Tobacco NtUBC1 and NtUBQ2 enhance salt tolerance by reducing sodium accumulation and oxidative stress through proteasome activation in Arabidopsis.

The ubiquitin/proteasome system plays a crucial role in the regulation of plant responses to environmental stress. Here, we studied the involvement of the UBC1 and UBQ2 genes encoding a ubiquitin conjugating enzyme (E2) and ubiquitin extension protein, respectively, in the response to salt stress. Our results showed that the constitutive expression of tobacco NtUBC1 and NtUBQ2 in Arabidopsis thaliana improved salt tolerance, along with the lower Na+ level and higher K+/Na+ ratio compared to control plants. Moreover, the expression levels of sodium transporters, including AtHKT1 (High-Affinity K+ Transporter1) and AtSOS1 (Salt Overly Sensitive 1), were higher in NtUBC1- and NtUBQ2-Arabidopsis. However, the transcript level of AtNHX1 (Na+/H+ Exchanger 1) was similar between control and transgenic plants. After salt exposure, the activity of the 26S proteasome markedly increased in NtUBC1- and NtUBQ2-expressing plants; however, ubiquitinated protein levels decreased compared to control plants. Furthermore, higher activity of antioxidant enzymes and lower ROS production were observed in UBC1- and UBQ2-expressing plants. We further challenged atubc1, atubc2, and atubq2 single mutants and atubc1ubc2 double mutant lines with salt stress; interestingly, the salt sensitivity and sodium levels of the studied mutants were enhanced, while the potassium levels were reduced. However, the atubc1ubc2 double mutant illustrated a more severe phenotype than the single mutants, probably due to the redundant function of UBC1 and UBC2 in Arabidopsis. Taken together, NtUBC1 and NtUBQ2 enhance salt tolerance by enhancing 26S proteasome activity and reducing Na+ accumulation, ROS, and ubiquitinated/salt-denatured proteins.

Shear Bond Strength and Fluoride Release of a Universal Adhesive: An In-Vitro Study on Primary Teeth.

This investigation aimed to assess the shear bond strength and fluoride-releasing capabilities of Clearfil Universal Bond Quick (Kuraray Noritake Dental Inc., Tokyo, Japan). Forty-four extracted primary molars were divided into two groups, and the enamel substrate was prepared for evaluating shear bond strength. Scotchbond (3M ESPE) and Clearfil UBQ were used to bond composite-to-enamel substrates in each group (n = 22). Shear bond strength was measured using a universal testing device and compared. Sixteen discs (6 mm diameter and 3 mm thickness) were fabricated from each Clearfil UBQ, Fuji IX, and Fuji II LC. Over the course of 30 days, each materials' fluoride release was examined and compared using ion analysis. Results revealed that Clearfil UBQ had statistically similar shear bond strength to Scotchbond. Between the three materials, Clearfil UBQ had the lowest fluoride release at baseline (0.11 ± 0.25) and the lowest cumulative fluoride release (0.12-0.27 ppm) over 30 days. Fuji IX had the highest fluoride release at baseline (19.38 ± 2.50) and cumulatively (40.87 ± 4.03 ppm), followed by Fuji II LC. We conclude that Clearfil UBQ and Scotchbond showed comparable bond strengths to the enamel. Fluoride release was seen in Clearfil UBQ in the initial two days of the 30-day period. The amount of fluoride release was significantly less than with glass ionomer cements.

Drought and Oxidative Stress in Flax (Linum usitatissimum L.) Entails Harnessing Non-Canonical Reference Gene for Precise Quantification of qRT-PCR Gene Expression.

Flax (Linum usitatissimum L.) is a self-pollinating, annual, diploid crop grown for multi-utility purposes for its quality oil, shining bast fiber, and industrial solvent. Being a cool (Rabi) season crop, it is affected by unprecedented climatic changes such as high temperature, drought, and associated oxidative stress that, globally, impede its growth, production, and productivity. To precisely assess the imperative changes that are inflicted by drought and associated oxidative stress, gene expression profiling of predominant drought-responsive genes (AREB, DREB/CBF, and ARR) was carried out by qRT-PCR. Nevertheless, for normalization/quantification of data obtained from qRT-PCR results, a stable reference gene is mandatory. Here, we evaluated a panel of four reference genes (Actin, EF1a, ETIF5A, and UBQ) and assessed their suitability as stable reference genes for the normalization of gene expression data obtained during drought-induced oxidative stress in flax. Taking together, from the canonical expression of the proposed reference genes in three different genotypes, we report that EF1a as a stand-alone and EF1a and ETIF5A in tandem are suitable reference genes to be used for the real-time visualization of cellular impact of drought and oxidative stress on flax.

Evaluation of stability and validation of reference genes for RT-qPCR expression studies in rice plants under water deficit.

Many studies use strategies that allow for the identification of a large number of genes expressed in response to different stress conditions to which the plant is subjected throughout its cycle. In order to obtain accurate and reliable results in gene expression studies, it is necessary to use reference genes, which must have uniform expression in the majority of cells in the organism studied. RNA isolation of leaves and expression analysis in real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. In this study, nine candidate reference genes were tested, actin 11 (ACT11), ubiquitin conjugated to E2 enzyme (UBC-E2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta tubulin (ß-tubulin), eukaryotic initiation factor 4α (eIF-4α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin TIP41 (TIP41-Like) and cyclophilin, in two genotypes of rice, AN Cambará and BRS Querência, with different levels of soil moisture (20%, 10% and recovery) in the vegetative (V5) and reproductive stages (period preceding flowering). Currently, there are different softwares that perform stability analyses and define the most suitable reference genes for a particular study. In this study, we used five different methods: geNorm, BestKeeper, ΔCt method, NormFinder and RefFinder. The results indicate that UBC-E2 and UBQ5 can be used as reference genes in all samples and softwares evaluated. The genes ß-tubulin and eIF-4α, traditionally used as reference genes, along with GAPDH, presented lower stability values. The gene expression of basic leucine zipper (bZIP23 and bZIP72) was used to validate the selected reference genes, demonstrating that the use of an inappropriate reference can induce erroneous results.

Screening and validation of reference genes for quantitative RT-PCR analysis in Ampelopsis grossedentata / 中草药

Objective: To screen reference genes for real time quantitative PCR (qRT-PCR) research in Ampelopsis grossedentata. Methods: On the basis of the conserved sequences among plant species, six candidate reference genes (including Actin, 18 S-rRNA, GAPDH, α-Tubulin, β-Tubulin, and UBQ) were cloned from A. grossedentata by RT-PCR in this study. The expression stability of each reference gene in different tissues (shoot tip, young leaf, mature leaf, old leaf, stem, and root) were analyzed by three softwares (GeNorm, NormFinder, and BestKeeper), followed by validation of the expression pattern of AgPAL by qRT-PCR. Results: Actin, 18 S-rRNA, and GAPDH expressed most stably in all samples and were suitable for reference genes, which were further confirmed by the transcript level analysis result of AgPAL in different tissues. Conclusion: This is the first report on the screening and validation of reference genes for qRT-PCR in A. grossedentata, which benefits future studies on gene expression in this species.

Screening and analysis of Hc-ubq and Hc-gst related to desiccation survival of infective Haemonchus contortus larvae.

Infective Haemonchus contortus larvae (L3s) are able to protect themselves from desiccation. To explore the molecular mechanisms of desiccation survival, mRNA differential display RT-PCR was used to screen differentially expressed genes in L3s upon desiccation, followed by RNAi experiments to define gene functions. In this, 58 differentially expressed transcripts were obtained. Among these, the BF-U01A and CH-U02A fragments represent genes with the highest identity percentage in bioinformatic analysis. They were named Hc-ubq and Hc-gst based on their respective homologous ubiquitin in Caenorhabditis elegans and glutathione S-transferase in H. contortus. Quantitive RT-PCR results indicated that they were both up-regulated in desiccated L3s. Hc-ubq and Hc-gst RNAi in H. contortus showed reduced survival rate of L3s, with unchanged locomotion behavior. Homologous Ce-ubq-2 and Ce-gst-7 RNAi in C. elegans also displayed higher larval death rate. These results suggest that ubq and gst may play important roles in nematode desiccation tolerance. Our study analyzed desiccation resistance related genes in H. contortus L3s, and revealed significant research implications on the mechanisms behind nematode desiccation survival.

Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in Arabidopsis and rice (Oryza sativa) roots.

Ca(2+) signatures are central to developmental processes and adaptive responses in plants. However, high-resolution studies of Ca(2+) dynamics using genetically encoded Ca(2+) indicators (GECIs) such as Yellow Cameleon (YC) proteins have so far not been conducted in important model crops such as rice (Oryza sativa). We conducted a comparative study of 35S and ubiquitin-10 (UBQ10) promoter functionality in Arabidopsis thaliana and O. sativa plants expressing the Ca(2+) indicator Yellow Cameleon 3.6 (YC3.6) under control of the UBQ10 or 35S promoter. Ca(2+) signatures in roots of both species were analyzed during exposure to hyperpolarization/depolarization cycles or in response to application of the amino acid glutamate. We found a superior performance of the UBQ10 promoter with regard to expression pattern, levels and expression stabilities in both species. We observed remarkable differences between the two species in the spatiotemporal parameters of the observed Ca(2+) signatures. Rice appeared in general to respond with a lower maximal signal amplitude but greatly increased signal duration when compared with Arabidopsis. Our results identify important advantages to using the UBQ10 promoter in Arabidopsis and rice and in T-DNA mutant backgrounds. Moreover, the observed differences in Ca(2+) signaling in the two species underscore the need for comparative studies to achieve a comprehensive understanding of Ca(2+) signaling in plants.

Association of UBQ-8i polymorphism with Alzheimer's disease in Caucasians: a meta-analysis.

BACKGROUND: Several studies have reported an association between the UBQ-8i (rs12344615) polymorphism of the UBQLN1 gene and risk of Alzheimer's disease (AD), but these findings remain controversial. In this study, a meta-analysis was carried out to investigate the relationship between UBQ-8i polymorphism and AD risk and a possible synergy with apolipoprotein E (APOE)ε4 gene status. METHODS: Case-control studies were selected from PubMed, Medline and Embase (Ovid) databases. The potential association was evaluated by odds ratios (ORs) with 95% confidence intervals (CIs). Data were analyzed with Stata version 11.0. RESULTS: A total of 4679 AD cases and 9928 controls were included in the study. There was no evidence of heterogeneity between studies or publication bias in the meta-analysis. There were no significant differences among the examined genetic models. In the analysis stratified by age of onset, a significant association was detected in the late onset AD group under the allele (OR = 1.12, 95% CI: 1.01-1.24), heterozygote (OR = 1.15, 95% CI: 1.02-1.30) and dominant (OR = 1.13, 95% CI: 1.00-1-26) models. However, UBQ-8i polymorphism was not associated with a higher risk for AD among APOEε4 carriers. CONCLUSION: The results suggest that UBQ-8i polymorphism may contribute to AD susceptibility, but does not synergize with APOEε4 status to increase AD risk.

Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1.

Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice.

S-like ribonucleases (S-like RNases) are homologous to S-ribonucleases (S-RNases), but are not involved in self-incompatibility. In dicotyledonous plants, S-like RNases play an important role in phosphate recycling during senescence and are induced by inorganic phosphate-starvation and in response to defense and mechanical wounding. However, little information about the functions of the S-like RNase in monocots has been reported. Here, we investigated the expression patterns and roles of an S-like RNase gene, OsRNS4, in abscisic acid (ABA)-mediated responses and phytochrome-mediated light responses as well as salinity tolerance in rice. The OsRNS4 gene was expressed at relatively high levels in leaves although its transcripts were detected in various organs. OsRNS4 expression was regulated by salt, PEG and ABA. The seedlings overexpressing OsRNS4 had longer coleoptiles and first leaves than wild-type seedlings under red light (R) and far-red light (FR), suggesting negative regulation of OsRNS4 in photomorphogenesis in rice seedlings. Moreover, ABA-induced growth inhibition of rice seedlings was significantly increased in the OsRNS4-overexpression (OsRNS4-OX) lines compared with that in WT, suggesting that OsRNS4 probably acts as a positive regulator in ABA responses in rice seedlings. In addition, our results demonstrate that OsRNS4-OX lines have enhanced tolerance to high salinity compared to WT. Our findings supply new evidence on the functions of monocot S-like RNase in regulating photosensitivity and abiotic stress responses.

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions.