Proteomic analysis of pollination-induced corolla senescence in petunia

Abstract

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petuniaxhybrida 'Mitchell Diploid' corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence.

Fig. 1.

Protein changes during petunia corolla development and senescence. (A) Pollination accelerates petunia flower senescence. Petunia×hybrida ‘Mitchell Diploid’ (MD) unpollinated (U) flowers at 0, 24, 48, and 72 h and pollinated (P) flowers at 24, 48, and 72 h. (B) 2-D gel profiling of protein changes corresponding to corolla senescence. Representative 2-D gels of petunia corolla proteome variation during pollination-induced senescence. The first dimension was performed using 200 μg total soluble proteins on linear gradient IPG strips with pH 5–8. Second dimension separation was conducted on 12% SDS-PAGE gels and proteins were visualized using GelCode blue staining. Approximately 600 spots were detected on each gel. Three biological replicates at each time point were conducted.

Fig. 2.

Representative 2-DE comparisons of unpollinated and pollinated gels at 72 h. (A) All of the down-regulated proteins are indictated with arrows on the 72 U gels. (B) All of the up-regulated proteins are indicated with arrows on 72 P gels. The spot numbers are the same as the sample numbers (without the dash in the middle) in Tables 2 and 3. The assigned identities of up- and down-regulated proteins and their expression data are also shown in Tables 2 and 3.

Fig. 3.

Biological processes classification of up- (A) and down-regulated (B) proteins. Forty-six up- and 26 down-regulated proteins were assigned to putative biological process categories according to the TAIR GO annotation database (http://www.arabidopsis.org/tools/bulk/go/index.jsp).

Fig. 4.

Hierarchical clustering analysis of the 73 differentially expressed proteins. Protein expression levels were presented as the Log ratio relative to the 0 h unpollinated reference. Colours ranging from green to red represent protein expression from the highest level of down-regulation to the highest level of up-regulation, respectively. Black colour indicates no change compared to 0 h. Two main clusters were formed, representing up- and down-regulated proteins during senescence. The similarities of protein expression patterns represented by the distance of tree branches are shown on the left side. The spot sample numbers with putative protein identification are indicated on the right side of the heat map.

Fig. 5.

Changes in relative protein abundance during corolla development and senescence. Protein expression patterns of each of the 14 subclusters from the hierarchical tree in Fig. 4 are shown by one representative protein. Spots that were up- or down-regulated are indicated with arrows on the 2-D gels. The solid and dotted lines indicate the protein expression changes in pollinated and unpollinated corollas from 0 h to 72 h, respectively.

Fig. 6.

Relative protein abundance of selected proteins and the corresponding gene expression in petunia corollas after pollination. Protein abundance of all isoforms corresponding to the protein of interest are presented as the spot volume from the replicated 2-DE gels (n=3) and gene expression was quantified using real-time PCR (n=3) as described in the methods. Error bars represent ±SD.