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The root apical meristem of Arabidopsis is established post-embryonically as the main source of root cells, and its activity is maintained by complex bidirectional signaling between stem cells and mature cells. The receptor-like kinases GASSHO1 (GSO1) and GSO2 have been shown to regulate aerial epidermal function and seedling growth in Arabidopsis.Here we show that gso1; gso2 seedlings also have root growth and patterning defects. Analyses of mutant root morphology indicate abnormal numbers of cells in longitudinal files and radial cell layers, as well as aberrant stem cell division planes. gso1; gso2 double mutants misexpress markers for stem cells and differentiated root cell types. In addition, gso1; gso2 root growth defects, but not marker missexpression or patterning phenotypes, are rescued by growth on media containing metabolizable sugars.We conclude that GSO1 and GSO2 function together in intercellular signaling to positively regulate cell proliferation, differentiation of root cell types, and stem cell identity. In addition, GSO1 and GSO2 control seedling root growth by modulating sucrose response after germination.

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.

The extracellular proton activity along primary roots of Phleum pratense L. was measured using proton-selective microelectrodes. Removal of the root cap caused a reduction of the proton influx in the transitional region between the meristem and the apical elongation zone of the vertical root and inhibited the development of pH differences between the physically upper and lower flanks of the gravistimulated root. Disruption of the actin filament system of the root with 5 mmol m-3 cytochalasin D did not result in an altered proton flux and pH pattern compared with untreated vertical control roots, but inhibited the gravity-induced development of pH differences between the physically upper and lower root flanks as well as gravitropic curvature. These results provide evidence that pH changes following gravistimulation are induced by a signal transmitted from the root cap and that the actin filament system is involved in the gravity perception/transduction mechanism.

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied 3H-IAA and 3H-ABA during maize root gravitropism and examined the relative basipetal movement of 3H-IAA and 3H-ABA applied to the caps of vertical roots. Lateral movement of 3H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of 3H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of 3H-IAA was greater than that of 3H-ABA. Basipetal movement of label from 3H-IAA or 3H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2-4 mm from the tip with substantial levels found 7-8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of 3H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from 3H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.

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.

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

Root growth and cell differentiation were analysed in lentil seedlings grown (1) in microgravity (F microg), (2) on the 1 x g centrifuge (F1 x g), (3) in microgravity and placed on the 1 x g centrifuge for 4 h [F(microg + 1 x g)], (4) on the 1 x g centrifuge and placed in microgravity for 4 h [F(1 x g + microg)]. In microgravity, there were strong oscillations of the root tip, even when the seedlings were grown first on the 1 x g centrifuge [F(1 x g + microg)]. In the [F(microg + 1 x g)] sample, the roots grown in microgravity were oblique with respect to the 1 x g acceleration when the seedlings were placed on the centrifuge. They were therefore gravistimulated. However, root length was similar in the 4 samples after 29 h of growth and growth rate of the root was the same between 25 h and 29 h although it appeared to be slightly greater in the [F(microg + 1 x g)] sample. Cell elongation was analysed as a function of the distance from the root cap junction. Cell length was similar in the seedlings grown in microgravity or on the 1 x g centrifuge. The transfer from the 1 x g centrifuge to microgravity [F(1 x g + microg)] did not modify cell elongation in the roots. Cell length in the roots which were grown in microgravity and gravistimulated [F(microg + 1 x g)] was different from that observed in microgravity but this was only due to gravistimulation. Thus, gravity does not have an effect on cell elongation when the roots are strictly oriented in the vertical position but it does as soon as the root tip deviates from this orientation.

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.

The response of plant roots to gravity has fascinated many botanists since the early days of plant physiology and much research has been devoted to the elucidation of the sequence of events between the physical reception of gravity and the visible growth response. In the last few decades the ideas on the graviresponse of roots have changed profoundly and much progress has been made in understanding parts of the process. One of the reasons for writing this review was my curiosity to know what has happened since the time I myself was involved in the study of root geotropism, as it was called, about 30 years ago. Some excellent reviews have appeared since then, e.g. Audus (1975), Jackson & Barlow (1981) and Moore & Evans (1986), which were more restricted in scope and, moreover, there have been several fascinating developments. The aim of this review is to discuss briefly all aspects of the graviresponse of roots and the progress made in understanding during the last three decades. Some data on other plant organs are included where appropriate.

