Overexpression of Switchgrass Histone H3 tagged with YFP Fusion Genes Trigger Programmed Cell Death in Nicotiana benthamiana

Switchgrass is one of most promising energy crops, where the breeding for biofuel production is just recent. Aneuploidy is common in switchgrass indicating its genome instability in nature, which makes it difficult for studying switchgrass genetics and breeding selection. The genome instability of switchgrass could be related to the abnormal centromeres that interact with mitotic spindle causing selective chromosome lost. CENH3 and canonical H3 are the important components of nucleosomes; however, both histone H3 and CENH3 genes of switchgrass are largely uninvestigated. We identified 18 putative switchgrass histone H3 genes in silico.  Thirteen unique histone H3 genes, including six H3.1 genes, four H3.3 genes, three H3.3-like genes and one CENH3 gene were cloned from switchgrass cDNA and genomic DNA. The H3.1 genes have no introns, the H3.3 and H3.3-like genes have one or three introns, while the CENH3 gene has six introns. One histone H3.3 and the CENH3 gene were fused with YFP and transiently expressed in the Nicotiana benthamiana (N. benthamiana) leaves.  Both of the histone H3.3 and the CENH3 proteins were localized into plant nucleus. Unexpectedly, the overexpression of Histone H3-YFP and CENH3-YFP proteins could trigger programmed cell death (PCD) of the transformed plant cells. Deletion analysis of the Histone H3 and CENH3 genes revealed that the N-terminal tail is essential and sufficient to trigger PCD in plant cells. Our results provide foundation for further studying the variations of histone H3 and CENH3 genes and test if they contribute to genome instability in switchgrass.