In applied transgenic research as well as in agriculture there is an increasing need for high-throughput analyses of plants for genotypic selection or to identify the purity of seed stocks, e.g. for transgenic contaminations or the identification of pathogens. We developed and optimised conditions for the isolation of DNA from single seeds using an automated high-throughput protocol. Our results show that the system provided is capable of isolating DNA from any tested seed source. Furthermore, seeds remain capable of germinating during the homogenisation procedure. Quantification of endogenous and transgenic sequences by Real-Time PCR revealed that the prepared DNA is suitable in quality and quantity for multiple PCR analyses with both SYBR Green and hybridisation probe detection. The described method will be a useful tool for routine analyses like screening of large populations or the specific detection of genetically modified organisms (GMO).

There is an increasing need for high-throughput analyses of plants and food samples for the presence of specific DNA sequences, e.g. transgenic contaminations. We developed and optimized conditions for the automated isolation of DNA from several maize tissues and various edibles containing maize using the MagNA Pure LC system (Roche Applied Science). Our results show that the system provided is capable of isolating DNA from any tested source. Quantification of an endogenous gene by LightCycler real-time PCR revealed that the DNA is suitable in quality and quantity for multiple PCR analyses.

Carbon assimilation in maize follows the C(4) mechanism. This requires the tissue-specific and light-induced expression of a set of different genes involved in CO(2) fixation as well as adaptations in the leaf anatomy including a reduced distance between vascular bundles compared to C(3) plants. However, several maize tissues exist with larger bundle distances and there is significant evidence that CO(2) fixation follows the C(3) mechanism in these tissues. We isolated maize C(3) and C(4) tissues and quantified the accumulation of mRNAs encoding PEPC, ME, the small subunit of Rubisco, and PPDK. For this, primer systems for the specific and sensitive detection by real-time PCR were established. The observed patterns show the expected distribution for foliar leaf tissues. Also in total husk leaves, all transcripts under investigation were detected, albeit at a lower level. When mesophyll cells which are located distant from bundles were isolated from husk leaves, only accumulation of RbcS was observed. Comparing the expression of two genes encoding for isoenzymes of the small subunit of RbcS in the different tissues differential patterns of relative transcript abundance were observed. Transcripts for the DOF1 transcription factor involved in the activation of photosynthetic genes in maize were found in leaf tissues performing both C(4) and C(3) photosynthesis with highest accumulation levels in C(4) mesophyll cells, whereas the homologous DOF2 gene was not expressed in any of the investigated samples. The results provide novel insights into the regulation of C(3) and C(4) carbon fixation pathways in maize.

Expression of genes necessary to perform C4 photosynthesis in maize is activated by light. It is not known how this activation is regulated on the chromatin level in vivo. We analysed alterations in the chromatin structure of the promoter of the C4-specific isoform of phosphoenolpyruvate carboxylase (PEPC) after illumination of seedlings. A protocol was established that facilitates the preparation of nuclei from maize leaves with intact chromatin structure and resistance to DNA degradation during prolonged incubation at high temperatures. The presence of non-spliced transcripts from the C4-PEPC gene in the nuclei was demonstrated by RT-PCR. The chromatin was partially digested with restriction endonucleases. Quantitative PCR analyses revealed a clear increase in the accessibility of the promoter chromatin to restriction dependent on illumination of the seedlings. The data indicate chromatin reorganization at the C4-PEPC promoter during activation.

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