IC4R001-lncRNA-2012-22308482

Project Title

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

The Background of This Project

• Hybrid rice has greatly contributed to the global increase of rice productivity.Amajorcomponentthatfacilitatedthedevelopmentof 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.
• Photoperiod regulates a large array of processes underlying growth and development of plants. It has been known for centuries that flowering of plants is regulated by day length. In recent years, many genes for photoperiod-controlled flowering have been identified in a number of plants, leading to characterization of pathways in flowering regulation. However, photoperiod regulation of other developmental processes has been studied in far less detail than flowering. It has been reported in several plants that male fertility is also sensitive to photoperiod; acriticaldaylengthisrequiredfornormalpollendevelopment. However, nothing is known about the molecular control of the photoperiod regulation of male fertility, despite the tremendous progress in recent years in genetic and molecular understanding of male reproductive development in many plant species.

Figure 1 Mapping and cloning of the pms3.

• Hybridricehasgreatlycontributedtotheglobal increaseofrice productivity. A spontaneous mutant exhibiting photo-period-sensitive male sterility (PSMS) was found in 1973 in a japonica rice (Oryza sativa ssp. japonica) variety Nongken 58 (referred to as 58N) in Hubei Province, China. Intensive studies of the mutant (58S) established that its pollen fertility is regulated by day length; it is completely sterile when grown under long-day conditions, whereas pollen fertility varies when it is grown under short-day conditions (SI Appendix, Fig. S1); and the critical stage forphotoperiod induction comesbetween secondary branch differentiation and microsporogenesis during panicle development, suggesting that photoperiod regulation of PSMS is independent of photoperiod flowering. For hybrid rice breeding, PSMS rice can be used to propagate itself under short days, and to produce hybrid seeds by interplanting it with normal fertile lines under long-day conditions. Moreover, PSMS rice has a broad spectrum of restoration; all of the normal rice varieties tested can restore the fertility of F 1 hybrids. Two-line hybrids developed using this PSMS germplasm have occupied millions of hectaresofricefieldsmostlyinChinaformorethanadecade. PSMS germplasms have also been found and explored in several other crops.

Plant Culture & Treatment

• 58N and 1514 are japonica rice varieties. 58S is a spontaneous mutant from 58N and DH80 is a doubled haploid line derived from a cross between 58S and 1514. The rice plants examined under natural field conditions were grown in normal rice growing seasons in the Experimental Station of Huazhong Agricultural University, Wuhan, China. Following Zhang and Yuan, for natural long-day conditions, seeds of Nongken 58 and Nongken 58S as well as the transgenic plants were sown in a seed bed in mid-April, and seedlings were transplanted to the field in mid-May, ensuring the day-length at young panicle developing stage to be longer than 14 h. For natural short-day conditions, seeds were sown in mid-June, and seedlings transplanted to the field in mid-July, which placed young panicle developing stage at day-length of ~13.5 h.

[[File:|1000px|thumb|center|Figure 1 Box plots for relative phenotypic values. The median of each trait is shown as the horizontal bar in the box, and the upper and lower sides of a box represent the first and third quartile values of the distribution, respectively. Whiskers extended to 1.5 times the interquartile range (box size) or to the maximum/minimum values.]]

Research Findings

• It was determined previouslythat pms3 on chromosome 12 wasthe original mutation that changed 58N to 58S (40–44). Using an F 2 population of 7,000 plants from across between 58S and DH80,adoubled haploidline derived from a cross between 58S and 1514 (a normal japonica variety), which were examined for fertility under natural field long-day conditions in Wuhan, the pms3 locus was delimited to a 28.4 -kb DNA fragment between two molecular markers LJ25 and LK40 (Figure. 1A). We sequenced this 28.4-kb region and found seven polymorphic sites between 58N and DH80 and a SNP resulting from substituting guanine(G)in 58N with cytosine(C) in 58S (Figure.1B). We developed four additional molecular markers, which resolved pms3 to a 12-kb region between M1 and M4 (Figure. 1A).
• Although there were three predicted genes in the 28.4-kb region (LOC_12g36010, LOC_12g36020, and LOC_12g36030), the targeted 12-kb region contained only part of LOC_12g36030 (Figure. 1C). LOC_12g36030 encodes three alternatively spliced transcripts supported by full-length cDNAs, AK071577, AK100176, and AK111270(http://cdna01.dna.affrc.go.jp/cDNA/), each of which had two exons. The ORF was in the first exon that was shared among all three transcripts, whereas the second exon was specific to each transcript.
• We prepared four constructs containing overlapping genomic DNA fragments covering the 12-kb region by digesting clone 117H5 froma BAC library of 58N constructed in our laboratory (Figure. 1D) and subcloning into the vector pCAMBIA1301. We transformed the constructs into 58S. We also overexpressed the three alternative transcripts (AK071577, AK100176, and AK111270) in both 58N and58S(SIAppendix,Fig.S2A).Atleast 15independent T 0 plants were produced for each of the construct/genotype combinations. Surprisingly, no statistically significant difference was detected in spikelet fertility between the transgene-positive and-negative plants in the T 0 and/or T 1 progenies under long-day conditions in any of the construct/genotype combinations.
• Because the targeted 12-kb region contained only part of LOC_12g36030 (Figure. 1 D and E), we speculated that there might be other transcripts from this region that regulate PSMS.To check this possibility, we designed 24 pairs of oligonucleotide primers to cover the genomic region ∼3kb in length surrounding the SNP site. Three unique transcripts were identified from the two strands (Figure.1E),which we named Transcript-1 to -3. RT-PCR showed that none of the three transcripts had an intron. Transcript-1 and -2 were transcribed from the sense strand relative to AK111270. Transcript-1 was 1,236 bases in length, and its 5′ end had an 110-base overlap with the 3′ end of AK111270. The 1,268-base sequence of Transcript-2 was located upstream of Transcript-1 and overlapped with the second exon of AK111270 in its entire length. Transcript-3, 1,088 bases in length, was transcribed from the complementary strand, overlapping with Transcript-1 by 688 bases. Thus, this region had a complex transcription pattern producing multiple partial overlapping transcripts. This complexity may also explain why transformation with the genomic fragments could not recover male fertility.

Labs working on this Project

• National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research
• College of Life Science and Technology, Huazhong Agriculture University, Wuhan 430070, China

Corresponding Author

• Qifa Zhang (qifazh@mail.hzau.edu.cn)