Transketolase (TK) catalyzes reactions in the Calvin cycle and the oxidative pentose phosphate pathway (OPPP) and produces erythrose-4-phosphate, which is a precursor for the shikimate pathway leading to phenylpropanoid metabolism. To investigate the consequences of decreased TK expression for primary and secondary metabolism, we transformed tobacco with a construct containing an antisense TK sequence. The results were as follows: (1) a 20 to 40% reduction of TK activity inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis. The inhibition of photosynthesis became greater as irradiance increased across the range experienced in growth conditions (170 to 700 mol m Ϫ 2 sec Ϫ 1 ). TK almost completely limited the maximum rate of photosynthesis in saturating light and saturating CO 2 . (2) Decreased expression of TK led to a preferential decrease of sugars, whereas starch remained high until photosynthesis was strongly inhibited. One of the substrates of TK (fructose-6-phosphate) is the starting point for starch synthesis, and one of the products (erythrose-4-phosphate) inhibits phosphoglucose isomerase, which catalyzes the first reaction leading to starch. (3) A 20 to 50% decrease of TK activity led to decreased levels of aromatic amino acids and decreased levels of the intermediates (caffeic acid and hydroxycinnamic acids) and products (chlorogenic acid, tocopherol, and lignin) of phenylpropanoid metabolism. (4) There was local loss of chlorophyll and carotene on the midrib when TK activity was inhibited by Ͼ 50%, spreading onto minor veins and lamina in severely affected transformants. (5) OPPP activity was not strongly inhibited by decreased TK activity. These results identify TK activity as an important determinant of photosynthetic and phenylpropanoid metabolism and show that the provision of precursors by primary metabolism colimits flux into the shikimate pathway and phenylpropanoid metabolism. INTRODUCTIONThe interaction between primary and secondary pathways is an important but poorly understood aspect of plant metabolism. Phenylpropanoids represent an important class of secondary metabolites, with roles in plant structure, defense, and signaling (Dixon and Paiva, 1995). They are derived from aromatic amino acids, which are synthesized via the shikimate pathway in the plastid. Up to 20% of the total carbon in a plant passes through this pathway (Jensen, 1985). Even though there have been numerous studies of transformants with altered expression of genes that encode enzymes in the Calvin cycle, glycolysis, the tricarboxylic acid cycle, and the oxidative pentose phosphate pathway (OPPP) (Stitt and Sonnewald, 1995;Stitt, 1999), the consequences for phenylpropanoid metabolism or other secondary pathways have not been explored. The presence of redundant enzymes and pathways for carbohydrate breakdown and glycolysis (Dennis, 1987; Dennis et al., 1997;Stitt, 1998) and the absence of marked phenotypic changes after alterations in the expression of key enzymes, including phosphofructokinase (Burrell et...
Transketolase (TK) catalyzes reactions in the Calvin cycle and the oxidative pentose phosphate pathway (OPPP) and produces erythrose-4-phosphate, which is a precursor for the shikimate pathway leading to phenylpropanoid metabolism. To investigate the consequences of decreased TK expression for primary and secondary metabolism, we transformed tobacco with a construct containing an antisense TK sequence. The results were as follows: (1) a 20 to 40% reduction of TK activity inhibited ribulose-1,5-bisphosphate regeneration and photosynthesis. The inhibition of photosynthesis became greater as irradiance increased across the range experienced in growth conditions (170 to 700 mol m Ϫ 2 sec Ϫ 1 ). TK almost completely limited the maximum rate of photosynthesis in saturating light and saturating CO 2 . (2) Decreased expression of TK led to a preferential decrease of sugars, whereas starch remained high until photosynthesis was strongly inhibited. One of the substrates of TK (fructose-6-phosphate) is the starting point for starch synthesis, and one of the products (erythrose-4-phosphate) inhibits phosphoglucose isomerase, which catalyzes the first reaction leading to starch. (3) A 20 to 50% decrease of TK activity led to decreased levels of aromatic amino acids and decreased levels of the intermediates (caffeic acid and hydroxycinnamic acids) and products (chlorogenic acid, tocopherol, and lignin) of phenylpropanoid metabolism. (4) There was local loss of chlorophyll and carotene on the midrib when TK activity was inhibited by Ͼ 50%, spreading onto minor veins and lamina in severely affected transformants. (5) OPPP activity was not strongly inhibited by decreased TK activity. These results identify TK activity as an important determinant of photosynthetic and phenylpropanoid metabolism and show that the provision of precursors by primary metabolism colimits flux into the shikimate pathway and phenylpropanoid metabolism. INTRODUCTIONThe interaction between primary and secondary pathways is an important but poorly understood aspect of plant metabolism. Phenylpropanoids represent an important class of secondary metabolites, with roles in plant structure, defense, and signaling (Dixon and Paiva, 1995). They are derived from aromatic amino acids, which are synthesized via the shikimate pathway in the plastid. Up to 20% of the total carbon in a plant passes through this pathway (Jensen, 1985). Even though there have been numerous studies of transformants with altered expression of genes that encode enzymes in the Calvin cycle, glycolysis, the tricarboxylic acid cycle, and the oxidative pentose phosphate pathway (OPPP) (Stitt and Sonnewald, 1995;Stitt, 1999), the consequences for phenylpropanoid metabolism or other secondary pathways have not been explored. The presence of redundant enzymes and pathways for carbohydrate breakdown and glycolysis (Dennis, 1987; Dennis et al., 1997;Stitt, 1998) and the absence of marked phenotypic changes after alterations in the expression of key enzymes, including phosphofructokinase (Burrell et...
