Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998     

Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor

 Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity. Received: 30 August 1999 / Accepted: 6 December 1999     

Homologous and heterologous desensitization and synergy in pathways leading to the soybean oxidative burst

Because the H₂O₂ and O₂ ⁻ generated during a pathogen-triggered oxidative burst could either protect or destroy a besieged plant cell, their synthesis might be expected to be tightly regulated. We have examined the nature of this regulation as it is communicated between homologous and heterologous oxidative-burst pathways, using both chemical (oligogalacturonic acid, harpin, fensulfothion) and mechanical (osmotic stress) stimuli to induce the burst. We report here that the above three chemical elicitors attenuate a subsequent oxidative burst induced in cultured soybean (Glycine max L.) cells by either the same (homologous desensitization) or a different chemical elicitor (heterologous desensitization). Further, when the magnitude of the initial oxidative burst is maximal, the cells remain refractory to subsequent elicitation for at least 10 min and then revive their sensitivities to re-stimulation with a half-time of >20 min. Mechanical stimulation of the oxidative burst appears to be regulated by a different set of constraints. Although initiation of a mechanically induced burst leads to attenuation of a subsequent mechanically induced burst, the same mechanical stimulus is peculiarly unable to reduce a subsequent chemically induced burst. The converse is also true, suggesting that heterologous desensitization of the oxidative burst does not extend to mixed chemical and mechanical/osmotic stimuli. However, communication between these disparate forms of elicitation is still demonstrated to occur, since low-level chemical stimuli strongly synergize concurrent low-level osmotic stimuli and vice versa. Furthermore, the pattern of synergy changes dramatically if one stimulus is administered immediately prior to the other. Taken together, these data demonstrate that significant cross-talk occurs among the different signaling pathways of the oxidative burst and that the overall process is tightly regulated. Received: 10 January 2000 / Accepted: 22 February 2000     

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

An F₁ hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC₁ hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin. Received: 20 September 2000 / Accepted: 05 January 2001     

Increases in Intracellular pH and Ca2+ are Essential for K+ Channel Activation After Modest `Physiological' Swelling in Villus Epithelial Cells

We studied the relationship between changes in intracellular pH (pH _i ), intracellular Ca²⁺([Ca²⁺] _i ) and charybdotoxin sensitive (CTX) maxi-K⁺ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH <sub>i</sub> and [Ca<sup>2+</sup>] <sub>i</sub> by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH <sub>i</sub> alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH <sub>i</sub> and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH<sub>4</sub>Cl permitted complete RVD which could be inhibited by CTX. The rate of <sup>86</sup>Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na<sup>+</sup>-free medium. Modest swelling transiently increased [Ca<sup>2+</sup>] <sub>i</sub> and Ca<sup>2+</sup>-free medium or blocking alkalinization by MIA or Na<sup>+</sup>-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca<sup>2+</sup>-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca<sup>2+</sup>] <sub>i</sub> and volume changes became sensitive to CTX. We conclude that both alkalinization of pH <sub>i</sub> and increased [Ca<sup>2+</sup>] <sub>i</sub> observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K⁺ channels required for RVD. Received: 30 March 1999/Revised: 6 July 1999     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculataL. Walp. reveals extensive gene flow between wild and domesticated types

Amplified fragment length polymorphisms (AFLPs) were used to evaluate genetic relationships within cowpea [Vigna unguiculata (L.) Walp.] and to assess the organization of its genetic diversity. Nei’s genetic distances were estimated for a total of 117 accessions including 47 domesticated cowpea (ssp. unguiculata var. unguiculata), 52 wild and weedy annuals (ssp. unguiculata var. spontanea), as well as 18 perennial accessions of the wild subspecies pubescens, tenuis and alba. AFLP variation was also used to study genetic variation among and within domesticated and wild accessions based on their geographical origin (western, eastern and southern Africa). Wild annual cowpea (var. spontanea) (H _T =0.175) was more diverse than domesticated cowpea (H _T =0.108). Wild cowpea was more diverse in eastern (H _S =0.168) than in western Africa (H _S =0.129), suggesting an eastern African origin for the wild taxon. The AFLP data were consistent with earlier findings of a unique domestication event in cowpea in the northern part of the continent and suggested that domestication in eastern or southern Africa was unlikely. It did not allow a more precise localization of domestication due to extensive gene flow between wild and domesticated forms that has led to a large crop-weed complex distributed over the entire African continent. In addition, wild materials from northeastern Africa are still lacking. Overall, the superiority of the AFLP technique over isozymes resided in its ability to uncover variation both within domesticated and wild cowpea, and should be a powerful tool once additional wild material becomes available. Received: 11 September 2000 / Accepted: 14 June 2001     

A system to induce the deletion of gemomic sequences using R/RS site-specific recombination and the Ac transposon in transgenic rice plants

Recombinase encoded by the R gene of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce deletion or inversion of the DNA segment that is flanked by the RSs. The R gene under the control of the CaMV 35 S promoter was introduced into rice (Oryza sativa L.). R/RS-specific deletion was first demonstrated in transgenic rice callus carrying the R gene by transient introduction of a cryptic reporter gene that was designed to confer β-glucuronidase (GUS) expression once deletion between two RSs took place. The rice containing the R gene was subsequently crossed with transgenic rice carrying (I-RS/dAc-I-RS) T-DNA that contained RS sequences within the T-DNA and another RS in a modified Ac element that had been transposed to a new locus by Ac transposase. Deletion of the gemomic sequences flanked by the two RSs was detected by PCR analysis in somatic cells of F₂ plants. These results demonstrate a technical advance in that the R/RS recombination system, in combination with the Ac transposable element, can be used to generate deletion in rice chromosomes. Received: 30 June 2000 / Accepted: 16 October 2000     

Production of reactive oxygen species in Arabidopsis thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum: implications for basal resistance

The present understanding of ROS generation in the defence responseof Arabidopsis thaliana is reviewed. Evidence suggests thatthe apoplastic oxidative burst generated during basal resistanceis peroxidase-dependent. The ROS generated during this basalresistance may serve to activate NADPH oxidase during the R-gene-mediatedhypersensitive response. The processes involved in the productionof reactive oxygen species in A. thaliana cell suspension culturesin response to an elicitor from Fusarium oxysporum are investigatedin the present work. This system appears analogous to the productionof ROS during the basal resistance response in French bean,which is peroxidase-dependent. A panel of modulators effectivein other pathogen elicitor and plant cell systems has been usedto investigate the Arabidopsis signalling pathways and the plantcell responses involved. Thus as in other systems, an earlycalcium influx into the cytosolic compartment, a rapid effluxof K⁺ and Cl^–, and extracellular alkalinization of elicitedcell cultures has been found. However the alkalinization isnot sufficient to stimulate the apoplastic oxidative burst byitself, unlike in French bean, although vectorial ion fluxesare needed. A secretory component which is sensitive to monensinand N-ethylmaleimide and insensitive to brefeldin A may alsobe necessary for the release and provision of substrates forperoxidase-dependent generation of H₂O₂. Key words: Arabidopsis thaliana, calcium, elicitation, hydrogen peroxide, oxidative burst, secretion     

Incompatible pathogen infection results in enhanced reactive oxygen and cell death responses in transgenic tobacco expressing a hyperactive mutant calmodulin

Transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38) lines expressing a mutant calmodulin (VU-3) that hyperactivates NAD kinase exhibit an enhanced elicitor-stimulated oxidative-burst reaction (S.A. Harding et al., 1997, EMBO J. 16: 1137–1144). VU-3 transgenic tobacco was used in the present study to investigate the relationship between calmodulin signalling, the production of active oxygen species and cell death in response to infection with an incompatible pathogen. Following P. syringae pv. syringae 61 infection, suspension cells derived from VU-3 transgenic plants exhibited a stronger oxidative burst (3- to 4-fold higher primary and secondary burst reactions), greater media alkalinization (3-fold) and more rapid cell death (4-fold greater mortality at 20 h post infection) than did infected control tobacco cells. Infection of leaf tissues with P. syringae pv. syringae 61 also resulted in an enhanced cell death response compared to control tobacco tissues. This cell death response of VU-3 leaf tissues, but not control leaf tissues, was further enhanced by the presence of 50 μM salicylic acid, suggesting that this transgenic line is more sensitive to the effects of this agent. Overall, the data support the model that calmodulin signalling pathways are involved in the plant oxidative burst and contribute to the regulation of cell death in infected plant tissues undergoing the hypersensitive response. Received: 6 January 1998 / Accepted: 7 March 1998     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.)

A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt (Triticum spelta L.) and an approximately 250 year-old bread wheat (Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta ’Oberkulmer.’ The alleles from the historical bread wheat were most similar to those of modern T. aestivum cultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt. Received: 22 July 2000 / Accepted: 27 April 2001     

Analysis of genetic similarity detected by AFLP and coefficient of parentage among genotypes of sugar cane ( Saccharum spp.)

