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Rhizobia modulate root-hair-specific expression of extensin genes.

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5' flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.

Amino Acid Sequence↗

The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to Brassica S-locus glycoproteins.

Non-embryogenic carrot suspension cells secrete the EP1 glycoprotein. A cDNA clone encoding EP1 was isolated and sequenced. The EP1 sequence revealed a region of homology with Brassica S-locus glycoprotein genes, an Arabidopsis S-like gene and putative S-like receptor protein kinases from maize and Arabidopsis. EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot. In developing seeds, expression was most pronounced in both the inner and outer integument epidermis.

Amino Acid Sequence↗

Maize genes specifically expressed in the outermost cells of root cap.

The cells at the periphery of the root cap are continuously sloughed off from the root into the mucilage, and are thought to be programmed to die. By using subtractive hybridization/differential screening and a transmembrane-domain trapping screening strategy involving expression screening in transfected COS-7 cells, we isolated two related maize cDNAs (ZmRCP1 and ZmRCP2) that are specifically expressed in the outermost one to three cells of the cap. ZmRCP1 and ZmRCP2 are homologous proteins of 37 kDa mature polypeptides with a region of regularly-spaced Cys residues and putative N-terminal signal peptides, and represent members of a novel protein family which is conserved among angiosperm and gymnosperm.

Amino Acid Sequence↗

Cloning and characterization of two subunits of Arabidopsis thaliana nuclear cap-binding complex.

In this report we characterize two Arabidopsis thaliana proteins, named AtCBP20 and AtCBP80, that are homologues of human subunits of a nuclear cap-binding protein complex (CBC). AtCBP20 has a calculated molecular mass of 29.9 kDa, and AtCBP80 is a 96.5 kDa protein. AtCBP20 exhibits 68% identity and 82% similarity to human CBP20. Like its human homologue, AtCBP20 contains a canonical RNA binding domain (RBD) with single RNP2 and RNP1 motifs. In addition to the N-terminal part, which is similar to the human protein, AtCBP20 has a long C-terminus rich in arginine, glycine and aspartate residues. The second subunit of the Arabidopsis cap-binding complex, AtCBP80, shows 28% identity and 50% similarity to its homologue from HeLa cells. The protein contains a MIF4G domain at its N-terminus, the feature characteristic to all analyzed CBP80s. This domain, described also in eIF4G and NMD2 proteins, is thought to be involved in protein-protein and also in protein--RNA interactions. Both proteins AtCBP20 and AtCBP80 are encoded by single-copy genes in the A. thaliana genome. The AtCBP20 gene is located on chromosome V, and the AtCBP80 gene is encoded by chromosome II. Among introns identified in the AtCBP20 gene, we discovered an U12 type intervening sequence (an AT-AC intron). This intron is spliced out very efficiently in plants, but when isolated and tested for splicing in tobacco protoplasts, the efficiency of the U12 intron excision was low. Splicing efficiency of the U12 intron is improved by the addition of exon and intron sequences upstream or downstream of the U12 intron. AtCBP20 and AtCBP80 are constitutively expressed in all examined organs of A. thaliana, including roots, stems, leaves and flowers. Interestingly, the steady-state level of both transcripts seem to be very similar in all tissues analyzed.

Amino Acid Sequence↗

Cloning and functional characterization of a formin-like protein (AtFH8) from Arabidopsis.

The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.

Actin Cytoskeleton↗

Revegetation strategies for bauxite refinery residue: a case study of Alcan Gove in Northern Territory, Australia.

Alumina extraction from bauxite ore with strong alkali produces waste bauxite refinery residue consisting of residue sand and red mud. The amount and composition of refinery residue depend on the purity of the bauxite ore and extraction conditions, and differs between refineries. The refinery residue is usually stored in engineered disposal areas that eventually have to be revegetated. This is challenging because of the alkaline and sodic nature of the residue. At Alcan Gove's bauxite refinery in Gove, Northern Territory, Australia, research into revegetation of bauxite residue has been conducted since the mid-1970s. In this review, we discuss approaches taken by Alcan Gove to achieve revegetation outcomes (soil capping of refinery residue) on wet-slurry disposal areas. Problems encountered in the past include poor drainage and water logging during the wet season, and salt scalding and capillary rise during the dry season. The amount of available water in the soil capping is the most important determinant of vegetation survival in the seasonally dry climate. Vegetation cover was found to prevent deterioration of the soil cover by minimising capillary rise of alkalinity from the refinery residue. The sodicity and alkalinity of the residue in old impoundments has diminished slightly over the 25 years since it was deposited. However, development of a blocky structure in red mud, presumably due to desiccation, allows root penetration, thereby supplying additional water to salt and alkali-tolerant plant species. This has led to the establishment of an ecosystem that approaches a native woodland.

