Methodology And Strategy

Secretome analysis in plants is only beginning to emerge. A survey of limited studies shows that techniques for developing a highly confident secretome of plant cell/tissue have yet be fully standardized to ensure reproducibility from laboratory to laboratory. It is therefore important to establish a workflow for systematic secretome analysis of plant species. A general consideration to investigate plant secretome on a large scale should be: isolation of pure and intact secreted proteins, application of complementary proteomics approaches, development of high-resolution 2D reference map, high-quality MS and MS/MS spectra generation to assign peptide sequence with high accuracy, quantitative data on identified proteins, and database development and dissemination to the scientific community.

In vitro-cultured cell systems have widely been used to prepare secreted proteins. Though an in vitro system is easy to maintain and handle, the system does not represent the physiological processes of plants. Therefore, investigation of secretome in planta is essential. In the case of cultured cells, the culture filtrates are used for extraction of secreted proteins, whereas vacuum infiltration method has widely been used to extract secreted proteins in planta. The culture filtrates or vacuum infiltrates are centrifuged to obtain clear supernatant. The clear supernatant is generally freeze-dried in a vacuum lyophilizer, and the pellet resuspended in small volume of water (approximately 2 mL) is either dialyzed against suitable buffer like MES (10 mM, pH 5.8) or passed through a desalting column (for example, PD-10 with 5-kDa MW cutoff from Bio-Rad), followed again by vacuum lyophilization. The secreted proteins pellet thus prepared can be stored at -80°C until further use. The pellet has been used directly for 2-DGE studies [15] or subjected to TCA precipitation to remove other interfering impurities before conducting downstream analysis [16]. The prepared secreted proteins can be analyzed using 2-DGE- and MS-based proteomics approaches [15].

We found that the lyophilized pellet carries interfering impurities such as carbohydrates. These impurities should be removed prior to downstream proteomics analysis. We routinely use TCA and cetyltrimethylammonium bromide (CTAB) precipitation steps to remove interfering impurities (unpublished data; Jung et al., personal communication). CTAB precipitation seems to be necessary for secreted proteins derived from fungus-infected plants. Two-dimensional gels of protein samples prepared without CTAB step showed high background when stained with silver nitrate, masking low abundance proteins. High background might be due to carbohydrates in the protein sample. CTAB precipitation largely removes carbohydrates besides other interfering impurities. Therefore, we recommend performing TCA followed by CTAB precipitation steps to prepare in planta high-quality secreted proteins for secretome analysis.

All proteins identified using complementary proteomics approaches should be subjected for signal peptide analysis using SignalP prediction software [18, 21]. Prediction of transmembrane domains (TMDs) of proteins has also been used along with signal peptide prediction to predict the true secreted proteins [6]. These computational programs can be used to easily generate a comprehensive list of potentially secreted proteins in a given tissue or organism providing the availability of genome sequence and, if possible, gene expression data. However, proteomics-based (or other experimental) data are required to verify the computational generated data. In light of this view, Arabidopsis and rice are indeed excellent reference plants to begin investigating plant secretome.

6.3 A CASE STUDY: IN PLANTA AND IN VITRO PROTEIN PROFILES OF SOLUBLE AND SECRETED PROTEINS IN RICE

We present a case example of in planta and in vitro secretome analyses in rice. A 2D gel-based proteomics approach was applied to identify and quantify the secreted proteins (unpublished data; Jung et al., personal communication). As an example, selected areas of 2D profiles of in plant and in vitro experiments are shown in Figure 6.1. A comparative analysis of these 2D profiles indicates dramatic differences in the protein patterns of in planta and in vitro derived soluble and secreted proteins. Trypsin-digested protein spots were subjected to LC-MS/MS and database analyses to identify protein. Four protein spots marked in Figure 6.1 were identified as RuBisCO activase isoform precursor, glucan 1,3-^-glucosidase precursor, «-amylase isoform III, and aspartyl protease. Glucan 1,3-^-glucosidase precursor and «-amylase isoform III have previously been reported as secreted proteins. Inventory of all identified proteins reveals that secreted proteins are involved in a variety of cellular processes. Many novel and unknown proteins were also identified.

