Arabidopsis translation factor eEF1Bγ impacts plant development and is associated with heat-induced cytoplasmic foci.

The important role of translational control for maintenance of proteostasis is well documented in plants, but the exact mechanisms that coordinate translation rates during plant development and stress response are not well understood. In Arabidopsis, the translation elongation complex eEF1B consists of three subunits: eEF1Bα, eEF1Bß, and eEF1Bγ. While eEF1Bα and eEF1Bß have a conserved GDP/GTP exchange function, the function of eEF1Bγ is still unknown. By generating Arabidopsis mutants with strongly reduced eEF1Bγ levels, we revealed its essential role during plant growth and development and analysed its impact on translation. To explore the function of the eEF1B subunits under high temperature stress, we analysed their dynamic localization as green fluorescent protein fusions under control and heat stress conditions. Each of these fusion proteins accumulated in heat-induced cytoplasmic foci and co-localized with the stress granule marker poly(A)-binding protein 8-mCherry. Protein-protein interaction studies and co-expression analyses indicated that eEF1Bß physically interacted with both of the other subunits and promoted their recruitment to cytoplasmic foci. These data provide new insights into the mechanisms allowing for rapid adaptation of translation rates during heat stress response.

A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

Whole-proteome analysis of mesonephric-derived cancers describes new potential biomarkers.

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervix but occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155 EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%) but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLC & FATWO but was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%) but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

Inhibition of the antioxidant enzyme PRDX1 activity promotes MPP+-induced death in differentiated SH-SY5Y cells and may impair its colocalization with eEF1A2.

AIM: eEF1A2 is highly expressed in postmitotic cells and has been reported to interact with the antioxidant enzyme peroxiredoxin 1 (PRDX1). PRDX1 is involved in motor neuron differentiation. Here, we studied the relationship between eEF1A2 and PRDX1 during dopaminergic neuron differentiation, and examined their possible association in an oxidative stress model of Parkinson's disease (PD). MAIN METHODS: Expression of eEF1A2 and PRDX1 in SH-SY5Y cells at various durations of retinoic acid (RA) induction was detected using qRT-PCR, Western blotting and immunofluorescence. Neurons of 10-day differentiation were treated with the PRDX1 inhibitor H7, MPP+ and H7 plus MPP+. The cell viability, the amounts of apoptotic nuclei, DHE signals, and the expression of p53, p-Akt and p-mTOR were determined. The colocalization of eEF1A2 and PRDX1 was visualized using confocal microscopy. KEY FINDINGS: eEF1A2 gradually increased after RA-induced differentiation of SH-SY5Y cells, while PRDX1 protein gradually decreased. MPP+ treatment increased eEF1A2 in both undifferentiated and differentiated neurons; however, PRDX1 appeared to elevate only in mature neurons. The inhibition of the PRDX1 activity with H7 promoted MPP+-induced cell death, as evidenced by decreased cell viability, increased apoptotic nuclei, increased the DHE signal, and increased p53. However, H7 induced the activation of the prosurvival Akt and mTOR in MPP+-treated cells. Besides, a colocalization of eEF1A2 and PRDX1 was evidenced in MPP+-treated neurons. This colocalization was possibly prevented by inhibiting the PRDX1 activity, resulting in aggravated neuronal death. SIGNIFICANCE: Our results suggest that the possible association between eEF1A2 and PRDX1 may be a promising target for modifying neuronal death in PD.

Novel nannocystin A analogues as anticancer therapeutics: Synthesis, biological evaluations and structure-activity relationship studies.

