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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 response is unknown. Here, we used the mossPhyscomitrium patensas a model to gain insight into the role of RALF peptides in coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloproteases and some membrane proteins, including those involved in stress response, inPpRALF1, 2and3knockout (KO) lines. We found that knockout ofPpRALF2andPpRALF3genes resulted in increased resistance to bacterial and fungal phytopathogens -Pectobacterium carotovorumandFusarium solani, suggesting the role of these peptides in negative regulation of immune response inP. patens. The comparative transcriptome analysis ofPpRALF3KO and wild type plants underFusarium solaniinfection showed the clear difference in regulation of genes belonging to phenylpropanoid pathway and associated with cell wall modification and biogenesis between these genotypes. The follow-up analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress regulation, which suggests the role of RALFs in responses to different adverse conditions. Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to ancestral functions of RALF peptides in plant stress response.

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.

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 Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

Recent evidence shows that small open reading frame (smORF

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.

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

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.

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 Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

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 °C) and elevated temperature (28 °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 °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., The work describes mechanisms whereby proteomic, transcriptional, and metabolic changes associated with the methionine cycle may modulate temperature‐sensitive plant–virus interactions.

Manipulation of the nutrient concentration is an inexpensive and efficient method for increasing lipid and TAG accumulation in algal cells. However, high volumetric production requires finding a proper balance between the decrease of biomass production and the increase in the total lipid content. We isolated a strain of green microalga Bracteacoccus bullatus and increased its lipid content from 17 to 59% of biomass dry weight by manipulating of nitrogen and phosphorus content in the medium. The 10-fold reduction of the nitrogen and phosphorus concentration in the medium was the most efficient method of the lipid induction compared to nutrient deplete and high nutrient conditions. The oleic (48–64% mass of total fatty acids) and linoleic (14–24% mass of total fatty acids) acids dominated in the fatty acid profile, thus making this strain a suitable candidate for biodiesel production.

Effect of nitric oxide (NO) on phosphorylation of soluble proteins in the cell culture of wild-type Arabidopsis thaliana (L.) Heynh. (ecoptype Columbia, Col-0) was studied. Among the identified proteins whose phosphorylation was affected by the NO donor treatment, the enzymes of primary metabolism (glyceraldehyde-3-phosphate dehydrogenase, enolase) and regulatory proteins (14-3-3-like protein GF14ω, protein-disulfide isomerase-like protein, chaperonin-60α) were detected. The results clarify possible mechanisms of NO action on primary metabolism, cell cycle, and stress-induced responses of cultured plant cells.

Fucoxanthin is one of the most abundant carotenoids in nature. It is a major carotenoid in heterokont and haptophyte groups of algae that includes > 20 thousand species. Fucoxanthin also has many beneficial health effects including anticancer, antihypertensive, anti-inflammatory, and antiobesity effects. Microalgae are a promising source of fucoxanthin for commercial production. To find the most efficient fucoxanthin producing strains of microalgae, we isolated seven new strains of microalgae from multiple locations in Russia and Vietnam and studied their growth parameters and fucoxanthin content. Based on these results we isolated a novel strain of Mallomonas sp. (Synurophyceae) with the highest known content of fucoxanthin in biomass (26.6 mg g − 1 DW). The analysis of available data on heterokont and haptophyte algae shows that three groups of algae are the most promising for commercial production of fucoxanthin: diatoms (up to 21.67 mg g − 1 DW), Synurophyceae (up to 26.6 mg g − 1 DW) and Prymnesiophyceae (up to 18.23 mg g − 1 DW).

In addition to photosynthesis, chloroplasts perform a variety of important cellular functions in the plant cell, which can, for example, regulate plant responses to abiotic and biotic stress conditions. Under stress, intensive chloroplast protein remodeling and degradation can occur, releasing large numbers of endogenous peptides. These protein-derived peptides can be found intracellularly, but also in the plant secretome. Although the pathways of chloroplast protein degradation and the types of chloroplast proteases implicated in this process have received much attention, the role of the resulting peptides is less well understood. In this review we summarize the data on peptide generation processes during the remodeling of the chloroplast proteome under stress conditions and discuss the mechanisms leading to these changes. We also review the experimental evidence which supports the concept that peptides derived from chloroplast proteins can function as regulators of plant responses to (a)biotic stresses.

