Heterologous expression of LbbHLH36 from Lycium barbarum enhances carotenoid accumulation in tomato fruits via synergistic activation of biosynthesis and suppression of degradation.

Abstract

Carotenoids are critical determinants of nutritional quality in tomato (Solanum lycopersicum) fruits, and their biosynthesis is tightly regulated by the bHLH transcription factor family. In this study, we investigated the heterologous regulatory function of LbbHLH36, a gene isolated from Lycium barbarum L., in carotenoid accumulation and its responsiveness to abiotic stresses in tomato. The LbbHLH36 coding sequence was cloned into the pNULPGE700 vector and introduced into the model tomato cultivar 'Micro Tom' via Agrobacterium-mediated leaf disc transformation. Quantitative real-time PCR revealed that LbbHLH36 transcript levels peaked in mature fruits of transgenic lines and were associated with significant upregulation of key early biosynthetic genes, including SlPSY1, SlPSY2, SlPDS, and SlZDS. Enzymatic assays further demonstrated increased activities of the rate-limiting enzymes phytoene synthase (PSY) and phytoene desaturase (PDS), alongside a marked suppression of carotenoid cleavage dioxygenase 4 (CCD4) activity in transgenic plants. Metabolomic analyses confirmed that overexpression of LbbHLH36 substantially elevated total carotenoid, lycopene, and β-carotene contents in tomato fruits. Furthermore, LbbHLH36 expression was strongly induced by high temperature, low temperature, drought, and salt stress, suggesting its potential involvement in crosstalk between stress responses and primary metabolism. Collectively, our findings reveal that LbbHLH36 acts as a positive regulator of carotenoid accumulation in tomato through a dual mechanism that concurrently regulate biosynthetic flux and attenuates catabolic degradation, highlighting its value as a candidate gene for nutritional quality improvement in Lycium barbarum, tomato, and other Solanaceous crops.