SignalP prediction analysis of all identified proteins indicated that secreted proteins possessing signal peptides were significantly higher in an in vitro -prepared secreted protein population compared to in planta. A similar result was found in a comparative analysis of in planta - and in vitro-secreted proteins [16]. Lack of signature signal peptide in approximately 50% identified in planta-secreted proteins along with studies conducted in independent laboratories on the global level supports the notion that plant possesses yet unidentified secretory pathway(s) in addition to the ER/Golgi pathway. Evidence in this direction is emerging. It has been shown that improperly folded GFP is secreted via a nonclassical pathway in the extracellular space of Chinese hamster ovary cells [22]. Availability of large-scale protein localization tools and resources of knockout mutants for Arabidopsis and rice will help in dissecting the secretory pathway by understanding the subcellular localization of an experimentally identified secretory protein lacking signal peptide.

6.4 CONCLUSIONS

Proteomics of secreted proteins in plants is at an initial stage, but has still produced meaningful data. These data are due to continuous improvement in techniques and

In Planta In Vitro

Soluble Secreted Soluble Secreted

Soluble Secreted Soluble Secreted

RuBisCO activase Glucan 1,3-beta- Alpha-amylase Aspartyl protease small isoform glucosidase precursor isozyme III

precursor

RuBisCO activase Glucan 1,3-beta- Alpha-amylase Aspartyl protease small isoform glucosidase precursor isozyme III

precursor

FIGURE 6.1. In planta- and in wfro-derived soluble and secreted proteins reveal dramatic differences in their 2D protein profiles. Both soluble and secreted proteins were isolated from 3-week-old rice seedling leaves treated with 250 ppm Tween 20 buffer for 24 h (called in planta experiment) and rice callus cultured in liquid medium (2 N6 [24,25] ) for 5 days after replacing the culture medium with the fresh culture medium (called in vitro experiment), respectively. Rice callus was generated from the rice seed growing on the induction medium [3% (w/v) sucrose, 0.03% (w/v) casamino acid, proline (2.878 mg/L), CHU (3.981 g/L), 1 mL of 2,4-D (2 mg/mL ethanol), pH 5.8, 0.2% (w/v) gellan gum] for three weeks. Growing calli (0.5 to 1 g) were transferred into 50 mL induction liquid media to establish cell suspension culture. The cell suspension culture was maintained by subculturing (1 mL) into 50 mL of fresh induction medium once every week with gentle agitation (120 rpm) at 28°C. Isolated proteins (150|g) were separated on linear IPG strips (pH 4-7; 24 cm) in the first dimension followed by SDS-PAGE (12%) in the second dimension to develop 2D gel-secreted protein reference maps. Proteins were visualized with silver nitrate staining. The gel areas were selected from high-resolution 2D gels of an in planta- and in v/tro-experiments to display the drastic differences in protein profiles between not only the soluble and secreted proteins but also between in planta and in vitro secreted proteins. The pi and molecular mass range (in kDa) of the selected gels are given. Soluble and secreted 2D protein profiles of in planta experiments are displayed in A and B, respectively, whereas in vitro experiments are displayed in C and D, respectively. Results presented here are representative of four independent biological experiments. Protein spots identified by LC-MS/MS and database analyses are also given for one protein spot per 2D gel. Except RuBisCO activase isoform precursor and aspartyl protease, glucan 1,3-p-glucosidase precursor and a-amylase isoform III have been previously reported as secreted proteins, suggesting that the applied workflow for preparation and enrichment of secreted proteins is suitable for investigating the secret of rice secretome.

workflow to properly investigate the plant secretome at a global scale. In planta - and in vitro-collected secreted proteins show dramatic differences in the 2D protein patterns and in the number of secreted proteins with a signal peptide, and therefore these experimental approaches can together be used to dig dipper into the plant secretome. Accumulated data have also begun providing insight on the nature of secreted proteins in plants under pathogen invasion or due to chemical elicitors. These proteins are of two kinds: proteins carrying and lacking signal peptides. Proteins lacking signal peptides imply the presence of nonclassical ER/Golgi secretory pathways in plants.

Lastly, based on studies conducted to date on plant secretome, though very limited, we must emphasize that proteomics approaches can be used to identify secreted proteins and to reveal their function.

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