A vegetative storage protein homolog is expressed in the growing shoot apex of hybrid poplar

The ability of poplars (Populus deltoiaes Bartr. ex Marsh., and Populus trichocarpa Torr. and Gray) to sequester nitrogen in stems in preparation for winter has been associated with the massive accumulation of protein bodies in the bark and xylem ray parenchyma. These protein bodies contain a bark storage protein (BSP) that can account for up to 30% of the total soluble bark protein during the winter months. Perhaps the plant's ability to efficiently cycle nitrogen through BSP is an important aspect of its growth potential. Sequence analysis of BSP led to the identification of a leaf-associated homolog, win4, which was initially isolated because its transcript increased in abundance upon mechanical wounding. The goal of this work was to characterize this putative leaf-associated vegetative storage protein, and determine whether it might perform a storage role in vivo. Antibodies, produced against protein synthesized upon over-expression of the win4 coding region in Escherichia coli, were used to examine the relative abundance of WIN4 protein in response to supplemental nitrogen, and during development. The transcript and protein were most abundant in the youngest leaves and also increased with nitrogen fertilization. Immunolocalization of the protein was performed and showed that WIN4 was associated with cells surrounding the vasculature, and cells of the lower epidermis and stipules of immature leaves. Under moderate nitrogen fertilization regimes, WIN4 accounted for only about 2% of total soluble leaf protein; however, given the cellular specificity and enhancement with nitrogen, the protein is regulated in a. manner similar to other vegetative storage proteins. Since poplar is amenable to DNA transformation and regeneration, it is now possible to ask direct questions about the role these proteins play in nitrogen storage in rapidly expanding or in dormant tissue. This type of analysis could determine whether these proteins mainly ameliorate the toxic effects of excess nitrogen, if they are instrumental in controlling nitrogen allocation or if they simply represent an efficient method for sequestering this valuable nutrient.