Phosphorylation of <scp>MdWRKY70L</scp> by <scp>MdMPK6</scp>/<scp>02G</scp> mediates reactive oxygen accumulation to regulate apple fruit senescence

Apple (Malus domestica Borkh.) is a globally significant crop and a vital dietary component worldwide. During ripening, apples exhibit a longitudinal gradient, ripening first at the stalk cavity and extending towards the calyx concave. When the fruit is harvested at the right time or later, the stalk cavity of many varieties often shows over-ripening, that is, premature senescence such as peel browning, which diminishes fruit quality. This study examines the natural senescence process in 6-year-old 'Ruixue' apples by screening transcriptome data to uncover senescence-related genes and validate their molecular functions. Our analysis of antioxidant capacity and reactive oxygen species (ROS) in different peel regions revealed that malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msubsup><mml:mi>O</mml:mi> <mml:mn>2</mml:mn> <mml:mrow><mml:mo>-</mml:mo> <mml:mo>⋅</mml:mo></mml:mrow> </mml:msubsup> </mml:mrow> </mml:math> ) levels increased with senescence, where ROS-scavenging enzyme activity was notably reduced, especially in the stalk cavity (compared with the fruits in the stalk cavity at 120 days, the activities of SOD, POD, and CAT in stalk cavity of fruits at 205 days were significantly decreased in 65.4%, 82.7%, and 91.1%, respectively). Transcriptome clustering and enrichment analyses across developmental stages revealed MdWRKY70L, MdSAG101, and MdZAT12 as key regulators of peel senescence. MdWRKY70L could interact with MdSAG101/MdZAT12 both in vivo and in vitro, thereby mediating ROS accumulation in the peel and accelerating the fruit senescence process. Further in vitro and in vivo studies demonstrated that MdWRKY70L is phosphorylated at Ser199 by MdMPK6/02G, enhancing MdWRKY70L protein stability and promoting peel senescence. These findings offer insights for developing strategies to delay fruit senescence and improve postharvest quality control.