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PubMed1992

A rapid and efficient method for the screening of acid phosphatase 1 in young tomato seedlings, and for the identification of root-knot nematode species using miniaturized polyacrylamide gel electrophoresis.

A relatively rapid and highly sensitive miniaturized polyacrylamide gel electrophoresis technique is described for the analysis of certain isozymes from single cotyledons of tomato seedlings and from single females of the root-know nematode (Meloidogyne spp.). Homogenates from single tomato cotyledons (7, 14, 21, and 28 days old) were electrophoresed and stained for acid phosphatase 1 (Aps 1) activity. Cotyledons from plants of all the above age groups showed good Aps 1 activity. Nondestructive screening for tomato Aps 1 is therefore feasible, using very small samples, from as young as 7-day-old tomato seedlings. This could be of important use in expediting root-knot nematode resistance (based on the Aps 1-linked resistance gene Mi) screening for breeding programs, or F1 testing for seed production purposes. In addition, the mini-polyacrylamide gel electrophoresis technique was useful for determination of the Aps 1 allelic contribution to the total enzyme activity. The system was also used to detect malate dehydrogenase and esterase isozyme activity from single adult females of the four common root-knot nematodes, Meloidogyne arenaria, M. hapla, M. incognita, and M. javanica, with equally good results, enabling species discrimination.

Acid Phosphatase↗
PubMed1992

Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize.

We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 micromole) that did not affect root growth. Calmodulin antagonists (> or = 1 micromole) inhibited downward transport of label from 45Ca2+ across the caps of gravistimulated roots relative to the downward transport of 45Ca2+ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip (> or = 1 micromole) also decreased the relative downward movement of label from 3H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of 45Ca2+ in the root tip nor the upward movement of label from 3H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists > or = 1 micromole become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.

Biological Transport↗
PubMed1999

Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation

The occurrence and function of the side chains occurring in the rhamnogalacturonan I domain of pectic poly- saccharides have been investigated during carrot cell development using monoclonal antibodies to defined epitopes of (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan. Immunolocalization studies of carrot root apices indicated that cell walls in the central region of the meristem contained higher levels of (1-->5)-alpha-arabinan than the cell walls of surrounding cells. In contrast (1-->4)-beta-galactan was absent from the cell walls of the central meristematic cells but appeared abundantly at a certain point during root cap cell differentiation and also appeared in cell walls of differentiating stele and cortical cells. This developmental pattern of epitope occurrence was also reflected in a suspension-cultured carrot cell line that can be induced to switch from proliferation to elongation by altered culture conditions. (1-->4)-beta-galactan occurred at a low level in cell walls of proliferating cells but accumulated rapidly in cell walls following induction, before any visible cell elongation, while (1-->5)-alpha-arabinan was present in cell walls of proliferating cells but was absent from cell walls of elongated cells. Immunochemical assays of the cultured cells confirmed the early appearance of (1-->4)-beta-galactan during the switch from cell proliferation to cell elongation. Anion-exchange chromatography confirmed that (1-->4)-beta-galactan was attached to acidic pectic domains and also indicated that it was separate from a distinct homogalacturonan-rich component. These results indicate that the neutral components of pectic polysaccharides may have important roles in plant cell development.

Journal Article↗
PubMed2000

Functional characterization of the EMCV IRES in plants.

The translation of eukaryotic messenger RNA is typically dependent upon the presence of an m7GpppN cap structure at the 5' end of the transcript. However, several animal viruses, including the Picorna viruses, have been shown to exhibit cap-independent translation through the presence of an internal ribosome entry site or IRES. This IRES-mediated cap-independent internal translation initiation has been exploited to generate bicistronic transcripts that function in animal cells. Recently IRES elements have also been identified in a small number of vertebrate, insect and yeast cellular messenger RNAs although no such sequences have been identified in endogenous plant genes and there are no reports of animal virus derived IRES activity in plant cells. Here we have constructed a bicistronic gene containing both green fluorescent protein and luciferase open-reading frames separated by the encephalomyocarditis IRES element under the control of the CaMV 35S promoter. Northern analysis reveals expression of the bicistronic transcript and in vivo imaging of GFP and luciferase activities demonstrates the functional presence of both proteins. Western blot analysis confirms the independent translation of both reporter proteins. These data suggest that insertion of the encephalomyocarditis virus (EMCV) IRES element between two open-reading frames of a plant bicistronic transcript can mediate translation of the second open-reading frame. This activity is more apparent in the leaves, than in the roots, of transgenic seedlings carrying the bicistronic reporter gene construct.

