NCPbook: a comprehensive database of non-canonical peptides.

Non-canonical peptides (NCPs) are a class of peptides generated from regions previously thought of as non-coding, such as introns, 5' untranslated regions (UTRs), 3' UTRs, and intergenic regions. In recent years, the significance and diverse functions of NCPs have come to light, yet a systematic and comprehensive NCP database remains absent. Here, we developed NCPbook (https://ncp.wiki/ncpbook/), a database of evidence-supported NCPs, which aims to provide a resource for efficient exploration, analysis, and manipulation of NCPs. NCPbook incorporates data from diverse public databases and scientific literature. The current version of NCPbook includes 180,676 NCPs across 29 different species, evidenced by mass spectrometry (MS), ribosome profiling (Ribo-seq), or molecular experiments (ME). These NCPs are distributed across kingdoms, comprising 123,408 from 14 plant species, 56,999 from seven animal species, and 269 from eight microbial species. Furthermore, NCPbook encompasses 9,166 functionally characterized NCPs playing important roles in immunity, stress resistance, growth, and development. Equipped with a user-friendly interface, NCPbook allows users to search, browse, visualize, and retrieve data, making it an indispensable platform for researching NCPs in various plant, animal, and microbial species.

Ascorbate peroxidase 1 confers resistance to southern corn leaf blight in maize.

Southern corn leaf blight (SCLB), caused by Bipolaris maydis, is one of the most devastating diseases affecting maize production. However, only one SLCB resistance gene, conferring partial resistance, is currently known, underscoring the importance of isolating new SCLB resistance-related genes. Here, we performed a comparative proteomic analysis and identified 258 proteins showing differential abundance during the maize response to B. maydis. These proteins included an ascorbate peroxidase (Zea mays ascorbate peroxidase 1 (ZmAPX1)) encoded by a gene located within the mapping interval of a previously identified quantitative trait locus associated with SCLB resistance. ZmAPX1 overexpression resulted in lower H2 O2 accumulation and enhanced resistance against B. maydis. Jasmonic acid (JA) contents and transcript levels for JA biosynthesis and responsive genes increased in ZmAPX1-overexpressing plants infected with B. maydis, whereas Zmapx1 mutants showed the opposite effects. We further determined that low levels of H2 O2 are accompanied by an accumulation of JA that enhances SCLB resistance. These results demonstrate that ZmAPX1 positively regulates SCLB resistance by decreasing H2 O2 accumulation and activating the JA-mediated defense signaling pathway. This study identified ZmAPX1 as a potentially useful gene for increasing SCLB resistance. Furthermore, the generated data may be relevant for clarifying the functions of plant APXs.

Comparative proteomics combined with analyses of transgenic plants reveal ZmREM1.3 mediates maize resistance to southern corn rust.

Southern corn rust (SCR), which is a destructive disease caused by Puccinia polysora Underw. (P. polysora), commonly occurs in warm-temperate and tropical regions. To identify candidate proteins related to SCR resistance and characterize the molecular mechanisms underlying the maize-P. polysora interaction, a comparative proteomic analysis of susceptible and resistant maize lines was performed. Statistical analyses revealed 1489 differentially abundant proteins in the resistant line, as well as 1035 differentially abundant proteins in the susceptible line. After the P. polysora infection, the abundance of one remorin protein (ZmREM1.3) increased in the resistant genotype, but decreased in the susceptible genotype. Plant-specific remorins are important for responses to microbial infections as well as plant signalling processes. In this study, transgenic maize plants overexpressing ZmREM1.3 exhibited enhanced resistance to the biotrophic P. polysora. In contrast, homozygous ZmREM1.3 UniformMu mutant plants were significantly more susceptible to P. polysora than wild-type plants. Additionally, the ZmREM1.3-overexpressing plants accumulated more salicylic acid (SA) and jasmonic acid (JA). Moreover, the expression levels of defence-related genes were higher in ZmREM1.3-overexpressing maize plants than in non-transgenic control plants in response to the P. polysora infection. Overall, our results provide evidence that ZmREM1.3 positively regulates maize defences against P. polysora likely via SA/JA-mediated defence signalling pathways. This study represents the first large-scale proteomic analysis of the molecular mechanisms underlying the maize-P. polysora interaction. This is also the first report confirming the remorin protein family affects plant resistance to SCR.

