Search

Mass spectrometry imaging was applied on germinated barley for the detailed localization of metabolites in longitudinal and transversal seed sections. Among others, 20 m/z signals occurred in three regular peak clusters with specific, distinct localizations in embryo tissues. High resolution FT-ICR MS, MALDI-TOF MS/MS, and UHPLC-ESI MS/MS served for the identification and structural characterization of these compounds. Only five metabolites were published in their structures, namely the antifungal compounds hordatine A and B in non-glycosylated and glycosylated forms. All other non-identified cluster compounds were of hordatine-like structure and differed by systematic O-methylations, hydroxylations, and glycosylations. These differences in molecular structures correlated to distinct localization patterns within the embryo and might serve for the regulation of antifungal properties. Based on the structural investigations by mass spectrometry, an array of different hordatines that comprises the five published hordatines, 15 novel hordatine derivates and their six precursors could be localized in the embryo of germinated barley. Implications for the biosynthetic pathway and transport processes are discussed.

The lipopolysaccharides (LPSXcc) of the phytopathogenic bacteria Xanthomonas campestris pv. campestris (X.c.c.) were purified from an exopolysaccharide-deficient mutant strain. The isolated LPSXcc induced an oxidative burst reaction in cell-suspension cultures of the non-host plant tobacco (Nicotiana tabacum L.) SR1. The oxidative burst elicited by LPSXcc differed from that induced by yeast elicitor (YE), a cell wall preparation of baker's yeast. The LPSXcc-induced oxidative burst was characterised by a slow increase in H2O2 production and an extended decline. Both the LPSXcc- and YE-induced oxidative bursts were completely blocked by the NAD(P)H-oxidase inhibitor diphenylene-iodonium. When LPSXcc and YE were applied in combination, a synergistic effect and the establishment of refractory states in the generation of H2O2 were observed. The amount of cytosolic calcium was measured in transgenic tobacco cell cultures carrying the apoaequorin gene by coelenterazine-derived chemiluminescence. Whereas YE induced a calcium peak within 1 min after application, LPSXcc induced a long-term calcium signal without transients. To our knowledge this is the first report on the elicitation of an oxidative burst in plant cell cultures by isolated LPS of a phytopathogenic bacterium.

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 acidificatiton of the culture medium, instead of an alkalinization, but no oxidative burst.

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.