Regulation of Phosphoenolpyruvate Carboxylase Phosphorylation by Metabolites and Abscisic Acid during the Development and Germination of Barley Seeds  

Abstract

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPCspecific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by L-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca 2+ -independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by L-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.

Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth

“…The phosphorylation state of PPC was investigated using immunoblotting with an anti-phospho-PPC antibody (Feria et al, 2008). PPC protein levels were also measured on replicate immunoblots using an anti-PPC antibody to ensure even loading of protein in each lane (Fig.…”

“…Blots were developed using the ECL system (GE Healthcare). Immunoblot analysis was carried out using antisera to PPC (raised against PPC from K. fedtschenkoi, kindly supplied by Hugh G. Nimmo, University of Glasgow; Nimmo et al, 1986), and the phosphorylated form of PPC (raised against a phospho-PPC peptide from barley [Hordeum vulgare] and kindly supplied by Cristina Echevarría, Universidad de Sevilla; González et al, 2002;Feria et al, 2008), PPDK and phosphorylated PPDK (raised against maize [Zea mays] PPDK and kindly supplied by Chris J. Chastain, Minnesota State University, Moorhead; Chastain et al, 2000Chastain et al, , 2002, and a-and b-NAD-ME (raised against the Arabidopsis proteins, supplied by Maria F. Drincovich, Universidad Nacional de Rosario; Tronconi et al, 2008).…”

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi

“…Immunoblot analysis was performed using antisera to PPC (raised against PPC from K. fedtschenkoi, kindly supplied by H.G. Nimmo, University of Glasgow) (Nimmo et al, 1986) and the phosphorylated form of PPC (raised against a phospho-PPC peptide from barley [Hordeum vulgare] and kindly supplied by Cristina Echevarría, Universidad de Sevilla, Spain) (González et al, 2002;Feria et al, 2008).…”

Characterisation of seeds of a C4 phosphoenolpyruvate carboxylase‐deficient mutant of Amaranthus edulis

“…In germinating seeds, this post-translational regulation of PEPC has been described for wheat, barley and Sorghum vulgare (Osuna et al 1996(Osuna et al , 1999Nhiri et al 2000). Recent data have established that the phosphorylation of PEPC in barley seed is mainly regulated by the glucose-6phosphate ⁄ malate ratio and is not correlated with the amount of PEPC kinase activity (Feria et al 2008).…”

“…2000). Recent data have established that the phosphorylation of PEPC in barley seed is mainly regulated by the glucose‐6‐phosphate/malate ratio and is not correlated with the amount of PEPC kinase activity (Feria et al. 2008).…”