Peach Fruit Development: A Comparative Proteomic Study Between Endocarp and Mesocarp at Very Early Stages Underpins the Main Differential Biochemical Processes Between These Tissues

Abstract

Peach (Prunus persica) is an important economically temperate fruit. The development follows double sigmoid curve with four phases (S1-S4). We centered our work in the early development. In addition to S1, we studied the very early stage (E) characterized by the lag zone of the exponential growing phase S1, and the second stage (S2) when the pit starts hardening. "Dixiland" peach fruit were collected at 9 (E), 29 (S1), and 53 (S2) days after flowering (DAF) and endocarp and mesocarp were separated. There was a pronounced decrease in total protein content along development in both tissues. Quantitative proteomic allowed the identification of changes in protein profiles across development and revealed the main biochemical pathways sustaining tissue differentiation. Protein metabolism was the category most represented among differentially proteins in all tissues and stages. The decrease in protein synthesis machinery observed during development would be responsible of the protein fall, rather than a proteolytic process; and reduced protein synthesis during early development would reroute cell resources to lignin biosynthesis. These changes were accompanied by net decrease in total amino acids in E1-S1 and increase in S1-S2 transitions. Amino acid profiling, showed Asn parallels this trend. Concerted changes in Asn and in enzymes involved in its metabolism reveal that increased synthesis and decreased catabolism of Asn may conduct to an Asn increase during very early development and that the β-Cyano-Alanine synthase/β-Cyano-Alanine hydratase could be the pathway for Asn synthesis in "Dixiland" peach fruit. Additionally, photosynthetic machinery decays during early development in mesocarp and endocarp. Proteins related to photosynthesis are found to a higher extent in mesocarp than in endocarp. We conclude mesocarpic photosynthesis is possible to occur early on the development, first providing both carbon and reductive power and latter only reductive power. Together with proteomic, histological tests and anatomical analysis help to provide information about changes and differences in cells and cell-walls in both tissues. Collectively, this work represents the first approach in building protein databases during peach fruit development focusing on endocarp and mesocarp tissues and provides novel insights into the biology of peach fruit development preceding pit hardening.

Keywords: Prunus persica; asparagine; endocarp; fruit development; lignification; mesocarp; β–cyanoalanine hydratase; β–cyanoalanine synthase.

FIGURE 1

(A) Lignin staining during early developing of “Dixiland” peach fruit. Peach fruit were collected after 9 (E), 29 (S1) and 53 (S2) DAF and subjected to phloroglucinol–HCl staining to detect lignin deposition. Scale bars: 1 cm. S: seed; E: Endocarp; M: mesocarp; Ex: exocarp. Weight (B), protein quantification (C) and chlorophyll analysis (D) were conducted in mesocarp (orange) and endocarp (blue) from peach fruit at E, S1, and S2 stages. Whole and dotted lines, respectively, represent fresh and dry weight curves. Total protein and chlorophylls are expressed in a fresh weight basis. Within each tissues, values with different letters are statically significant different (p < 0.05).

FIGURE 2

Functional classification of differentially expressed proteins over very early stages of peach fruit development. Proteins from endocarp (A) and mesocarp (B) tissues were analyzed at E, S1, and S2. Blue bars correspond to proteins increased (positive values) and decreased (negative values) in S1 with respect to E. Red bars represent the number of proteins increased (positive values) and decreased (negative values) in S2 with respect to S1.

FIGURE 3

Overview of PDA in endocarp and mesocarp during the transitions from E to S1 and from S1 to S2 in relation to their correspondent metabolic pathways. Each square corresponds to a protein. Red and blue indicate lower and higher expression in the earlier stage of development, respectively, in a log2 basis. Scale bar is indicated at the top right of each figure. Images were generated using MapMan program (Usadel et al., 2009).

FIGURE 4

Distribution of variable proteins across very early stages of development within protein metabolism functional category. E1 to S1 and S1 to S2 transitions were analyzed in both endocarp and mesocarp. (A) Pie charts representing the total number of PDA distributed within “protein metabolism” GO terms subcategories. (B) Classification of PDA involved in protein biosynthesis in endocarp (left graph) and mesocarp (right graph). (C) Distribution of PDA participating in protein degradation in endocarp (left graph) and mesocarp (right graph). Blue bars correspond to proteins increased (positive values) and decreased (negative values) in S1 with respect to E. Red bars represent the number of proteins increased (positive values) and decreased (negative values) in S2 with respect to S1.

FIGURE 5

Functional classification of PDA in endocarp and in mesocarp. Positive and negative values represent the number of proteins increased and decreased, respectively, in mesocarp with respect to endocarp when proteomes of fruits at E (red bars), S1 (green bars) and S2 (yellow bars) were analyzed.

FIGURE 6

Amino acid profiling during early peach fruit development. (A) Total amino acid quantification in endocarp and mesocarp. Values represent the mean of six independent determinations. Error bars represent the standard deviation. Bars with at least one same letter are not statistically different within the same tissue (p < 0.001). (B) Pie charts showing the proportion of each amino acid in the endocarp and the mesocarp at the developmental stages E, S1, and S2. (C) Heat map showing the amount of each amino acid during development in the endocarp (e) and the mesocarp (m). The scale bar at the top of the figure represents the amount of each amino acid expressed in μg/GFW. Gray boxes indicate that the amino acid was not detected.

FIGURE 7

Asparagine metabolism in peach fruit. An overview of the metabolic pathways conducting to asparagine biosynthesis and metabolism is shown. Graphs in gray background represent the proteins profiles as assessed by nanoHPLC-MS (within each box the uniprot accession number of the protein is presented), graphs with a white background show the amounts of amino acids determined by PICT-HPLC and the graph with dotted background displays the relative expression of the transcript encoding β–Cyano-Alanine-hydratase analyzed by qRT-PCR. For each parameter and tissue, values with at least one same letter are not statistically different. Error bars represent the standard deviation. Enzymes in gray were not detected in the proteome of “Dixiland” peach fruit during the very early development. ND, not detected.

FIGURE 8

Calcofluor white staining of endocarpic (A) and mesocarpic (B) cells at E, S1, and S2. Magnification used: 60X. Scale bars: 25 μm. Bright field images of the interphases between endocarp and mesocarp (C). Magnification used: 20X.

FIGURE 9

Box plots of cell wall thickness of endocarpic (e) and mesocarpic (m) cell walls in sections of fruit at E, S1, and S2 stages. (A) Cell wall width was measured using confocal laser scanning microscopy images of aniline blue-stained sections collected in the blue channel. Cell wall width was measured using the program “Image J” (http://imagej.nih.gov/ij) in sections at 90° with respect to the perimeter of the wall. Plots were constructed with Sigma Plot Software. Since the distribution of the data is not parametric, the Kruskal-Wallis One Way Analysis of Variance on ranks was applied followed by the Dunn’s Method for All Pairwise Multiple Comparison Procedure. (B) Cell wall width measured using images collected in the red channel from the endocarp and mesocarp of fruits collected at S2. Cell walls from S2e are thicker than from S2m (p < 0.001, Mann–Whitney t-test). Boxes with different letters are statistically different.