The size and distribution of polysaccharides during their synthesis within the membrane system of maize root cells

Summary The polymers contained within pellets rich in either dictyosomes or the endoplasmic reticulum have been investigated. These were detected by the incorporation of radioactivity from D-[U-¹⁴C]glucose into the sugars in the intact root. Three types of material were obtained 1) water-soluble 2) soluble in chloroform 3) insoluble. The relative proportions of each of these 3 fractions were different in the 2 membrane preparations. The dictyosome pellet contained a high proportion of water-soluble material, some of the water-soluble polysaccharides contained in both membrane preparations had a molecular weight greater than 40,000. These polymers resembled the pectins and xylans deposited in the cell wall. Some of the insoluble material could be rendered soluble by incubation with proteolytic enzymes and all of this from the dictyosome pellet had a molecular weight greater than 4,000 whereas most of that obtained from the endoplasmic reticulum had a molecular weight less than 4,000 and it did not contain fucose.Abbreviations RER rough endoplasmic reticulum - GA golgi apparatus     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

The sites of synthesis and transport of extracellular polysaccharides in the root tissues of maize

1. Subcellular fractionation of maize roots resulted in the isolation of the following enriched fractions: cell wall, dictyosome, smooth-membrane and rough-microsomal fractions. In addition, extracellular polysaccharide of the root slime was isolated. 2. Maizeseedling roots were incubated in vivo with d-[U-(14)C]glucose, and the pattern of incorporation of radioactivity into the polysaccharides of each fraction was investigated. 3. The differentiation of maize-root cells with respect to the synthesis of specific extracellular polysaccharide directly relates to the polysaccharide synthesized and transported within the membrane system of the cell. A fucose-containing polysaccharide, characteristic only of root slime, was present only in the membrane system of the root-tip region of the root. Regions of typical secondary wall development within the root were characterized by an increased incorporation of radioactivity into xylose of polysaccharide within the membrane system. 4. The incorporation of radioactivity into glucan polymers in the membrane fractions was very low in all regions of the root. Since in regions of secondary wall development greater than 60% of all radioactive incorporation was into a glucan polymer, it can be inferred that this polymer, most probably cellulose, is not synthesized or transported within the compartments of the membrane system. It is suggested that synthesis of cellulose occurs at the surface of the plasmalemma. 5. Maize-root cells contained 40 times more rough endoplasmic reticulum than dictyosome membrane. The relative specific radioactivities of each fraction indicated that polysaccharide was concentrated in the region of the Golgi apparatus, which showed a 100% increase in specific radioactivity compared with the rough endoplasmic reticulum. The Golgi apparatus can thus be regarded as a localized focal point on the synthetic and transport system of polysaccharide by the intracellular membrane compartments.     

Dictyosome polarity and membrane differentiation in outer cap cells of the maize root tip

Outer rootcap cells of maize produce large numbers of secretory vesicles that ultimately fuse with the plasma membrane to discharge their product from the cell. As a result of the fusion, these vesicles contribute large quantities of membrane to the cell surface. In the present study, this phenomenon has been investigated using sections stained with phosphotungstic acid at low pH (PACP), a procedure in plant cells that specifically stains the plasma membrane. In the maize root tip, the PACP also stains the membranes of the secretory vesicles derived from Golgi apparatus to about the same density that it stains the plasma membrane. Additionally, the membranes of the secretory vesicles acquire the staining characteristic while still attached to the Golgi apparatus. The staining progresses across the dictyosome from the forming to the maturing pole, thus confirming the marked polarity of these dictyosomes. Interestingly, the PACP staining of Golgi apparatus is confined to the membranes of the secretory vesicles. It is largely absent from the central plates or peripheral tubules and provides an unambiguous example of lateral differentiation of membranes orthogonal to the major polarity axis. In the cytoplasm we could find no vesicles other than secretory vesicles bearing polysaccharide that were PACP positive. Even the occasional coated vesicle seen in the vicinity of the Golgi apparatus did not stain. Thus, if exocytotic vesicles are present in the maize root cap cell, they are formed in a manner where the PACP-staining constituent is not retained by the internalized membrane. The findings confirm dictyosome polarity in the maize root cap, provide evidence for membrane differentiation both across and at right angles to the major polarity axis, and suggest that endocytotic vesicles, if present, exclude the PACP-staining component.     

