Small open reading frames (smORFs, <100 amino acids) are a ubiquitous feature of eukaryotic and prokaryotic genomes. However, only a fraction of these smORFs encode functional proteins, known as microproteins. Functional microproteins have been found in all three domains of life, although due to limitations in their prediction and characterization, only a few have been described in plants. Therefore, the catalogization and functional analysis of plant smORF-encoded microproteins is far from complete. In this chapter, we present a bioinformatic pipeline designed to predict and validate coding smORFs in plant transcriptomes. The workflow comprises several key steps: transcriptome assembly, prediction of potentially coding smORFs, filtration of candidates, and validation of their translation through analysis of mass-spectrometry data.

Microproteins encoded by small open reading frames comprise the "dark matter" of proteomes. Although microproteins have been detected in diverse organisms from all three domains of life, many more remain to be identified, and only a few have been functionally characterized. In this comprehensive study of intergenic small open reading frames (ismORFs, 15-70 codons) in 5,668 bacterial genomes of the family Enterobacteriaceae, we identify 67,297 clusters of ismORFs subject to purifying selection. Expression of tagged Escherichia coli microproteins is detected for 11 of the 16 tested, validating the predictions. Although the ismORFs mainly code for hydrophobic, potentially transmembrane, unstructured, or minimally structured microproteins, some globular folds, oligomeric structures, and possible interactions with proteins encoded by neighboring genes are predicted. Complete information on the predicted microprotein families, including evidence of transcription and translation, and structure predictions are available as an easily searchable resource for investigation of microprotein functions.

Small secreted peptides (SSPs) play an important role in modulating immune responses in all land plants. However, the evolution of stress peptide signaling in different plant phyla remains poorly understood. Here, we compared the expression of SSP genes in the pathogen-induced transcriptomes of vascular and non-vascular plants. We found 13, 19, 15, and 28 SSP families that were differentially expressed during infection in Physcomitrium patens, Zea mays, Brassica napus, and Solanum tuberosum, respectively. A comparative study of peptide motifs and predicted three-dimensional structures confirmed the similarity of SSPs across the examined plant species. In both vascular and non-vascular plants. However, only the RALF peptide family was differentially regulated under infection. We also found that EPFL peptides, which are involved in growth and development processes in angiosperms, were differentially regulated in P. patens in response to pathogen infection. The search for novel immune-specific peptides revealed a family of PSY-like peptides that are differentially regulated during infection in P. patens. The treatment with synthetic tyrosine-modified and non-modified PSY, and PSY-like peptides, as well as recombinant EPFL and MEG, validated their roles in the immune response and growth regulation. Thus, our study showed the complex nature of SSP signaling and shed light on the regulation of SSPs in different plant lineages during infection.

Drought conditions impact plants at the morphological, physiological, and molecular levels. Plant tolerance to drought conditions is frequently associated with maintaining proteome stability, highlighting the significance of proteomic analysis in understanding the mechanisms underlying plant resilience. Here, we performed proteomic and peptidomic analysis of spring wheat (Triticum aestivum L.) under drought stress conditions. Using isobaric tags for relative and absolute quantitation (iTRAQ), we identified 497 and 157 differentially abundant protein (DAP) groups in leaves and roots, respectively. The upregulated DAP groups in leaves were primarily involved in stress responses, such as oxidative stress and heat response, whereas those in roots were associated with responses to water deprivation and sulfur compound metabolic processes. The analysis of the extracellular root peptidome revealed 2294 native peptides, including members of small secreted peptide (SSP) families. In the peptidomes of stress-induced plants, we identified 16 SSPs as well as peptides derived from proteins involved in cell wall catabolism, intercellular signaling, and stress response. These peptides represent potential candidates as regulators of drought responses. Our results help us to understand adaptation mechanisms and develop new agricultural technologies to increase productivity.

