Aux/IAA14 Regulates microRNA-Mediated Cold Stress Response in Arabidopsis Roots

Abstract

The phytohormone auxin and microRNA-mediated regulation of gene expressions are key regulators of plant growth and development at both optimal and under low-temperature stress conditions. However, the mechanistic link between microRNA and auxin in regulating plant cold stress response remains elusive. To better understand the role of microRNA (miR) in the crosstalk between auxin and cold stress responses, we took advantage of the mutants of Arabidopsis thaliana with altered response to auxin transport and signal. Screening of the mutants for root growth recovery after cold stress at 4 °C revealed that the auxin signaling mutant, solitary root 1 (slr1; mutation in Aux/IAA14), shows a hypersensitive response to cold stress. Genome-wide expression analysis of miRs in the wild-type and slr1 mutant roots using next-generation sequencing revealed 180 known and 71 novel cold-responsive microRNAs. Cold stress also increased the abundance of 26-31 nt small RNA population in slr1 compared with wild type. Comparative analysis of microRNA expression shows significant differential expression of 13 known and 7 novel miRs in slr1 at 4 °C compared with wild type. Target gene expression analysis of the members from one potential candidate miR, miR169, revealed the possible involvement of miR169/NF-YA module in the Aux/IAA14-mediated cold stress response. Taken together, these results indicate that SLR/IAA14, a transcriptional repressor of auxin signaling, plays a crucial role in integrating miRs in auxin and cold responses.

Keywords: Aux/IAA; auxin; cold stress; microRNA.

Figure 1

Screening of auxin mutants for root growth recovery response after cold stress: (A) percentage root growth recovery at 6 h after 12 h of cold stress; (B) percentage root growth recovery at 24 h after 12 h of cold stress. Root growth recovery was calculated against the root growth at 23 °C. Vertical bars represent mean ± S.E. Data are from at least three independent experiments (n = 3 or more) with 8–10 seedlings per treatment. Asterisks denote the statistical significance between control and treatment as judged by the Student’s t-test (* p < 0.05 and *** p < 0.001).

Figure 2

Auxin mutant slr1 shows hypersensitive response to cold stress: (A) Comparison of primary root elongation at optimal temperature (23 °C) and 12 h cold-stressed seedlings in wild type (Col-0) and slr1. Note that slr1 shows slower root growth recovery compared with wild type at all time points we tested. Vertical bars represent mean ± SE. Data are from at least three independent experiments (n = 3 or more) with 8–10 seedlings per treatment. slr1 root growth recovery at 4 °C was statistically significant at all time points as judged by the Student’s t-test. (B) Root phenotype of Col-0 and slr1 during 24 h recovery period after cold stress at 4 °C for 12 h. Tick marks indicate the starting point of the recovery at 23 °C. Scale bar = 10 mm.

Figure 3

Nucleotide length distribution in wild type (Col-0) and slr1. Nucleotide length distribution of small RNA libraries. The results are obtained from two independent biological replicates. Vertical bars represent mean ± SD. Asterisks represent the statistical significance between the treatments as judged by the Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 4

Known miR expression profiles in the root of wild type (Col-0) and slr1. The heat map shows the hierarchical cluster analysis of miRs regulated in the cold-stressed root of wild type and slr1. The color bars of the heatmap represent the gradient scale of normalized log2-TPM values for each miR. Red color indicates a high level of miR abundance, and green color indicates low abundance. The analysis was performed using two independent biological replicates.

Figure 5

Venn diagram analysis showing the overlap of miRs among four libraries. (A) Known miRs identified in auxin-mediated cold stress response. (B) Novel miRs identified in auxin-mediated cold stress response.

Figure 6

Validation of miR expression in response to cold stress. RT-qPCR validation of selected miRs from cold stress NGS library. Vertical bars represent the mean ± SE of three biological replicates. Asterisks represent the statistical significance between control and treatment as judged by the Student’s t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 7

Pie charts showing the classes of proteins targeted by identified miRs: (A) protein classes targeted by known miRs; (B) protein classes targeted by novel miRs.

Figure 8

Relative expression of miR169 precursors and NF-YA3, NF-YA5, and NF-YA8 in Col-0 and slr1 after 12 h of cold (4 °C) stress. Vertical bars represent the mean ± SE of three biological replicates. Asterisks denote the statistical significance between the Col-0 and slr1 at 4 °C as judged by the Student’s t-test (*** p < 0.001).