Study On The Localization Of Plastid-targeted Protein In Sperm Cell Without Plastid

The majority of plastid proteins are synthesized in the cytosol and transported into the plastid. Differently from other proteins, most of those plastid proteins are synthesized with a transit peptide at the N-terminal end. Interestingly, the mis-targeting of the plastid proteins into the mitochondrion has not been seen in plant cells until today, although the transit peptide is similar to the signaling peptide of mitochondrion-imported proteins at the contents of amino acid. In an in-vitro system, it was reported that some plastid precursors could be transported into mitochondria when the system only contained the mitochondria, and these precursor proteins could be rightly imported into plastid when adding the plastids into the system. These results indicate that the specificity of plastid protein transport is related to plastid. However, the phenomenon has not been tested in in-vivo plant cell. In this study, using the sperm cells that containing mitochondria but lacking plastid as an experimental system, we investigated the localization of the plastid proteins, and obtained the following results:1.Thro ugh constructing the fusion genes which contain plastid transit peptide at the N-terminal and red fluorescent protein (tdTomato) at the C-terminal, we expressed four fusion proteins in sperm cell under the control of sperm cell-specific promoter of HTR10. It was found that the fusion proteins of CPA-tdTomato, CPB-tdTomato and CPD-tdTomato displayed the punctate fluorescence, and the number of the fluorescent points is similar to that of mitochondria. These results stated that these three proteins could be targeted into mitochondria. Differently, the CPC-tdTomato did not show the punctate fluorescence, and mainly displayed the cytoplasmic location.2. We also analyzed the localization of five mitochondria and plastids dual-targeted proteins. We fused their transit peptide at the N-terminal of green fluorescent protein (GFP), and expressed them in sperm cell. The four fusion proteins of DTA-GFP, DTB-GFP, DTD-GFP, and DTE-GFP could locate at mitochondria except DTC-GFP. Interestingly, when fusing the DTE with tdTomato, we also observed its cytoplasmic localization, indicating this fusing protein had the cytoplasmic and mitochondrial localization.3. We also analyzed the localization of Tic32, a plastid membrane protein. Differently from the above proteins, this fusion protein of Tic32-tdTomato exhibited the endomembrane system location.In this study, our results show that the plastid proteins display mitochondrial, cytoplasmic and endomembrane localization, when expressed in the sperm cell without plastid. It indicates that plastid proteins can be translated, matured and mis-targeted, but not degraded even when no plastid is existent. These results will benefit us to understand the question of how plant cell localizes the plastid and mitochondrial proteins specifically.