*2.3. Cytosolic Pyruvate Kinases Respond to Cold Stress*

In contrast to the plastid-localized pyruvate kinases, the analysis of public available transcript data [15] revealed that the cytosolic PKs are induced in response to cold treatment (Figure 4A). According to these data, *cPK1* and *cPK2* expression is induced, whereas *cPK3* transcript levels are not altered. The strongest induction upon cold treatment was found for the isogenes *cPK4* and *cPK5*. However, regarding the microarray data, *cPK4* and *cPK5* expression cannot be considered separately, since respective data do not discriminate between both genes. Thus, we validated these data by using *Arabidopsis* lines carrying the respective GUS-promoter constructs that were sampled after cold treatment (Figure 5B). In *cPK4*-lines, the GUS signal was very low in untreated control plants, and it was strongly induced in the leaf vasculature of cold treated plants. *cPK5* expression appeared to also be increased; however, the observed effect was not as strong as in the *cPK4*-GUS line. Therefore, our results indicate that cytosolic pyruvate kinases are induced during cold treatment.

**Figure 5.** (**A**) Expression data of cytosolic and plastidic pyruvate kinases after cold treatment obtained from publicly available data source (https://genevestigator.com). Sixteen-day-old plants were transferred to the cold room (4 ◦C), and plant tissues were harvested at 24 h after onset of treatment. (**B**) To validate cold-induced expression of *cPK4* and *cPK5*, twenty-day-old *cPK4* and *cPK5* promoter GUS plants grown on soil were transferred to the cold room (4 ◦C) for three days. Control plants were grown under normal growth conditions. The oldest leaves of control and cold-treated plants were sampled, and the GUS activity was determined by histological staining. Representative images of one out of three different transgenic lines are shown.
