*2.2. Pyruvate Kinase Genes Show an Isoform-Specific Expression Pattern*

The high number of expressed cytosolic PK isoforms allows each gene candidate to potentially fulfill distinct tasks during plant glycolysis. To assess tissue-specific expression of respective cPK genes in dependence on the developmental status of the plant, a β-glucuronidase (GUS) reporter approach was taken. Promoters of the five *cPK* candidates were fused to GUS and expressed in *Arabidopsis thaliana*. Subsequently, the plant organs were stained and analyzed in detail. The GUS-staining results suggest that the cytosolic isogenes *cPK1, 2* and *3* are expressed in vegetative and reproductive tissues, as promoter-GUS activity was observed in leaves, roots and flowers of plants from diverse developmental stages (Figure 3, Figure 4, Appendix A Figure A3). However, for cPK4 and cPK5 a quite strong GUS signal was observed during seedling stage, and the same lines showed no or very low expression in tissues of mature plants under standard growth conditions.

**Figure 3.** Histochemical β-glucuronidase (GUS) localization in seedlings of the *cPK1*, *cPK2*, *cPK3*, *cPK4* and *cPK5* promoter-GUS constructs, one day after germination (DAG) up to ten DAG. Representative images of one out of three individual transgenic lines are shown. n.d. indicates developmental stages without detectable GUS activity. Bars indicate 0.5 mm.

Analysis of the first ten days of seedling development revealed clear differences in the expression of selected *cPK* isogenes (Figure 3). While the *cPK1* promoter lead to broadly abundant GUS activity in roots and leaves of seedlings from the first day on, respective lines for the *cPK2* and *cPK3* promoters showed comparable GUS signals only at later stages. Notably, *cPK1* promoter GUS lines displayed strong staining in the shoot apical meristem (Figure 3) and in the basal part of young leaves (Figure 2 promcPK1::GUS, 8 DAG). *cPK2* promoter GUS expression did not initiate in the rosette axis until ten days after germination. The *cPK3* promoter-induced expression was restricted to the root tip and was already visible on the first day after germination. From the third day on, the signal became abundant in the entire root and was also observed in the leaf axis and spatial related trichomes. In *cPK4*-promoter plants expression was observed from the first day on in the entire root and the leaf vasculature, and later in hydathodes and at the sides of emerging leaf buds (Figure 3, promcPK4::GUS, 8 DAG). In contrast, expression of *cPK5* started later and was mainly restricted to the cotyledon, leaf and root vasculature. As indicated before, promoter-GUS constructs for *cPK1*, *2* and *3* were expressed in the vasculature and mesophyll of rosette leaves with varying intensity depending on developmental stage of the leaf (Appendix A, Figure A3). In roots, *cPK1* appeared to be ubiquitously expressed, whereas GUS activity for *cPK2* and *cPK3* promoters was limited to confined areas. The *cPK2* promoter GUS lines showed blue stains restricted to primary roots (Figure 4N), while the *cPK3* promoter primarily lead to GUS activity in younger roots and root tips (Figure 4M). Promoters of both *cPK2* and *cPK3* drive GUS expression at sites of emerging lateral roots (Figure 4P,Q). In young flowers of *cPK1* promoter-GUS lines, no signal was observed, whereas fully developed flowers appeared to be ubiquitously stained in sepals, petals, filaments of stamen, style and the stigma tissue (Figure 4B,E,H). *cPK2* was also not expressed in young flowers, whereas in later stages, GUS-staining was limited to sepals and style (Figure 4C,F,I). In contrast, *cPK3* promoter-dependent GUS expression was already detected in the abscission zones of young flower buds (A), and later expression became apparent in the anthers as well (G).

**Figure 4.** Histochemical GUS localization in flower organs (**A**–**G**) and in roots (**K**–**Q**), for the *cPK1*-promoter-GUS (**B**,**E**,**H**,**K**), the *cPK2*-promoter-GUS (**C**,**F**,**I**,**L**,**N**,**P**) and the *cPK3*-promoter-GUS (**A**,**D**,**G**,**J**,**M**,**O**,**Q**) in 3-week-old plants (**A**–**D**) and 5-week-old plants (**E**–**J**), is illustrated in developing flowers (**B**–**D**) and in fully developed carpels (**E**–**G**) with stamen as well as entire flowers (**H**–**J**) of 5-week-old plants. Pictures show root tips of 3-week-old plants (**K**–**M**) and GUS expression in 5-week-old plants, restricted to primary roots for the *cPK2*-promoter-GUS (**N**) and stem cells in root tips for the *cPK3*-promoter-GUS (**O**). Expression at sites of emerging secondary roots for the *cPK2*- and *cPK3*-promoter-GUS (**P**,**Q**). Representative images of one out of three different transgenic lines are shown. n.d. indicates plant organs without detectable GUS activity. Bars indicate 0.5 mm.
