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Effects of rooting and tree growth of selected woodland species on cap integrity in a mineral capped landfill site.

The above and below ground growth of three tree species (Alnus glutinosa, Pinus nigra var. maritima and Acer pseudoplatanus) was studied on a containment landfill site at Waterford, Hertfordshire, UK. Tree root architecture was studied using soil inspection pits excavated next to 12 trees of each species and mapped in detail. Tree height was related to soil thickness over the compacted mineral cap. No roots entered the cap where soil thickness was 1.3 m, but a few roots, especially of alder, were observed within it when the soil cover was 1.0 m or less. Micromorphological analysis of undisturbed samples of the mineral cap suggested that roots exploited weaknesses in the cap rather than actively causing penetration into it. Alder roots were more tolerant of anaerobic conditions within the cap than the other species examined. The results confirm that mineral caps should be covered by 1.5 m of soil or soil-forming material if tree establishment is intended over a restored landfill site, unless protected by other parts of a composite capping system.

Environmental Pollution↗

Possible use of a 3-D clinostat to analyze plant growth processes under microgravity conditions.

A three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles was constructed, and various growth processes of higher plants grown on this clinostat were compared with ground controls, with plants grown on the conventional horizontal clinostat, and with those under real microgravity in space. On the 3-D clinostat, cress roots developed a normal root cap and the statocytes showed the typical polar organization except a random distribution of statoliths. The structural features of clinostatted statocytes were fundamentally similar to those observed under real microgravity. The graviresponse of cress roots grown on the 3-D clinostat was the same as the control roots. On the 3-D clinostat, shoots and roots exhibited a spontaneous curvature as well as an altered growth direction. Such an automorphogenesis was sometimes exaggerated when plants were subjected to the horizontal rotation, whereas the curvature was suppressed on the vertical rotation. These discrepancies in curvature between the 3-D clinostat and the conventional ones appear to be brought about by the centrifugal force produced. Thus, the 3-D clinostat was proven as a useful device to simulate microgravity.

Cell Wall↗

Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos.

The 5' fragment (1 647 bp) of the cotton glucuronosyltransferase gene (GhGlcAT1) was transcriptionally fused to the beta-glucuronidase (GUS) gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars (glucose and sucrose) as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from -281 to +30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element(s) for sucrose induction might be located between -635 and -281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugar-responsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGlcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGlcAT1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.

Base Sequence↗

Evaluation of the three-dimensional clinostat as a simulator of weightlessness.

Concerns regarding the reliability of slow-and fast-rotating uni-axial clinostats in simulating weightlessness have induced the construction of devices considered to simulate weightlessness more adequately. A new three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles has been constructed. In the clinostat, the rotation achieved with two motors is computer-controlled and monitored with encoders attached to the motors. By rotating plants three-dimensionally at random rates on the clinostat, their dynamic stimulation by gravity in every direction can be eliminated. Some of the vegetative growth phases of plants dependent on the gravity vector, such as morphogenesis, are shown to be influenced by rotation on the 3-D clinostat. The validity of 3-D clinostatting has been evaluated by comparing structural parameters of cress roots and Chara rhizoids obtained under real microgravity with those obtained after 3-D clinostatting. The parameters analyzed up to now (organization of the root cap, integrity and polarity of statocytes, dislocation of statoliths, amount of starch and ER) demonstrate that the 3-D clinostat is a valuable device for simulating weightlessness.

Plant Physiological Phenomena↗

The Ca2+ pump inhibitor, thapsigargin, inhibits root gravitropism in Arabidopsis thaliana.

Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in the sarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plant Thapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering root gravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 microM alter root gravitropism whereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugated thapsigargin (BODIPY FL thapsigargin) suggests that target sites for thapsigargin are located in intracellular organelles in the root distal elongation zone and the root cap, regions known to regulate root gravitropism.

Arabidopsis↗

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

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

Arabidopsis↗

WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis.

Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

Amino Acid Sequence↗

High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron.

The 3.6 kb of 5' flanking sequence, leader intron, and 0.7 kb of 3' sequence from the potato sucrose synthase gene Sus4-16 are sufficient to direct high-level expression in developing tubers, in basal tissues of axillary buds and shoots, and in meristems and caps of roots, and to confer sucrose inducibility in leaves. By examining a series of deletion and substitution constructs in transgenic potato plants, we found that this pattern of expression requires 5' flanking sequences both upstream and downstream of position -1500 and that sequences between positions -1500 and -267 are essential for sucrose induction. Replacement of the native 3' sequence with the nopaline synthase 3' sequence resulted in the loss of sucrose inducibility and of expression in basal tissues of axillary buds. A general decrease in expression in other tissues was also observed. Removal of the 1612-bp leader intron also had a dramatic effect on both the pattern and level of expression.

Base Sequence↗

Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function.

The transport of mineral ions into and out of tissues and cells is central to the life of plants. Ion transport and the plasma membrane transporters themselves have been studied using a variety of techniques. In the last 15 years, measurement of specific ion fluxes has contributed to the characterization of transport systems. Progress in molecular genetics is allowing gene identification and controlled expression of transporter molecules. However the molecular expression of transporter gene products must be characterized at the functional level. The ion-selective microelectrode technique to measure specific ion fluxes non-invasively is ideally suited to this purpose. This technique, its theory, its links with others and its application and prospects in plant science, are discussed. Ions studied include hydrogen, potassium, sodium, ammonium, calcium, chloride and nitrate. Applications discussed include: solute ion uptake by roots; gravitropism and other processes in the root cap, meristematic and elongation zones; Nod factor effect on root hairs; osmotic and salt stresses; oscillations; the effects of light and temperature. Studies have included intact roots, leaf mesophyll and other tissues, protoplasts and bacterial biofilms. A multi-ion capability of the technique will greatly assist functional genomics, particularly when coupled with imaging techniques, patch clamping and the use of suitable mutants.

Cell Membrane↗

The Arabidopsis locus RCB mediates upstream regulation of mitotic gene expression.

Transcriptional regulation of cell cycle regulatory genes, such as B-type cyclins, is tightly linked with the mitotic activity in the meristems. To study the regulation of a B-type cyclin gene, a targeted genetic approach was undertaken. An Arabidopsis line containing a fusion construct between the CYCB1;1 promoter and a bacterial beta-glucuronidase marker gene (uidA) was used in ethyl methanesulfonate mutagenesis. The mutants were screened for altered CYCB1;1::uidA expression patterns. In a reduced CYCB1;1 expression mutant (rcb), the CYCB1;1::uidA expression was severely affected, being excluded from the shoot and root apical meristems and leaf primordia and shifted to cells associated with root cap and stomata. In addition to the overall reduction of the endogenous CYCB1;1 transcript levels, other G2-to-M phase-specific genes were also down-regulated by the mutation. In the mutant plants, the inflorescence stem growth was reduced, indicating low meristem activity. Based on the altered CYCB1;1::uidA expression patterns in rcb root meristem, a model is proposed for RCB that mediates the tissue specificity of CYCB1;1 promoter activity.

Arabidopsis↗

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.

Cell Differentiation↗

'Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis uniflora.

Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. These propagules may detach from the roots, establishing new plants.

Argentina↗

An analysis of heterochromatin in maize root tips.

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15-20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.

Autoradiography↗

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea.

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.

Arabidopsis↗

Opposing effects of aluminum on inward-rectifier potassium currents in bean root-tip protoplasts.

Inward currents in root cap protoplasts of the aluminum-tolerant cultivar, Dade, of Phaseolus vulgaris L. were investigated using the whole-cell patch-clamp technique. The properties of these currents were similar to those seen in inward rectifying K+ channels in other plant tissues. Replacing bath K+ with Na+ nearly abolished the observed currents. Higher bath K+ concentrations increased inward currents. AlCl3 in pH 4.7 bath solutions caused inward K+ currents to activate more rapidly and at more positive voltages when compared with AlCl3 free solutions. In 10 microM AlCl3 the activated inward K+ currents were significantly larger than in the AlCl3-free solution at all voltages except at the most negative voltage of -174 mV and the least negative of -74 mV. In contrast, in 80 microM Al3+, when hyperpolarizing voltages were most negative, the inward K+ currents were inhibited relative to the currents in 10 microM AlCl3. Enhancement of inward K+ currents by AlCl3 is consistent with Al3+ binding to the external surface of the root cap protoplast, decreasing the surface charge, thus causing the channels to sense a more negative membrane potential. Inhibition of inward K+ currents with higher AlCl3 concentrations and more negative voltages is consistent with Al3+ block of K+ channels.

Aluminum↗

Organ-specific transcription of the rrn operon in spinach plastids.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

Base Sequence↗