Marcelo Nogueira do Amaral

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.

The Quest for More Tolerant Rice: How High Concentrations of IronAffect Alternative Splicing?

Rice (Oryza sativa L.) is a global staple food crop and an important model organism for plant studies. Recent reports have shown that alternative splicing is affected by many stressful conditions, suggesting its importance for the adaptation to adverse environments. Due to the little information on this subject, this study aimed to explore changes in splicing patterns that occur in response to high iron concentration in nutrient solutions. Here we quantified different kind of junctions and splicing events in the transcriptome of a relatively tolerant rice cultivar BRS Querencia, under iron excess with concentration of 300 mg L-1 Fe+2. Plants kept under standard conditions (control) presented 127,781 different splicing junctions, while stressed plants had 123,682 different junctions. Canonical (98.85% and 98.91%), semi-canonical (0.73% and 0.70%) and non-canonical (0.42% and 0.40%) junctions were found in control and stressed plants, respectively. Intron retention was the most frequent event (44.1% and 47.4%), followed by 3’ splice site (22.6% and 21.9%), exon skipping (18.9% and 17.3%) and alternative 5’ splice site (14.4% and 13.4%) in control and stressed plants, respectively. We also found 25 differentially expressed genes (five up and 20 down regulated) that are related to post-translational modifications. These results represent an important step in the understanding of how plant stress responses occur in an iron tolerant genotype, uncovering novel genes involved in iron stress response.

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.