Comparative proteomics analysis of proteins expressed in the I-1 and I-2 internodes of strawberry stolons

Abstract

Background: Strawberries (Fragaria ananassa) reproduce asexually through stolons, which have strong tendencies to form adventitious roots at their second node. Understanding how the development of the proximal (I-1) and distal (I-2) internodes of stolons differ should facilitate nursery cultivation of strawberries.

Results: Herein, we compared the proteomic profiles of the strawberry stolon I-1 and I-2 internodes. Proteins extracted from the internodes were separated by two-dimensional gel electrophoresis, and 164 I-1 protein spots and 200 I-2 protein spots were examined further. Using mass spectrometry and database searches, 38 I-1 and 52 I-2 proteins were identified and categorized (8 and 10 groups, respectively) according to their cellular compartmentalization and functionality. Many of the identified proteins are enzymes necessary for carbohydrate metabolism and photosynthesis. Furthermore, identification of proteins that interact revealed that many of the I-2 proteins form a dynamic network during development. Finally, given our results, we present a mechanistic scheme for adventitious root formation of new clonal plants at the second node.

Conclusions: Comparative proteomic analysis of I-1 and I-2 proteins revealed that the ubiquitin-proteasome pathway and sugar-hormone pathways might be important during adventitious root formation at the second node of new clonal plants.

Figure 1

The strawberry stolon. As shown, a stolon shoots away from the base of a strawberry plant. A clone is formed at a variable distance away from the parent at the second node concomitant with adventitious root formation.

Figure 2

Two-dimensional SDS-PAGE gels of the I-1 and I-2 proteomes. Proteins (300 μg) in I-1 and I-2 extracts were separated, in the first dimension by isoelectric focusing (pH 3-10) and in the second dimension by SDS-PAGE through 12.5% acrylamide gels. Proteins were visualized by silver staining. Circled proteins were identified by matrix-assisted laser desorption/ionization time-of-flight MS and database searches.

Figure 3

Venn diagram for the identified I-1 and I-2 proteins. The numbers and percentages of unique proteins (excluding isoforms) found for either or both internodes are given. Spot numbers for I-1 proteins are given first followed by spot numbers for the corresponding I-2 proteins in parentheses.

Figure 4

Number of proteins with sequences that matched those of organisms listed in the Swissprot Viridiplantae database. Over 50% of the proteins identified had sequences similar to annotated proteins from dicot species in the Viridiplantae database.

Figure 5

Pie charts classifying the identified I-1 and I-2 proteins according to biological function. The identified proteins were grouped according to their biological processes and are expressed in percentage.

Figure 6

Gene ontology classification of identified I-1 and I-2 proteins according to their subcellular location. Subcellular locations of the proteins were assigned according to the GO annotations and are expressed as percentages of the assigned proteins.

Figure 7

Protein abundance for nine metabolic pathways in I-1 and I-2. For carbon fixation, glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism, starch and sucrose metabolism, oxidative phosphorylation, ubiquitin-proteasome pathway; 7, 6, 4, 1, 1, 3, 6, and 5 enzymes were identified, respectively, corresponding to 15, 10, 6, 5, 5, 4, 6, and 6 2-DE spots.

Figure 8

Possible protein-protein interaction network among I-2 proteins derived using the Cytoprophet module of Cytoscape. Cytoprophet draws a network of potential interactions with probability scores and GO distances as edge attributes. Proteins are marked with UniProt ID names.

Figure 9

Model for adventitious root and clonal plant formation in I-2 that incorporates four regulated pathways. Five identified I-2 proteins were integrated into the model, and the possible PPIs are shown (dashed lines) based on the PPI network in Figure 8. (a) Anaphase-promoting complex (APC/C) is a ubiquitin ligase that plays a key role in the cell cycle. (b) Eukaryotic translation initiation factor 5A (EIF-5A) may interact with PUB34 to regulate cell division. (c) ACS7, when interacting with ubiquitin ligase, plays a central role in ethylene biosynthesis. (d) Important regulatory effects on plant growth and development have been reported for trehalose (Tre) and trehalose 6-phosphate (T6P). (CDC20: cell-division cycle protein 20; G6P: glucose 6-phosphate; TPP: trehalose-6-phosphate phosphatase