Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

[The growth and differentiation of root cap columella cells and the proper root grown in the stationary conditions and under clinorotation].

The results of light- and electron-microscopic investigations of root apices of Beta vulgaris 3-day-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown that ultrastructure and topography of organelles in root cap statocytes (graviperceptive cells) and in the cells of distal elongation zone clearly reflected the different direction in their growth and differentiation in space and time in dependence on specialization and functions. Cell growth and genetically determined differentiation occur similarly to control, although certain differences in ultrastructure are evident on metabolism changes.

Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi.

Acidification of intracellular compartments by the vacuolar-type H(+)-ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the V(1)V(o)-ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane V(o) sector was investigated. Expression of VHA-c1, monitored by promoter-driven beta-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wild-type seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the V(o) sector and V(1)V(o)-ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.

Gamma-irradiated onions as a biological indicator of radiation dose.

Post-irradiation identification and dose estimation are required to assess the radiation-induced effects on living things in any nuclear emergency. In this study, radiation-induced morphological/cytological changes i.e., number of root formation and its length, shooting length, reduction in mitotic index, micronuclei formation and chromosomal aberrations in the root tip cells of gamma-irradiated onions at lower doses (50-2000 cGy) are reported. The capabilities of this biological species to store the radiation-induced information are also studied.

Orientation of root hair growth is influenced by simulated microgravity.

We have tried to investigate the mechanisms supporting the plagiotropic growth (growth in parallel to the Earth) of root hairs in simulated microgravity. Our strategy to understand the regulation of such type of growth depends upon the study of cytoskeleton topography and calcium ions distribution in root hairs both in control and simulated microgravity.

Effect of microgravity on the cell cycle in the lentil root.

Characteristics of the cell cycle in cortical regions (0-0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the l-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in l-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could he due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.

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.

Modulation of statolith mass and grouping in white clover (Trifolium repens) growth in 1-g, microgravity and on the clinostat.

Current models of gravity perception in higher plants focus on the buoyant weight of starch-filled amyloplasts as the initial gravity signal susceptor (statolith). However, no tests have yet determined if statolith mass is regulated to increase or decrease gravity stimulus to the plant. To this end, the root caps of white clover (Trifolium repens) grown in three gravity environments with three different levels of gravity stimulation have been examined: (i) 1-g control with normal static gravistimulation, (ii) on a slow clinostat with constant gravistimulation, and (iii) in the stimulus-free microgravity aboard the Space Shuttle. Seedlings were germinated and grown in the BioServe Fluid Processing Apparatus and root cap structure was examined at both light and electron microscopic levels, including three-dimensional cell reconstruction from serial sections. Quantitative analysis of the electron micrographs demonstrated that the starch content of amyloplasts varied with seedling age but not gravity condition. It was also discovered that, unlike in starch storage amyloplasts, all of the starch granules of statolith amyloplasts were encompassed by a fine filamentous, ribosome-excluding matrix. From light micrographic 3-D cell reconstructions, the absolute volume, number, and positional relationships between amyloplasts showed (i) that individual amyloplast volume increased in microgravity but remained constant in seedlings grown for up to three days on the clinostat, (ii) the number of amyloplasts per cell remained unchanged in microgravity but decreased on the clinostat, and (iii) the three-dimensional positions of amyloplasts were not random. Instead amyloplasts in microgravity were grouped near the cell centers while those from the clinostat appeared more dispersed. Taken together, these observations suggest that changing gravity stimulation can elicit feedback control over statolith mass by changing the size, number, and grouping of amyloplasts. These results support the starch-statolith theory of graviperception in higher plants and add to current models with a new feedback control loop as a mechanism for modulation of statolith responsiveness to inertial acceleration.

Statocyte polarity and gravisensitivity in seedling roots grown in microgravity.

