Response of colored poplar ‘Xuanyang 2’ to shading and purification by tissue culture

Colored-leaf trees are widely used in landscapes, yet some cultivars show poor color stability and sensitivity to low light, often fading and re-greening, which reduces ornamental value. Investigating leaf color changes under shade is thus crucial for enhancing aesthetics. This study used the new poplar cultivar ‘Xuanyang 2’ ( Populus × euramericana ‘Xuanyang 2’), with four shading treatments: full sunlight (CK), 30% (S30), 50% (S50), and 80% (S80). Physiological, chloroplast ultrastructural, and transcriptomic analyses were integrated to examine low-light responses and tissue culture purification efficacy. Chlorophyll content was significantly higher in the green regions than in the yellow regions under all treatments and increased with increasing shade intensity. In contrast, chlorophyll content in the yellow regions initially increased and then decreased, reaching a maximum under S50. The yellow regions showed significantly lower net photosynthetic rate (Pn), stomatal conductance (Gs), initial fluorescence (F0), and maximum fluorescence (Fm) than the green regions. Shading differentially affected antioxidants and osmolytes in both regions. Notably, S80 elevated malondialdehyde (MDA) in the yellow regions versus full sunlight, indicating oxidative stress and concurrent re-greening. Transmission electron microscopy (TEM) observations revealed that chloroplasts in the yellow regions exhibited more irregular morphology and blurred membrane boundaries under full sunlight, indicating greater sensitivity to high light intensity. Under severe shading, thylakoid stacking increased locally, accompanied by osmiophilic granule accumulation. The Gene Graphical Model (GGM) identified four top-tier transcription factor families responsive to low-light stress: C2H2, MBF1, C2C2-CO-like, and HMG. Weighted Gene Co-expression Network Analysis (WGCNA), integrated with physiological data, revealed four modules significantly correlated with Pn, soluble sugar (SS), lightness ( L * ), and yellow-blue parameter ( b * ), identifying bZIP, C2H2, and MYB as hub transcription factors regulating chloroplast development and pigment synthesis. During tissue culture, the chimera senesced and died, while stable green plantlets regenerated. These remained green under full sunlight, suggesting that the chimeric trait may not be genetically stable. Shading induced regreening of yellow sectors and increased chlorophyll in green sectors of ‘Xuanyang 2’, accompanied by adaptive chloroplast changes. It promoted chlorophyll synthesis while inhibiting degradation, modulated pigment metabolism and light-harvesting protein gene expression, and differentially regulated MYB, C2C2, and bZIP transcription factors. Generational segregation of variegated traits suggests a possible non-genetic mechanism, with implications for tissue culture purification.