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Article

Nitric Oxide Promoted the Seed Germination of Cynanchum auriculatum under Cadmium Stress

by
Fang-Fang Liu
1,†,
Xuan-Huan Qiao
1,†,
Tao Yang
1,
Peng Zhao
1,
Zhi-Peng Zhu
1,
Jun-Hao Zhao
1,
Jia-Ming Luo
1,
Ai-Sheng Xiong
2,* and
Miao Sun
1,2,*
1
College of Marine and Biological Engineering, Yancheng Teachers University, Yancheng 224002, China
2
State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(1), 86; https://doi.org/10.3390/agronomy14010086
Submission received: 8 December 2023 / Revised: 26 December 2023 / Accepted: 28 December 2023 / Published: 29 December 2023
(This article belongs to the Special Issue Progress in Horticultural Crops - from Genotype to Phenotype)
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Abstract

:
Cynanchum auriculatum, an early food-medicine homologous plant native to Asia, possesses significant nutritional and health benefits. However, the presence of cadmium (Cd) in the soil poses a hazard to the germination and growth of C. auriculatum. As nitric oxide (NO) plays a vital role in plant resistance to heavy metal stress, we used three different concentrations of SNP treatment during the germination phase, aiming to alleviate the inhibitory effects of Cd stress on the seed germination of C. auriculatum. The results indicated that when compared to seeds treated with SNP concentrations of 0.2 mM and 0.8 mM, C. auriculatum seeds treated with 0.4 mM SNP exhibited an improved germination rate and germination index, as well as longer hypocotyl. Furthermore, in comparison to NOS-like, the SNP application stimulated the production of endogenous NO through NR catalysis. Additional investigations showed that the ABA level decreased while the GA level increased under normal conditions, while the SNP application enhanced the accumulation of both ABA and GA in C. auriculatum seeds under Cd stress. Histochemical staining and biochemical indicators demonstrated that SNP treatment enhanced the enzymatic activity of SOD, POD, and CAT, while inhibiting the production of hydrogen peroxide and superoxide anion. Moreover, SNP treatment resulted in increased α-amylase activity, which facilitated starch hydrolysis and the generation of soluble sugar. Ultimately, the seed vitality of C. auriculatum under Cd stress was promoted. Our findings present a theoretical framework for the application of SNP in the seed germination mechanism of C. auriculatum and establish the groundwork for comprehending the physiological role of NO under Cd stress.

1. Introduction

The global rise of environmental pollution problems, particularly the concern regarding cadmium (Cd) contamination in soil, has become increasingly acknowledged as a consequence of the rapid advancements in industry and agriculture [1]. Cd possesses a prolonged biological half-life, allowing it to persist in the human kidney for a considerable period of 10–30 years, causing significant harm to human health [2]. The majority of Cd pollution is assimilated into the human body through dietary sources like cereals, produce, and root crops, accounting for a significant ninety percent [3]. In plants, Cd pollution also affects a variety of life activities, including the absorption of essential elements, photosynthesis, respiration, and plant growth [4]. Recently, there has been a noticeable correlation between the growing human demand and the increasing necessity for medicinal plants [5]. However, the presence of Cd pollution within the soil has had a detrimental effect on the process of seed germination and subsequent seedling development in these medicinal plants due to the enrichment, accumulation, and transportation of Cd [6].
Cynanchum auriculatum, a member of the Cynanchum family, is a medicinal plant commonly found in China, Korea, and India [7]. It is widely recognized as a traditional Chinese medicinal food homologous herb, renowned for its medicinal properties and significant levels of steroids, acetophenones, terpenes, and alkaloids [8]. Recent studies have shown that C. auriculatum exhibits various therapeutic effects, such as enhancing immunity by improving the phagocytosis ability of macrophages [9], treating breast cancer by controlling the formation of breast CSCs [10], and the inhibition of neuron damage by reducing apoptosis and reactive oxygen species (ROS) [11]. However, our previous study revealed that soil contamination with Cd negatively affected the germination process of C. auriculatum, leading to a decreased germination rate. Therefore, it would be valuable to develop an effective method for germinating C. auriculatum seeds under Cd stress.
Recent research has demonstrated that nitric oxide (NO), an influential gaseous signaling molecule with the unique capability to freely permeate biofilms, possesses multiple biological functions in various plant physiological processes, including the regulation of plant growth and development [12], stomatal movement [13], respiration [14], and the biosynthesis and transduction of phytohormone [15]. In the seeds of Oryza sativa [16], Brassica chinensis [17], and Cicer arietinum [18], NO has been found to enhance the germination rate through various regulatory mechanisms, such as the combined actions of NO and photosensitive pigments [19], the interaction between NO and phytohormones [20], and the regulation of the “oxidative window” model by controlling ROS generation [21]. More interestingly, in the defense mechanism of the plant exposed to Cd pollution, NO is also involved in the response mechanism and plays a significant role as a signaling molecule in regulating the resistance. Through S-nitrosylation, NO plays a crucial role in regulating the activity and functionality of Cd response proteins [22], as well as interacting with superoxide anions to modify various biomolecules [23]. In addition, the reaction between NO and hydrogen sulfide leads to the production of thionirous acid, which in turn influences physiological processes and stress responses [24]. Furthermore, NO has the ability to bind to heme, resulting in the inhibition of bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) production, and the inhibition of soil pathogens through the NosP/NahK signaling pathway [25].
In Arabidopsis thaliana [26], Hordeum vulgare [27], and Capsicum annuum [28], it was found that increasing the endogenous NO level or administering exogenous NO enhanced the resistance of plants against Cd stress, suggesting that NO is a potential gas component to improve the resistance of plants to Cd stress. However, it remains unclear whether NO is effective for the resistance of C. auriculatum against Cd stress, or the underlying mechanism by which NO regulates the Cd resistance network of C. auriculatum. Thus, to investigate the role of NO in regulating the resistance of C. auriculatum against Cd stress, we treated C. auriculatum seeds with sodium nitroprusside (SNP, exogenous NO donor) and cadmium chloride (CdCl2, Cd stress) for 48 h, followed by the photography of the germination phenotype, as well as measured the germination rate, hypocotyl length, biomass, and seed viability. To explore whether exogenous NO affected the production of endogenous NO, we determined the endogenous NO level, the enzymatic activity of nitric oxide synthase-like (NOS-like) and nitrate reductase (NR), and the expression level of the NO biosynthesis gene. To explore the interaction between the NO and phytohormone of C. auriculatum seeds under Cd stress, the contents of abscisic acid (ABA) and gibberellin (GA) and the expression of phytohormone biosynthesis and signaling genes were measured. In addition, we determined the α-amylase activity, starch content, soluble sugar content, and the level of superoxide anion (O2−) and hydrogen peroxide (H2O2). We also visualized the accumulation of ROS through histochemical staining. To summarize, the current study has substantiated the impact of NO on the seed germination of C. auriculatum under Cd stress. Furthermore, these results provide preliminary findings regarding the most effective concentration of SNP that can be employed to enhance the seed biology of C. auriculatum.

2. Materials and Methods

2.1. Plant Materials and Treatments

The seeds of C. auriculatum variety ‘Binwu No. 1’ were collected from the C. auriculatum Planting Base located at Yancheng Teachers University (120°12′20″ E, 34°11′48″ N), Yancheng, China. Full, pest-free seeds were screened out and washed with ethanol (75%), sodium hypochlorite (10%), mercuric chloride (0.1%), and sterile water. Then, the C. auriculatum seeds were immersed in distilled water for a duration of 48 h at 30 °C. After that, the seed germination of C. auriculatum was carried out with between two layers of filter paper lined in sterile petri dishes.
To examine the influence of Cd stress on the seed germination of C. auriculatum, CdCl2 (Catalog No. 202908, purchased from Shanghai Sigma-Aldrich Co., Ltd., Shanghai, China) was added into the filter paper [29]. Considering the Cd exposure level in the soil, air, and water [30], the CdCl2 solution was used at the final concentration of 50 μM. Meanwhile, filter papers and the CdCl2 solution were regularly replaced every 48 h in the incubation medium to ensure a constant water potential [31]. To investigate the role of SNP in regulating the resistance of C. auriculatum against Cd stress, SNP solutions (Catalog No. PHR1423, purchased from Shanghai Sigma-Aldrich Co., Ltd., Shanghai, China) at various concentrations (0.2, 0.4, and 0.6 mM) were added into the filter paper, while ddH2O-treated seeds were set as the control group. According to previous research [32], the 0.1 mM SNP solution released an average of 0.65 μM min−1 of NO within 3 h. Each petri dish contained a total of 50 seeds and was placed in the thermostatic incubator at (25 ± 1) °C for 6 d, and the determination and photographic documentation of the germination rate occurred at 2-day intervals. The indication of germination was observed by subjecting the lower radicle to a measurement of 2 mm. The germination index of C. auriculatum seeds under different treatments was measured using the method [33].

2.2. Endogenous NO Content and the Activity of NO Biosynthesis Enzyme

To investigate the role of endogenous NO in regulating the resistance of C. auriculatum against Cd stress, the compound 4-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO, Catalog No. C221, purchased from Shanghai Sigma-Aldrich Co., Ltd., Shanghai, China) was used as a specific NO scavenger [34]. Given that the clearance of one molecule of NO is facilitated by one molecule of cPTIO, and cPTIO is concentration-dependent and time-sensitive [35], we applied a final concentration of 10 mM for the cPTIO solution. Following germination, the seeds of C. auriculatum underwent grinding in liquid nitrogen and subsequent homogenization. The resultant homogenized powder, weighing 200 mg, was utilized for the subsequent analysis. Additionally, in this study, each treatment included 3 biological replicates consisting of 20 seeds. Triphenyl tetrazolium chloride (TTC) was used to evaluate seed viability in petri dishes in addition to the germination experiment [36].
The endogenous NO content of C. auriculatum seeds was determined using the Total NO Assay kit (Catalog No. A012–1). In brief, the procedure involved combining the mixture with coenzyme, reaction enhancer, and nitrate reductase, and incubating it at 37 °C for 30 min. Following that, lactate dehydrogenase (LDH, 10 μL) and 10 μL of LDH buffer were added and incubated at 37 °C for an additional cycle. Griess reagent I and Griess reagent II were mixed in, and the OD540 value of the mixture was determined after incubating at 30 °C for 10 min. Furthermore, sodium nitrite with various concentrations (0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1, and 1 µM) was used to construct the standard curve. Additionally, the assessment of NOS-like and NR enzymatic activities was conducted using the Plant NOS ELISA (Catalog No. A014–2–2) and Plant NR ELISA (Catalog No. A096–1–1) kits, respectively. The kits were all procured from Nanjing Jiancheng Biological Engineering Co., Ltd. (Nanjing, China).

2.3. Endogenous ABA and GA Content

The preparation method for the determination of the endogenous phytohormone was referred to in the previous study [37]. The C. auriculatum seeds (1.0 g) were mixed with 2.0 mL of the extraction solution containing 100% methanol and ground using liquid nitrogen. The resulting mixture was left to stand at 4 °C for a duration of 12 h. Afterward, it was centrifugated at 4000 rpm for 15 min. The supernatant was then collected, and the aforementioned steps were repeated once. The resulting supernatant was combined and subjected to solid phase extraction using C18 and activated carbon. The supernatant was eluted using 100% methanol and purified using a 0.45 μm microporous filter to enable the determination of the phytohormone. Then, the contents of endogenous ABA and GA in C. auriculatum seeds were detected using the Plant ABA ELISA kit (Catalog No. 077235) and Plant GA ELISA kit (Catalog No. 072782), respectively. The above kits were all purchased from Shanghai Mlbio Biotech Co., Ltd. (Shanghai, China).

2.4. The Determination of Starch and Soluble Sugar and the Activity of α-amylase

Briefly, the initial step involved drying the seeds in an oven, followed by the grinding process with a grinder, and ultimately screening the resulting powder for the extraction. Sterile water (400 μL) was added to the powder, which was then gelatinized in a boiling water bath for 15 min. After cooling, a mixture of 400 μL perchloric acid solution (9.2 mM) and 600 μL sterile water was added to the powder. The mixture was thoroughly mixed and centrifuged at 4000 rpm for 10 min, and the extract was used to determine the starch content [38]. Meanwhile, the soluble sugar of C. auriculatum seeds was extracted with 80% ethanol [39]. The starch content and soluble sugar level of C. auriculatum seeds under different treatments were measured according to the phenol sulfuric acid method [40].
The α-amylase activity of C. auriculatum seeds was measured using the dinitrosalicylic (DNS) acid method [41] with minor modifications. C. auriculatum seeds (1.0 g) were meticulously ground and homogenized using liquid nitrogen. The mixture was then subjected to centrifugation at 3000 rpm for 10 min. Following this, the supernatant was heated to 70 °C for 15 min. The amylase liquid was subsequently cooled, and then combined with 2 mL of DNS and 1 mL of starch solution (10%). The resulting mixture was agitated thoroughly and subsequently boiled for 10 min. After being cooled in an ice bath, the colorimetry of the mixture at 540 nm was measured.

2.5. Histochemical Staining and Quantification of H2O2 and O2−

To detect the distribution of H2O2 in C. auriculatum seeds under different treatments, we used the diaminobenzidine (DAB) histochemical staining method as previously described [42]. Following the removal of the seed coat, the seeds were longitudinally incised into two segments with the sterilized scalpel. Then, these segments were submerged in the DAB solution (1.0%) at 30 °C for 1 h. Afterwards, the resultant stained phenotype of the seeds was recorded with a camera (EOS 200D, Canon). At the same time, the H2O2 content of C. auriculatum seeds was quantified using the Plant H2O2 ELISA kit (Catalog No. A064-1-1, purchased from Nanjing Jiancheng Biological Engineering Co., Ltd., Nanjing, China).
The O2− staining of C. auriculatum seeds was performed according to the method [43]. Following the removal of the seed coat using tweezers, the seeds were longitudinally divided into two sections and then submerged in the nitroblue tetrazolium (NBT) solution (0.02%) at 30 °C in darkness for 10 min. The stained seeds were then treated with 95% alcohol for several decolorization steps and captured using a camera. Meanwhile, the O2− generation rate of C. auriculatum seeds was measured using the previous method [44].

2.6. The Antioxidant Enzyme Activity of C. auriculatum Seeds

In brief, C. auriculatum seeds (1.0 g) were carefully sterilized and washed and meticulously transferred into a pre-cooled mortar. Subsequently, 50 mM of phosphate buffer (2.0 mL, pH = 7.8) was added to the homogenate at 4 °C. The mixture was then centrifuged at 12,000 rpm for 20 min, and the resulting supernatant was carefully preserved for further analysis of antioxidant enzyme activity. The enzymatic activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in C. auriculatum seeds were determined using the SOD assay kit (Catalog No. A001-1-1), POD assay kit (Catalog No. A084-3-1), and CAT assay kit (Catalog No. A007-1-1), respectively. Furthermore, these kits were procured from Nanjing Jiancheng Biological Engineering Co., Ltd., Nanjing, China.

2.7. Quantitative Real-Time PCR (qRT-PCR) Analysis

Based on our previous report, CaActin7 was used as the reference gene for quantifying the expression profiles of target genes [37]. In this study, NO synthesis genes (CaNOS-like, CaNR1) [45], ABA synthesis (xanthoxin dehydrogenase, CaABA2) [46] and signaling gene (SNF1-related protein kinase 2, CaSnRK2) [47], and GA synthesis (G protein alpha subunit 1, CaGα1) and signaling gene (Ga20 oxidase, CaGA20ox1) [48], whose expression profiles were determined using qRT-PCR (primer information in Supplementary Table S1) were used. The titanium One-Step RT-PCR kit (Catalog No. 639503, purchased from Beijing Takara Co., Ltd., Beijing China) was used for qRT-PCR amplification. Additionally, the relative expression levels of the target genes were calculated using the 2−ΔΔCt method [49] with CaActin7 as the reference gene.

2.8. Statistical Analysis and Data Visualization

In this study, we performed three biological and three technical repeats for each treatment to eliminate the accumulative error. To identify the statistical significance between the ddH2O, Cd, Cd + SNP, and Cd + cPTIO treatment groups, a statistical analysis was performed with a t-test and one-way ANOVA, while the data were visualized using the Graphpad Prism 8.0 software. In addition, the p values of ≤0.05 and ≤0.01 were considered to be significant (*) and very significant (**), respectively.

3. Results

3.1. SNP Treatment Promoted the Germination of C. auriculatum Seeds under Cd Stress

To investigate the effects of Cd stress on the seed germination of C. auriculatum, we treated sterilized C. auriculatum seeds with Cd stress in the CdCl2 (50 μM)-containing plates. As shown in Figure 1, compared to the control group (ddH2O treatment), Cd stress severely reduced the germination of C. auriculatum seeds during the 6-day culture cycle. However, under Cd stress, SNP treatments significantly promoted the germination of C. auriculatum seeds, of which 0.4 mM SNP-treated C. auriculatum seeds exhibited the best results.
At the same time, we measured the effects of Cd stress and SNP treatments on the germination rate, germination index, hypocotyl length, and biomass of C. auriculatum seeds. The data showed that during the 6-day culture cycle of the control group, the seed germination was initiated on the 4th day of sowing, with a germination rate of 92.67% and a germination index of 45.34%, respectively. In contrast, Cd stress significantly reduced the germination rate and germination index throughout the entire culture cycle. Meanwhile, under Cd stress, the SNP treatment groups showed a higher germination rate and germination index. Among these groups, the 0.4 mM SNP-treated C. auriculatum group showed the highest germination rate (80.67%) and germination index (41.74%) compared to the 0.2 mM and 0.6 SNP treatment groups. Additionally, the hypocotyl length of different treatment groups showed a similar trend (Table 1). These results indicated that the SNP treatment effectively alleviated the inhibitory effect of Cd stress on the germination of C. auriculatum seeds, with 0.4 mM being the most suitable concentration compared to other concentrations of SNP solutions.

3.2. The Role of NO on the Germination of C. auriculatum Seeds under Cd Stress

To verify the impact of the 0.4 mM SNP treatment on the germination of C. auriculatum seeds under Cd stress, we determined the seed viability of C. auriculatum under ddH2O treatment, Cd treatment, Cd + 0.4 mM SNP treatment, and Cd + 10 mM cPTIO treatment using the TTC staining method. The results showed that the highest staining degree was observed in ddH2O-treated seeds, followed by Cd + 0.4 mM SNP-treated seeds, Cd-treated seeds, and Cd + 10 mM cPTIO-treated seeds (Figure 2A). This indicated that the presence of 0.4 mM SNP not only enhanced the seed viability of C. auriculatum under Cd stress, but also highlighted the significant role of endogenous NO in the seed germination process of C. auriculatum.
To investigate the role of endogenous NO in the seed germination process of C. auriculatum under Cd stress, we determined the content of endogenous NO at four different time points. Throughout the 6-day culture cycle, the endogenous NO content of the control group increased from 0.22 nM kg−1 (0 d) to 1.52 nM kg−1 (4 d) and then decreased slowly, indicating that NO was positively involved in the germination process of the C. auriculatum seed. However, the NO content of C. auriculatum seeds under Cd stress was significantly higher than that of the control group at 4 d (increased by 15.13%) and 6 d (increased by 34.03%). At the same time, the highest NO content was noted in the Cd + 0.4 mM SNP treatment group, while the Cd + 10 mM cPTIO treatment group had the lowest level of NO content among all the groups (Figure 2B).
We also measured the enzymatic activities of NOS-like and NR to evaluate the impact of SNP treatment on endogenous NO biosynthesis in C. auriculatum seeds. Throughout the culture cycle, there were no significant differences in NR activity observed in the control group at four time points. However, the NR activity in C. auriculatum seeds under Cd stress increased by 74.11% at 6 d compared to the control group. Furthermore, treatment with Cd + 0.4 mM SNP resulted in a significant 1.44-fold increase in NR activity, while the NR activity in the Cd + 10 mM cPTIO treatment group was significantly lower than that of the Cd-treated group and Cd + 0.4 mM SNP-treated group (Figure 2C). In contrast, the activity of NOS-like showed no significant difference between different groups during the culture cycle, except for the Cd + 0.4 mM SNP treatment group (Figure 2D). Meanwhile, the expression profiles of CaNOS-like and CaNR1 were also evaluated at four time points and showed similar trends as the enzymatic activity (Figure 2E).

3.3. The Role of NO on the Accumulation of ABA and GA in C. auriculatum Seeds under Cd Stress

Since ABA and GA are involved in the germination process and abiotic stress resistance of plants, we determined the content of ABA and GA in C. auriculatum seeds under Cd stress. In the control group, the ABA content of C. auriculatum seeds gradually declined from 23.33 μg kg−1 (0 d) to 11.65 μg kg−1 (6 d). However, the ABA content of C. auriculatum seeds in other treatment groups were higher compared to the control group, with the Cd + 0.4 mM SNP treatment group exhibiting the highest ABA content (Figure 3A). At the same time, the GA content of C. auriculatum seeds under ddH2O treatment showed a gradual increasing trend, rising from 0.47 μg kg−1 (0 d) to 2.03 μg kg−1 (6 d). Compared to the control group, the GA content in other treatment groups was lower, with the Cd + 10 mM cPTIO treatment group having the lowest GA content (Figure 3B).
To further investigate the critical role of NO in regulating the biosynthesis and signal transduction of phytohormones, we measured the relative expression levels of genes related to the biosynthesis and signaling of ABA and GA. Compared to the control group, the expression of CaABA2 under Cd stress showed an overall upward trend, particularly in the Cd + 0.4 mM SNP treatment group (Figure 3C). A similar pattern was observed in the expression of CaSnRK2 (Figure 3D). In the biosynthesis and signaling of GA, it was found that the expression of CaGα1 (Figure 3E) and CaGA20ox1 (Figure 3F) under different treatments increased at 4 d and peaked at 6 d, among which the Cd + 0.4 mM SNP-treated samples showed the highest expression profiles.

3.4. The Role of NO in Starch Degradation of C. auriculatum Seeds under Cd Stress

To investigate the role of NO in starch degradation during the culture cycle, we determined the levels of starch and soluble sugar in C. auriculatum seeds under different treatments. The results showed that in the control group, the decrease in starch content was accompanied by an increase in soluble sugar content. Under Cd stress, the hydrolysis of starch was slower in the Cd + 10 mM cPTIO treatment group, whereas the Cd + 0.4 mM SNP treatment group exhibited lower starch content (Figure 4A). Meanwhile, at 4 d and 6 d, it was found that the soluble sugar content of Cd + 0.4 mM SNP-treated samples was higher compared to the Cd treatment group, while the Cd + 10 mM cPTIO-treated samples had the lowest soluble sugar content, particularly at 6 d (39.54 g kg−1) (Figure 4B).
Meanwhile, to further analyze the impact of Cd stress on the starch hydrolysis of C. auriculatum seeds, we measured the enzymatic activity of α-amylase. In the control group, α-amylase activity peaked at 4 d (56.17 × 103 U kg−1) and slightly decreased at 6 d. In other groups, the trend of changes in α-amylase activity was similar to the soluble sugar content. α-amylase activity was found to be the highest in the Cd + 0.4 mM SNP treatment group, whereas the lowest was assayed in the Cd + 10 mM cPTIO treatment group (Figure 4C).

3.5. The Role of NO on ROS Scavenging of C. auriculatum Seeds under Cd Stress

To explore the effects of NO on the antioxidant enzyme activity of C. auriculatum seeds under Cd stress, we measured the enzymatic activities of SOD, POD, and CAT throughout the culture cycle. As shown in Figure 5A, the SOD activity of the control group displayed a gradual increase, with the highest activity observed in the Cd + 0.4 mM SNP-treated samples at 6 d (57.17 × 103 U kg−1), which was 1.76 times higher than the control group. Overall, the POD activities of all groups peaked at 4 d after sowing, followed by a slight decline at 6 d. The Cd + 0.4 mM SNP treatment group showed the highest POD activity during the 6-day culture cycle (Figure 5B). Additionally, the CAT activities of all groups showed a notable upward trend, with changes in the CAT activity aligning with the variations of SOD and POD. The Cd + 0.4 mM SNP treatment group presented the highest CAT activity (Figure 5C).
To assess the impact of the increased activity of the antioxidant enzyme on the ROS generation of C. auriculatum seeds under Cd stress, we analyzed the distribution and the content of H2O2 and O2− throughout the culture cycle. As shown in Figure 6A, after 6 d of cultivation, the Cd + 0.4 mM SNP group exhibited a lower degree of brown and blue staining compared to the control group, while a greater degree of staining was observed in the Cd + 10 mM cPTIO group. Moreover, we quantified the production of H2O2 and O2− to evaluate the ROS-scavenging ability of NO. Compared to the Cd stress group, the Cd + 0.4 mM SNP treatment group exhibited a decrease in H2O2 content at 2 d (reduced by 25.05%), 4 d (reduced by 18.09%), and 6 d (reduced by 18.33%) (Figure 6B). Additionally, the O2− generation rate of the Cd + 0.4 mM SNP treatment group was slower, with a reduction of 0.91-fold at 2 d, 0.73-fold at 4 d, and 0.67-fold at 6 d (Figure 6C).

4. Discussion

The germination of crops like Solanum lycopersicum [50], Triticum aestivum [51], and Oryza sativa [52] is severely hindered by Cd contamination in soil. However, there is little research about the impact of Cd stress on the germination of C. auriculatum. The present study found that Cd stress induced the accumulation of ABA and GA, activated the production of H2O2 and O2−, inhibited the hydrolysis of starch, and decreased the seed viability, thereby inhibiting the germination of C. auriculatum seeds. Therefore, to stimulate seeds’ germination and establishment of C. auriculatum, we treated C. auriculatum seeds with the nitric oxide donor SNP. SNP has been used for alleviating the inhibitory effect of Cd on the seed germination and seedling growth of Oryza sativa [53] and Brassica oleracea [54], but the suitable concentration of SNP treatment varies among different plant species and developmental stages [34,45]. Nitraria tangutorum treated with 70 μM SNP had the best growth rate, while 100 μM SNP significantly inhibited the growth rate [55]. Brassica chinensis seeds pre-soaked with 10 μM SNP showed a higher germination rate, germination index, and vitality index under NaCl stress, while 200 μM SNP had no significant effect on the germination [17]. In this study, compared to concentrations of 0.2 mM and 0.6 mM, C. auriculatum seeds treated with 0.4 mM SNP exhibited a higher germination rate, germination index, and longer hypocotyl length, suggesting that the concentration of 0.4 mM SNP is more effective in promoting seed germination of C. auriculatum under Cd stress. Furthermore, excessively high concentrations of SNP in the environment can lead to nitrate poisoning in vertebrates [56], suggesting that using low concentrations of SNP avoids negative effects on other organisms in the ecological environment.
In plants, the production of NO depends on NR and the mitochondrial electron transport chain, or through the arginine pathway, similar to the NOS activity present in animals [57]. In Solanum tuberosum, SNP and cPTIO affected the metabolism and signal transduction of ABA by up-regulating and down-regulating NR activity, and ultimately controlled the dormancy mechanism of a potato tuber [58]. In Glycine max, NO was the key signaling molecule in static magnetic field-stimulated tolerance towards UV-B stress, and NOS may possibly be accountable for the triggered NO production in seedlings [59]. Our study revealed that the change in NR activity and CaNR1 expression corresponded to the variation in NO content, indicating that the promotion of endogenous NO synthesis by exogenous SNP was mainly dependent on the NR pathway, which was similar to findings in Daucus carota [45], Fragaria annassa [60], and Cucumis sativus [61]. In addition, NO is a crucial player in the leaf senescence of plants under abiotic stress [62], and it was observed that compared to young leaves, the NO level was higher in senescent leaves [63]. Given the association between nitrogen content and cardiovascular diseases [64], it also suggested that controlling for NO production helps to regulate early senescence in plants, while prolonged SNP treatment on C. auriculatum may even have a negative impact.
Moreover, further studies have shown that the intricate balance between ABA and GA plays a significant role in overcoming dormancy and facilitating the germination process [20]. During normal germination conditions, NO acts prior to ABA and GA to enhance the production of GA and suppress the production of ABA, thereby promoting optimal seed germination while preserving essential nutrients [65]. However, under heavy meal stress, the increased expression of GA20ox1 and accelerated GA accumulation counteracted the inhibition of abiotic stress in seed germination [66], leading to stomatal closure and enhanced plant resistance to abiotic stress [67]. Our data also showed that under normal conditions, the biosynthesis and signaling of the ABA pathway were repressed during the germination process. However, the increase in NO induced by SNP plays a protective role in ABA accumulation under Cd stress, while the removal of NO induced by cPTIO inhibits ABA accumulation. Furthermore, under Cd stress, the treatment of SNP significantly enhances the biosynthesis and signaling of GA in C. auriculatum seeds, which has also been confirmed in Oryza sativa [68,69] and Cucumis sativus [70].
Additionally, during the seed germination process, heavy metal stress is accompanied by ROS generation, which decreases the activity of antioxidant enzymes and increases oxidative damage [71]. While a moderate level of ROS is required for the seed germination process of plants, excess ROS directly harms the mitochondrial membrane structure and has detrimental effects [72]. During the germination process of Oryza sativa seeds under Cd stress, the application of SNP enhanced the activities of SOD, POD, CAT, and APX, while reducing the accumulation of H2O2 and MDA [53]. In this study, the results of the histochemical staining and biochemical indicators demonstrated that under Cd stress, the increase in the endogenous NO level by SNP treatment enhanced the activity of the antioxidant enzyme and inhibited the excessive generation of H2O2 and O2−. On the other hand, the decrease in endogenous NO content through cPTIO treatment had the opposite effect, indicating the significant role of NO in protecting the ROS scavenging system during the germination of C. auriculatum under Cd stress. Besides the ROS scavenging system, Cd stress also impeded the starch degradation by reducing the α-amylase activity of seeds, which led to the immobilization of starch and lack of nutrients [71]. Recent reports have shown that NO accelerated the starch hydrolysis to form soluble sugar by increasing the activity of α-amylase and β-amylase [73]. In Vigna radiata, drought stress stimulated the production of endogenous NO, while SNP treatment significantly up-regulated α-amylase activity and accelerated the formation of reducing sugar and soluble sugar, which alleviated the inhibition of seed germination [74]. Our findings showed that Cd stress inhibited the hydrolysis of starch and the production of soluble sugar, and cPTIO treatment further exacerbated this problem by weakening the α-amylase activity of C. auriculatum seeds. However, the utilization of SNP to enhance the endogenous NO level enhanced the α-amylase activity, facilitated starch hydrolysis and soluble sugar accumulation, consequently promoting seed germination under Cd stress, and similar results were also obtained in Triticum aestivum [75] and Glycine max [76]. Furthermore, it has been reported that SNP treatment on seeds significantly reduced the accumulation of Cd and stress markers in seedlings during the development process [53,77]. Therefore, the resistance of SNP-treated C. auriculatum plants against Cd stress during seedling development will be one of our future research directions. Furthermore, NO metabolism related genes and transcription factors can be further functionally characterized using genome sequencing [78], overexpression [79,80], and knockout [81] strategies.

5. Conclusions

Overall, the importance of NO in enhancing the resistance of C. auriculatum to Cd stress during germination was demonstrated. Among different concentrations, a 0.4 mM treatment of SNP was found to be particularly effective in increasing the germination rate and germination index and promoting hypocotyl growth. A further study showed that 0.4 mM SNP treatment promoted endogenous NO accumulation by enhancing the NR activity. This, in turn, affected the biosynthesis and signaling of ABA and GA, as well as the activities of SOD, POD, and CAT. Moreover, it inhibited the generation of H2O2 and O2- during germination. Together, these combined effects led to starch hydrolysis and the production of soluble sugar, resulting in the improved seed germination of C. auriculatum under Cd stress (Figure 7).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14010086/s1.

Author Contributions

F.-F.L. and X.-H.Q.: experimental design, writing—original draft, data analysis, investigation, methodology. T.Y., P.Z., Z.-P.Z., J.-H.Z. and J.-M.L.: investigation, data curation. A.-S.X. and M.S.: experimental design, writing—review and editing, methodology, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the Natural Science Foundation for Higher Education Institutions in Jiangsu Province (21KJB210007), the Key Research and Development Program of Jiangsu (BE2022386), and the Priority Academic Program Development of Jiangsu Higher Education Institutions Project (PAPD).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. Yang, S.; Yang, D.; Taylor, D.; He, M.; Liu, X.; Xu, J. Tracking cadmium pollution from source to receptor: A health-risk focused transfer continuum approach. Sci. Total Environ. 2023, 867, 161574. [Google Scholar] [CrossRef] [PubMed]
  2. Clemens, S.; Aarts, M.G.; Thomine, S.; Verbruggen, N. Plant science: The key to preventing slow cadmium poisoning. Trends Plant Sci. 2013, 18, 92–99. [Google Scholar] [CrossRef]
  3. Yan, J.; Wu, X.; Li, T.; Fan, W.; Abbas, M.; Qin, M.; Li, R.; Liu, Z.; Liu, P. Effect and mechanism of nano-materials on plant resistance to cadmium toxicity: A review. Ecotoxicol. Environ. Saf. 2023, 266, 115576. [Google Scholar] [CrossRef]
  4. Li, Y.; Ding, L.; Zhou, M.; Chen, Z.; Ding, Y.; Zhu, C. Transcriptional Regulatory Network of Plant Cadmium Stress Response. Int. J. Mol. Sci. 2023, 24, 4378. [Google Scholar] [CrossRef] [PubMed]
  5. Siadjeu, C.; Pucker, B. Medicinal plant genomics. BMC Genom. 2023, 24, 429. [Google Scholar] [CrossRef] [PubMed]
  6. Al-Khayri, J.M.; Banadka, A.; Rashmi, R.; Nagella, P.; Alessa, F.M.; Almaghasla, M.I. Cadmium toxicity in medicinal plants: An overview of the tolerance strategies, biotechnological and omics approaches to alleviate metal stress. Front. Plant Sci. 2022, 13, 1047410. [Google Scholar] [CrossRef] [PubMed]
  7. Jung, D.H.; Kim, H.Y.; Won, J.H.; Park, S.H. Development of a classification model for Cynanchum wilfordii and Cynanchum auriculatum using convolutional neural network and local interpretable model-agnostic explanation technology. Front. Plant Sci. 2023, 14, 1169709. [Google Scholar] [CrossRef]
  8. Wang, L.; Cai, F.; Zhao, W.; Tian, J.; Kong, D.; Sun, X.; Liu, Q.; Chen, Y.; An, Y.; Wang, F.; et al. Cynanchum auriculatum Royle ex Wight., Cynanchum bungei Decne. and Cynanchum wilfordii (Maxim.) Hemsl.: Current Research and Prospects. Molecules 2021, 26, 7065. [Google Scholar] [CrossRef]
  9. Wang, Y.; Han, J.; Yue, Y.; Wu, Y.; Zhang, W.; Xia, W.; Wu, M. Purification, structure identification and immune activity of a neutral polysaccharide from Cynanchum auriculatum. Int. J. Biol. Macromol. 2023, 237, 124142. [Google Scholar] [CrossRef]
  10. Zhen, X.; Choi, H.S.; Kim, J.H.; Kim, S.L.; Liu, R.; Ko, Y.C.; Yun, B.S.; Lee, D.S. Caudatin Isolated from Cynanchum auriculatum Inhibits Breast Cancer Stem Cell Formation via a GR/YAP Signaling. Biomolecules 2020, 10, 925. [Google Scholar] [CrossRef]
  11. Zhang, X.; Gao, M.; Rao, Z.; Lei, Z.; Zeng, J.; Huang, Z.; Shen, C.; Zeng, N. The antitumour activity of C(21) steroidal glycosides and their derivatives of Baishouwu: A review. J. Ethnopharmacol. 2022, 293, 115300. [Google Scholar] [CrossRef] [PubMed]
  12. Gupta, K.J.; Kaladhar, V.C.; Fitzpatrick, T.B.; Fernie, A.R.; Møller, I.M.; Loake, G.J. Nitric oxide regulation of plant metabolism. Mol. Plant 2022, 15, 228–242. [Google Scholar] [CrossRef] [PubMed]
  13. Van Meeteren, U.; Kaiser, E.; Malcolm Matamoros, P.; Verdonk, J.C.; Aliniaeifard, S. Is nitric oxide a critical key factor in ABA-induced stomatal closure? J. Exp. Bot. 2020, 71, 399–410. [Google Scholar] [CrossRef] [PubMed]
  14. Poderoso, J.J.; Helfenberger, K.; Poderoso, C. The effect of nitric oxide on mitochondrial respiration. Nitric Oxide 2019, 88, 61–72. [Google Scholar] [CrossRef] [PubMed]
  15. Xie, Z.; Yang, C.; Li, M.; Zhang, Z.; Wu, Y.; Gu, L.; Peng, X. Nitric Oxide Crosstalk With Phytohormone Is Involved in Enhancing Photosynthesis of Tetrastigma hemsleyanum for Photovoltaic Adaptation. Front. Plant Sci. 2022, 13, 852956. [Google Scholar] [CrossRef] [PubMed]
  16. Kumari, A.; Singh, P.; Kaladhar, V.C.; Manbir; Paul, D.; Pathak, P.K.; Gupta, K.J. Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice. Plant Cell Environ. 2022, 45, 178–190. [Google Scholar] [CrossRef]
  17. Ren, Y.; Wang, W.; He, J.; Zhang, L.; Wei, Y.; Yang, M. Nitric oxide alleviates salt stress in seed germination and early seedling growth of pakchoi (Brassica chinensis L.) by enhancing physiological and biochemical parameters. Ecotoxicol. Environ. Saf. 2020, 187, 109785. [Google Scholar] [CrossRef]
  18. Pandey, S.; Kumari, A.; Shree, M.; Kumar, V.; Singh, P.; Bharadwaj, C.; Loake, G.J.; Parida, S.K.; Masakapalli, S.K.; Gupta, K.J. Nitric oxide accelerates germination via the regulation of respiration in chickpea. J. Exp. Bot. 2019, 70, 4539–4555. [Google Scholar] [CrossRef]
  19. Beligni, M.V.; Lamattina, L. Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 2000, 210, 215–221. [Google Scholar] [CrossRef]
  20. Zhang, Y.; Wang, R.; Wang, X.; Zhao, C.; Shen, H.; Yang, L. Nitric Oxide Regulates Seed Germination by Integrating Multiple Signalling Pathways. Int. J. Mol. Sci. 2023, 24, 9052. [Google Scholar] [CrossRef]
  21. Bailly, C.; Merendino, L. Oxidative signalling in seed germination and early seedling growth: An emerging role for ROS trafficking and inter-organelle communication. Biochem. J. 2021, 478, 1977–1984. [Google Scholar] [CrossRef] [PubMed]
  22. Feng, J.; Chen, L.; Zuo, J. Protein S-Nitrosylation in plants: Current progresses and challenges. J. Integr. Plant Biol. 2019, 61, 1206–1223. [Google Scholar] [CrossRef]
  23. Domingos, P.; Prado, A.M.; Wong, A.; Gehring, C.; Feijo, J.A. Nitric oxide: A multitasked signaling gas in plants. Mol. Plant 2015, 8, 506–520. [Google Scholar] [CrossRef] [PubMed]
  24. Kolupaev, Y.E.; Yemets, A.I.; Yastreb, T.O.; Blume, Y.B. The role of nitric oxide and hydrogen sulfide in regulation of redox homeostasis at extreme temperatures in plants. Front. Plant Sci. 2023, 14, 1128439. [Google Scholar] [CrossRef] [PubMed]
  25. Anantharaman, S.; Guercio, D.; Mendoza, A.G.; Withorn, J.M.; Boon, E.M. Negative regulation of biofilm formation by nitric oxide sensing proteins. Biochem. Soc. Trans. 2023, 51, 1447–1458. [Google Scholar] [CrossRef]
  26. Besson-Bard, A.; Gravot, A.; Richaud, P.; Auroy, P.; Duc, C.; Gaymard, F.; Taconnat, L.; Renou, J.P.; Pugin, A.; Wendehenne, D. Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake. Plant Physiol. 2009, 149, 1302–1315. [Google Scholar] [CrossRef]
  27. Valentovicová, K.; Halusková, L.; Huttová, J.; Mistrík, I.; Tamás, L. Effect of cadmium on diaphorase activity and nitric oxide production in barley root tips. J. Plant Physiol. 2010, 167, 10–14. [Google Scholar] [CrossRef]
  28. Kaya, C.; Ashraf, M.; Alyemeni, M.N.; Ahmad, P. The role of nitrate reductase in brassinosteroid-induced endogenous nitric oxide generation to improve cadmium stress tolerance of pepper plants by upregulating the ascorbate-glutathione cycle. Ecotoxicol. Environ. Saf. 2020, 196, 110483. [Google Scholar] [CrossRef]
  29. Zhang, Z.X.; Wang, S.L.; Wang, J.; Zhang, C.; Liu, D.; Wang, C.; Xu, F.S. The overexpression of LOW PHOSPHATE ROOT 1 (LPR1) negatively regulates Arabidopsis growth in response to Cadmium (Cd) stress. Plant Physiol. Biochem. 2023, 196, 556–566. [Google Scholar] [CrossRef]
  30. Li, G.Y.; Yan, L.J.; Chen, X.M.; Lam, S.S.; Rinklebe, J.; Yu, Q.; Yang, Y.F.; Peng, W.X.; Sonne, C. Phytoremediation of cadmium from soil, air and water. Chemosphere 2023, 320, 138058. [Google Scholar] [CrossRef]
  31. Pegler, J.L.; Oultram, J.M.J.; Nguyen, D.Q.; Grof, C.P.L.; Eamens, A.L. MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana. Plants 2021, 10, 130. [Google Scholar] [CrossRef] [PubMed]
  32. Ederli, L.; Reale, L.; Madeo, L.; Ferranti, F.; Gehring, C.; Fornaciari, M.; Romano, B.; Pasqualini, S. NO release by nitric oxide donors in vitro and in planta. Plant Physiol. Biochem. 2009, 47, 42–48. [Google Scholar] [CrossRef] [PubMed]
  33. Zhang, Z.; Fan, J.; Wu, J.; Zhang, L.; Wang, J.; Zhang, B.; Wang-Pruski, G. Alleviating effect of silicon on melon seed germination under autotoxicity stress. Ecotoxicol. Environ. Saf. 2020, 188, 109901. [Google Scholar] [CrossRef] [PubMed]
  34. Sun, M.; Yang, X.L.; Zhu, Z.P.; Xu, Q.Y.; Wu, K.X.; Kang, Y.J.; Wang, H.; Xiong, A.S. Comparative transcriptome analysis provides insight into nitric oxide suppressing lignin accumulation of postharvest okra (Abelmoschus esculentus L.) during cold storage. Plant Physiol. Biochem. 2021, 167, 49–67. [Google Scholar] [CrossRef]
  35. D’Alessandro, S.; Posocco, B.; Costa, A.; Zahariou, G.; Schiavo, F.L.; Carbonera, D.; Zottini, M. Limits in the use of cPTIO as nitric oxide scavenger and EPR probe in plant cells and seedlings. Front. Plant Sci. 2013, 4, 340. [Google Scholar] [CrossRef]
  36. Qiao, J.; Liao, Y.; Yin, C.; Yang, X.; Tú, H.M.; Wang, W.; Liu, Y. Vigour testing for the rice seed with computer vision-based techniques. Front. Plant Sci. 2023, 14, 1194701. [Google Scholar] [CrossRef]
  37. Sun, M.; Zhu, Z.P.; Yu, J.X.; Wu, K.X.; Guo, Y.X.; Shen, M.; Liu, F.F.; Tang, X.H.; Kang, Y.J. Transcriptomic and physiological analysis reveal phytohormone and phenylpropanoid biosynthesis in root of Cynanchum auriculatum. Plant Growth Regul. 2023, 101, 67–85. [Google Scholar] [CrossRef]
  38. Maniglia, B.C.; Silveira, T.M.G.; Tapia-Blácido, D.R. Starch isolation from turmeric dye extraction residue and its application in active film production. Int. J. Biol. Macromol. 2022, 202, 508–519. [Google Scholar] [CrossRef]
  39. Zhu, Z.P.; Yu, J.X.; Qiao, X.H.; Yu, Z.F.; Xiong, A.S.; Sun, M. Hydrogen sulfide delays yellowing and softening, inhibits nutrient loss in postharvest celery. Sci. Hortic. 2023, 315, 111991. [Google Scholar] [CrossRef]
  40. Paraginski, R.T.; Colussi, R.; Dias, A.R.G.; da Rosa Zavareze, E.; Elias, M.C.; Vanier, N.L. Physicochemical, pasting, crystallinity, and morphological properties of starches isolated from maize kernels exhibiting different types of defects. Food Chem. 2019, 274, 330–336. [Google Scholar] [CrossRef]
  41. Visvanathan, R.; Qader, M.; Jayathilake, C.; Jayawardana, B.C.; Liyanage, R.; Sivakanesan, R. Critical review on conventional spectroscopic α-amylase activity detection methods: Merits, demerits, and future prospects. J. Sci. Food Agric. 2020, 100, 2836–2847. [Google Scholar] [CrossRef] [PubMed]
  42. Zhu, Z.P.; Yu, J.X.; Liu, F.F.; Zhu, D.W.; Xiong, A.S.; Sun, M. AeWRKY32 from okra regulates anthocyanin accumulation and cold tolerance in Arabidopsis. J. Plant Physiol. 2023, 287, 154062. [Google Scholar] [CrossRef]
  43. Jambunathan, N. Determination and detection of reactive oxygen species (ROS), lipid peroxidation, and electrolyte leakage in plants. Methods Mol. Biol. 2010, 639, 292–298. [Google Scholar] [CrossRef] [PubMed]
  44. Poór, P.; Kovács, J.; Borbély, P.; Takács, Z.; Szepesi, Á.; Tari, I. Salt stress-induced production of reactive oxygen- and nitrogen species and cell death in the ethylene receptor mutant Never ripe and wild type tomato roots. Plant Physiol. Biochem. 2015, 97, 313–322. [Google Scholar] [CrossRef] [PubMed]
  45. Sun, M.; Yang, T.; Qiao, X.H.; Zhao, P.; Zhu, Z.P.; Wang, G.L.; Xu, L.L.; Xiong, A.S. Nitric oxide regulates the lignification and carotenoid biosynthesis of postharvest carrot (Daucus carota L.). Postharvest Biol. Technol. 2024, 207, 112593. [Google Scholar] [CrossRef]
  46. Sun, M.; Yang, T.; Qiao, X.H.; Zhao, P.; Zhu, Z.P.; Su, K.Y.; Sun, C.W.; Xie, Z.B.; Zhang, S.S.; Xu, M.; et al. Hydrogen sulfide delays postharvest ripening of white fleshed strawberry by regulating phytohormone accumulation and ROS generation. Postharvest Biol. Technol. 2023, 205, 112536. [Google Scholar] [CrossRef]
  47. Belda-Palazón, B.; Adamo, M.; Valerio, C.; Ferreira, L.J.; Confraria, A.; Reis-Barata, D.; Rodrigues, A.; Meyer, C.; Rodriguez, P.L.; Baena-González, E. A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth. Nat. Plants 2020, 6, 1345–1353. [Google Scholar] [CrossRef]
  48. Hwang, O.J.; Back, K. Exogenous Gibberellin Treatment Enhances Melatonin Synthesis for Melatonin-Enriched Rice Production. Biomolecules 2022, 12, 198. [Google Scholar] [CrossRef]
  49. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 2008, 3, 1101–1108. [Google Scholar] [CrossRef]
  50. Lv, Y.; Zhao, Y.; He, Y.; Wang, J.; Zheng, Y.; Chen, X.; Huang, F.; Liu, J.; Yu, L. Synergistic effects of gamma-aminobutyric acid and melatonin on seed germination and cadmium tolerance in tomato. Plant Signal Behav. 2023, 18, 2216001. [Google Scholar] [CrossRef]
  51. Zhang, L.; Gao, B. Effect of Isosteviol on Wheat Seed Germination and Seedling Growth under Cadmium Stress. Plants 2021, 10, 1779. [Google Scholar] [CrossRef] [PubMed]
  52. Rizwan, M.; Ali, S.; Adrees, M.; Rizvi, H.; Zia-Ur-Rehman, M.; Hannan, F.; Qayyum, M.F.; Hafeez, F.; Ok, Y.S. Cadmium stress in rice: Toxic effects, tolerance mechanisms, and management: A critical review. Environ. Sci. Pollut. Res. Int. 2016, 23, 17859–17879. [Google Scholar] [CrossRef] [PubMed]
  53. He, J.; Ren, Y.; Chen, X.; Chen, H. Protective roles of nitric oxide on seed germination and seedling growth of rice (Oryza sativa L.) under cadmium stress. Ecotoxicol. Environ. Saf. 2014, 108, 114–119. [Google Scholar] [CrossRef] [PubMed]
  54. Ma, J.; Saleem, M.H.; Alsafran, M.; Jabri, H.A.; Mehwish; Rizwan, M.; Nawaz, M.; Ali, S.; Usman, K. Response of cauliflower (Brassica oleracea L.) to nitric oxide application under cadmium stress. Ecotoxicol. Environ. Saf. 2022, 243, 113969. [Google Scholar] [CrossRef]
  55. Gao, Z.; Zhang, J.; Zhang, J.; Zhang, W.; Zheng, L.; Borjigin, T.; Wang, Y. Nitric oxide alleviates salt-induced stress damage by regulating the ascorbate-glutathione cycle and Na(+)/K(+) homeostasis in Nitraria tangutorum Bobr. Plant Physiol. Biochem. 2022, 173, 46–58. [Google Scholar] [CrossRef]
  56. Chen, H.; Pang, Y.; Wei, Y.; He, X.; Zhang, Y.; Xie, L. Nitrate and sodium nitroprusside alter the development of Asian black-spined toads’ embryos by inducing nitric oxide production. Environ. Sci. Pollut. Res. Int. 2023, 30, 23060–23069. [Google Scholar] [CrossRef]
  57. Astier, J.; Gross, I.; Durner, J. Nitric oxide production in plants: An update. J. Exp. Bot. 2018, 69, 3401–3411. [Google Scholar] [CrossRef]
  58. Wang, Z.; Ma, R.; Zhao, M.; Wang, F.; Zhang, N.; Si, H. NO and ABA Interaction Regulates Tuber Dormancy and Sprouting in Potato. Front. Plant Sci. 2020, 11, 311. [Google Scholar] [CrossRef]
  59. Raipuria, R.K.; Kataria, S.; Watts, A.; Jain, M. Magneto-priming promotes nitric oxide via nitric oxide synthase to ameliorate the UV-B stress during germination of soybean seedlings. J. Photochem. Photobiol. B 2021, 220, 112211. [Google Scholar] [CrossRef]
  60. Kaya, C.; Ashraf, M.; Alyemeni, M.N.; Ahmad, P. Nitrate reductase rather than nitric oxide synthase activity is involved in 24-epibrassinolide-induced nitric oxide synthesis to improve tolerance to iron deficiency in strawberry (Fragaria × annassa) by up-regulating the ascorbate-glutathione cycle. Plant Physiol. Biochem. 2020, 151, 486–499. [Google Scholar] [CrossRef]
  61. Feng, Y.; Fu, X.; Han, L.; Xu, C.; Liu, C.; Bi, H.; Ai, X. Nitric Oxide Functions as a Downstream Signal for Melatonin-Induced Cold Tolerance in Cucumber Seedlings. Front. Plant Sci. 2021, 12, 686545. [Google Scholar] [CrossRef] [PubMed]
  62. Procházková, D.; Wilhelmová, N. Nitric oxide, reactive nitrogen species and associated enzymes during plant senescence. Nitric Oxide 2011, 24, 61–65. [Google Scholar] [CrossRef] [PubMed]
  63. Corpas, F.J.; Barroso, J.B.; Carreras, A.; Quirós, M.; León, A.M.; Romero-Puertas, M.C.; Esteban, F.J.; Valderrama, R.; Palma, J.M.; Sandalio, L.M.; et al. Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol. 2004, 136, 2722–2733. [Google Scholar] [CrossRef] [PubMed]
  64. Hong, C.; Zhu, H.; Zhou, X.; Zhai, X.; Li, S.; Ma, W.; Liu, K.; Shirai, K.; Sheerah, H.A.; Cao, J. Association of Blood Urea Nitrogen with Cardiovascular Diseases and All-Cause Mortality in USA Adults: Results from NHANES 1999–2006. Nutrients 2023, 15, 461. [Google Scholar] [CrossRef] [PubMed]
  65. Bethke, P.C.; Libourel, I.G.; Aoyama, N.; Chung, Y.Y.; Still, D.W.; Jones, R.L. The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy. Plant Physiol. 2007, 143, 1173–1188. [Google Scholar] [CrossRef] [PubMed]
  66. Bouteraa, M.T.; Mishra, A.; Romdhane, W.B.; Hsouna, A.B.; Siddique, K.H.M.; Saad, R.B. Bio-Stimulating Effect of Natural Polysaccharides from Lobularia maritima on Durum Wheat Seedlings: Improved Plant Growth, Salt Stress Tolerance by Modulating Biochemical Responses and Ion Homeostasis. Plants 2022, 11, 1991. [Google Scholar] [CrossRef]
  67. Malcheska, F.; Ahmad, A.; Batool, S.; Müller, H.M.; Ludwig-Müller, J.; Kreuzwieser, J.; Randewig, D.; Hänsch, R.; Mendel, R.R.; Hell, R.; et al. Drought-Enhanced Xylem Sap Sulfate Closes Stomata by Affecting ALMT12 and Guard Cell ABA Synthesis. Plant Physiol. 2017, 174, 798–814. [Google Scholar] [CrossRef]
  68. Liu, Y.S.; Tao, Y.; Yang, X.Z.; Liu, Y.N.; Shen, R.F.; Zhu, X.F. Gibberellic acid alleviates cadmium toxicity in rice by regulating NO accumulation and cell wall fixation capacity of cadmium. J. Hazard. Mater. 2022, 439, 129597. [Google Scholar] [CrossRef]
  69. Singh, P.K.; Chakrabarty, D.; Dwivedi, S.; Kumar, A.; Singh, S.P.; Sinam, G.; Niranjan, A.; Singh, P.C.; Chatterjee, S.; Majumdar, D.; et al. Nitric oxide-mediated alleviation of arsenic stress involving metalloid detoxification and physiological responses in rice (Oryza sativa L.). Environ. Pollut. 2022, 297, 118694. [Google Scholar] [CrossRef]
  70. Wu, P.; Kong, Q.; Bian, J.; Ahammed, G.J.; Cui, H.; Xu, W.; Yang, Z.; Cui, J.; Liu, H. Unveiling Molecular Mechanisms of Nitric Oxide-Induced Low-Temperature Tolerance in Cucumber by Transcriptome Profiling. Int. J. Mol. Sci. 2022, 23, 5615. [Google Scholar] [CrossRef]
  71. Seneviratne, M.; Rajakaruna, N.; Rizwan, M.; Madawala, H.; Ok, Y.S.; Vithanage, M. Heavy metal-induced oxidative stress on seed germination and seedling development: A critical review. Environ. Geochem. Health 2019, 41, 1813–1831. [Google Scholar] [CrossRef] [PubMed]
  72. Oracz, K.; Karpiński, S. Phytohormones Signaling Pathways and ROS Involvement in Seed Germination. Front. Plant Sci. 2016, 7, 864. [Google Scholar] [CrossRef] [PubMed]
  73. Hajihashemi, S.; Skalicky, M.; Brestic, M.; Pavla, V. Cross-talk between nitric oxide, hydrogen peroxide and calcium in salt-stressed Chenopodium quinoa Willd. At seed germination stage. Plant Physiol. Biochem. 2020, 154, 657–664. [Google Scholar] [CrossRef] [PubMed]
  74. Zhang, Y.; Su, J.; Cheng, D.; Wang, R.; Mei, Y.; Hu, H.; Shen, W.; Zhang, Y. Nitric oxide contributes to methane-induced osmotic stress tolerance in mung bean. BMC Plant Biol. 2018, 18, 207. [Google Scholar] [CrossRef] [PubMed]
  75. Zheng, C.F.; Jiang, D.; Liu, F.L.; Dai, T.B.; Liu, W.C.; Jing, Q.; Cao, W.X. Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity. Environ. Exp. Bot. 2009, 67, 222–227. [Google Scholar] [CrossRef]
  76. Basit, F.; Bhat, J.A.; Guan, Y.J.; Jan, B.L.; Tyagi, A.; Ahmad, P. Nitric oxide and spermine revealed positive defense interplay for the regulation of the chromium toxicity in soybean (Glycine max L.). Environ. Pollut. 2022, 308, 119602. [Google Scholar] [CrossRef]
  77. Kumar, D.; Manhas, R.K.; Ohri, P. Deciphering the growth promoting and stress curtailing role of nitric oxide in Meloidogyne incognita infested Solanum lycopersicum seedlings. Sci. Hortic. 2023, 319, 112147. [Google Scholar] [CrossRef]
  78. Li, M.Y.; Feng, K.; Hou, X.L.; Jiang, Q.; Xu, Z.S.; Wang, G.L.; Liu, J.X.; Wang, F.; Xiong, A.S. The genome sequence of celery (Apium graveolens L.), an important leaf vegetable crop rich in apigenin in the Apiaceae family. Hortic. Res. 2020, 7, 9. [Google Scholar] [CrossRef]
  79. Li, T.; Liu, J.X.; Deng, Y.J.; Duan, A.Q.; Liu, H.; Zhuang, F.Y.; Xiong, A.S. Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot. Hortic. Res. 2022, 9, uhac193. [Google Scholar] [CrossRef]
