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Unravelling the proteomic profile of rice meiocytes during early meiosis.

Collado-Romero M, Alós E, Prieto P - Front Plant Sci (2014)

Bottom Line: Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins.Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs.The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

View Article: PubMed Central - PubMed

Affiliation: Plant Breeding Department, Institute for Sustainable Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas Córdoba, Spain.

ABSTRACT
Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

No MeSH data available.


(A) Venn diagram of preliminary experiments comparing the proteins identified in a pull of full anthers and a pull of isolated meiocytes in meiosis (mix of different stages). (B)Venn diagram representing the proteins identified in rice meiocytes at early prophase I (leptotene, zygotene, and pachytene). The proteins identified at later stages (diplotene and diakinesis) were subtracted. Only peptides with 5–30 amino acids and a minimum of two peptides per protein were used for positive identification, and peptide FDR < 0.01. (C) Diagram of the biological functions associated to the 422 proteins identified only at early prophase I stages. The numbers of proteins associated to each function are in brackets.
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Figure 4: (A) Venn diagram of preliminary experiments comparing the proteins identified in a pull of full anthers and a pull of isolated meiocytes in meiosis (mix of different stages). (B)Venn diagram representing the proteins identified in rice meiocytes at early prophase I (leptotene, zygotene, and pachytene). The proteins identified at later stages (diplotene and diakinesis) were subtracted. Only peptides with 5–30 amino acids and a minimum of two peptides per protein were used for positive identification, and peptide FDR < 0.01. (C) Diagram of the biological functions associated to the 422 proteins identified only at early prophase I stages. The numbers of proteins associated to each function are in brackets.

Mentions: The protein extraction protocol consisted on several rounds of extraction with a phenol-based buffer followed by ammonium acetate precipitation. The quality and the complexity of the extracted proteins were checked by 1D-SDS-PAGE prior to nLC-MS/MS (Figure S2). The complexity of the meiocyte extracts was different to that found in roots, whole anthers either in early or late meiosis and lemma (floral somatic tissue). It is worth to mention that the band pattern of whole anthers at different meiotic stages was rather similar (lanes 3 and 4), but significantly different to the meiocytes extract (lane 2), suggesting that the isolation of rice meiocytes led to the enrichment on meiosis-related proteins. Indeed, preliminary nLC-MS/MS analyses comparing the protein composition from whole anthers and isolated meiocytes revealed that only 8% of the proteins found in whole anthers were also found in isolated meiocytes (Figure 4A), suggesting that an important effort isolating meiotic cells is required in order to isolate proteins that are expressed in the meiocytes. Finally, since the isolation of rice meiocytes was critical and time-consuming, the individual protein extracts from each stage were not checked in a 1D-SDS-PAGE and used only for nLC-MS/MS analysis.


Unravelling the proteomic profile of rice meiocytes during early meiosis.

Collado-Romero M, Alós E, Prieto P - Front Plant Sci (2014)

(A) Venn diagram of preliminary experiments comparing the proteins identified in a pull of full anthers and a pull of isolated meiocytes in meiosis (mix of different stages). (B)Venn diagram representing the proteins identified in rice meiocytes at early prophase I (leptotene, zygotene, and pachytene). The proteins identified at later stages (diplotene and diakinesis) were subtracted. Only peptides with 5–30 amino acids and a minimum of two peptides per protein were used for positive identification, and peptide FDR < 0.01. (C) Diagram of the biological functions associated to the 422 proteins identified only at early prophase I stages. The numbers of proteins associated to each function are in brackets.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109522&req=5

Figure 4: (A) Venn diagram of preliminary experiments comparing the proteins identified in a pull of full anthers and a pull of isolated meiocytes in meiosis (mix of different stages). (B)Venn diagram representing the proteins identified in rice meiocytes at early prophase I (leptotene, zygotene, and pachytene). The proteins identified at later stages (diplotene and diakinesis) were subtracted. Only peptides with 5–30 amino acids and a minimum of two peptides per protein were used for positive identification, and peptide FDR < 0.01. (C) Diagram of the biological functions associated to the 422 proteins identified only at early prophase I stages. The numbers of proteins associated to each function are in brackets.
Mentions: The protein extraction protocol consisted on several rounds of extraction with a phenol-based buffer followed by ammonium acetate precipitation. The quality and the complexity of the extracted proteins were checked by 1D-SDS-PAGE prior to nLC-MS/MS (Figure S2). The complexity of the meiocyte extracts was different to that found in roots, whole anthers either in early or late meiosis and lemma (floral somatic tissue). It is worth to mention that the band pattern of whole anthers at different meiotic stages was rather similar (lanes 3 and 4), but significantly different to the meiocytes extract (lane 2), suggesting that the isolation of rice meiocytes led to the enrichment on meiosis-related proteins. Indeed, preliminary nLC-MS/MS analyses comparing the protein composition from whole anthers and isolated meiocytes revealed that only 8% of the proteins found in whole anthers were also found in isolated meiocytes (Figure 4A), suggesting that an important effort isolating meiotic cells is required in order to isolate proteins that are expressed in the meiocytes. Finally, since the isolation of rice meiocytes was critical and time-consuming, the individual protein extracts from each stage were not checked in a 1D-SDS-PAGE and used only for nLC-MS/MS analysis.

Bottom Line: Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins.Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs.The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

View Article: PubMed Central - PubMed

Affiliation: Plant Breeding Department, Institute for Sustainable Agriculture, Agencia Estatal Consejo Superior de Investigaciones Científicas Córdoba, Spain.

ABSTRACT
Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

No MeSH data available.