DNA, Recombinant↗
PubMed1988

Lectin-enhanced accumulation of manganese-limited Rhizobium leguminosarum cells on pea root hair tips.

The ability of Rhizobium leguminosarum 248 to attach to developing Pisum sativum root hairs was investigated during various phases of bacterial growth in yeast extract-mannitol medium. Direct cell counting revealed that growth of the rhizobia transiently stopped three successive times during batch culture in yeast extract-mannitol medium. These interruptions of growth, as well as the simultaneous autoagglutination of the bacteria, appeared to be caused by manganese limitation. Rhizobia harvested during the transient phases of growth inhibition appeared to have a better attachment ability than did exponentially growing rhizobia. The attachment characteristics of these manganese-limited rhizobia were compared with those of carbon-limited rhizobia (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986, and J. Bacteriol. 169:4294-4301, 1987). In contrast to the attachment of carbon-limited cells, accumulation of manganese-limited rhizobia (cap formation) was already in full progress after 10 min of incubation; significantly delayed by 3-O-methyl-D-glucose, a pea lectin haptenic monosaccharide; partially resistant to sodium chloride; and partially resistant to pretreatment of the bacteria with cellulase. Binding of single bacteria to the root hair tips was not inhibited by 3-O-methyl-D-glucose. Whereas attachment of single R. leguminosarum cells to the surface of pea root hair tips seemed to be similar for both carbon- and manganese-limited cells, the subsequent accumulation of manganese-limited rhizobia at the root hair tips is apparently accelerated by pea lectin molecules. Moreover, spot inoculation tests with rhizobia grown under various culture conditions indicated that differences in attachment between manganese- and carbon-limited R. leguminosarum cells are correlated with a significant difference in infectivity in that manganese-limited rhizobia, in contrast to carbon-limited rhizobia, are infective. This growth-medium-dependent behavior offers and explanation for the seemingly conflicting data on the involvement of host plant lectins in attachment of rhizobia to root hairs of leguminous plants. Sym plasmid-borne genes do not play a role in manganese-limitation-induced attachment of R. leguminosarum.

Carbon↗
PubMed2003

[Distribution of microfilaments in detached corn root cap cells under fluorescent microscope].

The detached root cap cells from maize seedlings comprised of round-shaped cells, ellipse-shaped cells and longitude-shaped cells. By using fluorescein-iso-thiocyanate-phalloidin(FITC-Ph) as fluorescent probe and treatment with cytochalasin B (CB) or HMC toxin, a host-specific toxin from Bipolaris (Helminthosporium) maydis race C, the distribution and variation of microfilaments (MFs) in detached cells were investigated. The results were as follows (i) the round-shaped cells had intense fluorescence, but the network of MFs was not distinct. There was clear MFs network in the cytoplasm of both ellipse-shaped and longitude-shaped cells. The distribution of MFs in detached cells seemed to be relevant to their shape and vigor. (ii) the fluorescence of detached cells of Charrua cytoplasmic male sterility(cms-C) maize was decreased after treatment with HMC-toxin. The cause of this outcome was unclear. We only obtained the pictures of the distorted protoplast membrane and dead cells owing to treatment with HMC-toxin. The distribution of MFs of detached cells of Normal(N) cytoplasmic maize was not affected by HMC-toxin, and their protoplasts shank slightly. (iii) CB could change the distribution of MFs in detached cells of both cms-C maize and N maize to disordered arrangement.

Actin Cytoskeleton↗
PubMed2005

PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots.

