Analysis of the proteome released from Lupinus albus seeds at early germination stages

Upon imbibition, the quiescent dry seed rapidly resumes metabolic activity. Cell structures, mRNAs and enzymes necessary for this initial activation are already present within the mature dry seed. In particular, hydrolytic enzymes are synthesized to mobilize the stored reserves and to favour radicle extension through the structures surrounding the embryo. This latter event marks the end of germination and the onset of seedling growth. Although the general mechanisms of germination have been extensively studied, some aspects are still obscure. During the first steps of germination (i.e. 3-4 days) Lupinus albus, a legume seed, releases various kinds of proteins outside the fully viable seeds. The type and quantities of the polypeptides accumulated in the medium vary continuously during the germination, indicating a modulated mechanism and a functional role of these proteins, never described in detail before. Aim of the present work is to study the time course of the protein release and to characterize the polypeptides accumulated into the germinating medium by 2D electrophoresis and mass spectrometry. This would help to unveil the role of these polypeptides and the mechanisms of their release outside the seed. Lupinus albus (var. Multitalia) seeds have been vernalized overnight in deionized water, at 4\ub0 C. Then the seeds hav e been manually dehulled, soaked in deionized water (1 mL/seed) and kept at 21\ub0 C under mild shaking. The incubation time has been extended up to 72 hours. Two experimental settings have been performed. In the first, aliquots of the medium have been withdrawn at given times and the concentrations of either the ionic potassium and the released proteins have been determined (Figure 1). In the second setting, the water medium has been collected and replaced every 24 hours. In this case, the proteins have been analyzed by two-dimensional (2-D) IEF/SDS-PAGE and characterized by mass spectroscopy. Figure 2 shows the protein profile of the medium after 24 hours of incubation. The full set of results obtained indicate that the release of polypeptides in the germination medium is not caused by a passive leakage from the seed but rather it is driven by a specific mechanism, as shown by the release/reabsorption curve of ionic potassium. Moreover, the characteristic potassium curve matched that one of protein secretion into the medium. The electrophoretic pattern appeared relatively simple in term of composition, being formed by only few types of spots. Some of them are released immediately after the onset of germination (before 24 hours) and are accumulated for the whole time considered; other polypeptides appeared and/or disappeared in the subsequent timing (48 and 72 hours). The overall quantity of proteins released in the medium decreased with time. In conclusion, the fact that some polypeptides are specifically released outside the seed during the early stages of germination indicate a well defined role, possibly in the defence mechanisms against microbial pathogens