OsmiRNA5488 Regulates the Development of Embryo Sacs and Targets OsARF25 in Rice (Oryza sativa L.)

Abstract

Small RNAs are a class of non-coding RNAs that typically range from 20 to 24 nucleotides in length. Among them, microRNAs (miRNAs) are particularly important regulators for plant development. The biological function of the conserved miRNAs has been studied extensively in plants, while that of the species-specific miRNAs has been studied in-depth. In this study, the regulatory role of a rice-specific OsmiRNA5488 (OsmiR5488) was characterized with the miR5488-overexpressed line (miR5488-OE) and miR5488-silenced line (STTM-5488). The seed-setting rate was notably reduced in miR5488-OE lines, but not in STTM-5488 lines. Cytological observation demonstrated the different types of abnormal mature embryo sacs, including the degeneration of embryo sacs and other variant types, in miR5488-OE lines. The percentage of the abnormal mature embryo sacs accounted for the reduced value of the seed-setting rate. Furthermore, OsARF25 was identified as a target of OsmiR5488 via RNA ligase-mediated 3'-amplifification of cDNA ends, dual luciferase assays, and transient expression assays. The primary root length was decreased with the increases in auxin concentrations in miR5488-OE lines compared to wild-type rice. Summarily, our results suggested that OsmiR5488 regulates the seed-setting rate and down-regulates the targeted gene OsARF25.

Keywords: IAA; embryo sac; miRNA; rice; seed-setting rate.

Figure 1

qRT-PCR analysis of relative expression levels of OsmiR5488 in different tissues from wild-type Nipponbare (NIP). Data are mean ± s.e.m. (n = 3 plants each with three technical replicates).

Figure 2

Phenotype, expression level of OsmiR5488, and seed-setting rate of the transgenic lines of miR5488-OE and STTM-5488. (A) Similar phenotypes among WT, miR5488-OE, and STTM-5488 plants at the mature growth stage. (B) Seed-setting rate of WT, miR5488-OE, and STTM-5488 lines. Data are means (±s.e.m.) (n = 20 plants, **, p < 0.01). (C) The change in expression level of OsmiR5488 in embryo sacs of WT, miR5488-OE, and STTM-5488 lines.

Figure 3

Analysis of the fertility of mature pollens and embryo sacs in miR5488 transgenic lines. (A–E) Pollen fertility; green arrow indicated the sterile pollen; bar = 10 μm. (F–J) Pollen vitality; green arrow indicated the non-vitalitious pollen; bar = 10 μm. (K) Normal embryo sac. (L) Degeneration of embryo. (M) Abnormal number of polar nuclei. (N) Abnormal embryo sac with degenerated egg apparatus. (O) Small embryo sac. Bar = 40 μm in (K–O). (P) Percentage of pollen fertility. (Q) Percentage of pollen vitality. (R) Percentage of normal or abnormal embryo sac.

Figure 4

The down-regulation of OsARF25 by OsmiR5488. (A) The change in expression levels of OsARF25 in root. (B) The change in expression levels of OsARF25 in mature embryo sac. Asterisks indicated significant differences by Student’s t-test, **, p < 0.01.

Figure 5

The identification of OsARF25 as a targeted gene by OsmiR5488. (A) Gene structures of OsARF25 and the cleavage site of OsARF25 confirmed by sequencing 8 clones out of 16. (B) Schematic diagram of OsmiR5488 or OsARF25 constructs for dual LUC assay. (C) Dual LUC assays indicated that OsARF25 was targeted by OsmiR5488. The RLUC/FLUC values were obtained with at least five replicates. **, p  <  0.01 by Student’s t test. (D) Schematic diagram of OsmiR5488 or OsARF25 constructs for transient expression assays. (E) The GFP transient expression assays showed that OsARF25 was targeted by OsmiR5488. Bars: 40 μm.

Figure 6

Comparison of the response of primary root length to IAA between miR5488-OE and wild-type Nip lines. (A) Primary root length of Nip after 4-day treatment with 10 μM IAA. Bar, 1 cm. (B) Primary root length of miR5488-OE after 4-day treatment with 10 μM IAA. Bar, 1 cm. (C) Statistical comparison of primary root length between WT and miR5488-OE after 4-day treatment with different concentrations of IAA. Values were shown as the mean ± SD of 20–30 seedlings, The experiment was repeated at least three times with similar results, **, p < 0.01.