Gehlen J, Panstruga R, Smets H, Merkelbach S, Kleines M, Porsch P, Fladung M, Becker I, Rademacher T, Häusler R E, Hirsch H J
Institut für Biologie I (Botanik/Molekulargenetik), Rheinisch Westfälische Technische Hochschule, Aachen, Germany.
Plant Mol Biol. 1996 Dec;32(5):831-48. doi: 10.1007/BF00020481.
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.
将谷氨酸棒杆菌（cppc）、大肠杆菌（eppc）或三脉叶黄耆（fppc）的磷酸烯醇式丙酮酸羧化酶（PEPC）基因转移到马铃薯中。通过蛋白质免疫印迹分析鉴定出产生外源PEPC的植物再生植株。温室中生长的eppc和fppc植株中测得的最大PEPC活性与对照植株相比提高了一倍。对于cppc，可选择出一个PEPC活性提高四倍的转基因株系。在乙酰辅酶A（一种已知的原核PEPC激活剂）存在的情况下，观察到活性水平提高了六倍。在无菌培养条件下生长的cppc植株中，PEPC活性甚至更高。与分别在不存在或存在乙酰辅酶A的情况下测得的对照相比，PEPC活性分别提高了6倍或12倍。在烟草原生质体中进行瞬时表达也获得了类似的结果。马铃薯叶片提取物中的谷氨酸棒杆菌PEPC（PEPC C.g.）显示出其特征性的K(m)（PEP）值。对PEPC高表达的植株以及表达马铃薯反义（anti-sppc）基因的植物细胞系进行了植物生长情况检测。在无菌培养条件下，cppc植物细胞系的生长速率明显降低，而反义sppc株系的生长速率与对照相似或略高于对照。cppc植株中的苹果酸水平升高，反义植株中的苹果酸水平降低。对照植株与过表达PEPC C.g.的转化体或反义sppc植株之间在光合电子传递或稳态CO2同化方面没有显著差异。然而，长时间的黑暗处理导致PEPC含量较低的植株中光合电子传递的诱导延迟。在高光质子通量密度下光照45分钟后测定黑暗中的CO2释放速率，cppc植株中该速率显著提高，反义sppc植株中该速率略有降低。当根据光下线粒体呼吸的估计速率校正CO2同化速率时，在低CO2条件下测定的CO2同化所需电子在PEPC较高的转化体中略低，而PEPC降低的转化体表现出明显升高的电子需求。PEPC活性高的植株（cppc）中的CO2补偿点保持不变，但在反义植物细胞系中可能会升高。反义植株中的气孔开放延迟，但与对照相比，过表达PEPC C.g.的植株中气孔开放加速。
