Gerard J. Niemann

Differences in relative growth rate in 11 grasses correlate with differences in chemical composition as determined by pyrolysis mass spectrometry

SummaryEleven grass species varying in potential relative growth rate (RGR) were investigated for differences in chemical composition by pyrolysis mass spectrometry. The spectral data revealed correlations between RGR and the relative composition of several biopolymers. Species with a low potential RGR contained relatively more cell wall material such as lignin, hemicellulose, cellulose, polysaccharide-bound ferulic acid and hydroxyproline-rich protein, whereas species with a high potential RGR showed relatively more cytoplasmic elements such as protein (other than those incorporated in cell walls) and sterols.

Free and cell wall-bound phenolics and other constituents from healthy and fungus-infected carnation (Dianthus caryophyllus L.) stems

Phenolic compounds and related substances from carnation stems infected with Fusarium oxysporum f.sp. dianthi were investigated. Healthy stems contained, esterified to wall polysaccharides, among others benzoic, p-hydroxybenzoic, vanillic, trans p-coumaric, cis and trans ferulic, 3-methoxy-4-hydroxy-n-chlorophenyl propionic and (in large amounts) dihydroferulic acid. Fungal infection affected the concentrations of these phenolic acids and induced the accumulation of two types of anthranilic acid derivatives. One group of those compounds included the dianthramides which were further characterized by HPLC-MS. A representative of the other group was identified as 2,2′-dicarboxy-5,5′-dihydroxy-N,N-diphenylamine, a new natural product.

The anthranilamide phytoalexins of the caryophyllaceae and related compounds

Abstract After elicitation, carnations and some other Caryophyllaceae accumulate a large array of phytoalexins and stress metabolites which have a unique chemical structure, derived from anthranilic acid. Only some Gramineae contain related compounds. An overview is given of the presently known chemical structures, their biosynthesis and metabolism, and their fungitoxicity, localization in the plant and relation with plant resistance.

Geographic trends in flavone-glycosylation genes and seed morphology in EuropeanSilene pratensis (Caryophyllaceae)

Three of the loci controlling isovitexin glycosylation inSilene pratensis are polymorphic and show geographic trends which are compared with geographic trends in seed morphology (and other phenotypic characters) as demonstrated by multivariate analysis. Various lines of evidence support the hypothesis thatS. pratensis spread into Europe from at least two genetically differentiated sources.S. dioica, by contrast, shows little interpretable geographic variation in morphology or flavonoid content.

The geographic distribution of flavone-glycosylating genes in Silene pratensis (Rafn.) Godron and Gren. (Caryophyllaceae)

In Silene pratensis three loci (g, gl and fg) control the glycosylation of isovitexin. Three alleles are known for both the g-locus (g, gGand gX) and the gl-locus (gl, glAand glR); for the fg-locus there are only two alleles (fg and Fg). The distribution of these alleles over 285 European populations of S. pratensis has been investigated. It was concluded that there are three different chemical races within S. pratensis in Europe. The first race contains the populations in western and southern Europe and displays high frequencies of gG, gl and fg. The frequencies of gGand glRare very high in the second chemical race, which can be found in the USSR, Scandinavia and eastern Poland. The third chemical race occurs in central Europe and in this race the frequencies of both g and glRare high, Fg has low to moderate frequencies in the second and third groups. The alleles glAand gXare seldom found in S. pratensis, but are present in the closely related S. dioica. They do occur with low frequencies in some populations of S. pratensis, possibly as a result of hybridization with S. dioica.