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.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

In angiosperms, growth of the root apex is determined by the quiescent centre. All tissues of the root proper and the root cap are derived from initial cells that surround this zone. The diversity of cell lineages originated from these initials suggests an interesting variation of the displacement velocity within the root apex. However, little is known about this variation, especially in the most apical region including the root cap. This paper shows a method of determination of velocity field for this region taking the Arabidopsis root apex as example. Assuming the symplastic growth without a rotation around the root axis, the method combines mathematical modelling and two types of empirical data: the published velocity profile along the root axis above the quiescent centre, and dimensions of cell packet originated from the initials of epidermis and lateral root cap. The velocities, calculated for points of the axial section, vary in length and direction. Their length increases with distance from the quiescent centre, in the root cap at least twice slower than in the root proper, if points at similar distance from the quiescent centre are compared. The vector orientation depends on the position of a calculation point, the widest range of angular changes, reaching almost 90°, in the lateral root cap. It is demonstrated how the velocity field is related to both distribution of growth rates and growth-resulted deformation of the cell wall system. Also changes in the field due to cell pattern asymmetry and differences in slope of the velocity profile are modelled. Electronic supplementary material The online version of this article (doi:10.1007/s00425-012-1707-x) contains supplementary material, which is available to authorized users.

A collaborative study involving laboratories in six countries was initiated under the sponsorship of the International Programme on Chemical Safety (IPCS) to determine the sensitivity, efficiency and reliability of the Vicia faba root tip meristem chromosomal aberration assay using a standardized protocol. The six Laboratories that participated in this study were located in the Slovak Republic, India, Japan, Poland, Sweden and the USA. All laboratories adhered to a standardized protocol for the Vicia faba chromosomal aberartion assay. Four coded chemicals, azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH) were tested with the Vicia faba chromosomal aberration assay. Of the four chemicals, three (MH, AG and MNU) were found to be clastogenic and gave a concentration related response. However, the results of NaN3 were equivocal which might be explained by the stability of NaN3. The conclusions from this study suggest that the Vicia faba chromosomal aberration bioassay is an efficient and reliable short-term bioassay for the rapid screening of chemicals for clastogenicity.

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 intensility 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 strcuture. 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.

The tips of roots and shoots commonly show lateral movements as they grow forwards. These occur as both circumnutations (with long periods and large amplitudes) and micronutations (with short periods and small amplitudes). Their properties are reviewed, with emphasis on roots, and possible ways in which they could be regulated are discussed. The mechanisms could include long-range controls (for circumnutations) that depend on transmissible signals using steps common to gravitropism, and short-range controls (for micronutations) that operate within the elongation zone. The former are a property of the apex as a whole, while the latter may be confined to localized groups of cells. Simulation of nutations is presented with a view to isolating key physiological processes. However, this approach is limited by the current inadequate understanding of the growth mechanisms involved.

In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

The root system is a crucial determinant of plant growth potential because of its important functions, e.g. uptake of water and nutrients, structural support and interaction with symbiotic organisms. Elucidating the molecular mechanism of root development and functions is therefore necessary for improving plant productivity, particularly for crop plants, including rice (Oryza sativa). As an initial step towards developing a comprehensive understanding of the root system, we performed a large-scale transcriptome analysis of the rice root via a combined laser microdissection and microarray approach. The crown root was divided into eight developmental stages along the longitudinal axis and three radial tissue types at two different developmental stages, namely: epidermis, exodermis and sclerenchyma; cortex; and endodermis, pericycle and stele. We analyzed a total of 38 microarray data and identified 22,297 genes corresponding to 17,010 loci that showed sufficient signal intensity as well as developmental- and tissue type-specific transcriptome signatures. Moreover, we clarified gene networks associated with root cap function and lateral root formation, and further revealed antagonistic and synergistic interactions of phytohormones such as auxin, cytokinin, brassinosteroids and ethylene, based on the expression pattern of genes related to phytohormone biosynthesis and signaling. Expression profiling of transporter genes defined not only major sites for uptake and transport of water and nutrients, but also distinct signatures of the radial transport system from the rhizosphere to the xylem vessel for each nutrient. All data can be accessed from our gene expression profile database, RiceXPro (http://ricexpro.dna.affrc.go.jp), thereby providing useful information for understanding the molecular mechanisms involved in root system development of crop plants.