Genomes contain millions of short (Physcomitrella patens(moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of ten plant species examined. Mass spectrometry analysis of proteomic and peptidomic datasets suggested that 584 sORFs located on distinct parts of mRNAs and long non-coding RNAs (lncRNAs) are translated, including 73 conservative sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Alternative splicing is likely involved in the excision of translatable sORFs from such transcripts. We identified a group of sORFs homologous to known protein domains and suggested they function as small interfering peptides. Functional analysis of candidate lncRNA-encoded peptides showed it to be involved in regulating growth and differentiation in moss. The high evolutionary rate and wide translation of sORFs suggest that they may provide a reservoir of potentially active peptides and their importance as a raw material for gene evolution. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Ethylene, being one of five classical plant phytohormones is involved in regulation of numerous physiological processes. There are contradictory data about the effect of ethylene on the cell growth and division; although it is accepted that in culture flasks, the content of ethylene rises to a few tens of μL/L and production of ethylene is associated with the periods of active growth of the cells in vitro. We revealed a strong correlation (r = 0.96) between ethylene production and specific rate of dry weight accumulation in suspension cell cultures of Ajuga turkestanica, heterotrophic and mixotrophic strains of Arabidopsis thaliana, Beta vulgaris, Euonymus maximoviczianus, Medicago sativa, Panax ginseng, and Triticum timopheevii. In heterotrophic cell culture of A. thaliana, the peaks and general shape of the curves describing dynamics of ethylene production, the number of S-phase cells, and specific rate of increase in cell number coincided in log phase and in the phase of growth deceleration. Pretreatment of subculture inoculum with 100 μL/L ethylene caused doubling of S-phase cell number after 3-h-long culturing in fresh nutrient medium. It was found that exogenous ethylene affects the number of S-phase cells only when the level of endogenously produced ethylene is low.

Research performed over the last few years identified nitric oxide (NO) as an intracellular signaling molecule involved in regulation of plant physiological processes at all stages of the life cycle. Nevertheless, some extremely important aspects of NO biology are still far from being clarified. There exist different points of view on NO formation and utilization in plants. The mechanisms of perception and transduction of the NO signal are not yet fully understood, and the origin of specificity underlying coordinated activation of responses to NO remains unresolved. It is reasonable to expect that the deep knowledge of NO functioning in animals may provide some keys to these questions. Such a comparative analysis is a way to reveal similarities and emphasize the differences in the current understanding of the NO role in plants. The present lecture highlights these aspects of NO functioning.

Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5�ethynyl�2'�deoxyuridine (E dU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combi� nation with click reaction adapted for plant cells appeared, and they are help for a monitoring Speriod of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.Arabidopsis thaliana, Chlamydomonas reinhardtii, Synechocystis, Vigna radiata, cell cycle, cell cul� ture, nucleotides, Speriod, protoplasts, flow cyto metry, thymidine kinase, fluorescence microscopy

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.

To investigate the mechanisms of Ni(2+) effects on initiation and maintenance of polar cell growth, we used a well-studied model system-germination of angiosperm pollen grains. In liquid medium tobacco pollen grain forms a long tube, where the growth is restricted to the very tip. Ni(2+) did not prevent the formation of pollen tube initials, but inhibited their subsequent growth with IC(50) = 550 μM. 1 mM Ni(2+) completely blocked the polar growth, but all pollen grains remained viable, their respiration was slightly affected and ROS production did not increase. Addition of Ni(2+) after the onset of germination had a bidirectional effect on the tubes development: there was a considerable amount of extra-long tubes, which appeared to be rapidly growing, but the growth of many tubes was impaired. Studying the localization of possible targets of Ni(2+) influence, we found that they may occur both in the wall and in the cytoplasm, as confirmed by specific staining. Ni(2+) disturbed the segregation of transport vesicles in the tips of these tubes and significantly reduced the relative content of calcium in the aperture area of pollen grains, as measured by X-ray microanalysis. These factors are considered being critical for normal polar cell growth. Ni(2+) also causes the deposition of callose in the tips of the tube initials and the pollen tubes that had stopped their growth. We can assume that Ni(2+)-induced disruption of calcium homeostasis can lead to vesicle traffic impairment and abnormal callose deposition and, consequently, block the polar growth.