A vast pool of lineage-specific microproteins encoded by long non-coding RNAs in plants.

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.

Effect of Magnetic and Electric Field on the Orientation of Rare-Earth-Containing Nematics.

We report rare-earth-containing metallomesogens with newly synthesized ligands represented by the ß-diketone 1-(4-(4-propylcyclohexyl)phenyl)octane-1,3-dione (CPDk3-5) and the Lewis base 5,5'-bis(heptadecyl)-2,2'-bipyridine (bpy17-17). The stoichiometry of the complexes is [Ln(CPDk3-5)3bpy17-17], where Ln is a trivalent rare-earth ion (La, Sm, Eu, Gd, Tb, Dy, Ho, Tm, and Yb). Although the ligands themselves do not form any mesophase, the respective metal complexes produce nematic and smectic A phases. The mesogenic rare-earth complexes were characterized by NMR, MS, POM, DSC, X-ray diffraction, magnetic susceptibility measurements, and dielectric spectroscopy. The metal complexes display a remarkably large magnetic anisotropy in the mesophase. These nematic liquid crystals can, therefore, be easily aligned by an external low-threshold magnetic field.

Influence of Eu(III) Complexes Structural Anisotropy on Luminescence of Doped Conjugated Polymer Blends.

Europium(III) complexes that are the anisometric analogues of a well-known Eu(DBM)3Phen (DBM = dibenzoylmethane; Phen = 1,10-phenanthroline) complex were synthesized. Quantum-chemical calculations of the excited states of new Eu(III) complexes and some conjugated polymers showed that poly(N-vinylcarbazole) provides the most efficient energy transfer in polymer films doped with synthesized Eu(III) complexes. These new composite films were prepared, and their luminescence properties were studied. The results of experimental data confirmed theoretical prediction about significant influence introduced in molecular structure long terminal alkyl chains and cyclohexane on the luminescence efficiency of "Ln(III)-polymer" composite. As the result a substantial contribution of complex anisometry to the luminescence efficiency of composite was revealed due to the increase of the complex limit concentration before self-quenching more than twofold.

Influence of structural anisotropy on mesogenity of Eu(III) adducts and optical properties of vitrified films formed on their base.

A new series of europium adducts with the general formula Eu(CPDk3-CnH2n+1)3Phen, where CPDk3-CnH2n+1 denotes ß-diketones and Phen is 1,10-phenanthroline, was synthesized. The obtained mesogenic complexes were heated to the temperatures of the isotropic liquid state and then cooled. The complexes having short CH3 and C2H5 substituents crystallized upon cooling, and the complexes with longer substituents from C3H7 to C6H13 underwent a glass transition with the formation of optically transparent amorphous films. Inside the series the complexes with C7H15 and C8H17 substituents exhibited a unusual smectic C mesomorphism for lanthanidomesogens. On the basis of quantum-chemical simulations and the results of small-angle X-ray scattering the dependence between the anisotropy of Eu(III) complexes with various ligand environments and their supramolecular organization was found. The synthesized Eu(III) complexes in the solid state show intense red photoluminescence upon irradiation by ultraviolet light (λmax - 337 nm).

Hormonal Signaling during dPCD: Cytokinin as the Determinant of RNase-Based Self-Incompatibility in Solanaceae.

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects-petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD.

RALF peptides modulate immune response in the moss Physcomitrium patens.

Background: RAPID 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. Conclusion: Thus, 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.

Quantitative proteomic dataset of the moss Physcomitrium patens PSEP3 KO and OE mutant lines.

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].

Transcriptomic Reprogramming, Alternative Splicing and RNA Methylation in Potato (Solanum tuberosum L.) Plants in Response to Potato Virus Y Infection.

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.

Direct RNA sequencing dataset of SMG1 KO mutant Physcomitrella (Physcomitrium patens).

Nonsense-mediated mRNA decay (NMD) is a system that controls the quality of mRNA transcripts in eukaryotes by degradation of aberrant transcripts in a pioneer round of translation. In mammals, NMD targets one-third of mutated, disease-causing mRNAs and ∼10% of unmutated mRNAs, facilitating appropriate cellular responses to environmental changes [1]. In plants, NMD plays an important role in development and regulating abiotic and biotic stress responses [2]. The transcripts with premature termination codons (PTCs), upstream ORFs or long 3'-UTRs can be targeted to NMD. It was shown that alternative splicing plays a crucial role in regulation of NMD triggering, for example, by the introduction of a PTC in transcripts. Therefore, the correct identification of mRNA isoforms is a key step in the study of the principles of regulation of the cell transcriptome by the NMD pathway. Here, we performed long-read sequencing of Physcomitrella (Physcomitrium patens) mutant smg1Δ line 2 native transcriptome by Oxford Nanopore Technology (ONT). The smg1Δ is a knockout (KO) mutant deficient in SMG1 kinase is a key component of NMD system in plants and animals [3]. RNA was isolated with Trizol from 5 day old protonemata and sequenced using kit SQK-RNA002, flow cells FLO-MIN106 and a MinION device (Oxford Nanopore Technologies Ltd., UK (ONT)) in three biological repeats. Basecalling was performed with Guppy v.4.0.15. The presented transcriptomes give advantages in the identification and functional characterization of RNA transcripts that are direct targets of the Nonsense-mediated mRNA decay system.

Optical and structural characteristics of PMMA films doped with a new anisometric EuIII complex.

A new film material capable of transforming UV radiation into visible light was obtained from a highly anisometric EuIII complex with organic ligands in a polymethylmethacrylate (PMMA) matrix and then structurally characterized. An important advantage of the synthesized complex is its good solubility in organic solvents such as dichloromethane, chloroform, THF, toluene, etc. The ligand environment (flexible alkyl and cyclohexyl substituents) of the EuIII complex was selected to prevent crystallization, to inhibit the formation of defects in the structure of films and to provide its uniform distribution in the polymer during polymerization. As a result we obtain an EuIII complex of the film with remarkable thermal behavior: the complex melts to isotropic liquid without decomposition, it supercools at ambient temperature and it forms a stable amorphous material at low (up to -30°C) temperatures. The films were prepared by two methods: bulk polymerization and spin coating. A comparison of the differences of luminescent and optical characteristics of micro- and nanosized PMMA films doped with the anisometric EuIII complex is given. Based on X-ray powder diffraction and small-angle scattering data, it has been supposed that the association of EuIII complex molecules occurs in the voids of the PMMA matrix and is accompanied by the formation of a nanocrystalline phase. For films obtained by spin coating, a deeper microphase separation is demonstrated than by bulk polymerization. The dimensional characteristics of the nano-associates were determined and a correlation between the method of preparation and the type of distribution of the EuIII complex in the PMMA matrix is established.

Pilot satellitome analysis of the model plant, Physcomitrellapatens, revealed a transcribed and high-copy IGS related tandem repeat.

Satellite DNA (satDNA) constitutes a substantial part of eukaryotic genomes. In the last decade, it has been shown that satDNA is not an inert part of the genome and its function extends beyond the nuclear membrane. However, the number of model plant species suitable for studying the novel horizons of satDNA functionality is low. Here, we explored the satellitome of the model "basal" plant, Physcomitrellapatens (Hedwig, 1801) Bruch & Schimper, 1849 (moss), which has a number of advantages for deep functional and evolutionary research. Using a newly developed pyTanFinder pipeline (https://github.com/Kirovez/pyTanFinder) coupled with fluorescence in situ hybridization (FISH), we identified five high copy number tandem repeats (TRs) occupying a long DNA array in the moss genome. The nuclear organization study revealed that two TRs had distinct locations in the moss genome, concentrating in the heterochromatin and knob-rDNA like chromatin bodies. Further genomic, epigenetic and transcriptomic analysis showed that one TR, named PpNATR76, was located in the intergenic spacer (IGS) region and transcribed into long non-coding RNAs (lncRNAs). Several specific features of PpNATR76 lncRNAs make them very similar with the recently discovered human lncRNAs, raising a number of questions for future studies. This work provides new resources for functional studies of satellitome in plants using the model organism P.patens, and describes a list of tandem repeats for further analysis.

The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation.

Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level.