Investigation of the effects of nitrogen starvation and drought stresses on microRNA expressions in Arabidopsis thaliana

MicroRNAs (miRNAs) which involve in non-coding RNAs has generally 20-22 nucleotides long and they have responsibility for regulation of gene expression. miRNAs have important roles in the development of animals and plants and in many biological processes. miRNA mediated gene regulation occur in two different ways: Inhibition of translation of target mRNA or degredation of target mRNA. Gene expression is regulated by miRNAs which are discovered in Caenorhabditis elegans. miRNAs target genes and transcription factors include growth and development processes of plants in various stress conditions. It is known that several stres factors such as drought, salt and starvation, which effect development and growth in plants, change miRNAs expression and regulate target genes expression. In this thesis, it is aimed to investigate the changes in miRNA expression levels by drought treatment and nitrogen starvation, which are two different stress conditions affecting significantly development and growth in plants. It was targeted to establish Arabidopsis thaliana with plant tissue culture techniques, to achieve RNA isolation from these plants and to synthesize cDNA with specific primers to various miRNAs. Differences and changes in expression of targeted miRNAs were analyzed by Real Time Quantitative Polymerase Chain Reaction (qRTPCR) method. In addition, growth index and ion leakages were analyzed by Tukey Test and ion leakages had no significant data for all callus tissue under different stress conditions. miRNA expressions were analyzed by One Way ANOVA Tukey Test and nearly all miRNAs that selected had significant results. From growth index rates, it is inferred that there is a significant change under 7-day applications of drought (****p<0.0001) and combination (***p<0.0006) stress compared to control samples, while under nitrogen deficiency conditions no significant changes in weight were observed. In addition, there was a significant difference between nitrogen starvation (*p<0.0124) and combination stress (**p<0.0024) compared to control conditions of 14-day application. As a result of One way ANOVA Tukey Test analysis of miRNA expressions, all of the 7 and 14-day applications of miR165a-3p, miR165b samples showed significant downregulation, while miR167a-5p, miR167b showed decrease in all 7-day stress applications and no significant change in 14-day applications. miR167c-5p expression decreased for 7-day applications of drought and combination stress while significantly increasing for 14-day nitrogen starvation and decreasing for drought and combination samples. Significant upregulation of miR167d during 7 and 14-day nitrogen starvation applications, and downregulation during 7-day combination, 14-day drought and combination conditions were observed. Expressions of miR169b-5p, miR169c, significantly decreased as a result of 7-day combination and 14-day drought and combination stress, while miR169h-n, miR169d,e,f-5p,g-5p showed no significant change under any of the stress applications. During all of the 7-day stress conditions, miR319a,b expressions were downregulated, when for 14-day samples only drought conditions resulted in downregulation. Strong downregulation of miR319c was observed in callus tissues that were treated with 7-day drought and combination stress, along with upregulation for 14-day nitrogen starvation and downregulation for the remaining drought and combination conditions. miR396a-5p expression under 7-day stress application were found to be non-significant, all 14day samples displayed significant decrease. For all 7-day samples under every stress application, miR396b was downregulated, when 14-day combination samples showed upregulation. Under 7-day drought and combination applications significant decrease of miR399a expression and under 14-day drought stress a significant increase was determined. For miR399b expression under 7-day stress conditions, no significant change was observed while expressions of all 14-day samples strongly decreased. miR399d exhibited downregulation for all stress conditions applied for 7 or 14 days. While miR399e expression increased in 14-day nitrogen starvation samples, it was downregulated under 7 and 14-day drought and combination stresses. Expression level of miR399f significantly dropped under 7-day nitrogen starvation and combination stress conditions, where the remaining samples displayed no significant change. miR827 expression was downregulated for 7-day applications of all stress conditions, while significantly increasing under 14-day nitrogen starvation. Aside from the changes of expression under nitrogen starvation and drought stresses, miRNAs for which the expression analyses were performed in this study, are also shown to be variable under the combination of two stress conditions, additionally, the changes occurring in stress responses between 7 and 14-day applications are believed to be valuable to elucidate the associated molecular mechanisms of miRNAs.