Nannocystin A is a novel 21-membered macrocyclic lactam that targets eukaryotic translation elongation factor 1α (eEF1A) and displays potent antiproliferative activities. Herein, a series of nannocystin A analogues were synthesized and their structure-activity relationship (SAR) were established based on the MTT assay and western blotting analysis. The SAR enabled us to identify a structurally simplified nannocystin A analogue LQ18, which exhibited potent antiproliferative activities with IC50 values ranging from 4.3 to 48 nM against the tested cell lines, and inhibited eEF1A1 expression in A549 cell line. LQ18 arrested cell cycle at G2 phase and induced A549 cell apoptosis via up-regulation of caspase-3, caspase-9 and bax protein expressions in a dose-dependent manner, while it significantly decreased the bcl-2 expression. Collectively, these data demonstrated that LQ18 could be a promising lead for the development of structurally novel eEF1A1 inhibitor for cancer treatment.

The toad fly Lucilia bufonivora: its evolutionary status and molecular identification.

The blow fly genus Lucilia is composed largely of saprophages and facultative myasis agents, including the economically important species Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) and Lucilia sericata (Meigen). Only one species is generally recognized as an obligate agent of myiasis, Lucilia bufonivora Moniez, and this is an obligate parasite of toads. Lucilia silvarum (Meigen), a sister species, behaves mainly as a carrion breeder; however, it has also been reported as a facultative parasite of amphibians. Morphologically, these species are almost identical, and historically this has led to misidentification, taxonomic ambiguity and a paucity of studies of L. bufonivora. In this study, dipterous larvae were analysed from toad myiasis cases from the U.K., The Netherlands and Switzerland, together with adult specimens of fly species implicated in amphibian parasitism: L. bufonivora, L. silvarum and Lucilia elongata Shannon (from North America). Partial sequences of two genes, cox1 and ef1α, were amplified. Seven additional blow fly species were analysed as outgroups. Bayesian inference trees of cox1, ef1α and a combined-gene dataset were constructed. All larvae isolated from toads were identified as L. bufonivora and no specimens of L. silvarum were implicated in amphibian myiasis. This study confirms L. silvarum and L. bufonivora as distinct sister species and provides unambiguous molecular identification of L. bufonivora.

A Non-Flagellated Member of the Myxogastria and Expansion of the Echinosteliida.

Myxogastria (also called Myxomycetes or plasmodial slime-moulds) are mostly known through their usually conspicuous fruiting bodies. Another unifying trait is the presence of a facultative flagellate stage along with the obligate amoeboid stage. Here we show with two-gene phylogenies (SSU rRNA and EF-1alpha genes) that the incertae sedis, non-flagellate Echinosteliopsis oligospora belongs to the dark-spore clade (Fuscisporidia) of the Myxogastria. In addition, we confirm that Echinostelium bisporum, firstly described as a protostelid, belongs to the Echinosteliida, which are divided into three major clades and are paraphyletic to the remaining Fuscisporidia.

Direct detection of Tritrichomonas foetus in cattle genital fluid trough loop mediated isothermal amplification of elongation factor 1 alpha 1.

Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100-1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3-235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions.

First detection of Endogone ectomycorrhizas in natural oak forests.

The order Endogonales in Mucoromycotina, an early divergent lineage of fungi, includes ectomycorrhizal (EM) fungi. This order is therefore considered a key taxon for elucidation of the evolution of EM associations. Recent studies have revealed high diversity of EM lineages of Basidiomycota and Ascomycota; however, EM associations of Endogonales and its relatives remain largely unknown. In this study, EM root tips with a unique fungal sheath, with aseptate and highly branched hyphae of variable widths, were identified in Quercus acutissima and Quercus crispula forests in the temperate zone of Japan. The mycobionts were confirmed as Endogone sp., which were placed as a sister clade of Endogone pisiformis, based on phylogenetic analyses of the small and large subunits of the nuclear ribosomal RNA and elongation factor-1α genes. This is the first report of EM of Endogone in natural forests of the Northern Hemisphere and the first finding on Quercus.

Occurency of Giardia duodenalis assemblages in river water sources of Black Sea, Turkey.

