Proteogenomics-based functional genome research: approaches, applications, and perspectives in plants.

Proteogenomics (PG) integrates the proteome with the genome and transcriptome to refine gene models and annotation. Coupled with single-cell (SC) assays, PG effectively distinguishes heterogeneity among cell groups. Affiliating spatial information to PG reveals the high-resolution circuitry within SC atlases. Additionally, PG can investigate dynamic changes in protein-coding genes in plants across growth and development as well as stress and external stimulation, significantly contributing to the functional genome. Here we summarize existing PG research in plants and introduce the technical features of various methods. Combining PG with other omics, such as metabolomics and peptidomics, can offer even deeper insights into gene functions. We argue that the application of PG will represent an important font of foundational knowledge for plants.

SWATH-MS-based proteogenomic analysis reveals the involvement of alternative splicing in poplar upon lead stress.

Alternative splicing (AS) regulates gene expression and increases proteomic diversity for the fine tuning of stress responses in plants, but the exact mechanism through which AS functions in plant stress responses is not thoroughly understood. Here, we investigated how AS functions in poplar (Populus trichocarpa), a popular plant for bioremediation, in response to lead (Pb) stress. Using a proteogenomic analysis, we determine that Pb stress induced alterations in AS patterns that are characterized by an increased use of nonconventional splice sites and a higher abundance of Pb-responsive splicing factors (SFs) associated with Pb-responsive transcription factors. A strong Pb(II)-inducible chaperone protein, PtHSP70, that undergoes AS was further characterized. Overexpression of its two spliced isoforms, PtHSP70-AS1 and PtHSP70-AS2, in poplar and Arabidopsis significantly enhances the tolerance to Pb. Further characterization shows that both isoforms can directly bind to Pb(II), and PtHSP70-AS2 exhibits 10-fold higher binding capacities and a greater increase in expression under Pb stress, thereby reducing cellular toxicity through Pb(II) extrusion and conferring Pb tolerance. AS of PtHSP70 is found to be regulated by PtU1-70K, a Pb(II)-inducible core SF involved in 5'-splice site recognition. Because the same splicing pattern is also found in HSP70 orthologs in other plant species, AS of HSP70 may be a common regulatory mechanism to cope with Pb(II) toxicity. Overall, we have revealed a novel post-transcriptional machinery that mediates heavy metal tolerance in diverse plant species. Our findings offer new molecular targets and bioengineering strategies for phytoremediation and provide new insight for future directions in AS research.

Secondary Metabolites of Osmanthus fragrans: Metabolism and Medicinal Value.

Osmanthus fragrans (scientific name: Osmanthus fragrans (Thunb.) Lour.) is a species of the Osmanthus genus in the family Oleaceae, and it has a long history of cultivation in China. O. fragrans is edible and is well known for conferring a natural fragrance to desserts. This flowering plant has long been cultivated for ornamental purposes. Most contemporary literature related to O. fragrans focuses on its edible value and new species discovery, but the functional use of O. fragrans is often neglected. O, fragrans has many properties that are beneficial to human health, and its roots, stems, leaves, flowers and fruits have medicinal value. These characteristics are recorded in the classics of traditional Chinese medicine. Studies on the metabolites and medicinal value of O. fragrans published in recent years were used in this study to evaluate the medicinal value of O. fragrans. Using keywords such as metabolites and Osmanthus fragrans, a systematic and nonexhaustive search of articles, papers and books related to the medicinal use of Osmanthus fragrans metabolites was conducted. Fifteen metabolites were identified through this literature search and classified into three categories according to their properties and structure: flavonoids, terpenes and phenolic acids. It was found that the pharmacological activities of these secondary metabolites mainly include antioxidant, anticancer, anti-inflammatory and antibacterial activities and that these metabolites can be used to treat many human diseases, such as cancer, skin diseases, cardiovascular diseases, and neurological diseases. Most of the reports that are currently available and concern the secondary metabolites of Osmanthus fragrans have limitations. Some reports introduce only the general classification of compounds in Osmanthus fragrans, and some reports introduce only a single compound. In contrast, the introduction section of this paper includes both the category and the functional value of each compound. While reviewing the data for this study, the authors found that the specific action sites of these compounds and their mechanisms of action in plants are relatively weak, and in the future, additional research should be conducted to investigate this topic further.

Alternative Splicing and Its Roles in Plant Metabolism.

Plant metabolism, including primary metabolism such as tricarboxylic acid cycle, glycolysis, shikimate and amino acid pathways as well as specialized metabolism such as biosynthesis of phenolics, alkaloids and saponins, contributes to plant survival, growth, development and interactions with the environment. To this end, these metabolic processes are tightly and finely regulated transcriptionally, post-transcriptionally, translationally and post-translationally in response to different growth and developmental stages as well as the constantly changing environment. In this review, we summarize and describe the current knowledge of the regulation of plant metabolism by alternative splicing, a post-transcriptional regulatory mechanism that generates multiple protein isoforms from a single gene by using alternative splice sites during splicing. Numerous genes in plant metabolism have been shown to be alternatively spliced under different developmental stages and stress conditions. In particular, alternative splicing serves as a regulatory mechanism to fine-tune plant metabolism by altering biochemical activities, interaction and subcellular localization of proteins encoded by splice isoforms of various genes.

SWATH-MS Proteomic Approach to Discover Novel Protein Targets and Pathways in Response to Abscisic Acid.

SWATH-MS proteomic approaches enable the identification and quantification of thousands of proteins within a single profiling experiment, which is useful for the identification of genes regulated by abscisic acid (ABA) in a high-throughput manner. Here we describe the experimental procedures for protein extraction, digestion, peptides desalting, followed by the establishment of a DDA spectrum database and DIA-based SWATH detection and protein quantification. This method is able to identify and quantify proteins involved in ABA metabolism, signal perception and transduction with high accuracy and reproducibility.

Deficiency in flavonoid biosynthesis genes CHS, CHI, and CHIL alters rice flavonoid and lignin profiles.

Lignin is a complex phenylpropanoid polymer deposited in the secondary cell walls of vascular plants. Unlike most gymnosperm and eudicot lignins that are generated via the polymerization of monolignols, grass lignins additionally incorporate the flavonoid tricin as a natural lignin monomer. The biosynthesis and functions of tricin-integrated lignin (tricin-lignin) in grass cell walls and its effects on the utility of grass biomass remain largely unknown. We herein report a comparative analysis of rice (Oryza sativa) mutants deficient in the early flavonoid biosynthetic genes encoding CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), and CHI-LIKE (CHIL), with an emphasis on the analyses of disrupted tricin-lignin formation and the concurrent changes in lignin profiles and cell wall digestibility. All examined CHS-, CHI-, and CHIL-deficient rice mutants were largely depleted of extractable flavones, including tricin, and nearly devoid of tricin-lignin in the cell walls, supporting the crucial roles of CHS and CHI as committed enzymes and CHIL as a noncatalytic enhancer in the conserved biosynthetic pathway leading to flavone and tricin-lignin formation. In-depth cell wall structural analyses further indicated that lignin content and composition, including the monolignol-derived units, were differentially altered in the mutants. However, regardless of the extent of the lignin alterations, cell wall saccharification efficiencies of all tested rice mutants were similar to that of the wild-type controls. Together with earlier studies on other tricin-depleted grass mutant and transgenic plants, our results reflect the complexity in the metabolic consequences of tricin pathway perturbations and the relationships between lignin profiles and cell wall properties.

Comparative transcriptome analysis of coleorhiza development in japonica and Indica rice.

BACKGROUND: Coleorhiza hairs, are sheath-like outgrowth organs in the seeds of Poaceae family that look like root hair but develop from the coleorhiza epidermal cells during seed imbibition. The major role of coleorhiza hair in seed germination involves facilitating water uptake and nutrient supply for seed germination. However, molecular basis of coleorhiza hair development and underlying genes and metabolic pathways during seed germination are largely unknown and need to be established. RESULTS: In this study, a comparative transcriptome analysis of coleorhiza hairs from japonica and indica rice suggested that DEGs in embryo samples from seeds with embryo in air (EIA) as compared to embryo from seeds completely covered by water (CBW) were enriched in water deprivation, abscisic acid (ABA) and auxin metabolism, carbohydrate catabolism and phosphorus metabolism in coleorhiza hairs in both cultivars. Up-regulation of key metabolic genes in ABA, auxin and dehydrin and aquaporin genes may help maintain the basic development of coleorhiza hair in japonica and indica in EIA samples during both early and late stages. Additionally, DEGs involved in glutathione metabolism and carbon metabolism are upregulated while DEGs involved in amino acid and nucleotide sugar metabolism are downregulated in EIA suggesting induction of oxidative stress-alleviating genes and less priority to primary metabolism. CONCLUSIONS: Taken together, results in this study could provide novel aspects about the molecular signaling that could be involved in coleorhiza hair development in different types of rice cultivars during seed germination and may give some hints for breeders to improve seed germination efficiency under moderate drought conditions.

SWATH-MS based quantitive proteomics reveal regulatory metabolism and networks of androdioecy breeding system in Osmanthus fragrans.

BACKGROUND: The fragrant flower plant Osmanthus fragrans has an extremely rare androdioecious breeding system displaying the occurrence of males and hermaphrodites in a single population, which occupies a crucial intermediate stage in the evolutionary transition between hermaphroditism and dioecy. However, the molecular mechanism of androdioecy plant is very limited and still largely unknown. RESULTS: Here, we used SWATH-MS-based quantitative approach to study the proteome changes between male and hermaphroditic O. fragrans pistils. A total of 428 proteins of diverse functions were determined to show significant abundance changes including 210 up-regulated and 218 down-regulated proteins in male compared to hermaphroditic pistils. Functional categorization revealed that the differentially expressed proteins (DEPs) primarily distributed in the carbohydrate metabolism, secondary metabolism as well as signaling cascades. Further experimental analysis showed the substantial carbohydrates accumulation associated with promoted net photosynthetic rate and water use efficiency were observed in purplish red pedicel of hermaphroditic flower compared with green pedicel of male flower, implicating glucose metabolism serves as nutritional modulator for the differentiation of male and hermaphroditic flower. Meanwhile, the entire upregulation of secondary metabolism including flavonoids, isoprenoids and lignins seem to protect and maintain the male function in male flowers, well explaining important feature of androdioecy that aborted pistil of a male flower still has a male function. Furthermore, nine selected DEPs were validated via gene expression analysis, suggesting an extra layer of post-transcriptional regulation occurs during O. fragrans floral development. CONCLUSION: Taken together, our findings represent the first SWATH-MS-based proteomic report in androdioecy plant O. fragrans, which reveal carbohydrate metabolism, secondary metabolism and post-transcriptional regulation contributing to the androdioecy breeding system and ultimately extend our understanding on genetic basis as well as the industrialization development of O. fragrans.

Phylogenetic Comparison and Splicing Analysis of the U1 snRNP-specific Protein U1C in Eukaryotes.

As a pivotal regulator of 5' splice site recognition, U1 small nuclear ribonucleoprotein (U1 snRNP)-specific protein C (U1C) regulates pre-mRNA splicing by interacting with other components of the U1 snRNP complex. Previous studies have shown that U1 snRNP and its components are linked to a variety of diseases, including cancer. However, the phylogenetic relationships and expression profiles of U1C have not been studied systematically. To this end, we identified a total of 110 animal U1C genes and compared them to homologues from yeast and plants. Bioinformatics analysis shows that the structure and function of U1C proteins is relatively conserved and is found in multiple copies in a few members of the U1C gene family. Furthermore, the expression patterns reveal that U1Cs have potential roles in cancer progression and human development. In summary, our study presents a comprehensive overview of the animal U1C gene family, which can provide fundamental data and potential cues for further research in deciphering the molecular function of this splicing regulator.

Global proteome response to Pb(II) toxicity in poplar using SWATH-MS-based quantitative proteomics investigation.

Lead (Pb) toxicity is a growing serious environmental pollution that threatens human health and crop productivity. Poplar, as an important economic and ecological forest species, has the characteristics of fasting growth and accumulating heavy metals, which is a powerful model plant for phytoremediation. Here, a novel label-free quantitative proteomic platform of SWATH-MS was applied to detect proteome changes in poplar seedling roots following Pb treatment. In total 4388 unique proteins were identified and quantified, among which 542 proteins showed significant abundance changes upon Pb(II) exposure. Functional categorizations revealed that differentially expressed proteins (DEPs) primarily distributed in specialized biological processes. Particularly, lignin and flavonoid biosynthesis pathway were strongly activated upon Pb exposure, implicating their potential roles for Pb detoxification in poplar. Furthermore, hemicellulose and pectin related cell wall proteins exhibited increased abundances, where may function as a sequestration reservoir to reduce Pb toxicity in cytoplasm. Simultaneously, up-regulation of glutathione metabolism may serve as a protective role for Pb-induced oxidative damages in poplar. Further correlation investigation revealed an extra layer of post-transcriptional regulation during Pb response in poplar. Overall, our work represents multiply potential regulators in mediating Pb tolerance in poplar, providing molecular targets and strategies for phytoremediation.

SWATH-MS-Based Proteomics: Strategies and Applications in Plants.

Most applied proteomic approaches require labeling steps. Recent technological advances provide an alternative label-free proteomics approach: SWATH-MS. This powerful tool is now widely used in animal studies but has drawn far less attention in plants. Here we summarize how this promising technology can be applied to facilitate functional analysis in plant research.

Systematic characterization of the branch point binding protein, splicing factor 1, gene family in plant development and stress responses.

BACKGROUND: Among eukaryotic organisms, alternative splicing is an important process that can generate multiple transcripts from one same precursor messenger RNA, which greatly increase transcriptome and proteome diversity. This process is carried out by a super-protein complex defined as the spliceosome. Specifically, splicing factor 1/branchpoint binding protein (SF1/BBP) is a single protein that can bind to the intronic branchpoint sequence (BPS), connecting the 5' and 3' splice site binding complexes during early spliceosome assembly. The molecular function of this protein has been extensively investigated in yeast, metazoa and mammals. However, its counterpart in plants has been seldomly reported. RESULTS: To this end, we conducted a systematic characterization of the SF1 gene family across plant lineages. In this work, a total of 92 sequences from 59 plant species were identified. Phylogenetic relationships of these sequences were constructed, and subsequent bioinformatic analysis suggested that this family likely originated from an ancient gene transposition duplication event. Most plant species were shown to maintain a single copy of this gene. Furthermore, an additional RNA binding motif (RRM) existed in most members of this gene family in comparison to their animal and yeast counterparts, indicating that their potential role was preserved in the plant lineage. CONCLUSION: Our analysis presents general features of the gene and protein structure of this splicing factor family and will provide fundamental information for further functional studies in plants.

Alternative splicing and its regulatory role in woody plants.

Alternative splicing (AS) is an important post-transcriptional process to enhance proteome diversity in eukaryotic organisms. In plants, numerous reports have primarily focused on AS analysis in model plant species or herbaceous plants, leading to a notable lack of research on AS in woody plants. More importantly, emerging evidence indicates that many important traits, including wood formation and stress resistance, in woody plants are controlled by AS. In this review article, we summarize the current progress of all kinds of AS studies in different tree species at various stages of development and in response to various stresses, revealing the significant role played by AS in woody plants, as well as the similar properties and differential regulation within their herbaceous counterparts. Furthermore, we propose several potential strategies to facilitate the functional characterization of splicing factors in woody plants and evaluate a general pipeline for the systematic characterization of splicing isoforms in a complex AS regulatory network. The utilization of genetic studies and high-throughput omics integration approaches to analyze AS genes and splicing factors is likely to further advance our understanding of AS modulation in woody plants.

Flavonoids are indispensable for complete male fertility in rice.

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.

Phylogenetic comparison of 5' splice site determination in central spliceosomal proteins of the U1-70K gene family, in response to developmental cues and stress conditions.

