Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress

Abstract

Tobacco (Nicotiana tabacum L.) is more acclimated to cadmium (Cd) uptake and preferentially enriches Cd in leaves than other crops. MicroRNAs (miRNAs) play crucial roles in regulating expression of various stress response genes in plants. However, genome-wide expression of miRNAs and their target genes in response to Cd stress in tobacco are still unknown. Here, miRNA high-throughput sequencing technology was performed using two contrasting tobacco genotypes Guiyan 1 and Yunyan 2 of Cd-sensitive and tolerance. Comprehensive analysis of miRNA expression profiles in control and Cd treated plants identified 72 known (27 families) and 14 novel differentially expressed miRNAs in the two genotypes. Among them, 28 known (14 families) and 5 novel miRNAs were considered as Cd tolerance associated miRNAs, which mainly involved in cell growth, ion homeostasis, stress defense, antioxidant and hormone signaling. Finally, a hypothetical model of Cd tolerance mechanism in Yunyan 2 was presented. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in Cd tolerance.

Figure 1

Length distribution of small RNAs in the roots of control (open bars) and Cd-treated (filled bars) Guiyan 1 (A) and Yunyan 2 (B) plants. Y-axis: the percentage of small RNA reads. G − Cd, G + Cd, Y − Cd and Y + Cd correspond to hydroponically grown tobacco of the cultivars Guiyan 1 and Yunyan 2 grown in basic nutrition solution (BNS) or BNS + 50 μM Cd.

Figure 2. Root transcriptome profiles of Cd stress-responsive known miRNAs in tobacco.

Venn diagrams show the number of miRNAs regulated by Cd treatment (50 μM Cd stress for 5 days) and overlap between the two genotypes: Guiyan 1 (G) and Yunyan 2 (Y). The data are overlaps of miRNAs in Guiyan 1, which were down-regulated (down) (A), no change (NC) (B) and up-regulated (up) (C) in Yunyan 2. Within each genotype, fold change (Cd vs control) is log₂N, where changes in log₂N ≥ 1.5 are considered as up-regulated, between 0 < |log₂N| < 1.5 are considered as unchanged and log₂N ≤ -1.5 are considered as down-regulated, p < 0.01.

Figure 3. Precursor secondary structure prediction of five novel miRNAs differently expressed in Guiyan 1 (G) and Yunyan 2 (Y) in response to Cd stress.

Green bars indicate the mature miRNAs, black letters show complementary base pairing (including mismatches).

Figure 4. Validation expression patterns of six miRNAs identified in Guiyan 1 (G) and Yunyan 2 (Y) in response to Cd stress by qRT-PCR (RT).

Seq represents high-throughput sequencing. The actin gene was used as a constitutive internal control for qRT-PCR.

Figure 5. Gene ontology (GO) analysis of target genes of known and novel miRNAs identified in Guiyan 1 (G) and Yunyan 2 (Y).

The y-axis indicates number of targeted genes in each GO category, the x-axis provides a definition of the GO terms.

Figure 6. A hypothetically integrated schematic diagram of the mechanism involved in Cd tolerance in tobacco Yunyan 2.

miRNAs labelled with red, black, green and white circles (Guiyan 1) and triangles (Yunyan 2) are up-regulated, unaltered, down-regulated and undetected in response to Cd stress, respectively. SPL, squamosa promoter binding like protein; GRF, growth-regulating factor; NSF, N-ethylmaleimide sensitive fusion protein; NTH23, N. tabacum homeobox 23; EF-1 alpha, elongation factor 1-alpha; CNGC, Cyclic nucleotide-gated calmodulin-binding ion channel; CBF/NF-Y, CCAAT-binding transcription factor; ACRE4, Avr9/Cf-9 rapidly elicited protein 4; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; BC, biotin carboxylase; GPI, glucose-6-phosphate isomerase; TGA10, bZIP factor; CYP, cytochromes P450; E2, ubiquitin carrier protein; PPase, serine/threonine protein phosphatase; SCMT, S-adenosyl-methionine-sterol-C-methyltransferase; EIL1, ethylene -stabilized transcription factor.