Selection of candidate reference genes and validation for real-time PCR studies in rice plants exposed to low temperatures.

Rice is a cereal that presents a great ability to adapt to different soil and climate conditions. However, as it is a tropical crop with C3 metabolism, it performs better in warm temperatures with high solar radiation. Tolerance to stress caused by low temperatures is a highly complex process that involves various metabolic pathways and cellular compartments, resulting in general or specific effects on plant growth and development. In order to observe the true effect of a particular stress on genetic expression, reference genes need to be chosen for real-time PCRs, the expression levels of which should remain stable independent of the situation imposed. In this paper, the expression stability was evaluated of the actin 11 (ACT11), ubiquitin-conjugating enzyme 2 (UBC-E2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta tubilin (ß-Tubulin), eukaryotic initiation factor 4α (eIF-4-α), eukaryotic initiation factor 1α (eIF-1-α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin (TIP41), and cyclophilin genes, in two rice genotypes cultivated in low temperature (13°C) conditions in vegetative stage (V4). The analysis material (leaves) was collected after 0, 6, 24, 48, and 72 h of exposure to the stress. In this study, the geNorm, BestKeeper, ΔCt, NormFinder, and RefFinder methods were used to evaluate the expression stability of the candidate reference genes. The results revealed that the most indicated genes for all the analysis methods were UBQ10 and UBQ5 for BRS Bojuru and BRS Pampa, respectively. On the other hand, the eIF-1-α gene presents the least expression stability and is not indicated for studies of rice plants subjected to low temperatures. The validation with the antioxidant system genes SODCc1-Cu/Zn, CATC, APX2, and GR2 confirmed the importance of using previously tested normalizing genes for adequate real-time PCR results.

Changes in gene expression and catalase activity in Oryza sativa L. under abiotic stress.

Different rice (Oryza sativa L.) genotypes were subjected to high salinity and low temperature (150 mM NaCl and 13°C, respectively) for 0, 6, 24, 48, or 72 h. We evaluated the simultaneous expression of the genes OsCATA, OsCATB, and OsCATC, correlated gene expression with enzyme activity, and verified the regulation of these genes through identification of cis-elements in the promoter region. The hydrogen peroxide content increased in a tolerant genotype and decreased in a sensitive genotype under both stress conditions. Lipid peroxidation increased in the tolerant genotype when exposed to cold, and in the sensitive genotype when exposed to high salinity. Catalase activity significantly increased in both genotypes when subjected to 13°C. In the tolerant genotype, OsCATA and OsCATB were the most responsive to high salinity and cold, while in the sensitive genotype, OsCATA and OsCATC responded positively to saline stress, as did OsCATA and OsCATB to low temperature. Cis-element analysis identified different regulatory sequences in the catalase promoter region of each genotype. The sensitive genotype maintained a better balance between hydrogen oxyacid levels, catalase activity, and lipid peroxidation under low temperature than the resistant genotype. OsCATA and OsCATB were the most responsive in the salt-tolerant genotype to cold, OsCATA and OsCATC were the most responsive to saline stress, and OsCATA and OsCATB were the most responsive to chilling stress in the sensitive genotype. There were positive correlations between catalase activity and OsCATB expression in the tolerant genotype under saline stress and in the sensitive genotype under cold stress.

Expression of LTP genes in response to saline stress in rice seedlings.

Saline stress is one of the primary factors limiting increased rice productivity in the southern region of Brazil. Farming can be affected by salinity that is due to both the origin of the soils as well as the irrigation water. Lipid transfer proteins (LTPs) have many physiological functions, including in the response to saline stress. Therefore, the objective of this study was to quantify the relative expression of 11 genetic isoforms that encode LTP1-type proteins in rice genotypes tolerant and sensitive to saline stress in the vegetative period. When the plants reached development stage V4, alternating irrigation was started with nutritive solution and water containing 150 mM NaCl. The LTP7 gene showed an increase in expression by 13.81-fold after 96 h of stress exposure in the saline-tolerant group, whereas the LTP10 gene expression level was increased by 71.10-fold after 96 h in the saline-sensitive group. The LTP26, LTP23, and LTP18 genes showed increased expression in both genotypes; however, the expression levels and response times were different. Thus, LTP7 and LTP10 showed the highest response to salinity. The LTP18, LTP23, and LTP26 genes were negatively correlated with the response to salinity.

Evaluation of reference genes for RT-qPCR studies in the leaves of rice seedlings under salt stress.

To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (ß-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitin-conjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results.