Intron-containing genes have the ability to generate multiple transcript isoforms by splicing, thereby greatly expanding the eukaryotic transcriptome and proteome. In eukaryotic cells, precursor mRNA (pre-mRNA) splicing is performed by a mega-macromolecular complex defined as a spliceosome. Among its splicing components, U1 small nuclear ribonucleoprotein (U1 snRNP) is the smallest subcomplex involved in early spliceosome assembly and 5'-splice site recognition. Its central component, named U1-70K, has been extensively characterized in animals and yeast. Very few investigations on U1-70K genes have been conducted in plants, however. To this end, we performed a comprehensive study to systematically identify 115 U1-70K genes from 67 plant species, ranging from algae to angiosperms. Phylogenetic analysis suggested that the expansion of the plant U1-70K gene family was likely to have been driven by whole-genome duplications. Subsequent comparisons of gene structures, protein domains, promoter regions and conserved splicing patterns indicated that plant U1-70Ks are likely to preserve their conserved molecular function across plant lineages and play an important functional role in response to environmental stresses. Furthermore, genetic analysis using T-DNA insertion mutants suggested that Arabidopsis U1-70K may be involved in response to osmotic stress. Our results provide a general overview of this gene family in Viridiplantae and will act as a reference source for future mechanistic studies on this U1 snRNP-specific splicing factor.

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.

SWATH-MS-facilitated proteomic profiling of fruit skin between Fuji apple and a red skin bud sport mutant.

BACKGROUND: Apple is one of the most popular fruit crops world-wide and its skin color is an important quality consideration essential for commercial value. However, the strategy on genetic breeding for red skin apple and the genetic basis of skin color differentiation is very limited and still largely unknown. RESULTS: Here, we reported a bud sport mutant of Fuji apple with red skin color and enhanced anthocyanins accumulation. Quantitative SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) proteomics investigations revealed proteome changes in the apple red skin bud mutation and a total of 451 differentially expressed proteins were identified in apple skin. The mutant showed significantly increased expression levels of photosynthesis-related proteins, stress-related proteins as well as anthocyanins biosynthesis pathway. On the other hand, substantial downregulation of mitogen-activated protein kinase 4 (MAPK4) and mevalonate kinase (MVK) were detected, indicating a promising role for the red skin color development in the mutant. Furthermore, we also hypothesize that a post-transcriptional regulation of the skin color formation occurs in the mutant through the advanced SWATH-MS analysis. CONCLUSION: Our work provides important information on the application of proteomic methods for analysing proteomes changes in Fuji apple and highlights a clade of regulatory proteins potentially contributing for the molecular breeding of fruit skin color.

Comparative metabolite profiling of two switchgrass ecotypes reveals differences in drought stress responses and rhizosheath weight.

MAIN CONCLUSION: Rhizosheath comprises soil that adheres firmly to roots. In this study, two ecotypes of switchgrass with different rhizosheath sizes after drought stress were analyzed which showed metabolic differences under drought conditions. The rhizosheath comprises soil that adheres firmly to roots by a combination of root hairs and mucilage and may aid in root growth under soil drying. The aim of this work is to reveal the potential metabolites involved in rhizosheath formation under drought stress conditions. Panicum virgatum L. (switchgrass), which belongs to the Poaceae family, is an important biofuel and fodder crop in drought areas. Five switchgrass ecotypes (cv. Alamo, cv. Blackwake, cv. Summer, cv. Cave-in-Rock and cv. Kanlow) have a broad range of rhizosheath weight under drought conditions. For two selected ecotypes with contrast rhizosheath weight (cv. Alamo and cv. Kanlow), root hair length and density, lateral root number, root morphological parameters were measured, and real-time qRT-PCR was performed. Gas chromatography mass spectrophotometry (GC-MS) was used to determine the primary metabolites in the shoots and roots of selected ecotypes under drought stress conditions. The change trends of root hair length and density, lateral root number and related gene expression were consistent with rhizosheath weight in Alamo and Kanlow under drought and watered conditions. For root morphological parameters, Alamo grew deeper than Kanlow, while Kanlow exhibited higher values for other parameters. In this study, the levels of amino acids, sugars and organic acids were significantly changed in response to drought stress in two switchgrass ecotypes. Several metabolites including amino acids (arginine, isoleucine, methionine and cysteine) and sugars (kestose, raffinose, fructose, fucose, sorbose and xylose) in the large soil-sheathed roots of Alamo and Kanlow were significantly increased compared to small or no soil-sheathed roots of Alamo and Kanlow. Difference in rhizosheath size is reflected in the plant internal metabolites under drought stress conditions. Additionally, our results highlight the importance of using metabolite profiling and provide a better understanding of rhizosheath formation at the cellular level.