KNOX, short for KNOTTED1-like HOMEOBOX, belongs to a class of homeobox genes that encode homeodomain proteins. These proteins act as master transcription factors, binding to specific cis-regulatory elements in the DNA of target genes and switching entire developmental programs on or off. In plants, KNOX genes are famous for their role in maintaining the shoot apical meristem, the reservoir of stem cells from which all above-ground organs emerge. They also participate in secondary metabolism and stress adaptation, making them a compelling target for any researcher trying to understand how a tree balances growth with survival. Yet, until this study, no genome-wide identification of the KNOX family had ever been reported for E. ulmoides.

The research team, led by Liu Jun along with co-corresponding authors Kou Jiefeng and Chen Suiqing, mined the genomic database of E. ulmoides and identified eight KNOX proteins, which they named EuKNOX. Phylogenetic analysis, comparing these proteins with their counterparts in the model plant Arabidopsis, showed that the EuKNOX family is distributed across five subgroups, each reflecting a distinct evolutionary lineage. The genes are unevenly scattered across six chromosomes, a pattern that hints at a dynamic history of duplication, loss and rearrangement during the species’ long evolutionary journey.

Gene structure analysis added another layer of insight. The EuKNOX genes contain between three and four introns, and remarkably, members of the same subgroup tend to share similar intron numbers. This conservation of gene architecture within subgroups suggests that structural features have been maintained through millions of years of evolution, likely because they carry functional importance. Prediction of cis-regulatory elements in the promoter regions of these genes revealed that EuKNOX genes are wired to respond to hormones, light and stress, providing a molecular blueprint for how the tree coordinates its developmental and environmental responses.

Expression data from RNA sequencing, a technique that measures the activity of thousands of genes simultaneously, revealed striking tissue-specific patterns. Five of the eight EuKNOX genes were highly expressed in the xylem, the woody tissue responsible for water transport and structural support, suggesting a central role in wood formation. The expression levels of most EuKNOX genes were also higher in female flowers than in male flowers, pointing to sex-specific regulatory roles in reproduction. Beyond wood and flowers, the researchers found that EuKNOX genes contribute to bark, fruit and leaf development, to the biosynthesis of Eu-rubber, the natural rubber that gives the tree its economic value, and to leaf-color formation, the pigment changes that give the species its ornamental appeal.

To test how these genes respond to environmental challenges, the team turned to quantitative real-time PCR, a sensitive method for measuring gene expression with high precision. The results were dramatic. Under salt stress induced by sodium chloride, most EuKNOX genes were strongly activated, with one gene, EuKNOX5, showing an extraordinary 343-fold increase in expression at 24 hours after treatment. This level of induction is among the most extreme responses reported for a developmental gene under abiotic stress and suggests that EuKNOX5 may be a key player in the tree’s salt-tolerance machinery.

Gibberellic acid, a hormone that promotes stem elongation and germination, also induced the expression of most EuKNOX genes, consistent with their known role in growth regulation. In contrast, methyl jasmonate, a signaling molecule involved in plant defense, suppressed the expression of most EuKNOX genes, revealing an antagonistic relationship between growth promotion and defense activation. Under drought stress and abscisic acid treatment, the EuKNOX genes displayed a more nuanced pattern, with expression first increasing, then decreasing, and then increasing again, a wave-like response that may reflect the tree’s attempt to balance water conservation with continued growth under fluctuating conditions.

The significance of these findings extends far beyond basic biology. E. ulmoides is valued not only for its medicinal bark, known as Eucommiae Cortex in traditional Chinese medicine, but also for its trans-polyisoprene rubber, which is found in its bark, leaves and fruit and holds promise as a sustainable alternative to tropical rubber. Understanding which genes control rubber biosynthesis, wood quality and stress tolerance could accelerate the genetic improvement of this species, allowing breeders to select trees that produce more rubber, grow faster and withstand drought, salinity and other stresses intensified by climate change.

The study also provides a foundation for future functional investigations. By systematically identifying the KNOX superfamily and characterizing their expression under multiple stresses, the researchers have created a roadmap for follow-up experiments, such as gene knockout, overexpression and promoter-reporter assays, that could pinpoint exactly which EuKNOX genes control which traits. The work was supported by the Henan Province Natural Science Foundation, the Joint Fund for Science and Technology Research of Henan Province, and the Henan Province Modern Agricultural Industry Technology System, reflecting the strategic importance of this species to regional agriculture and forestry.

As climate pressures mount and the demand for sustainable biomaterials grows, studies like this one remind us that the answers to modern agricultural challenges may lie in the ancient genetic machinery of trees that have survived for millions of years. The EuKNOX genes, quietly orchestrating growth, rubber production and stress survival in a living fossil, may soon become targets for the next generation of crop and forest improvement, bridging the gap between evolutionary biology and practical applications in a warming world.

Subject of Research: Evolution and regulatory roles of the KNOX gene in Eucommia ulmoides in growth, development, and multiple stress responses

Article Title: Evolution and regulatory roles of the KNOX gene in Eucommia ulmoides in growth, development, and multiple stress responses

Article References: Evolution and regulatory roles of the KNOX gene in Eucommia ulmoides in growth, development, and multiple stress responses. (n.d.). https://doi.org/10.1186/s12870-026-10141-4

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10141-4

Keywords: Evolution, regulatory, roles, KNOX, gene, Eucommia, ulmoides, growth, development, multiple, stress, responses