Lysine 2-hydroxyisobutyrylation is a recently identified protein post-translational modification that is known to affect the association between histone and DNA. However, non-histone protein lysine 2-hydroxyisobutyrylation remains largely unexplored. Utilizing antibody-based affinity enrichment and nano-HPLC/MS/MS analyses of 2-hydroxyisobutyrylation peptides, we efficaciously identified 9,916 2-hydroxyisobutyryl lysine sites on 2,512 proteins in developing rice seeds, representing the first lysine 2-hydroxyisobutyrylome dataset in plants. Functional annotation analyses indicated that a wide variety of vital biological processes were preferably targeted by lysine 2-hydroxyisobutyrylation, including glycolysis/gluconeogenesis, TCA cycle, starch biosynthesis, lipid metabolism, protein biosynthesis and processing. Our finding showed that 2-hydroxyisobutyrylated histone sites were conserved across plants, human, and mouse. A number of 2-hydroxyisobutyryl sites were shared with other lysine acylations in both histone and non-histone proteins. Comprehensive analysis of the lysine 2-hydroxyisobutyrylation sites illustrated that the modification sites were highly sequence specific with distinct motifs, and they had less surface accessibility than other lysine residues in the protein. Overall, our study provides the first systematic analysis of lysine 2-hydroxyisobutyrylation proteome in plants, and it serves as an important resource for future investigations of the regulatory mechanisms and functions of lysine 2-hydroxyisobutyrylation.

In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus.