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.

Food security is a major global issue because of the growing population and decreasing land area. Rice (Oryza sativa L.) is the most important staple cereal crop in the world. Application of nitrogen (N) fertilizer has improved crop yield in the world during the past five decades but with considerable negative impacts on the environment. New solutions are therefore urgently needed to simultaneously increase yields while maintaining or preferably decreasing applied N to maximize the nitrogen use efficiency (NUE) of crops. Plant NUE is inherently complex with each step (including N uptake, translocation, assimilation, and remobilization) governed by multiple interacting genetic and environmental factors. Based on the current knowledge, we propose some possible approaches enhancing NUE, by molecular manipulation selecting candidate genes and agricultural integrated management practices for NUE improvement. Developing an integrated research program combining approaches, mainly based on whole-plant physiology, quantitative genetics, forward and reverse genetics, and agronomy approaches to improve NUE, is a major objective in the future.

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I₂ staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins.

Lysine succinylation has been recognized as a post-translational modification (PTM) in recent years. It is plausible that succinylation may have a vaster functional impact than acetylation because of bulkier structural changes and more significant charge differences on the modified lysine residue. Currently, however, the quantity and identity of succinylated proteins and their corresponding functions in cereal plants remain largely unknown. In this study, we estimated the native succinylation occupancy on lysine was between 2% to 10% in developing rice seeds. Eight hundred fifty-four lysine succinylation sites on 347 proteins have been identified by a thorough investigation in developing rice seeds. Six motifs were revealed as preferred amino acid sequence arrangements for succinylation sites, and a noteworthy motif preference was identified in proteins associated with different biological processes, molecular functions, pathways, and domains. Remarkably, heavy succinylation was detected on major seed storage proteins, in conjunction with critical enzymes involved in central carbon metabolism and starch biosynthetic pathways for rice seed development. Meanwhile, our results showed that the modification pattern of in vitro nonenzymatically succinylated proteins was different from those of the proteins isolated from cells in Western blots, suggesting that succinylation is not generated via nonenzymatic reaction in the cells, at least not completely. Using the acylation data obtained from the same rice tissue, we mapped many sites harboring lysine succinylation, acetylation, malonylation, crotonylation, and 2-hydroxisobutyrylation in rice seed proteins. A striking number of proteins with multiple modifications were shown to be involved in critical metabolic events. Given that these modification moieties are intermediate products of multiple cellular metabolic pathways, these targeted lysine residues may mediate the crosstalk between different metabolic pathways via modifications by different moieties. Our study exhibits a platform for extensive investigation of molecular networks administrating cereal seed development and metabolism via PTMs. Graphical Abstract Highlights Nonenzymatically Ksu proteins shown different pattern from native cell Ksu proteins. Motif preference of Ksu proteins was associated with different biological processes. Up to 67 developing rice seeds proteins contain PTMs of Kac, Ksu, Kcr, Kmal, and Khib. Some lysine residues of the key pathway enzymes are modified by succinylation.