Limits...
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.


Stereomicroscopy and light microscopy images of rice anthers and isolated meiocytes. (A) Stereomicroscopy observations of rice anthers suspended in a solution PBS and 1% proteinases inhibitors on a multi-well slide. (B) Inset in (A) shows the meiocytes bag (arrowed) from a sectioned rice anther. (C) Rice isolated meiocytes under the stereomicroscope. (D) Light microscopy observations of rice isolated meiocytes in a Neubauer chamber. Bar represents 500 μm in (A,B), and 20 μm in (C,D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109522&req=5

Figure 3: Stereomicroscopy and light microscopy images of rice anthers and isolated meiocytes. (A) Stereomicroscopy observations of rice anthers suspended in a solution PBS and 1% proteinases inhibitors on a multi-well slide. (B) Inset in (A) shows the meiocytes bag (arrowed) from a sectioned rice anther. (C) Rice isolated meiocytes under the stereomicroscope. (D) Light microscopy observations of rice isolated meiocytes in a Neubauer chamber. Bar represents 500 μm in (A,B), and 20 μm in (C,D).

Mentions: In this work we developed a method to isolate rice meiocytes at different stages of early meiosis for the identification of the proteins involved in early meiotic events by nLC-MS/MS (summarized in Figure 1). First, fixed rice panicles were screened for the identification of the meiotic stage by checking one anther per flower. Rice anthers in prophase I stages were usually shorter than 800 μm and moreover, no significant differences in size were observed among the different stages of prophase I. Therefore, checking one anther per flower was necessary to unequivocally identify the meiotic stage (Figure 2). Since all the anthers in the same flower are synchronized, the remaining five anthers were immediately collected for the isolation of meiocytes (Figure 3). The number of flowers in a rice panicle at different stages of early meiosis varied from 1 to 6 at each stage of prophase I. Panicles from at least 25 rice plants (representing between 80 and 300 anthers, depending on the meiotic stage) were needed to obtain a minimum of 1.5 μg of protein necessary for each proteomic analysis. The protein yield ranged from 50 to 110 ng per flower. The isolation of meiocytes at diplotene was arduous since the number of flowers at this stage was scarce because of the rapid transition from diplotene to zygotene. Indeed, in other organisms such as Saccharomyces cerevisiae, the synaptonemal complex disassembly is so fast that diplotene is not even apparent (Dresser and Giroux, 1988). However, the protein yield per rice flower in diplotene was sufficient enough (1.5 μg) 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)

Stereomicroscopy and light microscopy images of rice anthers and isolated meiocytes. (A) Stereomicroscopy observations of rice anthers suspended in a solution PBS and 1% proteinases inhibitors on a multi-well slide. (B) Inset in (A) shows the meiocytes bag (arrowed) from a sectioned rice anther. (C) Rice isolated meiocytes under the stereomicroscope. (D) Light microscopy observations of rice isolated meiocytes in a Neubauer chamber. Bar represents 500 μm in (A,B), and 20 μm in (C,D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Stereomicroscopy and light microscopy images of rice anthers and isolated meiocytes. (A) Stereomicroscopy observations of rice anthers suspended in a solution PBS and 1% proteinases inhibitors on a multi-well slide. (B) Inset in (A) shows the meiocytes bag (arrowed) from a sectioned rice anther. (C) Rice isolated meiocytes under the stereomicroscope. (D) Light microscopy observations of rice isolated meiocytes in a Neubauer chamber. Bar represents 500 μm in (A,B), and 20 μm in (C,D).
Mentions: In this work we developed a method to isolate rice meiocytes at different stages of early meiosis for the identification of the proteins involved in early meiotic events by nLC-MS/MS (summarized in Figure 1). First, fixed rice panicles were screened for the identification of the meiotic stage by checking one anther per flower. Rice anthers in prophase I stages were usually shorter than 800 μm and moreover, no significant differences in size were observed among the different stages of prophase I. Therefore, checking one anther per flower was necessary to unequivocally identify the meiotic stage (Figure 2). Since all the anthers in the same flower are synchronized, the remaining five anthers were immediately collected for the isolation of meiocytes (Figure 3). The number of flowers in a rice panicle at different stages of early meiosis varied from 1 to 6 at each stage of prophase I. Panicles from at least 25 rice plants (representing between 80 and 300 anthers, depending on the meiotic stage) were needed to obtain a minimum of 1.5 μg of protein necessary for each proteomic analysis. The protein yield ranged from 50 to 110 ng per flower. The isolation of meiocytes at diplotene was arduous since the number of flowers at this stage was scarce because of the rapid transition from diplotene to zygotene. Indeed, in other organisms such as Saccharomyces cerevisiae, the synaptonemal complex disassembly is so fast that diplotene is not even apparent (Dresser and Giroux, 1988). However, the protein yield per rice flower in diplotene was sufficient enough (1.5 μg) 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.