Application of an antibody chip for screening differentially expressed proteins during peach ripening and identification of a metabolon in the SAM cycle to generate a peach ethylene biosynthesis model

Abstract

Peach (Prunus persica) is a typical climacteric fruit that produces ethylene rapidly during ripening, and its fruit softens quickly. Stony hard peach cultivars, however, do not produce large amounts of ethylene, and the fruit remains firm until fully ripe, thus differing from melting flesh peach cultivars. To identify the key proteins involved in peach fruit ripening, an antibody-based proteomic analysis was conducted. A mega-monoclonal antibody (mAb) library was generated and arrayed on a chip (mAbArray) at a high density, covering ~4950 different proteins of peach. Through the screening of peach fruit proteins with the mAbArray chip, differentially expressed proteins recognized by 1587 mAbs were identified, and 33 corresponding antigens were ultimately identified by immunoprecipitation and mass spectrometry. These proteins included not only important enzymes involved in ethylene biosynthesis, such as ACO1, SAHH, SAMS, and MetE, but also novel factors such as NUDT2. Furthermore, protein-protein interaction analysis identified a metabolon containing SAHH and MetE. By combining the antibody-based proteomic data with the transcriptomic and metabolic data, a mathematical model of ethylene biosynthesis in peach was constructed. Simulation results showed that MetE is an important regulator during peach ripening, partially through interaction with SAHH.

Keywords: Plant development; Protein-protein interaction networks.

Fig. 1. Construction of the peach mAb library and screening of differentially expressed proteins in ripening peach fruit with a mAbArray chip.

a Peach protein antigens used to generate the mAb library. SDS-PAGE analysis of proteins extracted from peach fruit that were used as antigens for antibody library construction. Proteins were extracted from the mesocarp of the CN13 and CN16 cultivars at ripening stages S3, S4 I, S4 II, and S4 III. Twenty micrograms of protein was separated by SDS-PAGE and stained with Coomassie blue. b–d Quality of the antibody chip analyzed by hybridization. b Antibody chip after hybridization with Cy5-labeled goat anti-mouse IgG, and the Cy5 signal was scanned. c Magnified image of one block. The red circles and block indicate the positive controls (biotin-labeled BSA), and the green circles indicate the negative controls. d Diagram of mAb arrangement on the chip. e The peach mAbArray chip was used to screen for differentially expressed proteins in CN13 fruit between fruit-ripening stages S3 and S4 III. The differences in fluorescence intensities between peach fruit from stages S3 and S4 III within a small area of the chip are presented. f Correlation matrix of the replicated samples. g Volcano plot depicting the analysis of the differentially expressed proteins. The p value cut-off was 0.05 (y-axis), while the abundance fold-change cut-off was >1.5 or <−1.5 (x-axis). Significantly and nonsignificantly differentially expressed proteins are indicated in blue and red, respectively. h Western blot (WB) analysis of the differentially expressed proteins identified by LC-MS/MS. CN13 fruit samples harvested during stages S3 and S4 III were analyzed

Fig. 2. Analysis of enzyme abundance and metabolite changes during peach ripening.

a WB analysis of SAMs, MetE, ACO1, and SAHH in CN13 and CN16 during ripening stages S3–S4 III. Actin-1 was used as a protein-loading control for the WB analyses of SAMs MetE and ACO1, and actin-2 was used as the control for SAHH. b Diagram of the recycling of methionine via the SAM and Yang cycles. The major enzymes and metabolites involved in sulfur group recycling through the SAM cycle and ethylene synthesis through the Yang cycle are indicated. Monoclonal antibodies recognizing the enzymes in blue were obtained in this study. c–f Measurement of metabolites in the SAM cycle during ripening stages in peach. The contents of c DL-homocysteine (Hcy), d l-methionine (Met), e S-(5′-adenosyl)-l-homocysteine (SAH), and f S-(5′-adenosyl)-l-methionine (SAM) were measured. Values are means ± SDs, n = 3. The significance of the differences was analyzed by Student’s t-test. ***p < 0.001; **p < 0.01; *p < 0.05

Fig. 3. Detection of protein–protein interaction between MetE and SAHH.

a Yeast two-hybrid analysis of the interaction between MetE and SAHH. Yeast transformants were grown on QDO medium (SD-Ade/-His/-Leu/-Trp) (left panel) or QDO medium supplemented with X gal (right panel). pGBKt7/pGADT7-T is a positive control. b BIFC analysis of the interaction between MetE and SAHH. The combined constructs (top to bottom) are PSPYNE-35S-MetE/PSPYCE-35S-SAHH, PSPYNE-35S/PSPYCE-35S (negative control), and PSPYNE-35S-bZIP63/PSPYCE-35S-bZIP63 (positive control). Bar = 32 μm

Fig. 4. Functional analyses of differentially expressed genes during fruit ripening in peach.

a Clustering analysis showing the coexpression modules identified by WGCNA. The different modules identified are color labeled. b Functional enrichment analysis of differentially expressed genes. The upregulated functions (filled circles) and downregulated (filled diamonds) functions are displayed with respect to their significance (p < 0.02). Different colors correspond to the ratio of genes in each category. c Gene expression during the ripening process in the melting flesh (MF) cultivar CN13 and the stony hard (SH) cultivar CN16

Fig. 5

Biochemical network of the ethylene biosynthesis pathway during fruit ripening identified via proteomic analysis with the mAbArray

Fig. 6. In silico dynamics of ethylene biosynthesis and emission in fruit under various conditions during fruit ripening.

a In MF cultivars, IAA initiates ethylene biosynthesis, and the simulations were compared with experimental measurements (magenta bar: IAA concentration; green bar: ethylene emission rates of CN13). b The expression of YUC11 in SH fruit during fruit ripening is silenced. The simulation of ethylene emission is compared with experimental measurements (green bar: ethylene emission rates of CN16). c In MF cultivars, the identified transcriptional feedback of ACO1 is absent. d In MF cultivars, the identified protein–protein interaction between MetE and SAHH is absent