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. 2015 Jun 4:6:410.
doi: 10.3389/fpls.2015.00410. eCollection 2015.

Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

Maria Barciszewska-Pacak  1 , Kaja Milanowska  1 , Katarzyna Knop  1 , Dawid Bielewicz  1 , Przemyslaw Nuc  1 , Patrycja Plewka  1 , Andrzej M Pacak  1 , Franck Vazquez  2 , Wojciech Karlowski  3 , Artur Jarmolowski  1 , Zofia Szweykowska-Kulinska  1
Affiliations

Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

Maria Barciszewska-Pacak et al. Front Plant Sci. .

Abstract

Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species.

Keywords: abiotic stress; gene expression; miRNA; pri-miRNA.

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Figures

Figure 1
Figure 1
Differential expression of Arabidopsis thaliana pri-miRNAs (white background) and mature miRNAs (gray background) under different abiotic stress conditions. Blue, red, and green colors indicate pri-miRNAs and mature miRNAs unchanged, upregulated and downregulated, respectively. (A) Upper panel depicts 30%SWC and 20%SWC drought stress (D30 and D20, respectively), middle panel shows half an hour and 6 h heat stress (H 0.5; H 6), lower panel shows 250 mM salinity (NaCl+), (B) upper panel presents copper deficiency (Cu−) and 10 μM copper excess (Cu+), lower panel depicts 10 μM cadmium excess (Cd+) and sulfur deficiency (S−). The fold change-based numbers of up- and downregulated pri-miRNAs and mature miRNAs shown in Venn diagrams were suggested by a two-tailed Student t test (p ≤ 0.05) for pri-miRNA RT-qPCR analyses (modified mirEX high throughput real-time PCR platform, Bielewicz et al., , http://comgen.pl/mirex2/) and a One-Way Anova test (p ≤ 0.05) for mature miRNA analyses by small RNA NGS, respectively.
Figure 2
Figure 2
Arabidopsis thaliana miRNAs revealed by Northern hybridization as affected under 30%SWC mild drought, 20%SWC severe drought, 0.5 h heat, 6 h heat, and salinity stress conditions. Only new stress responsive miRNAs unknown until now are shown. The white lines separate signals from miRNAs and U6snRNA loading control probed on the same blots. H 0 and H 6 samples were run on the same gel but not next to each other as indicated by the white separating lines between all signals probed. The star marks NGS revealed miRNAs with no statistic significance and the maintained tendency of expression level change as seen in Northern hybridization. Symbols representing various stresses are marked as described in the Figure 1.
Figure 3
Figure 3
Arabidopsis thaliana miRNAs revealed by Northern hybridization as affected under copper deficiency, copper excess, cadmium excess, and sulfur deficiency stress conditions. Only new stress responsive miRNAs unknown until now are shown. The white lines separate signals from miRNAs and U6snRNA loading control probed on the same blots. Control (Ctrl) and stress (Cu−, Cu+, Cd+, S−) samples were run on the same gel but not next to each other as indicated by the white separating lines between all signals probed. The star marks NGS revealed miRNAs with no statistic significance and the maintained tendency of expression level change as seen in Northern hybridization. Symbols representing various stresses are marked as described in the Figure 1.
Figure 4
Figure 4
General stress-responsive miRNAs. (A) General abiotic stress responsive Arabidopsis thaliana miRNAs 319a/b, 319b.2, and 400 revealed by Northern hybridization under different abiotic stresses. The white lines separate signals from miRNAs and U6snRNA loading control as well as both signals for the following stress samples: NaCl+ and Cu− (for miR319a/b), control (Ctrl) and Cu− (for miR319b.2), Cd+ and H 0 (for miR400), all probed on the same blots. (B) The structure of the TBL10 and the predicted as well as 5′RACE identified slicing sites within its mRNAs. (C) The agarose gels showing the miR319b.2 directed 3′-TBL10 mRNA cleavage products in wt plants and ΔmiR319b mutant plants. Arrow points to the expected length of the 5′RACE product. (D) The RT-qPCR of the TBL10 expression in wt plants under different abiotic stress conditions. Values on the chart are shown as the mean ± SD relative expression level from three independent experiments. Symbols representing various stresses are marked as described in the Figure 1.
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