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.
Key Terms and Concepts
1. Tetraploidization
Tetraploidization is the process where an organism gains four complete sets of chromosomes (4X) instead of the usual two (2X). This occurs naturally or through lab methods like chemical treatment. In plants, tetraploidization often leads to larger leaves, flowers, and fruits. For example, the study found tetraploid Changshan Huyou citrus had thicker leaves and bigger fruits. This process is important in agriculture to develop hardier crops or seedless varieties through breeding.
2. Polyploids
Polyploids are organisms with more than two sets of chromosomes. Many plants, including wheat and strawberries, are naturally polyploid. Polyploidy can improve stress tolerance or fruit quality. In citrus, polyploids like tetraploids are used to breed triploid (seedless) fruits. The study compares diploid (2X) and tetraploid (4X) citrus to understand how polyploidy affects growth and metabolism.
3. Autopolyploids
Autopolyploids are polyploids created by duplicating chromosomes from the same species. For example, tetraploid Changshan Huyou in the study arose from natural duplication of its diploid genome. Autopolyploids often show thicker tissues and adaptability to environmental stress. They are useful in breeding because they retain traits of the original species while offering new characteristics.
4. Allopolyploids
Allopolyploids form when two different species hybridize and combine their chromosomes. For example, combining citrus and related genera can create hybrids with traits from both parents. Unlike autopolyploids, allopolyploids benefit from genetic diversity. They are important for developing crops with hybrid vigor, such as disease-resistant citrus varieties.
5. Metabolome
The metabolome refers to the complete set of small molecules (metabolites) in an organism, like sugars, amino acids, and flavonoids. Studying the metabolome helps researchers understand how plants respond to changes, such as tetraploidization. In the study, tetraploid citrus had altered levels of alkaloids and flavonoids, impacting its medicinal value.
6. Transcriptome
The transcriptome is the full set of RNA molecules produced by genes in a cell. It shows which genes are active under specific conditions. The study analyzed the transcriptome of tetraploid citrus to identify genes linked to larger fruits or stress tolerance. This helps pinpoint genetic pathways affected by polyploidy.
7. Differentially Accumulated Metabolites (DAMs)
DAMs are metabolites that increase or decrease significantly between groups. In the study, tetraploid citrus peels had lower flavonoids but higher alkaloids compared to diploids. Identifying DAMs reveals how polyploidy changes a plant’s chemical composition, which can influence its nutritional or pharmaceutical uses.
8. Principal Component Analysis (PCA)
PCA is a statistical method to simplify complex data by highlighting patterns. In the study, PCA visualized differences in metabolite profiles between diploid and tetraploid citrus tissues. It showed that peels and segment membranes changed more than juice sacs after tetraploidization.
9. Flow Cytometry
Flow cytometry is a technique to analyze cell properties, like DNA content, using lasers. The study used it to confirm tetraploidy by measuring doubled DNA in leaf cells. This method is fast and accurate for identifying polyploid plants.
10. Guard Cells
Guard cells are pairs of cells on leaf surfaces that control gas exchange by opening/closing stomata (pores). Tetraploid citrus had larger guard cells but fewer stomata, which may reduce water loss. This adaptation helps plants survive drought or salinity.
11. Palisade Parenchyma
Palisade parenchyma is a layer of tightly packed cells in leaves where photosynthesis occurs. Tetraploid citrus had thicker palisade layers, which may improve light capture and growth. This structural change is common in polyploids.
12. Spongy Parenchyma
Spongy parenchyma is a loose layer of cells beneath the palisade layer in leaves. It allows gas exchange for photosynthesis. Tetraploid citrus had thicker spongy tissue, possibly enhancing carbon dioxide absorption.
13. Flavonoids
Flavonoids are plant compounds with antioxidant and anti-inflammatory properties. Citrus peels are rich in flavonoids like neohesperidin. The study found tetraploid juice sacs had higher flavonoid levels, which could boost their health benefits.
14. Alkaloids
Alkaloids are nitrogen-containing compounds, often with medicinal effects. Tetraploid citrus peels accumulated more alkaloids like clausine K, which may have antibacterial properties. Alkaloids contribute to the plant’s pharmaceutical value.
15. Coumarins
Coumarins are aromatic compounds with roles in plant defense and human medicine (e.g., blood thinners). Tetraploid citrus had fewer coumarins, which might affect its resistance to pests or diseases.
16. Terpenoids
Terpenoids are a large class of plant chemicals, including essential oils and hormones. Tetraploid peels had more terpenoids like deacetylnomilin, which can influence flavor and stress responses.
17. Huanglongbing (HLB)
HLB, or “citrus greening,” is a deadly bacterial disease spread by insects. Tetraploid rootstocks may resist HLB better by limiting bacterial spread. This study suggests tetraploid citrus could help combat HLB outbreaks.
18. Triploid Breeding
Triploid breeding creates seedless fruits by crossing diploid (2X) and tetraploid (4X) plants. Tetraploid Changshan Huyou can serve as a parent to produce triploid citrus, which is desirable for commercial fruit production.
19. Nucellar Seedlings
Nucellar seedlings grow from maternal tissue, not fertilized embryos, producing clones of the parent plant. Tetraploid citrus can generate nucellar seedlings, ensuring consistent traits in rootstocks or new varieties.
20. PEROXIDASE
Peroxidase is an enzyme that breaks down toxins and protects cells from damage. In tetraploid peels, peroxidase genes were linked to higher amino acid levels, possibly improving stress tolerance.
21. CYTOCHROME P450 (CYP450)
CYP450 enzymes detoxify chemicals and synthesize metabolites like alkaloids. Their upregulation in tetraploid citrus explains increased medicinal compound production.
22. FERULATE-5-HYDROXYLASE (F5H)
F5H is an enzyme in lignin and flavonoid biosynthesis. In tetraploid juice sacs, F5H activity correlated with higher flavonoid levels, enhancing antioxidant properties.
23. Gene Ontology (GO) Analysis
GO analysis categorizes genes by their roles (e.g., “stress response” or “metabolism”). The study used GO to show tetraploid genes were enriched in metabolic pathways, explaining altered fruit chemistry.
24. Kyoto Encyclopedia of Genes and Genomes (KEGG)
KEGG maps genes to biological pathways. The study found tetraploid DEGs were linked to “phenylpropanoid biosynthesis,” a pathway for making flavonoids and lignin.
25. Single Nucleotide Polymorphism (SNP)
SNPs are single-letter changes in DNA that distinguish individuals. The study used SNPs to confirm the tetraploid’s genetic similarity to its diploid parent, ruling out hybridization. SNPs help track genetic diversity in breeding programs.
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
