Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots

Abstract

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

Keywords: Cadmium stress; Raphanus sativus; degradome; high-throughput sequencing; microRNAs; transcriptome..

Fig. 1.

Size distribution of small RNAs in Cd-free (CK) and Cd-treated (Cd200) libraries from radish roots. (This figure is available in colour at JXB online.)

Fig. 2.

Sizes and abundance of identified known miRNA families from radish. (A) Distribution of known miRNA family size in radish. (B) Counts of each known miRNA family in radish. (This figure is available in colour at JXB online.)

Fig. 3.

Validation and comparative relative expression of differentially expressed known (A) and novel (B) miRNAs between the CK and Cd200 libraries in radish by qRT-PCR. (This figure is available in colour at JXB online.)

Fig. 4.

Target plots (t-plots) of miRNA targets in different categories confirmed by degradome sequencing. The normalized signature abundance throughout the length of the indicated transcripts is shown. Representative t-plots for class I (A), class II (B), and class III (C) categories are shown. Blue arrows indicate signatures consistent with miRNA-directed cleavage. The solid lines and dot in miRNA:mRNA alignments indicate matched RNA base pairs and GU mismatch, respectively. On the left of the t-plots, the cleavage sites and normalized signature abundance are shown in the left and right column, respectively.

Fig. 4.

Target plots (t-plots) of miRNA targets in different categories confirmed by degradome sequencing. The normalized signature abundance throughout the length of the indicated transcripts is shown. Representative t-plots for class I (A), class II (B), and class III (C) categories are shown. Blue arrows indicate signatures consistent with miRNA-directed cleavage. The solid lines and dot in miRNA:mRNA alignments indicate matched RNA base pairs and GU mismatch, respectively. On the left of the t-plots, the cleavage sites and normalized signature abundance are shown in the left and right column, respectively.

Fig. 5.

Target plots (t-plots) for a set of Cd-responsive miRNA targets confirmed by degradome sequencing. (A) rsa-miR156, (B) rsa-miR159, (C) rsa-miR166, (D) rsa-miR393. Blue arrows indicate signatures consistent with miRNA-directed cleavage. The solid lines and dot in miRNA:mRNA alignments indicate matched RNA base pairs and GU mismatch, respectively.

Fig. 6.

qRT-PCR validation of known Cd-responsive miRNAs in radish. (A–F) and (G–L) Known and novel miRNAs, respectively. Small RNAs (<200 nt) were extracted from radish root treated or not with Cd (0, 1, 6, 12, 24, and 48h). The amount of expression was normalized to the level of 5.8S rRNA. The normalized miRNA levels at 0h were arbitrarily set to 1. Different letters indicate significant differences at P<0.05 according to Duncan’s multiple range tests.