A Small Decrease of Plastid Transketolase Activity in Antisense Tobacco Transformants Has Dramatic Effects on Photosynthesis and Phenylpropanoid Metabolism

Abstract

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...

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