Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Detection of Charge-Neutral Near-Equilibrium Processes at Na-Metal Electrodes by Electrochemical Microcalorimetry

Investigations on electrochemical kinetics usually rely on the measurement of current or potential as a function of time. Charge-neutral process steps or side reactions are naturally disguised in the electrical signals and have only indirect impact. However, all processes will contribute to heat evolution. In this work, heat absorption/liberation is measured as a function of time for pulsed Na deposition/dissolution on a Na-electrode in a 1 m NaPF₆/diglyme solution, in addition to the standard electrochemical signals. While potential and current transients both exhibited sharp rectangular shapes, indicating instantaneous electrochemical Na deposition or dissolution on the time scale of the pulse (10 ms), heat absorption or liberation continued up to about 0.5 s after the pulse. Since heat evolution is to large extent reversible, this corresponded to entropy changes in the absence of external electric current flow, pointing to a reversible, charge-neutral chemical process accompanying Na deposition or dissolution. From the observed entropy changes, it is suggested that upon Na deposition solvated Na⁺ ions are instantaneously transferred into the outer layers of the solid electrolyte interphase, followed by slow desolvation.     

Vegetation characteristics and eco-hydrological processes in a pristine mire in the Ob River valley (Western Siberia)

Relations between vegetation characteristics and eco-hydrological processes were assessed in a pristine mire in the valley of the Ob River (Western Siberia). Along a transect from the terrace scarp to the river, field data were collected on vegetation composition, peat stratigraphy, peat chemistry, hydrology and hydrochemistry. Based on floristic composition, eight vegetation communities were distinguished. Hydraulic head measurements were used to obtain an indication of groundwater flow directions. The water balance of the mire was calculated with a two-dimensional steady-state numerical groundwater model. Water types were defined based on cluster analysis of hydrochemical data. The results revealed that the dominant hydrological factor in the Ob mire is the discharge of groundwater, which supplies about threefold more water than net precipitation. Although the discharge flux decreases with increasing distance from the terrace scarp, high water levels and a “groundwater-like” mire water composition were observed in the major part of the study site. Precipitation and river water play only a minor role. Despite dilution of discharging groundwater with rainwater, spatial differences in pH and solute concentrations of the surficial mire water are small and not reflected in the vegetation composition. Although small amounts of silt and clay were found in the peat in the proximity of the river, indicating the occurrence of river floods in former times, no river-flood zone could be recognized based on hydrochemical characteristics or vegetation composition. A comparison of the Ob mire with well-studied and near-natural mires in the Biebrza River valley (Poland) revealed substantial differences in both vegetation characteristics and the intensity and spatial pattern of eco-hydrological processes. Differences in the origin and ratios of water fluxes as well as a dissimilar land use history would seem to be key factors explaining the differences observed.     

Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution

With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.