Aluminum↗

Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot.

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.

Amino Acid Sequence↗

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

Arabidopsis Proteins↗

The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize.

Al-induced release of Al-chelating ligands (primarily organic acids) into the rhizosphere from the root apex has been identified as a major Al tolerance mechanism in a number of plant species. In the present study, we conducted physiological investigations to study the spatial and temporal characteristics of Al-activated root organic acid exudation, as well as changes in root organic acid content and Al accumulation, in an Al-tolerant maize (Zea mays) single cross (SLP 181/71 x Cateto Colombia 96/71). These investigations were integrated with biophysical studies using the patch-clamp technique to examine Al-activated anion channel activity in protoplasts isolated from different regions of the maize root. Exposure to Al nearly instantaneously activated a concentration-dependent citrate release, which saturated at rates close to 0.5 nmol citrate h(-1) root(-1), with the half-maximal rates of citrate release occurring at about 20 microM Al(3+) activity. Comparison of citrate exudation rates between decapped and capped roots indicated the root cap does not play a major role in perceiving the Al signal or in the exudation process. Spatial analysis indicated that the predominant citrate exudation is not confined to the root apex, but could be found as far as 5 cm beyond the root cap, involving cortex and stelar cells. Patch clamp recordings obtained in whole-cell and outside-out patches confirmed the presence of an Al-inducible plasma membrane anion channel in protoplasts isolated from stelar or cortical tissues. The unitary conductance of this channel was 23 to 55 pS. Our results suggest that this transporter mediates the Al-induced citrate release observed in the intact tissue. In addition to the rapid Al activation of citrate release, a slower, Al-inducible increase in root citrate content was also observed. These findings led us to speculate that in addition to the Al exclusion mechanism based on root citrate exudation, a second internal Al tolerance mechanism may be operating based on Al-inducible changes in organic acid synthesis and compartmentation. We discuss our findings in terms of recent genetic studies of Al tolerance in maize, which suggest that Al tolerance in maize is a complex trait.

Adaptation, Physiological↗

Immunocytolocalization of tryptophan decarboxylase in Catharanthus roseus hairy roots.

We investigated the intracellular distribution of tryptophan decarboxylase (TDC) (EC 4.1.1.28) in Catharanthus roseus hairy roots using immunofluorescence and immunogold techniques. TDC was detected by immunofluorescence localization in the cytosol and in the apoplastic region of the meristematic cells of the roots, with a slight enrichment in the epidermal cells of the root cap and in the meristematic region. In the enlargement zone, TDC was localized only in the first three layers of the cortex. In the maturation zone, the enzyme was not present. Immunogold studies confirmed that the enzyme was localized in the cytosol of the meristematic region, and intense gold labeling was found in the apoplastic zone. A protein fraction isolated from the apoplastic zone and assayed for TDC activity showed high activity.

Aromatic-L-Amino-Acid Decarboxylases↗

Happy end in sight after 70 years of controversy.

Differential auxin transport from the columella to lateral root cap cells as a result of root gravitropism has recently been shown using a GFP-based auxin biosensor. Together with the recent finding of gravity-dependent localization of an auxin efflux carrier, these results strongly support the Cholodny-Went hypothesis for the tropic response, which has been disputed for 70 years.

Biosensing Techniques↗

Nonvascular, Symplasmic Diffusion of Sucrose Cannot Satisfy the Carbon Demands of Growth in the Primary Root Tip of Zea mays L.