MicroRNA 399 as a potential integrator of photo-response, phosphate homeostasis, and sucrose signaling under long day condition.

BACKGROUND: Photoperiod-sensitivity is a critical endogenous regulatory mechanism for plant growth and development under specific environmental conditions, while phosphate and sucrose signaling processes play key roles in cell growth and organ initiation. MicroRNA399 is phosphate-responsive, but, whether it has roles in other metabolic processes remains unknown. RESULTS: MicroRNA399 was determined to be sucrose-responsive through a microRNA array assay. High levels of sucrose inhibited the accumulation of microRNA399 family under phosphate starvation conditions in Arabidopsis thaliana. Similarly, exogenous sucrose supplementation also reduced microRNA399 expression in maize at developmental transition stages. RNA sequencing of a near-isogenic line(photoperiod-sensitive) line and its recurrent parent Huangzao4, a photoperiod-insensitive line, was conducted at various developmental stages. Members of microRNA399 family were down-regulated under long-day conditions in the photoperiod-sensitive near-isogenic line that accumulated more sucrose in vivo compared with the control line Huangzao4. CONCLUSION: MicroRNA399s may play central roles in the integration of sucrose sensing and photoperiodic responses under long day conditions in maize.

Long photoperiod affects the maize transition from vegetative to reproductive stages: a proteomic comparison between photoperiod-sensitive inbred line and its recurrent parent.

Maize (Zea mays L.) is a typical short-day plant that is produced as an important food product and industrial material. The photoperiod is one of the most important evolutionary mechanisms enabling the adaptation of plant developmental phases to changes in climate conditions. There are differences in the photoperiod sensitivity of maize inbred lines from tropical to temperate regions. In this study, to identify the maize proteins responsive to a long photoperiod (LP), the photoperiod-insensitive inbred line HZ4 and its near-isogenic line H496, which is sensitive to LP conditions, were analyzed under long-day conditions using isobaric tags for relative and absolute quantitation. We identified 5259 proteins in maize leaves exposed to the LP condition between the vegetative and reproductive stages. These proteins included 579 and 576 differentially accumulated proteins in H496 and HZ4 leaves, respectively. The differentially accumulated proteins (e.g., membrane, defense, and energy- and ribosome-related proteins) exhibited the opposite trends in HZ4 and H496 plants during the transition from the vegetative stage to the reproductive stage. These results suggest that the photoperiod-associated fragment in H496 plants considerably influences various proteins to respond to the photoperiod sensitivity. Overall, our data provide new insights into the effects of long-day treatments on the maize proteome, and may be useful for the development of new germplasm.

Dual functions of the ZmCCT-associated quantitative trait locus in flowering and stress responses under long-day conditions.

BACKGROUND: Photoperiodism refers to the ability of plants to measure day length to determine the season. This ability enables plants to coordinate internal biological activities with external changes to ensure normal growth. However, the influence of the photoperiod on maize flowering and stress responses under long-day (LD) conditions has not been analyzed by comparative transcriptome sequencing. The ZmCCT gene was previously identified as a homolog of the rice photoperiod response regulator Ghd7, and associated with the major quantitative trait locus (QTL) responsible for Gibberella stalk rot resistance in maize. However, its regulatory mechanism has not been characterized. RESULTS: We mapped the ZmCCT-associated QTL (ZmCCT-AQ), which is approximately 130 kb long and regulates photoperiod responses and resistance to Gibberella stalk rot and drought in maize. To investigate the effects of ZmCCT-AQ under LD conditions, the transcriptomes of the photoperiod-insensitive inbred line Huangzao4 (HZ4) and its near-isogenic line (HZ4-NIL) containing ZmCCT-AQ were sequenced. A set of genes identified by RNA-seq exhibited higher basal expression levels in HZ4-NIL than in HZ4. These genes were associated with responses to circadian rhythm changes and biotic and abiotic stresses. The differentially expressed genes in the introgressed regions of HZ4-NIL conferred higher drought and heat tolerance, and stronger disease resistance relative to HZ4. Co-expression analysis and the diurnal expression rhythms of genes related to stress responses suggested that ZmCCT and one of the circadian clock core genes, ZmCCA1, are important nodes linking the photoperiod to stress tolerance responses under LD conditions. CONCLUSION: Our study revealed that the photoperiod influences flowering and stress responses under LD conditions. Additionally, ZmCCT and ZmCCA1 are important functional links between the circadian clock and stress tolerance. The establishment of this particular molecular link has uncovered a new relationship between plant photoperiodism and stress responses.

Overexpression of ZmMAPK1 enhances drought and heat stress in transgenic Arabidopsis thaliana.

Mitogen-activated protein kinase (MAPK) signal transduction cascades play a crucial role in the response to extracellular stimuli in eukaryotes. A number of MAPK family genes have been isolated in plants, but the maize MAPK genes have been little studied. Here, we studied the role of maize MAP kinase 1 (ZmMAPK1) using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes and physiological parameter analysis. Our physiological parameter analysis suggested that over-expression ZmMAPK1 can increase proline content and decrease malondialdehyde content under drought, and prevent chlorophyll loss and the production of scavenger reactive oxygen species under heat stress. The resistance characteristics of the over-expression of ZmMAPK1 were associated with a significant increase in survival rate. These results suggest that ZmMAPK1 plays a positive role in response to drought and heat stress in Arabidopsis, and provide new insights into the mechanisms of action of MAPK in response to abiotic stress in plants.

Phosphoproteomic analysis of the resistant and susceptible genotypes of maize infected with sugarcane mosaic virus.

Protein phosphorylation plays a pivotal role in the regulation of many cellular events. No information is yet available, however, on protein phosphorylation in plants in response to virus infection. In this study, we characterized phosphoproteomes of resistant and susceptible genotypes of maize (Zea mays L.) in response to Sugarcane mosaic virus (SCMV) infection. Based on isotope tags for relative and absolute quantification technology, TiO2 enrichment method and LC-MS/MS analysis, we identified 65 and 59 phosphoproteins respectively, whose phosphorylation level regulated significantly in susceptible and resistant plants. Some identified phosphoproteins were shared by both genotypes, suggesting a partial overlapping of the responsive pathways to virus infection. While several phosphoproteins are well-known pathogen response phosphoproteins, virus infection differentially regulates most other phosphoproteins, which has not been reported in literature. Changes in protein phosphorylation status indicated that response to SCMV infection encompass a reformatting of major cellular processes. Our data provide new valuable insights into plant-virus interactions.

Valid application of western blotting.

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.

Readthrough events in plants reveal plasticity of stop codons.

Stop codon readthrough (SCR) has important biological implications but remains largely uncharacterized. Here, we identify 1,009 SCR events in plants using a proteogenomic strategy. Plant SCR candidates tend to have shorter transcript lengths and fewer exons and splice variants than non-SCR transcripts. Mass spectrometry evidence shows that stop codons involved in SCR events can be recoded as 20 standard amino acids, some of which are also supported by suppressor tRNA analysis. We also observe multiple functional signals in 34 maize extended proteins and characterize the structural and subcellular localization changes in the extended protein of basic transcription factor 3. Furthermore, the SCR events exhibit non-conserved signature, and the extensions likely undergo protein-coding selection. Overall, our study not only characterizes that SCR events are commonly present in plants but also identifies the recoding plasticity of stop codons, which provides important insights into the flexibility of genetic decoding.

