Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

Root primordia and endogenous auxin in maize roots cultured in vitro

Quantitative analyses of indol-3yl-acetic acid (I aa ) in Zea mays L. (cv. LG 11) root segments cultured in vitro were performed by gas chromatography-mass spectrometry with selected ion monitoring. The root extracts were first purified by highperformance liquid chromatography. Root primordia initiation in intact and decapitated roots showed different patterns: decapitation strongly enhanced primordia initiation in their first 10 mm. During the culture (5 days), I aa content decreased in both intact and decapitated roots. No correlation was found between the level of endogenous auxin and the numher of root primordia initiated from either intact or decapitated maize root segments.     

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an olgosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.     

Development of nodule bacteria in soil

The rate of growth of a population of nodule bacteria in soil and the time it takes for the cell generation depend on the content and accessibility of necessary nutrient substances. The periods of adaptation, active growth, and stabilization of the population number in the course of growth of a population are typical of soddy-podzolic continuous-cultivated soil. The duration of each period is determined by a sum of ecological factors.     

Development of the Drosophila genital disc requires interactions between its segmental primordia

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. We show that Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc.     

The course of root differentiation from root primordia in poplar stems

Upon rooting of poplar stem cuttiags the total inductive stimulation does not take place, but the individual root primordia maintain a relative independence. This becomes evident by various time parameters of their differentiation. Under experimental conditions, in dormant cuttings from one year individuals of the hybrid I 214 the activation of the first root primordia occurs after 24 h, the pre-emergent development of the roots formed was completed after 72 h. The activated root primordium is divided into two regions. In the distal region with the predominating cell division the root apex with histogens is formed by the action of initials. From peripheral cell layers of the distal region the so-called “Wurzeltasche” develops which covers the root cap. Due to cell elongation in the proximal region the root apex is pushed up towards the stem surface. The beginning differentiation of the connective vascular tissue is a preparative step for the connection of the vascular system of the developing root with the secondary vascular system of the maternal stem. Following the penetration of the root through the peripheral stem tissues this connection is realized with progressing development. In the developed root the protoxylem elements differentiate continuously and acropetally in direct continuity with tracheids of the basal connective region.     

Organ culture of craniofacial primordia

Organ culture systems have proven extremely useful techniques in studies that investigate the process of normal and abnormal development. The explant of tissues into an organ culture system is one of the few techniques that maintains three-dimensional cellular interactions under conditions that simultaneously permit controlled experimental manipulation in vitro. In this article we outline a procedure for growing "faces" in culture. In this system, the facial primordia prepared from embryonic mice or chicks can be maintained in culture for up to 7--8 days. During this time, the facial primordia continue to grow, differentiate, fuse and develop into organized structures that closely resemble those observed in situ. The procedure is relatively simple, requiring only a stable substratum, culture medium, sufficient oxygenation and incubation of the organ system at 37 degrees C. The advantages and disadvantages of the procedure are presented, along with detailed methods to help troubleshoot some of the common pitfalls of organ culture systems.     

Shoot meristem differentiation in shoot primordia culture (SPC) obtained from root primordia culture (RPC) of Solanum lycopersicoides Dun.

We present a unique example of conversion of the morphogenesis type (from rhisogenesis to shoot organogenesis) in in vitro cultures of Solanum lycopersicoides. Liquid shoot primordia cultures (SPC) were obtained from root primordia culture (RPC) on two kinds of MS-based media with BA. The first SMS₈, contained a full set of organic compounds; the second, ²SMS₈, was devoid of organic compounds except sucrose and edamine. Two weeks after passage of RPC onto both the media, disintegration of root primordia and cell aggregates began. After 8 weeks of cultivation on SMS₈ and ²SMS₈ media, SPC aggregates developed from meristematic cells located near the vascular tissue of disintegrating RPC aggregates. Initiation of shoot meristems started from meristematic cells centers which were localised under the surface of the newly formed SPC aggregates. The change in the type of morphogenesis occurred faster on medium SMS₈, but the SPC on medium ²SMS₈ was characterized by more frequent formation of shoots and plants (45% of aggregates, in the case of SPC on medium SMS₈, and 90% in the case of SPC on medium ²SMS₈). This fact was correlated with the structural organization of the meristematic centers. Our results indicate that an RPC system has high morphogenetic potential due to the continual presence of meristematic cells. The change in the type of morphogenesis was followed by a rebuilding of the aggregate structure on the basis of the meristematic cells already existing in RPC, which gave rise to SPC aggregates from which shoot meristems developed.     

Chondrogenesis and myogenesis in micromass cultures of mesenchyme from mouse facial primordia

Summary The face develops from small buds of tissue positioned around the primitive mouth. The chondrogenic and myogenic cell populations contained within these facial primordia in mouse embryos have been investigated in short-term micromass culture. Chondrogenesis occurred in frontonasal mass mesenchyme from E11-E13 embryos, in maxillary mesenchyme from E12.5 embryos and was absent in mandibular mesenchyme. Myogenesis was greatest in mandibular mesenchyme, moderate in maxillary mesenchyme and low in the frontonasal mass. When compared with chick embryos the mouse facial primordia have lower chondrogenic potential, which in the case of the frontonasal mass may be related to the relative outgrowth of the primordia in the two species. Chondrogenesis in the mouse mandibular mesenchyme may be affected by the presence of a large population of odontogenic mesenchyme. The behavior of myogenic cell populations is related to the pattern of the musculature of the face, as the mandible contains the most muscle, the maxilla some, and the frontonasal mass none. However, the presence of myoblasts in early mesenchyme of all primordia may indicate that, as with chick, facial primordia are initially seeded with muscle cells and that the size of the cell population is subsequently controlled according to the development of the musculature within the primordia.     

Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species

The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.     

Development and function ofAzospirillum-inoculated roots

Summary The surface distribution ofAzospirillum on inoculated roots of maize and wheat is generally similar to that of other members of the rhizoplane microflora. During the first three days, colonization takes place mainly on the root elongation zone, on the base of root hairs and, to a lesser extent, on the surface of young root hairs.Azospirillum has been found in cortical tissues, in regions of lateral root emergence, along the inner cortex, inside xylem vessels and between pith cells. Inoculation of several cultivars of wheat, corn, sorghum and setaria with several strains ofAzospirillum caused morphological changes in root starting immediately after germination. Root length and surface area were differentially affected according to bacterial age and inoculum level. During the first three weeks after germination, the number of root hairs, root hair branches and lateral roots was increased by inoculation, but there was no change in root weight. Root biomass increased at later stages. Cross-sections of inoculated corn and wheat root showed an irregular arrangement of cells in the outer layers of the cortex. These effects on plant morphology may be due to the production of plant growth-promoting substances by the colonizing bacteria or by the plant as a reaction to colonization. Pectic enzymes may also be involved. Morphological changes had a physiological effect on inoculated roots. Specific activities of oxidative enzymes, and lipid and suberin content, were lower in extracts of inoculated roots than in uninoculated controls. This suggests that inoculated roots have a larger proportion of younger roots. The rate of NO^– ₃, K⁺ and H₂PO^– ₄ uptake was greater in inoculated seedlinds. In the field, dry matter, N, P and K accumulated at faster rates, and water content was higher inAzospirillum-inoculated corn, sorghum, wheat and setaria. The above improvements in root development and function lead in many cases to higher crop yield.     

Differentiation of myogenic cells in micromass cultures of cells from chick facial primordia

Antibodies to the myosin heavy chains of striated muscle were used to trace myogenic differentiation in the developing face and in cultures of cells from the facial primordia of chick embryos. In the intact face, myogenic cells differentiate first in the mandibular primordia and can be detected at stage 28. The early muscle blocks contain both fast and slow classes of myosin heavy chains. At stages 20 and 24, no myogenic cells are found in any of the facial primordia. However, when the cells are placed in micromass (high density) cultures, myogenic cells differentiate, revealing the presence of potentially myogenic cells in all the facial primordia. The number of myogenic cells bears no consistent relationship to the extent and pattern of chondrogenesis. Therefore the ability of the cell populations of the facial primordia to differentiate into cartilage when placed in culture is independent of the muscle cell lineage. The facial primordia represent a mixed cell population of neural crest and mesodermal cells from at least as early as stage 18.     

Genetic properties of abortive products resulting from the protoplast fusion in yeasts

Summary Protoplast fusion was carried out by using a routine technique with various auxotrophic strains of Saccharomyces diastaticus and Schizosaccharomyces pombe, and abortive fusion products were observed as small colonies which appeared more frequently than large prototrophic colonies. Sixty abortive fusion products retained one or more auxotrophic characters derived from S. diastaticus, one of the strains used in the protoplast fusion. Several hybrids were obtained between the abortive products and S. cerevisiae, and the segregants of these hybrids showed many aberrant tetrads with regard to some genetic markers. These segregation patterns would be likely to result if the segregating characters were in the trisomic condition +/+/-. The results indicate that (1) the abortive fusion products are an alien monosome additional (AMA) haploid containing the genome of S. diastaticus and only one chromosome of S. pombe. (2) The additional chromosome of S. pombe, which is integrated with the genome of Saccharomyces, can be stably transmitted to the progeny.     

Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis

During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle.     

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.