A microanalytical approach to plant tissue characterization: A comparative study of healthy and fungus-infected carnation by pyrolysis-mass spectrometry

Abstract Minute samples of xylem from healthy and Fusarium oxysporum f.sp. dianthi-infected stems of carnation cultivars differing in degree of resistance to F. oxysporum were investigated by in-source pyrolysis low voltage EI mass spectrometry and by pyrolysis under ammonia CI conditions. Healthy xylem of both cultivars showed PY-MS spectra with markers for cellulose, hemicelluloses, phenolic acids, and a mixed guaiacyl-syringyl lignin. At different stages of infection visibly infected, brown coloured, xylem was separated under the microscope from healthy-looking parts. The PY-MS spectra of the diseased xylem of susceptible ‘Lena’ were entirely different from those of healthy(-looking) areas, showing a drastic change in lignin pyrolysis product distributions. In most cases practically all syringyl constituents, both mono- and dimeric, had disappeared. In some cases also an overall decrease in relative intensity of the lignin pyrolysis products was observed. Invasion of the fungus thus induced a major change of cell wall composition as a result of lignin degradation along different pathways: demethoxylation of syringyl units, the major pathway in ‘Lena’, and propenyl side chain oxidation. The PY-MS spectra of infected ‘Novada’ generally showed the overall oxidation of lignin and less demethoxylation. The ‘Novada’ spectrum also showed m/z 239, 287, and an increase of 257 and 271, which are molecular ions indicative of the phytoalexins dianthalexin and the dianthramides methoxydianthramide S, hydroxydianthramide B and methoxydianthramide B. The compounds appeared comparatively volatile and therefore could be desorbed as a thermal extract before other pyrolysis fragments appear. The compounds were practically absent in adjacent, healthy looking, xylem. Low voltage EI MS of methanol extracts of about 2 mm3 of infected ‘Novada’ xylem confirmed these data and in a single case also showed evidence for hydroxyanthranilic acid (m/z 153) which is attributed to degradation of dianthramine. Spectra of thermal and methanolic extracts were quite similar.

Differential chemical allocation and plant adaptation: A Py-MS Study of 24 species differing in relative growth rate

The chemical composition of leaves of 24 wild species differing in potential relative growth rate (RGR) was analysed by pyrolysis-mass spectrometry. The variation in RGR significantly correlated with differences in chemical composition: slow-growing species were richer in glucan-based polysaccharides and in C16:0 fatty acid, whereas fast growing ones contained more protein (other than those incorporated in cell walls) and chlorophyll, sterols and diglycerides. Other, apparently significant correlations, e.g. for pentose-based hemicellulose and for guaiacyl lignin appeared solely based on a group separation between mono- and dicotyledonous species.Considering the eleven monocotyledonous and thirteen dicotyledonous species separately, correlations were found in addition to the previously mentioned general ones. Within the group of the monocotyledons the low-RGR species were significantly enriched in pentose-based hemicellulose, ferulic acid and (hydroxy)proline-rich cell wall protein and nearly significant in guaiacyl and syringyl lignin, fast-growing species contained more potassium. Within the group of the dicotyledons slow-growing species were enriched in triterpenes and aliphatic wax esters.In general, the monocotyledons contained more cell wall material such as pentose-based hemicellulose, ferulic acid, glucans (including cellulose) and guaiacyl-lignin, and also more aliphatic wax esters, than the dicotyledons. The dicotyledons, on the other hand, contained somewhat more protein than the grasses.Per unit weight of cell wall, the amount of (hydroxy)proline- rich protein in low-RGR species was comparatively low. A higher investment of cell wall proteins to explain the low rate of photosynthesis per unit of leaf nitrogen of slow-growing species as suggested by Lambers and Poorter (1992), therefore, seems unlikely.

