Complete Mitochondrial Genomes of Nedyopus patrioticus: New Insights into the Color Polymorphism of Millipedes.

There has been debate about whether individuals with different color phenotypes should have different taxonomic status. In order to determine whether the different color phenotypes of Nedyopus patrioticus require separate taxonomic status or are simply synonyms, here, the complete mitochondrial genomes (mitogenomes) of two different colored N. patrioticus, i.e., red N. patrioticus and white N. patrioticus, are presented. The two mitogenomes were 15,781 bp and 15,798 bp in length, respectively. Each mitogenome contained 13 PCGs, 19 tRNAs, 2 rRNAs, and 1 CR, with a lack of trnI, trnL2, and trnV compared to other Polydesmida species. All genes were located on a single strand in two mitogenomes. Mitochondrial DNA analyses revealed that red N. patrioticus and white N. patrioticus did not show clear evolutionary differences. Furthermore, no significant divergence was discovered by means of base composition analysis. As a result, we suggest that white N. patrioticus might be regarded as a synonym for red N. patrioticus. The current findings confirmed the existence of color polymorphism in N. patrioticus, which provides exciting possibilities for future research. It is necessary to apply a combination of molecular and morphological methods in the taxonomy of millipedes.

The First Complete Mitochondrial Genome of the Genus Litostrophus: Insights into the Rearrangement and Evolution of Mitochondrial Genomes in Diplopoda.

This study presents the complete mitochondrial genome (mitogenome) of Litostrophus scaber, which is the first mitogenome of the genus Litostrophus. The mitogenome is a circular molecule with a length of 15,081 bp. The proportion of adenine and thymine (A + T) was 69.25%. The gene ND4L used TGA as the initiation codon, while the other PCGs utilized ATN (A, T, G, C) as the initiation codons. More than half of the PCGs used T as an incomplete termination codon. The transcription direction of the L. scaber mitogenome matched Spirobolus bungii, in contrast to most millipedes. Novel rearrangements were found in the L. scaber mitogenome: trnQ -trnC and trnL1- trnP underwent short-distance translocations and the gene block rrnS-rrnL-ND1 moved to a position between ND4 and ND5, resulting in the formation of a novel gene order. The phylogenetic analysis showed that L. scaber is most closely related to S. bungii, followed by Narceus magnum. These findings enhance our understanding of the rearrangement and evolution of Diplopoda mitogenomes.

The role of the ABF1 gene in regulation of Cd-induced hormesis in Arabidopsis thaliana.

Hormesis is important in plant performance in contaminated environments, but the underlying genetic mechanisms are poorly understood. This study aimed at mining key genes in regulating Cd-induced hormesis in Arabidopsis thaliana and verifying their biological function. Hormesis of fresh weight, dry weight, and root length occurred at concentrations of 0.003-2.4, 0.03-0.6, and 0.03-0.6 µM Cd, respectively. Superoxide dismutase and catalase activities, and chlorophyll content displayed inverted U-shaped curves, indicating that the antioxidant defense system and photosynthesis system played roles in hormesis. Based on KEGG pathway analysis with the trend chart of differentially expressed genes and weighted correlation network analysis, the key gene ABF1 in the metabolic pathway of abscisic acid was identified. Subsequently, genetic experiments with wild, overexpressing, and knockdown lines of A. thaliana were conducted to further verify the biological function of ABF1 involving Cd-induced hormesis in A. thaliana. The results revealed that the resistance capability of the overexpressing type to Cd stress was significantly enhanced and implicated that the ABF1 gene is essential for Cd-induced hormesis in A. thaliana. Mining key genes that regulate Cd-induced hormesis in plants and stimulate them could have a transformative impact on the phytoremediation of metal-contaminated environments.

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.

Alternative Splicing Analysis Revealed the Role of Alpha-Linolenic Acid and Carotenoids in Fruit Development of Osmanthus fragrans.

Alternative splicing refers to the process of producing different splicing isoforms from the same pre-mRNA through different alternative splicing events, which almost participates in all stages of plant growth and development. In order to understand its role in the fruit development of Osmanthus fragrans, transcriptome sequencing and alternative splicing analysis was carried out on three stages of O. fragrans fruit (O. fragrans "Zi Yingui"). The results showed that the proportion of skipping exon events was the highest in all three periods, followed by a retained intron, and the proportion of mutually exclusive exon events was the lowest and most of the alternative splicing events occurred in the first two periods. The results of enrichment analysis of differentially expressed genes and differentially expressed isoforms showed that alpha-Linolenic acid metabolism, flavonoid biosynthesis, carotenoid biosynthesis, photosynthesis, and photosynthetic-antenna protein pathways were significantly enriched, which may play an important role in the fruit development of O. fragrans. The results of this study lay the foundation for further study of the development and maturation of O. fragrans fruit and further ideas for controlling fruit color and improving fruit quality and appearance.

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.

SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

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.

ABA Mediates Plant Development and Abiotic Stress via Alternative Splicing.

Alternative splicing (AS) exists in eukaryotes to increase the complexity and adaptability of systems under biophysiological conditions by increasing transcriptional and protein diversity. As a classic hormone, abscisic acid (ABA) can effectively control plant growth, improve stress resistance, and promote dormancy. At the transcriptional level, ABA helps plants respond to the outside world by regulating transcription factors through signal transduction pathways to regulate gene expression. However, at the post-transcriptional level, the mechanism by which ABA can regulate plant biological processes by mediating alternative splicing is not well understood. Therefore, this paper briefly introduces the mechanism of ABA-induced alternative splicing and the role of ABA mediating AS in plant response to the environment and its own growth.

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.

High-Throughput Sequencing Reveals the Regulatory Networks of Transcriptome and Small RNAs During the Defense Against Marssonina brunnea in Poplar.

MicroRNAs are implicated in the adjustment of gene expression in plant response to biotic stresses. However, the regulatory networks of transcriptome and miRNAs are still poorly understood. In the present study, we ascertained the induction of genes for small RNA biosynthesis in poplar defense to a hemibiotrophic fungus Marssonina brunnea and afterward investigated the molecular regulatory networks by performing comprehensive sequencing analysis of mRNAs and small RNAs in M. brunnea-inoculated leaves. Differentially expressed genes in M. brunnea-infected poplar are mainly involved in secondary metabolisms, phytohormone pathways, the recognition of pathogens, and MAPK pathway in the plant, with real-time quantitative PCR (qPCR) validating the mRNA-seq results. Furthermore, differentially expressed miRNAs, such as MIR167_1-6, MIR167_1-12, MIR171_2-3, MIR395-13, MIR396-3, MIR396-16, MIR398-8, and MIR477-6, were identified. Through psRobot and TargetFinder programs, MIR167-1-6, MIR395-13, MIR396-3, MIR396-16, and MIR398-8 were annotated to modulate the expression of genes implicated in transportation, signaling, and biological responses of phytohormones and activation of antioxidants for plant immunity. Besides, validated differentially expressed genes involved in lignin generation, which were phenylalanine ammonia-lyase, ferulate-5-hydroxylase, cinnamyl alcohol dehydrogenase, and peroxidase 11, were selected as targets for the identification of novel miRNAs. Correspondingly, novel miRNAs, such as Novel MIR8567, Novel MIR3228, Novel MIR5913, and Novel MIR6493, were identified using the Mireap online program, which functions in the transcriptional regulation of lignin biosynthesis for poplar anti-fungal response. The present study underlines the roles of miRNAs in the regulation of transcriptome in the anti-fungal response of poplar and provides a new idea for molecular breeding of woody plants.

Effect of Alternate Wetting and Drying Irrigation on the Nutritional Qualities of Milled Rice.

Alternate wetting and drying (AWD) irrigation has been widely used to save irrigation water during rice production when compared to the traditionally continuous flooding (CF). Although the influence of AWD on water-saving potential and grain yield has been studied before, its detailed effect on grain nutritional quality in milled rice remains relatively unexplored. In this study, AWD could maintain grain yield as compared with CF. Thus, we undertook efforts to compare the nutritional traits of milled rice irrigated with AWD and CF regimes. A targeted metabolome assay on milled rice identified 74 differentially accumulated metabolites (DAMs) with 22 up- and 52 down-accumulated metabolites under AWD vs. CF. Clustering of the metabolite content obtained in this assay suggested that most of the metabolites showing significant changes belonged to "lipids," "alkaloids," and "phenolic acids." In addition, total protein, starch, lipid, and amino acids content were measured to correlate it with the differential accumulation of specific metabolites detected in the metabolome. Overall, the data suggested that AWD may improve the nutritional performance of milled rice by increasing amino acids and phenolic acids and decreasing lipids and alkaloids. Our study provides research proof for the need for the optimization of irrigation to optimize rice nutritional qualities.

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.