AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading.

RNA interference (RNAi) is mediated by small, 20-24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs via the action of ARGONAUTE (AGO) proteins. High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of cytoplasmic sRNAs, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Expression of selected miRNAs in transient and transgenic systems validated their altered loading abilities implying that this process is controlled by information associated with the diverse miRNA precursors. We also showed that the availability of AGO proteins is a limiting factor determining the loading efficiency of miRNAs. Our data reveal the existence of a regulatory checkpoint determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.

Suppression of NB-LRR genes by miRNAs promotes nitrogen-fixing nodule development in Medicago truncatula.

Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen-associated molecular patterns and the resistance genes with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB-LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB-LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen-fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB-LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB-LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB-LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N-fixing nodule.

Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants.

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis.

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB(Ser86Asp) Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light-induced nuclear import and interaction of phyB(Ser86Asp)-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

The circadian clock-associated small GTPase LIGHT INSENSITIVE PERIOD1 suppresses light-controlled endoreplication and affects tolerance to salt stress in Arabidopsis.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycle. The small GTPase LIGHT INSENSITIVE PERIOD1 (LIP1) is a circadian clock-associated protein that regulates light input to the clock. In the absence of LIP1, the effect of light on free-running period length is much reduced. Here, we show that in addition to suppressing red and blue light-mediated photomorphogenesis, LIP1 is also required for light-controlled inhibition of endoreplication and tolerance to salt stress in Arabidopsis (Arabidopsis thaliana). We demonstrate that in the processes of endoreplication and photomorphogenesis, LIP1 acts downstream of the red and blue light photoreceptors phytochrome B and cryptochromes. Manipulation of the subcellular distribution of LIP1 revealed that the circadian function of LIP1 requires nuclear localization of the protein. Our data collectively suggest that LIP1 influences several signaling cascades and that its role in the entrainment of the circadian clock is independent from the other pleiotropic effects. Since these functions of LIP1 are important for the early stages of development or under conditions normally experienced by germinating seedlings, we suggest that LIP1 is a regulator of seedling establishment.

A reduced-function allele reveals that EARLY FLOWERING3 repressive action on the circadian clock is modulated by phytochrome signals in Arabidopsis.

Arabidopsis thaliana EARLY FLOWERING3 (ELF3) is essential for the generation of circadian rhythms. ELF3 has been proposed to restrict light signals to the oscillator through phytochrome photoreceptors, but that has not been explicitly shown. Furthermore, the genetic action of ELF3 within the clock had remained elusive. Here, we report a functional characterization of ELF3 through the analysis of the elf3-12 allele, which encodes an amino acid replacement in a conserved domain. Circadian oscillations persisted, and unlike elf3 null alleles, elf3-12 resulted in a short circadian period only under ambient light. The period shortening effect of elf3-12 was enhanced by the overexpression of phytochromes phyA and phyB. We found that elf3-12 was only modestly perturbed in resetting of the oscillator and in gating light-regulated gene expression. Furthermore, elf3-12 essentially displayed wild-type development. We identified targets of ELF3 transcriptional repression in the oscillator, highlighting the action at the morning gene PSEUDO-RESPONSE REGULATOR9. Taken together, we identified two separable roles for ELF3, one affecting the circadian network and the other affecting light input to the oscillator. This is consistent with a dual function of ELF3 as both an integrator of phytochrome signals and a repressor component of the core oscillator.

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Partners in time: EARLY BIRD associates with ZEITLUPE and regulates the speed of the Arabidopsis clock.

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.

Ecotype-specific blockage of tasiARF production by two different RNA viruses in Arabidopsis.

Arabidopsis thaliana is one of the most studied model organisms of plant biology with hundreds of geographical variants called ecotypes. One might expect that this enormous genetic variety could result in differential response to pathogens. Indeed, we observed previously that the Bur ecotype develops much more severe symptoms (upward curling leaves and wavy leaf margins) upon infection with two positive-strand RNA viruses of different families (turnip vein-clearing virus, TVCV, and turnip mosaic virus, TuMV). To find the genes potentially responsible for the ecotype-specific response, we performed a differential expression analysis of the mRNA and sRNA pools of TVCV and TuMV-infected Bur and Col plants along with the corresponding mock controls. We focused on the genes and sRNAs that showed an induced or reduced expression selectively in the Bur virus samples in both virus series. We found that the two ecotypes respond to the viral infection differently, yet both viruses selectively block the production of the TAS3-derived small RNA specimen called tasiARF only in the virus-infected Bur plants. The tasiARF normally forms a gradient through the adaxial and abaxial parts of the leaf (being more abundant in the adaxial part) and post-transcriptionally regulates ARF4, a major leaf polarity determinant in plants. The lack of tasiARF-mediated silencing could lead to an ectopically expressed ARF4 in the adaxial part of the leaf where the misregulation of auxin-dependent signaling would result in an irregular growth of the leaf blade manifesting as upward curling leaf and wavy leaf margin. QTL mapping using Recombinant Inbred Lines (RILs) suggests that the observed symptoms are the result of a multigenic interaction that allows the symptoms to develop only in the Bur ecotype. The particular nature of genetic differences leading to the ecotype-specific symptoms remains obscure and needs further study.

