Letícia Carvalho Benitez

Análise multivariada da divergência genética de genótipos de arroz sob estresse salino durante a fase vegetativa

Soil salinity is a limiting factor for rice cultivation, especially in the early stages of development and the flowering period. The use of sources of poor quality water for irrigation results in the accumulation of salts in the soil, causing major toxicity in culture. A solution to the problem would be the introduction of varieties with tolerance to high salinity. Hus the aim of this work is to evaluate genetic divergence among rice genotypes, aiming at the selection of genotypes tolerant to salinity during the vegetative phase. Seeds of 10 rice genotypes were grown in vitro on MS medium supplemented with 0 and 136 mM NaCl. After 21 days, six morphological characters were evaluated and the results subjected to multivariate analysis. The methods of Tocher, based on Mahalanobis distance, and graphic dispersion of canonic variables followed the same pattern of clustering structure, forming six groups. The characteristic of shoot fresh weight was the largest contributor to the genetic dissimilarity between genotypes by the method of Singh, while the other two canonic variables were sufficient to account for 91.27% of observed variation. Under the experimental conditions tested, the genotypes showed different degrees of salinity tolerance, while Colossus BRS, BRS Bojuru and BR IRGA 410, belonging to the groups three and four, were those who were more tolerant genotype and Moti, belonging to two what was more sensitive to salt stress.

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 (β-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 β-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.

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.

Transcriptome profiling of rice seedlings under cold stress

Rice (Oryza sativa L.) is one of the most important species for food production worldwide, besides being an excellent genetic model among the grasses. Cold is one of the major abiotic factors reducing rice yield, primarily affecting germination and reproduction phases. Currently, the RNAseq technique allows the identification of differential expressed genes in response to a given treatment, such as cold stress. In the present work, a transcriptome (RNAseq) analysis was performed in the V3 phase for contrasting genotypes Oro (tolerant) and Tio Taka (sensitive), in response to cold (13°C). A total of 241 and 244M readings were obtained, resulting in the alignment of 25.703 and 26.963 genes in genotypes Oro and Tio Taka respectively. The analyses revealed 259 and 5579 differential expressed genes in response to cold in the genotypes Oro and Tio Taka respectively. Ontology classes with larger changes were metabolic process ~27%, cellular process ~21%, binding ~30% and catalytic activity ~22%. In the genotype Oro, 141 unique genes were identified, 118 were common between Oro and Tio Taka and 5461 were unique to Tio Taka. Genes involved in metabolic routes of signal transduction, phytohormones, antioxidant system and biotic stress were identified. These results provide an understanding that breeding for a quantitative trait, such as cold tolerance at germination, several gene loci must be simultaneously selected. In general, few genes were identified, but it was not possible to associate only one gene function as responsible for the cultivar tolerance; since different genes from different metabolic routes were identified. The genes described in the present work will be useful for future investigations and for the detailed validation in marker assisted selection projects for cold tolerance in the germination of rice.

Validation of reference genes for RT-qPCR studies in Stevia rebaudiana in response to elicitor agents

Stevia rebaudiana is an important source of natural steviol glycosides and is of increasing interest in various fields of study. Therefore, understanding the molecular processes regulating its metabolism is of great importance. In this study, the stability of seven reference genes (18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1-α, Malate dehydrogenase, and Ubiquitin) under the effect of three stress-related elicitors (methyl jasmonate, salicylic acid, and spermidine) was evaluated in stevia plants. We used RefFinder software, which makes use of the four main currently available algorithms for reference gene selection: geNorm, NormFinder, BestKeeper, and the Comparative ∆Ct method. The results indicated that Ubiquitin and Actin can be used as reference genes under all tested experimental conditions. The genes, 18S ribosomal RNA, traditionally used as a reference gene, along with Calmodulin, showed the lowest stability. The expression of Deoxyxylulose-5-phosphate synthase and Kaurenoic acid hydroxylase genes was used to confirm the validated reference genes, showing that inadequacy of the reference gene may lead to erroneous results. This is the first study on the stability of reference genes in Stevia rebaudiana plants, and is of great relevance for further analysis of the gene expression of the steviol glycoside biosynthetic pathway.

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 (β-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.