Results of the electron-microscopic investigation of root apices of Arabidopsis thaliana 3-, 5- and 7-days-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown the similarity in the root apex cell ultrastructure in control and under clinorotation. At the same time there were some differences in the ultrastructure of statocytes and the distal elongation zone under clinorotation. For the first time the sensitivity of ER-bodies, which are derivatives of GER and contain beta-glucosidase, to the influence of simulated microgravity was demonstrated by increased quantity and area of ER-bodies at the cell section as well as by higher variability of their form under clinorotation. A degree of these changes correlated with the duration of clinorotation. On the basis of experimental data a protective role of ER-bodies in adaptation of plants to microgravity is supposed.

We used a video digitizer system to (a) measure changes in the pattern of longitudinal surface extension in primary roots of maize (Zea mays L.) upon application and withdrawal of auxin and (b) compare these patterns during gravitropism in control roots and roots pretreated with auxin. Special attention was paid to the distal elongation zone (DEZ), arbitrarily defined as the region between the meristem and the point within the elongation zone at which the rate of elongation reaches 0.3 of the peak rate. For roots in aqueous solution, the basal limit of the DEZ is about 2.5 mm behind the tip of the root cap. Auxin suppressed elongation throughout the elongation zone, but, after 1 to 3 h, elongation resumed, primarily as a result of induction of rapid elongation in the DEZ. Withdrawal of auxin during the period of strong inhibition resulted in exceptionally rapid elongation attributable to the initiation of rapid elongation in the DEZ plus recovery in the main elongation zone. Gravistimulation of auxin-inhibited roots induced rapid elongation in the DEZ along the top of the root. This resulted in rapid gravitropism even though the elongation rate of the root was zero before gravistimulation. The results indicate that cells of the DEZ differ from cells in the bulk of the elongation zone with respect to auxin sensitivity and that DEZ cells play an important role in gravitropism.

Roots of Pisum sativum L. and Zea mays L. were exposed to different moisture gradients established by placing both wet cheesecloth (hydrostimulant) and saturated aqueous solutions of various salts in a closed chamber. Atmospheric conditions with different relative humidity (RH) in a range between 98 and 86% RH were obtained at root level, 2 to 3mm from the water-saturated hydrostimulant. Roots of Silver Queen corn placed vertically with the tips down curved sideways toward the hydrostimulant in response to approximately 94% RH but did not respond positively to RH higher than approximately 95%. The positive hydrotropic response increased linearly as RH was lowered from 95 to 90%. A maximum response was observed at RH between 90 and 86%. However, RH required for the induction of hydrotropism as well as the responsiveness differed among plant species used; gravitropically sensitive roots appeared to require a somewhat greater moisture gradient for the induction of hydrotropism. Decapped roots of corn failed to curve hydrotropically, suggesting the root cap as a major site of hydrosensing.

The sedimentation of starch-filled plastids is thought to be the primary mechanism by which gravity is perceived in roots. Following gravity perception, auxin redistribution toward the lower flank of roots, initiated in the root cap, is believed to play a role in regulation of the gravity response. Amyloplast sedimentation and auxin flux, however, have never been directly linked. The overall aim of this study was to investigate the relationship among plastid sedimentation, gravitropism and auxin flux. Our data show that pgm-1 roots respond to gravity at one-third the rate of wild-type (WT) roots. Maintaining the root tip at a constant angle using image analysis coupled to a rotating stage resulted in a constant rate of response regardless of the angle of tip orientation in pgm-1 mutants, in contrast to the responses of WT and pin3-1 mutants, which showed increasing response rates as the tip was constrained at greater angles. To indirectly visualize auxin flux following reorientation, we generated a pgm-1 mutant line expressing the DR5::GFPm reporter gene. In WT roots a GFP gradient was observed with a maximum along the lower flank, whereas pgm-1 roots formed a GFP maximum in the central columella but lacked any observable gradient up to 6 h following reorientation. Our study suggests that the relationship between root cap angle and gravitropic response depends upon plastid sedimentation-based gravity sensing and supports the idea that there are multiple, overlapping sensory response networks involved in gravitropism.

International audience; Roots of many plants are known to produce large numbers of ‘border’ cells that play a central role in root protection and the interaction of the root with the rhizosphere. Unlike border cells, border-like cells were described only recently in the model plant Arabidopsis thaliana and other Brassicaceae species and very little is known about the functional properties of border-like cells as compared with ‘classical’ border cells. To stimulate discussion and future research on this topic, the function of border cells and the way border-like cells are organized, maintained, and possibly involved in plant protection is discussed here.

Maize (Zea mays L.) root cap cells secrete a large variety of compounds including proteins via an amorphous gel structure called mucilage into the rhizosphere. In the present study, mucilage secreted by primary roots of 3-4 day old maize seedlings was collected under axenic conditions, and the constitutively secreted proteome was analyzed. A total of 2848 distinct extracellular proteins were identified by nanoLC-MS/MS. Among those, metabolic proteins (approximately 25%) represented the largest class of annotated proteins. Comprehensive sets of proteins involved in cell wall metabolism, scavenging of reactive oxygen species, stress response, or nutrient acquisition provided detailed insights in functions required at the root-soil interface. For 85-94% of the mucilage proteins previously identified in the relatively small data sets of the dicot species pea, Arabidopsis, and rapeseed, a close homologue was identified in the mucilage proteome of the monocot model plant maize, suggesting a considerable degree of conservation between mono and dicot mucilage proteomes. Homologues of a core set of 12 maize proteins including three superoxide dismutases and four chitinases, which provide protection from fungal infections, were present in all three mucilage proteomes investigated thus far in the dicot species Arabidopsis, rapeseed, and pea and might therefore be of particular importance.

SummaryThe formation of different tissue systems in multicellular organisms depends on the activity of groups of undifferentiated cells called stem cells [1]. In vascular plants, the three principal tissue systems—dermal, ground, and vascular—are derived from specific groups of stem cells that are laid down during embryogenesis [2, 3]. We show here that SCHIZORIZA (SCZ) is necessary for the early establishment of the stem cells that produce the ground tissue in the embryonic root meristem. Our results show that SCZ expression in stem cells is sufficient to maintain these cells in an undifferentiated stem cell state. Furthermore, we show that embryos that lack SCZ and SCARECROW (SCR) functions do not form a ground tissue because they do not develop ground tissue stem cells. We demonstrate that the formation of a complex tissue system requires the interaction between specific stem cell fate regulatory genes and patterning genes. Our study reveals a new function for a member of the heat-shock transcription factor family in stem cell development [4] and provides a molecular framework to understand stem cell formation during embryogenesis.

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

Data are presented of a comparative analysis on rhizogenesis in the Arabidopsis thaliana tissue culture growing in a solid nutrient medium under stationary conditions, clinastatic conditions and microgravity. Tissue samples weighing 100 mg. were set in the Petri dishes and placed in a horizontal slow clinostate /2 revs/min/. After 14 days of growth they were analyzed. On clinostating the number of roots formed from the callus cells was approximately one half the control. The formed root cap manifested no essential differences, in comparison with the stationary control, in the number of layers and cell sizes in its layers. In callusogenic roots, formed from clinostated cells, differentiation including root cap cells, proceeds without noticeable deviations from the norm. At the same time, gravireceptor cells do not function under these conditions. This is clearly displayed at a structural level in the location of amyloplasts-statoliths throughout the cytoplasm. The callus cell cultures experienced microgravity for 8 days. The number of formed roots under the influence of this factor was 36% relative to the stationary control. Root cap formation was abnormal. Gravireceptor cells did not formed under microgravity.

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dzeta2 gene (PLDzeta2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldzeta2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldzeta2 mutant roots. In root hydrotropism experiments, pldzeta2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDzeta2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDzeta2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.

Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud. were evaluated by light and electron microscopy to determine the distribution of organelle sedimentation towards gravity. Roots of Limnobium are strongly gravitropic. The rootcap consists of only two layers of cells. Although small amyloplasts are present in the central cap cells, no sedimentation of any organelle, including amyloplasts, was found. In contrast, both amyloplasts and nuclei sediment consistently and completely in cells of the elongation zone. Sedimentation occurs in one cell layer of the cortex just outside the endodermis. Sedimentation of both amyloplasts and nuclei begins in cells that are in their initial stages of elongation and persists at least to the level of the root where root hairs emerge. This is the first modern report of the presence of sedimentation away from, but not in, the rootcap. It shows that sedimentation in the rootcap is not necessary for gravitropic sensing in at least one angiosperm. If amyloplast sedimentation is responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the elongation zone and not the rootcap. These data do not necessarily conflict with the hypothesis that sensing occurs in the cap in other roots, since Limnobium roots are exceptional in rootcap origin and structure, as well as in the distribution of organelle sedimentation. Similarly, if nuclear sedimentation is involved in gravitropic sensing, then nuclear mass would function in addition to, not instead of, that of amyloplasts.

Low-oxygen stress imposed by field waterlogging is a serious impediment to plant germination and growth. Plants respond to waterlogging with a complex set of physiological responses regulated at the transcriptional, cellular, and tissue levels. The Arabidopsis(Arabidopsis thaliana) NAC domain-containing gene ANAC102was showntobeinduced under 0.1%oxygen within 30 min in both roots and shoots as well as in 0.1% oxygen-treated germinating seeds. Overexpression of ANAC102 altered the expression of a number of genes, including many previously identified as being low-oxygen responsive. Decreasing ANAC102 expression had no effect on global gene transcription in plants but did alter expression patterns in low-oxygen-stressed seeds. Increasing ordecreasing the expression of ANAC102 did not affect adult plant survival oflow-oxygen stress. Decreased ANAC102 expression significantly decreased germination efficiency following a 0.1% oxygen treatment, but increased expression had no effecton germination. This protective roleduring germination appearedto bespecificto low-oxygen stress, implicating ANAC102 as an important regulator of seed germination under flooding. © 2009 American Society of Plant Biologists.

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.

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.

The ultrastructure and symplastic transport function of Ectodesmata-like (ED-like) of the root cap cells of Zea mays, during the detaching stage, were reported by using fluorescence and electron microscopy. It was described the process that plasmodesmata (PD) were gradually stretched and changed into ED-like. It was discovered that the diameter of appressed endoplasmic reticulum (AER) in PD became thinner while the ED-like still remained some structures of PD. By using fluorescence probe incubating, 457Da Lucifer Yellow (LYCH) which was impermeable to the membrane, could enter the root cap cells through ED-like. The results proved that ED-like still retained physiological activity and kept the symplastic transport function during a period time. When the root tissues were pre-treated by cytochalasin D (CD), Phalloidin and 2,3-butanedione, 2-monoxime (BDM) and then combined with fluorescence probe detecting, the results showed that F-actin and myosin might take part in the regulation of the substance translocation of the ED-like.

Summary The C18 ketone (5E,7E)-6-methyl-8-(2,6,6-trimethylcyclohex-1-enyl)octa-5,7-dien-2-one (D’orenone) has been postulated to be an early cleavage product of b-carotene en route to trisporic acids; these act as morphogenetic factors during the sexual reproduction of zygomycetes. Here we report that D’orenone blocks the highly polarized tip growth of root hairs, causing tip growth to stop completely within a few minutes. Importantly, external auxin reverses the effects of D’orenone on root hairs. Further analysis revealed that D’orenone lowers the auxin concentration in trichoblasts via PIN2-mediated auxin efflux to below the critical levels essential for root hair growth. D’orenone specifically increases PIN2 protein abundance without affecting PIN2 transcripts, and the PIN2 expression domain enlarges and shifts basipetally, resulting in more active auxin transport. The observation that D’orenone does not interfere with the root hair growth in roots of null mutant lines provides additional evidence that PIN2 is its specific target.

