence of atmospheric convection and mixing.


The estimate of gravisensitivity

G.Perbal and D. Driss-Ecole
Université Pierre et Marie Curie, 4, Place Jussieu, F-75252 Paris Cedex 05, France

The dose-response curve of the gravitropic reaction was often used to estimate gravisensitivity. It was proposed that the response varies as a linear function of the logarithm of the dose of gravistimulus. As this model fitted correctly most of the data obtained, the presentation time (minimal time of stimulation to provoke a slight curvature) was calculated by extrapolating down to zero curvature the straight line representing the response as a function of the logarithm of the stimulus. In the present review, it is shown that the logarithmic model does not fit the experimental data as well as the hyperbolic model. In the hyperbolic model, there is theoretically no presentation time since the curve passes through the origin. We propose a new method to estimate gravisensitivity which is based on the study of graviresponsiveness for threshold doses of gravistimulation.

GRAVITATIONAL EFFECTS ON METABOLISM AND GENE EXPRESSION OF ARABIDOPSIS THALIANA CELL CULTURES

E. Magel, R-M. Maier, M. Martzivanou, M. Ecke, R. Hampp
University of Tübingen, Physiological Ecology of Plants, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

Suspensions of cell cultures of Arabidopsis thaliana (cv. columbia) were exposed to altered g-forces due to centrifugation (1 to 10g) and in the course of sounding rocket flights (less than 1g). Under reduced g-forces the energy status (ATP/ADP ratio) was increased. This confirmed earlier results obtained with tobacco protoplasts. A 2-min-feeding of labelled glucose at different stages during a sounding rocket flight showed clear changes in the labelling pattern of products of metabolic conversion of glucose. Most interesting is the appearance of spot which could be identified as an amino acid, most probably lysine. One of the key enzymes for supplying carbon skeletons for amino acid synthesis in plants is phosphoenolpyruvate carboxylase (PEPC). When cell cultures of A. thaliana were exposed to g-forces from 1 to 10g, the transcript of the gene coding for PEPC was significantly increased in amount. The change in expression was maximal after 60 min at 5 to 6g, an increase in transcript was, however, already detectable after an exposure to 7g for 15 min. We thus assume that g-forces above a threshold of about 5g as well as below 1g are sensed by plant cells in general, causing distinct metabolic responses, which obviously in part are regulated by gene expression.

THE NOVEL ARABIDOPSIS GENE RHA1 IS A POSSIBLE TRANSDUCER OF GRAVITATIONAL SIGNALS IN PLANTS ROOTS

F. Migliaccio, S. Piconese, M. Fagiano, and C. Rosi
Institute of Plant Biochemistry and Ecophysiology; Consiglio Nazionale delle Ricerche, via Salaria Km 29.300; 00016 Monterotondo (Rome) Italy

Recently, a new Arabidopsis mutant, that shows reduced gravitropic response and auxin sensitivity in the roots, has been characterized. The mutant was named rha1 for the additional characteristic of showing, when grown on a vertically set agar plate, reduced or inverted chirality, seen as a slanting of the roots toward the right-hand. The mutation is recessive and segregates following a mendelian mechanism. The gene resulted tagged for a T-DNA insert. Taking advantage of the insert, through TAIL-PCR techniques, a fragment of the tag left border, together with a fragment of the gene were cloned and by comparison with the data in the Gene-Bank it was found that the T-DNA is inserted in the promoter region of a new gene, belonging to the Heath Shock Factors (HSF). The gene maps on the chromosome 5 of Arabidopsis, close and above the RFLP marker mi61. In addition, through RT-PCR we obtained confirmation that the gene is expressed in the wild-type, but not in the mutant. This HSF seems to be deprived of the additional hydrophobic repeat C, which is normally present, but lacking in a HSF from budding yeast and the human HSF4. Comparative analysis of the sequences indicated that the gene shows a high degree of homology, in the DNA binding motif, with different HSFs from plants (Arabidopsis, tomato, mays, soybean), yeast, Caenorhabdites, Drosophila, mouse, and humans.
We consider particularly interesting the fact that RHA1 belongs to the HSF family of genes, to which many important functions in the physiology of cells and tissues have recently been ascribed. We hypothesize that RHA1 could be involved in the transduction of gravitropic signals in plant roots, possibly in connection with the action and/or the transport of the plant hormone auxin. It could be thus one of the sought elements of the gravitropic signal transduction pathway. In particular, it could be, as shown for the HSF2 from humans, involved in the activation of the PP2A phosphatase (Hong Y. and Sargent K.D., 1999, J. Biol.Chem. 274,
12967- 70), which in turn has been shown to be involved in the transport of auxin in Arabidopsis roots (Garbers C. et al. 1996, EMBO J. 15, 2115-2124). Our next step will be the study of the expression of the gene RHA1, through techniques of in-situ hybridization and RT-PCR, on clinostates and in space.