Morphogenesis of the root cap in adventitious roots of Salix viminalis

The preformed root primordia in stems of Salk viminalis L. consist of undifferentiated cells. Forty-eight hours after activation of the primordia in cuttings a root cap meristem was initiated four to five cell tiers from the surface of the primordia. The cells distal to the meristem divided only in an anticlinal plane, while in the meristem they divided mostly periclinally but sometimes anticlinally. After 72 hours a columella was established and the amyloplasts began to sediment in response to gravity. Shortly after this stage the roots began to bend slightly downward, probably as a geo-tropic response. Six days after activation the root cap consisted of up to 15 tiers of cells. The ultrastucture of the cap cells just prior to emergence was studied in more detail. The plastids in the cells adjoining the root proper were typical proplastids. Distal to this cell tier starch accumulated in the plastids. In the fifth tier the amyloplasts were fully sedimented to the lowermost cell walls. The amount of ER increased with the distance from the initial cells and most of it was located at the distal periclinal cell wall. The nucleus and the vacuoles in the geo-sensitive cells occurred in the space above the sedimented amyloplasts. The cytoplasm was less electron opaque than in the initial cells and the mitochondria had more cristae. In the distal cells of the columella and the lateral root cap secretion of mucilage seemed to have started. Numerous large dictyosomes were associated with large vesicles containing a fibrillar or granular material. The plasmalemma lining the distal periclinal cell wall had separated from the wall. A fibrillar material was present between the plasmalemma and the wall and also in intercellular spaces outside the root cap.     

Positional effect of cell inactivation on root gravitropism using heavy-ion microbeams

When primary root apical tissues of Arabidopsis thaliana were irradiated by heavy-ion microbeams with 120 microm diameter, strong inhibition of root elongation and curvature were observed at the root tip. Irradiation of the cells that become the lower part of the root cap after gravistimulation showed strong inhibition of root curvature, whereas irradiation of the cells that become the upper part of the root cap after gravistimulation did not show severe damage in either root curvature or root growth. Further analysis using smaller area microbeams with 40 microm diameter indicated that the greatest inhibition of curvature occurred at the root tip and the next greatest inhibition occurred in the cells in the lower part of the root cap. These results indicate not only that the root tip and columella cells are the most sensitive sites for root gravity, but also that signalling of root gravity would go through the lower part of the cap cells after perception.     

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.     

Changes in cytosolic pH within Arabidopsis root columella cells play a key role in the early signaling pathway for root gravitropism

Ratiometric wide-field fluorescence microscopy with 1',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran demonstrated that gravistimulation leads to rapid changes in cytoplasmic pH (pHc) in columella cells of Arabidopsis roots. The pHc of unstimulated columella cells in tiers 2 and 3, known sites of graviperception (E.B. Blancaflor, J.B. Fasano, S. Gilroy [1998] Plant Physiol 116: 213-222), was 7.22 +/- 0.02 pH units. Following gravistimulation, the magnitude and direction of pHc changes in these cells depended on their location in the columella. Cells in the lower side of tier 2 became more alkaline by 0.4 unit within 55 s of gravistimulation, whereas alkalinization of the cells on the upper side was slower (100 s). In contrast, all cells in tier 3 acidified by 0.4 pH unit within 480 s after gravistimulation. Disrupting these pHc changes in the columella cells using pHc modifiers at concentrations that do not affect root growth altered the gravitropic response. Acidifying agents, including bafilomycin A1, enhanced curvature, whereas alkalinizing agents disrupted gravitropic bending. These results imply that pHc changes in the gravisensing cells and the resultant pH gradients across the root cap are important at an early stage in the signal cascade leading to the gravitropic response.     

A morphometric analysis of cellular differentiation in root caps ofCucurbita peop

In order to quantify the ultrastructural changes that occur during cellular differentiation in an “open” type of root cap, we have performed a morphometric analysis of the ultrastructures of calyptrogen, columella, and peripheral cells of the root cap ofCucurbita pepo. The relative volumes of nuclei, nucleoli, and mitochondria decrease as cells move (i.e., differentiate) through the root cap. Before cells are sloughed from the cap, the relative volume of the vacuole increases by 250%. The relative volumes of plastids and plastid starch increase as calyptrogen cells differentiate into columella cells, but decrease as columella cells differentiate into peripheral cells. Dictyosomal volumes increase only as columella cells differentiate into peripheral cells. These results indicate that the five cell types comprising the root cap ofC.pepo are each characterized by a unique structure, and that the ultrastructural changes associated with cellular differentiation in root caps are organelle specific. These results are discussed relative to the functions of the various cell types of the root cap.     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Gravity signal transduction in primary roots

AIMS: The molecular mechanisms that correlate with gravity perception and signal transduction in the tip of angiosperm primary roots are discussed. SCOPE: Gravity provides a cue for downward orientation of plant roots, allowing anchorage of the plant and uptake of the water and nutrients needed for growth and development. Root gravitropism involves a succession of physiological steps: gravity perception and signal transduction (mainly mediated by the columella cells of the root cap); signal transmission to the elongation zone; and curvature response. Interesting new insights into gravity perception and signal transduction within the root tip have accumulated recently by use of a wide range of experimental approaches in physiology, biochemistry, genetics, genomics, proteomics and cell biology. The data suggest a network of signal transduction pathways leading to a lateral redistribution of auxin across the root cap and a possible involvement of cytokinin in initial phases of gravicurvature. CONCLUSION: These new discoveries illustrate the complexity of a highly redundant gravity-signalling process in roots, and help to elucidate the global mechanisms that govern auxin transport and morphogenetic regulation in roots.     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Negative gravitropic response of roots directs auxin flow to control root gravitropism

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.     

The Involvement of Glucose-6-Phosphatase in Mucilage Secretion by Root Cap Cells of Zea mays

In order to determine the involvement of glucose-6-phosphatasein mucilage secretion by root cap cells, we have cytochemicallylocalized the enzyme in columella and peripheral cells of rootcaps of Zea mays. Glucose-6-phosphatase is associated with theplasmalemma and cell wall of columella cells. As columella cellsdifferentiate into peripheral cells and begin to produce andsecrete mucilage, glucose-6-phosphatase staining intensifiesand becomes associated with the mucilage and, to a lesser extent,the cell wall. Cells being sloughed from the cap are characterizedby glucose-6-phosphatase staining being associated with thevacuole and plasmalemma. These changes in enzyme localizationduring cellular differentiation in root caps suggest that glucose-6-phosphataseis involved in the production and/or secretion of mucilage byperipheral cells of Z. mays. Zea mays, corn, glucose-6-phosphatase, columella cell, peripheral cell, mucilage, secretion, cytochemistry     

Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.     

Root apical organization inArabidopsis thaliana 1. Root cap and protoderm

Summary We investigated the development of the root cap and protoderm inArabidopsis thaliana root tips.A. Thaliana roots have closed apical organization with the peripheral root cap, columella root cap and protoderm developing from the dermatogen/calyptrogen histogen. The columella root cap arises from columella initials. The initials for the peripheral root cap and protoderm are arranged in a collar and the initiation event for these cells occurs in a sequential pattern that is coordinated with the columella initials. The resulting root cap appears as a series of interconnected spiraling cones. The protoderm, in three-dimensions, is a cylinder composed of cell files made up of packets of cells. The number of cell files within the protoderm cylinder increases as the root ages from one to two weeks. The coordinated division sequence of the dermatogen/calyptrogen and the increase in the number of protoderm cell files are both features of post-embryonic development within the primary root meristem.Abbreviations RCP root cap/protoderm - CI columella initial - PI protoderm initial     

The Root Cap: Cell Dynamics, Cell Differentiation and Cap Function

The root cap is a universal feature of angiosperm, gymnosperm, and pteridophyte roots. Besides providing protection against abrasive damage to the root tip, the root cap is also involved in the simultaneous perception of a number of signals – pressure, moisture, gravity, and perhaps others – that modulate growth in the main body of the root. These signals, which originate in the external environment, are transduced by the cap and are then transported from the cap to the root. Root gravitropism is one much studied response to an external signal. In the present paper, consideration is given to the structure of the root cap and, in particular, to how the meristematic initial cells of both the central cap columella and the lateral portion of the cap which surrounds the columella are organized in relation to the production of new cells. The subsequent differentiation and development of these cells is associated with their displacement through the cap and their eventual release, as “border cells”, from the cap periphery. Mutations, particularly in Arabidopsis, are increasingly playing a part in defining not only the pattern of genetic activity within different cells of the cap but also in revealing how the corresponding wild-type proteins relate to the range of functions of the cap. Notable in this respect have been analyses of the early events of root gravitropism. The ability to image auxin and auxin permeases within the cap and elsewhere in the root has also extended our understanding of this growth response. Images of auxin distribution may, in addition, help extend ideas concerning the positional controls of cell division and cell differentiation within the cap. However, firm information relating to these controls is scarce, though there are intriguing suggestions of some kind of physiological link between the border cells surrounding the cap and mitotic activity in the cap meristem. Open questions concern the structure and functional interrelationships between the root and the cap which surmounts it, and also the means by which the cap transduces the environmental signals that are of critical importance for the growth of the individual roots, and collectively for the shaping of the root system.     

