Matthias Fladung

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.

Genetic variants of Panicum maximum (Jacq.) in C4 photosynthetic traits

Summary During the evolution of the C 4 type of photosynthesis, the key C 4 enzymes levels have become increased, together with the development of the typical C 4 «Kranz»-anatomy. The existence in nature of C 3 –C 4 intermediate species suggest that C 4 plant variants hindered in their capacity to fix CO 2 may exist. Seeds of the C 4 plant Panicum maximum Qacq.) were mutagenized using ethylmethanesulfonate (EMS) and putative variants were isolated in the M 2 generation by visual inspection, considering morphology and pigmentation of the plant as criteria for variant selection. Variant plants and their progenies, when available, were characterized by photosynthetic, photorespiratory and leaf morphological parameters. The variants isolated were midribless ( mbl ), large interveinal space-1 and -2 ( lis1 , lis2 ), abnormal bundle sheath ( abs ), high number of small veins-1 and -2 ( hsv1 , hsv2 ) and variegated leaf-1 ( var1 ). The variant lis1 was a short plant with leaves smaller than in the wildtype, had a leaf lamina with a crinkly surface and a lighter green color. Photosynthetically, lis1 indicates a clear, but not extreme, regression from C 4 to C 3 photosynthesis represented by a COz compensation value around 8 μL · L -1 , which was correlated in the leaf lamina with an increase in the distance between small veins. The leaf anatomy of the variant abs showed several alterations, including doublets of veins, veins without bundle sheath (BS cells), additional BS cells outside the veins or large BS cells participating in two bundle sheaths. The variant var1 had a variegated phenotype with stripes of yellow-green and white tissues alternating in the leaf laminae. In yellow-green sectors the chloroplasts were absent only in BS cells, the adjacent mesophyll cells were less pigmented than similar ones present in non-variant sectors. Based on anatomical alterations induced by the mutations isolated and characterized in this paper, it is concluded that in P. maximum the establishment of the vascular system is under complex genetic control: all variants interfere with shape, organisation and distribution of midvein, lateral and small veins.

Constitutive or light-regulated expression of the rolC gene in transgenic potato plants has different effects on yield attributes and tuber carbohydrate composition

Tetraploid potato clones, transgenic for the rolC gene of Agrobacterium rhizogenes under control of the light-inducible ribulose bisphosphate carboxylase small subunit promoter (rbcS-rolC), were compared, with respect to yield attributes and tuber carbohydrates, with transformed and untransformed controls and with 35S-rolC transgenic potato plants. In rbcS-rolC plants, the expression of the rolC gene was located mainly in leaves, while in 35S-rolC plants transcripts were detected as well in shoots and roots. Phenotypically, rbcS-rolC transgenic plants were found to be slightly reduced in plant size with a few more tillers than control plants. Photosynthetic rate and chlorophyll content were significantly lower in all rolC transgenic plants irrespective of the type of construct used. Tuber yield was not significantly different between controls and rbcS-rolC transgenic plants, but was reduced in the 35S-rolC transformants. Sucrose level was unchanged in all rolC clones investigated, whereas fructose content was significantly enhanced in 35S-rolC transformants, but not in the plants expressing the rolC gene in aerial plant parts only. In both types of rolC transgenic plants, glucose content was lower than in controls, resulting in a significant reduction of reducing sugar in tubers. The results suggest a hormonal influence on the carbohydrate composition of potato tubers.

Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides

Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.

Effect of varying environments on photosynthetic parameters of C3, C3-C4 and C4 species of Panicum

SummaryCO2 exchange characteristics and the activity of the carboxylating enzymes phosphoenolpyruvate carboxylase (PEP-C, E.C. 4.1.1.31) and ribulose 1,5-bisphosphate carboxylase (RuBP-C, E.C. 4.1.1.39) during one year in the greenhouse and at two levels of light and temperature in growth chambers were determined in the C3-C4 intermediate species P. milioides Nees ex. Trin. These results were compared with those of P. bisulcatum Thumb. (C3) and P. maximum Jacq. (C4). Under all tested conditions, and even when the influence of leaf surface temperature on photosynthetic rates and CO2 compensation points were measured, the biochemical and physiological behaviour of the C3-C4 intermediate was more similar to that of the C3 plant than the C4 species. The C4 plant P. maximum, however, responded positively, mainly in terms of PEP-C activity and photosynthetic rate, to the regime of high light and temperature. The results presented indicate that in the C3-C4Panicum grown in high light and temperature no direct relationships between a low CO2 compesation point and superior growth are evident. It has still to be clarified why in nature a photosynthetic-photorespiratory pathway leading to an intermediate CO2 compensation value has evolved in P. milioides.

Antibacterial Resistance of Transgenic Potato Plants Producing T4 Lysozyme

Transgenic potato plants have been produced that express and secrete the foreign bacteriophage T4 lysozyme. The chimeric barley a-amylase signal peptide — T4 lysozyme gene is driven by the CaMV 35S promoter. Low level expression can be detected at mRNA and protein level. Subcellular localization analysis demonstrates secretion of the foreign protein to the intercellular spaces. Biological in vitro and greenhouse experiments demonstrate that tuber maceration following inoculation with Erwinia carotovora atroseptica in transgenic tissue is significantly reduced in comparison to control tissues. Moreover, explants from inoculated transgenic tuber pieces show sprouting and growth without development of disease symptoms. Expression and secretion of the foreign lysozyme obviously leads to a reduced susceptibility of the transgenic potato plants towards the phytopathogenic bacterium.

Developmental Studies on Photosynthetic Parameters in C3, C3 - C4 and C4 Plants of Panicum

Summary Mature, half unfolded, and not unfolded leaves of Panicum bisulcatum , a C 3 plant, P. milioides , a C 3 -C 4 intermediate plant and P. maximum , a C 4 plant, were examined in respect to leaf anatomy, photosynthetic rates, CO 2 compensation points, phosphoenolpyruvate carboxylase (PEP-C, E.C. 4.1.1.31) and ribulose 1,5-bisphosphate carboxylase (RuBP-C, E.C. 4.1.1.39) activities. In cross sections of all leaf stages examined for the C 3 and for the C 3 -C 4 species no visible changes in the leaf anatomy were observed. In the C 4 plant the characteristic centrifugal arrangement of the chloroplasts in the bundle sheath cell, found in mature leaves, was not found in all not unfolded leaves. In the C 3 and the C 3 -C 4 species no significant changes in photosynthetic rates and CO 2 compensation points during leaf development were found, while in the C 4 plant an increase of the photosynthetic rates and a decrease of the CO 2 compensation points were measured. This seems to be due to the increase of the PEP-C activity during leaf maturation in the C 4 plant, while in the C 3 and C 3 -C 4 plants a decrease of PEP-C and an increase of RuBP-C activity was observed. The study of other photosynthetic tissues, e.g. stems and panicles, revealed an absence of distinctive morphological characteristics related to the type of photosynthesis. Nevertheless, physiological and enzymatic differences between the stems and the panicles of the different Panicum species were found.