Exogenous melatonin improves growth in hulless barley seedlings under cold stress by influencing the expression rhythms of circadian clock genes

Materials and growth condition

The hulless barley used in the study was Kunlun 12, which was a generous gift from Dr. Tao He (Qinghai University, Xining, Qinghai province, China). The seeds were first rinsed in running tap water for 10 min, and then soaked in 75% alcohol for 30 s. Subsequently, the seeds were sterilized (Cai et al., 2018) with 0.1% HgCl₂ for 10 min, and thoroughly washed 5 times with plenty of sterile water. Base on some previous studies on melatonin (Han et al., 2017) and Fig. S1, the sterilized seeds were then separately soaked in 1 µM melatonin (MT) or distilled water (mock) for 12 h. The hulless barley seeds were then transplanted into 7 cm length * 7 cm wide * 10 cm height dark seedling boxes with sterile water-moistened medical gauze and a clear plastic cover. Germination was carried out in the dark at 25 °C for the following 2 d. Then, the culture condition was switched to constant light photoperiodic illumination provided by cool white fluorescent light with a density of 100 µMol m⁻² s⁻¹ for a week. Subsequently, the seedlings were randomly divided into three groups and transplanted to individual growth chambers at various temperatures of 25 °C (Normal), 15 °C (Low temperature) or 5 °C (Cold) and the same 100 µMol m⁻² s⁻¹12 h/12 h light/dark photoperiodic illumination condition for 2 d. To avoid the effects of diurnal light changes, the illumination for all the growth chamber was then switched to 100 µMol m⁻² s⁻¹constant light by cool white fluorescent light during the following experimental procedure. According to different processing conditions, the experimental materials were divided into the following 6 groups: 25 °C group, 25 °C+MT group, 15 °C group, 15 °C+MT group, 5 °C group and 5 °C+MT group. A more detail description is given in Fig. S2. Starting from the time point at which the light was on (Circadian Time, CT₀), about 100 mg leave tissues were harvested every 4 h continuously during the following 3 days. All the plant samples, isolated at each time point for subsequent analysis, were immediately stored at −80 °C after a brief deep freezing in liquid nitrogen.

Effect of exogenous melatonin on the germination rate of hulless barley seeds

After treatment as described above by 1 µM melatonin or distilled water, the hulless barley seeds were randomly divided into mock or MT group. The seed germination rates of the different tests were determined at 25 °C.

Determination of the effects of melatonin treatment on plant vegetative growth

After treatment as described above by 1 µM melatonin or distilled water, the seedlings were randomly divided into three groups and transplanted to individual growth chambers at various temperatures of 25, 15 or 5 °C and the same 100 µMol m⁻² s⁻¹12 h/12 h light/dark photoperiodic illumination condition for 5 d. Then, their morphological indices, such as root length, leaf length, fresh or dry weight of each plantlet were measured.

Determination of endogenous melatonin contents in hulless barley seedlings

An acetone–methanol method of Pape & Lüning (2006)) was used to assay endogenous melatonin contents in 1.0 g leaf tissue from each individual seedling that harvested at CT₀ and CT₇₂. First, the leaf tissue sample of the hulless barley was fully homogenized to a fine powder in liquid nitrogen with a mortar and pestle. Then, 5 ml of extraction buffer (mixture of acetone, methanol and water at a volume ratio of 89:10:1) was added to the powder, and the grinding of the tissue homogenate was continued on ice for 5 min in the dark. Subsequently, the supernatant was collected by centrifugation at 4,500 g for 5 min in a refrigerated centrifuge and transferred to a new centrifuge tube containing 0.5 mL of 1% trichloroacetic acid (TCA) to precipitate the protein. Ultimately, the melatonin content in the supernatant from the different groups of hulless barley seedlings was determined using an Extraction-Melatonin ELISA kit (RE 54021; IBL International GmbH, Hamburg, Germany). To avoid the degradation of melatonin by natural light, all the experiments involving this compound were conducted in a dark room.

