Functional validation of choline monooxygenase genes in banana A and B genomes

【Objective】Osmotic stress is a major environmental constraint that severely limits plant growth, development, and agricultural productivity. Glycine betaine (GB), a compatible osmolyte synthesized in response to abiotic stresses, plays a pivotal role in osmotic adjustment, with choline monooxygenase (CMO) acting as the rate-limiting enzyme in its biosynthetic pathway. Banana (Musa spp.), a globally significant crop with diverse genomic compositions derived from M. acuminata (A genome) and M. balbisiana (B genome), exhibits genotype-specific variations in stress tolerance. Previous studies have identified structural and functional differences in CMO genes across banana genotypes, suggesting potential divergence in their roles during stress adaptation. This study aims to functionally validate CMO genes derived from the A and B genomes of banana, elucidate their contributions to osmotic stress tolerance, and explore the genomic basis for stress resilience in different banana genotypes. 【Methods】Four banana genotypes [Zhanjiang AA (AA genome), Brazilian banana (AAA genome), Guangdong plantain (AAB genome), and Fenjiao (ABB genome)] were selected for their distinct genomic compositions. Tissue- cultured seedlings at the five- leaf stage were used for gene cloning and stress treatments. CMO genes were amplified using genotype-specific primers designed based on priorsequencing data. The four CMO gene coding sequences (CMO- A, CMO- H, CMO- B1 and CMO- B2) were recombined into the pYES2-NTB yeast expression vector and the pGFPGUSplus plant overexpression vector by homologous recombination method. The yeast transformation experiment was carried out by lithium acetate conversion method. Yeast functional complementation assays were conducted using INVSC1 (for osmotic, salt, and low-temperature stress) and ycf1 (for heavy metal stress) strains. Transgenic Nicotiana benthamiana lines overexpressing banana CMO genes were generated through Agrobacterium-mediated transformation (GV3101 strain) and validated by PCR and hygromycin resistance screening. For stress assays, yeast transformants were grown in SG-Ura media containing 1.0 mol· L- 1 NaCl (salt stress), 1.0 mol·L-1 mannitol (osmotic stress), 40 µmol·L-1 CdCl2 (heavy metal stress), or incubated at 18 ℃ (low-temperature stress). Growth was monitored via serial dilution spot assays. In tobacco, seed germination rates and root elongation were evaluated under 150 mmol · L- 1 mannitol-induced osmotic stress, with or without 20 mmol · L- 1 exogenous GB pretreatment. Subcellular localization of CMO-GFP fusion proteins was transiently expressed in tobacco leaves, and visualized via confocal microscopy using chloroplast autofluorescence (640 nm excitation).【Results】Overexpression of banana CMO genes significantly enhanced yeast tolerance to multiple stresses. Under 1.0 mol· L- 1 NaCl, 1.0 mol · L- 1 mannitol, and 18 ℃ conditions, when INVSC1 strains expressing CMO-A (AA genome), CMO-H (hybrid A/B genome), CMO-B1, and CMO-B2 (B genome) were compared with the empty vehicle control, the survival rate of INVSC1-CMO recombinant yeast significantly increased. No significant differences were observed among the four CMO variants under these stresses. Under 40 µmol·L-1 CdCl2, ycf1 strains expressing CMO-H and CMO-B1 showed superior growth, and colony growth was significantly better than that of ycf1-CMO-A and ycf1-CMO-B2 recombinant yeasts, respectively, indicating enhanced heavy metal tolerance linked to B genome- derived CMO variants. Osmotic stress (150 mmol·L-1 mannitol) reduced seed germination rates in wild-type (WT) tobacco to 42.3%, whereas transgenic lines overexpressing CMO- B1 and CMO- B2 maintained germination rates of 68.9% and 71.2%, respectively—a 30%-33% improvement over WT. Exogenous GB further increased germination rates to 85%-89% in transgenic lines, demonstrating synergistic effects between endogenous and exogenous GB. Root elongation under osmotic stress revealed genotype-specific effects: CMO-B1 and CMO-B2 transgenic seedlings exhibited root lengths of 2.57 cm, 1.43- fold longer than WT (1.80 cm), whereas CMO-A and CMO-H lines showed no significant improvement (2.03-2.07 cm). Confocal microscopy confirmed that all four CMO-GFP fusion proteins were localized exclusively to chloroplasts, as evidenced by complete overlap with chloroplast autofluorescence. This finding aligns with the role of CMO in chloroplastic GB biosynthesis.【Conclusion】B genome- derived CMO genes (CMO- B1/B2) confer superior osmotic/heavy metal stress tolerance compared to A genome variants, highlighting functional diversification shaped by genomic ancestry. Chloroplastic localization underscores their role in stress-responsive GB synthesis. These findings identify CMO-B1/B2 as key targets for breeding stressresilient banana cultivars and provide a molecular framework for understanding A/B genome functional divergence. This study pioneers the functional dissection of CMO allelic diversity in banana, linking genomic ancestry (A vs B) to stress-responsive gene performance. The development of a dual-model validation system (yeast and tobacco) provides a scalable framework for screening stress-tolerance genes in polyploid crops. Additionally, the chloroplast-specific localization of CMO resolves long-standing ambiguities about its subcellular activity in monocots, offering insights for engineering GB biosynthesis pathways in non-accumulator species.