The endoplasmic reticulum and the Golgi structures in maize root cells

Maize root tips were fixed in potassium permanganate, embedded in epoxy resin, sectioned to show silver interference color, and studied with the electron microscope. All the cells were seen to contain an endoplasmic reticulum and apparently independent Golgi structures. The endoplasmic reticulum is demonstrated as a membrane-bounded, vesicular structure comparable in many aspects to that of several types of animal cells. With the treatment used here the membranes appear smooth surfaced. The endoplasmic reticulum is continuous with the nuclear envelope and, by contact at least, with structures passing through the cell wall. The nuclear envelope is characterized by discontinuities, as previously reported for animal cells. The reticula of adjacent cells seem to be in contact at or through the plasmodesmata. Because of these contacts the endoplasmic reticulum of a given cell appears to be part of an intercellular system. The Golgi structures appear as stacks of platelet-vesicles which apparently may, under certain conditions, produce small vesicles around their edges. Their form changes markedly with development of the cell.     

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

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

Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.     

Characterization of the hexose transport system in maize root tips

Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.     

The effect of hydrostatic pressure on structural organization of maize root cells

We analyzed the effect of hydrostatic pressure at the 2–4 MPa level on the growth and organization of maize seedlings cells. At a pressure value of 2 MPa, we observed clarification of mitochondrial matrix, clusterization of endoplasmic reticulum elements with formation of ergastoplasm sites, and an increase of the number of small vacuoles in the cytoplasm. On the trans-side of dictyosomes, we revealed accumulation of vesicles with an electron-dense content, which indicates changes of transport in the endomembranous system. A decrease in the electronic density of tonoplast was accompanied by a reduction of the osmophilic inclusion amount in vacuoles. An elevation of pressure up to 4 MPa has been shown to cause destructive alterations in the tonoplast. None of the variants showed any destruction of the plasmalemma integrity, which was confirmed by staining with Evans blue. At a fall of the pressure to an atmospheric value, the frequency of structural disturbances of the tonoplast membrane was proportional to the pressure difference: minimum at a drop from 2 to 0 MPa and maximum at a drop from 4 to 0 MPa. Thus, it was concluded that the endomembranous system was among the targets of the pressure effect on the plant cells.     

Modelling the branching growth fractal pattern of the maize root system

Using the technique of L-systems, a growth model of the maize root system is developed. From the observation of the root systems developed under various soil density in eight root boxes, a spatial hierarchy of growth rules was extracted. The rules were divided into three categories: a meta-rule for describing features of an entire root system, a branching growth rule and a tip elongation rule. Some variations in the entire features of the root system, such as the outline and the root distribution, were confirmed by observation, and then the respective meta-rules were re-defined. The branching properties of first- and second-order lateral roots were statistically almost equal in the observations, and this lead us to set up a single stochastic branching growth rule. Tip elongation movement was not observed here; its rule had to be assumed by reference to data in the literature. A single set of branching growth and tip elongation rules were coupled with the respective meta-rules corresponding to the root samples observed, where a small scale rule was loosely governed by a large scale rule. Computer simulations offered optimized drawings of the observed root systems, and they also reproduced a typical anisotropic power distribution of roots similar to those observed.     

The fine structure of the cells that perceive gravity in the root tip of maize

Summary Within the root cap, in maize, the cells believed to be responsible for the perception all possess large well-developed amyloplasts. They also have normal mitochondria and Golgi bodies, normal rough-surfaced ER with a very striking pattern of distribution, few free ribosomes, walls with an abnormal reticulate encrusting material, irregularly distributed plasmodesmata and an as yet unidentified fine quadruple membranous system. All of these features are discussed in relation to the role of the cells in perception.     

Directional secretion by the Golgi bodies in maize root cells

Summary In the root caps of many plant species, the outer few layers of cells secrete a polysaccharide mucus. This mucus probably derives from the breakdown of the starch in the amyloplasts. Is then fed through the Golgi bodies and, in vesicular form, reaches and is discharged through the plasmamembrane. Once in the wall it accumulates briefly between the plasmalemma and the wall and finally passes through the wall to the outside of the root. Its function is probably to facilitate the passage of the root through the soil by lubricating the movement of the root tip around and between the soil particles. Our observations show that the pattern of this discharge is not random, but that the mucus accumulates only against that wall which is or will be the outer tangential. The implication of this directional movement and possible suggestions for its control are discussed.     

