Identification of boron-deficiency-responsive microRNAs in Citrus sinensis roots by Illumina sequencing

Abstract

Background: Boron (B)-deficiency is a widespread problem in many crops, including Citrus. MicroRNAs (miRNAs) play important roles in nutrient deficiencies. However, little is known on B-deficiency-responsive miRNAs in plants. In this study, we first identified miRNAs and their expression pattern in B-deficient Citrus sinensis roots by Illumina sequencing in order to identify miRNAs that might be involved in the tolerance of plants to B-deficiency.

Results: We isolated 52 (40 known and 12 novel) up-regulated and 82 (72 known and 10 novel) down-regulated miRNAs from B-deficient roots, demonstrating remarkable metabolic flexibility of roots, which might contribute to the tolerance of plants to B-deficiency. A model for the possible roles of miRNAs in the tolerance of roots to B-deficiency was proposed. miRNAs might regulate the adaptations of roots to B-deficiency through following several aspects: (a) inactivating reactive oxygen species (ROS) signaling and scavenging through up-regulating miR474 and down-regulating miR782 and miR843; (b) increasing lateral root number by lowering miR5023 expression and maintaining a certain phenotype favorable for B-deficiency-tolerance by increasing miR394 expression; (c) enhancing cell transport by decreasing the transcripts of miR830, miR5266 and miR3465; (d) improving osmoprotection (miR474) and regulating other metabolic reactions (miR5023 and miR821). Other miRNAs such as miR472 and miR2118 in roots increased in response to B-deficiency, thus decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots.

Conclusions: Our work demonstrates the possible roles of miRNAs and related mechanisms in the response of plant roots to B-deficiency.

Figure 1

Relative abundances of selected known miRNAs in B-deficient and control roots revealed by qRT-PCR. Bars represent mean ± SD (n = 3). Significant differences were tested between control and B-deficient roots for the same miRNA. Different letters above the bars indicate a significant difference at P < 0.05. All the values were expressed relative to the control roots.

Figure 2

GO of the predicted target genes for 122 (22) differentially expressed known (novel) miRNAs. Categorization of miRNAs target genes was performed according to cellular component (A), molecular function (B) and biological process (C).

Figure 3

Effects of B-deficiency on root concentrations of anthocyanin (A) and flavonoids (B). Bars represent mean ± SD (n = 7). Significant differences was tested between B-deficient and control roots. Different letters above the bars indicate a significant difference at P < 0.05.

Figure 4

Effects of B-deficiency on proline concentration (A) and proline dehydrogenase activity (B) in roots. Bars represent mean ± SD (n = 4). Different letters above the bars indicate a significant difference at P < 0.05.

Figure 5

Effects of B-deficiency on GDH-NAD activity (A) and GDH-NADH activity (B) in roots. Bars represent mean ± SD (n = 4 or 6). Different letters above the bars indicate a significant difference at P < 0.05.

Figure 6

A proposed model for the possible roles of miRNAs in the tolerance of Citrus sinensis roots to B-deficiency.