Space experiments have offered a unique opportunity to analyse the mechanism of gravisensing in plant roots. It has been shown that the strict structural polarity of statocytes observed on the ground is perturbed in microgravity: the amyloplasts move towards the proximal half of the cell and, at least in some cases, the nucleus becomes located further away from the (proximal) plasma membrane. It has thus been demonstrated that the amyloplasts do not move freely in the cytoplasm. Experiments using cytochalasin B (or D) have indicated that these organelles are attached to the actin network, probably by motor proteins. These findings have led to a new hypothesis on gravisensing the basis of which is that the tension in the actin filaments resulting from interaction with the statoliths would be transmitted to stretch-activated ion channels located in the plasma membrane (Sievers et al., 1991, In: Lloyd (ed) The cytoskeletal basis of plant growth and form, Academic Press, London New York, pp 169-182). Recently, it has been shown that the sensitivity of roots grown under 1 g conditions in orbit is less than that of roots grown in microgravity or under simulated weightlessness on clinostats. Since the location of the amyloplasts in microgravity is different from that in 1 g, the greater sensitivity observed could be due to different tensions in the actin network.

Gravitropic reaction in the growth of tea roots.

In Japan, tea (Camellia sinenis (L.) Kuntze) seedlings are propagated by cutting. A root system of clonal plants by cutting consists of adventitious roots and lateral roots. Most of the roots grow horizontally, which results in a shallow distribution of the root system. Such a shallow root system could be one of the factors contributing to the deterioration of nutrient uptake and resistance to water stress. Gravitropism of the roots is considered to be a decisive factor that controls the depth of a root system. The authors have investigated changes in the growth direction of roots to gravitative stimulus, using several kinds of roots (seminal roots, lateral roots and adventitious roots). Furthermore, amyloplasts in the root-cap cells, which are considered to be an equipment sensing gravistimulus, were observed. Seminal roots prominently showed orthogravitropism and contained many amyloplast particles in their root cap cells. Most lateral and adventitious roots showed plagiogravitropism, growing in an angle to gravistimulus, and lacked observable amyloplast particles in their root cap cells. The results suggest that the shallowing of root systems of elonal tea plants could be attributed to a gravitropic reaction of the adventitious and lateral roots composing the root system. There could also be a close relationship between the growth direction of roots and the presence of amyloplasts in root-cap cells.

Changes in IAA responsiveness in the elongation region of graviresponding mung bean roots.

IAA responsiveness of sections of root tissue taken from the top and bottom of mung bean roots was assessed prior to and at varying times following gravistimulation. Prior to gravistimulation, root tissue sections from the sides of the elongation zone responded similarly to IAA. After gravistimulation (within 5 min), root sections from the bottom of the elongation zone became more responsive to IAA than sections collected from the upper side of the elongation zone. The change in IAA responsiveness of these tissue sections was transient with root sections from both the top and bottom of the elongation zone again exhibiting similar responsiveness to IAA following 15 minutes of gravistimulation. These studies also examined if the root tip is required for the gravity-induced shift in IAA responsiveness in the tissues of the elongation zone. The IAA responsiveness of top and bottom sections of the elongation zone from decapped mung bean roots was assessed at varying times following gravistimulation. The responsiveness to IAA of top and bottom sections changed rapidly in decapped roots, just as had been previously found for intact roots. Although the alteration in responsiveness was transient in decapped roots (just as intact roots), the time it took for the sections to recover previous responsiveness to IAA was extended. The results suggest that the initial growth response of graviresponding roots may be due to a change in the IAA responsiveness of tissues in the elongation zone and not an asymmetric accumulation of IAA on the lower side of the elongation zone. The results also indicate that the gravity-induced shift in IAA responsiveness in the elongation zone occurs independently of the root cap, suggesting that the cells in the elongation region can perceive and respond to gravity independently of the root cap during the initial phases of the gravity response.

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells.

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.

Transduction of the gravity stimulus in the root statocyte.