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

We examined the kinetics of electrotropic curvature in solutions of low electrolyte concentration using primary roots of maize (Zea mays L., variety Merit). When submerged in oxygenated solution across which an electric field was applied, the roots curved rapidly and strongly toward the positive electrode (anode). The strength of the electrotropic response increased and the latent period decreased with increasing field strength. At a field strength of 7.5 volts per centimeter the latent period was 6.6 minutes and curvature reached 60 degrees in about 1 hour. For electric fields greater than 10 volts per centimeter the latent period was less than 1 minute. There was no response to electric fields less than 2.8 volts per centimeter. Both electrotropism and growth were inhibited when indoleacetic acid (10 micromolar) was included in the medium. The auxin transport inhibitor pyrenoylbenzoic acid strongly inhibited electrotropism without inhibiting growth. Electrotropism was enhanced by treatments that interfere with gravitropism, e.g. decapping the roots or pretreating them with ethyleneglycol-bis-[beta-ethylether]-N,N,N',N' -tetraacetic acid. Similarly, roots of agravitropic pea (Pisum sativum, variety Ageotropum) seedlings were more responsive to electrotropic stimulation than roots of normal (variety Alaska) seedlings. The data indicate that the early steps of gravitropism and electrotropism occur by independent mechanisms. However, the motor mechanisms of the two responses may have features in common since auxin and auxin transport inhibitors reduced both gravitropism and electrotropism.

Root caps of primary, secondary, and seminal roots of Z. mays cv. Kys secrete large amounts of mucilage and are in close contact with the root all along the root apex. These roots are strongly graviresponsive. Secondary and seminal roots of Z. mays cv. Ageotropic are also strongly graviresponsive. Similarly, their caps secrete mucilage and closely appress the root all along the root apex. However, primary roots of Z. mays cv. Ageotropic are non-responsive to gravity. Their caps secrete negligible amounts of mucilage and contact the root only at the extreme apex of the root along the calyptrogen. These roots become graviresponsive when their tips are coated with mucilage or mucilage-like materials. Peripheral cells of root caps of roots of Z. mays cv. Kys contain many dictyosomes associated with vesicles that migrate to and fuse with the plasmalemma. Root-cap cells of secondary and seminal (i.e. graviresponsive) roots of Z. mays cv. Ageotropic are similar to those of primary roots of Z. mays cv. Kys. However, root-cap cells of primary (i.e. non-graviresponsive) roots of Z. mays cv. Ageotropic have distended dictyosomal cisternae filled with an electron-dense, granular material. Large vesicles full of this material populate the cells and apparently do not fuse with the plasmalemma. Taken together, these results suggest that non-graviresponsiveness of primary roots of Z. mays cv. Ageotropic results from the lack of apoplastic continuity between the root and the periphery of the root cap. This is a result of negligible secretion of mucilage by cells along the edge of the root cap which, in turn, appears to be due to the malfunctioning of dictyosomes in these cells.

We tested whether the first response to gravistimulation is an asymmetry in the root tip that results from differential growth of the rootcap itself. The displacement of markers on the rootcap surface of maize (Zea mays L. cv. Merit) roots was quantified from videotaped images using customized software. The method was sensitive enough to detect marker displacements down to 15 microns and root curvature as early as 8 min after gravistimulation. No differential growth of the upper and lower sides of the cap occurred before or during root curvature. Fewer than a third of all gravistimulated roots developed an asymmetrical outline of the root tip after curvature had started, and this asymmetry did not occur in the rootcap itself. Our data support the view that the regions of gravitropic sensing and curvature are spatially separate during all phases of gravitropism in maize roots.

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.

Primary roots of the mutant 'Ageotropic' cultivar of Zea mays are nonresponsive to gravity. Their root caps secrete little or no mucilage and touch the root only at the extreme apex. A gap separates the cap and root at the periphery of the cap. Applying mucilage from normal roots or substances with a consistency similar to that of mucilage to tips of mutant roots causes these roots to become strongly graviresponsive. Gravicurvature stops when these substances are removed. Caps of some mutants secrete small amounts of mucilage and are graviresponsive. These results indicate that (a) the lack of graviresponsiveness in the mutant results from disrupting the transport pathway between the cap and root, (b) movement of the growth-modifying signal from the cap to the root occurs via an apoplastic pathway, and (c) mucilage is necessary for normal communication between the root cap and root in Zea mays cv Ageotropic.

The location of the nucleus in statocytes or lentil roots grown: 1), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots crown on the clinostat there was a distance of 0.47 micrometers (horizontal clinorotation) and or 0.76 micrometers (vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 micrometers from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.

7 pages, 5 figures.-- PMID: 18023916 [PubMed].-- Available online 19 November 2007., Recently, curvature was described as a new trait useful in the analysis of root apex shape. Treating the root profile as a geometric curve revealed that root apex curvature values are lower in ethylene-insensitive mutants (Cervantes E, Tocino A. Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development. J Plant Physiol 2005;162:1038–45). This fact suggests that curvature is regulated by ethylene. In this work, we have determined the curvature values in embryonic roots of wild-type Columbia as well as in ethylene signal-transduction mutants, and found smaller values in embryos of the mutants. We also report on the evolution of root curvature during early development after seed germination. The line Lt16b that expresses GFP in the cell wall has allowed us to investigate the evolution of curvature values in three successive cell layers of seedling roots by confocal microscopy. Treatment of seedlings with norbornadiene resulted in lower curvature values.

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root region (0-5 mm) and decreased in the root regions susceptible to rhizobia (5-20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.

The effects of microgravity and ethylene on morphology and ultrastructural organization of mitochondria in root statocytes of soybean seedlings grown for 6 days on the board of the space shuttle Columbia during the STS-87 mission were investigated. The spaceflight seedlings and the ground-grown control seedlings were grown in BRIG (Biological Research in Canister) in the presence of KMnO4 to remove ethylene. It was revealed that irrespectively of KMnO4 treatment the mitochondria in the spaceflight seedlings were characterized by round or oviform and by low electron density of organelle matrix, whereas the organelles in the ground controls were polymorphic in shape and had higher electron density of matrix. The possible mechanisms of morphological and ultrastructural rearrangements of mitochondria that may be involved in adaptation processes of soybean seedlings to microgravity conditions are discussed.

Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% ± 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection.

Arsenic exposure is associated with various diseases and cancers. By using Allium-root micronucleus (MN) assay, possible genotoxicity of sodium arsenite (0.3-100 mg/l) and arsenic trioxide (0.05-50 mg/l) was evaluated in this study. Our results showed that arsenic compounds induced MN formation concentration-dependently. Exposure to 0.5-20 mg/l arsenic trioxide or to 1-100 mg/l sodium arsenite caused MN significantly in meristematic cells and daughter cells of Allium roots. A time-course study revealed that MN increased significantly after a short term (1 h) exposure to 10 mg/l sodium arsenite, demonstrating an effective rapid response. Arsenic compounds also caused mitotic delay and a concentration-dependent decrease in mitotic index. Results of the present study suggest that Allium-root MN assay is a simple, efficient and reproducible method for the genotoxicity monitoring of arsenic water contamination.

Based on the growth tensor method an unsteady field of growth rates for developing root apex is presented. Maps of growth rates distribution as well as simulations in which the field was applied to initially uniform grid of points are presented. In the simulations, the grid undergoes deformation that resembles new root formation in its axial plane. Four variants of field operation on the grid are shown.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.

The survival of terrestrial plants depends upon the capacity of roots to obtain water and nutrients from the soil. Directed growth of roots in relation to a gradient in moisture is called hydrotropism and begins in the root cap with the sensing of the moisture gradient. Even though the lack of sufficient water is the single-most important factor affecting world agriculture, there are surprisingly few studies on hydrotropism. Recent genetic analysis of hydrotropism in Arabidopsis has provided new insights about the mechanisms that the root cap uses to perceive and respond simultaneously to moisture and gravity signals. This knowledge might enable us to understand how the root cap processes environmental signals that are capable of regulating whole plant growth.

To elucidate the rapid and plagiotropic growth of hairy root induced by A. rhizogenes, a root apex was investigated with respect to it's amyloplast deposition, activity of alpha-amylase and glucose content. The amyloplasts distributed in the hairy roots were fewer than those of the adventitious root. Since auxin availability is enhanced in hairy roots, it could affect the statolith degradation by elevating alpha-amylase activity so that the energy requirement for rapid growth could be fulfilled as represented of glucose content. Consequently, it is suggested the overall decrease of starch grains could result in the lack of gravi-response in hairy roots.