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FIGURE 22.2. Nuclear proteome map of chickpea. The nuclear proteins were resolved onto (A) pH 4-7 with 150 |g of protein and (B) pH 6-11 with 100 |g of protein. IEF was performed on 13-cm IPG strips before being separated by SDS-PAGE on 12.5% gel. Protein spots marked by arrows were identified by LC-ESI-MS/MS.

Protein folding

Translation

Protein folding

Translation

Protein degradation

Metabolism

DNA replication and transcription fs (17%)

FIGURE 22.3. Functional classification of chickpea nuclear proteins. The MS-identified proteins were distributed among different protein classes based on their putative function in the organelle.

Signaling and gene □ regulation

Metabolism

Protein degradation

DNA replication and transcription fs (17%)

FIGURE 22.3. Functional classification of chickpea nuclear proteins. The MS-identified proteins were distributed among different protein classes based on their putative function in the organelle.

The proteins involved in signaling and gene regulation (36%) were found to be the most abundant. The proteins in this class include FCA, an RNA-binding protein. It is a plant-specific nuclear protein that is involved in flowering time control as well as ABA signaling [23]. The glycolytic enzymes, PGK and aldolase, were present as multiple spots in the chickpea nuclear proteome. These proteins are characteristic nuclear proteins in most or all eukaryotes [22]. They may have important secondary roles not directly related to carbon metabolism. Because they are known to contain a nucleotide-binding site, they may therefore bind to DNA or RNA in the nucleus. Kinases are important signaling components in any proteome—for example, Ser/Thr kinase. It is known that the majority of 14-3-3 molecules are present in the cytoplasm; however, in the absence of bound ligands, 14-3-3 homes to the nucleus [24]. The TFs are important components in any nuclear proteome, although many of these would have escaped detection because the expression level for TFs is generally not very high. Nucleocytoplasmic shuttling in turn is necessary to ensure that all these components are available at the right time in the life of a cell, and RAN-binding protein (Ras-related protein in the nucleus) acts as a major regulator of this process [25]. It is interesting to note that proteins involved in signaling and gene regulation dominated other categories, reflecting the role of nucleus in gene expression and regulation. In other organelles, like chloroplast and mitochondria, the largest percentage of proteins were reported to be involved in energy production, either in electron transport or in ATP production [26].

The second-largest category comprised proteins involved in DNA replication and transcription. Glycine-rich RNA-binding proteins (GRPs) are the predominant proteins in this category. These proteins contain two distinct domains: an amino-terminal RNA-binding domain and a Gly-rich carboxy-terminal domain. These proteins were also found to be a part of the Arabidopsis nucleolar proteome [19]. GAPDH is a known nuclear-resident protein. Besides its glycolytic activity, GAPDH is a multifunctional protein with both cytoplasmic and nuclear functions, one of which is as a tRNA-binding protein participating in RNA export [27]. Asp carbamoyltransferase (ATCase) is a protein involved in de novo pyrimidine biosynthesis pathway. The ATCase activity is virtually absent from "isotonic nuclei" but present in nuclei isolated in hyperosmotic sucrose media [28].

Structural proteins represent the third set of nuclear proteins. These proteins are known to be major constituents of the cytoskeleton in eukaryotic cells and are involved in chromatin remodeling and related processes [29-31]. Actin is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription [32]. Histones are the chief protein components of chromatin. They act as spools around which DNA winds, enabling the compaction necessary to fit the large genomes of eukaryotes inside cell nuclei. Matrix or scaffold attachment regions (MARs or SARs) are involved in looping of open chromatin fibers. The interaction of chromatin with the nuclear matrix via MARs on the DNA is considered to be of fundamental importance for higher-order chromatin organization and regulation of gene expression. MAR-binding filament-like protein 1 (MFP1) with a filament protein-like structure is a good candidate for a MAR-binding constituent of the nuclear filaments [33].

Proteins involved in translational machinery are standard in case of any nuclear proteome. In our study, 5% of the total identified proteins belong to this category. Eukaryotic EF-1a plays a pivotal role in protein biosynthesis, present mainly in the cytoplasm, but a small population of eEF-1a molecules has been previously identified in the nucleus, where it forms a complex with zinc finger protein [34]. The molecular chaperones account for 5% of the total nuclear proteome of chickpea; this includes chaperonin 60 and DnaK-type molecular chaperone (HSP70). Under normal circumstances, HSP70 is present mainly in the cytosol, but it translocates to the nucleus and nucleolus during physiological stress to prevent random aggregation of proteins [35]. Another important category of proteins identified are presumably involved in degradation mechanism. Several metabolism-related proteins have also been identified across all the plant nuclear proteomes, apart from the proteins involved in transport. The miscellaneous class of proteins makes up the rest of the chickpea nuclear proteome.

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