Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs.

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

Genetic modification of plant secondary metabolite pathways using transcriptional regulators.

Plant secondary metabolism is the source of many natural products with diverse applications, including pharmaceuticals, food colors, dyes and fragrances. Functions in plants include attraction of pollinating insects and protection against pests and pathogens. An important regulatory step in secondary metabolism is transcription of the biosynthetic genes. The aim of this chapter is to discuss results and opportunities concerning modification of secondary metabolism using transcriptional regulators. The transcriptional regulation of two well-studied secondary pathways, the phenylpropanoid pathway and its flavonoid branch, and the terpenoid indole alkaloid biosynthetic pathway, are reviewed. Some examples of successful engineering of these pathways via transcriptional regulators are discussed.

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.

Preserving the (water) harvest: effective water use in agriculture.

The alternative to increasing the world's irrigated area by an estimated 30% to secure food security for all, seems to be limited irrigation expansion and consequently higher food prices and probably food shortages. This paper explores other options for ensuring food security. It discusses meaningful similarities between innovative approaches for land and water management in rainfed and irrigated agriculture. The focus is on innovative approaches to increase yields in sub-Saharan Africa and South Asia. Innovative technologies, such as improved tillage practices and water harvesting are important. But at least as important are the processes by which new agricultural practices are developed, improved and extended. In the end it comes down to human inventiveness.

ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism.

Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.

Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos.

We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti-MAPK (mitogen-activated protein kinase) antibody. Rehydration of the embryo induced a decrease in activity of this 45 kDa MAPK-like protein kinase. In addition, activity of a MBP kinase of the same molecular weight was subsequently found to be induced. This second MBP kinase activity could not be immunoprecipitated with the anti-MAPK antibody and was induced only in germinating embryos, not in dormant embryos.

B-type granule containing protrusions and interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression.

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch granules in wheat endosperm cells was studied by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) after expression and targeting of fluorescent protein into amyloplasts. Both techniques demonstrated the presence of protrusions emanating from A-type granules-containing amyloplasts and the presence of B-type starch granules in these evaginations. Moreover, CLSM recordings demonstrated the interconnection of the amyloplasts by these protrusions, suggesting a possible role of these protrusions in interplastid communication.

Subcellular differences in post-translational modification of barley 14-3-3 proteins.

Expression and post-translational modification of barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, were investigated using isoform-specific antibodies. Although all three isoforms were shown to be present in the cytosolic, the nuclear and the microsomal cell fractions, differences in post-translational modification were identified for the different cell fractions. Germination-related modifications of 14-3-3 proteins were observed in the cytosol and the microsomal fraction, but not in the nucleus. In vitro proteolytic cleavage of 14-3-3 proteins using trypsin suggests that for 14-3-3A this change was caused by proteolytic cleavage of the unconserved C-terminal region.

Isolation and partial characterization of thermostable isoperoxidases from potato (Solanum tuberosum L.) tuber sprouts.

Peroxidases (POD; EC 1.11.1.7) can cross-link cell wall polymers and may have an impact on the final textural quality of potato tubers. Because heat treatments are important during processing, the thermal properties of isoPODs from soluble and ionically and covalently bound fractions were studied from both potato tubers and sprouts. For both tissues, the ionically bound fraction was the most thermostable; approximately 20% of POD activity remained after a heat treatment of 10 min at 90 degrees C (for sprouts). The temperature profile of the ionically bound sprout fraction appeared to be nonlinear and suggested the presence of a very thermostable POD, which still showed activity after a heat treatment at 100 degrees C. Visualization by using isoelectric focusing confirmed the occurrence of a thermostable isoPOD with an IEP of 9.5, which displayed regeneration of activity after heat inactivation. This cationic POD was further purified by chromatography techniques, and by SDS-PAGE its molecular mass was estimated at 38 kDa.

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene.

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.

Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos.

The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process.

A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.

Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

Function of chitin oligosaccharides in plant and animal development.

In plant development chitin oligosaccharides have been studied intensively as part of the communication between leguminous plants and Rhizobium bacteria. The Rhizobium bacteria synthesize and secrete lipochitin oligosaccharides (LCOs) to induce the development of a root nodule, in which the bacteria will infiltrate to start a symbiotic relation with the plant. Here we will give an overview of the biosynthetic route used by the bacteria to synthesize these LCOs. Perception by the plant will also be discussed as well as early responses to the LCOs. By working with the genes from the biosynthetic route, other genes were identified that share homology with the chitin synthase genes from Rhizobium. These genes are now isolated from human, mouse, chick, Xenopus and zebrafish and can be divided into three classes. They are mainly expressed during early development at the same stage as chitin oligosaccharide synthase activity can be detected. A controversy has been risen about their biochemical activity and will be further discussed here.

Molecular cloning and characterization of an inorganic pyrophosphatase from barley.

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.

Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(B1-->4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta1-->4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta1-->4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish.

Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

Chitin oligosaccharides can induce cortical cell division in roots of Vicia sativa when delivered by ballistic microtargeting.

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules that can induce nodule organogenesis in the cortex of legume roots in a host-specific way. The multi-unsaturated fatty acyl and the O-acetyl moieties of the LCOs of Rhizobium leguminosarum biovar viciae were shown to be essential for obtaining root nodule induction in Vicia sativa plants. We have used ballistic microtargeting as a novel approach to deliver derivatives of the nodulation signal molecules inside the roots of V. sativa. This method offers the unique ability to introduce soluble compounds into the tissue at a small area. The mitogenic effect of microtargeting of chitin oligosaccharides, including an analysis of the influence of the chain length and modifications, was tested in a qualitative assay. The role of a cell division factor from the root stele, uridine, has also been examined in these experiments. The results show that O-acetylated chitin oligosaccharides can induce root cortical cell divisions when delivered by microtargeting. For this effect it is essential that uridine is co-targeted. The foci of cortical cell division were often similar to root nodule primordia. Anatomical examination also revealed chimeric structures that share characteristics with lateral root and nodule primordia. Our data favour a model in which the oligosaccharide moiety of the rhizobial LCO induces cortical cell division and the fatty acyl moiety plays a role in transport of the LCO into the plant tissue.