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Plants have acquired sets of highly regulated and complex signaling pathways to respond to unfavorable environmental conditions during evolution. Calcium signaling, as a vital mechanism, enables plants to respond to external stimuli, including abiotic and biotic stresses, and coordinate the basic processes of growth and development. In the present study, two calcium sensor families, CBL and CIPK, were investigated in a halophyte plant, Aeluropus littoralis, with a comprehensive analysis. Here, six AlCBL genes, and twenty AlCIPK genes were studied. The analysis of the gene structure and conserved motifs, as well as physicochemical properties, showed that these genes are highly conserved during evolution. The expression levels of AlCBL genes and AlCIPK genes were evaluated under salt stress in leaf and root tissue. Based on the real-time RT-PCR results, the AlCIPK gene family had a higher variation in mRNA abundance than the AlCBL gene family. AlCIPK genes were found to have a higher abundance in leaves than in roots. The results suggest that the correlation between AlCBL genes and AlCIPK is tissue-specific, and different correlations can be expected in leaves and roots. Based on these correlations, AlCIPK3.1–AlCBL4.1 and AlCIPK1.2–AlCBL4.4 can be co-expressed in the root tissue, while AlCBL10 has the potential to be co-expressed with AlCIPK5, AlCIPK26, and AlCIPK12.3 in the leaf tissue. Our findings reveal valuable information on the structure and function of calcium sensor families in A. littoralis, a halophyte plant, that can be used in future research on the biological function of CBLs and CIPKs on salt stress resistance.

Plants have acquired sets of highly regulated and complex signaling pathways to respond to unfavorable environmental conditions during evolution. Calcium signaling, as a vital mechanism, enables plants to respond to external stimuli, including abiotic and biotic stresses, and coordinate the basic processes of the growth and development. In the present study, the calcium sensor families, including CBL and CIPK, were investigated in the halophyte plant, Aeluropus littoralis, with a comprehensive analysis. Here, six AlCBL genes, and twenty AlCIPK genes were presented. The analysis of the gene structure and conserved motifs, as well as physicochemical properties, showed that these genes are highly conserved during evolution. The expression levels of AlCBL genes and AlCIPK genes were evaluated under salt stress in leaf and root tissue. Based on the real-time RT-PCR results, AlCIPK gene family had a higher variation in mRNA abundance compared to the AlCBL gene family. The AlCIPKs were found to have a higher abundance in leaves than in roots. The results suggest that the interaction pattern of AlCBL genes with AlCIPK is tissue-specific, and different interactions can be expected in leaves and roots. Based on these patterns AlCIPK3.1 - AlCBL4.1 and AlCIPK1.2 - AlCBL4.4 can interact in root tissue, while the AlCBL10 has the potential to interact with the AlCIPK5, AlCIPK26 and AlCIPK12.3 in the leaf tissue. These findings provide valuable information on the structure and function of calcium sensor families in Aeluropus littoralis a halophyte plant, for future research on the biological function of CBLs and CIPKs on salt stress resistance.

Background: Rhizoctonia solani AG1 IA is an important pathogen of rice (Oryza sativa L.) that causes rice sheath blight (RSB). Since control of RSB by conventional measures has failed, novel strategies like application of plant growth-promoting rhizobacteria (PGPR) can be an efficient alternative. Method and Results: mRNA sequences of rice were retrieving from NCBI for candidate reference genes selction, and seven candidate reference genes (RGs), namely 18SrRNA, ACT1, GAPDH2, UBC5, RPS27, eIF4aand CYP28, were selected for their stability in real-time quantitative PCR (RT-qPCR). Different algorithms were exploited, Delta Ct, geNorm, NormFinder, BestKeeper, and Comprehensive ranking by RefFinder, to evaluate RT-qPCR of rice in tissues infected with R. solani and treated with the PGPR strains, Pseudomonas saponiphilia and Pseudomonas protegens, and potassium silicate (KSi) alone or in combination with each PGPR strain. RGs stability was affected by each treatment and treatment-specific selection was approved and validated for nonexpressor of PR-1(NPR1) for each treatment. Conclusion: Overall, ACT1 was the most stable RG with R. solani infection alone, GAPDH2 with R. solani infection plus KSi, UBC5 with R. solani infection plus P. saponiphilia, and eIF4a with R. solani infection plus P. protegens. Both ACT1 and RPS27 were the most stable with the combination of KSi and P. saponiphilia, while PRS27 was the most stable with the combination of KSi and P. protegens

