Klaus Biehler

Targeted inactivation of the smallest plastid genome-encoded open reading frame reveals a novel and essential subunit of the cytochrome b(6)f complex

The smallest conserved open reading frame in the plastid genome, ycf6, potentially specifies a hydrophobic polypeptide of only 29 amino acids. In order to determine the function of this reading frame we have constructed a knockout allele for ycf6. This allele was introduced into the tobacco plastid genome by chloroplast transformation to replace the wild‐type ycf6 allele. Homoplasmic Δycf6 plants display a photosynthetically incompetent phenotype. Whereas the two photosystems are intact and physiologically active, we found that the electron transfer from photosystem II to photosystem I is interrupted in Δycf6 plants. Molecular analyses revealed that this block is caused by the complete absence of the cytochrome b6f complex, the redox‐coupling complex that interconnects the two photosystems. Analysis of purified cytochrome b6f complex by mass spectroscopy revealed the presence of a protein that has exactly the molecular mass calculated for the Ycf6 protein. This suggests that Ycf6 is a genuine subunit of the cytochrome b6f complex, which plays a crucial role in complex assembly and/or stability. We therefore propose to rename the ycf6 reading frame petN.

Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower, bean and maize to water stress

Gas exchange and fluorescence measurements of attached leaves of water stressed bean, sunflower and maize plants were carried out at two light intensities (250 μmol quanta m-2s-1 and 850 μmol quanta m-2s-1). Besides the restriction of transpiration and CO2 uptake, the dissipation of excess light energy was clearly reflected in the light and dark reactions of photosynthesis under stress conditions. Bean and maize plants preferentially use non-photochemical quenching for light energy dissipation. In sunflower plants, excess light energy gave rise to photochemical quenching. Autoradiography of leaves after photosynthesis in 14CO2 demonstrated the occurrence of leaf patchiness in sunflower and maize but not in bean. The contribution of CO2 recycling within the leaves to energy dissipation was investigated by studies in 2.5% oxygen to suppress photorespiration. The participation of different energy dissipating mechanisms to quanta comsumption on agriculturally relevant species is discussed.

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Mass-spectrometric measurements of 16O2 and 18O2 were made to compare the rates of light-dependent O2 evolution and uptake by Chlamydomonas reinhardtii Dang. grown in air (0.035% CO2; low-Ci cells) or CO2-enriched air (5% CO2; high-Ci cells) at pH 5.5 and 8.0. While at pH 5.5, no differences were observed in the isotopic O2-gas exchange of high- and low-Ci cells, at pH 8.0 the rates of true O2 evolution and uptake were considerably higher in low-Ci than in high-Ci cells. The enhanced rates of O2 uptake and evolution by low-Ci cells were completely inducible within 6 h after transferring high-Ci cells to ambient air. At pH 8.0, O2 uptake in the light was inhibited by 2 μM 3-(3,4-dichlorophenyl)-1,1 dimethylurea in both types of alga, but this effect was more pronounced in low-Ci than in high-Ci cells.When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO2 concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-Ci than in high-Ci cells. When high-Ci cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50% of the level observed with low-Ci cells. These results are consistent with an enhanced operation of O2 photoreduction which could provide energy to the inorganic-carbon-concentrating mechanism via pseudo-cyclic photophosphorylation.

Regulation of nitrate reductase transcript levels by glutamine accumulating in the leaves of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of Arabidopsis thaliana, and by glutamine provided via the roots

Abstract. The regulation by glutamine of the leaf transcript level corresponding to the Arabidopsis thaliana (L.) Heynh. nitrate reductase gene nia2 was examined using a novel approach: we took advantage of the ability of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of A. thaliana to accumulate glutamine in the leaves when illuminated under conditions that favour photorespiration. The accumulation of glutamine in gluS mutant leaves and the concomitant decline in the leaf glutamate pool were not correlated with a reduction in the foliar nia2 transcript level. This result indicates that glutamine may not exert a negative control of the leaf nia2 transcript pool. The pattern of diurnal nia2 mRNA oscillation did not change upon illumination of the gluS mutant in air, although the leaf glutamine level remained high during the diurnal cycle. The amplitude of the diurnal fluctuation in nia2 transcript abundance, therefore, does not seem to depend on the size of the leaf glutamine pool (which normally fluctuates in opposite phase). This result also appears to argue against a role of glutamine as an effective repressor of nia2 transcript accumulation. The application of a solution containing 100 mM glutamine to the roots of A. thaliana resulted in an increase in the leaf glutamine level and in a decrease in the leaf nia2 transcript level. Net CO2 uptake and chlorophyll fluorescence quenching by attached leaves of A. thaliana were determined as a control of the physiological status of the plants and remained unaffected by the glutamine treatment. However, there was a decrease in the foliar nitrate level. The negative effect on the nia2 transcript pool exerted by exogeneous glutamine may, therefore, be explained as a result of the down-regulation of nitrate-uptake permeases in the roots by glutamine.