A total of 420 environmental water samples and 120 drinking water samples from 45 different sampling sites of the Black Sea in Turkey were collected between 2012 and 2014. Genomic DNA was isolated from all the investigated water samples and comparativelly analyzed by Loop-mediated isothermal amplification (LAMP) of the elongation factor 1 Alfa (EF1α) gene, and by nested Polymerase Chain Reaction (nPCR) of the small subunit (SSU) rRNA and semi-nested PCR (snPCR) of the glutamate dehydrogenase gene (GDH). 141 (58.7%), 125 (52.1%) and 120 (50%) samples respectivelly were positive by each method. Out of 240 environmental samples collected from 25 sites of Samsun Province have been found positive for G. duodenalis by LAMP, nPCR and snPCR, respectively. 55 (30.5%), 50 (27.8%) and 47 (26.1%) of 180 environmental samples collected from 20 other sampling sites of Giresun Province were positive for Giardia by LAMP, nPCR and snPCR, respectively. Five PCR products from different samples of the Giresun Province and 10 other samples from the Samsun Province were found positive for G. duodenalis assemblage B. Five PCR products from Giresun Province and 5 samples from Samsun Province were found positive for G. duodenalis assemblage A. This is the first report about G. duodenalis assemblages A and B from water samples investigations in Black Sea of Turkey.

Taxonomy and Phylogeny of Polyporus Group Melanopus (Polyporales, Basidiomycota) from China.

Melanopus is a morphological group of Polyporus which contains species with a black cuticle on the stipe. In this article, taxonomic and phylogenetic studies on Melanopus group were carried out on the basis of morphological characters and phylogenetic evidence of DNA sequences of multiple loci including the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (EF1-α), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), and ß-tubulin gene sequences (ß-tubulin). The phylogenetic result confirmed that the previously so-called Melanopus group is not a monophyletic assemblage, and species in this group distribute into two distinct clades: the Picipes clade and the Squamosus clade. Four new species of Picipes are described, and nine new combinations are proposed. A key to species of Picipes is provided.

Eukaryotic translation elongation factor-1 alpha is associated with a specific subset of mRNAs in Trypanosoma cruzi.

BACKGROUND: Regulation of gene expression in trypanosomatids is mainly posttranscriptional. Tight regulation of mRNA stability and access to polysomes allows Trypanosoma cruzi to adapt to different environmental conditions during its life cycle. Posttranscriptional regulation requires association between mRNAs and specific proteins to form mRNP complexes. Proteins that lack a canonical RNA-binding domain, such as eukaryotic elongation factor-1α (EF-1α), may also associate with mRNPs. EF-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including RNA transport, the cell cycle, and apoptosis. RESULTS: In a previous study, EF-1α was found associated with mRNP-forming mRNAs in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. This finding suggested the possibility that EF-1α has a non-canonical function. Thus, we investigated the dynamics of EF-1α in association with T. cruzi epimastigote mRNAs under normal and stressed nutritional conditions. EF-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. The protein is cytoplasmically localized with a granular pattern in all forms analyzed. Following puromycin treatment, EF-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. We next characterized the EF-1α-associated mRNAs in unstressed and stressed epimastigotes. We observed that specific subsets of mRNAs were associated with EF-1α-mRNPs in unstressed or stressed epimastigotes. Some mRNAs were identified in both physiological conditions, whereas others were condition-specific. Gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, ATP and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding. CONCLUSION: These results indicate that in T. cruzi, as in other eukaryotes, EF-1α may play a non-canonical cellular role. We observed the enrichment of functionally related transcripts bound to EF-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of EF-1α mRNP in stress response.

Characterisation of translation elongation factor eEF1B subunit expression in mammalian cells and tissues and co-localisation with eEF1A2.

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.

Independent overexpression of the subunits of translation elongation factor complex eEF1H in human lung cancer.

