Salt stress is one of the most problematic abiotic stress affecting plants in agriculture worldwide. In saline soils, plants try to neutralize the effects of salt stress by physiological changes, leading to the decreasing of both oxidative and osmotic stresses (Stassinos et al. 2021). Melatonin, an indoleamine widely found in animals and plants, is considered as a candidate phytohormone that affects responses to a variety of biotic and abiotic stresses, such as salt stress (Wei et al. 2015; Li et al. 2019). In the present study, exogenous melatonin treatment alleviated the inhibition of trifoliate orange seedling growth under 150 mmol/L NaCl stress to a certain extent, which is in line with the earlier result as reported by Zhang et al. (2014) that melatonin treatment could improve the germination ability of Pennisetum alopecuroides (Linn.) seeds and alleviate the negative effect of salt stress on its growth. In addition, exogenous melatonin treatment can effectively promote dry matter accumulation, leaf elongation rate and alleviate the inhibition of plant height under salt stress, which imply that melatonin improved plant resistance to salt stress through osmotic regulation (Li et al. 2019). Also, melatonin promotes soybean growth, seed production, and stresses (salt and drought) tolerance by regulating cell division, photosynthesis, carbohydrate metabolism, fatty acid biosynthesis, and ascorbate metabolism (Wei et al. 2015).
When plants are under stress, the photosynthetic rate and the level of chlorophyll of leaves were decreases (Harizanova and Koleva-Valkova, 2019). The main medium of plant photosynthesis is leaf, and the amount of chlorophyll content directly affects the ability of plant to carry out photosynthesis (Demming and Adams, 1996). Salt stress not only affect the synthesis of chlorophyll but also accelerate the decomposition of chlorophyll, resulting in the decrease of chlorophyll content (Schreiber et al. 1998). Our results showed that NaCl stress led to the degradation of chlorophyll in leaves, and exogenous melatonin alleviated the damage of NaCl stress on chlorophyll A and chlorophyll B in trifoliate orange leaves, as similarly reported by Kostopoulou et al. (2014) in citurs. Salt stress could also decrease the net photosynthetic rate of plants, reduce the synthesis of organic matter, and ultimately affect the normal growth of plants (Centritto et al. 2003). In cotton, salt stress significantly inhibited the seedlings growth and biomass accumulation, and decreased leaf area and the indexes of Pn, Gs, Ci and Tr, which is in line with our results (He et al. 2005). In this study, salt stress decreased leaf Pn, Gs, Ci and Tr while melatonin increased them partially, which is agreement with previous work in cucumber (Harizanova and Koleva-Valkova, 2019). The possible mechanism is that melatonin can improve the content of chlorophyll, promote the activity of PSII and effectively regulate the photosynthesis of plant leaves under salt stress, resulting in improve the salt tolerance of plants.
Chlorophyll fluorescence is an effective probe of photosynthesis, through which almost all changes of photosynthesis can be detected (Mimuro et al. 1999). Chlorophyll fluorescence contains PSII reaction center actual photochemical efficiency (φPSII), PSII effective light quantum yield (Fv’/Fm’), photochemical quenching coefficient (qP) and non-photochemical quenching coefficient (NPQ) (Farzad et al. 2007). This study showed that salt stress markedly increased NPQ while significantly decreased φPSII, Fv’/Fm’ and qP. Furthermore, melatonin alleviated the increase and decrease degree of them. As actual photochemical efficiency of the PSII reaction center, the decrease of φPSII indicated that the photosynthetic electron decreased under carbon fixation, resulting in the decrease of net photosynthetic rate, which explained the mechanism of salt stress inhibiting photosynthesis (Han et al. 2010). The decrease of Fv’/Fm’ is due to the fact that photosynthetic electron transfer is not carried out in time after light capture, but directly consumes light energy through chlorophyll transformation, which leads to the decrease of PSII (Shibata et al. 2012). The index of qP reflects the degree to which the photoreaction center is effectively applied in the photochemical reaction. The decrease of qP indicated that PSII could not transfer photosynthetic electrons smoothly after being damaged, and the effective reaction light decreased (Havaux et al. 1991). NPQ is a non-photochemical quenching index, reflecting the process of chlorophyll absorption and transformation, and is an effective heat dissipation element used to resist light damage (Tietz et al. 2017). In this experiment, NaCl stress leaded to an increase in NPQ, resulting in a decrease in PSII and photosynthetic rate. Melatonin increased the index of φPSII, Fv’/Fm’ and qP and decrease the NPQ in trifoliate orange seedlings under salt stress, implying that melatonin can effectively improve PSII photochemical efficiency and photosynthetic rate of leaves, which is consistent with previous studies on maize and celery (Ye et al. 2016). Thus, melatonin can improve the photosynthetic capacity of plants under NaCl stress.
IAA plays an important role in regulating plant growth under adverse stresses (Iqbal and Ashraf, 2007; Zhang et al. 2013, 2018, 2019). As an cytokinin (CTK), ZR has been reported to have the ability to enhance plant salt tolerance and temperature stress (Javid et al. 2011). GAs are an essential for many plants in response to abiotic stress and also take part in plant growth and development (Colebrook et al. 2014). BRs, a kind of steroid hormones, are necessary for plant growth and development, and can tolerate environmental stresses by inducing antioxidant activities (Bajguz and Piotrowska-Niczyporuk, 2014). IAA could significantly enhanced the tolerance of salt stress in maize (Kaya et al. 2013). Our study showed that there was a significant decrease in root IAA levels of melatonin and non-melatonin trifoliate orange seedlings under the salt stress versus the non-salt stress. At the same time, melatonin treatment notably increased root IAA concentration. The result is similar to the findings of Liu et al. (2016). Furthermore, IAA is closely related to the growth and development of plant roots (Liu et al. 2018). Therefore, the melatonin effect on IAA is effectively associated with melatonin-induced growth improvement, root modification and salt tolerance. However, the salt stress significantly increased root ZR and GAs levels, while melatonin has no effect with ZR and GAs in this study. Perhaps there has no interaction between melatonin and ZR or GAs. In this study, melatonin did not significantly alter root BRs concentration in non-salt and salt trifoliate orange seedlings. Perhaps in salt stress, the regulation of melatonin to other phytohormones is sufficient for plants to resist salt stress. So, melatonin increased plant salt tolerance mainly through interaction with auxin.