Illuminating the mechanisms of inflorescence architecture of grain crops that feed our world may strengthen the goal towards sustainable agriculture. Lateral spikelet development of barley (Hordeum vulgare L.) is such an example of a floral architectural trait regulated by VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1, syn. HvHOX1). Its lateral spikelet-specific expression and the quantitative nature of suppressing spikelet development were previously shown in barley. However, the mechanistic function of this gene and its paralog HvHOX2 on spikelet development is still fragmentary.Here, we show that these duplicated transcription factors (TFs) have contrasting nucleotide diversity in various barley genotypes and several Hordeum species. Despite this difference, both proteins retain their basic properties of the homeodomain leucine zipper class I family of TFs. During spikelet development, these genes exhibit similar spatiotemporal expression patterns yet with anticyclic expression levels. A gene co-expression network analysis suggested that both have an ancestral relationship but their functions appear antagonistic to each other, i.e., HvHOX1 suppresses whereas HvHOX2 rather promotes spikelet development. Our transgenic promoter-swap analysis showed that HvHOX2 can restore suppressed lateral spikelets when expression levels are increased; however, at its low endogenous expression level, HvHOX2 appears dispensable for spikelet development. Collectively, this study proposes that the dosage of the two antagonistic TFs, HvHOX1 and HvHOX2, influence spikelet development in barley.

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3′-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.

Background: The use of wild plant species or their halophytic relatives has been considered in plant breeding programs to improve salt and drought tolerance in crop plants. Aeluropus littoralis serves as halophyte model for identification and isolation of novel stress adaptation genes. This species is described as perennial monocot grass. A. littoralis grows in damp or arid areas, often salt-impregnated places and waste land in cultivated areas. A. littoralis can survive where the water salinity is periodically high and tolerate high salt concentrations in the soil up to 1100 mM sodium chloride. Therefore, it serves as valuable genetic resource to understand molecular mechanisms of stress-responses in monocots. The knowledge can potentially be used for improving tolerance to abiotic stresses in economically important crops. Several morphological, anatomical, ecological, and physiological traits of A. littoralis have been investigated so far and also the transfer of stress related genes to other species resulted in enhanced stress resistance. After watering with salt water the grass is able to excrete salt via its salt glands. Meanwhile, a number of ESTs (expressed sequence tag), genes and promoters induced by the salt and drought stresses were isolated, sequenced and annotated at a molecular level. Results: Here we describe the genome sequence and structure of A. littoralis analyzed by whole genome sequencing and histological analysis. The chromosome number was determined to be 20 (2n = 2X = 20), absence of B chromsomes shown, and the genome size calculated to be 354 Megabasepairs.Conclusions: This genomic information provided here, will support the functional investigation and application of novel genes improving salt stress resistance in crop plants.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

Summary Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome‐wide association study (GWAS) with a transcriptome‐wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re‐sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate‐to‐high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate‐to‐high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis‐acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wx a) explained the intermediate‐to‐high GI variation. A SNP (C˃T) at exon‐10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI . The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non‐starch polysaccharide pathway in lowering GI.

Summary Plant‐specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY‐YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators.Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana.Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET‐specific differentially expressed genes and mostly independently characteristic, ET‐specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects.The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1 Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA.

Nitrogen (N) is a macronutrient determining crop yield. The application of N fertilisers can substantially increase the yield, but excess use also causes the nitrate pollution of water resources and increases production costs. Increasing N use efficiency (NUE) in crop plants is an important step to implement low-input agricultural systems. We used Arabidopsis thaliana as model system to investigate the natural genetic diversity in traits related to NUE. Natural variation was used to study adaptive growth patterns and changes in gene expression associated with limited nitrate availability. A genome-wide association study revealed an association of eight SNP markers on Chromosome 1 with shoot growth under limited N. The identified linkage disequilibrium (LD) interval includes the DNA sequences of three cysteine/histidine-rich C1 domain proteins in tandem orientation. These genes differ in promoter structure, methylation pattern and expression level among accessions, correlating with growth performance under N deficiency. Our results suggest the involvement of epigenetic regulation in the expression of NUE-related traits.

