Nuclear proteome response to cell wall removal in rice (Oryza sativa)

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.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Polycomb Gene Regulation in Rice Endosperm Development and Global Analysis of Protein Acetylation in Rice

Cereal endosperm represents half of all human food calories and serves as the primary feedstock for livestock. The regulatory mechanism of cereal endosperm 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. In addition, the enzyme activities of all the plant polycomb complexes have not been demonstrated in vitro. 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 and the gene expression was not regulated by imprinting. Genetic studies showed that a severe reduction of OsFIE2 expression led to completely endospermree seeds and a moderated reduction of OsFIE2 expression resulted in smaller seeds and loss of seed dormancy. Genome wide ChIP-seq analyses found that a large number of endosperm specific regulatory genes and storage nutrient metabolic pathway genes were directly regulated by H3K27me3 modification in the rice endosperm. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism different from that in Arabidopsis. In this dissertation, lysine acetylation, an important posttranslational modification in rice was also studied. 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 global analysis of lysine acetylation and a large number of proteins of diverse biological function in mammalian and bacterial cells have been shown to be acetylated. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in plants, especially in cereal crops. Here we report a large scale study of lysine acetylation in rice. We identified 112 lysine acetylated sites on 80 proteins with diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated nonhistone proteins. Overall, our results suggest that lysine acetylation may constitute an important regulatory mechanism for a large number of proteins including both histones and nonhistone proteins

Polycomb Gene Regulation in Rice Endosperm Development and Global Analysis of Protein Acetylation in Rice

Cereal endosperm represents half of all human food calories and serves as the primary feedstock for livestock. The regulatory mechanism of cereal endosperm 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. In addition, the enzyme activities of all the plant polycomb complexes have not been demonstrated in vitro. 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 and the gene expression was not regulated by imprinting. Genetic studies showed that a severe reduction of OsFIE2 expression led to completely endospermree seeds and a moderated reduction of OsFIE2 expression resulted in smaller seeds and loss of seed dormancy. Genome wide ChIP-seq analyses found that a large number of endosperm specific regulatory genes and storage nutrient metabolic pathway genes were directly regulated by H3K27me3 modification in the rice endosperm. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism different from that in Arabidopsis. In this dissertation, lysine acetylation, an important posttranslational modification in rice was also studied. 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 global analysis of lysine acetylation and a large number of proteins of diverse biological function in mammalian and bacterial cells have been shown to be acetylated. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in plants, especially in cereal crops. Here we report a large scale study of lysine acetylation in rice. We identified 112 lysine acetylated sites on 80 proteins with diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated nonhistone proteins. Overall, our results suggest that lysine acetylation may constitute an important regulatory mechanism for a large number of proteins including both histones and nonhistone proteins

Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa).

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

Polycomb Group Gene <em>OsFIE2</em> Regulates Rice (<em>Oryza sativa</em>) Seed Development and Grain Filling via a Mechanism Distinct from <em>Arabidopsis</em>

<div><p>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 <i>Arabidopsis</i>, 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 <i>OsFIE2</i>-polycomb complex using tandem affinity purification and demonstrated its specific H3 methyltransferase activity. We found that the <i>OsFIE2</i> gene product was responsible for H3K27me3 production specifically <i>in vivo</i>. Genetic studies showed that a reduction of <i>OsFIE2</i> 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 <i>OsFIE2</i> 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 <i>OsFIE2</i>. Our results suggest that <i>OsFIE2</i>-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism highly different from that in <i>Arabidopsis</i>.</p> </div