Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds1

Abstract

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C 4 PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103-and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32 P-labeled inorganic phosphate. In vitro phosphorylation by a Ca 2؉ -independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme's velocity and decreased its sensitivity to L-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca 2؉ -independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C 4 mesophyll protoplasts. These collective data support the hypothesis that this Ca 2؉ -independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.

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

“…This has been investigated in developing seeds of the leguminous species Vicia faba (Golombek et al, 1999) and during castor oil seed development, as mentioned above (Tripodi et al, 2005;Gennidakis et al, 2007;Murmu andPlaxton, 2007: Uhrig et al, 2008). In barley (Hordeum vulgare), wheat (Triticum aestivum), and sorghum seeds, our results established that PEPC phosphorylation occurred in situ during early germination, and the dedicated Ca 2+ -independent PEPC kinase appeared to be present (Osuna et al, 1996(Osuna et al, , 1999Nhiri et al, 2000). However, in contrast to the leaf context, the PEPC kinase was constitutive during barley seed imbibition, and the cascade controlling its activity by protein synthesis was not operative (Osuna et al, 1999).…”

“…In barley (Hordeum vulgare), wheat (Triticum aestivum), and sorghum seeds, our results established that PEPC phosphorylation occurred in situ during early germination, and the dedicated Ca 2+ -independent PEPC kinase appeared to be present (Osuna et al, 1996(Osuna et al, , 1999Nhiri et al, 2000). However, in contrast to the leaf context, the PEPC kinase was constitutive during barley seed imbibition, and the cascade controlling its activity by protein synthesis was not operative (Osuna et al, 1999). This led us to study when and how the kinase was accumulated during seed development.…”

“…2B). Two immunologically related subunits have also been identified in developing and germinating wheat seeds (Osuna et al, 1996;González et al, 1998), castor oil seed (Sangwan et al, 1992;Uhrig et al, 2008), and during the germination of barley and sorghum seeds (Osuna et al, 1999;Nhiri et al, 2000). However, whether these different subunits (108 and 103 kD in barley, 110 and 107 kD in castor oil seed) are combined to form a heterotetrameric structure in vivo (as in banana [Musa spp.…”

“…However, this scenario is not probable, since only phosphorylation by the Ca 2+ -independent PEPC kinase alters PEPC sensitivity to malate (Duff et al, 1996;Nhiri et al, 1998;Ogawa et al, 1998). Furthermore, during imbibition of barley seeds, PEPC phosphorylation was not affected by the Ca 2+ -dependent protein kinase inhibitor W7 (Osuna et al, 1999). The comparison of the in vitro PEPC kinase activity (Fig.…”

“…The PEPC Kinase Is Subject to Metabolite Control in Seeds PEPC kinase activity is mainly controlled at the transcriptional level in most plant organs and tissues (for review, see Nimmo, 2003), with the notable exception of germinating seeds (Osuna et al, 1999). Other mechanisms acting at the posttranslational level have been reported to affect PEPC kinase activity: in vitro thioredoxin-mediated changes of PEPC kinase activity, but this has still to be shown in planta (Tsuchida et al, 2004;Murmu and Plaxton, 2007); a metabolic control, both in vitro (Echevarría et al, 1994;Murmu and Plaxton, 2007) and in situ by feeding mesophyll protoplasts with metabolite effectors ; and, finally, a heat-sensitive 100-kD protein inhibitor interacting with the kinase in extracts from maize and Kalanchoe fedtschenkoi leaves (Nimmo et al, 2001).…”