Journal
JOURNAL OF EXPERIMENTAL BOTANY
Volume 67, Issue 11, Pages 3523-3536Publisher
OXFORD UNIV PRESS
DOI: 10.1093/jxb/erw186
Keywords
Germination; monoubiquitination; phosphorylation; phosphoenolpyruvate carboxylase; post-translational modifications; seed development; seeds; Sorghum bicolor; tissue-specific gene expression
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Funding
- Ministerio de Economia y Competitividad [AGL2012-35708]
- 'La Junta de Andalucia' grant [P06-CVI-02186, BIO298]
- 'Plan Propio de la Universidad de Sevilla'
- Natural Science and Engineering Research Council of Canada
- Queens's Research Chairs program
- Chinese Academy of Sciences
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This work documents the remarkable versatility and complexity of in vivo post-translational modifications (monoubiquitination and phosphorylation) of different plant-type phosphoenolpyruvate carboxylase (PEPC) isoenzymes during the life cycle of sorghum seeds.Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.
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