Photosynthesis and fluorescence quenching, and the mRNA levels of plastidic glutamine synthetase or of mitochondrial serine hydroxymethyltransferase (SHMT) in the leaves of the wild-type and of the SHMT-deficient stm mutant of Arabidopsis thaliana in relation to the rate of photorespiration

Abstract.The regulation by photorespiration of the transcript level corresponding to plastidic glutamine synthetase (GS-2) was investigated in the leaves of Arabidopsis thaliana (L.) Heynh.. Photorespiration was suppressed by growing the plants in an atmosphere containing 300 Pa CO2. Suppression of photorespiration was demonstrated by the ability of the conditionally lethal serine hydroxymethyltransferase (SHMT)-deficient stm mutant of A. thaliana to grow normally under these conditions. In contrast to previous studies with bean or pea that were performed at very high CO2 partial pressure (2–4 kPa; Edwards and Coruzzi, 1989, Plant Cell 1: 241–248; Cock et al., 1991, Plant Mol Biol 17: 761–771), suppression of photorespiration during growth of A. thaliana in an atmosphere with 300 Pa CO2 had no effect on the leaf GS-2 transcript level. In the short term, neither suppression of photorespiration induced by the transfer of air-grown A. thaliana plants into a CO2-enriched atmosphere, nor an increase in the rate of photorespiration achieved by the transfer of high-CO2-grown A. thaliana plants into air resulted in a change in the GS-2 mRNA level. The absence of photorespiratory ammonium release in leaves of the stm mutant had no effect on the GS-2 transcript level. Overall, our data argue against a control by photorespiration of the A. thaliana leaf GS-2 mRNA pool. In contrast, regulation of the leaf SHMT mRNA level may involve a negative feedback effect of at least one metabolite derived from the glycine/serine conversion during photorespiration, as indicated by the overexpression of SHMT transcripts in the leaves of the stm mutant.

Treating urinary tract infections due to MDR E. coli with Isothiocyanates – a phytotherapeutic alternative to antibiotics?

BACKGROUND Multidrug-resistant (MDR) bacteria are increasingly causing urinary tract infections (UTI), which has been linked to frequent use of antibiotics. Alternative treatment regimens are urgently needed and natural isothiocyanates (ITC) may represent one. ITCs are natural plant products found in nasturtium (Tropaeoli majoris herba) and horseradish (Armoraciae rusticanae radix). PURPOSE The objectives were to (1) assess the antimicrobial effects of nature-identical ITCs for UTI treatment caused by uropathogenic E. coli (UPEC), (2) to evaluate a potential influence of antimicrobial resistance on ITC susceptibility, and (3) to test whether ITCs affect UPEC penetration into human uroepithelial cells. METHODS We tested 217 clinical UPEC isolates, 54.5% of which were classified as MDR, for susceptibility against ITCs. ITC susceptibility testing was performed by broth dilution using a mixture of three synthetic ITCs. Internalization was tested using human T-24 bladder carcinoma cells in an internalization assay co-incubated with UPEC (n = 5) and ITCs. RESULTS The mean minimal inhibitory concentration (MIC) 90 was 0.17 mg/ml, showing very high susceptibility against ITCs. Interestingly, MDR E. coli were significantly less susceptible than non-MDR strains (p = .01). Internalization of UPEC was decreased by 31.9% in the mean when treated with ITCs. Overall, ITCs exerted a strong antimicrobial activity against clinical UPEC isolates and reduced internalization into uroepithelial cells. CONCLUSION ITCs might present a promising treatment alternative for UTIs, expressing both high antimicrobial activity as well as blocking the pathogenic process of human cell penetration by UPEC. Clinical studies, however, are needed to confirm activity of ITCs in UTIs in vivo.