Study The Function Of PtCDD, A Ca²⁺ Dependent Nuclease, That Is Involved In Secondary Xylem Development In Populus Tomentosa

The study of wood development is the hot spot of the core of plant developmental biology, it has the typical characteristic of programmed cell death. Its development process involves initiation in the vascular cambium, rapid cell expansion, deposition of the secondary cell wall, and finally cell death. Nucleases play a crucial role in the final stages of xylem cell death. Recently, the studies have shown that the nucleases which belong SNc-domain gene family, involved in mature unisexual flowers and involved in secondary xylem development in Eucommia ulmoides, but it did not have been found any nucleases involved in secondary xylem development in poplar. Thus, the study on the function of nucleases in secondary xylem development has important theoretical value in aspects of the secondary xylem development mechanism. Here, we focus on the important role of PtCDD gene in the secondary xylem development in populus. The main results are as follows:The sequence analysis of PtCDD gene showed that it belongs to the typical SNc domain containing gene family. RT-PCR results showed that the gene were expressed in roots, stems, leaves, staminate inflorescence, pistillate inflorescence and pollen. Further tests showed that PtCDD was expressed in the phloem region and strongly expressed in cambium and immature/differentiating xylem. The PtCDDpromoter::GUS chimeric construct(PPtCDD::GUS) was transformed into populus plants, GUS staining analysis found that GUS activity specifically expressed mainly in vascular tissue. Stem slices observed that GUS activity specifically expressed in the phloem, cambium and immature xylem. The pattern of PtCDD gene expression showed that PtCDD may involve in vascular tissue development.Through sequences analysis of PtCDD gene promoter, the promoter region contains many components associated with hormone and abiotic stress response. PPtCDD::GUS transgenic poplar plants were treated by GA, ABA, SA, drought and NaCl, we found that the GUS activity was affected by these factors. These results suggested that the expression of PtCDD gene was higher sensitivity to the hormone response and environment stress.For the localization of PtCDD, PtCDD and GFP fusion protein was used to the protein in vivo observation, it could accurately obtain the localization of PtCDD. The study results found that the C-terminal of PtCDD protein could influence the protein localization. Through GFP-PtCDD and nucleus marker RFP-IIP co-localization, the results showed that the post-translational GFP-PtCDD fusion protein migrated as a cluster wrapping the nucleus.To study the in vivo function of PtCDD in plant tissue, transformation of PtCDD, the Ca2+ ions binding sites mutant of PtCDD, and the active site of the SNase domain mutant of PtCDD into tobacco leaves by Agrobacterium-mediated transient expression experiments. The results showed that mRNA transcription level were increased 3 d after injection. Transient over-expression of PtCDD triggered the chlorotic phenotype after 3 days of co-cultivation, whereas empty vector, 35S::PtCDD(Ca2+ ions binding sites mutant) and 35S::PtCDD(SNase domain mutant) did not impair N. benthamiana leaves at the same time. Transient expression of PtCDD triggers cell death after two weeks, and others have not any change. The results suggest that PtCDD gene is a Ca2+ ions dependent DNase, and have SNc domain activity, which could trigger cell death in vivo degradation of genomic DNA.In the PtCDD over-expression transgenic 84 K populous, RT-PCR results show that the expression of PtCDD is significant rise. Phenotype analysis found that the differentiation capacity of lateral root was influence. Stem slices observed that PtCDD over-expression transgenic plants had more TEs in the third internodes compared with those of wt stem. The transgenic plants had more cell layers and more the detection of TUNEL signal in the 5th internodes than the wild type controls. In the per unit area of 7th internodes and 9th internodes, the transgenic plants had more TEs numbers than the wild type controls. These results suggested that PtCDD over-expression confers enhanced cell death, and promote TEs differentiation in Populus tomentosa.Through expression silencing of homologous gene of PtCDD by VIGS, we could analysis the biology function of PtCDD gene. RT-PCR analysis revealed the expression of homologous gene of PtCDD was knock-down, phenotype analysis showed the development of tobacco plant was retardation, and the auto-fluorescence of lignin was weak. These results suggest that reduced the expression of homologous gene of PtCDD, may affect the xylem secondary wall formation.In this study, the expression patterns of PtCDD, subcellular localization of PtCDD, transient expression of PtCDD triggers cell death in tobacco leaves and the function of PtCDD promote the xylem development, these results provide evidence that Pt CDD gene plays a key role in the PCD during secondary xylem development. PtCDD gene is responsible for a gradual degradation of genomic DNA and trigger cell death. Finally, continued to formation of mature wood.