Early Sr2+-induced effects on membrane potential,proton pumping- and ATP hydrolysis-activities of plasma membrane vesicles from maize root cells

When released in plant environment, strontium (Sr²⁺) can be absorbed predominantly by the plant roots. As the plasma membrane of root cells is amongst the first barriers encountered by Sr²⁺ during its soil/plant transfer and the main entry point of Sr²⁺ into the roots, the main objective of this work aimed to enlighten on some of the Sr²⁺-induced effects at this level in Zea mays L. cv. “Liberal”.Thus this study focused on the Sr²⁺-induced changes on membrane potential of cortical root cells and on proton fluxes in maize roots, in order to determine whether the activity of some of the ion transport systems present in the plasma membrane of maize root cell could be among the first targets of Sr²⁺. We focused in particular on the plasma membrane H⁺-ATPase, known to be one of the major transport systems found in the plasmalemma where it generates a proton motive force (contributing to membrane potential maintaining, and providing energy for ion transport through membrane).The data presented here showed that Sr²⁺ triggered an early and transient membrane depolarisation whose magnitude and duration were dependent on the Sr²⁺-concentration. The time course pattern of a second longer lasting depolarisation could be examined in perspective with the Sr²⁺-induced decrease of the spontaneous proton extrusion observed in root tissues, suggesting a relationship between Sr²⁺-effects on membrane potential and H⁺ excretion. Furthermore, the inhibitory effect exerted by Sr²⁺ on the fusicoccin (FC)-enhanced proton extrusion strongly suggested an inhibition of the plasma membrane H⁺-ATPase. This hypothesis was supported by the inhibition induced by Sr²⁺ on proton pumping- and ATP hydrolysis-activities measured in plasma membrane vesicles (PMV) prepared from maize roots.Taken together the data reported here evidence that, with however a lower efficiency, Sr²⁺ behaved in a quite similar way to Ca²⁺ when inhibiting the H⁺-ATPase activity, and suggest that Sr²⁺ could partially mimic Ca²⁺ onto regulation of the H⁺-ATPase activity.     

The longevity and activity of the primary root of maize

The longevity of the main root cylinder and the laterals of the primary root of maize plants was determined under controlled greenhouse conditions by means of nuclear staining with acridine orange. The cortex of the main root was found to be alive for the whole life-span of the plant, whereas the life-span of the root hairs was only 2 to 3 days as evidenced by electronmicroscopical examination of cell integrity. The onset of senescence of laterals was observed at the older part of the main root at the 6-leaf stage of the plant. Senescence of 1st and 2nd order laterals commenced near the root tip a few days after their protrusion and advanced towards the basal region of the root. In any root segment death of the cortex cells preceeded that of the stele. At the late grain filling stage all laterals along the main root exhibited advanced senescence, but stainable nuclei were seen in the root tissues of the basal part of 1^st order laterals (both cortex and stele) as well as of the 2^nd order laterals which emerged from that root segment. The pattern of the dying of the root tissue is discussed with regard to the P-nutrition of the shoot system by the primary root.     

A simulation model of the three-dimensional architecture of the maize root system

In order to study the nutrient and water uptake of rootsin situ, we need a quantitative three-dimensional dynamic model of the root system architecture. The present model takes into account current observations on the morphogenesis of the maize root system. It describes the root system as a set of root axes, characterised by their orders and their inter-node of origin. The evolution of the simulated pattern is achieved by three processes, occuring at each time step: emission of new primary root axes from the shoot, growth and branching of existing root axes. The elongation of an axis depends on its order, inter-node and local growing conditions. Branches appear acropetally at a specified distance from the apex and from former branches, along ranks facing xylem poles, with a branching angle specific to their order and inter-node. From the three-dimensional branched patterns simulated by the model, various outputs, such as root profiles or cross-section maps can be computed, compared to observed data and used as inputs in uptake models. A number of examples of such possible outputs are presented.     

Contribution of a plasma membrane redox system to the superoxide production by wheat root cells

Summary Wound stress activated wheat root cells to produce oxygen radicals. The production was accompanied by an increased permeability for potassium ions and a depolarization of the plasma membrane. Various electron donors, such as the nonpenetrating donor potassium ferrocyanide as well as NADH and NADPH, caused the amplification of superoxide production by root cells. The -generating system in wheat root cells was found to be considerably sensitive to diphenylene iodonium, which is generally considered as a suicide inhibitor of neutrophil NADPH oxidase, and to other inhibitors of flavoprotein activity, chlorpromazine and quinine. The xenobiotic compound amidopyrine caused activation of the -generating system, which was depressed by DPI. The -generating system in root cells was shown to be significantly dependent on calcium content. Calcium loading of the root cells induced a powerful increase of the superoxide release. Data obtained indicate that superoxide generation is one of the early events of the wound stress response. Redox systems of the plasma membrane may be involved in the superoxide production in response to wound stress and detoxification of xenobiotic compounds in root cells.Abbreviations DPI diphenylene iodonium - MP membrane potential - superoxide anion radical - ROS reactive-oxygen species - SOD superoxide dismutase