DNA damage response (DDR), a complex network of cellular pathways that cooperate to sense and repair DNA lesions, is regulated by several mechanisms, including microRNAs. As small, single-stranded ...RNA molecules, miRNAs post-transcriptionally regulate their target genes by mRNA cleavage or translation inhibition. Knowledge regarding miRNAs influence on DDR-associated genes is still scanty in plants. In this work, an in silico analysis was performed to identify putative miRNAs that could target DDR sensors, signal transducers and effector genes in wheat. Selected putative miRNA-gene pairs were tested in an experimental system where seeds from two wheat mutant lines were irradiated with 50 Gy and 300 Gy gamma(γ)-rays. To evaluate the effect of the treatments on wheat germination, phenotypic and molecular (DNA damage, ROS accumulation, gene/miRNA expression profile) analyses have been carried out. The results showed that in dry seeds ROS accumulated immediately after irradiation and decayed soon after while the negative impact on seedling growth was supported by enhanced accumulation of DNA damage. When a qRT-PCR analysis was performed, the selected miRNAs and DDR-related genes were differentially modulated by the γ-rays treatments in a dose-, time- and genotype-dependent manner. A significant negative correlation was observed between the expression of tae-miR5086 and the RAD50 gene, involved in double-strand break sensing and homologous recombination repair, one of the main processes that repairs DNA breaks induced by γ-rays. The results hereby reported can be relevant for wheat breeding programs and screening of the radiation response and tolerance of novel wheat varieties.
•Knowledge of miRNA regulation on genes involved in the DNA Damage Response (DDR) pathway is still limited in plants.•Several miRNAs and DDR-related genes are differentially modulated by γ-rays during wheat germination in a time- and genotype-dependent manner.•An indirect validation of miRNA-target gene relation is provided for the tae-miR5086-RAD50 pair.•The gathered results are relevant for future wheat breeding programs and screening of new varieties with improved radiation response.
Early blight disease of tomato is one of the most devastating biotic stresses worldwide, and in Iran, Alternaria alternata is one of the most predominant species causing the disease. In the current ...study, a diverse collection of 35 tomato genotypes and implication of 5 SlWRKYs and 7 PR genes as well as enzymatic activity were evaluated on resistant and susceptible cultivars through real-time polymerase chain reaction at transplanting and maturing stages and by measuring product formation using spectrophotometry. The results indicated that the expression of these antifungal genes in 14 genotypes at two growth stages after inoculation with A. alternata highly enhanced by 1–50-fold. There was also significant upregulation of WRKYs and PRs genes among the resistant tomato varieties in comparison to susceptible and control varieties at both stages. These findings demonstrate the varieties that showed increased or decreased SlWRKY1 expression also displayed similar changes in the expression of PR1 and PR2 genes. Furthermore, the differential expression patterns of SlWRKY1 and SlWRKY11 were consistent with PR7 and PDF1.2 expression patterns. The analysis of enzymatic activity of PR2 and PR3 proteins, β-1,3-glucanase, and chitinase showed the highest level of activity in resistant inoculated genotypes against A. alternata. Therefore, the current findings suggest the possible involvement of these transcription factors in the increased expression of PR genes in response to A. alternata infection.
•Some tomato varieties are resistant to early blight disease caused by A. alternata.•The expression of defense-related genes provides EB disease resistance.•Chitinase and glucanase contribute to resistance to EB disease in tomato.•Expression and enzymatic activity of chitinase and glucanase are directly related.
Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used molecular techniques, often implemented to allow for the detection and quantitation of gene expression ...because of its high sensitivity, specificity, and reproducibility. However, reliable and comparable relative quantitative results using qRT-PCR require the application of appropriate reference genes in order to eliminate non-biological variations caused by initial RNA templates, efficiency of cDNA synthesis, and laboratory procedures. However, previous studies have reported that the stability of many reference genes may vary across species, tissue types, cell lines, developmental stages, and experimental treatments, yielding inaccurate or incorrect gene expression results. Therefore, the selection and validation of stable reference genes for different tissues from a specific species are especially important for obtaining accurate target gene expression results. The striped jack, Pseudocaranx dentex, belonging to the order Perciformes and family Carangidae, is a pelagic migratory fish with high nutritional value. This fish has already received extensive attention in global aquaculture production and is regarded as a candidate species for far-reaching marine aquaculture in China. Given this, there are currently a large number of fairly extensive molecular biology and genetics studies of P. dentex underway, which in turn have increased the demand for quantitative gene expression analysis by qRT-PCR in these animals. However, few studies have evaluated the reference genes for this species. Thus, the objective of this study was to identify suitable reference genes in different tissues of P. dentex, in an effort to provide the necessary tools to support subsequent gene expression pattern analysis.We evaluated nine commonly used reference genes, including beta actin (β-actin), ribosomal protein L13 (RPL13), elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase (HPRT), peptidylprolyl isomerase A (PPIA), beta 2-microglobulin (β2M), beta tubulin (TUB), and serine/threonine-protein phosphatase 2A catalytic subunit (PP2A) using qRT-PCR analysis across various P. dentex tissues. These evaluations included the study of their expression stability across ten tissues, including the brain, gill, heart, intestine, kidney, liver, spleen, stomach, slow-twitch muscle, and fast-twitch muscle, from three adult individuals of P. dentex using four independent methods, namely BestKeeper, NormFinder, geNorm, and RefFinder. These results were then validated in the qRT-PCR profiling of the myoblast determination protein 1 (myod1) gene in both muscle (slow-twitch and fast-twitch muscles) and non-muscle tissues (kidney and gills) using the various recommended reference genes or their combinations.Expression analysis showed that RPL13 was the most highly expressed gene in these samples, followed by EF-1α, β2M, β-actin, PPIA, HPRT, TUB, and PP2A, whereas GAPDH was the most weakly expressed across all ten P. dentex tissues. In addition, all nine candidate reference genes exhibited relatively inconsistent variations in Ct value across various tissues, with the BestKeeper stability assay, which uses standard deviation (SD) and coefficient of variation (CV) to score the candidates, suggesting that these genes could be ordered from most stable to least stable as follows: EF-1α > RPL13 > PP2A > PPIA > HPRT > β2M > TUB > GAPDH > β-actin. NormFinder suggested that the ranking was best described as follows: RPL13 > EF-1α > HPRT > TUB > PP2A > PPIA > β2M > GAPDH > β-actin. Evaluations using geNorm, which is based on the idea that the lower the expression stability value (M), the better the stability of gene expression, suggested that the expression stability of these genes is best described as follows: RPL13 = EF-1α > β-actin > GAPDH > PP2A > TUB > PPIA > HPRT > β2M. Finally, RefFinder analysis showed that the comprehensive stability ranking of each gene was RPL13 > EF-1α > PP2A > HPRT > PPIA > TUB > β2M > β-actin > GAPDH. In addition, all of the paired coefficients of variation, Vn/(n+1), reference genes had little effect on the V value. The combination of the two best reference genes is a valid normalization strategy, and the results can be used to correct the expression levels of various target genes. Given this, we can conclude that RPL13 and EF-1α are the two best reference genes for adult tissues. These outcomes were then validated in a myod1 expression assay in the slow-twitch muscle, fast-twitch muscle, kidney, and gills. These experiments revealed no significant differences in relative outcomes when using RPL13, EF-1α, and their combination as reference genes, whereas significant differences were identified when using the three least stable genes β2M, β-actin and GAPDH. This result confirmed that stability evaluation using the four methods was both necessary and effective.Based on our evaluations, we recommend that RPL13, EF-1α, and their combination are the ideal reference gene combinations for all previously evaluated adult tissue types in P. dentex, as verified by four individual algorithms. The results of this study provide the basis for improved standardization of qRT-PCR and transcriptomic evaluations in this species, which in turn should support more accurate evaluation of functional genes and provide technical support for the comprehensive and systematic molecular evaluation of more mature P. dentex samples.
