Role of calcium in gravity perception of plant roots

Calcium ions may play a key role in linking graviperception by the root cap to the asymmetric growth which occurs in the elongation zone of gravistimulated roots. Application of calcium-chelating agents to the root cap inhibits gravitropic curvature without affecting growth. Asymmetric application of calcium to one side of the root cap induces curvature toward the calcium source, and gravistimulation induces polar movement of applied ⁴⁵Ca²⁺ across the root cap toward the lower side. The action of calcium may be linked to auxin movement in roots since 1) auxin transport inhibitors interfere both with gravitropic curvature and gravi-induced polar calcium movement and 2) asymmetric application of calcium enhances auxin movement across the elongation zone of gravistimulated roots. Indirect evidence indicates that the calcium-modulated regulator protein, calmodulin, may be involved in either the transport or action of calcium in the gravitropic response mechanism of roots.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Physiological, biochemical and molecular processes associated with gravitropism in roots of maize.

This research aims to characterize regulation of the principal cytosolic protein kinases in maize, cultivar 'Merit' root tips, since much evidence indicates that stimuli which modulate the gravitropic response in this system act through regulation of activity of these enzymes. To this end, we have cloned a maize protein kinase belonging to a group of plant protein kinases with a catalytic domain similar in primary structure to the second messenger-regulated protein kinases known in animal and fungal systems. However, both the unique structural features conserved among plant protein kinases in this group, and lack of evidence for cyclic nucleotide signalling in plants point to operation of a novel protein kinase regulatory mechanism in plants. In order to test effects of possible regulators on protein kinase activity, we developed a sensitive method for detecting regulation of autophosphoryl labelling of protein kinases in unfractionated maize protein extracts. Regulation of protein kinase autophosphorylation in these extracts was different from that known in animals and fungi, further suggesting operation of unique protein kinase regulatory mechanisms in plants. Previous research has shown that light, or factors modulated by light, regulate plant protein kinase activity. We found that protein kinase activity was co-immunoprecipitated with the plant photoreceptor phytochrome, and was associated with phytochrome by high-affinity chemical interactions. Far-red reversibility of red-light regulation of phytochrome phosphorylation by the associated protein kinase indicates that it may modulate or transduce the light signals which lead to gravitropic sensitivity in 'Merit' maize.     

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.     

Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence.

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes. A rise in [Ca2+]i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in [Ca2+]i. The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.     

Agravitropic mutant for the study of hydrotropism in seedling roots

Roots have been shown to respond to a moisture gradient by positive hydrotropism. Agravitropic mutant plants are useful for the study of the hydrotropism in roots because on Earth hydrotropism is obviously altered by the gravity response in the roots of normally gravitropic plants. The roots are able to sense water potential gradient as small as 0.5 MPa mm⁻¹. The root cap includes the sensing apparatus that causes a differential growth at the elongation region of roots. A gradient in apoplastic calcium and calcium influx through plasmamembrane in the root cap is somehow involved in the signal transduction mechanism in hydrotropism, which may cause a differential change in cell wall extensibility at the elongation region. We have isolated an endoxy loglucan transferase (EXGT) gene that is strongly expressed in pea roots and appears to be involved in the differential growth in hydrotropically responding roots. Thus, it is now possible to study hydrotropism in roots by comparing with or separate from gravitropism. These results also imply that microgravity conditions in space are useful for the study of hydrotropism and its interaction with gravitropism.     

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.     

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

Calcium movements and the cellular basis of gravitropism.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.     

Gravisensing in single-celled systems: characean rhizoids and protonemata.

Gravitropically tip-growing cell types are attractive unicellular model systems for investigating the mechanisms and the regulation of gravitropism. Especially useful for studying the mechanisms of positive and negative gravitropic tip-growth are characean rhizoids and protonemata. They originate from the same cell type, show the same overall cell shape, cytoplasmic zonation, arrangement of actin and microtubule cytoskeleton, use statoliths for gravisensing, but show opposite gravitropism. In both cell types, actin microfilaments are complexly organized in the apical dome,where a dense spherical actin array is colocalized with spectrin-like epitopes and a unique endoplasmic reticulum aggregate, the structural center of the Spitzenk?rper. The opposite gravitropic responses seem to be based on differences in the actin-organized anchorage of the Spitzenk?rper and the actin-mediated transport of statoliths. In negatively gravitropic (upward bending) protonemata, the statoliths-induced drastic upward shift of the cell tip is preceded by a relocalization of dihydropyridine-binding calcium channels and of the apical calcium gradient to the upper flank (bending by bulging). Such relocalizations have not been observed in positively gravitropically responding (downward growing) rhizoids in which statoliths sedimentation is followed by differential flank growth (bending by bowing). This paper reviews the current knowledge and hypotheses on the mechanisms of the opposite gravitropic responses in characean rhizoids and protonemata.     

Calcium gradient in plant cells with polarized growth in simulated microgravity.

Plant cells characterized by apical growth, for example, root hairs and apical cells of moss protonema, are a convenient model to address the problem of gravity response mechanisms including initiation of cell polarity. The fluorescent calcium probe, chlorotetracycline, allowed us to display the calcium distribution gradient in these cells. Irradiation by red light led to a sharp decrease in the Ca2+ ion activity in cells. During clinostatting in darkness the pattern of calcium influx and distribution changes inconsiderably as compared with control; in root hairs calcium is detected mainly in their apices and bases as in control. Addition of chlorpromazine to the medium probably increases the influx and accumulation of Ca2+ ions. Under data obtained confirm speculations on the Ca2+ ion functional role for the apical growth of plant cells and may suggest the participation of gravity in redistribution or activation of ion channels, calcium channels included, in the plasmalemma.     

Possible mechanisms of plant cell wall changes at microgravity.

Space and clinostatic experiments revealed that changes of plant cell wall structure and its function depend on type of tissue and duration of influence. It was shown that clinostat conditions reproduce the part of weightlessness biological effects. It is established that various responses of wall structural-metabolic organization occur at microgravity: changes of cell walls ultrastructure and organelles structure; decrease of synthesis of primary plant cell wall; rearrangements of polysaccharides content. It is shown that mechanisms of plant cell wall changes at microgravity are connected with decrease of cellulose crystallization, activation of pectolytic enzymes and rearrangement of calcium balance of apoplast and cytoplasm.     

Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation.     

Biological effects due to weak magnetic field on plants.

Throughout the evolution process, Earth's magnetic field (MF, about 50 microT) was a natural component of the environment for living organisms. Biological objects, flying on planned long-term interplanetary missions, would experience much weaker magnetic fields, since galactic MF is known to be 0.1-1 nT. However, the role of weak magnetic fields and their influence on functioning of biological organisms are still insufficiently understood, and is actively studied. Numerous experiments with seedlings of different plant species placed in weak magnetic field have shown that the growth of their primary roots is inhibited during early germination stages in comparison with control. The proliferative activity and cell reproduction in meristem of plant roots are reduced in weak magnetic field. Cell reproductive cycle slows down due to the expansion of G1 phase in many plant species (and of G2 phase in flax and lentil roots), while other phases of cell cycle remain relatively stable. In plant cells exposed to weak magnetic field, the functional activity of genome at early pre-replicate period is shown to decrease. Weak magnetic field causes intensification of protein synthesis and disintegration in plant roots. At ultrastructural level, changes in distribution of condensed chromatin and nucleolus compactization in nuclei, noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids in meristem cells were observed in pea roots exposed to weak magnetic field. Mitochondria were found to be very sensitive to weak magnetic field: their size and relative volume in cells increase, matrix becomes electron-transparent, and cristae reduce. Cytochemical studies indicate that cells of plant roots exposed to weak magnetic field show Ca2+ over-saturation in all organelles and in cytoplasm unlike the control ones. The data presented suggest that prolonged exposures of plants to weak magnetic field may cause different biological effects at the cellular, tissue and organ levels. They may be functionally related to systems that regulate plant metabolism including the intracellular Ca2+ homeostasis. However, our understanding of very complex fundamental mechanisms and sites of interactions between weak magnetic fields and biological systems is still incomplete and still deserve strong research efforts.     

Growth restoration in azuki bean and maize seedlings by removal of hypergravity stimuli.