The amyloplasts of root statocytes are considered to be the perceptors of gravity. However, their displacement and the starch they contain are not required for gravisensing. The mechanism of the transduction of gravistimulus remains therefore controversial. It is well known that the amplitude of the stimulus is dependent upon the intensity of the acceleration and the inclination of the root with respect to gravity. This strongly supports the hypothesis that the stimulus results in a mechanical effect (pressure or tension) on a cellular structure. Three cellular components are proposed as possible candidates for the role of transducer: the actin filaments, the endoplasmic reticulum and the plasma membrane with its ion channels. Recent results obtained in the frame of the IML 1 Mission of Spacelab show that the endoplasmic reticulum should rather be responsible for the termination of the stimulus. The contacts of amyloplasts with the distal ER could therefore be involved in the regulation of root growth.

Correlations between changes in electrical parameters and changes in cell elongation rates in gravistimulated roots.

The earliest changes in growth rate following the gravistimulation of roots occur in a special group of cells between the meristem and the elongating region of the root. This zone is called the postmitotic isodiametric growth (PIG) zone and consists of cells which have ceased dividing and are expanding isodiametrically. Upon gravistimulation cells along the upper side of the PIG zone begin elongating rapidly and this accounts for much of the early growth asymmetry. There is rapid (< 30 s) hyperpolarization of cells on the upper side of the PIG zone as well as rapid uptake of potassium from the stele. We propose that there is a relationship between the rate of hydrogen ion efflux and the extent of membrane hyperpolarization in the PIG zone and that such changes in potential are an early indication of impending changes in growth performance. Although the development of auxin asymmetry in the cap and its transmission to the elongating region is considered to be the controlling factor in root gravitropism, auxin asymmetry in the cap develops only after 30 min, about the same as the lag before initiation of curvature. Although this dilemma may be partly resolved by the location of the PIG zone close to the cap, alternative explanations such as gravi-detection by the PIG zone or very rapid (electrical?) signal transmission from the cap to the PIG zone need to be considered.

Clover development during spaceflight: a model system.

The development of legume root nodules was studied as a model system for the examination of gravitational effects on plant root development. In order to examine whether rhizobial association with clover roots can be achieved in microgravity, experiments were performed aboard the KC-135 parabolic aircraft and aboard the sounding rocket mission Consort 3. Binding of rhizobia to roots and the initial stages of root nodule development successfully occurred in microgravity. Seedling germination experiments were performed in the sliding block device, the Materials Dispersion Apparatus, aboard STS-37. When significant hydration of the seeds was achieved, normal rates of germination and seedling development were observed.

[Microgravity and root gravitropism]. / Microgravite et gravitropisme racinaire.

Space experiments permit to understand better some phases of the gravitropic reaction which occurs when the orientation of the root changes in the gravitational field. In gravisensing cells (statocytes in the root cap), the nucleus is attached to the cell periphery, close to the plasma membrane, by actin filaments. The location of the amyloplasts (statoliths) depends also greatly on these elements of the cytoskeleton. A short period in microgravity (5 min.) modifies the location of the nucleus and of the amyloplasts in the statocytes. The tensions exerted by these very dense organelles on the actin network disappear and this network undergoes a relaxation. The kinetics of gravitropic curvature is also better understood. In fact, gravitropic reaction is regulated by a mechanism depending on gravity. In roots grown in space, then stimulated for 1 h on a 1 g centrifuge, and replaced in microgravity, the regulation limiting the curvature does not occur. It is hypothesized that the sedimentation of the amyloplasts on the endoplasmic reticulum placed at the basal pole of the statocytes could be responsible for this regulation. The contacts between these two organelles should have also a role in root growth. This hypothesis will be tested in our next space experiment (July 94). The experiments in near weightlessness also permit to determine the presentation time which is the duration of stimulation necessary to evoke a slight but significant curvature. Presentation time is 27 s. This short period allows a slight movement of the amyloplasts only (around 0.45 micrometer). The sequence of events leading to the curvature of the root is now well established: the first signal is the separation of the endoplasmic reticulum and the amyloplasts, when the root is subjected to a change in orientation. It is followed by the pressure of these organelles on the actin network which transmits this mechanical effect to the plasma membrane. The transduction of the effect occurs then by the activation of the ions channels (Ca++) and the carrier of a growth inhibitor (auxin), both located in the plasma membrane. This growth inhibitor provokes an asymmetrical growth in the distal part of the meristem and the proximal part of the cell elongation zone. At last, when the root tip reaches the direction of gravity, the amyoloplasts sediment on the endoplasmic reticulum and induce a signal of termination of the curavature.

