Heinz-Josef Hirsch

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C3 plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C3 photosynthesis.

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

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 Km(PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing and 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.

Solanum tuberosum double transgenic expressing phosphoenolpyruvate carboxylase and NADP-malic enzyme display reduced electron requirement for CO2 fixation

Abstract The cDNA of the NADP-dependent malic enzyme gene Me 2 from the C 3 plant Flaveria pringlei was used for expression in Escherichia coli and Solanum tuberosum . A chimeric GST-Me 2 gene complemented a malic enzyme deficient E. coli mutant. Two potato lines were transformed with Me 2-cDNA constructs, one line already overexpressing the phospho enol pyruvate carboxylase gene ( ppc ) from Corynebacterium glutamicum . Both genes were under the control of the constitutive 35S CaMV promoter. Increased levels of malic enzyme (ME) were found in chloroplasts of transformants. Western blot analysis indicated that the ME transit sequence was cleaved. Expression of both genes led to a significantly reduced electron requirement for apparent CO 2 assimilation (e/A) at higher temperature. At low temperatures (15°C) 11 electrons per CO 2 assimilated (e/A) were measured in controls, single transformants ( ppc or Me 2) and double transformants ( ppc and Me 2). However, when leaf temperature was raised to 36°C electron requirement of the double transformants (15 e/A) was 65% of controls or single transformants (23 e/A). Thus, the temperature dependent increase in electron requirement was reduced in the double transformants suggesting a suppression in the oxygenation reaction of Rubisco and with it presumably in the rate of photorespiratory CO 2 release which is more marked at high light and high temperatures.

Cloning, sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC-Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.

Expression and chloroplast-targeting of active phosphoenolpyruvate synthetase from Escherichia coli in Solanum tuberosum

Abstract Solanum tuberosum was transformed with a chimeric gene consisting of the constitutive 35S CaMV promoter, a potato ribulose bisphosphate carboxylase (Rubisco) small subunit transit sequence (rbcS), and the phosphoenolpyruvate synthetase gene (ppsA) from E. coli . Transgenic plants were regenerated producing (phospho enol pyruvate-) PEP-synthetase in amounts as much as 0.1% of total soluble protein. Western blot analysis indicated that most of the protein is located in the chloroplasts and that the transit sequence is cleaved off. Electron microscopy of leaves revealed that PEP-synthetase specific immunogold labeling was most pronounced over the chloroplast matrix occurring in between the thylakoid stacks. PEP-synthetase activity was detected in isolated chloroplasts of transgenic plants. Chloroplast morphology and starch production in leaves were affected.

Higher Plants as Bioreactors. Gene Technology with C3-Type Plants to Optimize CO2 Fixation for Production of Biomass and Bio-Energy

The application of biomass as a renewable energy source is currently a matter of lively discussions. Higher plants use photosynthesis as a non-exhaustible source for production of organic compounds. Virtually all life on earth depends on the energy provided by this process. Despite its importance, the photosynthetic process is associated with several flaws that limit the productivity of plants under agricultural growth conditions. The first part of this article describes these inefficiencies and strategies that were developed by plants and algae during evolution to overcome the problem. In the second part, an example of a successful bioengineering approach towards higher plant productivity is presented. Prospects for the use of higher plants in energy production are discussed.