Expression Landscape of circRNAs in Arabidopsis thaliana Seedlings and Adult Tissues.

RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Arabidopsis thaliana cbp80, c2h2, and flk Knockout Mutants Accumulate Increased Amounts of Circular RNAs.

Circular RNAs (circRNAs) are the products of the non-canonical splicing of pre-mRNAs. In contrast to humans and animals, our knowledge of the biogenesis and function of circRNAs in plants is very scarce. To identify proteins involved in plant circRNA generation, we characterized the transcriptomes of 18 Arabidopsis thaliana knockout mutants for genes related to splicing. The vast majority (>90%) of circRNAs were formed in more than one variant; only a small fraction of circRNAs was mutant-specific. Five times more circRNA types were identified in cbp80 and three times more in c2h2 mutants than in the wild-type. We also discovered that in cbp80, c2h2 and flk mutants, the accumulation of circRNAs was significantly increased. The increased accumulation of circular transcripts was not accompanied by corresponding changes in the accumulation of linear transcripts. Our results indicate that one of the roles of CBP80, C2H2 and FLK in splicing is to ensure the proper order of the exons. In the absence of one of the above-mentioned factors, the process might be altered, leading to the production of circular transcripts. This suggests that the transition toward circRNA production can be triggered by factors sequestering these proteins. Consequently, the expression of linear transcripts might be regulated through circRNA production.

A Tale of Two Families: Whole Genome and Segmental Duplications Underlie Glutamine Synthetase and Phosphoenolpyruvate Carboxylase Diversity in Narrow-Leafed Lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been supplied with advanced genomic resources and, as such, has become a well-known model for molecular evolutionary studies within the legume family-a group of plants able to fix nitrogen from the atmosphere. The phylogenetic position of lupins in Papilionoideae and their evolutionary distance to other higher plants facilitates the use of this model species to improve our knowledge on genes involved in nitrogen assimilation and primary metabolism, providing novel contributions to our understanding of the evolutionary history of legumes. In this study, we present a complex characterization of two narrow-leafed lupin gene families-glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC). We combine a comparative analysis of gene structures and a synteny-based approach with phylogenetic reconstruction and reconciliation of the gene family and species history in order to examine events underlying the extant diversity of both families. Employing the available evidence, we show the impact of duplications on the initial complement of the analyzed gene families within the genistoid clade and posit that the function of duplicates has been largely retained. In terms of a broader perspective, our results concerning GS and PEPC gene families corroborate earlier findings pointing to key whole genome duplication/triplication event(s) affecting the genistoid lineage.

Transcriptome-derived investigation of biosynthesis of quinolizidine alkaloids in narrow-leafed lupin (Lupinus angustifolius L.) highlights candidate genes linked to iucundus locus.

Unravelling the biosynthetic pathway of quinolizidine alkaloids (QAs), regarded as antinutritional compounds of narrow-leafed lupin (NLL) seeds, is fundamental to best exploit NLL as food or feed. We investigated 12 candidate genes connected to QA biosynthesis, selecting them by transcriptomic and genomic approaches, from the landscape of genes differentially expressed in leaves of the high- and low-alkaloid NLL accessions. Linkage analysis enabled the assessment of the location of the candidate genes in relation to iucundus, a major locus of unknown identity, that confers reduced QA content in seeds. The key finding was the identification of APETALA2/ethylene response transcription factor, RAP2-7, cosegregating with the iucundus locus and located within a region with highly significant QTLs that affect QA composition. We additionally identified a 4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) gene involved in L-lysine biosynthesis as being closely linked to iucundus. The distributed location of other remaining candidates (including previously known QA genes) across different linkage groups, also indirectly supports the transcription factor as a possible regulator of lupin alkaloid biosynthesis. Our findings provide crucial insight into QA biosynthesis in NLL. Additionally, we evaluated and selected appropriate reference genes for qRT-PCRs to analyse the expression levels of QA genes in NLL.

Legume Cytosolic and Plastid Acetyl-Coenzyme-A Carboxylase Genes Differ by Evolutionary Patterns and Selection Pressure Schemes Acting before and after Whole-Genome Duplications.

Acetyl-coenzyme A carboxylase (ACCase, E.C.6.4.1.2) catalyzes acetyl-coenzyme A carboxylation to malonyl coenzyme A. Plants possess two distinct ACCases differing by cellular compartment and function. Plastid ACCase contributes to de novo fatty acid synthesis, whereas cytosolic enzyme to the synthesis of very long chain fatty acids, phytoalexins, flavonoids, and anthocyanins. The narrow leafed lupin (Lupinus angustifolius L.) represents legumes, a plant family which evolved by whole-genome duplications (WGDs). The study aimed on the contribution of these WGDs to the multiplication of ACCase genes and their further evolutionary patterns. The molecular approach involved bacterial artificial chromosome (BAC) library screening, fluorescent in situ hybridization, linkage mapping, and BAC sequencing. In silico analysis encompassed sequence annotation, comparative mapping, selection pressure calculation, phylogenetic inference, and gene expression profiling. Among sequenced legumes, the highest number of ACCase genes was identified in lupin and soybean. The most abundant plastid ACCase subunit genes were accB. ACCase genes in legumes evolved by WGDs, evidenced by shared synteny and Bayesian phylogenetic inference. Transcriptional activity of almost all copies was confirmed. Gene duplicates were conserved by strong purifying selection, however, positive selection occurred in Arachis (accB2) and Lupinus (accC) lineages, putatively predating the WGD event(s). Early duplicated accA and accB genes underwent transcriptional sub-functionalization.

Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.

Six Medicago truncatula Dicer-like protein genes are expressed in plant cells and upregulated in nodules.

KEY MESSAGE: Here we report the existence of six putative Dicer-like genes in the Medicago truncatula genome. They are ubiquitously expressed throughout the plant and significantly induced in root nodules. Over the past decade, small noncoding RNAs (sncRNA) have emerged as widespread and important regulatory molecules influencing both the structure and expression of plant genomes. One of the key factors involved in sncRNA biogenesis in plants is a group of RNase III-type nucleases known as Dicer-like (DCL) proteins. Based on functional analysis of DCL proteins identified in Arabidopsis thaliana, four types of DCLs were distinguished (DCL1-4). DCL1 mainly produces 21 nt miRNAs. The products generated by DCL2, DCL3, and DCL4 belong to various classes of siRNAs that are 22, 24 and 21 nt in length, respectively. M. truncatula is a model legume plant closely related to many economically important cultivable species. By screening the recent M. truncatula genome assembly, we were able to identify three new DCL genes in addition to the MtDCL1-3 genes that had been earlier characterized. The newly found genes include MtDCL4 and two MtDCL2 homologs. We showed that all six M. truncatula DCL genes are expressed in plant cells. The first of the identified MtDCL2 paralogs encodes a truncated version of the DCL2 protein, while the second undergoes substantial and specific upregulation in the root nodules. Additionally, we identified an alternative splicing variant of MtDCL1 mRNA, similar to the one found in Arabidopsis. Our results indicate that DCL genes are differently activated during Medicago symbiosis with nitrogen fixing bacteria and upon pathogen infection. In addition, we hypothesize that the alternative splicing variant of MtDCL1 mRNA may be involved in tissue-specific regulation of the DCL1 level.

Cross talk between cytokine and hyperthermia-induced pathways: identification of different subsets of NF-κB-dependent genes regulated by TNFα and heat shock.

Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions.

Crosstalk between HSF1 and HSF2 during the heat shock response in mouse testes.

Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellular stress, while HSF2 becomes activated during development and differentiation, including spermatogenesis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heat shock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mechanisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heat shock response. For this purpose we used chromatin immunoprecipitation and the proximity ligation approaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32 °C) or heat shocked (at 38 °C or 43 °C) spermatocytes, which are cells the most sensitive to hyperthermia. At the physiological temperature or after mild hyperthermia at 38 °C, the sharing of binding sites for both HSFs was observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermia at 43 °C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of promoter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence of HSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resulted in a decreased number of such complexes and they were barely detected after strong hyperthermia at 43 °C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermatogenic cells. However, temperature elevation causes remodeling of chromatin binding and interactions between HSFs are disrupted. This potentially affects the regulation of stress response and contributes to the heat sensitivity of these cells.

Translational and structural analysis of the shortest legume ENOD40 gene in Lupinus luteus.

Two early nodulin 40 (enod40) genes, ENOD40-1, the shortest legume ENOD40 gene, and ENOD40-2, were isolated from Lupinus luteus, a legume with indeterminate nodules. Both genes were expressed at similar levels during symbiosis with nitrogen-fixing bacteria. ENOD40 phylogeny clustered the L. luteus genes with legumes forming determinate nodules and revealed peptide similarities. The ENOD40-1 small ORF A fused to a reporter gene was efficiently expressed in plant cells, indicating that the start codon is recognized for translation. The ENOD40-1 RNA structure predicted based on Pb(II)-induced cleavage and modeling revealed four structurally conserved domains, an absence of domain 4 characteristic for legumes of indeterminate nodules, and interactions between the conserved region I and a region located upstream of domain 6. Domain 2 contains Mg(II) ion binding sites essential for organizing RNA secondary structure. The differences between L. luteus and Glycine max ENOD40 RNA models suggest the possibility of a switch between two structural states of ENOD40 transcript.

Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (LlA) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg.L(-1) of culture, respectively. The enzymes are heat-stable up to 60 degrees C and show both isoaspartyl dipeptidase and l-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase genes in young wheat plants.

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase (ACCase) gene families at the mRNA level was analyzed in developing wheat (Triticum aestivum) plants. The major plastid ACCase mRNA level is high in the middle part of the plant and low in roots and leaf blades. An alternative plastid ACCase transcript initiated at a different promoter and using an alternative 5' splice site for the first intron accumulates to its highest level in roots. Cytosolic ACCase mRNA also consists of two species, one of which is present at approximately a constant level, whereas the other accumulates to a high level in the lower sheath section. It is likely that different promoters are also responsible for the two forms of cytosolic ACCase mRNA. The abundances of cytosolic and plastid ACCase mRNAs in the sheath section of the plant are similar. ACCase protein level is significantly lower in the leaf blades, in parallel with changes in the total ACCase mRNA level. Homoeologous ACCase genes show the same expression patterns and similar mRNA levels, suggesting that none of the genes was silenced or acquired new tissue specificity after polyploidization.