Exploring the nuclear proteome ofMedicago truncatulaat the switch towards seed filling

Abstract

SummaryDespite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatinmodifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.

Post-Genomics Studies of Developmental Processes in Legume Seeds

“…For the separated seed tissues, the citations refer to transcriptome and proteome analyses in soybean (Le et al, 2007) and M. truncatula (Boudet et al, 2006;Zhang et al, 2006;Gallardo et al, 2007). For the seed organelles, the citations refer to proteome analyses of pea mitochondria (Bardel et al, 2002) and M. truncatula nuclei (Repetto et al, 2008). The citation referring to the comparison of transcriptome and proteome datasets is Gallardo et al (2007) and that referring to data integration is Goffard and Weiller (2006).…”

“…These analyses have established reference maps of the protein complement of developing or mature seed in model and crop species, allowing for the identification of species-specific features (Agrawal et al, 2008;Gallardo et al, 2008). The methods have been applied to total protein extracts, or in some cases to subcellular fractions, as for example in the analysis of nuclear proteins (Repetto et al, 2008). By sampling the nuclear proteome just before seed filling, the most numerous class of nuclear proteins detected was found to be that involved in ribosome biogenesis.…”

Bioinformatics Applied to Proteomics

“…As an example of high technological potential of expression proteomics, in the last ten years plant proteomics research has been conducted in several land species achieving a high degree of knowledge of the dynamics of the proteome in many model plants (Agrawal & Rakwal, 2005;Baerenfaller et al, 2008;Grimplet et al, 2009;Komatsu, 2008;Plomion et al, 2006) and thereafter translating this knowledge in other species whose genome sequence is still under construction. The most successful studies are those which use separation of subcellular compartments (Haynes & Roberts, 2007;Dunkley et al, 2006;Agrawal et al, 2010) such as mitochondria (Heazlewood et al, 2005), chloroplast , endoplasmic reticulum (Maltman et al, 2007), peroxisomes (Fukao et al, 2002), plastoglobules (Grennan, 2008), vacuoles (Jaquinod et al, 2007), nucleus (Repetto et al, 2008) since they contain a limited number of proteins thus helping the protein identification. Since 30 years, the greater part of research into the plant proteome has utilized twodimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS-PAGE) for the protein separation step, which is usually followed by protein identification by mass spectrometry (MS).…”

Aislamiento y caracterización de genes MADS-box en Medicago truncatula: duplicaciones génicas y subfuncionalización en el linaje euAGAMOUS

“…Está estrechamente relacionada con la leguminosa forrajera más importante a nivel mundial, la alfalfa (Medicago sativa) y ha llamado la atención como sistema modelo genómico y molecular para estudios 6 biológicos de las leguminosas (Cook, 1999;Oldroyd y Geurts, 2001), debido a sus características peculiares: genoma pequeño (~500-550 Mpb) y diploide (2n=16), ciclo de vida corto (2-3 Medicago truncatula es un modelo valioso para el estudio de muchos aspectos de la biología de las leguminosas que son únicos para esta familia de plantas. Cuestiones importantes de simbiosis, patogénesis, biología de las semillas, desarrollo radicular, metabolismo secundario (Oldroyd y Geurts, 2001) y últimamente genómica, transcriptómica, proteómica y metabolómica (Benedito et al, 2008;Repetto et al, 2008;Farag et al, 2008) se están estudiando en esta planta modelo.…”

“…Está estrechamente relacionada con la leguminosa forrajera más importante a nivel mundial, la alfalfa (Medicago sativa) y ha llamado la atención como sistema modelo genómico y molecular para estudios Medicago truncatula es un modelo valioso para el estudio de muchos aspectos de la biología de las leguminosas que son únicos para esta familia de plantas. Cuestiones importantes de simbiosis, patogénesis, biología de las semillas, desarrollo radicular, metabolismo secundario (Oldroyd y Geurts, 2001) y últimamente genómica, transcriptómica, proteómica y metabolómica (Benedito et al, 2008;Repetto et al, 2008;Farag et al, 2008) se están estudiando en esta planta modelo.Además, el género Medicago está relacionado con un buen número de leguminosas de cultivo importantes como la alfalfa, guisante, haba, garbanzo, lenteja, trébol, lotus, soja y judía común (Doyle et al, 1996). Es notable el alto nivel de macrosintenia entre…”