The DnaJ-like zinc finger domain protein ORANGE localizes to the nucleus in etiolated cotyledons of Arabidopsis thaliana

Vitamin A deficiency (VAD) is a worldwide health problem. Overexpression of the DnaJ-like zinc finger domain protein ORANGE (OR) is a novel strategy for the biofortification of pro-vitamin A carotenoids in different staple crops to alleviate VAD. In plants, OR triggers the differentiation from non-pigmented plastids into carotenoid-accumulating plastids. There are different reports on the subcellular localization of this protein in either chloroplasts or the nucleus, both of which were supported by confocal observation and protein-protein interaction results. In this work, we studied the subcellular localization of OR in the cotyledons of germinating seedlings whose plastids were transitioning from non-pigmented proplastids into carotenoid-accumulating etioplasts in the dark, and then into chloroplasts upon illumination. Our Western blot analysis identified two bands of the Arabidopsis OR protein (AtOR) from the chloroplast fraction of the mature leaves (i.e., a 34-kDa form corresponding to the full-length peptide and a 30-kDa form suggesting the removal of the N-terminal chloroplast transit peptide). We found that the full-length AtOR was predominantly localized in the nucleus in etiolated cotyledons, although its abundance decreased upon illumination. Our bioinformatics analysis indicated a nuclear localization signal (NLS) after the N-terminal chloroplast transit peptide. When we substituted different N-terminal regions of AtOR with the green fluorescent protein, our confocal observations demonstrated that this NLS was sufficient to target AtOR to the nucleus. Our results demonstrate that AtOR is a dual-targeted protein that mainly localizes in the nucleus in etiolated cotyledons.