Heinrich P. Fock

The production of secondary metabolites by Digitalis lanata during CO2 enrichment and water stress

Abstract The influence of atmospheric CO2 enrichment and water stress on the production of biomass and cardioactive substances by the woolly foxglove Digitalis lanata was investigated. Carbon dioxide enrichment (1000 ppm) had a ‘fertilizing’ effect in that both biomass and cardenolide content increased to about 160% of the control. The yield of the pharmacologically relevant major product, digoxin, significantly increased following enrichment, whereas two other compounds decreased. Water stress, in the physiological range, reduced fresh weight more than either cardenolide content or dry weight. The amount of digitoxigenin was considerably reduced, whereas the other cardenolides, including digoxin, were less affected. CO2-enriched plants, which were also subjected to drought, exhibited mixed responses. We conclude from these investigations that not only primary, but also secondary metabolism is influenced by variations of the environment. Possible ecological consequences of changes in secondary metabolism due to atmospheric CO2 enrichment and water stress are discussed.

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans.

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.

Effects of water stress on the gas exchange, the activities of some enzymes of carbon and nitrogen metabolism, and on the pool sizes of some organic acids in maize leaves.

Water-stressed maize (Zea mays L.) leaves showed a large decrease in leaf conductance during photosynthesis. Net CO2 uptake and evaporation declined fast at mild stress (ψ=−0.6 to −1.0 MPa) and slower at more severe stress (ψ=−1.0 to -1.2 MPa), whereas the CO2 concentration in the intercellular spaces (Ci) did not drop to the CO2 compensation point. The activities of the enzymes of photosynthetic carbon metabolism tested in this study dropped by approx. 30% at ψ=-1.2 MPa. Glutamine synthetase activity was unaffected by water stress, whereas the activity of nitrate reductase was almost completely inhibited. The decline of enzyme activities in relation to ψ was correlated with a concomitant decrease in the content of total soluble protein of the stressed leaves. The total leaf pools of malate, pyruvate and oxaloacetate decreased almost linearly in relation to ψ, thus obviously contradicting the almost constant Ci. In comparison to the controls (ψ=0.6 MPa) the content of citrate and isocitrate increaed markedly at ψ=-0.9 MPa and decreased again at ψ=-1.2 MPa.

Comparative Studies on the Photorespiratory Nitrogen Metabolism in Wheat and Maize Leaves

Summary Release and refixation of ammonia during photorespiratory glycine decarboxylation was examined in illuminated wheat and maize leaves by feeding 15 mM [ 14 C, 15 N]glycine or/plus 5 mM methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, through the cut leaf base. The rates of net CO 2 assimilation (0.27 mmol g fw −1 h −1 in wheat and 0.40 mmol g fw −1 h -1 in maize leaves) and photorespiration determined from gas exchange measurements (0.1 mmol g fw −1 h −1 in wheat and 0 mmol g fw −1 h −1 in maize leaves) were not affected by glycine uptake (8–12 μmol g fw −1 h −1 ), 14 C of glycine accumulated in serine and neutral and acidic photosynthetic products. 15N was predominantly found in alanine, serine, ammonia, glutamine and glutamate. MSO caused an accumulation of NH 3 and inhibited the incorporation of 15N into glutamine and glutamate. Kinetics of 14 C and 15 N incorporation and concentration changes of NH 3 and amino acids were consistant with the operation of the photorespiratory nitrogen cycle in both plants. From NH 3 accumulation during MSO treatments or from concentration changes of glycine during glycine feedings, minimal rates of photorespiration were calculated to be 1–4 % of net CO 2 fixation. An analysis of the 15N abundance of the total amino acid pools led to higher estimates of ammonia release in the light (up to 24 % of CO 2 assimilation in wheat leaves). These rates of NH 3 release compare reasonably well with the rates of CO 2 evolution in the light.

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 μmol m−2 s−1 PAR during incubation with the leaf base in 2 mM 15NH4Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. 15N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO2 assimilation (8.3 and 17.4 μmol m−1 s−1 at 150 and 1350 μmol m−2 s−1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% 15N. In the high light regime the amino acids contained up to 15% 15N, whereas in low light 15N enrichments were small (up to 6%). The kinetics of 15N incorporation indicated that NH3 was firstly assimilated into glutamine and then into glutamate. After 15 min 15N was also found in glycine, serine and alanine. At high light intensity nearly half of the 15N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant 15N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.

Studies on the Photorespiratory Carbon and Nitrogen Metabolism of Glycine, Glutamate and Glutamine in Wheat Leaves

The ammonia released from glycine during photorespiration is reassimilated by glutamine synthetase (GS) and glutamate synthase (GOGAT, Woo et al. 1978, Keys et al. 1978). Studies on leaf discs infiltrated with 15N-glycine (Woo et al. 1982) indicated the occurence of this pathway in the light even though photosynthesis would presumbly be inhibited under these conditions. Evidence from treatments of leaves with inhibitors (Platt and Anthon 1981, Kaiser and Lewis 1980) or from studies on mutants lacking ferredoxin glutamate synthase (Somerville and Ogren 1980) or mitochondrial serine hydroxymethyltransferase (Somerville and Ogren 1981) indicated the occurence of the proposed pathway for photorespiratory N recycling. In recent studies we have fed 15N-glycine and inhibitors through the leaf base (Berger and Fock 1983 a,b) and demonstrated the operation of the photorespiratory N cycle in maize leaves. This study compares the pattern of 15N incorporation into the intermediates of the photorespiratory N cycle from 15N labelled glycine, glutamate and glutamine and supplies estimates for the recycling of photorespiratory ammonia in the GS/GOGAT pathway.