  80. Feng, K.; Xing, G.M.; Liu, J.X.; Wang, H.; Tan, G.F.; Wang, G.L.; Xu, Z.S.; Xiong, A.S. AgMYB1, an R2R3-MYB factor, plays a role in anthocyanin production and enhancement of antioxidant capacity in celery. Veg. Res. 2021, 1, 2. [Google Scholar] [CrossRef]
  81. Duan, A.Q.; Liu, H.; Shu, S.; Xiong, A.S. CRISPR/Cas9-mediated precise targeted mutagenesis of phytoene desaturase in celery. Hortic. Res. 2022, 9, uhac162. [Google Scholar] [CrossRef]
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Figure 1. The effects of Cd stress and multiple concentrations of SNP treatment on the germination phenotype of C. auriculatum seeds.
Figure 1. The effects of Cd stress and multiple concentrations of SNP treatment on the germination phenotype of C. auriculatum seeds.
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Figure 2. The effects of 0.4 mM SNP treatment on the biosynthesis of endogenous NO in C. auriculatum seeds under Cd stress. (A) Seed viability of C. auriculatum under the treatments of Cd + 10 mM cPTIO, Cd stress, Cd + 0.4 mM SNP, and ddH2O at 6 d using the TTC staining method. (B) Endogenous NO content, (C) NR activity, (D) NOS-like activity, and (E) the expression profiles of CaNR1 and CaNOS-like of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
Figure 2. The effects of 0.4 mM SNP treatment on the biosynthesis of endogenous NO in C. auriculatum seeds under Cd stress. (A) Seed viability of C. auriculatum under the treatments of Cd + 10 mM cPTIO, Cd stress, Cd + 0.4 mM SNP, and ddH2O at 6 d using the TTC staining method. (B) Endogenous NO content, (C) NR activity, (D) NOS-like activity, and (E) the expression profiles of CaNR1 and CaNOS-like of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
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Figure 3. The effects of 0.4 mM SNP treatment on the biosynthesis and signaling of ABA and GA of C. auriculatum seeds under Cd stress. (A) ABA content, (B) GA content, as well as the expression levels of (C) CaABA2, (D) CaSnRK2, (E) CaGα1, and (F) CaGA20ox1 of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
Figure 3. The effects of 0.4 mM SNP treatment on the biosynthesis and signaling of ABA and GA of C. auriculatum seeds under Cd stress. (A) ABA content, (B) GA content, as well as the expression levels of (C) CaABA2, (D) CaSnRK2, (E) CaGα1, and (F) CaGA20ox1 of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
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Figure 4. The effects of 0.4 mM SNP treatment on starch hydrolysis of C. auriculatum seeds under Cd stress. (A) Starch content, (B) soluble sugar content, and (C) α–amylase activity of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
Figure 4. The effects of 0.4 mM SNP treatment on starch hydrolysis of C. auriculatum seeds under Cd stress. (A) Starch content, (B) soluble sugar content, and (C) α–amylase activity of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
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Figure 5. The effects of 0.4 mM SNP treatment on the activity of antioxidant enzyme of C. auriculatum seeds under Cd stress. (A) SOD activity, (B) POD activity, and (C) CAT activity of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
Figure 5. The effects of 0.4 mM SNP treatment on the activity of antioxidant enzyme of C. auriculatum seeds under Cd stress. (A) SOD activity, (B) POD activity, and (C) CAT activity of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with * indicating a significance level of p ≤ 0.05, and ** indicating a very significant level of p ≤ 0.01.
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Figure 6. The effects of 0.4 mM SNP treatment on ROS scavenging of C. auriculatum seeds under Cd stress. (A) DAB and NBT staining seeds at 6 d, (B) H2O2 content, and (C) O2− generation rate of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with ** indicating a very significant level of p ≤ 0.01.
Figure 6. The effects of 0.4 mM SNP treatment on ROS scavenging of C. auriculatum seeds under Cd stress. (A) DAB and NBT staining seeds at 6 d, (B) H2O2 content, and (C) O2− generation rate of C. auriculatum seeds under different treatments during the culture cycle. The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with ** indicating a very significant level of p ≤ 0.01.
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Figure 7. The potential mechanism of 0.4 mM SNP treatment enhancing the germination of C. auriculatum seeds under Cd stress.
Figure 7. The potential mechanism of 0.4 mM SNP treatment enhancing the germination of C. auriculatum seeds under Cd stress.
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Table 1. The effect of Cd stress and multiple concentrations of SNP treatment on germination rate, germination index, and hypocotyl length of C. auriculatum seeds.
Table 1. The effect of Cd stress and multiple concentrations of SNP treatment on germination rate, germination index, and hypocotyl length of C. auriculatum seeds.
DayddH2O TreatmentCd StressSNP Treatment under Cd Stress
Cd + 0.2 mM SNPCd + 0.4 mM SNPCd + 0.6 mM SNP
Germination rate
200000
482.67 ± 1.626.67 ± 0.13 **9.33 ± 0.45 **12.67 ± 0.34 **8.56 ± 0.29 **
692.67 ± 5.2114.11 ± 0.31 **75.33 ± 4.62 **80.67 ± 4.96 **71.25 ± 3.75 **
Germination index
200000
441.34 ± 1.133.18 ± 0.06 **4.42 ± 0.07 **6.40 ± 0.04 **4.17 ± 0.07 **
646.36 ± 1.216.67 ± 0.25 **35.70 ± 1.06 **40.74 ± 1.65 **34.28 ± 1.25 **
Hypocotyl length
200000
43.44 ± 0.051.10 ±0.01 **2.51 ± 0.02 **2.90 ± 0.02 **2.32 ± 0.01 **
67.74 ± 0.121.81 ± 0.02 **5.85 ± 0.11 **6.33 ± 0.18 **5.32 ± 0.15 **
The values were expressed as the means ± standard error of triplicate assays, and the significance level for differences between groups was denoted by asterisks, with ** indicating a very significant level of p ≤ 0.01.
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MDPI and ACS Style

Liu, F.-F.; Qiao, X.-H.; Yang, T.; Zhao, P.; Zhu, Z.-P.; Zhao, J.-H.; Luo, J.-M.; Xiong, A.-S.; Sun, M. Nitric Oxide Promoted the Seed Germination of Cynanchum auriculatum under Cadmium Stress. Agronomy 2024, 14, 86. https://doi.org/10.3390/agronomy14010086

AMA Style

Liu F-F, Qiao X-H, Yang T, Zhao P, Zhu Z-P, Zhao J-H, Luo J-M, Xiong A-S, Sun M. Nitric Oxide Promoted the Seed Germination of Cynanchum auriculatum under Cadmium Stress. Agronomy. 2024; 14(1):86. https://doi.org/10.3390/agronomy14010086

Chicago/Turabian Style

Liu, Fang-Fang, Xuan-Huan Qiao, Tao Yang, Peng Zhao, Zhi-Peng Zhu, Jun-Hao Zhao, Jia-Ming Luo, Ai-Sheng Xiong, and Miao Sun. 2024. "Nitric Oxide Promoted the Seed Germination of Cynanchum auriculatum under Cadmium Stress" Agronomy 14, no. 1: 86. https://doi.org/10.3390/agronomy14010086

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