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The role of the distal elongation zone in the response of maize roots to auxin and gravity.

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.

Gravitation↗

Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity.

Despite the extensive study of plant gravitropism, there have been few experiments which have utilized hypergravity as a tool to investigate gravisensitivity in flowering plants. Previous studies have shown that starch-deficient mutants of Arabidopsis are less sensitive to gravity compared to the wild-type (WT). In this report, the question addressed was whether hypergravity could restore the sensitivity of starch-deficient mutants of Arabidopsis. The strains examined include a WT, a starchless mutant and a reduced-starch mutant. Vertical orientation studies with dark-grown seedlings indicate that increased centrifugal acceleration improves orientation relative to the acceleration vector for all strains, even the WT. For starchless roots, growth of seedlings under constant 5 g acceleration was required to restore orientation to the level of the WT at 1 g. In contrast, approximately 10 g was required to restore the orientation of the starchless mutant hypocotyls to a WT level at 1 g. Examination of plastid position in root cap columella cells of the starchless mutant revealed that the restoration of gravitropic sensitivity was correlated with the sedimentation of plastids toward the distal cell wall. Even in WT plants, hypergravity caused greater sedimentation of plastids and improved gravitropic capability. Collectively, these experiments support the hypothesis of a statolith-based system of gravity perception in plants. As far as is known, this is the first report to use hypergravity to study the mechanisms of gravitropism in Arabidopsis.

Arabidopsis↗

Transposon tagging and the study of root development in Arabidopsis.

The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

Arabidopsis↗

The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient.

The actin cytoskeleton has been implicated in regulating plant gravitropism. However, its precise role in this process remains uncertain. We have shown previously that disruption of the actin cytoskeleton with Latrunculin B (Lat B) strongly promoted gravitropism in maize roots. These effects were most evident on a clinostat as curvature that would exceed 90 degrees despite short periods of horizontal stimulation. To probe further the cellular mechanisms underlying these enhanced gravity responses, we extended our studies to roots of Arabidopsis. Similar to our observations in other plant species, Lat B enhanced the response of Arabidopsis roots to gravity. Lat B (100 nm) and a stimulation time of 5-10 min were sufficient to induce enhanced bending responses during clinorotation. Lat B (100 nm) disrupted the fine actin filament network in different regions of the root and altered the dynamics of amyloplasts in the columella but did not inhibit the gravity-induced alkalinization of the columella cytoplasm. However, the duration of the alkalinization response during continuous gravistimulation was extended in Lat B-treated roots. Indirect visualization of auxin redistribution using the DR5:beta-glucuronidase (DR5:GUS) auxin-responsive reporter showed that the enhanced curvature of Lat B-treated roots during clinorotation was accompanied by a persistent lateral auxin gradient. Blocking the gravity-induced alkalinization of the columella cytoplasm with caged protons reduced Lat B-induced curvature and the development of the lateral auxin gradient. Our data indicate that the actin cytoskeleton is unnecessary for the initial perception of gravity but likely acts to downregulate gravitropism by continuously resetting the gravitropic-signaling system.

Actins↗

Contribution of root cap mucilage and presence of an intact root cap in maize (Zea mays) to the reduction of soil mechanical impedance.

BACKGROUND AND AIMS: The impedance to root growth imposed by soil can be decreased by both mucilage secretion and the sloughing of border cells from the root cap. The aim of this study is to quantify the contribution of these two factors for maize root growth in compact soil. METHODS: These effects were evaluated by assessing growth after removing both mucilage (treatment I -- intact) and the root cap (treatment D -- decapped) from the root tip, and then by adding back 2 micro L of mucilage to both intact (treatment IM -- intact plus mucilage) and decapped (treatment DM -- decapped plus mucilage) roots. Roots were grown in either loose (0.9 Mg m(-3)) or compact (1.5 Mg m(-3)) loamy sand soils. Also examined were the effects of decapping on root penetration resistance at three soil bulk densities (1.3, 1.4 and 1.5 Mg m(-3)). KEY RESULTS: In treatment I, mucilage was visible 12 h after transplanting to the compact soil. The decapping and mucilage treatments affected neither the root elongation nor the root widening rates when the plants were grown in loose soil for 12 h. Root growth pressures of seminal axes in D, DM, I and IM treatments were 0.328, 0.288, 0.272 and 0.222 MPa, respectively, when the roots were grown in compact soil (1.5 Mg m(-3) density; 1.59 MPa penetrometer resistance). CONCLUSIONS: The contributions of mucilage and presence of the intact root cap without mucilage to the lubricating effect of root cap (percentage decrease in root penetration resistance caused by decapping) were 43 % and 58 %, respectively. The lubricating effect of the root cap was about 30 % and unaffected by the degree of soil compaction (for penetrometer resistances of 0.52, 1.20 and 1.59 MPa).

Adhesives↗

Influence of microgravity on cellular differentiation in root caps of Zea mays.

We launched imbibed seeds of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on cellular differentiation in root caps. The influence of microgravity varied with different stages of cellular differentiation. Overall, microgravity tended to 1) increase relative volumes of hyaloplasm and lipid bodies, 2) decrease the relative volumes of plastids, mitochondria, dictyosomes, and the vacuome, and 3) exert no influence on the relative volume of nuclei in cells comprising the root cap. The reduced allocation of dictyosomal volume in peripheral cells of flight-grown seedlings correlated positively with their secretion of significantly less mucilage than peripheral cells of Earth-grown seedlings. These results indicate that 1) microgravity alters the patterns of cellular differentiation and structures of all cell types comprising the root cap, and 2) the influence of microgravity on cellular differentiation in root caps of Zea mays is organelle specific.

Cell Differentiation↗

Changes in the distribution of plastids and endoplasmic reticulum during cellular differentiation in root caps of Zea mays.

In calyptrogen cells of Zea mays, proplastids are distributed randomly throughout the cell, and the endoplasmic reticulum (ER) is distributed parallel to the cell walls. The differentiation of calyptrogen cells into columella statocytes is characterized by the following sequential events: (1) formation of ER complexes at the distal and proximal ends of the cell, (2) differentiation of proplastids into amyloplasts, (3) sedimentation of amyloplasts onto the distal ER complex, (4) breakdown of the distal ER complex and sedimentation of amyloplasts to the bottom of the cell, and (5) formation of sheets of ER parallel to the longitudinal cell walls. Columella statocytes located in the centre of the cap each possess 4530 +/- 780 micrometers2 of ER surface area, an increase of 670 per cent over that of calyptrogen cells. The differentiation of peripheral cells correlates positively with (1) the ER becoming arranged in concentric sheets, (2) amyloplasts and ER becoming randomly distributed, and (3) a 280 per cent increase in ER surface area over that of columella statocytes. These results are discussed relative to graviperception and mucilage secretion, which are functions of columella and peripheral cells, respectively.

Cell Differentiation↗

Effects of abscisic acid and xanthoxin on elongation and gravitropism in primary roots of Zea mays.

We examined the involvement of abscisic acid (ABA) and xanthoxin (Xan) in maize root gravitropism by (1) testing the ability of ABA to allow positive gravitropism in dark-grown seedlings of the maize cultivar LG11, a cultivar known to require light for positive gravitropism of the primary root, (2) comparing curvature in roots in which half of the cap had been excised and replaced with agar containing either ABA or indole-3-acetic acid (IAA), (3) measuring gravitropism in roots of seedlings submerged in oxygenated solutions of ABA or IAA and (4) testing the effect of Xan on root elongation. Using a variety of methods of applying ABA to the root, we found that ABA did not cause horizontally-oriented primary roots of dark-grown seedlings to become positively gravitropic. Replacing half of the root cap of vertically oriented roots with an agar block containing ABA had little or no effect on curvature relative to that of controls in which the half cap was replaced by a plain agar block. Replacement of the removed half cap with IAA either canceled or reversed the curvature displayed by controls. When light-grown seedlings were submerged in ABA they responded strongly to gravistimulation while those in IAA did not. Xan (up to 0.1 mM) did not affect root elongation. The results indicate that ABA is not a likely mediator of root gravitropism and that the putative ABA precursor, Xan, lacks the appropriate growth-inhibiting properties to serve as a mediator of root gravitropism.

Abscisic Acid↗

Applied indol-3yl-acetic acid on the cap and auxin movements in gravireacting maize roots.

The graviresponsiveness of intact and primary maize roots kept horizontally in darkness and humid air is analysed. A precise local application of IAA is possible when using resin beads (diameter: 0.45 +/- 0.05 mm) loaded with IAA. The beads are placed on the upper or lower sides of the caps. They significantly change the root gravireaction. The effect of IAA is discussed in terms of its possible level in the growing and gravibending zones and its transport (acropetal, lateral and basipetal) respectively in the stele, the cap and the cortex of the elongating root.

Biological Transport↗

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-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

Cell Differentiation↗

Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH).

Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as sources of beneficial bacteria.

Agriculture↗

Purification and immunolocalization of an annexin-like protein in pea seedlings.

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

Amino Acid Sequence↗

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center.

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zeca mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development.

Amino Acid Sequence↗

Gravitropism in roots of intermediate-starch mutants of Arabidopsis.

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.

Arabidopsis↗

Amyloplast sedimentation and organelle saltation in living corn columella cells.

Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

Cytoplasmic Streaming↗

Regulation of phytochrome message abundance in root caps of maize.

In many cultivars of maize (Zea mays L.) red light affects root development via the photomorphogenetic pigment phytochrome. The site of perception for the light is the root cap. In the maize cultivar Merit, we investigated phytochrome-mediated events in the cap. We established that the message encoded by the phyA1 gene was most abundant in dark-grown tissue and was asymmetrically distributed in the root cap, with greatest expression in the cells which make up the central columella core of the cap. Phytochrome message was negatively autoregulated in a specific region within the root cap. This autoregulation was sensitive to very-low-fluence red light, and thus was characterized as a phytochrome-mediated, very-low-fluence event. The kinetics of message reaccumulation in the dark were also examined and compared to the kinetics of the light requirement for root gravitropism in this cultivar. Similarly, the degree of autoregulation present in two other maize cultivars with different light requirements for gravitropic sensitivity was investigated. It appears that the Merit cultivar expresses a condition of hypersensitivity to phytochrome-mediated light regulation in root tissues. We conclude that phytochrome regulates many activities within the cap, but the degree to which these activities share common phytochrome-mediated steps is not known.

Darkness↗

Changes in vacuolation in the root apex cells of soybean seedlings in microgravity.

Changes in the vacuolation in root apex cells of soybean (Glycine max L. [Merr.]) seedlings grown in microgravity were investigated. Spaceflight and ground control seedlings were grown in the absence or presence of KMnO4 (to remove ethylene) for 6 days. After landing, in order to study of cell ultrastructure and subcellular free calcium ion distribution, seedling root apices were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer and 2% (w/v) glutaraldehyde, 2.5% (w/v) formaldehyde, 2% (w/v) potassium antimonate K[Sb(OH)6] in 0.1 M K2HPO4 buffer with an osmolarity (calculated theoretically) of 0.45 and 1.26 osmol. The concentrations of ethylene in all spaceflight canisters were significantly higher than in the ground control canisters. Seedling growth was reduced in the spaceflight-exposed plants. Additionally, the spaceflight-exposed plants exhibited progressive vacuolation in the root apex cells, particularly in the columella cells, to a greater degree than the ground controls. Plasmolysis was observed in columella cells of spaceflight roots fixed in solutions with relatively high osmolarity (1.26 osmol). The appearance of plasmolysis permitted the evaluation of the water status of cells. The water potential of the spaceflight cells was higher than the surrounding fixative solution. A decrease in osmotic potential and/or an increase in turgor potential may have induced increases in cell water potential. However, the plasmolysed (i.e. non-turgid) cells implied that increases in water potential were accompanied with a decrease in osmotic potential. In such cells changes in vacuolation may have been involved to maintain turgor pressure or may have been a result of intensification of other vacuolar functions like digestion and storage.

Calcium↗

Interactions between red light, abscisic acid, and calcium in gravitropism.

The effect of red light on orthogravitropism of Merit corn (Zea mays L.) roots has been attributed to its effects on the transduction phase of gravitropism (AC Leopold, SH Wettlaufer [1988] Plant Physiol 87:803-805). In an effort to characterize the orthogravitropic transduction system, comparative experiments have been carried out on the effects of red light, calcium, and abscisic acid (ABA). The red light effect can be completely satisfied with added ABA (100 micromolar) or with osmotic shock, which is presumed to increase endogenous ABA. The decay of the red light effect is closely paralleled by the decay of the ABA effect. ABA and exogenous calcium show strong additive effects when applied to either Merit or a line of corn which does not require red light for orthogravitropism. Measurements of the ABA content show marked increases in endogenous ABA in the growing region of the roots after red light. The interpretation is offered that red light or ABA may serve to increase the cytoplasmic concentrations of calcium, and that this may be an integral part of orthogravitropic transduction.

Abscisic Acid↗