Despite the economical importance of sugar cane, until the present-date no studies have been carried out to determine the correlation of the molecular-based genetic similarity (GS) and the coefficient of parentage (f)-estimates generated for cultivars. A comprehensive knowledge of the amount of genetic diversity in parental cultivars, could improve the effectiveness of breeding programmes. In this study, amplified fragment length polymorphism (AFLP) and pedigree data were used to investigate the genetic relationship in a group of 79 cultivars (interspecific hybrids), used as parents in one of the Brazilian breeding programmes, and four species of Saccharum (Saccharum sinense, Saccharum barberi and two of Saccharum officinarum) . The objectives of this study were to assess the level of genetic similarity among the sugar-cane cultivars and to investigate the correlation between the AFLP-based GS and f, based on pedigree information. Twenty one primer combinations were used to obtain the AFLP molecular markers, generating a total of 2,331 bands, of which 1,121 were polymorphic, with a polymorphism rate, on average, of 50% per primer combination. GSs were determined using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method using arithmetic average (UPGMA). AFLP-based GS ranged from 0.28 to 0.89, with a mean of 0.47, whereas f ranged from 0 to 0.503, with a mean of 0.057. Cluster analysis using GS divided the genotypes into related subgroups suggesting that there is important genetic relationship among the cultivars. AFLP-based GS and f were significantly correlated (r = 0.42, P < 0.001), thus the significance of this r value suggests that the AFLP data may help to more-accurately quantify the degree of relationship among sugar-cane cultivars. Received: 27 November 2000 / Accepted: 27 April 2001     

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae

Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%). Received: 27 April 1999 / Accepted: 5 June 1999     

Early evolution of the chromosomal structure of Triticum turgidum–Aegilops ovata amphiploids carrying and lacking the Ph1 gene

After two selfing generations of two different Triticum turgidum –Aegilops ovata amphiploids carrying the Ph1 gene, or lacking it (ph1c mutant), karyotypes of their offspring were scored by GISH (genomic in situ hybridization). On average, the chromosome number was lower than expected (56 chromosomes) on the basis of the parental constitutions (T. turgidum, AABB, 2n=4x=28; Ae. ovata, M^oM^oU^oU^o, 2n=4x=28). The lost chromosomes belonged to the wild Aegilops species. The two families differed greatly by their number of intergenomic translocations, also detected by GISH. The ph1c family showed nine translocations over 12 plants while only one translocation was observed in the Ph1 family. All exchanges involved either the M^o and U^o chromosomes or the M^o and wheat chromosomes, the size of the exchanged segment ranging from 3% to 36% of the total chromosome length. The results suggest an epistatic effect of the ph1c deletion over the genetic diploidizing system that operates in Ae. ovata since translocated chromosomes are most-likely derived from homoeologous recombination. The potential of these results for wheat breeding programmes is also considered. Received: 28 November 2000 / Accepted: 20 March 2001     

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca²⁺ channels. Inhibition of the oxidative response impaired Cl⁻ efflux, K⁺ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.     

The Lipid A substructure of the Sinorhizobium meliloti lipopolysaccharides is sufficient to suppress the oxidative burst in host plants

Medicago sativa (alfalfa), Medicago truncatula and Nicotiana tabacum cell suspension cultures, responding to elicitation with the production of reactive oxygen species (ROS), were used to analyse the suppressor (and elicitor) activity of lipopolysaccharides (LPS) of the symbiotic soil bacterium Sinorhizobium meliloti. In order to identify the epitopes of the LPS molecule recognized by the plant, S. meliloti mutants defective in LPS biosynthesis and hydrolytically obtained Lipid A were analysed for biological activity. Lipopolysaccharides isolated from Sinorhizobium meliloti mutants 6963 (altered core region) and L994 (no long-chain fatty acid) showed the same ability to suppress the oxidative burst in host plant cell cultures as the wild-type LPS. Lipid A also displayed the same suppressor activity. By contrast, rhizobial LPS, but not Lipid A, was active as an inducer of the oxidative burst reaction in cell cultures of the nonhost Nicotiana tabacum. In host plants of Sinorhizobium meliloti the Lipid A part is sufficient to suppress the oxidative burst, but in non-host plants at least some sugars of the LPS core region are required to induce defence reactions.