Luisa Abruzzi de Oliveira

Reference Genes for the Normalization of Gene Expression in Eucalyptus Species

Abstract Gene expression analysis is increasingly important in biological research, with reverse transcription–quantitative PCR (RT–qPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT–qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus.

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.)

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Differential expression of cysteine desulfurases in soybean

BackgroundIron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance.ResultsHere we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins.ConclusionsOur results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression.

Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L.

Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

Mitochondrial iron-sulfur cluster genes in Eucalyptus

Iron-sulfur [Fe-S] clusters are prosthetic groups required to maintain life processes including respiration, photosynthesis, metabolic reactions, sensing, signaling,and gene regulation. In plants the biogenesis of Fe-S proteins is compartmentalized and adapted to specific needs of the eukaryotic and photosynthetic cell. Although critical to so many fundamental metabolic pathways and drastically affecting plant adaptability and productivity, Fe-S proteins were never investigated in woody species. Eucalyptus grandis is an important economical tree widely cultivated in subtropical regions which suffers under low temperature stress. Here we describe a transcriptional analysis of the E. grandis NFS1, ISU1 and ISA1, three genes involved in the biogenesis of [Fe-S] clusters. Microarray analyses were carried out for the comparison of global gene expression in leaves and vascular tissues (xylem) of E. grandis and vascular tissues of E. globulus. In general, leaves from E. grandis demonstrated higher expression of these genes than xylem. EgrISU1 had a constitutive expression in E. grandis, but its expression pattern was higher in this species than in E. globulus xylem. Differences observed in the relative gene expression profile between xylem tissues from the two Eucalyptus species suggest that these genes may be implicated in the contrasting phenotypic characteristics of their wood. The response of these genes to a series of hormonal and stress signals over E. grandis seedlings was also evaluated by RT-qPCR. After the chilling treatment of seedlings, EgrNFS1 and EgrISU1 showed 6 to 8-fold and 0.6 to 1.7-fold increase respectively; andEgrISA1 exhibited a drastic 69 to 114-fold increase. These data are the same observed in Arabidopsis microarrays available in GeneVestigator database. These results suggest that (i) NFS1 and ISA1 may be related to the cellular response to stress caused by chilling, and (ii) the increase in the expression is probably due to sulfur metabolism. A time-course chilling experiment was also carried out. The ISU1 gene expression was higher in the first two hours of treatment and decreased right after that period. The ISA1 gene, which showed the highest expression in the previous experiment, didn’t show significant differences in the expression pattern during the 16 hours of chilling, as well as the NFS1 gene. Our data indicated that Fe-S proteins are possibly involved in the recovery of plants after chilling stress.