Nonvascular, symplasmic transport of sucrose (Suc) was investigated theoretically in the primary root tip of maize (Zea mays L. cv WF9 x Mo 17) seedlings. Symplasmic diffusion has been assumed to be the mechanism of transport of Suc to cells in the root apical meristem (R.T. Giaquinta, W. Lin, N.L. Sadler, V.R. Franceschi [1983] Plant Physiol 72: 362-367), which grow apical to the end of the phloem and must build all biomass with carbon supplied from the shoot or kernel. We derived an expression for the growth-sustaining Suc flux, which is the minimum longitudinal flux that would be required to meet the carbon demands of growth in the root apical meristem. We calculated this flux from data on root growth velocity, area, and biomass density, taking into account construction and maintenance respiration and the production of mucilage by the root cap. We then calculated the conductivity of the symplasmic pathway for diffusion, from anatomical data on cellular dimensions and the frequency and dimensions of plasmodesmata, and from two estimates of the diffusive conductance of a plasmodesma, derived from independent data. Then, the concentration gradients required to drive a growth-sustaining Suc flux by diffusion alone were calculated but were found not to be physiologically reasonable. We also calculated the hydraulic conductivity of the plasmodesmatal pathway and found that mass flow of Suc solution through plasmodesmata would also be insufficient, by itself, to satisfy the carbon demands of growth. However, much of the demand for water to cause cell expansion could be met by the water unloaded from the phloem while unloading Suc to satisfy the carbon demands of growth, and the hydraulic conductivity of plasmodesmata is high enough that much of that water could move symplasmically. Either our current understanding of plasmodesmatal ultrastructure and function is flawed, or alternative transport mechanisms must exist for Suc transport to the meristem.

Journal Article↗

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.

Conjugation, Genetic↗

Rhizophores in Rhizophora mangle L: an alternative interpretation of so-called ''aerial roots''.

Rhizophora mangle L., one of the most common mangrove species, has an aerial structure system that gives it stability in permanently swampy soils. In fact, these structures, known as "aerial roots" or "stilt roots", have proven to be peculiar branches with positive geotropism, which form a large number of roots when in contact with swampy soils. These organs have a sympodial branching system, wide pith, slightly thickened cortex, collateral vascular bundles, polyarch stele and endarch protoxylem, as in the stem, and a periderm produced by a phellogen at the apex similar to a root cap. They also have the same type of trichosclereid that occurs in the stem, with negative geotropism, unlike true Rhizophora roots, which do not form trichosclereids at all. On the other hand, these branches do not form leaves and in this respect they are similar to roots. These peculiar branches are rhizophores or special root-bearing branches, analogous to those found in Lepidodendrales and other Carboniferous tree ferns that grew in swampy soils.

Brazil↗

Identification of an isozyme form of protein synthesis initiation factor 4F in plants.

We showed previously that wheat germ extracts contain two forms of protein synthesis initiation factor 4F that have very similar functional properties (Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232). One form, designated eIF-4F, is a complex containing two subunits, p220 and p26. The other form, designated eIF-(iso)4F, is a complex containing two subunits, p82 and p28, which are antigenically distinct from the subunits of eIF-4F. Both the p26 subunit of eIF-4F and the p28 subunit of eIF-(iso)4F are m7G cap-binding proteins. In this investigation, affinity-purified antibodies to the p220 and p26 subunits of wheat germ eIF-4F and to the p82 and p28 subunits of wheat germ eIF-(iso)4F were used to determine if isozyme forms of eIF-4F are present in maize and cauliflower. Extracts from wheat germ, maize root tips, and cauliflower inflorescences were partially purified by adsorption on m7GTP-Sepharose and elution with m7GTP (MGS eluate). Analysis by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies to the subunits of the wheat germ factors showed that the MGS eluate from maize contains polypeptides that react with antibodies to the p82 and p28 subunits of wheat eIF-(iso)4F, as well as polypeptides that react with antibodies to the p220 and p26 subunits of wheat eIF-4F. The MGS eluate from cauliflower also contains polypeptides that reacted with antibodies to the subunits of wheat eIF-(iso)4F. These results indicate that both maize and cauliflower contain the isozyme form of eIF-4F. In addition, it was found that the factors in the MGS eluate from maize support polypeptide synthesis in a system from wheat deficient in eIF-4F and eIF-(iso)4F, whereas the factors in the MGS eluate from cauliflower support polypeptide synthesis only to a small extent.

