Profiling the Abiotic Stress Responsive microRNA Landscape of Arabidopsis thaliana

Abstract

It is well established among interdisciplinary researchers that there is an urgent need to address the negative impacts that accompany climate change. One such negative impact is the increased prevalence of unfavorable environmental conditions that significantly contribute to reduced agricultural yield. Plant microRNAs (miRNAs) are key gene expression regulators that control development, defense against invading pathogens and adaptation to abiotic stress. Arabidopsis thaliana (Arabidopsis) can be readily molecularly manipulated, therefore offering an excellent experimental system to alter the profile of abiotic stress responsive miRNA/target gene expression modules to determine whether such modification enables Arabidopsis to express an altered abiotic stress response phenotype. Towards this goal, high throughput sequencing was used to profile the miRNA landscape of Arabidopsis whole seedlings exposed to heat, drought and salt stress, and identified 121, 123 and 118 miRNAs with a greater than 2-fold altered abundance, respectively. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was next employed to experimentally validate miRNA abundance fold changes, and to document reciprocal expression trends for the target genes of miRNAs determined abiotic stress responsive. RT-qPCR also demonstrated that each miRNA/target gene expression module determined to be abiotic stress responsive in Arabidopsis whole seedlings was reflective of altered miRNA/target gene abundance in Arabidopsis root and shoot tissues post salt stress exposure. Taken together, the data presented here offers an excellent starting platform to identify the miRNA/target gene expression modules for future molecular manipulation to generate plant lines that display an altered response phenotype to abiotic stress.

Keywords: Arabidopsis thaliana; RT-qPCR; abiotic stress; drought stress; heat stress; miRNA target gene expression; microRNAs (miRNAs); salt stress.

Figure 1

Phenotypic and physiological consequence of heat, drought and salt stress treatment of 15-day-old wild-type Arabidopsis whole seedlings. (A) Phenotypes displayed by 15-day old wild-type Arabidopsis whole seedlings post a 7-day treatment regime with heat, drought or salt stress, compared to non-stressed seedlings of the same age (left panel). Scale bar = 1.0 centimeter (cm) on larger sized panels and 0.5 cm on the superimposed images of a single representative seedling. (B) Whole seedling fresh weight of heat, drought (mannitol) and salt stressed Arabidopsis compared to their non-stressed counterparts of the same age. (C) Rosette diameter of 15-day-old Arabidopsis whole seedlings post 7-day exposure to heat, drought (mannitol) and salt stress compared to the non-stressed control. (D) Anthocyanin accumulation in heat, drought (mannitol) and salt stressed Arabidopsis whole seedlings compared to non-stressed whole seedlings of the same age (15 days). (E) RT-qPCR assessment of the expression of the stress induced gene, Δ1-PYRROLINE-5-CARBOXYLATE SYNTHETASE1 (P5CS1; AT2G39800) expression in 15-day-old Arabidopsis whole seedlings post a 7-day heat, drought (mannitol) and salt stress treatment regime compared to the abundance of the P5CS1 transcript in non-stressed Arabidopsis whole seedlings of the same age. (B–E) Error bars represent the standard deviation of four biological replicates and each biological replicate consisted of a pool of six individual plants. The presence of an asterisk above a column represents a statistically significantly difference between the stress treated sample and the non-stressed control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 2

Profiling of the miRNA landscape of heat, drought and salt stressed 15-day-old wild-type Arabidopsis whole seedlings. (A) Red (up) and blue (down) shaded tiles represent a Log2 fold change in abundance of the Arabidopsis miRNA sRNAs detected via high throughput sequencing (see Supplemental Table S1 for the normalized read numbers used to determine fold change values). (B) The number of miRNA sRNAs determined to have a greater than 2-fold change in abundance in heat, drought and salt stressed 15-day-old wild-type Arabidopsis whole seedlings compared to the abundance of each detected miRNA sRNA in non-stressed control plants of the same age. Red colored up arrows indicate the number of miRNAs with elevated abundance under each assessed stress, blue colored down arrows represent the number of miRNAs with reduced abundance post stress treatment and green colored up/down arrows state the number of miRNA sRNAs with a differing abundance trend between the individual stress treatments.

