K E Y W O R D S
rice;
melatonin; NaCl stress; AsA-GSH cycle; nitrogen metabolism
1 | INTRODUCTION
Rice (Oryza sativa L.) is a widely cultivated food crop. Food
security is of vital importance for ensuring the survival of human
beings worldwide. However, salt stress severely affects rice yield
globally (Zörb et al., 2019; Liu et al., 2022; Sengupta & Majumder,
2010). Rice is a moderately salt-sensitive crop, and salt stress can
induce the accumulation of ROS, such as superoxide anions
(O2-·) and hydrogen peroxide
(H2O2), in rice seedlings. Excess ROS
usually weakens various physiological functions of cells, resulting in a
decrease in the photosynthetic rate, blockage of protein synthesis,
disturbance of the nitrogen metabolism pathway, and cell death in severe
cases (Michard & Simon, 2020; Ullah et al.,
2019).
Therefore, low levels of ROS can be used as a signaling factor for rice
to respond to stress. However, the accumulation of large amounts of ROS
seriously affects the normal growth and development of rice.
Melatonin (N-acetyl-5-methoxytryptamine, MT) is an indole compound that
plays an important role in life activities and is widely found in most
animals and plants (Tan et al., 2012). Owing to the successful isolation
of the pineal gland of cattle in 1958, the pineal hormone was identified
and named melatonin (Lerner et al., 1958). However, trace amounts of
melatonin were not found in plants until 1995 (Hattori et al., 1995;
Dubbels et al., 1995). Based on an in-depth study, melatonin is an
important antioxidant that plays an important role in promoting seedling
growth (Liu et al., 2021; Samanta et al., 2021), increasing
photosynthetic rate (Sezer et al., 2021; Yan et al., 2021), and
enhancing metabolic activity (Yu et al., 2018; Wang et al., 2021).
Salt stress disrupts the balance of ROS in plants, leading to disorders
of plant physiology and metabolism. To restore ROS content to normal
levels, the antioxidant mechanism of ROS scavenging is necessary. The
ascorbate-glutathione (AsA-GSH) cycle is an important link in plant
antioxidant mechanisms that can effectively eliminate the accumulation
of ROS in plants (Ye et al., 2015). Ascorbic acid (AsA), glutathione
(GSH), ascorbic acid oxidase (AAO), dehydroascorbic acid reductase
(DHAR), glutathione reductase (GR), and glutathione peroxidase (GPX) are
key components of the plant AsA-GSH cycle (Vasseur et al.,
2011).
Other antioxidants, including polyamines, proline, and glycine betaine,
also contribute to the stabilization of ROS balance. Under the catalysis
of key enzymes of the AsA-GSH cycle, excess
H2O2 and
O2-· in the plant are converted into
water, which is finally absorbed and utilized by plant cells. According
to related studies, melatonin can reduce the ROS content in plants by
increasing the activities of key enzymes in the AsA-GSH cycle of sugar
beets and resisting oxidative damage caused by stress (Zhang et al.,
2021). Melatonin can also regulate the expression of key enzyme genes in
the AsA-GSH cycle to affect the activities of key enzymes and enhance
the tolerance of plants to adverse stress (Lv et al., 2019; Ma et al.,
2016).
Nitrogen metabolism is a necessary normal metabolic activity in the life
process of plant cells, and its pathway plays a certain role in all
functional regions of plants (Duan et al., 2018). Plants absorb
nitrogen-containing ions in the soil and convert them into nitrogen,
which is stored in plants and then used by plants as nitrate nitrogen
and ammonium nitrogen. Key enzymes involved in nitrogen metabolism play
important roles in nitrogen assimilation. Nitrate reductase (NR) is the
first key enzyme and an important rate-limiting enzyme that catalyzes
the reduction of NO3- to
NO2- in nitrogen metabolism. Glutamine
synthetase (GS) and glutamate synthase (GOGAT) constitute the GS/GOGAT
pathway, which participates in the primary assimilation and
re-assimilation of ammonia and is an indispensable enzyme in ammonia
assimilation (Wang, 2013). Under salt stress, the nitrogen metabolism
pathway is hindered, mainly by changing the expression of key nitrogen
metabolism enzyme genes, ultimately affecting the activities of key
nitrogen metabolism enzymes (Debouba et al.,
2013).
Exogenous melatonin was reported to induce the expression of key enzyme
genes for nitrogen metabolism in maize and alfalfa under drought stress,
and enhance the activities of key enzymes in nitrogen metabolism,
thereby ensuring the normal progress of nitrogen metabolism (Ren et al.,
2021; Antoniou et al., 2017).
