Study On Regulating Protein Maturation Efficiency In Sperm Cells

Sperm cells in angiosperms are present in pollen vegetative cells,and are transmited to the embryo sac through pollen tubes,thus realizing the fusion with egg cells to form zygotes.Previous studies have shown that some mRNAs,derived from sperm cells,are involved in the development of the zygotes,but the protein translated from the mRNAs do not appear in sperm cells.In this study,through studying that the heterologously expressed GFP in sperm cells did not exhibit visible green fluorescence.We found that sperm cells had a mechanism to regulate protein maturation efficiency at the folding level,which provides a new perspective for understanding the results of previous studies.In Arabidopsis transgenic line ProHTR10:H2B-GFP,the sperm cells exhibited bright green fluorescent signal,whereas the sperm cells of transgenic line ProHTR10:GFP did not.This indicates that the quantity of mature GFP protein is obviously lower than that of H2B-GFP mature protein in sperm cells.Since the phenomenon of the fluorescent difference was present in nearly all the transgenic lines of ProHTR10:H2B-GFP and ProHTR10:GFP,it excluded the possibility that the difference in mature protein amount resulted from the regulation at transcriptional level.Similarly,the sperm cells of the ProTIP5:H2B-GFP transgenic lines and the ProTIP5:GFP transgenic lines showed similar fluorescence phenomenon,indicating that promoters did not cause the difference of mature protein content.Meanwhile,the fusion proteins of EF1g-GFP and eIF1-GFP also showed visible green fluorescent signal,so the reason for the low GFP protein content is not related to its localization in the cytoplasm.Furthermore,when using the egg cell specific promoter proDD45 and pollen vegetative cell specific promoterproLat52 to specifically drive the GFP and eIF1-GFP expression in the egg cells and pollen vegetative cells,we observed that the two transgenic lines exhibited similar fluorescence intensity in egg cells and pollen grains,indicating that the low content of the GFP mature protein was sperm cells specific.Because the nucleotide sequences of ProHTR10:EF1g-GFP and ProHTR10:GFP are different in the vicinity of the start codon(AUG),the different amount between the mature protein EF1g-GFP and the mature protein GFP may attribute to the different efficiency of translation initiation.We thus have produced three kinds of transgenic lines,ProHTR10:GFP108-EF1g-GFP,ProHTR10:GFP75-EF1g-GFP,and ProHTR10:GFP36-EF1g-GFP.Compared with the transgenic line ProHTR10:GFP,they have similar nucleotide sequences near the start codon(AUG).We observed that the sperm cells of these three kinds of plants had similar GFP fluorescence intensity to that in sperm cells of ProHTR10:EF1g-GFP,but completely different fluorescence intensity from that in sperm cells of ProHTR10:GFP.These results suggested that the translation initiation efficiency,which is affected by the nucleotide sequence near the start codon,was not the reason to result in the low content of the mature GFP protein.Furthermore,the sperm cells in the transgenic lines ProHTR10:H2B18-GFP and ProHTR10:EFlg18-GFP did not show visible green fluorescent signal,and were similar to those in the transgenic lines ProHTR10:GFP,further suggesting that the low content of mature GFP protein did not result from the low efficiency in protein translation initiation.However,when immunostaining was performed to the sperm cells of ProHTR10:GFP and ProHTR10:eIF1-GFP with the GFP antibody,we observed similar fluorescent signals in the sperm cells of both transgenic lines.This indicates that the sperm cells of the two transgenic lines have similar total amount of GFP protein.Based on these results,we conclude that the mechanism regulating the efficiency of protein maturation at the folding level is present in sperm cells.These results show for the first time that sperm cells have a mechanism that regulates the maturation of protein at the level of folding.This mechanism benefits some genes expressed in sperm cells to exist as mRNAs not protein which mainly function in early zygotic development.It provides new insights for us on understanding the development of sperm cells in angiosperms.