Quantitative real-time PCR (qPCR) analysis and rhythm analysis of circadian clock genes in hulless barley seedlings

Total RNA from plant tissue samples was extracted with an RNAiso Plus kit (Takara, Dalian, China). Then, the extracted RNA samples were used to as templates for the subsequent reverse transcription reactions carried out using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). The synthesized cDNAs were purified using the MiniBEST DNA Fragment Purification Kit Ver. 4.0 (Takara), and stored at −80 °C freezers until the subsequent qPCR analyses were performed.

Using primers specific for the different circadian clock genes listed in Table S1 in supplement file, all the qPCR reactions were performed on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). A TB Green Premix Ex Taq™ II kit (Takara) was used to perform the amplifications by a two-step method. All samples were first pre-denatured at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s, annealing and elongation at 56 °C for 30 s with a read of fluorescence intensity from SYBR™ Green at the end of elongation process in each reaction. The Cq value for the qPCR amplification of individual samples is expressed as the mean value ± standard deviation (SD) from 3 independent biological replicates.

The Cq value data from 72 h time-course plant samples was uploaded to the online platform for data sharing and period analysis, BioDare2, and processed by a Spectrum Resampling (SR) method with a threshold of period length ranged from 18 to 34 h and a RAE (relative amplitude error) value (http://www.biodare.ed.ac.uk/) (Ramos-Sánchez et al., 2017; Zielinski et al., 2014). All the samples series with a feedback period data out of this setting threshold after the online processing, were screened as arrhythmia by BioDare2 automatically. Detailed circadian period and RAE (relative amplitude error) analysis results were shown in Figs. S3 and S4.

Determination of chlorophyll and carotenoid content in leaves of hulless barley under different temperature conditions

Fresh leaf tissue samples from hulless barley seedlings in an amount of 0.1 g,were harvested in a set of time-course experiments at CT₀, CT₁₂,CT ₂₄, CT₃₆, CT₄₈,CT₆₀ and CT₇₂. Chlorophyll (Chl) and carotenoid contents in these leaf tissue samples were measured following the method of Lichtenthaler and Wellburn with slight modifications (Lichtenthaler & Wellburn, 1983). After homogenization in 20 mL of 80% acetone (v/v) using a pre-cooled mortar and pestle, the homogenate of the leaf tissue was centrifuged at 8,000 g for 10 min. Then, three mL of the supernatant from each plant tissue sample was taken to measure the absorbance at the wavelengths of 663, 646 and 470 nm on a UV/VIS T6 spectrophotometer (Persee Analytics, Beijing, China). The contents of chlorophyll and carotenoid were calculated using the Lichtenthaler and Wellburn formula (Lichtenthaler & Wellburn, 1983) and expressed as mg g⁻¹FW.

Determination of free proline, soluble sugars and malondialdehyde (MDA) contents in leaves of hulless barley seedlings under different temperature conditions

Proper amounts of leaf tissues of hulless barley seedlings under different temperature conditions were harvested, and used to determine the intracellular levels of free proline, soluble sugars and MDA. The free proline in leaf tissues was extracted and determined using the Mahdavian’s method (Mahdavian, Ghaderian & Schat, 2016) with minor modifications. Briefly, 0.5 g of hulless barley seedling leaves was thoroughly homogenized in five mL of 3% sulfosalicylic acid solution (m/v), and centrifuged at 8,000 g for 10 min. Then, two mL of the supernatant were transferred into a test tube containing a mixture of two mL glacial acetic acid and two mL of acid ninhydrin, and the test tube was incubated in a water bath at 95 °C for 1 h. Subsequently, the red-colored reaction products were extracted and purified with four mL of toluene. The optical density (OD) value of the recovered toluene phase solution was measured at the wavelength of 520 nm. The proline content of each plant sample was determined using a standard curve for proline and expressed as µmol g⁻¹ FW.

The soluble sugars content was measured using the anthrone method (Azarmi et al., 2015). Precisely, 0.5 g of the leaf tissues was harvested and thoroughly homogenized in five mL of 80% ethanol and centrifuged at 8,000 g for 15 min. Then, 0.1 mL of the supernatant was accurately aspirated and mixed with three mL of anthrone. The reaction mixture was incubated in a boiling water bath for 30 min and then cooled on ice. The OD₆₂₅ value was recorded for each plant samples and used to calculate the soluble sugars content using a standard curve of soluble sugars reagent and expressed as mg g ⁻¹ FW.

The MDA content of each hulless barley leaf tissue sample was determined using the thiobarbituric acid assay (Nahar et al., 2015). Briefly, the leaf tissue (0.5 g) was thoroughly homogenized in 5 ml of TCA, and centrifuged at 8,000 g for 10 min. Then, 2 ml of the supernatant was aspirated and mixed with 2 ml of a 0.5% thiobarbituric acid solution. The reaction mixture was incubated in a 95 °C water bath for 30 min, and then immediately cooled on ice. After centrifugation at 5,000 g for 10 min, a supernatant aliquot was taken to measure the absorbance at 450, 532 and 600 nm. The MDA content was calculated according to the method reported by Nahar (Nahar et al., 2015) and expressed in µmol g⁻¹ FW.

Statistical analysis

All growth data were subjected to one-way analysis of variance (ANOVA) using the SigmaStat V3.5 software (Systat Software, San Jose, CA, USA). Duncan’s multiple range test was used to assess the difference between treatments at a significance level of P < 0.05. For gene expression analysis, data analyses involving multiple comparisons were performed using Student’s t-test and the SPSS 22.0 software (SPSS Inc., Chicago, IL, USA).^∗P <  0.05,^∗∗P <  0.01.

Seed germination and growth assay

The low temperature stress inhibited the growth of hulless barley seedlings, while melatonin pretreatment alleviated this inhibition to a certain extent (Fig. 1). At first, the germination rate of hulless barley seeds was 57% at 25 °C conditions (Fig. 2A). The germination rate analysis was carried out in our laboratory at Xi’an, Shaanxi, China with a location of 108.96° East longitude and 34.28° North latitude, an altitude of 405 m and an air pressure of 97 KPa. When the hulless barley seeds were pretreated with 1 µM exogenous melatonin, the germination rate was significantly increased to 63% (Figs. 2A–2B).

We also determined the effects of exogenous melatonin on leaf length, primary root length, fresh weight and dry weight of hulless barley seedlings under different temperature conditions. The results revealed that exogenous melatonin significantly promoted the growth of leaf length and primary root length and increased fresh weight and dry weight of hulless barley seedlings (Figs. 2C–2F). In particular, under the condition of cold stress, application of exogenous melatonin markedly alleviated the suppressive effects of low environmental temperature on the vegetative growth of either the hulless barley seedlings leaves, or their roots. These results indicated that a suitable concentration of melatonin could confer significant physiological protection on the barley seedlings when they were exposed to cold stress. Meanwhile, It is interesting that the dry weight of hulless barley at 5 °C is greater than that at 25 and 15 °C, which was same as previous study in hulless barley under salt stress (Ma et al., 2018) .

The rhythmic expression pattern of the circadian clock gene of hulless barley under cold stress

The expression patterns of the circadian clock genes of hulless barley seedlings were determined at 25, 15 and 5 °C. At 25 °C, the expression of the circadian clock genes, including HvCCA1, HvTOC1, HvGI, HvLUX, HvPRR59, HvPRR73 and HvPRR95 maintained a robust circadian rhythmic profile for three days. Unexpectedly, unlike previous reports in barley and A. thaliana, the expression of HvELF3 in hulless barley exhibited an arrhythmic phenotype. At 15 °C, the expression of the morning clock gene HvCCA1 was not affected by the low temperature signals and showed a similar rhythmic expression pattern to that at 25 °C, while the total mRNA abundance of the night clock genes HvTOC1 and HvGI were significantly downregulated. In contrast, the expression levels of genes encoding key components of the evening complex (EC) of circadian clock in plants, such as HvLUX and HvELF3, were significantly upregulated. With respect to the PRR genes, although the expression levels of HvPRR59, HvPRR73 and HvPRR95 were not affected by exposure to 15 °C (low temperature), the phase of their rhythmic expression pattern was shifted forward. Consistent with expectations, the expression of all the evaluated circadian clock genes was significantly suppressed, and showed arrhythmic phenotypes under cold stress conditions at 5 °C. (Fig. 3, Fig. S3 and Fig. S4).

Effects of melatonin on the rhythmic expression pattern of circadian clock genes in hulless barley

The effects of exogenous melatonin on the rhythmic expression pattern of the circadian clock genes under various temperature conditions were evaluated after soaking the seeds of hulless barley in a 1 µM melatonin solution. The results showed that at 25 °C, exogenous melatonin remarkably induced the expression level of HvCCA1, the crucial morning clock gene of the plant endogenous circadian clock, to nearly 30-fold higher than that of untreated mock seedlings. Additionally, exogenous melatonin also slightly inhibited the expression level of HvTOC1, which is the key gene in the evening loop of the plant circadian clock. PRR genes in hulless barley displayed diverse responses to the exogenous melatonin treatments. The expression of HvPRR73 was induced by 2∼4 folds, while that of HvPRR59 and HvPRR95 was significantly inhibited by 1 µM melatonin. Regarding other evening loop genes, HvELF3 expression was slightly induced by exogenous melatonin, but the expression of HvLUX and HvGI was suppressed by melatonin to various degrees. In general, exogenous melatonin was found to affect the rhythmic expression patterns of the circadian clock genes by influencing their amplitudes in various degrees at 25 °C. As well as, exogenous melatonin had influenced the period length of clock gene expression at 25 °C in different manners. As shown in Fig. S3, exogenous melatonin treatment significantly decreased the period length of HvTOC1 (about 2.5h), while remarkably increased the period length of HvPRR59 (2.7h) and HvGI (1.9 h). (Fig. 4, Fig. S3 and Fig. S4).

Under 15 °C (low temperature) conditions, the expression levels of the HvCCA1, HvPRR59, HvPRR95, HvTOC1, HvLUX and HvELF3 genes in hulless barley seedlings that had been pretreated with 1 µM exogenous melatonin, were substantially suppressed compared with those of the seedlings at 25 °C. Among all tested circadian clock genes, the rhythmic expression of some genes was not affected, such as those of HvPRR59 and HvPRR95. However, the peak phase of some others was delayed, such as HvPRR73 and HvGI, whereas the peak phase of the evening loop genes HvTOC1 and HvLUX was shifted forward. The circadian rhythmic expression of morning gene HvCCA1 was completely disrupted and showed an arrhythmic pattern in melatonin pretreated hulless barley seedlings (RAE >0.6). These results indicated that exogenous melatonin inhibited the expression of most circadian clock genes and influenced the phase of some other clock genes under low temperature conditions. Additionally, these results also suggested that the night clock genes (such as HvTOC1 and HvLUX) were the potential targets for melatonin pretreatment at 15 °C. Interestingly, the period of all rhythmically expressed gene was extended by exogenous melatonin at 15 °C (Fig. 5, Figs. S3 and S4).

Under 5 °C (cold stress) conditions, the expression of the HvPRR73, HvPRR59, HvPRR95, HvLUX, HvELF3 and HvGI genes was strongly suppressed by 1 µM melatonin treatments and showed arrhythmic phenotype in their time course expression patterns. Remarkably, exogenous melatonin treatment was able to restore the rhythmic expression of the HvCCA1 and HvTOC1 gene to some extent, although their amplitudes were much lower than the 25 °C group. It was also worth noting that the circadian oscillation of HvCCA1 and HvTOC1 peaked at the subjective night phase under exogenous melatonin treatment. These results indicated that under cold stress, the appropriate concentration of exogenous melatonin treatment could restore the rhythmicity of the expression of the core circadian clock genes that encode the pacemaker of plant circadian clock in hulless barley (Fig. 6, Figs. S3 and S4).

Determination of the chlorophyll and carotenoid contents in leaves of hulless barley

Under conditions at 25 °C, the treatment with 1 µM melatonin reduced the chlorophyll and carotenoid contents in the leaves of hulless barley seedlings at most time points, though an unexpected increase of these two photosynthetic pigments by 44% and 35% more than the mock seedlings was observed at CT₄₈ in a time course experiment that lasted 3 d.

At 15 °C, the accumulation of chlorophyll and carotenoids showed very similar fluctuation patterns in hulless barley. In addition, at 15 °C, the exogenous melatonin significantly increased the relative amounts of cytosolic chlorophyll and carotenoids in seedling leaves after CT₃₆. For example, triggered by the exogenous melatonin, the accumulation of chlorophyll contents in leaves peaked at the CT₃₆ and CT₆₀ time points by 27% and 45%, respectively, compared to the mock seedlings.

Exogenous melatonin could also increase the contents of chlorophyll and carotenoids in the leaves of hulless barley under cold stress conditions after CT₂₄ at 5 °C. For example, compared to 5 °C group, treatment with 1 µM melatonin also significantly increased chlorophyll accumulation by 49% and 35% at CT₃₆ and CT₆₀, respectively in a time course experiment. These results confirmed that the proper amount of exogenous melatonin contributed to provide a better protection to these key pigments of their photosynthetic systems under low temperature and cold stress conditions (Figs. 7A–7F).

Determination of the MDA, soluble sugar and free proline content in leaves of hulless barley

MDA is a stable product of membrane lipid peroxidation, which always occurs during various abiotic stress-response processes in plants. The relative content of MDA in cells has been used either as a general intracellular indicator to ealuate the damage of plant cells under stresses, or as a common physiological indicator to estimate different resistance abilities of individual plant species under various environmental stresses (Leshem, 1987). In this study, no noticeable changes in the MDA contents of stressed hulless barley leaves under both 15 °C and 5 °C conditions were found when compared to that of the seedlings under 25 °C (Figs. 7G–7I). Nevertheless, the contents of intracellular MDA also changed significantly in all the seedlings that have been treated by 1 µM melatonin. In particular, at 5 °C, the treatment with melatonin markedly decreased the production of MDA in hulless barley leaves to between 7 and 57% at different time points (Figs. 7G–7I).

The soluble sugar content of hulless barley leaves increased significantly with when the environmental temperature dropped to 15 °C or 5 °C, as shown in Fig. 7. However, the treatment of 1 µM exogenous melatonin notably reduced the amount of soluble sugars in hulless barley seedlings under 15 ° C and 5 °C conditions. In particular, the soluble sugar content in the melatonin-treated seedlings decreased 26% to that of the mock seedlings under the same cold stress conditions at 5 °C. These results provided direct evidence for exogenous melatonin had enhanced the stress-resistance of hulless barley seedlings on the physiological levels under cold stress (Figs. 7J–7L).

Various environmental abiotic stresses, such as low temperature, high salt and excessive heavy metals always induced the accumulation of free proline in plant cells as a compatible osmoprotectant (Ashraf & Foolad, 2007). In this study, no significant changes in the free proline content in mock hulless barley seedlings were detected under various temperature conditions. When hulless barley seeds were pretreated with 1 µM exogenous melatonin, the free proline content in hulless barley seedlings was reduced to 53% at 15 °C, meanwhile exogenous melatonin did not significantly influence the proline content in hulless barley seedlings at 25 °C and 5 °C (Figs. 7M–7O).