Silvia Graciele Hülse de Souza

Nitrogen Starvation, Salt and Heat Stress in Coffee (Coffea arabica L.): Identification and Validation of New Genes for qPCR Normalization

Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.

Transcriptional modulation of genes encoding nitrate reductase in maize ( Zea mays ) grown under aluminum toxicity

The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4 . With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1 , ZmNR2 , ZmNR3 , and ZmNR4 genes in leaves, 30-day-old hybrid maize BRAS 3010 plants were irrigated with a solution of Al 2 (SO 4 ) 3 .18H 2 O for 16 days. The transcriptional levels of ZmNR2 , ZmNR3 , and ZmNR4 and NR activity will exhibit standard changes similar in the leaves, where, from the second week of stress onwards, there was a decrease in enzymatic activity and in the accumulation of transcripts. An increase ZmNR1 mRNA levels were observed, indicating that this gene may be associated with other metabolic pathways. This study resulted in the identification and characterization of different genes that encode NR and are involved in nitrogen metabolism in maize, in which the ZmNR2 , ZmNR3 , and ZmNR4 genes regulate the activity of NR in response to aluminum stress. The characterization of these genes may help in our understanding of the genetic-molecular and physiological mechanisms of maize subjected to aluminum stress. Keywords: Abiotic stress, Al, gene expression, nitrogen, metal toxicity, mineral nutrition

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.