A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice

Abstract

Hybrid rice has greatly contributed to the global increase of rice productivity. A major component that facilitated the development of hybrids was a mutant showing photoperiod-sensitive male sterility (PSMS) with its fertility regulated by day length. Transcriptome studies have shown that large portions of the eukaryotic genomic sequences are transcribed to long noncoding RNAs (lncRNAs). However, the potential roles for only a few lncRNAs have been brought to light at present. Thus, great efforts have to be invested to understand the biological functions of lncRNAs. Here we show that a lncRNA of 1,236 bases in length, referred to as long-day-specific male-fertility-associated RNA (LDMAR), regulates PSMS in rice. We found that sufficient amount of the LDMAR transcript is required for normal pollen development of plants grown under long-day conditions. A spontaneous mutation causing a single nucleotide polymorphism (SNP) between the wild-type and mutant altered the secondary structure of LDMAR. This change brought about increased methylation in the putative promoter region of LDMAR, which reduced the transcription of LDMAR specifically under long-day conditions, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PSMS. Thus, a lncRNA could directly exert a major effect on a trait like a structure gene, and a SNP could alter the function of a lncRNA similar to amino acid substitution in structural genes. Molecular elucidating of PSMS has important implications for understanding molecular mechanisms of photoperiod regulation of many biological processes and also for developing male sterile germplasms for hybrid crop breeding.

Fig. 1.

Mapping and cloning of the pms3. (A) Genetic and physical maps of pms3. (B) Polymorphisms identified by comparative sequencing of a 28.4-kb mapping region in 58S, DH80, and 58N. The triangle represents an insertion, and vertical thin bars indicate single nucleotide substitutions relative to 58N. (C) Predicted genes in the pms3 region according to the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/). (D) Genomic fragments used for making constructs for transformation. (E) Transcripts in both strands detected in the target region. The black arrows represent exons and the dotted lines represent introns. The quadrangular legend represents the single SNP between 58N and 58S.

Fig. 2.

Expression analysis of transcripts identified in the pms3 region. (A) Expression profile of transcripts in 58N (tissue description in SI Appendix, Table S2). Numbers after gene names represent the number of cycles in RT-PCR amplification. (B) Comparative expression analysis of the transcripts between 58N and 58S under both long-day (LD) and short-day (SD) conditions in young panicles at four developing stages: stage3, secondary branch primordium differentiation; stage4, pistil/stamen primordium differentiation; stage5, pollen mother cell formation; and stage6, pollen meiosis. All three transcripts were quantified using a common check. Significant differences detected between LD-58N and LD-58S are indicated by **P < 0.01 or *P < 0.05, respectively. Data are means ± SEM (n = 4). (C) Diurnal expression patterns of Transcript-1 under LD and SD conditions in 58N and 58S. The black bars indicate the dark period, and the white bars indicate the light period. The numbers on the Top of the bars indicate hours of the day. Data are means ± SEM (n = 4). (D) DNA methylation levels (%) of the 1.5-kb genome region, including 450-bp putative promoter and the entire LDMAR region (1,050 bp), from young panicles of 58N and 58S grown under LD conditions. (E) DNA methylation levels (%) of the 500-bp genome region (−200 bp to +300 bp of LDMAR) from leaves of plants grown under LD conditions and young panicles of plants under SD conditions, for 58N and 58S.

Fig. 3.

The effect of overexpressing Transcript-1 (LDMAR) on male fertility. (A) Expression level of Transcript-1 (Top) in transgene-positive and -negative plants (Bottom) in a T₁ family. Data are means ± SEM (n = 3). (B) Fertility scores of transgene-positive and -negative plants in three T₁ families. There are 58 positive and 26 negative segregants from three T₁ families. Data are means ± SEM. (C) Whole plants of transgene-negative (Left) and -positive (Right) segregants from T₁ family. (D) Anther of transgene-negative (Left) and -positive (Right) plants. (Scale bars, 0.5 mm.) (E) Panicles of transgene-negative (Left, mostly sterile spikelets) and -positive (Right, mostly fertile) plants. (F) Pollen grains of transgene-negative (Top, sterile) and -positive (Bottom, fertile) plants. (Scale bars, 50 μm.)