Hypergravity stimuli, gravitational acceleration of more than 1 x g, decrease the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls by decreasing the cell wall extensibility via an increase in the molecular mass of matrix polysaccharides. An increase in the pH in the apoplastic fluid is hypothesized to be involved in the processes of the increase in the molecular mass of matrix polysaccharides due to hypergravity. However, whether such physiological changes by hypergravity are induced by normal physiological responses or caused by physiological damages have not been elucidated. In the present study, we examined the effects of the removal of hypergravity stimuli on growth and the cell wall properties of azuki bean and maize seedlings to clarify whether the effects of hypergravity stimuli on growth and the cell wall properties are reversible or irreversible. When the seedlings grown under hypergravity conditions at 300 x g for several hours were transferred to 1 x g conditions, the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls greatly increased within a few hours. The recovery of growth rate of these organs was accompanied by an immediate increase in the cell wall extensibility, a decrease in the molecular mass of matrix polysaccharides, and an increase in matrix polysaccharide-degrading activities. The apoplastic pH also decreased promptly upon the removal of hypergravity stimuli. These results suggest that plants regulate the growth rate of shoots reversibly in response to hypergravity stimuli by changing the cell wall properties, by which they adapt themselves to different gravity conditions. This study also revealed that changes in growth and the cell wall properties under hypergravity conditions could be recognized as normal physiological responses of plants. In addition, the results suggest that the effects of microgravity on plant growth and cell wall properties should be reversible and could disappear promptly when plants are transferred from microgravity to 1 x g. Therefore, plant materials should be fixed or frozen on orbit for detecting microgravity-induced changes in physiological parameters after recovering the materials to earth in space experiments.     

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.     

Efficacy of an ototoxic aminoglycoside (gentamicin) on the differentiation of the inner ear of cichlid fish.

Previous investigations revealed that the growth of fish inner ear otoliths depends on the amplitude and the direction of gravity, thus suggesting the existence of a (negative) feedback mechanism. In the course of these experiments, it was shown that altered gravity both affected otolith size (and thus the provision of the proteinacious matrix) as well as the incorporation of calcium. It is hitherto unknown, as of whether sensory hair cells are involved either in the regulation of otolith growth or in the provision of otolithic material (such as protein or inorganic components) or even both. The ototoxic aminoglycoside gentamicin (GM) damages hair cells in many vertebrates (and is therefore used for the treatment of Meniere's disease in humans). The present study was thus designed to determine as of whether vestibular sensory cells are needed for otolith growth by applying GM in order to induce a (functionally relevant) loss of these cells. Developing cichlid fish Oreochromis mossambicus were therefore immersed in 120 mg/l GM for 10 or 21 days. At the beginning and at the end of the experimental periods, the fish were incubated in the calcium-tracer alizarin complexone (AC). After the experiment, otoliths were dissected and the area grown during GM-exposure (i.e., the area enclosed by the two AC labellings) was determined planimetrically. The results showed that incubating the animals in a GM-solution had no effect on otolith growth, but the development of otolith asymmetry was affected. Ultrastructural examinations of the sensory hair cells revealed that they had obviously not been affected by GM-treatment (no degenerative morphological features observed). Overall, the present results suggest that hair cells are not affected by GM concerning their possible role in (general) otolith growth, but that these cells indeed might have transitionally been impaired by GM resulting in a decreased capacity of regulating otolith symmetry.     

Characterization of a novel gravitropic mutant of morning glory, weeping2

In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 (we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT (PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.     

The role of calcium ions in cytological effects of hypogravity

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema $\text{Funaria}$$\text{hygrometrica}$ grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.     

Gravitropism of axial organs in multicellular plants.

Gravitropism of plant organs such as roots, stems and coleoptiles can be separated into four distinct phases: 1. perception (gravity sensing), 2. transduction of a signal into the target region and 3. the response (differential growth). This last reaction is followed by a straightening of the curved organ (4.). The perception of the gravitropic stimulus upon horizontal positioning of the organ (1.) occurs via amyloplasts that sediment within the statocytes. This conclusion is supported by our finding that submerged rice coleoptiles that lack sedimentable amyloplasts show no graviresponse. The mode of signal transduction (2.) from the statocytes to the peripheral cell layers is still unknown. Differential growth (3.) consists of a cessation of cell expansion on the upper side and an enhancement of elongation on the lower side of the organ. Based on the facts that the sturdy outer epidermal wall (OEW) constitutes the growth-controlling structure of the coleoptile and that growth-related osmiophilic particles accumulate on the upper OEW, it is concluded that the differential incorporation of wall material (presumably glycoproteins) is causally involved. During gravitropic bending, electron-dense particles ('wall-loosening capacity') accumulate on the growth-inhibited upper OEW. It is proposed that the autotropic straightening response, which is in part due to an acceleration of cell elongation on the curved upper side, may be attributable to an incorporation of the accumulated particles ('release of wall-loosening capacity'). This novel mechanism of autotropic re-bending and its implications for the Cholodny-Went hypothesis are discussed.