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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.

Long non-coding RNA (lncRNA) refers to an over 200 nt functional RNA molecule that will not be translated into protein. Previously thought to be dark matters of the genome, lncRNAs have been gradually recognized as crucial gene regulators. Although tremendous progress has been made in animals and human, the study of lncRNAs in plant is still in its infancy. Here, we reviewed the biogenesis and regulation mechanisms of lncRNAs and summarized the achievements that have been made in plant lncRNA identification and functional characterization. Genome-wide identification has uncovered large amount of lncRNAs in Arabidopsis, Rice, Maize and Wheat, and more information from other plant species will be expected with the aid of deep sequencing technologies. Similar to other species, LncRNA-mediated gene regulation also widely exists in plants, even though only a few functionally characterized examples are available. Up to now, at least four divergent lncRNA-mediated regulation mechanisms have been unraveled, including target mimicry, transcription interference, PRC2 associated histone methylation and DNA methylation. lncRNAs may be involved in the regulation of flowering, male sterility, nutrition metabolism, biotic and abiotic stress response in plants.

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.

The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis) fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

MADS-box transcription factors are known for their roles in plant growth and development. The regulatory mechanisms of spatial and temporal specific expression of MADS-box genes and the function of MADS-box genes in other biological processes are still to be explored. Here, we report that OsMADS6 is highly expressed in flower and endosperm in Oryza sativa (rice). In addition to displaying a homeotic organ identity phenotype in all the four whorls of the flowers, the endosperm development is severely affected in its mutant. At least 32% of the seeds lacked starch filling and aborted. For seeds that have starch filling and develop to maturity, the starch content is reduced by at least 13%. In addition, the seed shape changes from elliptical to roundish, and the protein content increases from 12.1 to 15.0% (P < 0.05). Further investigation shows that ADP-glucose pyrophosphorylase genes, encoding the rate-limiting step enzyme in the starch synthesis pathway, are subject to the regulation of OsMADS6. Chromatin immunoprecipitation (ChIP)-PCR analyses on the chromatin of the OsMADS6 gene find that H3K27 is trimethylated in tissues where OsMADS6 is silenced, and that H3K36 is trimethylated in tissues where OsMADS6 is highly activated. Point mutation analysis reveals that leucine at position 83 is critical to OsMADS6 function.

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa). We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.

Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.

Cereal endosperm represents 60% of the calories consumed by human beings worldwide. In addition, cereals also serve as the primary feedstock for livestock. However, the regulatory mechanism of cereal endosperm and seed development is largely unknown. Polycomb complex has been shown to play a key role in the regulation of endosperm development in Arabidopsis, but its role in cereal endosperm development remains obscure. Additionally, the enzyme activities of the polycomb complexes have not been demonstrated in plants. Here we purified the rice OsFIE2-polycomb complex using tandem affinity purification and demonstrated its specific H3 methyltransferase activity. We found that the OsFIE2 gene product was responsible for H3K27me3 production specifically in vivo. Genetic studies showed that a reduction of OsFIE2 expression led to smaller seeds, partially filled seeds, and partial loss of seed dormancy. Gene expression and proteomics analyses found that the starch synthesis rate limiting step enzyme and multiple storage proteins are down-regulated in OsFIE2 reduction lines. Genome wide ChIP–Seq data analysis shows that H3K27me3 is associated with many genes in the young seeds. The H3K27me3 modification and gene expression in a key helix-loop-helix transcription factor is shown to be regulated by OsFIE2. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism highly different from that in Arabidopsis., Author Summary Rice is the staple food for over half of the world's population and an important feedstock for livestock. The rice grain is mainly endosperm tissue. The regulatory mechanism of rice endosperm development is still largely unknown thus far. Understanding the underlying mechanism will lead to crop yield and quality improvement in the long term, besides gaining new knowledge. Polycomb complex is a protein complex with a potential role in endosperm development according to prior publications. In this manuscript, we purified the rice OsFIE2-polycomb protein complex and demonstrated the enzyme activity of the complex. Genetic studies showed that a reduction of polycomb group gene OsFIE2 expression led to smaller seeds, partially filled seeds, and seed germination before seed maturation. Gene expression and proteomics analyses found that the starch synthesis rate limiting step enzyme and multiple storage proteins are down-regulated while a key transcription factor is up-regulated in OsFIE2 reduction lines. In addition, we identified many loci in the rice genome whose histone proteins are modified by the polycomb complex enzyme via a method called ChIP–Seq. Our results demonstrate that OsFIE2-polycomb complex positively regulates rice grain development via a mechanism distinct from that in Arabidopsis and provide new insight into the regulation of rice grain development.

MADS-box transcription factors are known for their roles in plant growth and development. The regulatory mechanisms of spatial and temporal specific expression of MADS-box genes and the function of MADS-box genes in other biological processes are still to be explored. Here, we report that OsMADS6 is highly expressed in flower and endosperm in Oryza sativa (rice). In addition to displaying a homeotic organ identity phenotype in all the four whorls of the flowers, the endosperm development is severely affected in its mutant. At least 32% of the seeds lacked starch filling and aborted. For seeds that have starch filling and develop to maturity, the starch content is reduced by at least 13%. In addition, the seed shape changes from elliptical to roundish, and the protein content increases from 12.1 to 15.0% (P0.05). Further investigation shows that ADP-glucose pyrophosphorylase genes, encoding the rate-limiting step enzyme in the starch synthesis pathway, are subject to the regulation of OsMADS6. Chromatin immunoprecipitation (ChIP)-PCR analyses on the chromatin of the OsMADS6 gene find that H3K27 is trimethylated in tissues where OsMADS6 is silenced, and that H3K36 is trimethylated in tissues where OsMADS6 is highly activated. Point mutation analysis reveals that leucine at position 83 is critical to OsMADS6 function.