Mapping the Functional Roles of Cap Cells in the Response of Arabidopsis Primary Roots to Gravity

The cap is widely accepted to be the site of gravity sensing in roots because removal of the cap abolishes root curvature. Circumstantial evidence favors the columella cells as the gravisensory cells because amyloplasts (and often other cellular components) are polarized with respect to the gravity vector. However, there has been no functional confirmation of their role. To address this problem, we used laser ablation to remove defined cells in the cap of Arabidopsis primary roots and quantified the response of the roots to gravity using three parameters: time course of curvature, presentation time, and deviation from vertical growth. Ablation of the peripheral cap cells and tip cells did not alter root curvature. Ablation of the innermost columella cells caused the strongest inhibitory effect on root curvature without affecting growth rates. Many of these roots deviated significantly from vertical growth and had a presentation time 6-fold longer than the controls. Among the two inner columella stories, the central cells of story 2 contributed the most to root gravitropism. These cells also exhibited the largest amyloplast sedimentation velocities. Therefore, these results are consistent with the starch-statolith sedimentation hypothesis for gravity sensing.     

Recovery of gravitropic response during regeneration of root caps does not require developed columella cells and sedimentation of amyloplasts

Correlations between regeneration of the root cap and recovery of a gravitropic response were studied using primary roots of Phaseolus vulgaris. After removal of various lengths of the root tip a gravistimulus was continuously given to the root. The statistical analysis of data showed that recovery of the gravitropic response was gradually delayed as the length of the tips removed increased. This suggested that the columella cells of the root cap were involved in gravitropism. When the root cap was completely removed, the roots did not respond to gravistimuli for the first 15 h and began to reorient their growth direction at 20 h. At this time, the columella cells had just begun to regenerate and had immature amyloplasts which did not sufficiently form a sediment. These results suggest that other systems of perception exist in plant cells in addition to the amyloplast-based model of graviperception.     

Characterization of hydrotropism: the timing of perception and signal movement from the root cap in the agravitropic pea mutant ageotropum

In this study, ageotropum pea mutant was used to determine the threshold time for perception of an osmotic stimulation in the root cap and the time requirement for transduction and transmission of the hydrotropic signal from the root cap to the elongation region. The threshold time for the perception of an osmotic stimulation was compared to current estimates of threshold times for graviperception in roots. The time required for transduction and transmission in the hydrotropic response of ageotropum was compared to the time requirement in the gravity response of Alaska pea roots. We determined that threshold time for perception of an osmotic stimulation in the root cap is very rapid, occurring in less than 2 min following the application of sorbitol to the root cap. Furthermore, a single 5 min exposure of sorbitol to the root cap fully induced a hydrotropic response. We also found that transduction and transmission of an osmotic stimulus requires 90-120 min for movement from the root cap to more basal tissues involved in differential growth leading to root curvature. The very rapid threshold time for perception of root hydrotropism is similar to those times reported for root gravitropism. However, the time required for the transduction and transmission of an osmotic stimulation from the root cap is significantly longer than the time required in gravitropism. These results suggest that there must exist some differences between root hydrotropism and gravitropism in either the rate or mechanisms of transduction and transmission of the tropistic signal from the root cap.     

Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.     

Preformation of root primordia in shoots and root morphogenesis in Salix viminalis

Root primordia are formed in the stems of Salix viminalis L. during normal growth. Some of these primordia are produced at definite sites in the nodes. The initiation and early structural and ultrastructural development of the nodal primordia were studied in young shoots. In the fourth node below the terminal leaf cluster some parenchyma cells situated at the lateral leaf gaps formed a small group of initial cells. Derivatives of the newly formed interfascicular cambium added cells to that group, in which later on cell divisions in various directions occurred resulting in the formation of a root primordium. Root morphogenesis was studied in cuttings from one-season-old stems. The cells in the dormant primordia contained many lipid bodies but only a small amount of starch. After the cuttings had been 24 hours in water starch was accumulating in the plastids and lipid bodies were seen in the vacuoles. 48 hours after activation cell divisions occurred throughout the primordia and a layered apical mer-istem was organized. After 96 hours a root cap with amyloplasts was formed and the procambium was well developed. The amyloplasts were sedimented in response to gravity. After six days the first roots were ready to emerge from the stems. Their root caps had a well developed columella and endodermal and pericyclic cells were recognizable.     

Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis

The plant root cap mediates the direction of root tip growth and protects internal cells. Root cap cells are continuously produced from distal stem cells, and the phytohormone auxin provides position information for root distal organization. Here, we identify the Arabidopsis thaliana auxin response factors ARF10 and ARF16, targeted by microRNA160 (miR160), as the controller of root cap cell formation. The Pro(35S):MIR160 plants, in which the expression of ARF10 and ARF16 is repressed, and the arf10-2 arf16-2 double mutants display the same root tip defect, with uncontrolled cell division and blocked cell differentiation in the root distal region and show a tumor-like root apex and loss of gravity-sensing. ARF10 and ARF16 play a role in restricting stem cell niche and promoting columella cell differentiation; although functionally redundant, the two ARFs are indispensable for root cap development, and the auxin signal cannot bypass them to initiate columella cell production. In root, auxin and miR160 regulate the expression of ARF10 and ARF16 genes independently, generating a pattern consistent with root cap development. We further demonstrate that miR160-uncoupled production of ARF16 exerts pleiotropic effects on plant phenotypes, and miR160 plays an essential role in regulating Arabidopsis development and growth.