Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.
Plant lipid transfer proteins (LTPs) constitute a family of small proteins recognized as being extracellular. In agreement with this notion, several lines of evidence have shown the apoplastic localization of HaAP10, a LTP from Helianthus annuus dry seeds. However, HaAP10 was recently detected intracellularly in imbibing seeds. To clarify its distribution, immunolocalization experiments were performed during the course of germination and confirmed its intracellular localization upon early seed imbibition. Further assays using a hydrophobic dye, FM4-64, inhibitors of vesicular traffic, and immunolocalization of the pectin rhamnogalacturonan-II, allowed the conclusion that endocytosis is activated as soon as seed imbibition starts. Furthermore, this study demonstrated that HaAP10 is endocytosed throughout imbibition. Biochemical and cellular approaches indicate that the intracellular fraction of this LTP appears associated with oil bodies and some evidence also suggest its presence in glyoxysomes. So, HaAP10 is apoplastic in dry seeds and upon imbibition is rapidly internalized and relocalized to organelles involved in lipid metabolism. The results suggest that HaAP10 may be acting as a fatty acid shuttle between the oil body and the glyoxysome during seed germination. This concept is consistent with the initial proposition that LTPs participate in the intracellular transfer of lipids which was further denied based on their apparent extracellular localization. This report reveals for the first time the relocalization of a lipid transfer protein and opens new perspectives on its role.
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