Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions

Abstract

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

Keywords: Arabidopsis; Zea mays L.; extracellular vesicles; microRNA; phytochrome; signal transduction.

Figure 1

Relative levels of the transcripts of miR165a (A), AGO1 (B), and AGO10 (C) in maize leaves under different light conditions. Abbreviations: R, irradiation by red light; FR, irradiation by far-red light; and R + FR, irradiation by far-red light after red light. The data represent the means of three biological repeats ± SD (p ≤ 0.05), different letters indicate significant differences.

Figure 2

Contents of the transcripts of pri-miR165a (A) and pre-miR165a (B) in maize leaves under different light conditions. Abbreviations are the same as in Figure 1. The data represent the means of three biological repeats ± SD (p ≤ 0.05), different letters indicate significant differences.

Figure 3

Relative levels of miR165 in leaves of A. thaliana plants under different light conditions: wild-type (A), phytochrome A knockout (B), and phytochrome B knockout (C). Abbreviations are the same as in Figure 1 and Figure 2. The data represent the means of three biological repeats ± SD (p ≤ 0.05), different letters indicate significant differences.

Figure 4

Phytochrome-dependent changes in the interfering complex with miR165a (A), and distribution of the interfering complex with miR165a (B), in maize leaf cells. Dark blue spots are nuclei; orange color corresponds to the fluorescent glow of the miR165a + ROX probe.

Figure 5

Assessment of the formation of the DNA-DNA-miR165a triplex structure in maize leaf cells upon irradiation of plants by light of different wavelengths.

Figure 6

Typical images of extracellular vesicle fractions P40 and P100 taken using Olympus CX41 microscope with 1000× magnification. P40 is a fraction of vesicles obtained after differential centrifugation at a speed of 40,000 g, having a size of >0.170 µm. P100 is a fraction of vesicles isolated after centrifugation at a speed of 100,000 g and having a size of <0.147 µm.

Figure 7

The content of miR165a in vesicles P40 (A) and P100 (B) of maize leaves under different light conditions. Light gray color—on the surface of vesicles; orange color—inside the vesicles. Abbreviations are the same as in Figure 1, Figure 2 and Figure 3. The data represent the means of three biological repeats ± SD (p ≤ 0.05).

Figure 8

Changes in the content of mature miR165a in maize leaf cells upon irradiation with light of different wavelengths. Mechanism of formation of mature miR165a in maize leaves under irradiation with light of different wavelengths is not associated with a change in the intensity of synthesis of miR165a from its precursors. The absence of changes in the amount of pri- and pre-miRNAs when plants are irradiated with red and far-red light indicates that the transcriptional activity of the gene of this microRNA and the precursor gene do not change under these conditions. Fluctuations in the content of free miR165a in maize leaf cells may be associated with its participation in the process of RNA interference during the formation of the RISC-miR165a complex.

Figure 9

The mechanism of regulation of the light-dependent miR165a content via the phytochrome system.

Figure 10

Participation of miR165a in phytochrome signal transduction in maize leaves upon changes in light conditions.