Transcriptome analysis reflects the status quo of transcribed genetic code present in the form of mRNA, which helps to infer biological processes and unravel metabolic status. Despite the increasing adoption of RNA-Seq technique in recent years, transcriptome analysis using the microarray platform remains the gold standard technique, which offers a simpler, more cost-effective, and efficient method for high-throughput gene expression profiling. In this chapter, we described a streamlined transcriptomic analyses pipeline employed to study developing rice grains that can also be applied to other tissue samples and species. We described a novel RNA extraction method that obviates the problem introduced by high-starch content during rice grain development that usually leads to reduction in RNA yield and quality. The detailed procedure of microarray analysis involved in cDNA synthesis, cRNA labeling, microarray hybridization, slide scanning, feature extraction to QC validation has been described. The description of a newly developed Indica- and Japonica-specific microarray slides developed from the genome information of subpopulation to study gene expression of 60,000 genes has been highlighted. The downstream bioinformatics analyses including expression QTL mapping and gene regulatory network analyses were mentioned.

DNA methylation is an important biomarker for gene activity. It contributes to gene silencing and is involved in regulating various seed developmental processes in plants. Many of these processes are involved in important traits associated with aspects of grain quality. A reliable, fast, and cheap method is the estimation of DNA methylation utilizing methylation sensitive restriction enzymes (MSRE) and quantitative real-time PCR (qPCR) for selected candidate regions. The presented method can be used to confirm an effect of RNAi constructs on their target genes or trans-activity. Analysis of promoter regions can contribute to estimation of gene activity and related traits.

DNA methylation is an important biomarker for gene activity. It contributes to gene silencing and is involved in regulating various seed developmental processes in plants. Many of these processes are involved in important traits associated with aspects of grain quality. A reliable, fast, and cheap method is the estimation of DNA methylation utilizing methylation sensitive restriction enzymes (MSRE) and quantitative real-time PCR (qPCR) for selected candidate regions. The presented method can be used to confirm an effect of RNAi constructs on their target genes or trans-activity. Analysis of promoter regions can contribute to estimation of gene activity and related traits.

Transcriptome analysis reflects the status quo of transcribed genetic code present in the form of mRNA, which helps to infer biological processes and unravel metabolic status. Despite the increasing adoption of RNA-Seq technique in recent years, transcriptome analysis using the microarray platform remains the gold standard technique, which offers a simpler, more cost-effective, and efficient method for high-throughput gene expression profiling. In this chapter, we described a streamlined transcriptomic analyses pipeline employed to study developing rice grains that can also be applied to other tissue samples and species. We described a novel RNA extraction method that obviates the problem introduced by high-starch content during rice grain development that usually leads to reduction in RNA yield and quality. The detailed procedure of microarray analysis involved in cDNA synthesis, cRNA labeling, microarray hybridization, slide scanning, feature extraction to QC validation has been described. The description of a newly developed Indica- and Japonica-specific microarray slides developed from the genome information of subpopulation to study gene expression of 60,000 genes has been highlighted. The downstream bioinformatics analyses including expression QTL mapping and gene regulatory network analyses were mentioned.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species.

Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8’-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

Terminal drought stress during grain filling is the major abiotic factor that limits crop yield in barley. The mother plant acclimates to the environment and perceives signals that result in a change of the physiological state within the grain and therefore affect the seed development and germination of the next generation. Small regulatory RNAs have been described to be involved in plant drought stress response by suppressing the respective target genes. Based on their origin and function, these small RNAs are classified as micro RNAs (miRNA), short interfering RNAs (siRNA) or heterochromatic small interfering RNA (hc-siRNA). In addition, 24mer sized hc-siRNAs are associated with RNA directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). The analysis of hc-siRNA by small RNA sequencing in barley caryopses after imposition of terminal drought stress allowed the identification of stress specific 24mers. Based on the sequence homology of the siRNAs to the promoter region of CYTOKININ-OXIDASE 2.1 (HvCKX2.1), this putative target gene was selected for further investigation. Terminal drought stress leads to an increased level of DNA methylation at the HvCKX2.1 promoter and the seeds derived from drought stressed plants showed faster shoot emergence. Accumulation of cytokinin ribosides, which are the known substrates of cytokinin-oxidase, can explain the observed phenotype of faster shoot emergence from seeds of drought stressed mother plants. Analysis of transgenic plants with modulated levels of abscisic acid (ABA) in the grain confirmed the ABA/drought stress responsive ProHvCKX2.1 methylation and correlation with shoot emergence speed.

Exploring genes with impact on yield-related phenotypes is the preceding step to accomplishing crop improvements while facing a growing world population. A genome-wide association scan on leaf blade area (LA) in a worldwide spring barley collection (Hordeum vulgare L.), including 125 two- and 93 six-rowed accessions, identified a gene encoding the homeobox transcription factor, Six-rowed spike 1 (VRS1). VRS1 was previously described as a key domestication gene affecting spike development. Its mutation converts two-rowed (wild-type VRS1, only central fertile spikelets) into six-rowed spikes (mutant vrs1, fully developed fertile central and lateral spikelets). Phenotypic analyses of mutant and wild-type leaves revealed that mutants had an increased leaf width with more longitudinal veins. The observed significant increase of LA and leaf nitrogen (%) during pre-anthesis development in vrs1 mutants also implies a link between wider leaf and grain number, which was validated from the association of vrs1 locus with wider leaf and grain number. Histological and gene expression analyses indicated that VRS1 might influence the size of leaf primordia by affecting cell proliferation of leaf primordial cells. This finding was supported by the transcriptome analysis of mutant and wild-type leaf primordia where in the mutant transcriptional activation of genes related to cell proliferation was detectable. Here we show that VRS1 has an independent role on barley leaf development which might influence the grain number.

In plants, RNA-directed DNA methylation (RdDM) and related transcriptional gene silencing (TGS) involve members of the suppressor of variegation 3-9-homologous (SUVH) group of putative histone methyltransferases. Utilizing a reverse genetic approach in Arabidopsis thaliana, we demonstrate that two closely related SUVH members, SUVH2 and SUVH9, act partially non-redundant in RdDM. DNA methylation, transcript accumulation and association with histone modifications were analyzed at the endogenous RdDM target AtSN1 (a SINE-like retroelement) in suvh2 and suvh9 single as well as suvh2suvh9 double mutants. SUVH2 was found to be required for full DNA methylation at AtSN1 in early seed development and was also higher expressed in seeds than at later developmental stages. SUVH9 had its impact on RdDM later during vegetative development of the plant and was also higher expressed during that stage than at earlier developmental stages. The strongest reduction of RdDM at AtSN1 was found in suvh2suvh9 double mutant plants. Histone 3-lysine 9-dimethylation (H3K9me2) associated with AtSN1 was reduced only in the simultaneous absence of functional SUVH2 and SUVH9. Thus, SUVH2 and SUVH9 functions in RdDM and TGS are overlapping in spite of some developmental specialization. Pol V specific transcripts were reduced in suvh2suvh9 plants. This might indicate a role of these SUVH proteins in Pol V complex recruitment. Electronic supplementary material The online version of this article (doi:10.1007/s11103-012-9934-x) contains supplementary material, which is available to authorized users.

In plants, a particular class of small non-coding RNAs, short interfering RNAs, can serve as a signal to induce cytosine methylation at homologous genomic DNA regions in the nucleus. If the targeted DNA regions have promoter function, this RNA-directed DNA methylation (RdDM) can result in transcriptional gene silencing (TGS). RNA-directed transcriptional gene silencing of transgenes provides a versatile system for the study of epigenetic gene regulation in plants. In our experimental setup in Arabidopsis thaliana, transcription of a promoter-inverted repeat provides a RNA signal that triggers de novo cytosine methylation and TGS of a homologous nopaline synthase promoter (proNOS) in trans. Utilising this two component transgene system in a forward-genetic screen for “suppressor of silencing” mutations, we were able to identify new candidates for factors involved in RdDM of transgenic as well as endogenous target regions.