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid-reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2002

A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination.

Xyloglucan endotransglycosylases (XETs) modify xyloglucans, major components of primary cell walls in dicots. A cDNA encoding an XET (LeXET4) was isolated from a germinating tomato (Lycopersicon esculentum Mill.) seed cDNA library. DNA gel blot analysis showed that LeXET4 is a single-copy gene in the tomato genome. LeXET4 mRNA was strongly expressed in germinating seeds, was much less abundant in stems, and was not detected in roots, leaves or flower tissues. During germination, LeXET4 mRNA was detected in seeds within 12 h of imbibition with maximum mRNA abundance at 24 h. Tissue prints showed that LeXET4 mRNA was localized exclusively to the endosperm cap region. Expression of LeXET4 was dependent on exogenous gibberellin (GA) in GA-deficient (gib-1 mutant) tomato seeds, while abscisic acid, a seed germination inhibitor, had no effect on LeXET4 mRNA expression in wild-type seeds. LeXET4 mRNA disappeared after radicle emergence, even though degradation of the lateral endosperm cell walls continued. The temporal, spatial and hormonal regulation pattern of LeXET4 gene expression suggests that XET has a role in endosperm cap weakening, a key process regulating tomato seed germination.

Abscisic Acid↗
PubMed1996

Genetic and contig map of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis thaliana.

In the course of the isolation of the MONOPTEROS (MP) gene, required for primary root formation in Arabidopsis thaliana, a yeast artificial chromosome (YAC) contig encompassing approximately 2200 kilobases corresponding to 5.5 cM on the top arm of chromosome 1 was established. Forty-six YAC clones were characterized and 12 new restriction fragment length polymorphism (RFLP) markers are presented. Three new codominant amplified polymorphic sequence (CAPS) markers were generated that enabled high resolution genetic mapping and correlation of physical and genetic distances along the contig. The map contributes to the completion of a physical map of the Arabidopsis genome and should facilitate positional cloning of other genes in the region as well as studies on genome organization. We also present another set of 11 physically linked probes, as well as mapping data for additional RFLP markers within a broader interval of 10.4 cM.

Arabidopsis↗
PubMed1998

Analysis of changes in relative elemental growth rate patterns in the elongation zone of Arabidopsis roots upon gravistimulation.

Although Arabidopsis is an important system for studying root physiology, the localized growth patterns of its roots have not been well defined, particularly during tropic responses. In order to characterize growth rate profiles along the apex of primary roots of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) we applied small charcoal particles to the root surface and analyzed their displacement during growth using an automated video digitizer system with custom software for tracking the markers. When growing vertically, the maximum elongation rate occurred 481 +/- 50 microns back from the extreme tip of the root (tip of root cap), and the elongation zone extended back to 912 +/- 137 microns. The distal elongation zone (DEZ) has previously been described as the apical region of the elongation zone in which the relative elemental growth rate (REGR) is < or = 30% of the peak rate in the central elongation zone. By this definition, our data indicate that the basal limit of the DEZ was located 248 +/- 30 microns from the root tip. However, after gravistimulation, the growth patterns of the root changed. Within the first hour of graviresponse, the basal limit of the DEZ and the position of peak REGR shifted apically on the upper flank of the root. This was due to a combination of increased growth in the DEZ and growth inhibition in the central elongation zone. On the lower flank, the basal limit of the DEZ shifted basipetally as the REGR decreased. These factors set up the gradient of growth rate across the root, which drives curvature.

Arabidopsis↗
PubMed2006

Transcription profile analyses identify genes and pathways central to root cap functions in maize.

