Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage

Abstract

The cleistogamous flower sheds its pollen before opening, forcing plants with this habit to be almost entirely autogamous. Cleistogamy also provides a means of escape from cereal head blight infection and minimizes pollen-mediated gene flow. The lodicule in cleistogamous barley is atrophied. We have isolated cleistogamy 1 (Cly1) by positional cloning and show that it encodes a transcription factor containing two AP2 domains and a putative microRNA miR172 targeting site, which is an ortholog of Arabidopsis thaliana AP2. The expression of Cly1 was concentrated within the lodicule primordia. We established a perfect association between a synonymous nucleotide substitution at the miR172 targeting site and cleistogamy. Cleavage of mRNA directed by miR172 was detectable only in a noncleistogamous background. We conclude that the miR172-derived down-regulation of Cly1 promotes the development of the lodicules, thereby ensuring noncleistogamy, although the single nucleotide change at the miR172 targeting site results in the failure of the lodicules to develop properly, producing the cleistogamous phenotype.

Fig. 1.

The lodicules of cleistogamous and noncleistogamous barley. (A) The lodicule [lo] located at the base of the stamen [st] open the spikelet by pushing apart the lemma [le] and palea [pa]. (B) A noncleistogamous and (C) a cleistogamous barley spike at anthesis. The lodicules of (D) a noncleistogamous and (E) a cleistogamous barley. A section of the spikelet in (F) a noncleistogamous and (G) a cleistogamous barley. The carpel [cp] is surrounded by the other floral organs. (Scale bars: D and E, 800 μm; F and G, 200 μm.)

Fig. 2.

Positional cloning of Cly1. (A) A genetic map of the critical region of the long arm of barley chromosome 2H constructed from a Azumamugi (AZ) x Kanto Nakate Gold (KNG) F₂ population. Barley EST sequence BF623536 is homologous to that of rice AP2. BACs M191K21 and M601E22 harbor Cly1, which encodes a transcription factor with two AP2 domains and a putative miR172 targeting site. The AZ and KNG Cly1 sequences differ from one another by a single nucleotide site within the target site (responsible for the Cly1.a and cly1.b alleles) and by a second nucleotide located upstream of the coding region (P101AP25’). (B) Sequence comparisons between alleles of the putative miR172 target site indicate that it is variation at the second and/or third variable positions (red stars), rather than at the first position (blue star), which is responsible for cleistogamy. (C) Lodicule size at the yellow anther stage and at anthesis in a 274 accession germplasm set. Noncleistogamous cultivars are marked by blue spots, and cleistogamous ones by red spots.

Fig. 3.

Expression of Cly1. (A) The 3′ end of Cly1, including Exon 10 which carries the putative miR172 target site (red bar). The two regions targeted by RT-PCR shown by arrows. The zigzag line indicates the 5′ RACE RNA oligo-adapter ligated to cleaved mRNA. The probe used for in situ hybridization is also shown. (B) Cly1 expression in an immature spike at the awn primordium stage of the noncleistogamous cultivar AZ, as detected by in situ hybridization with an anti-sense (Left) and sense (Right) Cly1 3′ UTR transcript. (C) A higher magnification of the presentation shown in (B). [lo] lodicule, [st] stamen. (D) Cly1 expression in the cleistogamous cultivar KNG. (E) Cly1 expression in an immature spike during development. The constitutively expressed actin gene was used as a control. Dark gray bars represent AZ and light gray bars KNG. The numbers below each bar refer to the developmental stage assayed (1, lemma primordium stage; 2, stamen primordium stage; 3, awn primordium stage; 4, white anther stage; 5, green anther stage; 6. yellow anther stage; 7, spike at anthesis). (F) Modified 5′ RACE ligations from (A) were amplified by nested PCR. (G) Cleavage at the miR172 site within Cly1. The 5′ termini of miR172-guided mRNA cleavage were determined by cloning and sequencing of the amplicon generated in (F). A rice miR172 sequence was aligned with the Cly1 mRNA sequences. Vertical arrows indicate the inferred 5′ termini of miR172-guided cleavage, and the number above each arrow indicates the proportion of clones showing these sites. (Scale bars: B, 500 μm; C and D, 250 μm.).

Fig. 4.

A phylogenetic analysis of the Cly1 sequence suggests that the KNG (cleistogamous) allele cly1.b evolved from the AZ (noncleistogamous) Cly1.a allele. The immediate ancestor of SV230 type cleistogamous cultivars remains unknown. Hordeum vulgare ssp. spontaneum accession OUH602 was used as the outgroup to construct this phylogeny.