title: New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.
abstract: 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.
authors.affiliation: Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes nº 6, 41012 Sevilla, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ruiz-Ballesta
authors.affiliation: Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes nº 6, 41012 Sevilla, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Baena
authors.affiliation: Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes nº 6, 41012 Sevilla, Spain.
authors.forename: Jacinto
authors.initials: J
authors.lastname: Gandullo
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, China State Key Laboratory of Crop Genetics and Germplasm Enhancement, Soybean Research Institute, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
authors.forename: Liqun
authors.initials: L
authors.lastname: Wang
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, China.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: William Charles
authors.initials: WC
authors.lastname: Plaxton
authors.affiliation: Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avda Reina Mercedes nº 6, 41012 Sevilla, Spain echeva@us.es.
authors.forename: Cristina
authors.initials: C
authors.lastname: Echevarría
mesh_terms: D018525:Germination; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D045868:Sorghum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D053719:Tandem Mass Spectrometry; D054875:Ubiquitination
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D010730:Phosphoenolpyruvate Carboxylase
keywords: Germination; Sorghum bicolor; monoubiquitination; phosphoenolpyruvate carboxylase; phosphorylation; post-translational modifications; seed development; seeds; tissue-specific gene expression.