In a recent study published in BMC Plant Biology in 2025, researchers explored how natural tetraploidization affects Changshan Huyou (Citrus changshan-huyou), a citrus species native to China that is valued for its hardiness and medicinal properties.
The study compared tetraploid (4X) scions with conventional diploid (2X) plants and revealed significant differences in plant structure, metabolism, and gene expression.In simple terms, tetraploidization doubles the number of chromosomes, which can lead to larger cells and modified traits.
Confirmation of Tetraploidization
The investigation began with the confirmation that a naturally occurring Changshan Huyou seedling had become tetraploid. The researchers used flow cytometry—a technique that measures the DNA content of cells—to determine the ploidy level.
They observed that the tetraploid cells displayed a fluorescence intensity peak of about 50, which is double the value recorded for the diploid control. Furthermore, since the full genome of Changshan Huyou was unavailable, the scientists performed genome resequencing using a closely related citrus species as a reference.
The analysis showed that the diploid and tetraploid plants shared approximately 73.96% of their genetic sequence, strongly suggesting that the tetraploid emerged from a natural doubling of the diploid genome. These initial confirmations were crucial as they set the foundation for the subsequent comparison of the plants’ traits.
Morphological Changes in Changshan Huyou
When examining the physical traits of the plants, several clear differences emerged between tetraploid and diploid specimens. For instance, the tetraploid plants exhibited rounder and thicker leaves than the diploids. Detailed measurements indicated that the average leaf width in tetraploids was about 6.47 centimeters, with a lower leaf index of 1.57.
Under the microscope, the tetraploid leaves showed a thicker upper epidermis, measured at approximately 16.07 micrometers, and larger palisade parenchyma cells, averaging around 79.58 micrometers.
In addition, the spongy parenchyma cells were significantly larger, with an average size of about 254.36 micrometers, and the midrib—essentially the leaf’s central vein—was notably wider, measuring around 1386.69 micrometers compared to 864.17 micrometers in diploids.
Although the density of stomata (the tiny openings on the leaf surface) was lower in tetraploids, each guard cell was longer and wider, which may help the plant regulate water loss more effectively.
Moreover, the flowers of the tetraploid plants exhibited larger petals, stamens, and ovules, though the number of these floral organs remained similar to that in diploids.
Pollen grains from tetraploids were also larger, even though their staining viability was slightly lower—about 85.03% viability with an in vitro germination rate of approximately 33.43%. Despite this small decline in pollen performance, the tetraploids maintain enough reproductive viability for breeding purposes.
Metabolic Reprogramming of Tetraploid Fruits
Another significant aspect of the study was the evaluation of the fruits’ chemical composition. The researchers used an advanced UPLC-MS/MS system to analyze three different fruit tissues—peels, juice sacs, and segment membranes—and identified a total of 2064 metabolites.
These metabolites were grouped into 13 primary categories, including amino acids, nucleotides, phenolic acids, flavonoids, lignans, and coumarins. Overall, the data revealed that many metabolites, especially flavonoids, lignans, and coumarins, were present in lower levels in the tetraploid fruits compared to the diploids.
For example, in the peels of the tetraploid fruits, most of the differentially accumulated metabolites (DAMs) were downregulated. However, not all changes were negative.
The study also identified a significant increase in some compounds: in the tetraploid peels, 2-quinoline increased by 10.2-fold, L-asparagine by 4.1-fold, and the terpenoid deacetylnomilin by 2.84-fold. Similarly, in the juice sacs, important flavonoids such as neohesperidin, hyperin, and rhamnetin-3-O-glucoside were upregulated by approximately 2.45-fold, 2.46-fold, and 2.53-fold respectively.
In the segment membranes, other flavonoids as well as alkaloids like betaine (which increased by 2.55-fold) were found in higher concentrations. These shifts in metabolite levels indicate that natural tetraploidization triggers a reprogramming of the plant’s chemical pathways. Consequently, this affects not only the fruit’s taste and nutritional quality but also its potential medicinal properties.
Gene Expression and Its Impact on Metabolism
To understand the molecular basis behind these metabolic changes, the researchers conducted an extensive transcriptomic analysis. They generated a massive dataset of 125.18 gigabases of clean sequence data from 18 cDNA libraries, with over 89.22% of the bases scoring a Q30 or above—an indicator of high-quality sequencing.
The principal component analysis (PCA) of the gene expression data showed that the juice sacs of tetraploid and diploid fruits had similar profiles, whereas the peels and segment membranes displayed significant differences.
In total, 700, 422, and 514 differentially expressed genes (DEGs) were identified in the peels, juice sacs, and segment membranes respectively. Most of the DEGs in the peels and segment membranes were downregulated in tetraploids, while the juice sacs exhibited a predominance of upregulated genes.
Importantly, several key enzymes were found to be upregulated in the tetraploid fruits. For example, cytochrome P450 (CYP450), ferulate-5-hydroxylase (F5H), and flavonoid 3’-monooxygenase (F3’H) were significantly increased in the juice sacs, suggesting an enhanced activity in the flavonoid biosynthetic pathway.
Furthermore, Pearson correlation analysis revealed that the upregulation of genes encoding peroxidase and CYP450 was closely linked to the increased accumulation of certain amino acids and alkaloids in the tetraploid peels.
These findings provide a clear molecular explanation for the altered metabolite profiles and demonstrate that tetraploidization leads to a significant genetic reprogramming that affects both the structure and function of the fruits.
Implications for Citrus Breeding
The findings from this study have important practical applications in the field of citrus breeding. One of the primary challenges in citrus cultivation is the production of seedless fruits, which are more appealing to consumers and are easier to process.
Tetraploid plants are valuable in breeding programs because they can be crossed with diploid plants to produce triploid hybrids that are often seedless or contain very few seeds.
Despite a slight reduction in pollen performance, the tetraploid Changshan Huyou maintained a pollen viability of around 85.03% and a germination rate of approximately 33.43%.
These values are sufficient for effective hybridization, making tetraploid Changshan Huyou an excellent candidate as a parental line in breeding programs aimed at developing new, improved citrus varieties with superior fruit quality and seedlessness.
Pharmaceutical and Agricultural Applications
In addition to its potential in breeding, the altered metabolic profile of tetraploid Changshan Huyou fruits holds promising implications for the pharmaceutical industry.
Changshan Huyou has long been recognized for its medicinal value, primarily due to its rich content of bioactive compounds such as flavonoids, volatile oils, and coumarins.
Although the study noted a reduction in some of these compounds in the tetraploid fruits, it also observed a significant increase in other metabolites, including specific alkaloids and amino acids with known health benefits.
For example, the increased levels of L-asparagine in the tetraploid peels could be valuable for reducing the formation of harmful compounds like acrylamide during food processing.
Likewise, the enhanced accumulation of flavonoids such as neohesperidin—which is known for its antioxidant properties and its potential to help regulate blood pressure—suggests that tetraploid fruits could serve as natural sources for health-promoting ingredients.
Moreover, the structural changes observed in tetraploid plants, such as thicker leaves with larger midribs and modified stomatal features, may contribute to improved resistance to environmental stresses.
These traits can lead to more stable yields even under challenging conditions such as drought, high salinity, or heavy metal exposure, thereby supporting more sustainable agricultural practices.
Integrating the Findings for a Holistic Understanding
Overall, the study on tetraploid Changshan Huyou offers an integrated view that combines morphological observations, metabolic profiling, and transcriptomic analysis.
The detailed measurements—from leaf thickness and midrib diameter to fold changes in key metabolites and the identification of hundreds of differentially expressed genes—provide a comprehensive picture of how tetraploidization affects the plant at multiple levels.
These integrated findings highlight the complex interplay between physical traits, chemical composition, and gene expression. As a result, they underscore how a single genetic event, such as genome doubling, can have far-reaching consequences for plant development and performance.
By understanding these interactions, scientists can better design breeding strategies that optimize desirable traits, whether it be for improved fruit quality, enhanced stress resistance, or increased production of bioactive compounds for pharmaceutical use.
Conclusion
In conclusion, the investigation into natural tetraploidization of Changshan Huyou reveals significant changes in plant morphology, metabolism, and gene expression that hold considerable promise for both agriculture and medicine.
The tetraploid plants demonstrate larger, thicker leaves, bigger flowers and fruits, and a restructured chemical profile that includes notable shifts in flavonoid, amino acid, and alkaloid levels. These changes are driven by clear genetic reprogramming, as evidenced by the extensive differentially expressed genes identified in the study.
Practically speaking, the tetraploid Changshan Huyou can serve as a valuable parental line for developing new, seedless citrus varieties through triploid hybridization. At the same time, the enhanced production of certain bioactive compounds makes these fruits attractive for pharmaceutical applications, offering natural ingredients that can promote health and well-being.
Moreover, the increased resilience of tetraploid plants under environmental stress suggests that they can contribute to more sustainable and stable agricultural production. Ultimately, this comprehensive study not only deepens our understanding of polyploidy in plants but also bridges fundamental research with practical applications, paving the way for innovations that benefit both producers and consumers in the future.
Reference: Huang, P., Xu, T., Wang, G. et al. Morphological and metabolic changes in Changshan Huyou (Citrus changshan-huyou) following natural tetraploidization. BMC Plant Biol 25, 301 (2025). https://doi.org/10.1186/s12870-025-06293-4
