# Exogenous melatonin alleviates copper stress in apple rootstock M9T337 by regulating the antioxidant system and carbon–nitrogen metabolism

## TL;DR

Exogenous melatonin helps apple rootstock resist copper stress by boosting antioxidants and balancing carbon-nitrogen metabolism.

## Contribution

This study reveals melatonin's novel role in mitigating copper stress in apple rootstock through antioxidant and metabolic regulation.

## Key findings

- Exogenous melatonin reduces oxidative damage and improves rootstock growth under copper stress.
- Melatonin enhances antioxidant enzyme activity and optimizes carbon-nitrogen metabolism in stressed plants.
- Melatonin modulates gene expression related to copper detoxification and nutrient transport.

## Abstract

Excess copper (Cu) in the soil has become a key environmental stress factor constraining the high quality and yield of apple. Melatonin (MT) has significant potential in regulating plant stress resistance. This study used apple rootstock M9T337 in a hydroponic experiment with four treatments: control (CK), 100 μmol·L−1 MT (MT), 30 μmol·L−1 CuSO4 (Cu), and Cu+MT. Isotopic labeling, noninvasive micro-test technology, and quantitative real-time PCR (qRT-PCR) were used to investigate the mechanisms by which MT affects the growth, antioxidant system, and carbon–nitrogen metabolism of apple rootstock under Cu stress. Copper stress inhibited seedling growth, deteriorated root morphology, decreased root activity, and led to carbon-nitrogen metabolism imbalance. Exogenous MT application alleviated these inhibitory effects. Compared to Cu treatment, Cu+MT treatment decreased H2O2 and malondialdehyde content and increased seedlings biomass, leaf superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase activities, net photosynthetic rate (Pn) and rubisco activity, and nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase activities and root tip NO3− flux transitions from efflux to uptake. Moreover, Cu+MT treatment increased 13C and 15N accumulation compared to Cu treatment. qRT-PCR showed that MT upregulated the expression of MT synthesis genes (e.g., MdTDC), copper detoxification genes (e.g., MdCCS), carbon metabolism genes (e.g., MdSUSY1), and nitrogen metabolism genes (e.g., MdNRT1.1), while downregulating the expression of copper absorption genes (e.g., MdCOPT2) and copper transport genes (e.g., MdYSL3). MT effectively alleviates Cu stress inhibition in apple rootstock by enhancing the antioxidant capacity, regulating key enzyme activities and gene expression in carbon–nitrogen metabolism, and optimizing the allocation of photosynthetic products and nitrogen. These results provide a theoretical basis for managing Cu pollution in apple orchards.