*3.4. Cytosolic Pyruvate Kinases are Vital for Energy Allocation*

Cytosolic PK is a key player in energy allocation, as it generates ATP in the cytosol, supplies pyruvate to the TCA cycle and thereby drives mitochondrial ATP synthesis. Cytosolic PK isoforms were strongly expressed in the leaf vasculature (Figure 6). This is expected since most cells of the vasculature are heterotrophic and depend on ATP generated via glycolysis and mitochondrial respiration. Furthermore, this enables fast access to carbohydrate substrates, for example sucrose, that is delivered from photosynthetic source tissues via the phloem sap. Our GUS experiments also revealed expression of *cPK1*, *cPK2* and *cPK3* in mesophyll cells. This expression became more pronounced at the end of the dark period, underlining the increased respiratory activity, which is normally present during the night. Furthermore, we observed distinct GUS expression in root tips, shoot apical meristems and leaf primordia (Figures 3 and 4A,K–Q), indicating a significant role of cPK isoforms in ATP and pyruvate provision to these young, developing and highly energy-consuming tissues.

#### *3.5. Pyruvate Kinase Enzymes are Di*ff*erently Regulated by Metabolites*

Several glycolytic enzymes underlie negative allosteric regulation by TCA cycle intermediates [33]. A major issue is to control the cellular NADH + H+/NAD<sup>+</sup> and ATP/ADP ratios in order to sustain tolerable physiological conditions. This is achieved by the pH-dependent balance between citrate and isocitrate formation, which determines the overall TCA cycle flux [34]. Our kinetic characterization demonstrated that all five cytosolic PK enzymes from *Arabidopsis thaliana* are strongly inhibited in the presence of citrate, confirming previous studies on PKs [33]. cPK1 underlies a further mechanism of control because its activity was affected by aspartate and glutamate (Table 2). This might provide a feedback control balancing the generation of carbon skeletons required for NH4 <sup>+</sup> assimilation in tissues highly active in amino acid biosynthesis. Furthermore, the activity of cPK1, cPK2 and cPK3 was negatively controlled by ATP, which suggests that these isoforms are inhibited in vivo under sufficient energy supply. Activities of cPK4 and cPK5 were not affected by ATP, indicating that both isoforms act independently of the energy status of the cell, for instance under stress-related conditions. In summary, our data show that *Arabidopsis thaliana* encodes five isoenzymes with individual regulatory properties, allowing a versatile system of cPK control.
