Fluence dependence of the ultraviolet-light-induced accumulation of chalcone synthase mRNA and effects of blue and far-red light in cultured parsley cells

The fluence dependence of the time course of accumulation of chalcone synthase mRNA in ultraviolet (UV)-light-irradiated cell suspension cultures of parsley (Petroselinum crispum) and the additional effects of blue and far-red light have been investigated. Variations of the UV fluence had no detectable influence on the initial rate of increase in mRNA amount or translational activity, nor on the preceding lag period of approximately 3 h, but strongly influenced the duration of the transient increase. The effects were the same whether the fluence rate or the time of irradiation was varied to obtain a given fluence. Blue-light pretreatment of the cells resulted in increased amounts of mRNA and abolished the apparent lag period. This effect remained cryptic without the subsequent UV-light treatment. Irradiation with long-wavelength far-red light following UV-light pulses shortened the duration of the mRNA accumulation period. This effect was not altered by a preceding blue-light treatment. Thus, three photoreceptors, a UV-B receptor, a blue-light receptor and phytochrome, participate in the regulation of chalcone synthase mRNA accumulation in this system.Abbreviations cDNA complementary DNA - UV ultraviolet - Pfr fai-red-absorbing form of phytochrome     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Differential usage of photoreceptors for chalcone synthase gene expression during plant development

Photoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult re-etiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the β-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.     

Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum

Ubiquitin is an omnipresent protein found in all eukaryotes so far analysed. It is involved in several important processes, including protein turnover, chromosome structure and stress response. Parsley (Petroselinum crispum) contains at least two active polyubiquitin (ubi4) genes encoding hexameric precursor proteins. The deduced amino acid sequences of the ubiquitin monomers are identical to one another and to ubiquitin sequences from several other plant species. Analysis of the promoter region of one ubi4 gene revealed putative regulatory elements. In parsley plants, the ubi4 mRNAs were the predominant ubiquitin mRNAs and were present at comparable levels in all plant organs tested. In cultured parsley cells, high levels of ubiquitin gene expression remained unaffected by heat shock, elicitor or light treatment.     

Differential patterns of phytoalexin accumulation and enzyme induction in wounded and elicitor-treated tissues ofPhaseolus vulgaris

In wounded cotyledons ofPhaseolus vulgaris L. the accumulation of the 5-hydroxy isoflavonoids kievitone and 2-hydroxygenistein precedes the major increases in the levels of the 5-deoxy compounds phaseollin and coumestrol. Increased phytoalexin levels are preceded by transient increases in the extractable activities of L-phenylalanine ammonia-lyase (EC 4.3.1.5.), chalcone synthase and chalcone isomerase (EC 5.5.1.6.). Accumulation of phytoalexins, above wounded control levels, is observed following treatment of excised cotyledons or hypocotyls with crude or fractionated elicitor preparations heat-released from the cell walls ofColletotrichum lindemuthianum. Chalcone synthase levels are also induced in cotyledons, although crude elicitor and all fractions suppress L-phenylalanine ammonia-lyase activity in both tissues. Kievitone is the major phytoalexin induced in cotyledons, whereas in hypocotyls phaseollin predominates. Patterns of phytoalexin accumulation have been studied in response to varying concentrations of the crude and fractionated elicitor; 5-hydroxy isoflavonoid accumulation is highly dependent upon elicitor concentration, the dose-response curves for kievitone accumulation showing maxima at around 1 g glucose equivalents per cotyledon, minima at 2–3 g equivalents and increasing induction at higher concentrations. Similar patterns are observed for L-phenylalanine ammonia-lyase and chalcone synthase levels, although the overall extent of these changes is masked by the high wound response. Accumulation of 5-deoxy isoflavonoids above control levels requires high elicitor concentrations; no experimental conditions were found under which phaseollin accumulated to higher levels than kievitone in cotyledons during the first 48 h after elicitation.Abbreviations CHS chalcone synthase - PAL L-phenylalanine ammonia-lyase     

Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole