Objectives The aim of the research was to investigate the effects of exogenous melatonin on endogenous melatonin synthesis, nitrogen metabolism key enzyme activities, nitrogen metabolism, and nitrogen transport gene expression in apple rootstock seedlings under nutrient stress.

Methods In this experiment, Malus hupehensis Rehd. seedlings were used as experimental materials for hydroponic experiments. M. hupehensis seedlings with 7−8 leaves were firstly cultured in 1/2 Hoagland nutrient solution for 12 days. Then the seedlings were divided into two groups, one group was added wtih 0.1 μmol/L melatonin in the nutrient solution and the other group wa not, and continued to grow for another 3 days. Each group was then further divided into two groups, one was supplied with 1/2 Hoagland nutrient solution, and the other was supplied with 1/20 Hoagland nutrient solution (nutrient stress), forming 4 treatments, as: 1/2 Hoagland nutrient solution (CK), 1/2 Hoagland nutrient solution+0.1 μmol/L melatonin (MCK), 1/20 Hoagland nutrient solution (ST), 1/20 Hoagland nutrient solution+0.1 μmol/L melatonin (MST). After 20 days of treatment, the leaf and root samples were collected for the measurement of melatonin content, nitrogen metabolism-related enzyme activities, melatonin synthesis enzymes, and N transport/metabolism related gene expression levels.

Results Compare with CK, ST treatment up-regulated the expression of melatonin synthesis-related genes MdTDC, MdT5H, MdAANAT and MdASMT in the leaves of M. hupehensis seedlings, and increased the endogenous melatonin content (P<0.05), decreased the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), Fd-glutamate synthetase (Fd-GOGAT) and NADH-glutamate synthetase (NADH-GOGAT) in the leaves and roots of M. hupehensis seedlings (P<0.05), and down-regulated the expression levels of nitrogen metabolism related genes NR, NiR, GS, Fd-GOGAT and NADH-GOGAT, while up-regulated the expression levels of nitrogen transport genes MdNRT1.1, MdNRT2.5, MdNRT2.7, MdAMT1.2, MdAMT1.5, and MdAMT2.1 in leaves. Compared with ST, MST treatment up-regulated the expression of the four melatonin synthesis-related genes in leaves, and increased the endogenous melatonin content by 3.10% (P<0.05), enhanced the activities of NR, NiR, GS and Fd-GOGAT in leaves and the activities of NiR, GS, Fd-GOGAT in roots of M.hupehensis seedlings. Enzyme activities related to N metabolism in leaves were increased by 65.62%, 51.49%, 59.63% and 17.53%, while those in roots were increased by 19.52%, 52.09% and 12.72%, respectively. Meanwhile, MST treatment up-regulated the leaf expression levels of the nitrogen metabolism-related genes NiR, GS, Fd-GOGAT and NADH-GOGAT to 2.16-, 2.08-, 2.22-, 1.71- and 1.19-fold, and root expression levels of 1.31-, 1.41-, 1.74- and 1.54-fold to those of ST treatment. In addition, melatonin significantly upregulated the expression levels of nitrogen transport genes MdNRT2.4, MdNRT2.5, MdNRT2.7, MdAMT1.2 and MdAMT2.1 in leaves of M. hupehensis seedlings under nutrient stress.

Conclusions Under nutrient stress, exogenous melatonin plant upregulates the gene expression related with synthesis of melatonin, so increases the melatonin content in leaves and roots, to alleviate nutrient stress by promoting nitrogen metabolism enzyme activity and upregulating the expression of nitrogen transport and metabolism related genes. The addition of exogenous melatonin strengthens the mitigation effect of plant to nutrient stress.