Histone H3.3-like Gene Specifically Expressed in the Vegetative Cell of Developing Lily Pollen

To investigate the progression of the cell cycle during pollen development, two clones of histone H3 genes, YAH3 and MPH3, were isolated from cDNA libraries of young anthers and mature pollen of Lilium longiflorum. Northern blot and reverse transcription-PCR (RT-PCR) analyses demonstrated that YAH3 transcripts were present in uninucleate microspores and generative cells at the postulated S phase of the cell cycle as well as in young anthers, meristematic root tips, and shoot apices that contained proliferating cells. YAH3 therefore appears to be a major type of histone H3 gene in the lily. In contrast, the expression of MPH3 was detected only during pollen development, and expression increased during the development of mid-bicellular pollen to mature pollen. The results of in situ hybridization revealed that the transcripts of MPH3 were specifically accumulated in the vegetative cell of developing bicellular pollen. The two histone H3s differed at eight amino acid positions, and the deduced amino acid sequence of MPH3 showed identity with histone H3.3, which is a replacement variant of histone H3. The localization of an MPH3-green fluorescent protein (GFP) fusion protein differed from that of YAH3-GFP in onion epidermal cells and tobacco BY-2 cells at stationary phase, which suggests the preferential ability of MPH3 to be incorporated into chromatin. MPH3 may be expressed replication independently in vegetative cells at the G₁ phase and be incorporated into highly active chromatin of the vegetative nucleus of bicellular pollen, in a manner similar to histone H3.3 in DROSOPHILA: