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PubMed2001

Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice.

A study of the diversity of endophytic bacteria present in seeds of a deepwater rice variety revealed the presence of seven types of BOX-PCR fingerprints. In order to evaluate the plant growth promoting potential the presence of nitrogenase, indole acetic acid production and mineral phosphate solubilization were estimated in the representative BOX-PCR types. The seven representatives of BOX-PCR types produced indole acetic acid, reduced acetylene and showed specific immunological cross-reaction with anti-dinitrogenase reductase antibody. Only four types showed mineral phosphate solubilizing ability. Comparison of cellulase and pectinase activities showed differences among different BOX-PCR types. PCR fingerprinting data showed that one strain isolated from the surface sterilized seeds as well as the aerial parts of the seedlings of rice variety showed low cellulase and pectinase but relatively high ARA. On the basis of 16S rDNA nucleotide sequence and BIOLOG system of bacterial identification, this strain was identified as Pantoea agglomerans. For studying the endophytic colonization this strain was genetically tagged with the reporter gene, gusA. Histochemical analysis of the seedling grown in hydroponics showed that the tagged strain colonized the root surface, root hairs, root cap, points of lateral root emergence, root cortex and the stelar region. Treatment of the roots with 2,4-D produced short thickened lateral roots which showed better colonization by P. agglomerans.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1995

Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus cerasifera.

We compared root system morphogenesis of micropropogated transplants of Prunus cerasifera L. inoculated with either of the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices or with the ericoid mycorrhizal species Hymenoscyphus ericae. All plants were grown in sand culture, irrigated with a nutrient solution that included a soluble source of phosphorus, for 75 days after transplanting. Arbuscular mycorrhizal colonization increased both the survival and growth (by over 100%) of transplants compared with either uninoculated controls or transplants inoculated with H. ericae. Arbuscular mycorrhizal colonization increased root, stem and leaf weights, leaf area, root length and specific leaf area, and it decreased root length/leaf area ratio, root/shoot weight ratio and specific root length. Both uptake of phosphorus and its concentration in leaves were increased by AM infection, although the time course of the relationships between intensity of AM infection and P nutrition were complex and suggested a role for factors other than nutrition. The time course for the development of infection varied. It was most rapid with G. mosseae, but it was ultimately higher with G. intraradices. None of the treatments significantly affected the lengths of adventitious roots or the first-, second- or third-order laterals that developed from them. Arbuscular mycorrhizal colonization increased the intensity of branching in all root orders with the effect being most obvious on first-order lateral roots where the number of branches increased from under 100 to over 300 brances m(-1). As a result, although first-order laterals made up 55% of the root systems of control plants, the comparable value was 36% in AM-infected plants. In contrast, second-order laterals represented 25% of control root systems, but 50% of AM-colonized root systems. Glomus intraradices but not G. mosseae increased root diameter. Anatomical studies revealed no changes in the overall form of the root tip, although there were changes in the diameter of the root cap, cell numbers and cell size. Hymenoscyphus ericae increased the duration of the metaphase index. Both AM fungal treatments increased the concentrations of soluble proteins in root extracts and modified the protein profiles by the elimination and addition of protein bands detected by PAGE analysis. We conclude that AM fungal inoculation influenced processes in the root system at different levels, but not all effects were due to improved P nutrition or increased physiological age.

Journal Article↗
PubMed2001

Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex.

Auxins represent an important class of plant hormone that regulate plant development. Plants use specialized carrier proteins to transport the auxin indole-3-acetic acid (IAA) to target tissues. To date, efflux carrier-mediated polar auxin transport has been assumed to represent the sole mode of long distance IAA movement. Localization of the auxin permease AUX1 in the Arabidopsis root apex has revealed a novel phloem-based IAA transport pathway. AUX1, asymmetrically localized to the plasma membrane of root protophloem cells, is proposed to promote the acropetal, post-phloem movement of auxin to the root apex. MS analysis shows that IAA accumulation in aux1 mutant root apices is impaired, consistent with an AUX1 phloem unloading function. AUX1 localization to columella and lateral root cap tissues of the Arabidopsis root apex reveals that the auxin permease regulates a second IAA transport pathway. Expression studies using an auxin-regulated reporter suggest that AUX1 is necessary for root gravitropism by facilitating basipetal auxin transport to distal elongation zone tissues.