The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD.Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31 555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60306-Da protein.For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate.High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.Despite the central importance of NAD to metabolism, the biochemistry and regulation of its de novo formation are still not completely clarified. In all known organisms examined to date the pyridine ring of the NAD molecule is synthesized via quinolinate (pyridine-2,3-dicarboxylate) ; quinolinate formation occurs via four different pathways depending on the organism [l -31.In Escherichia coli and Salmonella typhimurium quinolinate biosynthesis was suggested to be catalyzed by a quinolinate synthetase complex [4] from L-aspartate and dihydroxyacetone phosphate [2, 31. Up to six intermediates have been postulated for the conversion of L-aspartate and dihydroxyacetone phosphate into quinolinate [2], an exact reaction sequence, however, has not been established yet.The quinolinate synthetase complex consists of the two enzymes quinolinate synthetases A and B, which are the gene products of genes nadA and nadB. Quinolinate synthetase B has been identified and characterized as FAD-dependent L-aspartate oxidase [5]. It catalyses oxidation of L-aspartate to iminoaspartate which is condensed with dihydroxyacetone phosphate to quinolinate under the action of quinolinate synthetase A (Fig. 1). Correspondence to H. G. Quinolinate is further converted to nicotinic acid mononucleotide via decarboxylation and phosphoribosylation by the nadC gene product quinolinate phosphoribosyltransferase [6] (Fig. 1). nadC mutant strains are not able to convert and thus excrete quinolinate into the medium.The three structural genes, nadA, nadB and nadC have been located at 17 min, 56 min [7] and 1.5 min [8], respectively, on the E. coli genetic linkage map [9]; very similar locations (17 min, 55 min and 3 min respectively) were identified [lo] on the S. typhimurium genetic linkage map.In order to investigate the molecular biology of quinolinate biosynthesis we have cloned and sequen...
We have examined the biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) by human liver cathepsins B, H and L and the cathepsin B-like proteinase from malignant ascitic fluid. This study was carried out using two different methods: The first strategy was to follow the liberation of soluble proteins and peptides as a function of time at different pHs. Then the digestion products were characterized, as collagen IV, fibronectin and laminin fragments, using monospecific polyclonal antibodies and a quantitative dot-blot analysis. From these results, the ability of the four proteinases to digest "in vitro" intact bovine lens capsule in the physiological pH range is demonstrated. Cathepsin L is the most powerful against the three membrane components studied. As shown by electroelution and immunochemical quantitation, the digestion would be a consequence of proteinases binding to the capsule. With intact basement membrane as a substrate a "in vitro" molecular analysis of this digestion process was possible by these methods. On this basis, the "in vivo" secretion of cysteine proteinases during malignancy would be related to the local basement membrane dissolution associated with tumor invasion.
Transgenic tobacco plants expressing antisense RNA directed against the multigene family of the light-harvesting complex of photosystem II (LHCII) were raised and analyzed biochemically and physiologically. A partial 5' terminal sequence with 509 nucleotides complementary to cab (chlorophyll a/b binding protein) genes reduced the amount of transcript to almost undectectable levels. We demonstrated for endogenous genes that a 5' terminal sequence with only 52 to 105 nucleotides complementary to the transit sequence of cab can be equally efficient in gene repression. Chlorophyll content and chlorophyll a-to-chlorophyll b ratios of thylakoid membranes isolated from transgenic plants were unchanged in comparison with the wild type. Photosynthetic oxygen evolution and in vivo-measured chlorophyll fluorescence of the transformants showed that LHCII accumulates to normal levels. The reduced level of cab mRNA did not correlate with the amount of LHCII in thylakoids. This indicates that transcriptional regulation is not the rate-limiting step in the biogenesis of the LHCII apoprotein. The antenna size of photosystem II is therefore modulated by yet undiscovered posttranscriptional mechanisms.
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