**Background:** Environmental factors such as light spectral composition and temperature can influence the biosynthesis of secondary metabolites in plants by modulating photoreceptor activity. Phytochromes (PHYs), which sense red and far-red light and also act as temperature sensors, are key regulators of these processes. Green pepper fruits retain functional chloroplasts, photoreceptors, and photosynthetic activity, making them a useful model to study how post-harvest light and temperature treatments affect the accumulation of bioactive compounds such as carotenoids, capsaicinoids, and ascorbic acid. This study aimed to determine whether brief irradiation with red light (RL, 660 nm), far-red light (FRL, 730 nm), or combined RL+FRL, as well as low temperature (4°C), could enhance the nutritional quality of harvested green Capsicum annuum fruit.
**Methods:** Green fruits of C. annuum L. (Korean long green) were harvested and subjected to five treatments: (1) 4°C darkness, (2) 25°C darkness, (3) 25°C RL (660 nm, 70 µmol photons m⁻² s⁻¹), (4) 25°C FRL (730 nm, 70 µmol photons m⁻² s⁻¹), and (5) 25°C RL+FRL (35 + 35 µmol photons m⁻² s⁻¹). Samples were collected at 0 h (initial point), 24 h, and 72 h. Total chlorophylls and carotenoids were measured spectrophotometrically. Individual carotenoids were analyzed by HPLC on a reversed-phase column (Agilent Zorbax SB-C18). Ascorbate content was determined by HPLC with photodiode array detection. Capsaicinoids (capsaicin, dihydrocapsaicin, nonivamide) were quantified by HPLC on an Agilent Hypersil 5 AA-ODS column. Photochemical activity was assessed using a mini-PAM II fluorometer to measure maximum (Fv/Fm) and effective (Y(II)) quantum yields of photosystem II. Gene expression levels for PAL, CAM, CSY1, ACS, FATa, and KAS were measured by RT-PCR normalized to Actin1. All experiments were performed in three biological and three analytical replicates; significance was assessed by one-way ANOVA followed by Duncan's method (p < 0.05).
**Key Results:** Total carotenoid content increased most notably after 24 h of RL irradiation, reaching 0.51 g kg⁻¹ dry weight, which was more than 3.5 times higher than the initial value. After 72 h, the RL+FRL treatment resulted in a more than 2-fold increase in total carotenoids compared to the initial point. The most significant change in carotenoid composition occurred under FRL for 72 h, with an increase in the diversity of carotenoids and xanthophylls. Capsaicin alkaloid content increased markedly after 72 h of FRL irradiation, reaching more than 8 times the initial value. Low temperature (4°C) also significantly increased capsaicin and dihydrocapsaicin contents after both 24 h and 72 h. Ascorbate content increased 1.5-fold after 24 h of RL and 1.6- to 1.7-fold after 72 h in darkness and at 4°C, respectively. The maximum quantum yield (Fv/Fm) remained high under dark, FRL, and low-temperature conditions but decreased under RL and RL+FRL, with the most pronounced decline in the RL+FRL variant (Y(II) = 0.08 after 72 h). Gene expression analysis revealed that PAL expression increased more than 15-fold after 72 h of FRL and more than 12-fold after 72 h of RL. CAM expression increased 8-fold under FRL and 6-fold under RL at 72 h. KAS expression increased more than 10-fold upon cold treatment for 24 h and under FRL for 72 h. ACS expression increased almost 3-fold at low temperature after 24 h. The highest expressions of CAM, ACS, KAS, and CSY1 were observed at 4°C during the first 24 h.
**Clinical Implications:** This study demonstrates that short-term, post-harvest light treatments—particularly red light for boosting carotenoids and far-red light for boosting capsaicinoids—can substantially enhance the nutritional and bioactive compound content of green pepper fruit. Given the known anticancer and antioxidant properties of carotenoids and capsaicinoids, this approach offers a practical, non-chemical method to improve the health-promoting value of peppers before consumption. The findings also highlight the role of phytochrome inactivation (by FRL or low temperature) in upregulating alkaloid biosynthesis genes, providing a mechanistic basis for developing post-harvest processing strategies to enrich functional food ingredients.