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Gravity is a fundamental factor which affects all living organisms. Plant development is well adapted to gravity by directing roots downward and shoots upwards. For more than a century, plant biologists have been fascinated to describe the molecular mechanisms underlying the gravitropic response of plants. Important progress towards signal perception, transduction, and response has been made, but new tools are beginning to uncover the regulatory networks for gravitropic control. We summarise recent progress in study of gravitropism and discuss strategies to identify the molecular basis of the gravity response in Arabidopsis thaliana. This will put us on a road towards the molecular systems biology of the Arabidopsis gravitropic response.

The cellular structures of statocytes implicated in gravisensing in primary and lateral roots of Vigna angularis were compared. The statocytes of lateral roots already had small amyloplasts immediately after they emerged from the primary root. Although these amyloplasts sedimented, the lateral roots showed much weaker gravitropism than primary roots, at least until they reached a length of about 30 mm. The nuclei were usually positioned in the upper end of the statocytes in both types of roots. Electron microscopic surveys showed that many tubular elements of endoplasmic reticulum (ER) were frequently localized in the lower end of the statocyte and they sometimes diverged or curved, suggesting that the ER forms a large reticulate complex. It is worth noting that statocytes with a large ER complex were found much more frequently in primary roots than in lateral roots. The amyloplasts were not always settled on this complex but were very frequently under it, especially in the primary roots. In lateral roots, they were usually localized under the ER complex when they were present. Thus, it is suggested that the differential development and organization of the amyloplast-ER complex system is involved in the differential gravitropism of the two types of roots.

Root apical meristems are composed of two zones in which either formative or proliferative cell divisions occur. Within the formative zone, autoreproductive initial cells (a-cells) occupy distinctive locations. By means of graph-L-systems, the behavior of one such type of a-cells has been investigated, with particular reference to root caps within the developing primordia of lateral roots of Lycopersicon esculentum cultivated in vitro. Here, the a-cells constitute the "protoderm initials", cells which are found also in the root cap of many angiosperm species. A set of cuboidal (i.e., six-sided) a-cells develops early in the ontogeny of a lateral-root primordium. Then, according to both anatomical observations and theoretical simulations obtained by the application of graph-L-systems, sequential production of descendents from each a-cell leads to the formation of a new autoreproductive cell (a), a cap columella initial (c), and two mother cells (e and f) whose respective descendents differentiate as root epidermis and cap flank cells. In this graph-L-system, there is specification of the location of sister cells with respect to the three orthogonal directions of a cuboidal. In the early stage of root cap formation, only a few rounds of these formative cell divisions by each a-cell and its four types of descendents are required to provide the basic set of cells necessary for full cap development. After the lateral root emerges from the parent root, there may be a temporary cessation of the formative divisions of the a-cells which give rise to columella initials. Columella production is then supported entirely by its own independent set of autoreproductive c-initials. At the same time, division of the autoreproductive protoderm initial cell is directed towards maintaining the cap flank and the epidermal cell files. The regulation of the types of formative division by the a-cell may be represented by means of a division counter which may be specific for a given species.

It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a beta 1 integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the beta 1 integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known beta 1 integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.

Calcium has been implicated as a key component in the signal transduction process of root gravitropism. We measured cytoplasmic free calcium in protoplasts isolated from the elongation zone and cap of primary roots of light-grown, vertically oriented seedlings of Zea mays L. Protoplasts were loaded with the penta-potassium salts of fura-2 and indo-1 by incubation in acidic solutions of these calcium indicators. Loading increased with decreasing pH but the pH dependence was stronger for indo-1 than for fura-2. In the case of fura-2, loading was enhanced only at the lowest pH (4.5) tested. Dyes loaded in this manner were distributed predominantly in the cytoplasm as indicated by fluorescence patterns. As an alternative method of loading, protoplasts were incubated with the acetoxymethylesters of fura-2 and indo-1. Protoplasts loaded by this method exhibited fluorescence both in the cytoplasm and in association with various organelles. Cytoplasmic calcium levels measured using spectrofluorometry, were found to be 160 +/- 40 nM and 257 +/- 27 nM, respectively, in populations of protoplasts from the root cap and elongation zone. Cytoplasmic free calcium did not increase upon addition of calcium to the incubation medium, indicating that the passive permeability to calcium was low.

To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.

The peripheral root cap and protoderm in Arabidopsis thaliana are organized into modular packets of cells derived from formative T-divisions of the root cap/protoderm (RCP) initials and subsequent proliferative divisions of their daughter cells. Each module consists of protoderm and peripheral root cap packets derived from the same periclinal T-division event of an RCP initial. Anatomical analyses are used to interpret the history of extensively coordinated cell divisions producing this modular construction. Within a given layer of root cap, the columella and RCP initials divided in a centrifugal sequence from the innermost columella initials toward the RCP initials. All RCP initials in the lineages around the circumference of the root divided nearly simultaneously in "waves" to form one module prior to the next wave of initial divisions forming a younger module. The peripheral root cap and protoderm packets within each module completed four rounds of proliferative divisions in the axial plane to produce, on average, 16 cells per packet in the basalmost modules in axial view. Peripheral root cap and protoderm cells predominantly in the T-type (trichoblast) lineages also underwent radial divisions as they were displaced basipetally. The regularity in the cellular pattern within the modules suggests a timing mechanism controlling highly coordinated cell division in the initials and their daughter cells.

Indirect immunofluorescence, using monoclonal antibodies to actin and tubulin, applied to sections of root tips of Lepidium, Lycopersicon, Phleum, and Zea, revealed features of the cytoskeleton that were unique to the statocytes of their root caps. Although the cortical microtubules (CMTs) lay in dense arrays against the periphery of the statocytes, these same cells showed depleted complements of endoplasmic microtubules (EMTs) and of actin microfilament (AMF) bundles, both of which are characteristic of the cytoskeleton of other post-mitotic cells in the proximal portion of the root apex. The scarcity of the usual cytosketetal components within the statocytes is considered responsible for the exclusion of the larger organelles (e.g., nucleus, plastids, ER elements) from the interior of the cell and for the absence of cytoplasmic streaming. Furthermore, the depletion of dense EMT networks and AMF bundles in statocyte cytoplasm is suggested as being closely related to the elevated cytoplasmic calcium content of these cells which, in turn, may also favour the formation of the large sedimentable amyloplasts by not permitting plastid divisions. These latter organelles are proposed to act as statoliths due to their dynamic interactions with very fine and highly unstable AMFs which enmesh the statoliths and merge into peripheral AMFs-CMTs-ER-plasma membrane complexes. Rather indirect evidence for these interactions was provided by showing enhanced rates of statolith sedimentation after chemically-induced disintegration of CMTs. All these unique properties of the root cap statocytes are supposed to effectively enhance the gravity-perceptive function of these highly specialized cells.

The kinetics of MT [microtubule] reorientation in primary roots of Zea mays cv. Merit, were examined 15, 30, 45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4-5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10(-9) to 10(-6) M IAA. IAA treatment at 10(-8) M or less showed no major or consistent changes but 10(-7) M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10(-5) M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.

The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to "young" and "old", polysaccharide-slime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips of Arabidopsis and Nicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types. Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (approximately 30 nm) and are the least stained. Medial cisternae are narrower (approximately 20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4-6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network. Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.

Statocytes in root caps of Lepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a "younger" developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes of Lepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.

Antimonate staining procedures and energy dispersive X-ray micro-analytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.