Rice Proteome Research Articles

RiceProteomeDB (RPDB): a user-friendly database for proteomics data storage, retrieval, and analysis

Rice, feeding a significant portion of the world, poses unique proteomic challenges critical to agricultural research and global food security. The complexity of the rice proteome, influenced by various genetic and environmental factors, demands specialized analytical approaches for effective study. The central challenges in rice proteomics lie in developing custom methods suited to the unique aspects of rice biology. These include data preprocessing, method selection, and result validation, all of which are essential for advancing rice research. Our aim is to decode these proteomic intricacies to facilitate breakthroughs in strain improvement, disease resistance, and yield optimization, all vital for combating global food insecurity. To achieve this, we have created the RiceProteomeDB (RPDB), a React + Django database, offering a streamlined and comprehensive platform for the analysis of rice proteomics data. RiceProteomeDB (RPDB) simplifies proteomics data management and analysis. It offers features for data organization, preprocessing, method selection, result validation, and data sharing. Researchers can access processed rice proteomics data, conduct analyses, and explore experimental conditions. The user-friendly web interface enhances navigation and interaction. RPDB fosters collaboration by enabling data sharing and proper acknowledgment of sources, contributing to proteomics research and knowledge dissemination. Availability and implementation: Web application: http://riceproteome.plantprofile.net/. The web application’s source code, user’s manual, and sample data: https://github.com/dongu7610/Riceproteome.

Effects of rice blast biocontrol strain Pseudomonas alcaliphila Ej2 on the endophytic microbiome and proteome of rice under salt stress.

Soil salinity is a prevalent environmental stress in agricultural production. Microbial inoculants could effectively help plants to alleviate salt stress. However, there is little knowledge of the biocontrol strain Pseudomonas alcaliphila Ej2 mechanisms aiding rice plants to reduce the adverse effects caused by salt stress. We performed integrated field and greenhouse experiments, microbial community profiling, and rice proteomic analysis to systematically investigate the Ej2 mechanism of action. The results displayed that biocontrol strain Ej2 increased shoot/root length and fresh/dry weight compared with control under salt stress. Meanwhile, strain Ej2 has the ability to control rice blast disease and promote rice growth. Furthermore, the microbial community analysis revealed that the alpha-diversity of Ej2-inoculated plants was higher than the control plants, expect the Shannon index of the bacterial microbiome and the Ej2-inoculated samples clustered and separated from the control samples based on beta-diversity analysis. Importantly, the enriched and specific OTUs after Ej2 inoculation at the genus level were Streptomyces, Pseudomonas, Flavobacterium, and Bacillus. Moreover, we observed that Ej2 inoculation influenced the rice proteomic profile, including metabolism, plant-pathogen interactions, and biosynthesis of unsaturated fatty acids. These results provide comprehensive evidence that Ej2 inoculation induced the rice endophytic microbiome and proteomic profiles to promote plant growth under salt stress. Understanding the biocontrol strain effects on the endophytic microbiome and rice proteomics will help us better understand the complex interactions between plants and microorganisms under salt stress. Furthermore, unraveling the mechanisms underlying salt tolerance will help us more efficiently ameliorate saline soils.

Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

BackgroundLysine 2-hydroxyisobutyrylation (Khib), a newly identified post-translational modification, is known to regulate transcriptional activity in animals. However, extensive studies of the lysine 2-hydroxyisobutyrylome in plants and animals have yet to be performed.ResultsIn this study, using LC-MS/MS qualitative proteomics strategies, we identified 4163 Khib sites on 1596 modified proteins in rice (Oryza sativa) seedlings. Motif analysis revealed 10 conserved motifs flanking the Khib sites, and subcellular localization analysis revealed that 44% of the Khib proteins are localized in the chloroplast. Gene ontology function, KEGG pathway, and protein domain enrichment analyses revealed that Khib occurs on proteins involved in diverse biological processes and is especially enriched in carbon metabolism and photosynthesis. Among the modified proteins, 20 Khib sites were identified in histone H2A and H2B, while only one site was identified in histone H4. Protein-protein interaction (PPI) network analysis further demonstrated that Khib participates in diverse biological processes including ribosomal activity, biosynthesis of secondary metabolites, and metabolic pathways. In addition, a comparison of lysine 2-hydroxyisobutyrylation, acetylation, and crotonylation in the rice proteome showed that 45 proteins with only 26 common lysine sites are commonly modified by three PTMs. The crosstalk of modified sites and PPI among these PTMs may form a complex network with both similar and different regulatory mechanisms.ConclusionsIn summary, our study comprehensively profiles the lysine 2-hydroxyisobutyrylome in rice and provides a better understanding of its biological functions in plants.

Full-Length Transcript-Based Proteogenomics of Rice Improves Its Genome and Proteome Annotation.

Rice (Oryza sativa) molecular breeding has gained considerable attention in recent years, but inaccurate genome annotation hampers its progress and functional studies of the rice genome. In this study, we applied single-molecule long-read RNA sequencing (lrRNA_seq)-based proteogenomics to reveal the complexity of the rice transcriptome and its coding abilities. Surprisingly, approximately 60% of loci identified by lrRNA_seq are associated with natural antisense transcripts (NATs). The high-density genomic arrangement of NAT genes suggests their potential roles in the multifaceted control of gene expression. In addition, a large number of fusion and intergenic transcripts have been observed. Furthermore, 906,456 transcript isoforms were identified, and 72.9% of the genes can generate splicing isoforms. A total of 706,075 posttranscriptional events were subsequently categorized into 10 subtypes, demonstrating the interdependence of posttranscriptional mechanisms that contribute to transcriptome diversity. Parallel short-read RNA sequencing indicated that lrRNA_seq has a superior capacity for the identification of longer transcripts. In addition, over 190,000 unique peptides belonging to 9,706 proteoforms/protein groups were identified, expanding the diversity of the rice proteome. Our findings indicate that the genome organization, transcriptome diversity, and coding potential of the rice transcriptome are far more complex than previously anticipated.

A label-free proteomic approach differentiates between conventional and organic rice

Rice is a major cereal in the food industry. In this study, a quantitative label-free proteomic characterization of the organic rice proteome was performed; the proteome was compared to its conventional counterparts. As a result, a total of 114 proteins were significantly changed between the organic and conventional rice samples. Among them, 56 proteins were regarded as upregulated proteins and 58 were downregulated proteins in organic cultivated samples. Molecular function and bioinformatics analyses indicated that changes in the biosynthesis of secondary metabolites, metabolic pathways, and microbial metabolism in diverse environments contributed to the differences. These results were confirmed through the identification of 20 significantly changed metabolites. Specifically, these proteins mainly corresponded to carbohydrate metabolism, ribosome biogenesis, and the stress response. This in-depth study of proteins in rice extracts has implicated the role of those metabolic cycles involved in planting patterns, and represents a starting point in the selection of key proteins for rice authentication in the food industry.

Improvements to the Rice Genome Annotation Through Large-Scale Analysis of RNA-Seq and Proteomics Data Sets.

Rice (Oryza sativa) is one of the most important worldwide crops. The genome has been available for over 10 years and has undergone several rounds of annotation. We created a comprehensive database of transcripts from 29 public RNA sequencing data sets, officially predicted genes from Ensembl plants, and common contaminants in which to search for protein-level evidence. We re-analyzed nine publicly accessible rice proteomics data sets. In total, we identified 420K peptide spectrum matches from 47K peptides and 8,187 protein groups. 4168 peptides were initially classed as putative novel peptides (not matching official genes). Following a strict filtration scheme to rule out other possible explanations, we discovered 1,584 high confidence novel peptides. The novel peptides were clustered into 692 genomic loci where our results suggest annotation improvements. 80% of the novel peptides had an ortholog match in the curated protein sequence set from at least one other plant species. For the peptides clustering in intergenic regions (and thus potentially new genes), 101 loci were identified, for which 43 had a high-confidence hit for a protein domain. Our results can be displayed as tracks on the Ensembl genome or other browsers supporting Track Hubs, to support re-annotation of the rice genome.

Protein Phosphatase (PP2C9) Induces Protein Expression Differentially to Mediate Nitrogen Utilization Efficiency in Rice under Nitrogen-Deficient Condition.

Nitrogen (N) is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture. To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer. The identification or development of genotypes with high nitrogen utilization efficiency (NUE) which can grow efficiently and sustain yield in low N conditions is a possible solution. In this study, two isogenic rice genotypes i.e., wild-type rice kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene (PP2C9) were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE. 2D gel electrophoresis was used to perform the differential proteome analysis. In the leaf proteome, 30 protein spots were differentially expressed between the two isogenic lines under low N level which were involved in the process of energy, photosynthesis, N metabolism, signaling, and defense mechanisms. In addition, we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins. Furthermore, we showed that PP2C9TL exhibits higher NUE than WT due to higher activity of nitrate reductase. This study provides new insights on the rice proteome which would be useful in the development of new strategies to increase NUE in cereal crops.

Two-dimensional liquid chromatography separation and high resolution mass spectrometry analysis for proteome of rice leaves based on different extraction methods

A two-dimensional liquid chromatography (2D LC) system was developed to separate proteins from rice leaves, which was extracted by phenol method, followed by the analysis with linear trap quadrupole orbitrap mass spectrometry (LTQ/Orbitrap MS). After proteins were extracted with phenol method, the enzymolytic peptides were separated by offline two-dimensional RP-RP system and detected by LTQ/Orbitrap MS, yielding 2712 proteins. Liquid chromatography separation system (1D LC and 2D LC) and protein extraction methods (phenol method, sodium dodecyl sulfate method (SDS method) and trichloroacetic acid/acetone method (TCA/acetone method)) were compared. Proteins identified by 2D LC were 2712, 2415 and 1914 with the above three extraction methods, respectively. The proteins were 2.7-fold, 2.5-fold and 1.9-fold the number of proteins identified by 1D LC respectively. And in terms of 2D LC, the proteins identified by phenol method were 297 and 798 more than SDS method and TCA/acetone method, respectively. Some proteins with extreme properties, such as very acidic or basic protein and high relative molecular mass proteins, were only identified in phenol method. Furthermore, proteins, which were extracted by different extraction methods and separated by 2D LC, were classified according to biological functions. It was found that protein functions by the three extraction methods were complementary. However, phenol method had the most variety of functions. The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.

Impact of SNPs on Protein Phosphorylation Status in Rice (Oryza sativa L.).

Single nucleotide polymorphisms (SNPs) are widely used in functional genomics and genetics research work. The high-quality sequence of rice genome has provided a genome-wide SNP and proteome resource. However, the impact of SNPs on protein phosphorylation status in rice is not fully understood. In this paper, we firstly updated rice SNP resource based on the new rice genome Ver. 7.0, then systematically analyzed the potential impact of Non-synonymous SNPs (nsSNPs) on the protein phosphorylation status. There were 3,897,312 SNPs in Ver. 7.0 rice genome, among which 9.9% was nsSNPs. Whilst, a total 2,508,261 phosphorylated sites were predicted in rice proteome. Interestingly, we observed that 150,197 (39.1%) nsSNPs could influence protein phosphorylation status, among which 52.2% might induce changes of protein kinase (PK) types for adjacent phosphorylation sites. We constructed a database, SNP_rice, to deposit the updated rice SNP resource and phosSNPs information. It was freely available to academic researchers at http://bioinformatics.fafu.edu.cn. As a case study, we detected five nsSNPs that potentially influenced heterotrimeric G proteins phosphorylation status in rice, indicating that genetic polymorphisms showed impact on the signal transduction by influencing the phosphorylation status of heterotrimeric G proteins. The results in this work could be a useful resource for future experimental identification and provide interesting information for better rice breeding.

Elucidation of Complex Nature of PEG Induced Drought-Stress Response in Rice Root Using Comparative Proteomics Approach.

Along with many adaptive strategies, dynamic changes in protein abundance seem to be the common strategy to cope up with abiotic stresses which can be best explored through proteomics. Understanding of drought response is the key to decipher regulatory mechanism of better adaptation. Rice (Oryza sativa L.) proteome represents a phenomenal source of proteins that govern traits of agronomic importance, such as drought tolerance. In this study, a comparison of root cytoplasmic proteome was done for a drought tolerant rice (Heena) cultivar in PEG induced drought conditions. A total of 510 protein spots were observed by PDQuest analysis and 125 differentially regulated spots were subjected for MALDI-TOF MS-MS analysis out of which 102 protein spots identified which further led to identification of 78 proteins with a significant score. These 78 differentially expressed proteins appeared to be involved in different biological pathways. The largest percentage of identified proteins was involved in bioenergy and metabolism (29%) and mainly consists of malate dehydrogenase, succinyl-CoA, putative acetyl-CoA synthetase, and pyruvate dehydrogenase etc. This was followed by proteins related to cell defense and rescue (22%) such as monodehydroascorbate reductase and stress-induced protein sti1, then by protein biogenesis and storage class (21%) e.g. putative thiamine biosynthesis protein, putative beta-alanine synthase, and cysteine synthase. Further, cell signaling (9%) proteins like actin and prolyl endopeptidase, and proteins with miscellaneous function (19%) like Sgt1 and some hypothetical proteins were also represented a large contribution toward drought regulatory mechanism in rice. We propose that protein biogenesis, cell defense, and superior homeostasis may render better drought-adaptation. These findings might expedite the functional determination of the drought-responsive proteins and their prioritization as potential molecular targets for perfect adaptation.

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins.

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28-58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

Rice Proteomics and Beyond

Rice Proteomics and Beyond

Rice Proteomics and Beyond

Rice is the most widely consumed staple food for both developed as well as developing world, more so for Asia. According to the data of FAOSTAT (2012), rice has the third-highest worldwide production after sugarcane and maize, among all agricultural crops [1]. Developing countries account for 95% of the total rice production, with China and India contributing for nearly half of the world output [1].

The organelle-focused proteomes and interactomes in rice.

Protein subcellular localization has been a long-standing key problem in investigating proteins' function, which provides important clues for revealing their functions and aids in understanding their interactions with other biomolecules at the cellular level. Here, we systematically defined the organelle-focused proteomes and interactomes in Oryza sativa. A total of 83.42% of the whole rice proteome obtained their subcellular localizations based on manual annotation, manual adjustment and predictors' cross validation. The final organelle-focused interactomes were located in nine organelles. Furthermore, we discussed the cross talk bias between different organelles and the function organization accounting for nine organelles. Motif analysis illustrated the protein interaction bias in different organelles to implement certain biology functions. We exemplified the connection between functions and the overrepresented motifs in the organelle-focused interactomes and exemplified how to infer the functions of unknown proteins via expanding the overrepresented motifs.

Physiological and proteomic alterations in rice (Oryza sativa L.) seedlings under hexavalent chromium stress.

Rice plants employ two strategies to cope with Cr toxicity: immobilizing Cr ions into cell walls to reduce its translocation and activating antioxidant defense to mitigate Cr-induced oxidative stress. The investigation aimed at understanding the physiological and proteomic responses of rice seedlings to hexavalent chromium (Cr(6+)) stress was conducted using two rice genotypes, which differ in Cr tolerance and accumulation. Cr toxicity (200 µM) heavily increased the accumulation of H2O2 and [Formula: see text], enhanced lipid peroxidation, decreased cell viability and consequently inhibited rice plant growth. Proteomic analyses suggest that the response of rice proteome to Cr stress is genotype- and Cr dosage-dependent and tissue specific. Sixty-four proteins, which show more than fourfold difference under either two Cr levels, have been successfully identified. They are involved in a range of cellular processes, including cell wall synthesis, energy production, primary metabolism, electron transport and detoxification. Two proteins related to cell wall structure, NAD-dependent epimerase/dehydratase and reversibly glycosylated polypeptide were greatly up-regulated by Cr stress. Their enhancements coupled with callose accumulation by Cr suggest that cell wall is an important barrier for rice plants to resist Cr stress. Some enzymes involved in antioxidant defense, such as ferredoxin-NADP reductase, NADP-isocitrate dehydrogenase, glyoxalase I (Gly I) and glutamine synthetase 1 (GS1) have also been identified in response to Cr stress. However, they were only detected in Cr-tolerant genotype, indicating the genotypic difference in the capacity of activating the defense system to fight against Cr-induced oxidative stress. Overall, two strategies in coping with Cr stress in rice plants can be hypothesized: (i) immobilizing Cr ions into cell walls to reduce its translocation and (ii) activating antioxidant defense to mitigate Cr-induced oxidative stress.

Rice proteomics: A model system for crop improvement and food security

Rice proteomics has progressed at a tremendous pace since the year 2000, and that has resulted in establishing and understanding the proteomes of tissues, organs, and organelles under both normal and abnormal (adverse) environmental conditions. Established proteomes have also helped in re-annotating the rice genome and revealing the new role of previously known proteins. The progress of rice proteomics had recognized it as the corner/stepping stone for at least cereal crops. Rice proteomics remains a model system for crops as per its exemplary proteomics research. Proteomics-based discoveries in rice are likely to be translated in improving crop plants and vice versa against ever-changing environmental factors. This review comprehensively covers rice proteomics studies from August 2010 to July 2013, with major focus on rice responses to diverse abiotic (drought, salt, oxidative, temperature, nutrient, hormone, metal ions, UV radiation, and ozone) as well as various biotic stresses, especially rice-pathogen interactions. The differentially regulated proteins in response to various abiotic stresses in different tissues have also been summarized, indicating key metabolic and regulatory pathways. We envision a significant role of rice proteomics in addressing the global ground level problem of food security, to meet the demands of the human population which is expected to reach six to nine billion by 2040.

Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column

A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site inplantnuclear import factor importin-α

The specific recognition between the import receptor importin-α and the nuclear localization signals (NLSs) is crucial to ensure the selective transport of cargoes into the nucleus. NLSs contain 1 or 2 clusters of positively charged amino acids, which usually bind to the major (monopartite NLSs) or both minor and major NLS-binding sites (bipartite NLSs). In our recent study, we determined the structure of importin-α1a from rice (Oryza sativa), and made 2 observations that suggest an increased utilization of the minor NLS-binding site in this protein. First, unlike the mammalian protein, both the major and minor NLS-binding sites are auto-inhibited in the unliganded rice protein. Second, we showed that NLSs of the “plant-specific” class preferentially bind to the minor NLS-binding site of rice importin-α. Here, we show that a distinct group of “minor site-specific” NLSs also bind to the minor site of the rice protein. We further show a greater enrichment of proteins containing these “plant-specific” and “minor site-specific” NLSs in the rice proteome. However, the analysis of the distribution of different classes of NLSs in diverse eukaryotes shows that in all organisms, the minor site-specific NLSs are much less prevalent than the classical monopartite and bipartite NLSs.

Protein profile of rice (Oryza sativa) seeds

Seeds are the most important plant storage organ and play a central role in the life cycle of plants. Since little is known about the protein composition of rice (Oryza sativa) seeds, in this work we used proteomic methods to obtain a reference map of rice seed proteins and identify important molecules. Overall, 480 reproducible protein spots were detected by two-dimensional electrophoresis on pH 4–7 gels and 302 proteins were identified by MALDI-TOF MS and database searches. Together, these proteins represented 252 gene products and were classified into 12 functional categories, most of which were involved in metabolic pathways. Database searches combined with hydropathy plots and gene ontology analysis showed that most rice seed proteins were hydrophilic and were related to binding, catalytic, cellular or metabolic processes. These results expand our knowledge of the rice proteome and improve our understanding of the cellular biology of rice seeds.

Managing the green proteomes for the next decade of plant research

Managing the green proteomes for the next decade of plant research