Fluorescent chromatin tagging by the lacO operator/lac repressor system in Arabidopsis thaliana is useful to trace distinct chromatin domains in living cells. Nevertheless, the tandem repeats of the tagging system may alter the spatial organisation of chromatin within nuclei by increasing homologous pairing as well as association with heterochromatin. Efficient homologous pairing occurs if lacO repeat arrays of ∼10 kb are present at two loci, either on the same chromosome or on different chromosomes. DNA hypomethylation of lacO repeats results in reduced homologous pairing. Because, in plants, DNA methylation can serve as a signal for H3-lysine9-dimethylation (H3K9me2), and subsequently for non-CG-context DNA methylation, SET-domain histone methyltransferase and chromodomain dna methyltransferase 3 (cmt3) mutations were introgressed. In suvh4 suvh5 suvh6 and cmt3 mutants, H3K9me2 associated with lacO repeats is diminished, but homologous pairing persists. Thus, neither H3K9me2 nor CMT3-mediated non-CG methylation are required at wild-type level for homologous pairing of lacO repeat loci.

Aside from post-translational histone modifications and small RNA populations, the epigenome of an organism is defined by the level and spectrum of DNA methylation. Methyl groups can be covalently bound to the carbon-5 of cytosines or the carbon-6 of adenine bases. DNA methylation can be found in both prokaryotes and eukaryotes. In the latter, dynamic variation is shown across species, along development, and by cell type. DNA methylation usually leads to a lower binding affinity of DNA-interacting proteins and often results in a lower expression rate of the subsequent genome region, a process also referred to as transcriptional gene silencing. We give an overview of the current state of research facilitating the planning and implementation of whole-genome bisulfite-sequencing (WGBS) experiments. We refrain from discussing alternative methods for DNA methylation analysis, such as reduced representation bisulfite sequencing (rrBS) and methylated DNA immunoprecipitation sequencing (MeDIPSeq), which have value in specific experimental contexts but are generally disadvantageous compared to WGBS.

Repression of ABA degradation in transgenic barley alters the expression of genes involved in strigolactone biosynthesis, revealing cross-talk between ABA and strigolactone that affects the development of tillers., Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8’-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

Transcription of a nopaline synthase promoter (pNOS) inverted repeat provides an RNA signal that can trigger transcriptional gene silencing and methylation of pNOS promoters in trans. The degree of silencing is influenced by the local DNA features close to the target promoter integration sites. Among 26 transgenic Arabidopsis thaliana lines harbouring single copies of a T-DNA including a pNOS-NPTII reporter gene at different chromosomal loci, NPTII RNA levels showed limited variation. When challenged by the silencer transgene providing the pNOS RNA signal, reduction of the NPTII RNA levels in the F(1) generation varied by more than 100-fold, ranging from no reduction to reduction to

Background The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis—halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted. Results Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae. Conclusions Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A.littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed. Electronic supplementary material The online version of this article (doi:10.1186/s40709-016-0053-8) contains supplementary material, which is available to authorized users.

Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nucleotide long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing, while 24-nucleotide long siRNAs are involved in transcriptional gene silencing in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced post-transcriptional gene silencing. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C.elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in RGRs depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth.

Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function.

We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the ‘dicer-related-helicase’ from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF− cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF− cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.

„RNA Interferenz” steht seit 1998 fur einen vollig neuen Mechanismus des „Gene Silencing”, in dem mRNAs uber einen komplexen Mechanismus mit Hilfe doppelstrangiger RNA degradiert werden. Das schnell wachsende Verstandnis fur diese molekulare Maschinerie hat zusatzlich zu uberraschenden Erkenntnissen uber Virusabwehr, Chromatinorganisation und eine Klasse neuer Regulatoren, den miRNAs, gefuhrt. RNAi ist zu einem unverzichtbaren Werkzeug der Grundlagenforschung geworden. Medizinische Applikationen werden vorbereitet.