Affymetrix GeneChips arrayed with about one-half (~23K) of the rice genes were used to profile gene transcription activity in three tissues comprising the maize root tip; the proximal meristem (PM), the quiescent center (QC), and the root cap (RC). Here we analyze the gene transcription profile of the RC, compared to both the PM and the QC, from three biological replicates. In the RC, a total of 669 genes were identified as being differentially upregulated, and 365 differentially downregulated. Real-time quantitative RT-PCR analysis was used to confirm upregulated genes in the RC. In addition, using the technique of laser microdissection (LMD) we localized upregulated gene expression to the lateral RC cells. Taken as a whole, transcription profile analyses revealed the upregulation in the maize RC of clusters of genes linked to major metabolic processes and pathways, including: (1) transport, both the export of carbohydrates and the uptake of nutrients; (2) sensing and responding to (often stressful) biotic and abiotic environmental stimuli; (3) integrating the responses of at least 3 major growth regulators (auxin, ethylene, jasmonic acid); (4) processing the large amount of carbohydrate transported into the RC. Although the profile data are derived using heterologous rice GeneChips, with about half of the total rice gene set, this study, nevertheless, provides a genomic scale characterization of the entire RC, and serves as a new platform from which to advance studies of the network of pathways operating in the maize RC.

Arabidopsis↗
PubMed1998

Genomic organization and structure of alpha-hydroxynitrile lyase in cassava (Manihot esculenta Crantz).

Two clones with homology to the alpha-hydroxynitrile lyase (HNL) cDNA clone, MeHNL10, were isolatedfrom a lambdaEMBL3 cassava (Manihot esculenta Crantz) genomic library. Analysis of the sequences showed that both genomic clones contain HNL genes (MeHNL4, MeHNL24) which are interrupted by two introns. RT-PCR analysis of MeHNL4 shows that it is expressed at high levels in seedling roots and at lower levels in cotyledons and young leaves. The deduced amino acid sequences of MeHNL4, MeHNL10, and MeHNL24 show high sequence identity and homology to the HNL from Hevea brasiliensis whose tertiary structure has been solved at 1.9-A resolution by X-ray crystallography. This high homology allowed the construction of model structures for all of the cassava proteins using the MODELLER program. Homology modeling indicates that the short variable exon 2 encodes the "cap" region which is thought to influence the substrate specificity of the protein. Two hybrid proteins were modeled using the core alpha/beta domain of MeHNL10 and the cap region of either the Hevea HNL or a structurally related Zea protein of unknown function. This analysis suggests that changes in the active site can be engineered by swapping exons.

Aldehyde-Lyases↗
PubMed1997

Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis.

To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.

Arabidopsis↗
PubMed1999

Polygalacturonase and polygalacturonase inhibitor protein: gene isolation and transcription in Glycine max-Heterodera glycines interactions.

The cell wall acts as the first line of defense during pathogen invasion. Polygalacturonases (PGs) are a class of cell-wall-modifying enzymes with precise temporal and organ-specific expression. A 350-bp fragment with high homology to PGs was identified by differential display (DD) analysis of soybean cyst nematode (SCN) race 3 resistant PI 437654 and susceptible cultivar Essex. The fragment was strongly expressed in Essex, 2 days after inoculation (DAI). Complete coding sequences of two PG cDNAs, PG1 and PG2, were isolated by 3' and 5' rapid amplification of cDNA ends polymerase chain reaction (RACE PCR). PI 437654 and Essex had identical PG1 and PG2 sequences. A transversion from A to C created a PstI restriction site in the PG2 cDNA that was used to distinguish the two PG cDNAs by cleaved amplified polymorphic sequence (CAPS) analysis. A cDNA encoding a polygalacturonase-inhibitor protein (PGIP) that is 89% identical to the Phaseolus vulgaris PGIP was isolated from soybean roots by reverse transcription (RT)-PCR. Steady-state levels of PG and PGIP were investigated by RNA gel blot analysis in roots 1 to 5 DAI and in hypocotyls and leaves. Differences in the constitutive levels of PG mRNAs were observed in roots of different soybean genotypes. Steady-state levels of PG mRNAs were enhanced during compatible interactions with SCN and reduced in incompatible interactions and in mechanically wounded roots. Enhanced PGIP transcription was observed in response to mechanical wounding in both PI 437654 and Essex, but only in compatible interactions with SCN, suggesting uncoupling of PGIP functions in developmental and stress cues. Constitutive expression in incompatible interactions shows PGIP is not a factor in SCN resistance. Thus, the up-regulation of endogenous PG transcription in soybean roots early after SCN infection could facilitate successful parasitism by SCN.

Amino Acid Sequence↗
PubMed2004

[Ultrastructure of root cup meristematic cells of Cucumis sativus L. seedlings under inhibition of root growth by synthetic cyclopentene beta,beta'-triketone].

A study was made of the ultrastructure of root cap meristematic cells in the seedlings of Cucumis sativus L., whose growth was inhibited by the action of 2-acetyl-4-metylcyclopent-4-en-1,3-dione (AMCD). It is shown that the treatment of seeds by this substance at a concentration of 100 microg/ml leads to the main root inhibition by 73% and, as compared to the control, causes the highest changes in mitochondria that do not reach normal dimensions and undergo degeneration. AMCD caused significant changes in the cell walls that in test samples unusually became electron-dense. Deposits of osmiophylic substance, presumably consisting of terpenoid-like compounds, were often found in the cytoplasm of treated cells as well as in the extracellular space. In many cells, plasmalemma and amiloplast membranes underwent destructive changes. We discuss a possible relation between inhibitory effect of AMCD on the root growth, and its influence on the ultrastructure in investigated cells.

Cucumis sativus↗
PubMed1994

Carbohydrate binding activities of Bradyrhizobium japonicum. III. Lectin expression, bacterial binding, and nodulation efficiency.

In previous studies, evidence that the Bradyrhizobium japonicum lectin, designated BJ38, mediated the observed carbohydrate-specific binding activities of the bacteria, including the saccharide-specific adhesion to soybean root cells was presented. In the present study, it is found that both B. japonicum, as well as the purified BJ38, bind predominantly to young emergent root hairs of soybean roots and, to a much lesser extent, to the root cap, mature root hairs, epicotyl or hypocotyl regions. Thus, the region of preferential binding for both the bacteria and the isolated lectin coincide with the region of the soybean root most susceptible to B. japonicum infection. The importance of bacterial binding for the nodulation process was studied by comparing the nodulation efficiency of binding-deficient mutants N4 and N6 to the wild-type. These mutants had been shown to be defective in carbohydrate recognition, as represented by their diminished ability to bind to soybean roots. BJ38 was immunolocalized to one pole of the cell surface of wild-type B. japonicum, but no surface labeling could be detected on either mutant. Moreover, both N4 and N6 showed a substantial decrease in nodulation activity, relative to the wild-type. These results provide additional evidence that the carbohydrate binding activity of B. japonicum, most probably mediated by BJ38, may play an important role(s) in the initial phases of the infection process.

Bacterial Adhesion↗
PubMed1986

Perception of gravity in the lentil root.

The gravisensing cells (statocytes), responsible for the perception of gravity, are located in the center of the root cap. The statocytes contain voluminous amyloplasts (statoliths) which are capable of moving in the direction of gravity. The sedimentation of the amyloplasts is due to the starch grains, which are much denser than the surrounding cytoplasm. When the starch of these plastids is removed, the statoliths lose their mobility and the roots are no longer able to respond to gravity. When the roots grow in their normal position, the statoliths are sedimented on large aggregates of endoplasmic reticulum. The role of the association between these two types of organelles is still controversial. For some authors, the amyloplast-endoplasmic reticulum complex would play a role in the induction of the gravitropic response. For other authors, this complex would regulate gravitropic curvature and is more likely to be involved in the termination of the response. The goal of the 39F experiment (Biorack) was to dissociate this complex by growing lentil roots in microgravity and to stimulate them on a centrifuge in order to determine if they were still capable of curvature.

Cell Polarity↗
PubMed1994

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter.

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5' non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (-623 to -471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (-341 to -306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C + D + E, B + C + D, B + C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B + C + E is active only in vascular meristematic cells. Domain C (-216 to -158) seems to have a double regulatory role as construct B + E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.

Enzyme Induction↗