Arabidopsis↗
PubMed1986

Associative Nitrogen Fixation by Klebsiella spp.: Adhesion Sites and Inoculation Effects on Grass Roots.

Adhesion sites on grass roots for Klebsiella strains carrying type 3 or type 1 fimbriae or both were determined. Adhesion of the strains to the roots of Poa pratensis and Festuca rubra was highly localized; the bacteria adhered strongly to root hairs and with a markedly lower efficiency to the surface of the zone of elongation and to the root cap mucilage. No adhesion to the epidermal cells between root hairs was observed. The adhesion sites were identical for the type 3- and 1-fimbriated bacteria and for P. pratensis, F. rubra, and Trifolium pratense. Inoculation of P. pratensis seedlings with Klebsiella pneumoniae strain As resulted in morphological changes in plant roots. The roots of infected plants were heavily covered with root hairs, which often were deformed and branched.

Journal Article↗
PubMed1993

A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants.

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to -1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (-1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5' sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, -908H3/GUS and -185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from -909 to -1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to -185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.

Base Sequence↗
PubMed1998

Functional analysis and cell-specific expression of a phosphate transporter from tomato.

For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Amino Acid Sequence↗
PubMed2004

Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members.

1-Aminocyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, and ACS11) homodimers and one nonfunctional (ACS1) homodimer. Transgenic Arabidopsis lines were constructed expressing the beta-glucuronidase (GUS) and green fluorescence protein (GFP) reporter genes from the promoter of each of the gene family members to determine their patterns of expression during plant development. All genes, except ACS9, are expressed in 5-d-old etiolated or light-grown seedlings yielding distinct patterns of GUS staining. ACS9 expression is detected later in development. Unique and overlapping expression patterns were detected for all the family members in various organs of adult plants. ACS11 is uniquely expressed in the trichomes of sepals and ACS1 in the replum. Overlapping expression was observed in hypocotyl, roots, various parts of the flower (sepals, pedicle, style, etc.) and in the stigmatic and abscission zones of the silique. Exogenous indole-3-acetic acid (IAA) enhances the constitutive expression of ACS2, 4, 5, 6, 7, 8, and 11 in the root. Wounding of hypocotyl tissue inhibits the constitutive expression of ACS1 and ACS5 and induces the expression of ACS2, 4, 6, 7, 8, and 11. Inducers of ethylene production such as cold, heat, anaerobiosis, and Li(+) ions enhance or suppress the expression of various members of the gene family in the root of light-grown seedlings. Examination of GUS expression in transverse sections of cotyledons reveals that all ACS genes, except ACS9, are expressed in the epidermis cell layer, guard cells, and vascular tissue. Similar analysis with root tip tissue treated with IAA reveals unique and overlapping expression patterns in the various cell types of the lateral root cap, cell division, and cell expansion zones. IAA inducibility is gene-specific and cell type-dependent across the root tip zone. This limited comparative exploration of ACS gene family expression reveals constitutive spatial and temporal expression patterns of all gene family members throughout the growth period examined. The unique and overlapping gene activity pattern detected reveals a combinatorial code of spatio-temporal coexpression among the various gene family members during plant development. This raises the prospect that functional ACS heterodimers may be formed in planta.

Arabidopsis↗
PubMed1998

The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo.

In Arabidopsis, the root meristem originates from the hypophyseal cell and from an adjoining cell tier that is distinct at the heart stage of embryogenesis. We have analysed mutations in the HOBBIT (HBT) gene that is essential for root meristem formation. hbt embryos display incorrect hypophyseal cell development from the quadrant stage onward. At the heart stage, the adjoining cell tier of hbt embryos develops abnormally, in that the activation of cell division and the formation of a lateral root cap layer are disturbed. Strong hbt mutants give rise to seedlings that lack an anatomically recognisable quiescent centre and differentiated columella root cap cells, the cell types derived from the wild-type hypophysis. Furthermore, they have no mitotically active root meristem and lack a differentiated lateral root cap. Secondary roots of hbt mutants and roots obtained from cultured cells of hbt mutants have similar defects. Therefore the HBT gene is required for root meristem formation in different developmental contexts.

Arabidopsis↗
PubMed2002

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments.

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.

ADP-Ribosylation Factor 1↗
PubMed1999

Endoxyloglucan transferase cDNA isolated from pea roots and its fluctuating expression in hydrotropically responding roots.

We isolated an endoxyloglucan transferase cDNA (Ps-EXGT1) from the roots of an agravitropic pea mutant, ageotropum. The putative product of the cDNA was 34.1 kDa and consisted of 293 amino acid residues. The predicted amino acid sequence was 75.1-88.6% identical to those of EXGT genes in other plants. The Ps-EXGT1 cDNA was strongly expressed in elongating roots and stems but not in either mature stems or young leaves. In roots, the transcription level of Ps-EXGT1 was most abundant in the rapidly growing region. When root elongation was inhibited by a water stress, Ps-EXGT1 transcription was repressed. The roots curved hydrotropically due to differential growth of the cortical cells in the elongation zone when the root cap was exposed to a gradient of water potential; the length of the cells on the side of lower water potential was much longer than those on the side of higher water potential. The expression pattern of Ps-EXGT1 in the hydrotropically responding roots fluctuated between the side of the higher water potential and that of the lower water potential in the elongation zone. In other words, the accumulation of Ps-EXGT1 mRNA was much greater on the side of lower water potential than on that of higher potential just prior to the commencement of positive hydrotropism. When the roots started to curve slightly away from the side of higher water potential causing a rhythmic oscillatory movement [Takano et al. (1995) Planta 197: 410], there was more transcription of Ps-EXGT1 on the side of higher water potential. These results suggest that the transcription of Ps-EXGT1 is involved in cell growth and that this regulation of transcription plays a role in the differential growth of hydrotropically responding roots.

Amino Acid Sequence↗
PubMed1996

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant.

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 microM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 microM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.

Arabidopsis↗
PubMed2004

Lateral ABA transport in maize roots (Zea mays): visualization by immunolocalization.

The intensity of an ABA (abscisic acid) signal as a root-to-shoot signal, as well as its action on root hydraulic conductivity, strongly depends on the distribution of ABA during its radial transport across roots. Therefore ABA was visualized by immunolocalization with monoclonal ABA antibodies under conditions of lateral water flow induced by the application of a pressure gradient to the cut surface of the mesocotyl of maize seedlings. From the labelling of rhizodermis, hypodermis, cortical cells, and endodermis of roots of hydroponically (no exodermis) and aeroponically (with exodermis) grown seedlings it is concluded that the exodermis acts as a barrier to apoplastic transport that controls ABA uptake and efflux, but that the endodermis can easily be overcome via an apoplastic bypass. In longitudinal sections the strongest ABA signals originated from the root cap and the meristematic root tip, which is in agreement with the non-vacuolated cells of these tissues being an effective anion trap for ABA.

Abscisic Acid↗
PubMed1999

Immunogold localization of plant surface arabinogalactan-proteins using glycerol liquid substitution and scanning electron microscopy.

We have studied the spatial distributions of arabinogalactan-protein (AGP) epitopes on the surface of maize embryogenic calli and roots using monoclonal antibodies JIM4 and MAC207. For this purpose, a new immunogold-scanning electron microscopy (SEM) method was employed which is based on liquid substitution of samples with glycerol. Using this method, we were able to show that the AGP epitopes are distributed along callus and root surfaces and they decorate filamentous structures. In callus cells, the JIM4 epitope was specifically enriched in the outer extracellular layers covering compact clusters of embryogenic meristematic callus cells. In roots, the MAC207 epitope was abundant on the root epidermal surface corresponding to the outer root pellicle, but was only occasionally found on the mucilage layer covering the root cap cells. Silver-enhanced gold particles, indicating AGP epitopes, were often linearly arranged suggesting that AGPs associate with filamentous structures both on the surface of embryogenic calli and root epidermal cells. These results indicate that AGPs are components of the outer extracellular layers and networks that cover the surface of roots and cells undergoing somatic embryogenesis.