The effect of enzymatic removal of proteins from plant leaf material as studied by pyrolysis-mass spectrometry: detection of additional protein marker fragment ions

Abstract This analytical pyrolysis study investigates the effects of ethanol extraction and of enzymatic protein removal from leaf material of two grass species, Poa annua and Poa pratensis . After pyrolysis the leaf fragments were analysed by low-voltage electron impact (E1) and by ammonia chemical ionization (NH 3 Cl). Pronase-treated and untreated material were compared using multivariate analysis of the PyMS data. The resulting discriminant function spectra among others show a kind of ‘negative image’ of the PyMS spectrum of the plant proteins originally present in Poa leaves. In addition to known protein marker fragment ions several hithertoo unrecognised ones were apparent as well. The nitrogen concentration was determined for several plant fractions. Quantitative comparison of relative intensities of masses found by discriminant analysis led to selection of an additional group of protein marker fragments (m/z 54, 70, 84, 107, 130, 209, 225 and 243 for E1 and 70, 72, 75, 84, 86, 89, 125, 131, 136, 146, 165, 201, 226, 229, 244, 262, 281 and 295 for NH 3 Cl) which showed a significant correlation ( r 2 > 0.5) with the total nitrogen content in the Poa leaves. The origin of grass protein marker fragments was discussed in comparison with reference spectra of two new plant cell-wall proteins, a synthetic polyamine and of albumin. Enzymatic digestion, in addition, yielded a better exposure of the plant cell-wall skeleton, and, therefore, also of the biomacromolecule lignin. Using previously obtained wet chemical data response values were calculated for marker fragments comparative to the analysed content of proteins, polysaccharides and different lignins.

Differential response of four carnation cultivars to races 1 and 2 of Fusarium oxysporum f.sp. dianthi and to Phialophora cinerescens

The responses of four carnation cultivars to infection with races 1 or 2 of Fusarium oxysporum f.sp. dianthi and with Phialophora cinerescens were investigated. Four and nine days after stem inoculation with the different wilt pathogens, 5 cm stem segments were extracted and analysed for the accumulation of dianthalexins and dianthramides by HPLC, and approximately 1 mm3 blocks of xylem tissue were sampled and analysed by pyrolysis-mass spectrometry. Analysis of wilt development over a two-month period confirmed the differential responses of the cultivars to the pathogen isolates. The level of accumulation of the dianthramides was determined partly by the cultivar and partly by the invading pathogen and was not related to the level of symptoms developed. P. cinerescens induced the highest dianthramide concentrations, followed by F. oxysporum f.sp. dianthi, race 2. Race 1 elicited slight dianthramide accumulation and was less inhibited by them in vitro than was P. cinerescens. Pyrolysis-mass spectrometry indicated lignin degradation by all fungi and confirmed, in increasing order, induction of dianthramide phytoalexin accumulation in plants infected with F. oxysporum f.sp. dianthi race 1, race 2 and P. cinerescens. Irrespective of the pathogen, each inoculated cultivar showed a unique pattern of demethylation and degradation of pectin, degradation of lignin and hemicellulose, and changes in polysaccharide-bound phenolic acids.

Isozyme Variation in Silene pratensis: a Response to Different Environments

The isozymes of nine enzyme systems were screened and the frequencies of the flavone glycosylating genes were determined in an outdoor experiment with 70 populations of European S. pratensis and an indoor experiment with 30 populations of the same species. Cluster analysis (using Wards cluster criterion) were performed on all data sets. In the outdoor experiment, cluster analysis of the flavonoid data gave the same pattern that we obtained in an ealier survey of a larger set of populations and showed clearly that there are eight chemical races in European S. pratensis. No comparable geographic distributiion could be found in the isozyme data set, although the dendrogram showed two very clearly separated groups. The two groups represented the two years in which the populations were grown. This result indicates that isozyme variation in European S. pratensis is largely determined by environmental factors. Observations on changes in isozyme patterns during ontogeny and on differences between indoor and outdoor grown plants of the same F2 crosses confirm this. Differences in isozyme patterns can be caused by very small differencs in environment as is shown by the results of the indoor experiment, in which a slight gradient in environmental conditions was present in the greenhouses. The cluster analysis of the isozyme data from the indoor experiment revealed three distinct groups of populations that could be related to location within the greenhouses. As in the outdoor experiment, the dendrogram for the flavonoids gave the same geographic pattern as found with the earlier survey.