*3.3. Cytosolic Pyruvate Kinase Genes are Expressed in a Tissue-Specific and Developmental-Specific Manner*

Our GUS studies show a distinct tissue-specific expression of the five *cPK* isogenes, also supported by publicly available microarray data [15]. Considering the fact that the cPK isoforms underlie distinct metabolic control, it can be concluded that plants are able to fine-tune cell and tissue-specific metabolism by coordinated *cPK* expression, in order to closely match the energy requirements under different conditions.

Cytosolic *PK1* appeared to be ubiquitously expressed in all tissues and in all developmental stages. This stands in contrast to *cPK2* and *cPK3* expression, which started at a later stage of seedling development and was initially restricted to meristematic tissues, such as the root tips (Figure 3) and to the proliferation zone of young leaves. However, all three isoforms were expressed in later developmental stages. In contrast, *cPK4* expression was pronounced in roots and cotyledons during the first days of development (Figure 3). At a later stage, *cPK4* together with *cPK5* was expressed only under stress conditions (Figure 5). Seedling establishment might be considered as a stress situation, since the plants face limited carbon supply because of an inactive or not yet fully activated photosynthetic machinery. Accordingly, during this stage no carbon assimilation takes place, and the plants depend on the degradation of compounds that were supplied by the mother plant [21]. During germination, triacylglycerol (TAG) is degraded via β-oxidation, and the released fatty acids are converted to sucrose through the glyoxylate cycle and gluconeogenesis. Thereby PEP is formed from oxaloacetate by the cytosolic PEP carboxykinase (PEPCK) [22]. Possibly, conditions favoring gluconeogenic respiration, for example high ATP levels, restrict some cPK isoforms more than others. Interestingly, cPK4 and

cPK5 remained unaffected by ATP levels, while cPK1 and cPK3 were subjects of strong ATP inhibition (Figure 7). In general, gene expression is only one level of regulation and does not necessarily reflect the protein content in the respective tissue, since cPK has been reported also to be strongly regulated by protein degradation [14,23]. Furthermore, we demonstrated that full cPK activity depends on the formation of subgroup complexes (Figure 7). This leads to the possibility that a comparatively weakly expressed isoform that is part of a complex might still have an essential impact on total cPK activity. We induced the expression of cPK4 and cPK5-GUS via cold treatment, as we could not detect any expression under ordinary conditions. This result is in line with published microarray data [15]. Exposure to low temperatures enhances freezing tolerance, a versatile process involving various alterations in biochemical processes and global changes in gene expression that are accompanied by a shift in the composition of primary and secondary metabolites [24–27]. Under these conditions cell division and expansion is actively inhibited, leading to sink limitation and subsequent accumulation of carbohydrates [28]. The chilling response involves an acceleration in levels of γ-aminobutyric acid (GABA), proline and sucrose [29], metabolites that were shown to be involved in protecting membranes and proteins from freezing damage [30–32]. Under these conditions, cPKs have to maintain TCA cycle flux independent of the cytosolic ATP status, as GABA and proline depend on TCA cycle intermediates.
