Biochemical and Molecular Processes during Ripening and Over-ripening of Banana (Musa AAA Cavendish Subgroup) Fruit Exhibit Characteristics of Programmed Cell Death

Programmed cell death (PCD) has been well characterized in floral and vegetative tissues but little has been reported on fleshy fruits. The purpose of this study was to determine if ripening and over-ripening of fleshy fruit are accompanied by classic symptoms of PCD. Bananas were ethylene-treated at green stage and shipped overnight to the UF. Upon arrival they were stored at 20 °C and evaluated for biochemical and molecular changes. Fresh peel tissue was collected and stored in 4% paraformaldehyde in phosphate buffered saline (pH7.0) for microscopy and in situ cell death detection (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL). Fruit arrived at color stage 2, after 4 d they had reached color stage 4 and on day 8 fruit were at stage 7 with presence of senescence-related spots (SRS). Ethylene production increased within 24 h of storage and reached climacteric peak after day 2 (0.55 ± 0.01 ng kg^-1 s^-1). CO₂ production sharply increased on day 2, reaching a maximum on day 4 (31.4 ± 1.0 mg kg^-1 s^-1). Both Chroma and a* reached highest values at day 6, 48.14 ±0.27 and 48.11 ± 0.26, respectively. Pulp °Brix increased until day 6 when it reached a plateau while peel °Brix increased throughout the evaluation period. Total electrolyte leakage of peel tissue increased from 10% at day 0 to 30% at day 6. Among the nutrients evaluated for leakage, calcium leakage reached 65% by day 6. Colorimetric analysis of nuclease activity of peel tissue showed an increase from 108 ± 7.1 units kg^-1 FW s^-1 on day 0, reaching a maximum at day 4 of 359.4 ± 15.8 units kg^-1 FW s^-1. SDS-PAGE analysis for nuclease followed by staining detected three nucleases with apparent molecular mass of 22.5, 27 and 29 kDa and showed intensification by day 4. Total protease activity was 66.3 ± 1.94 units kg^-1 FW s^-1 at day 0 and reached a maximum of 88.04 ± 1.94 units kg^-1 FW s^-1 at day 6, and SDS-PAGE detected one major protease of 80 kDa. Light microscopy revealed morphological changes during ripening of the banana peel, particularly epidermal and subepidermal cells. Images of SRS on stage 7 peel revealed shrinkage of the surface layers, visually suggesting cell death. TUNEL-positive nuclei on the SRS confirmed DNA fragmentation. Ripening and over-ripening of banana peel do share biochemical and molecular processes previously described for programmed cell death in other plant systems.