Manja Božić1*, Dragana Ignjatović-Micić1, Nenad Delić1, Marko Mladenović1, Jelena Vančetović1, Bojana Banović Đeri2, Ana Nikolić1
1Maize Research Institute „Zemun Polje“, Slobodana Bajića 1, 11085 Belgrade, Serbia
2Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11042 Belgrade, Serbia
mbozic [at] mrizp.rs
Abstract
Micro RNAs (miRNAs) are known regulators of various processes in plants, including growth, development and stress responses. They achieve this through mRNA cleavage or translational inhibition, in a process called RNA interference. Herein, their role in chilling stress response in young maize seedlings (Zea mays L.) is examined, using high-throughput sequencing methods. Bringing light to all aspects of chilling stress response in maize is necessary since earlier sowing, during colder periods, is one of the most promising strategies of avoiding maize yield loss due to effects of climate change in these areas.
Sterilized seeds of two maize genotypes (tolerant – T and sensitive – S to low temperatures) were germinated in the dark for five days (optimal conditions), after which the 5-d old seedlings were exposed to chilling conditions for 6h (10° C). Samples for RNA isolation and cDNA library preparation were taken after the treatment ended, and single-end 50 bp sequencing was performed (Illumina® Novaseq 6000). The miRNAs were then filtered, mapped, identified and quantified using adequate bioinformatics tools; and the differential expression analysis was carried out using the DEGseq R package. The analysis was performed on 859 miRNAs, after previously executed TPM normalization using the MA-plot-based method with random sampling model (MARS). The threshold for significantly differential expression was set as the Bayesian adjusted p-value, or q-value < 0.01 and log2 fold change > 1.
A total of 612 were expressed differentially, but only 55 miRNAs were common for both genotypes and at the same time differentially expressed between control and treatment conditions – 40 novel and 15 known. Half of the common miRNAs showed the same expression patterns in both genotypes, while the other half did not. Among them, seven known miRNAs showed opposing expression patterns between the genotypes (zma-miR167b-3p zma-miR167e-3p, zma-miR159c-5p, zma-miR164g-3p, zma-miR166a-5p, zma-miR398a-3p, and zma-miR528a-3p). These miRNAs were shown to have a role in various abiotic stress responses, including drought, waterlogging, high salts – but not chilling. While the results point to their potential role in establishing chilling tolerance in maize seedlings, further research is necessary to confirm it and connect the miRNAs to their potential targets.
Keywords: maize, abiotic stress, chilling, high-throughput sequencing, miRNAs
