*4.3. Generation of Promoter-ß-Glucuronidase Fusion Constructs*

Promoter-β-glucuronidase (GUS) fusion constructs were generated in order to localize the expression of selected genes involved in growth and development of *Arabidopsis thaliana*. The 5 -flanking regions of cytosolic PK genes were cloned into the gateway binary vector pGWB3, which mediates GUS fusion [36]. Initially, ~1800 bp of the promoter regions were amplified from *Arabidopsis thaliana* genomic DNA by preparative PCR and introduced into gateway pENTR vectors. For the cPK1 promoter, the *Nco*I site was inserted into the 5 end forward primer prom\_cPK1\_*Nco*I\_for and the *Xma*I site into the 3 end reverse primer prom\_cPK1\_*Xma*I\_rev to facilitate T4-ligase (New England Biolabs)-mediated ligation into pENTR4 (Invitrogen, Carslbad, USA). Oligonucleotide sequences for cloning are provided in the Appendix A (Appendix A Table A1).

All other constructs were generated by TOPO cloning into the pENTR/D-TOPO vector (Invitrogen) according to the manufacturer's instructions using the following primer pairs for DNA amplification: for cPK2, prom\_cPK2\_TOPO\_for and prom\_cPK2\_TOPO\_rev; for cPK3, prom\_cPK3\_TOPO\_for and prom\_cPK3\_TOPO\_rev; for cPK4, prom\_cPK4\_TOPO\_for and prom\_cPK4\_TOPO\_rev; for cPK5, prom\_cPK5\_TOPO\_for and prom\_cPK5\_TOPO\_rev. The resulting pENTR constructs were transformed into the *E. coli* strain DH5α. After transformation, the *E. coli* were plated onto LB agar containing the respective antibiotics in order to select for successfully transformed colonies. Transformed colonies were confirmed via PCR and restriction digestion of isolated plasmid DNA as well as double-strand sequencing. The promoter fragments were cloned into the vector pGWB3 by LR reaction (LR-clonase, Invitrogen, Carslbad, CA, USA). The final plasmids were stably introduced into *Arabidopsis thaliana* plants by *Agrobacterium tumefaciens* (*A. tumefaciens*)-mediated transformation, and transformants were isolated following selection with the respective antibiotics on half-strength MS agar (Duchefa, 50 μg/mL kanamycin, 50 μg/mL hygromycin, 0.5% agarose).

## *4.4. Generation of 5x His-Tagged Fusion Constructs*

For heterologous expression of cytosolic PK proteins in *E. coli*, full-length cDNA was cloned into the pET16b vector (Novagen, Darmstadt, Germany) mediating N-terminal His-tag fusion. The cDNA of cPK2 and cPK3 was amplified with the following oligonucleotides carrying the restriction enzyme recognition sites for subsequent classical ligation into the destination vector for cPK2 cPK2\_*Nde*I\_for and cPK2\_*Nde*I\_rev, for cPK3 cPK3\_*Nde*I\_for and cPK3\_*BamH*I\_rev. In order to increase the DNA yield of cPK1, cPK4 and cPK5 amplicons, the cDNA was subcloned into the pENTR/D-TOPO vector (Invitrogen, Carslbad, USA) by TOPO cloning using primers cPK1\_TOPO\_for and cPK1\_TOPO\_rev for cPK1, cPK4\_TOPO\_for and cPK4\_TOPO\_rev for cPK4, and cPK5\_TOPO\_for and cPK5\_TOPO\_rev for cPK5. Oligonucleotide sequences for cloning are provided in the Appendix A (Appendix A Table A1). Amplified cDNA was digested with the indicated enzymes, purified by gel extraction and cloned into pET-16b by T4 Ligase (New England Biolabs, Ipswich, USA). Obtained plasmids were transformed into the *E. coli* strain BLR21, and the constructs were verified by colony PCR and restriction digestion.
