Fig 1.
Post-translational modifications identified in sugarcane histone Ss-H3.1 and Ss_H3.3 variant.
Amino acid residues covered by the peptides identified by the MS/MS analysis are indicated in red. The modification sites identified are shown on top of the sequence and the amino acid residues highlighted in blue (lysine), green (arginine), brown (serine), purple (threonine) and light blue (tyrosine). The first amino acid methionine was omitted from the sequence.
Fig 2.
Lysine acetylation in sugarcane histone H3.
(A) MS/MS spectrum of the doubly-charged ion at m/z 570.8407 corresponding to the H3 peptide prKacQLATKprAAR (residues 18–26) where K18 is acetylated. (B) Fragment ions of the recorded in MS/MS spectrum for the [M+2H]2+ ion (m/z 563.8325) matches to the peptide prKacQLATKacAAR acetylated at positions K18 and K23. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
Fig 3.
Serine/threonine O-acetylation in sugarcane histone H3.
(A) MS/MS spectrum of the [M+2H]2+ ion (m/z 556.3089) that matched the histone H3 peptide prKSacTGGKprAPR (residues 9–17) where S10 is acetylated. (B) MS/MS spectra of the doubly-charged precursor ion at m/z 598.8534 corresponding to H3T22 acetylation in the H3 peptide prKQLATacKprAAR (residues 18–26). Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
Fig 4.
Detection of H3K4 methylation in sugarcane.
(A) MS/MS fragmentation pattern recorded on the [M+2H]2+ ion at m/z 415.7401 that matches the histone H3 peptide (residues 3–8) prTKme1QTAR containing monomethyl K4. (B) MS/MS spectrum of the doubly-charged ion at m/z 394.7348 corresponding to the H3 peptide prTKme2QTAR. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 401.7426) that corresponds to the peptide prTKme3QTAR. Sequence of the peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal propionylation, product of the chemical derivatization, is indicated by pr.
Fig 5.
Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.
(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.
Fig 6.
Comparative analysis of post-translational modifications in the canonical Ss_H3.1 and Ss_H3.3 variant.
Relative amounts of the different modifications were calculated for the peptides corresponding to residues 27–40 of H3.1 (KSAPATGGVKKPHR) and H3.3 (KSAPTTGGVKKPHR). Only the most abundant isoforms are shown.
Fig 7.
Post-translational modifications identified in the histone H4 from sugarcane.
Amino acid residues identified by the nanoLC-MS/MS analysis are indicated in red. The modification sites identified are shown on top of the sequence and the amino acid residue highlighted in blue (lysine) and purple (threonine). The first amino acid methionine was omitted from the sequence.
Fig 8.
Lysine acetylation in sugarcane histone H4.
(A) MS/MS spectrum of the doubly charged ion at m/z 768.9466 that corresponds to the histone H4 peptide (residues 4–17) prGKprGGKacGLGKprGGAKprR containing acetyl K8. (B) MS/MS spectrum of the [M+2H]2+ ion at m/z 761.9385 matching the H4 peptide prGKacGGKprGLGKacGGAKprR where K5 and K12 are acetylated. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 754.9307) that corresponds to the peptide prGKacGGKacGLGKacGGAKprR where K5, K8 and K12 are acetylated. (D) The full acetylated peptide prGKacGGKacGLGKacGGAKacR at K5, K8, K12 and K16 was deduced from the MS/MS spectrum of the ion at m/z 747.9230. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
Fig 9.
Lysine methylation and threonine acetylation in sugarcane histone H4.
(A) MS/MS spectrum of the doubly charged ion at m/z 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]2+ ion at m/z 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
Fig 10.
Distribution patterns of histone post-translational modifications in sugarcane.
(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.