Evaluation of heat stress response in pepper (Capsicum annuum L.) seedlings under controlled environmental conditions using a high-throughput 3D multispectral phenotyping

• Chili pepper seedling responses to heat stress were analyzed under controlled environmental conditions. • Morphological and spectral traits were identified through non-destructive 3D imaging. • PCA and clustering revealed three distinct response patterns to heat stress among genotypes. • A novel PC distance-based approach quantified heat stress stability among genotypes. • The developed phenotyping method provides an efficient tool for large-scale heat tolerance screening in breeding programs. Climate change-driven heat stress presents a significant threat to global pepper production, highlighting the urgent need for efficient methods to assess heat tolerance in breeding programs. This study presents a robust approach for assessing heat stress responses in pepper integrating high-throughput phenotyping and multivariate analysis. Twenty pepper genotypes were evaluated under controlled temperature conditions (40/35 °C day/night) for 14 days using the TraitFinder system equipped a pair of 3D multispectral scanner. Principal component analysis (PCA) of morphological and spectral traits revealed progressive divergence between control and heat-treated groups, with the maximum separation observed at day 10 (ΔC = 2.05). Three distinct response groups were identified based on Euclidean distances in the PCA space: low response (five genotypes), moderate response (nine genotypes), and high response (six genotypes). The PC-based distance metric showed strong correlations with conventional stress tolerance indicators, including biomass retention ( r = 0.66) and root system maintenance ( r = 0.48). Notably, genotype 'Pep17 (GPC121710)' demonstrated enhanced growth under heat stress (26 % increase in 3D leaf area), while 'Pep06 (GPC003350)' showed marked growth reduction (27 % decrease). This study validated the integration of high-throughput phenotyping with PCA-based metrics for the quantitative assessment of heat stress responses. The method offers an efficient tool for identifying heat-tolerant pepper genotypes and holds potential for application to other crops and stress conditions, supporting climate resilience breeding programs.