Characterization and function of an E2-17 kDa (UBE2D) in an invertebrate Haliotis diversicolor supertexta.

Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homolog was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to class I E2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homolog and it may be involved in the immune response of abalone, Haliotis diversicolor supertexta.

BTL2 phospho-switch surveils plant immunity.

BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) is a co-receptor involved in the recognition of pattern-associated molecular patterns (PAMPs) via plasma membrane-localized pattern recognition receptors (PRRs). Absence of BAK1/SERK4 leads to the activation of autoimmunity in plants. Yu et al. recently showed that BAK-TO-LIFE 2 (BTL2) is required for the surveillance of BAK1/SERK4 integrity to maintain immune homeostasis.

Identification and expression localization of a Ran homologue in mollusc abalone, Haliotis diversicolor supertexta.

Ran protein is a central molecular in several key nuclear functions, including nucleocytoplasmic transport, cell-cycle progression and nuclear envelope assembly. In this study, we have isolated and characterized a Ran homologue from a gastropod abalone which we named ab-Ran. The full-length cDNA consists of 1239 bp with an ORF encoding a 220 amino acid protein. The deduced amino acid sequence of ab-Ran shows highly similar to that of other Ran members (84-88%). Moreover, the ab-Ran contains five conserved regions and four carboxy-terminal residues CAAX-box. RT-PCR analysis showed that the ab-Ran was ubiquitously expressed in abalone tissues. The intracellular localization examined by immunofluorescence and immunohistochemistry staining displayed that ab-Ran was largely concentrated in the nuclei and partially in the cytoplasm. To the best of our knowledge, this is the first identification and characterization of a Ran homologue in mollusk.

Robust expression and association of ZmCCA1 with circadian rhythms in maize.

In plants, the circadian clock is an endogenous mechanism that controls a wide range of biological processes. To date, as one of the key world crops, little is known about the molecular mechanism and components of the circadian clock in maize (Zea mays). In this study, we characterized the CIRCADIAN CLOCK ASSOCIATED1 gene of maize (ZmCCA1), an ortholog of CCA1 in Arabidopsis thaliana (AtCCA1). Quantitative real-time PCR analysis revealed that ZmCCA1 was expressed in leaves and stem apex meristems in a rhythmic pattern under long day and short day conditions, and its peak gene expression appeared during the morning. ZmCCA1 transcripts accumulated in all tissues evaluated, with higher levels in tassels and ears. Additionally, the expression of another photoperiod gene ZmTOC1 peaked 12 h after dawn on long days and at 10 h after dawn on short days. Subcellular localization analysis revealed that the ZmCCA1 protein is directed to the cell nucleus. Overexpression of ZmCCA1 in Arabidopsis reduced the expression levels of downstream genes, including GIGANTEA (AtGI), CONSTANS (AtCO), and FLOWERING LOCUST (AtFT), and resulted in longer hypocotyls and delayed flowering. Taken together, our data suggest that ZmCCA1 may be a core component of the circadian clock in maize.

Large-Scale Discovery of Non-conventional Peptides in Maize and Arabidopsis through an Integrated Peptidogenomic Pipeline.

Non-conventional peptides (NCPs), which include small open reading frame-encoded peptides, play critical roles in fundamental biological processes. In this study, we developed an integrated peptidogenomic pipeline using high-throughput mass spectra to probe a customized six-frame translation database and applied it to large-scale identification of NCPs in plants.A total of 1993 and 1860 NCPs were unambiguously identified in maize and Arabidopsis, respectively. These NCPs showed distinct characteristics compared with conventional peptides and were derived from introns, 3' UTRs, 5' UTRs, junctions, and intergenic regions. Furthermore, our results showed that translation events in unannotated transcripts occur more broadly than previously thought. In addition, we found that dozens of maize NCPs are enriched within regions associated with phenotypic variations and domestication selection, indicating that they potentially are involved in genetic regulation of complex traits and domestication in maize. Taken together, our study developed an integrated peptidogenomic pipeline for large-scale identification of NCPs in plants, which would facilitate global characterization of NCPs from other plants. The identification of large-scale NCPs in both monocot (maize) and dicot (Arabidopsis) plants indicates that a large portion of plant genome can be translated into biologically functional molecules, which has important implications for functional genomic studies.

