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.

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.

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.

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.

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.

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.

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.

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.

Global transcriptome analysis of the maize (Zea mays L.) inbred line 08LF during leaf senescence initiated by pollination-prevention.

In maize (Zea mays), leaf senescence acts as a nutrient recycling process involved in proteins, lipids, and nucleic acids degradation and transport to the developing sink. However, the molecular mechanisms of pre-maturation associated with pollination-prevention remain unclear in maize. To explore global gene expression changes during the onset and progression of senescence in maize, the inbred line 08LF, with severe early senescence caused by pollination prevention, was selected. Phenotypic observation showed that the onset of leaf senescence of 08LF plants occurred approximately 14 days after silking (DAS) by pollination prevention. Transcriptional profiling analysis of the leaf at six developmental stages during induced senescence revealed that a total of 5,432 differentially expressed genes (DEGs) were identified, including 2314 up-regulated genes and 1925 down-regulated genes. Functional annotation showed that the up-regulated genes were mainly enriched in multi-organism process and nitrogen compound transport, whereas down-regulated genes were involved in photosynthesis. Expression patterns and pathway enrichment analyses of early-senescence related genes indicated that these DEGs are involved in complex regulatory networks, especially in the jasmonic acid pathway. In addition, transcription factors from several families were detected, particularly the CO-like, NAC, ERF, GRAS, WRKY and ZF-HD families, suggesting that these transcription factors might play important roles in driving leaf senescence in maize as a result of pollination-prevention.

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.

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.

Comparative proteomic analysis of the shoot apical meristem in maize between a ZmCCT-associated near-isogenic line and its recurrent parent.

The ZmCCT, one of the most important genes affecting photoperiod response, delays flowering under long-day conditions in maize (Zea mays). In this study we used the isobaric tags for relative and absolute quantification (iTRAQ) technique-based proteomics approach to identify differentially expressed proteins between a near-isogenic line (NIL) and its recurrent parent, contrasting in alleles of ZmCCT. A total of 5,259 distinct proteins were identified. Among them, 386 proteins were differentially expressed between NIL-cml line (ZmCCT-positive) and H4 line (ZmCCT-negative). Functional categorization showed that the differentially proteins were mainly involved in energy production, photosynthesis, signal transduction, and cell organization and biogenesis. Our results showed that during shoot apical meristem (SAM) development cell division proteins, carbohydrate metabolism-related proteins, and flower inhibition-related proteins were more abundant in the ZmCCT-positive line than the ZmCCT-negative line. These results, taken together with morphological observations, showed that the effect of ZmCCT on flowering might be caused by its effect on one or all of these biological processes. Although the exact roles of these putative related proteins remain to be examined, our results obtained using the proteomics approach lead to a better understanding of the photoperiodicity mechanism in maize plants.

Comparative Proteomic Analysis of the Response of Maize (Zea mays L.) Leaves to Long Photoperiod Condition.

Maize (Zea mays L.), an important industrial material and food source, shows an astonishing environmental adaptation. A remarkable feature of its post-domestication adaptation from tropical to temperate environments is adaptation to a long photoperiod (LP). Many photoperiod-related genes have been identified in previous transcriptomics analysis, but proteomics shows less evidence for this mechanism of photoperiod response. In this study, we sampled newly expanded leaves of maize at the three- and six-leaf stages from an LP-sensitive introgression line H496, the donor CML288, LP-insensitive inbred line, and recurrent parent Huangzao4 (HZ4) grown under long days (15 h light and 9 h dark). To characterize the proteomic changes in response to LP, the iTRAQ-labeling method was used to determine the proteome profiles of plants exposed to LP. A total of 943 proteins differentially expressed at the three- and six-leaf stages in HZ4 and H496 were identified. Functional analysis was performed by which the proteins were classified into stress defense, signal transduction, carbohydrate metabolism, protein metabolism, energy production, and transport functional groups using the WEGO online tool. The enriched gene ontology categories among the identified proteins were identified statistically with the Cytoscape plugin ClueGO + Cluepedia. Twenty Gene Ontology terms showed the highest significance, including those associated with protein processing in the endoplasmic reticulum, splicesome, ribosome, glyoxylate, dicarboxylate metabolism, L-malate dehydrogenase activity, and RNA transport. In addition, for subcellular location, all proteins showed significant enrichment of the mitochondrial outer membrane. The sugars producted by photosynthesis in plants are also a pivotal metabolic output in the circadian regulation. The results permit the prediction of several crucial proteins to photoperiod response and provide a foundation for further study of the influence of LP treatments on the circadian response in short-day plants.

Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid.

Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA.

Quantitative iTRAQ-based proteomic analysis of phosphoproteins and ABA-regulated phosphoproteins in maize leaves under osmotic stress.

Abscisic acid (ABA) regulates various developmental processes and stress responses in plants. Protein phosphorylation/dephosphorylation is a central post-translational modification (PTM) in ABA signaling. However, the phosphoproteins regulated by ABA under osmotic stress remain unknown in maize. In this study, maize mutant vp5 (deficient in ABA biosynthesis) and wild-type Vp5 were used to identify leaf phosphoproteins regulated by ABA under osmotic stress. Up to 4052 phosphopeptides, corresponding to 3017 phosphoproteins, were identified by Multiplex run iTRAQ-based quantitative proteomic and LC-MS/MS methods. The 4052 phosphopeptides contained 5723 non-redundant phosphosites; 512 phosphopeptides (379 in Vp5, 133 in vp5) displayed at least a 1.5-fold change of phosphorylation level under osmotic stress, of which 40 shared common in both genotypes and were differentially regulated by ABA. Comparing the signaling pathways involved in vp5 response to osmotic stress and those that in Vp5, indicated that ABA played a vital role in regulating these pathways related to mRNA synthesis, protein synthesis and photosynthesis. Our results provide a comprehensive dataset of phosphopeptides and phosphorylation sites regulated by ABA in maize adaptation to osmotic stress. This will be helpful to elucidate the ABA-mediate mechanism of maize endurance to drought by triggering phosphorylation or dephosphorylation cascades.

Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress.

Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.

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.