Proteomic Analysis Of The Potato Tuber Life Cycle Kaisa M. Koistinen’s Group Institute of Applied Biotechnology, University of Kuopio Kuopio, Finland

Questions: How many proteins exist in a tuber? What types of proteins present in a tuber? What proteins are involved in the development of a tuber? Principal Component Analysis Hierarchical Clustering Analysis

Stage Physiological state 1 Non-swelling stolons (1–2 cm sub-apical portion) 2 Swelling stolons (1–2 cm sub-apical portion) 3a Small developing tubers (,1 cm diameter) 3b Small developing tubers (1–2 cm diameter) 3c Small developing tubers (2–2.5 cm diameter) 3d Small developing tubers (2.5–5 cm diameter) 4a Larger developing tubers (5–6 cm diameter; excised from plant and stored at 10 ℃ for 3 days) 4b Larger developing tubers (5–6 cm diameter; not excised but sampled at the same time as 4a) 5 Mature tubers (from senesced plants) 6a Mature tubers stored at 5℃ for 4 weeks 6b Mature tubers stored at room temperature for 4 weeks 7 Mature tubers (dormancy broken at 5 ℃ followed by 2 weeks at 10 ℃; sprouts ca. 1 cm long) 8 Mature tubers (fully sprouted in hydrated compost in the dark; sprouts 20 cm long)

Protein extraction and 2-DE: Proteins were extracted from ca. 1g freeze-dried powder or from ca. 3g powder from fresh tuber material under liquid nitrogen. The protein pellet was dissolved in 2-DE sample buffer containing 9.5M urea, 2% CHAPS, 1% DTT, 0.8% Bio-Lyte 3/10 ampholyte (Bio-Rad, Hercules, CA, USA). Protein was quantified using the Bio-Rad Protein Assay Dye reagent. A total of 150 mg protein was loaded on each 2-DE gel.

Image and data analysis: Gel image analysis was performed with PDQuest software (Bio-Rad). Protein spot quantities were normalized to the total quantity of valid spots to minimize possible errors due to differences in the amount of protein loaded and staining intensity.

Protein identification by HPLC-ESI-MS/MS: 2-DE gels were stained with silver according to Shevchenko et al., except that the gels were washed with a solution containing 40% ethanol and 5% acetic acid and then washed twice with water for 30 min. In-gel digestion was performed according to Koistinen et al. and the tryptic peptides were analyzed using two different MS systems. An automated spectral processing, peak list generation and database search from raw data acquired on an LCQ quadrupole IT mass spectrometer was performed using Xcalibur software v1.0 (Thermoquest, San Jose, CA, USA) and the SEQUEST algorithm.

PC scores for 2-DE of the potato tuber life cycle: PC1 vs. PC2 (A); PC1 vs. PC4 (B). Numbers for the stages correspond to those in Table 1. The percentage of the total variation accounted for by each principal component is shown in brackets.

Concluding remarks: In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Five well-defined protein expression patterns were found by hierarchical clustering. 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted. 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. The development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation.