miR172b–SNB Axis That Keeps Rice Flowers Closed Offers Clues for Cleistogamous Breeding
Background: The genetic basis of rice self-fertilization without flower opening
A study reports that when the microRNA miR172b is depleted in rice, regulation that suppresses SNB transcript accumulation is lifted, the lodicules that open the flower become smaller, and cleistogamy occurs. Researchers identified this regulatory axis by comparing a natural mutant with gene-edited lines. However, how much it reduces cross-pollination under actual field conditions has not yet been established.
Cleistogamy is self-fertilization while the flower remains closed. In rice and other grasses, lodicules swell during flowering and open the flower. When their development or swelling is restricted, the flower stays closed. This trait could help maintain varietal purity and reduce unintended gene flow.
Some previously identified cleistogamous rice mutants lose the trait at low temperatures or have lower seed-setting rates. This study focused on the natural mutant “lodiculeless spikelet” (ld), which was reported to have no clear disadvantages in major agronomic traits. The central question was what alters lodicule development.
Key findings: Linking MIR172b loss of function to release of SNB repression
Researchers crossed ld in the Ilpum rice background with Milyang 23, then analyzed 1,027 F2 plants and 1,673 F3 plants to narrow down the causal region. Sequence comparisons revealed a 4,572-base-pair deletion that included the entire MIR806a locus and the promoter region regulating MIR172b expression. To distinguish which of the two loci caused the trait, they separately edited each with CRISPR/Cas9. Cleistogamy was reproduced in lines with impaired MIR172b function, whereas lines lacking MIR806a developed normal lodicules.
Small RNA analysis of young panicles also showed that miR172b in ld had fallen to background detection levels. The same reduction was not found in other miR172 types. Of five APETALA2 (AP2)-family genes with miR172 target sites, only the SUPERNUMERARY BRACT (SNB) transcript was significantly increased in young panicles. This supports a link between release of SNB repression following miR172b deficiency and abnormal lodicule development.
To further test causality, researchers used prime editing to alter the miR172-binding site in SNB. They preserved the protein’s amino acid sequence while making the transcript evade miR172 repression. These PE-rSNB lines likewise showed increased SNB transcript levels and reduced lodicules. Tissue staining with a reporter gene showed overlapping MIR172b and SNB expression domains in the lodicules, supporting a regulatory relationship between the two genes.
Implications and outlook: Distinguishing mechanistic validation lines from breeding candidates
PE-rSNB also showed a defect in which the panicle failed to emerge normally from the flag leaf sheath. Researchers suggested that the strength or tissue-specific extent of SNB derepression may have differed from that in ld. Editing the SNB target site was useful for validating the mechanism, but this defect would need to be considered for breeding applications.
By contrast, in the ld lines examined, plant height, tiller number, and mature seed number per panicle were similar to those of the respective wild types. Grains became slightly shorter and wider, but thousand-grain weight—the weight of 1,000 grains—did not differ significantly. These findings support MIR172b loss-of-function alleles as potential resources for breeding cleistogamous rice. They do not establish that yields will be the same in every environment.
Which downstream genes SNB regulates, and whether it changes cell division, expansion, or identity, remain unresolved. The effect of cleistogamy on limiting gene flow also requires direct field evaluation. This study identifies a genetic target for controlling flower opening and a breeding candidate. Methods and results are available in the full Plant Physiology paper.
Cleistogamy, or self-fertilization of a closed flower, can limit unintended gene flow and may contribute to varietal purity in rice (Oryza sativa), with potential value for transgene containment. We previously identified the natural cleistogamous mutant lodiculeless spikelet (ld). Here, using map-based cloning, we show that ld carries a 4.6-kb deletion encompassing the entire MICRORNA806a (MIR806a) precursor and the upstream region of the MIR172b locus. CRISPR/Cas9-mediated editing of the MIR806a or MIR172b locus demonstrated that the loss of accumulation of miR172b, but not miR806a, is responsible for the cleistogamy phenotype of ld plants. Small RNA sequencing confirmed that miR172b is nearly absent in ld mutants. Among the five APETALA 2 (AP2)-like genes harboring miR172b target sites, only SUPERNUMERARY BRACT (SNB) transcripts accumulated to significantly higher levels in young ld panicles than the wild type. Prime editing of the miR172-binding site in SNB generated a miR172-resistant SNB transcript isoform that reproduced the vestigial lodicule phenotype, indicating that the derepression of SNB transcript accumulation is sufficient to alter lodicule development. Histological analysis of rice harboring the GUS reporter gene driven by the MIR172b or SNB promoter revealed a strong overlap between the MIR172b and SNB expression domains in developing lodicules, supporting their regulatory relationship. Together, these results identify the miR172b-mediated repression of SNB transcript abundance as an important regulatory module for lodicule development and cleistogamy in rice. The agronomically neutral ld alleles represent valuable genetic resources for developing cleistogamous rice cultivars.
Breeding cleistogamous rice requires assessing the closed-flower trait alongside normal panicle development and seed production. This study provides a basis for introducing MIR172b loss-of-function alleles into candidate varieties and selecting lines that retain major agronomic traits. In lines with direct editing of the miR172-binding site in SNB, a defect prevented panicles from emerging from the leaf sheath, showing that outcomes can vary with the target and scope of regulation even within the same regulatory axis.
A practical application would be to select candidate lines and then assess both cross-pollination rates and agronomic traits under actual field conditions. Cleistogamy may help maintain varietal purity or reduce transgene flow, but there is not yet a basis for concluding that cleistogamy alone can prevent gene flow completely.