Understanding of Histone H3 Phosphorylation and Acetylation on Enhancement of the Soybean Tolerance to Salinity Via Proteomic Profile.

摘要

Soybean (Glycine max (L.) Merrill) represents one of the most significant economical legume crops. However, conditions in almost all cultivated land are sub-optimal for plant growth. Boyer estimated that about 70% of the potential yield is lost as a result of unfavorable physiochemical environments, even in developed agricultural systems. This project tried to understand the defensive mechanisms/ responses of soybean upon the salinity stress for further improving its salinity tolerance.;The abundance dynamics of all available histone PTMs have been screened inside the soybean plants under salinity stress and demonstrated that in comparing with the control sample, H3 lysine acetylation (Lys Ac), H3 lysine 14 acetylation (H3K14Ac), H3 Serine 10 phosphorylation (H3S10Phos) increased under the salinity stress. Indicating that H3K14Ac and H3S10Phos might play important roles in soybean adaptation to salinity stress and they were chosen for exploring the mechanisms of soybean tolerance.;To investigate the nucleic factors (Proteins and genes) which interacted with the H3K14Ac, H3K9Ac and H3S10Phos, the precipitated proteins obtained from ChIP assay were further separated by SDS-PAGE and identified by MALDI-TOF/ TOF. Different proteins could be identified among all three PTMs. Results showed that H3S10Phos could recruit PHD finger family protein, which was previously found to interact with GNAT (a histone acetyltransferase for H3K14), it indicated that phosphorylation of H3S10 and acetylation of H3K14 have intrinsic relationship and happen in sequence. Besides, the acetylation of H3K9 and H3K14 cooperated during expression process of salinity inducible genes. Since the PHD finger domain containing protein can also interacts with Elongin A and ISWI, H3S10Phos might also contribute much to chromatin remodeling and transcription, while H3K14Ac could regulate the gene translation by recruiting translation elongation factor Ts.;To understand the detail pattern of histone PTMs' regulation mechanism, eight peptides were employed for the PTM peptide pull-down assay using the total root nucleic proteins as input. Results demonstrated that almost all the modified peptide could recruit the rubber elongation factor protein and peroxidase 1 precursor. However, there were also noticeable interaction patterns amongst them. For example, the H3K14Ac or combined H3K14Ac with S10P (S10pK14ac) could specifically interact with histone H2B, H4 and 14-3-3 (SGF14h, belongs to non epsilon class). Besides, the soybean ascorbate peroxidase (GmAPX) was always recruited by those peptides carrying phosphorylated S10.;In order to detect the locating position of H3K14Ac on the salinity inducible genes, chromatin immunoprecipitation (ChIP) was performed using the anti-H3K14Ac antibody and the soybean chromatin total extract. H3K14Ac was found predominantly located at promoter region of GmGST gene (codes for glutathione S-transferase). Besides, the specific location of H3K14Ac on this gene, two PTMs of its adjacent cites (anti-H3K9Ac, anti-H3S10Phos) were also confirmed using ChIP assay. Our results illustrate that H3K14Ac and H3K9Ac bind to the promoter region of this gene while H3S10Phos binds to the downstream region (P4) of the GmGST gene.;Based on these findings, a scheme is proposed to illustrate the functional roles of H3 N-terminal PTMs, GmGST and GmAPX in salinity stress-induced oxidative response in soybean plant. Initially, the accumulation of cellular ROS (such as H2O2) could up-regulate modification of H3S10Phos and H3K14Ac, which may active the expression of GmGST. Then the increased GmGST could either directly or indirectly activate downstream targets such as GmMDAR and GmAPX through the glutathione ascorbate cycle. Since the GmAPX has antioxidant effect, the accumulation of H 2O2 could be finally reduced. On the other hand, the over-accumulated GmAPX could transport into the nucleus and feedback to the GmGST expression system.