Epitopes↗
PubMed2001

Recovery of gravitropic response during regeneration of root caps does not require developed columella cells and sedimentation of amyloplasts.

Correlations between regeneration of the root cap and recovery of a gravitropic response were studied using primary roots of Phaseolus vulgaris. After removal of various lengths of the root tip a gravistimulus was continuously given to the root. The statistical analysis of data showed that recovery of the gravitropic response was gradually delayed as the length of the tips removed increased. This suggested that the columella cells of the root cap were involved in gravitropism. When the root cap was completely removed, the roots did not respond to gravistimuli for the first 15 h and began to reorient their growth direction at 20 h. At this time, the columella cells had just begun to regenerate and had immature amyloplasts which did not sufficiently form a sediment. These results suggest that other systems of perception exist in plant cells in addition to the amyloplast-based model of graviperception.

Fabaceae↗
PubMed2001

Developmental and thermal regulation of the maize heat shock protein, HSP101.

The plant heat stress protein, Hsp101, and the yeast ortholog, Hsp104, are required to confer thermotolerance in plants and yeast (Saccharomyces cerevisiae), respectively. In addition to its function during stress, Hsp101 is developmentally regulated in plants although its function during development is not known. To determine how the expression of Hsp101 is regulated in cereals, we investigated the Hsp101 expression profile in developing maize (Zea mays). Hsp101 protein was most abundant in the developing tassel, ear, silks, endosperm, and embryo. It was less abundant in the vegetative and floral meristematic regions and was present at only a low level in the anthers and tassel at anthesis, mature pollen, roots, and leaves. As expected, heat treatment resulted in an increase in the level of Hsp101 protein in several organs. In expanding foliar leaves, husk leaves, the tassel at the premeiosis stage of development, or pre-anthesis anthers, however, the heat-mediated increase in protein was not accompanied by an equivalent increase in mRNA. In contrast, the level of Hsp101 transcript increased in the tassel at anthesis following a heat stress without an increase in Hsp101 protein. In other organs such as the vegetative and floral meristematic regions, fully expanded foliar leaves, the young ear, and roots, the heat-induced increase in Hsp101 protein was accompanied by a corresponding increase in Hsp101 transcript level. However, anthers at anthesis, mature pollen, developing endosperm, and embryos largely failed to mount a heat stress response at the level of Hsp101 protein or mRNA, indicating that Hsp101 expression is not heat inducible in these organs. In situ RNA localization analysis revealed that Hsp101 mRNA accumulated in the subaleurone and aleurone of developing kernels and was highest in the root cap meristem and quiescent center of heat-stressed roots. These data suggest an organ-specific control of Hsp101 expression during development and following a heat stress through mechanisms that may include posttranscriptional regulation.

Amino Acid Sequence↗
PubMed2001

A resin embedding method for transparent teeth with ink-infiltrated pulp cavities.

This study was designed to evaluate whether resin embedded transparent teeth are as convenient to use as classical transparent teeth. For this purpose demineralized human teeth were divided into coronal and radical portions, and pulp tissue was extracted from the pulp chamber and root canals, into which drawing ink was injected. After dehydration, the specimens were made transparent in methyl salicylate and immersed in polyester resin. The divided portions were recombined at the polymerization. The resin embedded teeth maintained transparency and the black-stained pulp chamber and root canals showed morphological details. The resin embedded specimens could be handled manually and observed freely from any angle. Previously, transparent teeth have been observed in transparent media through a capped glass bottle. In this respect the resin embedding method is superior to the classical method. The new method will be helpful for investigating root canal morphology.

Dental Pulp↗
PubMed2004

The actin-interacting protein AIP1 is essential for actin organization and plant development.

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.

Actins↗
PubMed1987

Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips.

We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability. None of these Tn5 insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model cap formation as a two-step process is discussed.

Adhesins, Bacterial↗