Calcium movement, graviresponsiveness and the structure of columella cells and columella tissues in roots of Allium cepa L.

Roots of Allium cepa L. cv. Yellow are differentially responsive to gravity. Long (e.g. 40 mm) roots are strongly graviresponsive, while short (c.g. 4 mm) roots are minimally responsive to gravity. Although columella cells of graviresponsive roots are larger than those of nongraviresponsive roots, they partition their volumes to cellular organelles similarly. The movement of amyloplasts and nuclei in columella cells of horizontally-oriented roots correlates positively with the onset of gravicurvature. Furthermore, there is no significant difference in the rates of organellar redistribution when graviresponsive and nongraviresponsive roots are oriented horizontally. The more pronounced graviresponsiveness of longer roots correlates positively with (1) their caps being 9-6 times more voluminous, (2) their columella tissues being 42 times more voluminous, (3) their caps having 15 times more columella cells, and (4) their columella tissues having relative volumes 4.4 times larger than those of shorter, nongraviresponsive roots. Graviresponsive roots that are oriented horizontally are characterized by a strongly polar movement of 45Ca2+ across the root tip from the upper to the lower side, while similarly oriented nongraviresponsive roots exhibit only a minimal polar transport of 45Ca2+. These results indicate that the differential graviresponsiveness of roots of A. cepa is probably not due to either (1) ultrastructural differences in their columella cells, (2) differences in the rates of organellar redistribution when roots are oriented horizontally. Rather, these results indicate the graviresponsiveness may require an extensive columella tissue, which, in turn, may be necessary for polar movement of 45Ca2+ across the root tip.

Geoperception in primary and lateral roots of Phaseolus vulgaris (Fabaceae). III. A model to explain the differential georesponsiveness of primary and lateral roots.

Half-tipped primary and lateral roots of Phaseolus vulgaris bend toward the side of the root on which the intact half tip remains. Therefore, tips of lateral and primary roots produce growth effectors capable of inducing gravicurvature. The asymmetrical placement of a tip of a lateral root onto a detipped primary root results in the root bending toward the side of the root onto which the tip was placed. That is, the lesser graviresponsiveness of lateral roots as compared with primary roots is not due to the inability of their caps to produce growth inhibitors. The more pronounced graviresponsiveness of primary roots is positively correlated with the presence of columella tissues that are 3.8 times longer, 1.7 times wider, and 10.5 times more voluminous than the columellas of lateral roots. We propose that the lack of graviresponsiveness exhibited by lateral roots is due to the fact that they (i) produce smaller amounts of the inhibitor than primary (i.e., strongly graviresponsive) roots and (ii) are unable to redistribute the inhibitor so as to be able to create a concentration gradient sufficient to induce a pronounced gravitropic response.

[Geoperception in the lentil root cap]. / La perception geotropique dans la coiffe des racines de Lentille.

Previous analysis showed that, in its initial phase, the geotropic response of Lens culinaris L. roots cannot be explained by a simple action by sliding, pressure or contact of amyloplasts on a sensitive surface located along the longitudinal wall. In this study another mode of action is tested by considering the following parameters as functions of the roots inclination: (1) the distance (d) which the amyloplasts move; (2) their number of contacts (mean c) with parietal cytoplasm; (3) the variable (sin alpha) of the transversal component of the statolith weight (mean M x g sin alpha). It is shown that the initial rate of curvature (mean V), at the various angles, is related to the sedimentation of the amyloplasts by the equation mean V = a log b mean d mean c sin alpha (where a and b are constants). The results obtained prove that the geotropic stimulation is dependent upon the sine of the angle (alpha) of the root inclination and explain the sine rule deviation. The role of statoliths is discussed in the light of recent literature on growth inhibitors which are involved in the geotropic reaction.