Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only
Publication Acceptance Date: 4/6/1999
Publication Date: N/A
Technical Abstract: Accumulation of metal micronutrients in developing sink organs is due in large part to metal remobilization from mature leaves, via phloem translocation. However, very little is known about how, from which compartments, or in what form these nutrients are redistributed from source leaves via the phloem pathway. Our previous studies of Fe transport in three pea (Pisum sativum) genotypes, indicated that chelator(s) with stron metal-ligand stability at alkaline pH are involved in the phloem loading and transport of Fe. Using Lupinus albus plants fed with 59Fe, we also found a 13 kDa protein associated with 59Fe in the phloem sap, supporting our hypothesis that some phloem sap proteins serve as mobile metal chelators. In order to isolate potential metal chelators we employed immobilized metal affinity chromatography (IMAC) and SDS-PAGE techniques with pumpkin (Cucurbita pepo) phloem sap, or used HPLC separation of extracts from pea seed coats (the termination of the phloem transport pathway). IMAC columns with immobilized Ni(II) and Zn(II) were used to purify six high-affinity Ni(II)- and three high-affinity Zn(II)-chelating proteins from pumpkin phloem sap; these ranged from 4-18 kDa. HPLC separation of 59Fe-labeled pea seed coat-extracted proteins identified a 14 kDa and a 3 kDa protein, both with affinity for Fe. Through protein microsequencing and RT-PCR, we are cloning the genes encoding these metal chelating proteins and peptides. The resulting protein characterization and genomic information will be discussed in relation to the underlying mechanisms involved in phloem micronutrient translocation. This work was funded in part by USDA-ARS Cooperative Agreement No. 58-6250-1-003 and NSF Grant No. IBN-9630341 to MAG.