**2. Materials and methods**

### **2.1. Plant material**

*S. tuberosum*, accounting for up to 45% of the total soluble protein [1, 4–7]. However, the molecular mechanism that triggers the cleavage of the patatin along the tuber life cycle has not to date been identified. The regulatory mechanisms involved in the synthesis and degradation of storage proteins are better known in dry seeds [8–13]. Phosphorylation has proven to be a key regulator mechanism in the maturation, dormancy and germination of seed storage proteins (SSPs). Thus, reverse phosphorylation of the phytohormone abscisic acid (ABA) seems to play a crucial regulatory role in the synthesis of SSPs at the transcriptional level [11, 12]. More specifically, phosphoproteome studies in rapeseed and rice reported that cruciferins and cupins achieve higher levels of phosphorylation at the late maturation stage [9, 13]. In addition, mobilization of the major SSP in the common bean, phaseolin, was found to occur in germinating seeds through degradation of highly phosphorylated isoforms [10]. It suggests that degradation of SSPs in dry-to-germinating seed transition occurs through a phosphorylation-dependent

The past few years have witnessed a steady discovery of phosphorylated SSPs such as cruciferins, napins, cupins, legumins and vicilins [8–10, 14, 15], but no evidence of phosphorylated isoforms has been reported to date in patatin or other VSPs such as sporamins and ocatins. Therefore, elucidating the question of whether patatin can be phosphorylated is a mandatory initial step in follow-up research concerning the molecular processes underlying its mobilization. First of all, the term patatin applies to a group of glycoproteins encoded by a gene family constituted by ~10–18 genes per haploid genome, most of them organized as a single gene cluster at the end of the long arm of chromosome 8 [16–18]. Patatin gene family members exhibit a very high degree of nucleotide sequence identity [19, 20]. In addition, patatin is a family of immunologically indistinguishable isoforms with similar structural properties and thermal conformational stability [21, 22]. Overall, extensive heterogeneity in molecular mass (40–45 kDa) and isoelectric point (4.5–5.2) seems to be the most salient differential

The 2-DE has provided the most complete information about the heterogeneity in molecular

at three specific asparagine residues of the amino acid sequence by *N*-linked oligosaccharide side chains or glycans [21, 22, 25]. Charge differences among isoforms could be explained by variations in positively and negatively charged amino acids [22]. However, variable phosphorylation has potential to change the p*I* of proteins by substituting hydroxyl groups on amino acid residues with negatively charged phosphate groups [26]. Therefore, phosphorylation could be a plausible but unexplored factor contributes to explain charge heterogeneity

In this study, we undertook a proteomic approach addressed to the identification and mapping of phosphorylated isoforms of the patatin multigene family based on highresolution 2-DE. First, relatively abundant tuber proteins were successfully separated from low-abundance proteins by loading low amounts of total protein sample into 2-DE

) and isoelectric point (p*I*) of the patatin [23–25]. Specifically, a total of 17–23 spots

and/or p*I* were detected in 2-DE patatin profiles obtained from different

seem to be mainly due to differential N-glycosylation

regulatory mechanism.

66 Advances in Seed Biology

mass (*M*<sup>r</sup>

with variations in *M*<sup>r</sup>

molecular features among isoforms [21–25].

potato cultivars [25]. Variations in *M*<sup>r</sup>

among patatin isoforms on 2-DE gels.

Proteomic analyses were performed from mature potato tubers of cv. Kennebec (2n = 4x = 48). Larger pieces of lyophilized tuber were homogenized with a pre-cooled mortar and pestle. The samples were stored at −80°C until protein extraction. Four biological replicates were used for experiments.

#### **2.2. Protein extraction and quantification**

Total tuber proteins were extracted using the phenol extraction method. A 200 mg sample of lyophilized tuber was transferred to an extraction buffer (500 mM Tris-HCl, 500 mM EDTA, 700 mM sucrose, 100 mM KCl pH 8.0, 2% DTT and 1 mM PMSF). Tris-HCl (pH 6.6–7.9) saturated phenol was added and the phenol phase was collected using centrifuging (4500 rpm at 4°C). Protein precipitation solution of 0.1 M ammonium acetate in cold methanol was added. Protein pellet was washed with 0.1 M ammonium acetate and 10 mM DTT, and with 80% acetone and 10 mM DTT. The resuspended protein pellet was then diluted in lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS; 10 mM DTT, and 2% PharmalyteTM pH 3–10, GE Healthcare, Uppsala, Sweden). Protein concentration was evaluated using the commercial CB-X protein assay kit (G-Biosciences, St. Louis, MO, USA) according to the instructions of the manufacturer for interfering agent removal and use with a microplate reader. The bovine serum albumin (BSA) was used as standard protein to generate calibration curves.
