Enhancing proline turnover is the key physiological response of mature citrus leaves to fruiting

•Chlorosis of mature leaves caused by fruiting is related to N allocation plasticity.•Fruiting stimulated the transport of proline from mature leaves to phloem sap.•N supply affects leaf N allocation via N metabolism and consequently also C allocation.•N supply leads to a decrease in the content of TCA cycle intermediates. Mature leaves are the central organs involved in carbon (C) and nitrogen (N) metabolism to support the growth and development of citrus trees. These metabolisms may be greatly altered by fruiting and N supply, which can be verified by a typical field phenomenon in which leaf chlorosis appeared on leaves of fruiting branches but not on leaves of adjacent nonfruiting branches on citrus trees. To elucidate the underlying strategy of fruiting on C and N partitioning in citrus leaves, citrus trees were cultivated with low- and high-N supplies, and then leaves and phloem sap from fruiting and nonfruiting branches were analyzed. The results showed that fruiting drives N allocation but does not improve N assimilation while maintaining the nonstructural carbohydrate content of the leaves. Combined physiological analysis and nontargeted metabolomics showed that proline is a key metabolite of mature citrus leaves and phloem sap. Due to fruiting, the proline content in mature leaves and phloem sap decreased by 71∼73% and 17∼30%, respectively. Fruiting, followed by N supply, resulted in proline remobilization from mature leaves into phloem sap and enhanced the consumption of TCA cycle intermediates for N assimilation and energy generation. Catabolism of organic N, rather than N assimilation or C metabolism, is primarily accelerated in leaves of fruiting branches, becoming a major source of organic N (mainly proline) upon phloem transport to developing fruits. Furthermore, N supply strengthened N storage in leaves of both fruiting and nonfruiting branches and then facilitated proline turnover at higher levels, indicating the importance of timing for N management in orchards.