Identification of wild soybean miRNAs and their target genes responsive to aluminum stress

Abstract

Background: MicroRNAs (miRNAs) play important regulatory roles in development and stress response in plants. Wild soybean (Glycine soja) has undergone long-term natural selection and may have evolved special mechanisms to survive stress conditions as a result. However, little information about miRNAs especially miRNAs responsive to aluminum (Al) stress is available in wild soybean.

Results: Two small RNA libraries and two degradome libraries were constructed from the roots of Al-treated and Al-free G. soja seedlings. For miRNA identification, a total of 7,287,655 and 7,035,914 clean reads in Al-treated and Al-free small RNAs libraries, respectively, were generated, and 97 known miRNAs and 31 novel miRNAs were identified. In addition, 49 p3 or p5 strands of known miRNAs were found. Among all the identified miRNAs, the expressions of 30 miRNAs were responsive to Al stress. Through degradome sequencing, 86 genes were identified as targets of the known miRNAs and five genes were found to be the targets of the novel miRNAs obtained in this study. Gene ontology (GO) annotations of target transcripts indicated that 52 target genes cleaved by conserved miRNA families might play roles in the regulation of transcription. Additionally, some genes, such as those for the auxin response factor (ARF), domain-containing disease resistance protein (NB-ARC), leucine-rich repeat and toll/interleukin-1 receptor-like protein (LRR-TIR) domain protein, cation transporting ATPase, Myb transcription factors, and the no apical meristem (NAM) protein, that are known to be responsive to stress, were found to be cleaved under Al stress conditions.

Conclusions: A number of miRNAs and their targets were detected in wild soybean. Some of them that were responsive to biotic and abiotic stresses were regulated by Al stress. These findings provide valuable information to understand the function of miRNAs in Al tolerance.

Figure 1

Length distribution of the wild soybean samll RNAs obtained by high-throughput sequencing in two libraries.A) Size distribution of redundant sequences. B) Size distribution of unique sequences.

Figure 2

Precursors structures of six pairs of miRNA/miRNA*s strictly meet characteristics mentioned by Meyers et al. (A-F) The precursor sequences of PN-miR1507c/PN-miR1507c*, PN-miR862a/PN-miR862a-5p, PN-miR169c/PN-mir169c-p3, PN-miR390b/PN-mir390b-p3, PN-miR1509b/PN-mir1509b-p3 and PN-miR4415/PN-mir4415-p3, respectively.

Figure 3

Differential expressions of ten miRNAs that were responsive to Al stress .

Figure 4

T-plots of miRNA targets in the four different categories. The T-plots show the distribution of the degradome tags along the full-length of the target mRNA sequence (bottom). The red line represents the sliced target transcripts and is shown by an arrow. The alignments show the miRNA with a portion of its target sequence (top). Two dots indicate matched RNA base pairs; one dot indicates a GU mismatch. The arrow shows the cleavage site. (A) Example of the category I target Glyma18g03980 for gso-miR1509a. (B) Example of the category II target Glyma18g07890 for PN-miR169c. (C) Example of the category III target Glyma12g35720 for PN-miR319a. (D) Example of the category IV target Glyma13g04540 for gso-mir167a-p3. The categories were based on the relative abundance of the tags at the target sites.

Figure 5

GO analyses of the targets of the known and new miRNAs in Glycine soja. Blue bars indicate the enrichment of the GO terms in the miRNA targets in GO. Green bars indicate the percentage of the total annotated soybean genes that were mapped to the GO terms.

Figure 6

T-plots of the targets cleaved by the miR319 family. The T-plots show the distribution of the degradome tags along the full-length of the target mRNA sequence (bottom). The red line represents the sliced target transcripts and is shown by an arrow. The alignment shows the miRNA with a portion of its target sequence (top). The two dots indicate matched RNA base pairs; one dot indicates a GU mismatch. The arrow shows the cleavage site. (A, B, C, and D) Examples of different TCP transcription factors as targets for PN-miR319a. (E, F) Examples of different Myb transcription factors as targets for PN-miR319.