Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum

Abstract

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K(m) (PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing an antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.

The Role of Phosphoenolpyruvate Carboxylase during C4 Photosynthetic Isotope Exchange and Stomatal Conductance

“…The role of PEPC in stomatal opening was also confirmed in epidermal strips of C 3 plants using a PEPC inhibitor (Parvathi and Raghavendra, 1997;Asai et al, 2000). At the whole leaf level, the use of antisense and overexpression of PEPC in Solanum tuberosum also suggested that malate accumulation is involved in stomatal function (Gehlen et al, 1996). This showed that rates of stomatal opening increased in plants overexpressing PEPC and decreased in plants with reduced levels of PEPC.…”

“…This showed that rates of stomatal opening increased in plants overexpressing PEPC and decreased in plants with reduced levels of PEPC. However, low PEPC levels had no effect on steady-state stomatal conductance and overexpression of PEPC had only a marginal effect (Gehlen et al, 1996).…”

Flux Balance Analysis of Barley Seeds: A Computational Approach to Study Systemic Properties of Central Metabolism

“…In contrast, only a few reports exist on plants with decreased phosphoenolpyruvate carboxylase activity, and studies of knockout mutants have not been carried out to date. In the inhibition studies, transgenic potato plants with reduced phosphoenolpyruvate carboxylase activity (30% of wild-type activity [Rademacher et al, 2002] and 50% to 70% of wild-type activity [Gehlen et al, 1996]) revealed no significant effect on whole plant and tuber growth. However, metabolite analysis showing only little alteration in leaf metabolism suggests an insufficient reduction of phosphoenolpyruvate carboxylase activity to affect metabolism.…”

“…developing seeds and fruits) of C3 plants, phosphoenolpyruvate carboxylase plays an essential role in the anaplerotic replenishment of citrate cycle intermediates by providing precursors for several biosynthetic pathways, including the biosynthesis of amino acids (Chollet et al, 1996). Several groups have attempted to manipulate assimilate partitioning in heterotrophic tissues of C3 plants (Lebouteiller et al, 2007;Radchuk et al, 2007) or to improve CO 2 fixation in autotrophic tissues of C3 plants (Gehlen et al, 1996;Ku et al, 1999) by overexpressing phosphoenolpyruvate carboxylase. In contrast, only a few reports exist on plants with decreased phosphoenolpyruvate carboxylase activity, and studies of knockout mutants have not been carried out to date.…”

Overexpression of C4Phosphoenolpyruvate Carboxylase Increased Carbon Isotope Discrimination in Transgenic Rice Plants

“…Thus, as rates of PEP carboxylation are increased, more C0 2 is released than HC03 fixed. In potato, the respiration rate was increased as PEPC activity incresed (Gehlen et al, 1996;Hausler et al, 1999). Similarly, leaves of rice transformants of T3 generation with 20-fold more PEPC activity increased their respiration rate by up to 1.5 fold relative to non-transformed leaves (Agarie et al, 2001).…”