**3. Discussion**

## *3.1. Arabidopsis thaliana Five Cytosolic Pyruvate Kinases Form Two Functional Subgroups*

Various previous studies showed that plant PKs either localize to the cytosol or the plastid, whereby the subcellular distribution is of crucial relevance for enzyme activity. Plastidial PK isoforms are reported to be essential for biosynthesis of seed oil and the mobilization of storage compounds in *Arabidopsis thaliana* seedlings [5,19]. Cytosolic PKs on the contrary take over an important role in carbohydrate breakdown, cytosolic ATP biosynthesis and provision of pyruvate to the TCA cycle, as known from other plant and non-plant organisms [20]. Apart from their fundamental role in primary metabolism, reports on *Arabidopsis thaliana* PKs are limited to seed-expressed, plastid-localized isoforms [5]. In the present study, five potential cytosol-targeted PKs from *Arabidopsis thaliana* were identified based on phylogenetic studies and gene expression data [5] (Figure 1A). The prediction of cytosolic localization of cPK2 (At5g56350), cPK4 (At2g36580) and cPK5 (At3g52990) was confirmed by investigating YFP-fusion proteins in *Nicotiana benthamiana* leaf cells (Figure 2). Furthermore, PK activity was verified in vitro for all five isoforms since the isoenzymes were capable of hydrolyzing PEP into pyruvate and ATP (Table 1). Our observations suggest that within the subclade of cytosolic PKs, *cPK1*, *cPK2* and *cPK3* form a subgroup distinct from *cPK4* and *cPK5*, also displayed by structural characteristics of the genetic sequence. The specific roles for each subgroup were illustrated by GUS expression data, which showed entirely independent expression patterns for each isoform. Furthermore, the analysis of in vitro enzyme activity suggests regulatory properties of subunit complexes composed of cPK1/2, cPK2/3 or cPK4/5 isoforms, respectively (Figure 7).

## *3.2. Pyruvate Kinase Enzymes are Localized to the Cytosol*

Three out of five PK candidates were localized to the cytosol via confocal microscopy of YFP-fusion proteins (Figure 2). This is in agreement with predictions based on a bona fide alignment of PK proteins from other plants [5]. Since consensus sequence predictions for cPK1 and cPK3 identified no target peptide for a specific organelle, we presume that both PKs also target to the cytosol [16]. Furthermore, formation of complexes with cPK2, cPK4 and cPK5 bears regulatory property of PK activity, assuming both isoforms of an enzyme pair to target to the same compartment.
