Rainer Höfgen

Biochemical and Genetic-Analysis of Different Patatin Isoforms Expressed in Various Organs of Potato (Solanum-Tuberosum)

Abstract Patatin, a group of glycoproteins in potato ( Solanum tuberosum ), is encoded by a multigene family. The protein is found in mass amounts in potato tubers and considered to represent the main storage protein. However, unlike other storage proteins patatin displays enzymatic activity and furthermore, related proteins are found in lower amounts in organs of potato plants which do not serve as storage organs, i.e. stolons, roots and flowers. Evidence is presented showing differences in the enzymatic activities of patatin purified from these organs. The possible implications of these results are discussed. In a genetical approach two single isoforms derived from a class I resp. a class II patatin gene were expressed in transgenic tobacco plants under control of heterologous promoters in order to reduce the complexity of the gene family. The enzymatic activity of these single isoforms was compared to that of patatin of different organs. A class I specific isoform showed exactly the same activity profile as tuber derived patatin. A class II specific isoform showed a pattern different from both tuber and root patatin.

Manipulation of thiol contents in plants

Summary. As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5′-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3′-phosphoadenosine-5′-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and Jäger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are com-monly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.

Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) from Arabidopsis thaliana

SummaryCysteme synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms fromArabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively.AtCS-C andAtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (∼45.8 kD) and of 392 amino acids (∼ 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed thatAtCS-C is higher expressed in roots than in leaves whereas the expression ofAtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of theAtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase ofArabidopsis are able to complement a cysteine synthasedeficient mutant ofEscherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved thatAtCS-C encodes a mitochondrial form of cysteine synthase; first, import ofin vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers.

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.

Approaches towards understanding methionine biosynthesis in higher plants

Summary. Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality.This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value.

Expression of threonine synthase from Solanum tuberosum L. is not metabolically regulated by photosynthesis-related signals or by nitrogenous compounds

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper the isolation of a cDNA encoding threonine synthase is reported (TS; EC 4.2.99.2) from a leaf lambda ZAP II-library of Solanum tuberosum L. and the transcriptional regulation of the respective gene expression in response to metabolic changes. The pattern of expression of TS by feeding experiments of detached petioles revealed that TS expression is regulated neither by photosynthesis-related metabolites nor by nitrogenous compounds. The present study suggests that the regulation of the conversion of aspartate to threonine is not controlled at the transcript level of TS. The nucleotide and deduced amino acid sequences of potato TS show homology to other known sequences from Arabidopsis thaliana and microorganisms. TS is present as a low copy gene in the genome of potato as demonstrated in Southern blot analysis. When cloned into a bacterial expression vector, the cDNA did functionally complement the Escherichia coli mutant strain Gif41. TS transcript was found in all tissues of potato and was most abundant in flowers and source leaves.

Differences in sucrose-to-starch metabolism of Solanum tuberosum and Solanum brevidens

Abstract Sucrose-to-starch metabolism of the tuberising species Solanum tuberosum and that of the non-tuberising Solanum brevidens was studied using in vitro stem cuttings cultured under tuber inducing conditions. The shoots growing from axillary buds of S. brevidens and the in vitro induced stolons and tubers of S. tuberosum were characterised with respect to their carbohydrate composition by measuring the glucose, fructose, sucrose and starch contents. Expression of the genes encoding vacuolar- and cell-wall-bound invertases, vascular- and tuber-specific sucrose synthases was studied by Northern blot hybridisation. Enzyme activity patterns of alkaline and acid invertases, sucrose synthase, hexokinase and ADP-glucose pyrophosphorylase were determined in both species. Major differences between S. brevidens and S. tuberosum were detected in the starch content, in the expression of cell-wall-bound invertase (cwInv), tuber-specific sucrose synthase (SusyI) and in the activities of sucrose synthase and ADP-glucose pyrophosphorylase that were low in S. brevidens. Genes homologous to the cwInv and SusyI were detected in S. brevidens, however, expression of the SusyI, unlike in S. tuberosum, was not sucrose-inducible in the non-tuberising species. These data together with previous findings (Z. Banfalvi, A. Molnar, G. Molnar, L. Lakatos, L. Szabo, FEBS Lett. 383 (1996) 159–164) further support the idea that cell-wall-bound invertase, tuber-specific sucrose synthase and ADP-glucose pyrophosphorylase are important elements of regulatory and biosynthetic pathways resulting in storage starch synthesis and in the expression of tuber storage protein genes. The cwInv mRNA could be detected only in developing stolons and, thus, it can be a good molecular marker of stolon growth.