Molecular cloning and expression analysis of a beta-thymosin homologue from a gastropod abalone, Haliotis diversicolor supertexta.

The beta-thymosins are a family of highly conserved small peptides with multiple functions. In this study, we isolated the full-length cDNA of a beta-thymosin homologue from a gastropod abalone Haliotis diversicolor supertexta which we named ab-TMSB. The full-length cDNA of ab-TMSB consists of 499 bp with an ORF encoding a 43 amino acids protein. The deduced amino acid sequence of ab-TMSB shows 61-76% identity to other beta-thymosins and shares a conserved actin-binding domain. The phylogenetic analysis revealed that ab-TMSB was branched with Sycon raphanus beta-thymosin and clustered with Strongylocentrotus purpuratus beta-thymosin. Quantitative real-time PCR showed that ab-TMSB was ubiquitously expressed in abalone and highly expressed in hemocyte. Moreover, the expression level of ab-TMSB in hemocyte was upregulated after LPS challenge. Taken together, these results indicate that ab-TMSB is a beta-thymosin homologue and may be involved in the immune response of abalone H. diversicolor supertexta.

Comparative proteomic analysis of the maize responses to early leaf senescence induced by preventing pollination.

The aim of this study was to explore the molecular mechanisms of induced leaf senescence by preventing pollination in maize using a proteomic method combined with other physiological methods. An elite maize inbred line Yu816 was selected for evaluation of its senescence mechanism. Phenotypic and chlorophyll content analysis revealed that the onset of leaf senescence occurred earlier in non-pollinated (NONPOL) leaves than pollinated (POL) leaves. Leaf protein species of NONPOL and POL leaves were separately extracted and their proteomes were assessed using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. A total of 4371 protein species were identified, of which 809 exhibited differentially altered abundance (P < 0.05). The identified protein species were related to diverse functions including photosystems, plant hormones, cell death, oxidative degradation, and protein metabolism, suggesting a potential signaling cascade for ear leaf senescence induced by pollination prevention. In addition, leaf total soluble sugar and leaf starch contents were remarkably higher in NONPOL plants than in POL plants. These findings suggest that induced leaf senescence might be associated with nutrient remobilization. Our results reveal a network of molecular mechanisms at the protein level and provide some insights into the early senescence mechanism in higher plants. Biological significance: The coordination between growth and timing for senescence is critical for maize production. However, the molecular mechanism of induced leaf senescence by preventing pollination in maize remains to be further elucidated at the proteomic level. Herein, we revealed some new protein species that are involved in hormone signaling, glycometabolism, oxidation-reduction, protein degradation and photosystem breakdown, and other biological processes that were not previously known to be associated with leaf senescence. This is the first large-scale proteomics study to examine induced leaf senescence in maize by preventing pollination.

Genome-wide Association Studies in Maize: Praise and Stargaze.

Genome-wide association study (GWAS) has become a widely accepted strategy for decoding genotype-phenotype associations in many species thanks to advances in next-generation sequencing (NGS) technologies. Maize is an ideal crop for GWAS and significant progress has been made in the last decade. This review summarizes current GWAS efforts in maize functional genomics research and discusses future prospects in the omics era. The general goal of GWAS is to link genotypic variations to corresponding differences in phenotype using the most appropriate statistical model in a given population. The current review also presents perspectives for optimizing GWAS design and analysis. GWAS analysis of data from RNA, protein, and metabolite-based omics studies is discussed, along with new models and new population designs that will identify causes of phenotypic variation that have been hidden to date. The joint and continuous efforts of the whole community will enhance our understanding of maize quantitative traits and boost crop molecular breeding designs.