Bacterial genomes encompass numerous small open reading frames (smORFs), some of which encode functional microproteins or perform noncoding regulatory roles. The evolution of microproteins remains poorly understood, largely due to challenges in homology detection for these short sequences. To address this challenge, we constructed 36 957 orthologous groups of microproteins (microOGs) across 5668 Enterobacteriaceae genomes. Our pipeline identified dozens of novel, widely distributed microprotein families and refined conservation patterns for known ones. However, 86% of the microOGs are genus-specific and functionally uncharacterized, suggesting that enterobacteria harbor a pool of evolutionarily young, de novo-originated small genes. Nevertheless, the microprotein-encoding smORFs in the microOGs are preferentially adjacent to membrane transporter genes suggesting a role in regulating transport processes. MicroOGs formed closed pangenomes, indicative of a limited contribution to the noncore genome of enterobacteria, likely due to the limitations on the size of intergenic regions where microproteins could arise de novo and frequent loss of microprotein-encoding smORFs during bacterial evolution. Overall, we identified 4838 microOGs with clear signatures of de novo origin from noncoding sequences. Many of the microprotein-encoding smORFs overlap transcriptional regulatory signals or repetitive elements suggesting that the origin of microproteins is tied to selection for maintenance of regulatory sequences.

Here, we report our approach to peptidomic analysis of the plant model Physcomitrium patens. Intracellular and extracellular peptides were extracted under conditions preventing proteolytic digestion by endogenous proteases. The extracts were fractionated on size exclusion columns to isolate intracellular peptides and on reversed-phase cartridges to isolate extracellular peptides, with the isolated peptides subjected to LC-MS/MS analysis. Mass spectrometry data were analyzed for the presence of peptides derived from the known proteins or microproteins encoded by small open reading frames (<100 aa, smORFs) predicted in the moss genome. Experimental details are provided for each step.

Microproteins encoded by small open reading frames (smORFs) comprise the "dark matter" of proteomes. Although functional microproteins were identified in diverse organisms from all three domains of life, bacterial smORFs remain poorly characterized. In this comprehensive study of intergenic smORFs (ismORFs, 15-70 codons) in 5,668 bacterial genomes of the family Enterobacteriaceae, we identified 67,297 clusters of ismORFs subject to purifying selection. The ismORFs mainly code for hydrophobic, potentially transmembrane, unstructured, or minimally structured microproteins. Using AlphaFold Multimer, we predicted interactions of some of the predicted microproteins encoded by transcribed ismORFs with proteins encoded by neighboring genes, revealing the potential of microproteins to regulate the activity of various proteins, particularly, under stress. We compiled a catalog of predicted microprotein families with different levels of evidence from synteny analysis, structure prediction, and transcription and translation data. This study offers a resource for investigation of biological functions of microproteins.

— Recent studies demonstrated that short open reading frames (sORFs, <100 codons) can encode the peptides or microproteins that perform important functions in prokaryotic and eukaryotic cells. It was established that the products of sORF translation are involved in the regulation of many processes; for example, they modulate the activity of the mitochondrial respiratory chain or the activity of muscle cells in mammals. However, the identification and subsequent functional analysis of the peptides or microproteins encoded by sORF is a nontrivial task that requires the use of special approaches. The discovery of partner proteins for the studied peptide is one of the critical stages in functional analysis. This review considers the peculiarities of the analysis of the interactome of sORF-encoded microproteins and describes the approaches currently used for such studies.

BackgroundRAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress responses is unknown.Results: Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in the coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloprotease and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. The subsequent analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress conditions, implying the importance of RALFs in responding to various adverse conditions in bryophytes. We found that knockout of the PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens, Pectobacterium carotovorum and Fusarium solani, suggesting the role of these peptides in negative regulation of the immune response in P. patens. Comparing the transcriptomes of PpRALF3 KO and wild-type plants infected by F. solani showed that the regulation of genes in the phenylpropanoid pathway and those involved in cell wall modification and biogenesis was different in these two genotypes.ConclusionThus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to the ancestral functions of RALF peptides in plant stress response.

Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in controlling the temperature regulation of plant-virus interactions are poorly characterised. To elucidate these further, we analysed the responses of potato plants cv Chicago to infection by potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of which is known to significantly increase plant susceptibility to PVY. Using RNAseq analysis, we showed that single and combined PVY and heat-stress treatments caused dramatic changes in gene expression, affecting the transcription of both protein-coding and non-coding RNAs. Among the newly identified genes responsive to PVY infection, we found genes encoding enzymes involved in the catalysis of polyamine formation and poly ADP-ribosylation. We also identified a range of novel non-coding RNAs which were differentially produced in response to single or combined PVY and heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites. Finally, to gain more insights into the potential role of alternative splicing and epitranscriptomic RNA methylation during combined stress conditions, direct RNA nanopore sequencing was performed. Our findings offer insights for future studies of functional links between virus infections and transcriptome reprogramming, RNA methylation and alternative splicing.

Small open reading frames (<100 codons) that are located on long noncoding RNAs (lncRNAs) can encode functional microproteins. These microproteins are shown to play important roles in different cellular processes, such as cell proliferation, development and disease response [1], [2], [3], [4], [5], [6]. However, there are only a few known lncRNA-encoded functional microproteins in plants. One such microprotein that was named PSEP3, was identified in the moss Physcomitrium patens by mass-spectrometry analysis. 57-aa PSEP3 contains Low Complexity Region (LCR) enriched with proline. We have previously shown that PSEP3 is translated in protonemata and gametophores of P. patens, and its knockout (KO line) or overexpression (OE line) affects protonemata growth [7]. We performed a quantitative proteomic analysis of the mutant lines with PSEP3 knockout and overexpression. 7-days old protonemata of wild type (WT line) and both mutant lines (KO and OE) were collected and used for iTRAQ-based proteomic experiments. LC-MS/MS data were processed using PEAKS Studio v.8 software with protein identification based on a Phytozome protein database. More analysis of PSEP3 effects on plant growth can be obtained in the paper published in Nucleic Acid Research [8].

Nonsense-mediated RNA decay (NMD) mechanism controls the quality of eukaryotic mRNAs by degradation of aberrant transcripts with a premature stop codon (PTC) in a pioneer round of translation. Besides aberrant transcripts, up to 10% of normal mRNA transcripts can be regulated by NMD. As NMD machinery is associated with translation, this system takes part in proteome formation in eukaryotic cells [1,2]. However, no proteomic datasets of plants with deficient NMD system are currently available. Here, we provide an isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic dataset of the moss Physcomitrium patens smg1 knockout line. The kinase SMG1 is one of the key components of the NMD system in many organisms, including plants. 8-day old protonema of wild type and mutant lines was used for the iTRAQ experiment in three biological replicates. LC-MS/MS data were processed using PEAKS Studio v.8 Software with protein identification based on a Phytozome protein database. Differentially expressed protein groups up- and down-regulated in the smg1 knockout line were found in the resulting dataset. Presented data can improve our understanding of NMD functions in plants.

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen. We used spectrofluorometry and spectrophotometry to detect H2 O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, label-free quantification approach to study pollen proteome changes after H2 O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to the one provoked by H2 O2 . This effect is abolished by catalase treatment and ROS quencher MnTMPP. Inhibitory analysis indicates presumable participation of Ca2+ - and K+ -conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response we analyzed proteome alterations in H2 O2 -treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2 O2 and sensitivity of Ca2+ - and K+ -conducting channels to this ROS. Thus, H2 O2 .is one of the active substances in tobacco stigma exudate stimulating various physiological processes in germinating pollen.

Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in such regulatory effects remain largely uncharacterized. To provide more insight into the mechanisms whereby temperature regulates plant-virus interactions, we analysed changes in the proteome of potato cv. Chicago plants infected with potato virus Y (PVY) at normal (22 degrees C) and elevated temperature (28 degrees C), which is known to significantly increase plant susceptibility to the virus. One of the most intriguing findings is that the main enzymes of the methionine cycle (MTC) were down-regulated at the higher but not at normal temperatures. With good agreement, we found that higher temperature conditions triggered consistent and concerted changes in the level of MTC metabolites, suggesting that the enhanced susceptibility of potato plants to PVY at 28 degrees C may at least be partially orchestrated by the down-regulation of MTC enzymes and concomitant cycle perturbation. In line with this, foliar treatment of these plants with methionine restored accumulation of MTC metabolites and subverted the susceptibility to PVY at elevated temperature. These data are discussed in the context of the major function of the MTC in transmethylation processes.

Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of -0.15 MPa) to very high (-1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants.

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen. We used spectrofluorometry and spectrophotometry to detect H2 O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, and a label-free quantification approach to study pollen proteome changes after H2 O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2 O2 . This effect is abolished by catalase treatment and the ROS quencher, MnTMPP. Inhibitory analysis indicates probable participation of Ca2+ - and K+ -conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response, we analysed proteome alterations in H2 O2 -treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2 O2 and sensitivity of Ca2+ - and K+ -conducting channels to this ROS. Thus, H2 O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.

Plant-virus interactions are frequently influenced by elevated temperature, which often increases susceptibility to a virus, a scenario described for potato cultivar Chicago infected with potato virus Y (PVY). In contrast, other potato cultivars such as Gala may have similar resistances to PVY at both normal (22 °C) and high (28 °C) temperatures. To elucidate the mechanisms of temperature-independent antivirus resistance in potato, we analysed responses of Gala plants to PVY at different temperatures using proteomic, transcriptional and metabolic approaches. Here we show that in Gala, PVY infection generally upregulates the accumulation of major enzymes associated with the methionine cycle (MTC) independently of temperature, but that temperature (22 °C or 28 °C) may finely regulate what classes accumulate. The different sets of MTC-related enzymes that are up-regulated at 22 °C or 28 °C likely account for the significantly increased accumulation of S-adenosyl methionine (SAM), a key component of MTC which acts as a universal methyl donor in methylation reactions. In contrast to this, we found that in cultivar Chicago, SAM levels were significantly reduced which correlated with the enhanced susceptibility to PVY at high temperature. Collectively, these data suggest that MTC and its major transmethylation function determines resistance or susceptibility to PVY.

In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.

Plants utilize a plethora of peptide signals to regulate their immune response. Peptide ligands and their cognate receptors involved in immune signaling share common motifs among many species of vascular plants. However, the origin and evolution of immune peptides is still poorly understood. Here, we searched for genes encoding small secreted peptides in the genomes of three bryophyte lineages—mosses, liverworts and hornworts—that occupy a critical position in the study of land plant evolution. We found that bryophytes shared common predicted small secreted peptides (SSPs) with vascular plants. The number of SSPs is higher in the genomes of mosses than in both the liverwort Marchantia polymorpha and the hornwort Anthoceros sp . The synthetic peptide elicitors—AtPEP and StPEP—specific for vascular plants, triggered ROS production in the protonema of the moss Physcomitrella patens , suggesting the possibility of recognizing peptide ligands from angiosperms by moss receptors. Mass spectrometry analysis of the moss Physcomitrella patens , both the wild type and the Δ cerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

Pervasive transcription of eukaryotic genomes results in expression of long non-coding RNAs (lncRNAs) most of which are poorly conserved in evolution and appear to be non-functional. However, some lncRNAs have been shown to perform specific functions, in particular, transcription regulation. Thousands of small open reading frames (smORFs, <100 codons) located on lncRNAs potentially might be translated into peptides or microproteins. We report a comprehensive analysis of the conservation and evolutionary trajectories of lncRNAs-smORFs from the moss Physcomitrium patens across transcriptomes of 479 plant species. Although thousands of smORFs are subject to substantial purifying selection, the majority of the smORFs appear to be evolutionary young and could represent a major pool for functional innovation. Using nanopore RNA sequencing, we show that, on average, the transcriptional level of conserved smORFs is higher than that of non-conserved smORFs. Proteomic analysis confirmed translation of 82 novel species-specific smORFs. Numerous conserved smORFs containing low complexity regions (LCRs) or transmembrane domains were identified, the biological functions of a selected LCR-smORF were demonstrated experimentally. Thus, microproteins encoded by smORFs are a major, functionally diverse component of the plant proteome.