BACKGROUND: The constituents of stable multiprotein complexes are known to dissociate from the complex to play independent regulatory roles. The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bß, eEF1Bγ) were found overexpressed in different cancers. To gain the knowledge about novel cancer-related translational mechanisms we intended to reveal whether eEF1H exists as a single unit or independent subunits in different human cancers. METHODS: The changes in the expression level of every subunit of eEF1H in the human non-small-cell lung cancer tissues were examined. The localization of eEF1H subunits was assessed by immunohistochemistry methods, subcellular fractionation and confocal microscopy. The possibility of the interaction between the subunits was estimated by co-immunoprecipitation. RESULTS: The level of eEF1Bß expression was increased more than two-fold in 36%, eEF1Bγ in 28%, eEF1A in 20% and eEF1Bα in 8% of tumor specimens. The cancer-induced alterations in the subunits level were found to be uncoordinated, therefore the increase in the level of at least one subunit of eEF1H was observed in 52% of samples. Nuclear localization of eEF1Bß in the cancer rather than distal normal looking tissues was found. In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549. Unexpectedly, in the A549 nuclear fraction eEF1A lost the ability to interact with the eEF1B complex. CONCLUSIONS: The results suggest independent functioning of some fraction of the eEF1H subunits in human tumors. The absence of eEF1A and eEF1B interplay in nuclei of A549 cells is a first evidence for non-translational role of nuclear-localized subunits of eEF1B. We conclude the appearance of the individual eEF1B subunits in tumors is a more general phenomenon than appreciated before and thus is a novel signal of cancer-related changes in translation apparatus.

[Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)].

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

Tau mRNA is present in axonal RNA granules and is associated with elongation factor 1A.

The microtubule-associated protein tau is predominantly localized in the axonal compartment over the entire length of the axon in neurons. The mechanisms responsible for the localization of tau in axons at long distance from the cell body are not properly understood. Using fluorescence in situ hybridization, we show that tau mRNA is present in the central and distal parts of the axons of cultured rat cortical neurons. Axonal tau mRNA is associated with granules which are distributed throughout the entire length of the axon, including the growth cone. We also show that tau mRNA-containing axonal particles are associated with elongation factor 1A, a component of the protein translation machinery. The presence of tau mRNA in axons might be at least part of the process by which tau is localized to distal axons.

Novel protein identification methods for biomarker discovery via a proteomic analysis of periodontally healthy and diseased gingival crevicular fluid samples.

AIM: To identify possible novel biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals using high-throughput proteomic analysis. MATERIALS AND METHODS: Gingival crevicular fluid samples were collected from 12 CP and 12 periodontally healthy subjects. Samples were trypically digested with trypsin, eluted using high-performance liquid chromatography, and fragmented using tandem mass spectrometry (MS/MS). MS/MS spectra were analysed using PILOT_PROTEIN to identify all unmodified proteins within the samples. RESULTS: Using the database derived from Homo sapiens taxonomy and all bacterial taxonomies, 432 human (120 new) and 30 bacterial proteins were identified. The human proteins, angiotensinogen, clusterin and thymidine phosphorylase were identified as biomarker candidates based on their high-scoring only in samples from periodontal health. Similarly, neutrophil defensin-1, carbonic anhydrase-1 and elongation factor-1 gamma were associated with CP. Candidate bacterial biomarkers include 33 kDa chaperonin, iron uptake protein A2 and phosphoenolpyruvate carboxylase (health-associated) and ribulose biphosphate carboxylase, a probable succinyl-CoA:3-ketoacid-coenzyme A transferase, or DNA-directed RNA polymerase subunit beta (CP-associated). Most of these human and bacterial proteins have not been previously evaluated as biomarkers of periodontal conditions and require further investigation. CONCLUSIONS: The proposed methods for large-scale comprehensive proteomic analysis may lead to the identification of novel biomarkers of periodontal health or disease.

Identification and validation of rice reference proteins for western blotting.

Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen-antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR.

BACKGROUND: Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. RESULTS: Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD), actin (ACT), beta-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), histone H3 (H3), elongation factor 1-alpha (EF) and 18S rRNA (rRNA) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII) expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants. CONCLUSIONS: geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.).

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. RESULTS: Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. CONCLUSIONS: This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.