Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Abstract

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.

Figure 1

Molecular mapping of tms12-1(t). (A) Fine mapping of tms12-1(t) to a 5.8-kb region on chromosome 12. (B) Predicted gene/ORF composition of the mapped region in a BAC sequence, in which the only mutation within the mapped regions is indicated. (C) Fragments used for genetic complementation tests, of which PA-0.37 was linked to the promoter of the maize ubiquitin gene (Pubi).

Figure 2

Pollen fertility of transgenic NK58S and PA64S plants is restored by TMS12-1(t). (A, B) Fertile pollen of NK58S and PA64S plants grown under short-day (12 h) and low-temperature (22-23 °C) conditions during the MMC-meiosis stages. (C, D) Sterile pollen of NK58S and PA64S plants grown under sterility-inducing conditions (14-h day length, 25-30 °C). (E, F) Fertility-restored pollen of PA-10.4- and PA-9-transgenic T₁ NK58S plants, respectively. (G, H) Fertility-restored pollen of PA-2.4-transgenic T₁ plants of NK58S and PA64S, respectively. (I, J) Fertility-restored pollen of transgenic T₁ NK58S (I) and T₁ PA64S (J) plants overexpressing a 375-bp sequence (PA-0.37). All transgenic plants were grown under sterility-inducing conditions (14-h day length, 25-30 °C). Scale bars: 50 μm.

Figure 3

Identification of the small RNA and its precursor. (A) Confirmation of the small RNAs in NK58, NK58S, and overexpressing (P/TMS12-1-OE) plants by 3′ RACE and 5′ RACE assays and sequencing, by which the 5′ and 3′ termini of the small RNAs (shown by rectangles) were determined. The RNA samples were treated with poly(A) polymerase to polyadenylate the 3′ end of small RNAs for reverse transcription with a poly dT-adapter primer. The 5′ part (5′-ACTCACT-3′) of the specific primer is an adapted arbitrary sequence. (B) Identification of the 5′ and 3′ termini of the precursor RNA in an overexpressing plant (P-2 shown in Figure 5C) by 5′ and 3′ RACE assays and sequencing. (C) The 375-bp sequence from P/TMS12-1 used for overexpression in NK58S and PA64S, in which the transcribed sequence generating the processed intermediate RNA precursor (positions 106-241) is shown in italic and that generating the small RNA is in capital letters. Specific primers used for semi-nested PCR of the 5′ or 3′ RACE product are shown.

Figure 4

Prediction of the secondary structures of the small RNA precursors. (A) The secondary structure of the precursor from P/TMS12-1. (B) The secondary structure of the precursor from p/tms12-1. The small RNAs are located at positions 34-54 indicated by arrows and the SNP sites are shown by arrowheads. In secondary RNA structures, U residue can pair with G residue (shown with dots).

Figure 5

Expression patterns of osa-smR5864w and osa-smR5864m. (A) Expression patterns for osa-smR5864w in different organs of PA64. (B) Expression levels of osa-smR5864w (NK58 and PA64) and osa-smR5864m (NK58S and PA64S) in young panicles under sterility-inducing conditions. t-tests were performed between NK58 and NK58S and between PA64 and PA64S, with significant level of P < 0.01 (^**). (C) High levels of osa-smR5864w were detected in overexpressing transgenic plants (three independent plants were assayed for each of NK58S-OE and PA64S-OE). (D) Expression levels of osa-smR5864w and osa-smR5864m in young panicles of NK58 and NK58S under long-day (14 h, LD) and short-day (12 h, SD) conditions, respectively; and of PA64 and PA64S under high- temperature (27-30 °C, HT) and low-temperature (22-23 °C, LT) conditions (^* and ^** indicate P < 0.05 and P < 0.01, respectively). All qRT-PCR assays used the 5S RNA as an internal control for normalization, and the samples marked with S were used as standards (relative value=1) against which expression in the other samples was compared.