Plant growth must be regulated throughout the plant life cycle. The myeloblastosis (MYB) transcription factor (TF) family is one of the largest TF families and is involved in metabolism, lignin biosynthesis, and developmental processes. Here, we showed that OsMYB14, a rice R2R3-MYB TF, was expressed in leaves and roots, especially in rice culm and panicles, and that it localized to the nucleus. Overexpression of OsMYB14 (OsMYB14-ox) in rice resulted in a 30% reduction in plant height compared to that of the wild type (WT), while the height of the osmyb14-knockout (osmyb14-ko) mutant generated using the CRISPR/Cas9 system was not significantly different. Microscopic observations of the first internode revealed that the cell size did not differ significantly among the lines. RNA sequencing analysis revealed that genes associated with plant development, regulation, lipid metabolism, carbohydrate metabolism, and gibberellin (GA) and auxin metabolic processes were downregulated in the OsMYB14-ox line. Hormone quantitation revealed that inactive GA19 accumulated in OsMYB14-ox but not in the WT or knockout plants, suggesting that GA20 generation was repressed. Indole-3-acetic acid (IAA) and IAA-aspartate accumulated in OsMYB14-ox and osmyb14-ko, respectively. Indeed, real-time PCR analysis revealed that the expression of OsGA20ox1, encoding GA20 oxidase 1, and OsGH3-2, encoding IAA-amido synthetase, was downregulated in OsMYB14-ox and upregulated in osmyb14-ko. A protein-binding microarray revealed the presence of a consensus DNA-binding sequence, the ACCTACC-like motif, in the promoters of the OsGA20ox1 and GA20ox2 genes. These results suggest that OsMYB14 may act as a negative regulator of biological processes affecting plant height in rice by regulating GA biosynthesis and auxin metabolism.

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of β-glucosidases. Evaluation of previously characterized recombinant rice β-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher β-glucosidase activities on ABA-GE and gibberellin A₄ glucose ester (GA₄-GE). The β-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular β-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP‐binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%–100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two‐nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane‐localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5‐ to 2‐fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds.

Efficient sucrose transport and metabolism are vital for seed and pollen development in plants. Cell wall invertases (CINs) hydrolyze sucrose into glucose and fructose, maintaining a sucrose gradient in the apoplast of sink tissues. In rice, two CIN isoforms, OsCIN1 and OsCIN2, were identified as being specifically expressed in the anthers but not in pollen. Functional analyses through genetic crosses and mutant characterization showed that oscin1/2 double mutants exhibit a sporophytic male-sterile phenotype and produce shrunken seeds. This suggests that CIN activity is essential for proper pollen development and seed formation in rice. Observation of the progeny genotypes and phenotypes from various genetic crosses revealed that the phenotype of oscin1/2 seeds is determined by the genotype of the maternal tissue, indicating the critical role of CIN function in the apoplast between maternal and filial tissues for sucrose transport and metabolism. The CIN activity in the anthers and seeds of wild-type rice was found to be significantly higher—over 500-fold in the anthers and 5-fold in the seeds—than in the leaves, highlighting the importance of CIN in facilitating the efficient unloading of sucrose. These findings suggest that the fine-tuning of CIN activity in the apoplast, achieved through tissue-specific expression and CIN isoform regulation, plays a key role in determining the carbohydrate distribution across different tissues. Understanding this regulatory mechanism could provide opportunities to manipulate carbohydrate allocation to sink organs, potentially enhancing crop yields.

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.