**4. Materials and Methods**

#### *4.1. Assaying Tissue-Specific Pyruvate Kinase Expression by Promoter-GUS Fusion*

To analyze the localization of pyruvate kinase expression during plant growth and development β-glucuronidase (GUS) fusion constructs were stably transformed into *Arabidopsis thaliana*. The first 16 days after germination were investigated on seedlings grown on half strength MS (0.5% agarose), whereas samples of later developmental stages were taken from plants grown on soil in a growth chamber. For diurnal expression analysis plants were grown under short day conditions (16 h dark, 8 h light, 160 <sup>μ</sup>E·m−2·s−1). Histochemical staining of transgenic plants was performed by vacuum infiltration with staining solution (0.1M NaPO4 pH 7.2, 10 mM EDTA, 0.5 mM K3Fe[CN]6, 0.5 mM K4Fe[CN]6, 10% Triton x-100) supplied with 1 mM of the substrate of β-glucuronidase x-Gluc (5-bromo-4-chloro-3-indolyl *s*-d-glucuronic acid, solved in dimethylformamide) in an evacuated exsiccator according to the protocol established by Jefferson et al. (1987) [35]. For staining, the samples were incubated for 37 ◦C overnight and subsequently relieved from chlorophyll with 80% EtOH at 60 ◦C. Stained younger plants were documented with a Leica binocular (S8APO, equipped with an EC3 camera, Leica), and pictures were processed with the compatible LAS EZ imaging software (Leica). Older plants were photographed with a digital single-lens reflex camera (Sony α330). GUS-stained tissues and plants shown in this work represent the representative results of at least four independent lines for each construct.