Brassica↗

Expression pattern of transcripts encoding water channel-like proteins in Norway spruce (Picea abies).

With the aim of identifying cells and tissues with high expression of aquaporins (water channels) or homologous genes in Norway spruce (Picea abies), we report the expression patterns of such transcripts in seedlings, in roots of various ages, and in needles. In situ hybridization experiments with a conserved area of a tonoplast intrinsic protein (TIP) gene from P. abies gave high expression signals in differentiating vascular tissues and in the columella cells of the seedling root cap. High-staining signals were also seen in guard cells and in the bundle sheath cells of needles. Moreover, a slightly increased staining signal was seen in cells forming lateral roots as well as in adventitious roots formed from hypocotyl cuttings. By using PCR-based procedures we also identified a full-length aquaporin-like cDNA (mipr) from roots of two-week old seedlings. Sequence homology analysis of the gene suggests that it belongs to the TIP subgroup within the large MIP (major intrinsic protein) family. A phylogenetic analysis of the plant MIP family, including both plasmamembrane (PIP) and tonoplast intrinsic protein (TIP) from Picea, suggests that MIP subgroups evolved already 330 million years ago, as this is the dating of conifer and angiosperm divergence.

Amino Acid Sequence↗

A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia.

The frequency of lateral root initiation in tomato (Lycopersicon esculentum Mill cv. VFN8) seedling roots is increased over eightfold in response to 1.6 microM alpha-naphthalene-acetic acid (NAA). To identify genes that are activated during lateral root initiation, a cDNA library was made with RNA from roots treated with auxin and differentially screened with radioactive probes made from RNA isolated from treated and untreated roots. A cDNA clone, TR132, was identified that hybridized to a transcript that was induced within 4 h of auxin treatment and increased tenfold by 72 h. A gene (RSI-1) corresponding to the TR132 cDNA was cloned and characterized with regard to its nucleotide sequence, transcription start site and chromosomal map position. Approximately 1 kb of the 5' flanking DNA was linked to the beta-glucuronidase (GUS) protein coding region and tested for expression in transgenic tomato seedlings. GUS activity was observed in both lateral and adventitious root initials, including very early initials, and persisted until shortly after the lateral emerged from the parent tissue. In roots from seedlings with high activity, GUS expression was also observed in the root cap and vascular tissue. The predicted RSI-1 protein is rich in cysteine, lysine and proline, and includes an N-terminal region with characteristics of a signal peptide. The putative mature protein exhibits 79% amino acid identity to a protein encoded by a gene (GAST1) that is induced by gibberellic acid in tomato shoots.

Amino Acid Sequence↗

Isolated root caps, border cells, and mucilage from host roots stimulate hyphal branching of the arbuscular mycorrhizal fungus, Gigaspora gigantea.

Unlike previous reports that have shown that water soluble and volatile compounds from roots or root exudates play an important role in precolonization events during arbuscular mycorrhizal (AM) fungus-host root interactions (Bécard & Piché 1989, Giovannetti et al. 1993), the results shown here deal with particulate and viscous fractions isolated from host roots. Root caps and a slow sedimenting particulate fraction (SSPF) were rapidly isolated and separated from Ri T-DNA transformed carrot roots (D. carota) grown in liquid culture. In addition, border cells (BC) and mucilage were isolated from aseptically grown corn seedlings (Zea mays). Root caps, SSPF (composed mainly of small root cap fragments and some BCs), BCs, and mucilage all had an associated AM fungus hyphal branching stimulator. Root caps stored for 5 d at 4 degrees C appeared to either synthesize or slowly release the branching stimulator. Also, isolated root caps from roots grown in the absence of P contained more branch stimulating activity than those isolated from roots grown in the presence of P. Although the branching stimulation activity in particulate fractions was low compared to that of the exudate, the particulate fractions can stick to the root surface at considerable distances from the root tip. This may be significant during the infection and colonization of host roots at sites far removed from the primary location of exudation.

Daucus carota↗