Figure 3

Quantification of miRNA abundance via RT-qPCR analysis of 15-day-old wild-type Arabidopsis whole seedlings post exposure to heat, drought and salt stress treatment. (A–C) RT-qPCR assessment of miR169 (A), miR395 (B) and miR396 (C) abundance in heat stressed Arabidopsis whole seedlings. (D–F) RT-qPCR assessment of miR857 (D), miR156 (E) and miR399 (F) abundance in drought (mannitol) stressed Arabidopsis whole seedlings. (G–I) RT-qPCR assessment of miR169 (G), miR399 (H) and miR778 (I) abundance in salt stressed Arabidopsis whole seedlings. (J,K) RT-qPCR assessment of miR839 (J) and miR855 (K) abundance across heat, drought (mannitol) and salt stressed Arabidopsis whole seedlings. (A–K) Colored columns (green = non-stressed control; orange = heat stress; red = drought stress, and; blue = salt stress) represent RT-qPCR determined abundance of each quantified miRNA sRNA and the light (control) and dark grey (stress) shaded columns present the fold changes in miRNA abundance as determined via high throughput sequencing. Error bars represent the standard deviation of four biological replicates and each biological replicate consisted of a pool of six individual plants. The presence of an asterisk above a column represents a statistically significantly difference between the stress treated sample and the non-stressed control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 4

Determination of miRNA target gene expression via RT-qPCR analysis of 15-day-old wild-type Arabidopsis whole seedlings post exposure to heat, drought and salt stress treatment. (A–C) RT-qPCR assessment of NFYA5 (A), ATPS1 (B) and GRF7 (C) miRNA target gene expression in heat stressed Arabidopsis whole seedlings. (D–F) RT-qPCR assessment of LAC7 (D), SPL9 (E) and PHO2 (F) miRNA target gene expression in drought (mannitol) stressed Arabidopsis whole seedlings. (G–I) RT-qPCR assessment of NFYA5 (G), PHO2 (H) and SUVH6 (I) miRNA target gene expression in salt stressed Arabidopsis whole seedlings. (A–I) Colored columns (green = non-stressed control; orange = heat stress; red = drought stress, and; blue = salt stress) represent RT-qPCR quantified expression of a single target gene for each miRNA assessed via RT-qPCR analysis in Figure 3. Error bars represent the standard deviation of four biological replicates and each biological replicate consisted of a pool of six individual plants. The presence of an asterisk above a column represents a statistically significantly difference between the stress treated sample and the non-stressed control sample (p-value: * < 0.05; ** < 0.005; *** < 0.001).

Figure 5

Phenotypic and molecular assessment of the root and shoot tissues of 15-day-old wild-type Arabidopsis plants post the 7-day salt stress treatment regime. (A) Root and shoot architecture of 15-day-old wild-type Arabidopsis seedlings post a 7-day salt stress treatment (right panel) during which the growth media plates were orientated for vertical growth. Scale bar = 1.0 cm. (B) Primary root length of 15-day-old Arabidopsis whole seedlings cultivated on vertically oriented media growth plates that contained either standard Arabidopsis growth media (non-stressed control) or growth media that had been supplemented with 150 mM sodium chloride (stress treatment). (C) RT-qPCR assessment of the expression of the stress induced gene, P5CS1, expression in 15-day-old Arabidopsis root and shoot material post 7-day salt stress treatment compared to the abundance of the P5CS1 transcript in non-stress control plants of the same age. (D,E) RT-qPCR quantification of miR169 abundance (D) and NFYA5 target gene expression (E) in salt stressed Arabidopsis root and shoot tissues. (F,G) RT-qPCR quantification of miR399 abundance (F) and PHO2 target gene expression (G) in salt stressed Arabidopsis root and shoot tissues. (H,I) RT-qPCR quantification of miR778 abundance (H) and SUVH6 target gene expression (I) in salt stressed Arabidopsis root and shoot tissues. (B–I) Colored columns represent the values obtained for non-stressed control plants (green colored columns) and the salt stressed samples (blue colored columns). Error bars represent the standard deviation of four biological replicates and each biological replicate consisted of a pool of six individual plants. The presence of an asterisk above a column represents a statistically significantly difference between the salt stress sample and the non-stressed controls (p-value: * < 0.05; ** < 0.005; *** < 0.001).