The regulatory effects of melatonin on the antioxidant system of rice
and other crops under stress conditions have been widely reported (Jahan
et al.,
2020;
Zhang et al., 2021). However, only few studies sought to evaluate the
effects of melatonin on the rice AsA-GSH cycle and nitrogen metabolism
under salt stress. Further, the mechanism by which melatonin regulates
the rice AsA-GSH cycle and nitrogen metabolism remains unclear.
Therefore, in this study, exogenous melatonin was used to treat rice
seedlings under NaCl stress. Thereafter, the morphological,
physiological, and molecular indices were measured, and the regulatory
mechanism of exogenous melatonin on the expression of related enzyme
genes in the AsA-GSH cycle and nitrogen metabolism in rice seedlings
under NaCl stress was investigated.
2 | MATERIALS AND METHODS
2.1 | Rice material
The rice ‘glutinous rice 89-1’ was selected as the experimental material
and was obtained from the laboratory of Chongqing Engineering Research
Center of Specialty Crop Resource, Chongqing Normal University.
Glutinous rice 89-1 is an overwintering rice variety, as its axillary
buds are dormant during the winter and can germinate and regenerate the
following spring. The yield of overwintering regeneration season is
equivalent to that of current season (Deng et al., 2018; Chen et al.,
2021). After cleaning and disinfection, 100 rice seeds were selected and
placed in the germination box (10 rows × 10 columns), which was
transferred to a light incubator under the conditions of 16 h light/8 h
dark, 30 °C, and 70% humidity for 10 d. Seedlings were cultivated for
three- and one-heart periods, and seedlings with consistent growth were
used for the next treatment. The following seven experimental treatments
were administered: (1) control check treatment (CK): nutrient solution
treatment for 8 d; (2) NaCl treatment: nutrient solution treatment for 3
d and then 80 mmol·L-1 NaCl solution containing
nutrient solution treatment for 5 d; (3) melatonin pretreatment using
different concentrations (50, 100, 200, 400, and 800
μmol·L-1) of melatonin and nutrient solution for 3 d,
with the corresponding concentrations represented by MT50, MT100, MT200,
MT400, and MT800, respectively, and then treatment with 80
mmol·L-1 NaCl solution containing nutrient solution
for 5 d.
2.2 | Determination of seedling height, root
length, fresh weight, and dry weight
The plant height and root length of rice seedlings were measured with an
accurate scale ruler (cm), and the fresh and dry weights of the rice
seedlings were measured with an electronic balance (g).
2.3 | Determination of chlorophyll,
malondialdehyde (MDA), H2O2, and
O2- contents in seedlings
Chlorophyll, MDA, H2O2, and
O2- contents were determined according
to the method of Zhang and Li (2016); MDA content was determined
according to the absorbance at 450, 532, and 600 nm; and chlorophyll a
and b contents were determined via acetone extraction.
2.4 | Determination of AsA, GSH, AAO, DHAR,
GR, and GPX indices of seedlings
The contents of AsA and GSH were determined according to the method of
Griffith (1980); the activities of AAO and DHAR were determined at 265
nm following the method of Nakano and Asada (1981); and the activities
of GR and GPX were determined by the method of Nagalakshmi and Prasad
(2001) at an absorbance of 340 nm.
2.5 | Determination of nitrate-nitrogen
content, ammonium nitrogen content, NR, GS, and GOGAT indices of
seedlings
The nitrate and ammonium nitrogen contents in fresh leaves were
determined using the method of Gao (2000); the NR activity was
determined using the method described by Barro et al. (1991); and the
activities of GS and GOGAT were determined according to the method
described by Hao et al.
(2004).
2.6 | Determination of enzyme gene expression
for the AsA-GSH cycle and nitrogen metabolism in seedlings
2.6.1 | Design of RT-qPCR Primers
We downloaded the AsA-GSH cycle enzyme genes (OsGR3 andOsGPX1 ) and nitrogen metabolism enzyme genes (OsNR2 ,OsGS1 , and OsGOGAT1 ) of rice seedlings from NCBI
(https://www.ncbi.nlm.nih.gov)
and referred to the sequence of the gene, OsActin1 . Primers were
designed using Primer-BLAST sequence alignment. Thereafter, the
specificity of the primers was verified to obtain gene-specific primers,
and the sequence number of the genes and the size of the product were
obtained (Table 1).
TABLE 1 Primers for the
enzyme genes and reference gene in the AsA-GSH cycle and nitrogen
metabolism in rice seedlings