Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant.

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa.

We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors. Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation. The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed. This zone encompasses root hairs that have almost reached their maximal size. The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone. In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases. NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac). No correlation was found between Nod factor degradation and susceptibility. Degradation occurs both in the susceptible zone and in the mature zone. Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).

Sym2 of Pea Is Involved in a Nodulation Factor-Perception Mechanism That Controls the Infection Process in the Epidermis.

In pea (Pisum sativum) up to 50 nodulation mutants are known, several of which are affected in the early steps of the symbiotic interaction with Rhizobium sp. bacteria. Here we describe the role of the sym2 gene in nodulation (Nod) factor perception. Our experiments show that the sym2A allele from the wild pea variety Afghanistan confers an arrest in infection-thread growth if the Rhizobium leguminosarum bv viciae strain does not produce Nod factors with a NodX-mediated acetylation at their reducing end. Since the induction of the early nodulin gene ENOD12 in the epidermis and the formation of a nodule primordium in the inner cortex were not affected, we conclude that more than one Nod factor-perception mechanism is active. Furthermore, we show that sym2A-mediated control of infection-thread growth was affected by the bacterial nodulation gene nodO.

Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation.

Nod factors secreted by Rhizobium leguminosarum by, viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (+/-2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3, Nod factor perception and transduction leading to gene expression are unaffected.

Molecular genetic analysis of proline and tryptophan biosynthesis in Streptomyces coelicolor A3(2): interaction between primary and secondary metabolism--a review.

We studied the control of proline metabolism and tryptophan biosynthesis in Streptomyces coelicolor A3(2), because proline is involved in secondary metabolism [undecylprodigiosin (Red) biosynthesis] whilst tryptophan, to our knowledge, is not. Proline transport was constitutive in wild-type cells, as were the enzymes of proline catabolism. When we analysed proline biosynthesis, we discovered that growth in the presence of proline stimulated rather than repressed the biosynthetic genes. We isolated proline transport mutants and to our surprise discovered that such strains overproduced Red. As well as losing the ability to transport proline, they had lost, to differing extents, the ability to degrade proline. However, proline biosynthesis appeared to be unaffected. It appears that proline anabolism and catabolism in S. coelicolor A3(2) is in a state of dynamic equilibrium and that if this balance is disturbed, Red biosynthesis can act as a sink for excess proline. We cloned the trpD and the trpCBA clusters of S. coelicolor A3(2) and identified a promoter within the latter cluster. This promoter appeared not to be regulated by the presence or absence of exogenous tryptophan, but rather by the growth phase and/or the growth rate of the culture. It appears, therefore, that an amino acid biosynthetic pathway that is apparently not involved in secondary metabolism in the streptomycete is regulated at the genetic level--not by feedback repression, but rather by the overall physiological state of the cell.

Digging out roots: pattern formation, cell division, and morphogenesis in plants.

The analysis of plant development by genetic, molecular, and surgical approaches has accumulated a large body of data, and yet it remains a challenge to uncover the basic mechanisms that are operating. Early steps of development, when the zygote and its daughter cells organize the embryonic plant, are poorly understood despite considerable efforts toward the identification of relevant genes. Reported cases of genetic redundancy suggest that the difficulty in uncovering patterning genes may reflect overlapping gene activities. Our current knowledge on plant embryo development still leaves open whether mechanisms for axis formation and subsequent pattern formation are fundamentally different in animals and plants. Axis formation may follow the general principle of establishing a peripheral asymmetric cue and mobilizing the cytoskeleton toward this cue--in the case of plants possibly located in the cell wall--but the molecules involved may be entirely different. Embryonic pattern formation involves the establishment of different domains, but although there are candidates, it is not clear whether genes that define these domains are identified yet. Pattern formation continues postembryonically in the meristem, and the flexibility of this process may be explained by a feed-forward system of patterning cues originating from more mature cells. Control of cell division and differentiation, which is important in the meristems--regions of continuous development--has been studied intensively and appears to involve short-range signaling and transmembrane receptor kinase activation. Finally, although high importance of control of cell division rates and planes for plant morphogenesis have been often inferred, recent genetic studies as well as comparative morphological data point to a less decisive role of cell division and to global controls of as yet unknown nature.

The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression.

Transformation of tryptophan auxotrophs of Streptomyces coelicolor A3(2) and subsequent analysis have allowed the identification of four tryptophan biosynthetic genes. Subcloning, complementation of trp strains, nucleotide sequencing of 5.1 kb and 1.95 kb of DNA and subsequent homology comparisons identified the trpC, trpB and trpA genes and trpD gene respectively. The arrangement of genes in the trpCBA cluster is unusual in that trpC is separated by a small open reading frame, trpX, from the potentially translationally coupled trpB and trpA genes. Sequence analysis of the trpD gene revealed the presence of a large mRNA loop structure directly upstream of the trpD-coding region. S1 nuclease mapping studies of trpCXBA have revealed two major potential transcription start points, one just upstream of the trpC gene and the other located upstream of the trpX gene. S1 nuclease mapping of the trpD region revealed four fragment end-points. Quantitative S1 nuclease protection assays and a promoterless catechol dioxygenase reporter gene have revealed that the expression of all these genes is growth phase dependent and growth rate dependent, expression being maximal during early exponential phase and dropping off sharply in late exponential phase. This growth phase-dependent and growth rate-dependent regulation is the first reported in streptomycete primary metabolism.

Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue.

The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissue-specific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction.

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, involving a reinitiation of tip growth, and the formation of nodule primordia in Vicia sativa (vetch). Ethylene is a potent inhibitor of cortical cell division, an effect that can be counteracted by applying silver ions (Ag+) or aminoethoxy-vinylglycine (AVG). In contrast to the inhibitory effect on cortical cell division, ethylene promotes the formation of root hairs (which involves tip growth) in the root epidermis of Arabidopsis. We investigate the possible paradox concerning the action of ethylene, putatively promoting Nod factor induced tip growth whilst, at the same time, inhibiting cortical cell division. We show, by using the ethylene inhibitors AVG and Ag+, that ethylene has no role in the reinitiation of root hair tip growth induced by Nod factors (root hair deformation) in vetch. However, root hair formation is controlled, at least in part, by ethylene. Furthermore, we show that ACC oxidase, which catalizes the last step in ethylene biosynthesis, is expressed in the cell layers opposite the phloem in that part of the root where nodule primordia are induced upon inoculation with Rhizobium. Therefore, we test whether endogenously produced ethylene provides positional information controlling the site where nodule primordia are formed by determining the position of nodules formed on pea roots grown in the presence of AVG or Ag+.

Lipo-oligosaccharides of Rhizobium induce infection-related early nodulin gene expression in pea root hairs.

This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host-specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.