Arabidopsis thaliana circadian clock is regulated by the small GTPase LIP1.

BACKGROUND: At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS: The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS: LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Genome-Wide Identification of RNA Silencing-Related Genes and Their Expressional Analysis in Response to Heat Stress in Barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is an economically important crop cultivated in temperate climates all over the world. Adverse environmental factors negatively affect its survival and productivity. RNA silencing is a conserved pathway involved in the regulation of growth, development and stress responses. The key components of RNA silencing are the Dicer-like proteins (DCLs), Argonautes (AGOs) and RNA-dependent RNA polymerases (RDRs). Despite its economic importance, there is no available comprehensive report on barley RNA silencing machinery and its regulation. In this study, we in silico identified five DCL (HvDCL), eleven AGO (HvAGO) and seven RDR (HvRDR) genes in the barley genome. Genomic localization, phylogenetic analysis, domain organization and functional/catalytic motif identification were also performed. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to moderate, persistent or gradient heat stress treatments: transcriptional accumulation of siRNA- but not miRNA-based silencing factor was consistently detected. These results suggest that RNA silencing is dynamically regulated and may be involved in the coordination of development and environmental adaptation in barley. In summary, our work provides information about barley RNA silencing components and will be a ground for the selection of candidate factors and in-depth functional/mechanistic analyses.

Detection of Phytochrome Phosphorylation in Plants.

Posttranslational modification (PTM) of proteins occurs during or after translation and in most cases means covalent binding of a functional group to certain amino acid side chains. Among PTMs, phosphorylation is extensively studied for decades. During phosphorylation, a phosphate group is added to the target residue that is dominantly serine, threonine, and tyrosine in eukaryotes. The phosphate group attachment is catalyzed by kinases, whereas the removal of phosphate (dephosphorylation) is performed by phosphatases. Phosphorylation of phytochrome photoreceptors alters light signaling in multiple ways, thus the examination of this PTM is an expanding aspect of light signaling research. Although this chapter presents methods for detecting phosphorylated phytochrome B molecules, it can be applied on other phytochrome species. The first presented protocol of this chapter shows how the phosphorylation state of phytochrome photoreceptors can be monitored in a modified polyacrylamide gel electrophoresis system. The second protocol describes in detail how phosphorylated amino acids of a target molecule can be identified using mass spectrometry analysis.

Draft Genome Sequence of Mycolicibacterium sp. Strain CH28, a Potential Degrader of Diisopropyl Ether, Isolated from Pharmaceutical Wastewater.

Mycolicibacterium sp. strain CH28 is a novel bacterial isolate belonging to a group of rapidly growing mycobacteria. Here, we report the draft genome sequence of strain CH28 and provide insights into the genetic background of its potential diisopropyl ether-degrading capability.

Transcriptome reprogramming in the shoot apical meristem of CymRSV-infected Nicotiana benthamiana plants associates with viral exclusion and the lack of recovery.

In some plant-virus interactions plants show a sign of healing from virus infection, a phenomenon called symptom recovery. It is assumed that the meristem exclusion of the virus is essential to this process. The discovery of RNA silencing provided a possible mechanism to explain meristem exclusion and recovery. Here we show evidence that silencing is not the reason for meristem exclusion in Nicotiana benthamiana plants infected with Cymbidium ringspot virus (CymRSV). Transcriptome analysis followed by in situ hybridization shed light on the changes in gene expression in the shoot apical meristem (SAM) on virus infection. We observed the down-regulation of meristem-specific genes, including WUSCHEL (WUS). However, WUS was not down-regulated in the SAM of plants infected with meristem-invading viruses such as turnip vein-clearing virus (TVCV) and cucumber mosaic virus (CMV). Moreover, there is no connection between loss of meristem function and fast shoot necrosis since TVCV necrotized the shoot while CMV did not. Our findings suggest that the observed transcriptional changes on virus infection in the shoot are key factors in tip necrosis and symptom recovery. We observed a lack of GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) expression in tissues around the meristem, which likely stops virus replication and spread into the meristem.

Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary.

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.

Oncological hyperthermia: The correct dosing in clinical applications.

The problem with the application of conventional hyperthermia in oncology is firmly connected to the dose definition, which conventionally uses the concept of the homogeneous (isothermal) temperature of the target. Its imprecise control and complex evaluation is the primary barrier to the extensive clinical applications. The aim of this study was to show the basis of the problems of the misleading dose concept. A clear clarification of the proper dose concept must begin with the description of the limitations of the present doses in conventional hyperthermia applications. The surmounting of the limits the dose of oncologic hyperthermia has to be based on the applicability of the Eyring transition state theory on thermal effects. In order to avoid the countereffects of thermal homeostasis, the use of precise heating on the nanoscale with highly efficient energy delivery is recommended. The nano­scale heating allows for an energy­based dose to control the process. The main aspects of the method are the following: i) It is not isothermal (no homogeneous heating); ii) malignant cells are heated selectively; and iii) it employs high heating efficacy, with less energy loss. The applied rigorous thermodynamical considerations show the proper terminology and dose concept of hyperthermia, which is based on the energy­absorption (such as in the case of ionizing radiation) instead of the temperature­based ideas. On the whole, according to the present study, the appropriate dose in oncological hyperthermia must use an energy­based concept, as it is well­known in all the ionizing radiation therapies. We propose the use of Gy (J/kg) in cases of non­ionizing radiation (hyperthermia) as well.

Expansion of Capsicum annuum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important transcriptional and post-transcriptional regulators controlling a wide variety of physiological processes including fruit development. Data are, however, limited for their potential roles in developmental processes determining economically important traits of crops. The current study aimed to discover and characterize differentially expressed miRNAs and siRNAs in sweet pepper (Capsicum annuum) during fruit expansion. High-throughput sequencing was employed to determine the small regulatory RNA expression profiles in various fruit tissues, such as placenta, seed, and flesh at 28 and 40 days after anthesis. Comparative differential expression analyses of conserved, already described and our newly predicted pepper-specific miRNAs revealed that fruit expansion is accompanied by an increasing level of miRNA-mediated regulation of gene expression. Accordingly, ARGONAUTE1 protein, the primary executor of miRNA-mediated regulation, continuously accumulated to an extremely high level in the flesh. We also identified numerous pepper-specific, heterochromatin-associated 24-nt siRNAs (hetsiRNAs) which were extremely abundant in the seeds, as well as 21-nt and 24-nt phased siRNAs (phasiRNAs) that were expressed mainly in the placenta and the seeds. This work provides comprehensive tissue-specific miRNA and siRNA expression landscape for a developing pepper fruit. We identified several novel, abundantly expressing tissue- and pepper-specific small regulatory RNA species. Our data show that fruit expansion is associated with extensive changes in sRNA abundance, raising the possibility that manipulation of sRNA pathways may be employed to improve the quality and quantity of the pepper fruit.

Correction: Expansion of Capsicum annum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

[This corrects the article DOI: 10.1371/journal.pone.0200207.].

An allometric approach of tumor-angiogenesis.

Angiogenesis is one of the main supporting factors of tumor-progression. It is a complex set of interactions together with hypoxia and inflammation, regulating tumor growth. The objective of this study is to examine the effect of angiogenesis with an allometric approach applied to angiogenesis and the regulating factors. The results show that allometry has the potential to describe this aspect, including the sigmoid-like transport function. There are particular conditions under which the complex control maximizes the relative tumor mass. Linear growth of malignancy diameter with an allometric approach was proven.

Light perception and signalling in higher plants.

Plants monitor changes in the ambient light environment using sensory photoreceptor families: the phototropins and cryptochromes, which absorb UV-A or blue light; the phytochromes, which sense red/far-red light; and the UV-B photoreceptors, which have not yet been identified. Recent advances suggest that photoreceptor-induced signalling cascades regulate light-modulated gene expression. These regulatory networks interact at the levels of transcription, posttranslational modification and nucleo-cytoplasmic compartmentalisation.