Affymetrix ATH1 arrays, large-scale real-time reverse transcription PCR of ~ 2200 transcription factor genes and other gene families, and analyses of metabolites and enzyme activities were used to ...investigate the response of Arabidopsis to phosphate (Pi) deprivation and re-supply. Transcript data were analysed with MapMan software to identify coordinated, system-wide changes in metabolism and other cellular processes. Phosphorus (P) deprivation led to induction or repression of > 1000 genes involved in many processes. A subset, including the induction of genes involved in P uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of P-containing phospholipids with galactolipids and the repression of genes involved in nucleotide/nucleic acid synthesis, was reversed within 3 h after Pi re-supply. Analyses of 22 enzyme activities revealed that changes in transcript levels often, but not always, led to changes in the activities of the encoded enzymes in P-deprived plants. Analyses of metabolites confirmed that P deprivation leads to a shift towards the accumulation of carbohydrates, organic acids and amino acids, and that Pi re-supply leads to use of the latter. P-deprived plants also showed large changes in the expression of many genes involved in, for example, secondary metabolism and photosynthesis. These changes were not reversed rapidly upon Pi re-supply and were probably secondary in origin. Differentially expressed and highly P-specific putative regulator genes were identified that presumably play central roles in coordinating the complex responses of plants to changes in P nutrition. The specific responses to Pi differ markedly from those found for nitrate, whereas the long-term responses during P and N deprivation share common and non-specific features.
To extend plant benefits, three different concentrations of five Morinda citrifolia part was investigated on the collagen type II which is the primary collagen in human cartilage through the ...expression of the genes, COL2A1, COL-II and COLL2 regions in normal human dermal fibroblasts by qRT-PCR method. The results showed that 1) fibroblasts cultured in the presence of M. citrifolia extracts produced many times more collagen type II gene expression than control cells depending on the plant parts and concentrations, 2) the expression levels of the collagen type II gene stimulated by all fruit parts yields the higher percentages than leaves. Next, Morus alba leaf extract at a concentration expected to be a precursor protein source for collagen synthesis, working together with M. citrifolia stimulation, was added to the selected concentration indicating high expression in the M. citrifolia fruit experiments. The result showed various gene expression levels depended on the kind of gene and fruit part. Therefore, M. citrifolia fruits can benefit the creation of collagen type II with or without M. alba. The M. citrifolia fruit can be further benefited in product production for both the elderly and young for maintaining the typical structure and function of the skin, tendon, and bone.
The hypoxia-inducible transcription factors HIF-1 and HIF-2 regulate the response to hypoxia. Both proteins are dimers of an alpha subunit and a shared beta subunit. Under hypoxic conditions, the ...alpha subunits are stabilized, and the transactivation ability of the HIF-1 transcription factor is induced. Accordingly, assessment of HIF-1α protein levels and HIF transcriptional activity serve as an indirect indicator of hypoxia. In this series of protocols, I describe three methods to probe the HIF pathway.
Plants possess a well-organized protective network, wherein antioxidant enzymes play an important part in dealing with oxidative stress induced by over accumulation of ROS in plant cells. In the ...present study, a microcosm hydroponic experiment was performed to investigate the molecular modification of antioxidant enzymes at subcellular levels in rice seedlings in the presence of either trivalent Cr(III) or hexavalent chromium Cr(VI) using rice oligonucleotide microarray analysis. The results indicated that the production of ROS induced by Cr(III, VI) was concentration-dependent, Cr-specific and tissue-specific. Trivalent or hexavalent chromium exposure significantly (p < 0.05) altered the antioxidant enzymes activities in both rice tissues in comparison to control plants. In total, 41 genes were identified from the data of rice oligonucleotide microarray analysis. Under Cr(III) exposure, relatively higher expression of genes was observed in roots compared to those in shoots (p < 0.05), while gene expressions in both plant parts differed slightly during Cr(VI) exposure, implying different regulation and response strategies of plants against Cr(III) and Cr(VI). Subcellular localization indicated that genes encoding SOD, POD, APX, and GPX are mainly prevalent in the cytoplasm (30.77%), chloroplasts (29.23%), peroxisomes (10.77%) and mitochondria (9.23%), suggesting that cytoplasm and chloroplasts are the main sites responsible for scavenging ROS through enzymatic processes. Our study provides new insight into the roles of antioxidant enzymes in ROS metabolism at subcellular levels under Cr exposure.
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•Generation of ROS induced by Cr was concentration-dependent and tissue-specific.•Cr exposure altered the activities of antioxidant enzymes in both roots and shoots of rice plants.•41 antioxidant enzymatic genes corresponding to Cr were identified by microarray analysis.•A gene regulation diagram of eight antioxidant enzymes at the subcellular levels is proposed.•Genes encoding antioxidant enzymes are predominant in the cytoplasm and chloroplast.
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