Plant hoppers are pest herbivores that significantly affect the growth and productivity of rice worldwide. This study aimed to investigate the role of two chemicals, sodium nitroprusside (SNP), a nitric oxide inducer, and carboxy-PTIO potassium salt, a nitric oxide scavenger, under brown plant hopper (BPH) stress in rice. Screening tests were performed to evaluate various factors, including agronomic traits, relative water content, electrolytic leakage, gene expression, and nitric oxide (NO) production. Interestingly, the positive control and 100 µM cPTIO-treated seedlings showed decreased root and shoot growth, reduced relative water content, and induced electrolytic leakage under BPH stress. Similarly, the positive control and 100 µM cPTIO-treated seedlings exhibited decreased survival percentage and increased death rate under BPH stress. Gene expression analysis revealed that after 6, 12, and 24 h of BPH stress, the levels of OsNOA1 and OsEF-1α were significantly higher in the resistant rice lines Cheongcheong and Nagdong Double haploid (CNDH)-78 and CNDH-87, while OsNOA1 and OsNR were upregulated in the the susceptible rice lines CNDH 14 − 2 and CNDH 48 − 3. Moreover, the resistant lines exhibited increased NO content after 6, 12, and 24 h, and susceptible rice lines showed high NO content after 24 h of BPH stress. These findings suggest that the application of 100 µM SNP enhanced internal NO production and content, improved the overall agronomic traits, increased the relative water content, decreased electrolytic leakage, and protected rice from the harmful effects of BPHs.
Research Article
Enhancing rice crop resistance against brown plant hopper infestation through the foliar application of sodium nitroprusside
https://doi.org/10.21203/rs.3.rs-4322765/v1
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sodium nitroprusside
carboxy-PTIO potassium salt
brown plant hopper
nitric oxide
double haploid
Rice (Oryza sativa L.) is a cereal crop that feeds approximately half of the world’s population, particularly in Asia and other continents. It is grown in 114 countries and provides a primary source of income for more than 100 million households in Asia and Africa 1. White-backed plant hopper, Nilaparvata lugens Stal, and brown plant hopper (BPH) are detrimental phloem-feeding insect pests of rice. The first outbreak of these insect pests occurred in India in 1966 and ultimately spread to China, and subsequently to Japan and Korea. It has been reported that every year in China, these insect pests cause 10–20% of productivity loss in rice yields 2–5. BPH is a parasite that frequently attaches to the plant stem and penetrates the phloem using its stylet, distributing the translocation of assimilates and affecting plant growth and development. When a large number of BPH feed on a single plant, leaf dryness and stem wilting are common outcomes, a condition called hopper-burn 6.
BPH infestation also transmits two viruses, one of which is responsible for the disease of rice grassy stunt and the other for rugged stunt. Plants have evolved different defense mechanisms against insect herbivores and microbial pathogens. Ethylene, H2O2, jasmonic acid (JA), and salicylic acid are important signaling molecules in this process. Insect infestation is based on the cellular level, and most studies have only focused on insect chewing rather than sucking. Phloem-feeding insects are mainly responsible for promoting pathogenic infections, wounding, and water deficits in plants. Nitric oxide (NO) is a signaling molecule involved in many important physiological processes in plants, including seed germination, plant growth and development, maturation and senescence, root organogenesis, suppression of floral transition, and stomatal movement. It has also been shown to be involved in plants, apoptosis, and abiotic and biotic stresses 7–10. It has been thought that both phytohormones, such as JA, and NO can alter plant and animal defenses against abiotic and biotic stresses 11,12. NO can be generated through a nitrate- or nitrite-dependent pathway, which is catalyzed by nitrate reductase (NR) 13.
Studies have suggested that in Arabidopsis thaliana, nitrate reductase 1 and 2 (AtNIA1/NIA2) genes are responsible for the NR-dependent pathway 14,15. In the presence of oxygen and NADPH, a putative NO synthase (NOS) converts L-arginine into NO citrulline, which is an additional source of NO 16. Previous studies have shown that abscisic acid (ABA) can boost NO production in guard cells to regulate stomatal closure 17. Plants with AtNIA1 and AtNIA2 mutants showed poor stomatal closure because of the altered expression of essential genes involved in ABA signaling, and this poor stomatal closure could be reestablished by the application of exogenous NO 18. The AtNOS1 gene product sequence has been found to be similar to a protein involved in NO synthesis in the snail Helix pomatia. However, the recombinant AtNOS1 protein lacks in vitro NOS activity, raising the question of whether AtNOS1 is related to NOS. As a result, the gene was renamed NO-associated protein 1 (AtNOA1) 19. Therefore, AtNOS/AtNOA1 may function as a cGTPase instead of NOS 20. Despite its biochemical function, AtNOA1-dependent NO synthesis is believed to be involved in flowering, hormonal signaling, oxidative stress, pathogen defense, stomatal movement, and wounding 21–23. According to a recent study, expression of the rice gene OsNOS1/NOA1 improves salt tolerance and stress-related gene expression in the Arabidopsis nitric oxide-associated 1 mutant AtNOA1. It has been shown that OSNOA1 might function similarly to AtNOA 24. Other than the NOS and NR pathways, there is an indication that plants generate NO nonenzymatically under certain circumstances 25.
Agriculture is essential for providing food to the global population; however, open-field crops are susceptible to various biotic and abiotic stresses. Insects, pests, diseases, and water loss are some of the major factors that can result in significant crop yield loss. One approach to mitigate the negative effects of these stresses is the use of chemicals that can improve plant tolerance and resistance. In the current study, we investigated the effects of two chemicals, sodium nitroprusside (SNP), a nitric oxide inducer, and carboxy-PTIO (cPTIO), a nitric oxide scavenger, on the BPH-susceptible and -resistant rice lines. NO is a signaling molecule that plays a crucial role in plant defenses against biotic and abiotic stresses. SNP induces NO production, whereas cPTIO scavenges NO. This study aimed to determine whether increasing NO levels through SNP application could improve the tolerance of BPH-susceptible rice lines.
2.1 Plant materials and growth conditions
We selected four Cheongcheong and Nagdong Double haploid (CNDH) rice lines from the CNDH population, which was developed by the plant molecular breeding lab in 2011. Among the four selected lines, two were found to be BPH susceptible (CNDH 14-2, CNDH 48-3), and two were resistant lines (CNDH 78, CNDH 87) that were previously identified based on quantitative trait loci analysis. The rice seeds were pre-germinated for four days in small plastic bags with small holes for water to enter and incubated at 33°C. Three rectangular trays were filled with soil, and 12–15 germinated seeds from each line were planted in the trays and labeled (Figure 1).
2.2 Foliar spray with SNP and cPTIO on susceptible and resistant rice lines
We prepared seven small trays to screen for SNP application to susceptible and resistant rice lines under BPH stress. SNP solutions with concentrations of 25, 50, 100, 150, and 200 µM were prepared and foliar sprays were performed accordingly. The trays were labeled as follows: the negative control was non-sprayed, the positive control was sprayed with distilled water, and the remaining trays were subjected to foliar spraying with their respective concentrations of the SNP solution. After the initial screening, the rice seedlings were exposed separately to two different concentrations (100 and 200 µM) of SNP and cPTIO. The effects of these treatments were observed for seven days. After seven days of exposure to 100 and 200 µM of SNP and cPTIO, we observed the root and shoot length, mortality rate, electrolytic leakage (EL), and relative water content (RWC) of the seedlings.
2.3 Measurement of RWC and EL
The RWCs were determined for the seven-day-old seedlings after BPH infestation, as previously described 2, with slight modifications. After seven days of BPH infestation, ten seedlings were cut equally, and the fresh weight was measured using a balance. The seedlings were then suspended in deionized water and stored in a cool, dark area for 3 h. When the samples were fully hydrated, they were placed on dry clean tissue paper for 2 min, and the turgid samples were weighed. The samples were then incubated at 65°C for 24 h, and dry samples were weighed. The RWC of seedlings was determined using the following equation:
The EL was measured as previously described 26. Under BPH stress, the seedling samples were collected from the negative control, positive control, and samples treated with 100 and 200 µM of SNP and cPTIO. Seedling samples of equal size were collected in triplicate. The seedlings were rinsed with double-distilled water to eliminate any superficial electrolytes and then transferred to glass tubes containing 10 mL of deionized water. The tubes were kept at room temperature (25°C–27°C) for 24 h with gentle shaking, and the EL of each sample was measured using a portable conductivity meter, called electrolytic leakage 1 (EL1). The samples were autoclaved at 121°C for 20 min and allowed to cool to room temperature. The EL of each sample was measured again (EL2). The EL% was measured using the following equation:
2.4 Quantitative PCR (qPCR) profiling during BPH stress
The susceptible and resistant rice lines were exposed to BPH stress, whereas the control group remained unstressed. Leaf samples were collected at different time points (0, 6, 12, and 24 h) and immediately ground in liquid nitrogen. Total RNA was extracted from the samples using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA concentration was measured using a NanoDrop 2000 spectrometer (Thermo Scientific, Wilmington, DE, United States). First-stand cDNA was generated using a qPCR BIO cDNA Synthesis Kit with 400 ng of RNA. Quantitative real-time PCR analysis was performed using the StepOnePlus™ Real-Time PCR System, Life Technologies Holding Pty., Ltd. (Singapore), BioFact™ 2× Real-Time PCR Master Mix (including SYBR® Green I) that was obtained from www.bioft.com (South Korea), and gene-specific primers. The internal reference gene OsActin1 (accession no. AB047313) was used for normalization. The primer sequences used in this study are listed in Table 1.
2.5 Determination of NO content
Fourteen-day-old susceptible and resistant rice lines were infested with BPH for 0, 6, 12, and 24 h, and the plant material was harvested. The harvested plant material was ground using liquid nitrogen. Extraction was performed using 4 mL of 40 mM HEPES buffer (pH 7.2) per g of the plant tissue. The resulting mixture was centrifuged at 25,000 g × for 20 min. To expedite ultrafiltration, the supernatant was passed through a 0.45-µm filter. The NO content in the filtered supernatant was measured using the Total Nitric Oxide Assay Kit from Beyotome (China). The experiment was independently repeated three times.
NO was visualized in the rice leaves using the specific fluorescent probe DAF-FM DA (EMD Millipore Corp., 290 Concord Rd, Billerica, MA, USA, affiliated with Merck KGaA, Darmstadt, Germany, and made in Japan). After infestation for 24 h, leaf sections from the 14-day-old rice seedlings were incubated in the dark at 37°C in the presence of 20 mM HEPES-NaOH (pH 7.5) for 30 min. Subsequently, 10 µM of DAF-FM DA was added to the samples and incubated in the dark for another 30 min. The rice leaves were washed three times with HEPES-NaOH buffer and mounted for confocal laser scanning microscopy on the LSM 800 (Carl Zeiss, Oberkochen, Germany) using an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The images were processed and analyzed using the ZEN software.
2.6 Statistical analysis
Statistical analysis was performed using Tukey’s multiple comparison test using GraphPad Prism 9.0. Three replicate were performed for each treatment separately. A streak in the bar graphs represent significant difference and “ns” indicates no significant difference compared to the control group. All data is expressed as the mean ± standard deviation, and statistical significance was determined at p-values < 0.0001.
3.1 SNP application enhanced resistance in rice against BPH stress
In the current study, we applied different concentrations of SNP to both susceptible and resistant CNDH rice lines to evaluate their responses to BPH stress (Figure 1A-C). Based on the screening evaluation (Figure 2A), SNP application in the range of 100–200 µM significantly mitigated the feeding effects of BPH in both susceptible and resistant rice lines. However, we noted that the root growth was differentially regulated by SNP application. Susceptible rice lines showed a reduction in root growth compared to their respective negative control groups. In contrast, compared to the positive control group, the susceptible rice line, CNDH 14-2, exhibited a significant increase in root growth. In contrast, the other susceptible rice line, CNDH 48-3, showed a significant reduction in root growth compared to the positive control. Additionally, the resistant rice lines, CNDH 78 and CNDH 87, showed that SNP application at a concentration ranging from 100 to 200 µM increased the root growth compared to their respective negative and positive control groups (Figure 1A). However, different SNP concentrations exhibited varying effects on the shoot growth in the susceptible and resistant rice lines, and 100 µM SNP responded well to the feeding effect of BPH (Figure 1B).
3.2 Foliar application of SNP and cPTIO under BPH stress
In the current study, we applied both SNP and cPTIO at concentrations ranging from 100 to 200 µM, and evaluated the effects of these chemicals on both susceptible and resistant rice lines. We observed that cPTIO significantly affected the growth attributes of the rice seedlings compared to their respective negative control groups, whereas SNP application enhanced their growth parameters (Figure 2C and D). We found that root growth was significantly decreased in both susceptible rice lines compared to their respective negative control groups, whereas the root growth of the resistant rice lines showed no significant differences (Figure 2E). However, it was observed that the shoot growth was significantly decreased in both susceptible and resistant rice lines under both the positive control and 100 µM cPTIO application; however, 100 µM SNP application significantly enhanced shoot growth in both the susceptible and resistant rice lines (Figure 2F).
3.3 RWC and EL under BPH stress
Under BPH stress, both the positive control and the 100 µM cPTIO-treated seedlings showed a significant reduction in RWC by 20%–25% in both the susceptible CNDH lines and the resistant line CNDH-78, compared to the negative control group. However, the exogenous application of SNP led to a 15%–20% increase in the RWC in the BPH-susceptible rice lines (CNDH 14-2, CNDH 48-3) and resistant rice lines (CNDH-78 and CNDH-87) compared to the positive control group and 100 µM cPTIO-treated seedlings (Figure 3A). EL was significantly higher, ranging from 20% to 40%, in the positive control and the 100–200 µM cPTIO-treated rice lines than in their respective control groups. However, the application of 100 µM SNP minimized the EL to 10%–15% of their respective control groups (Figure 3B).
3.4 SNP application decreased mortality percentage under BPH stress
In the present study, we treated the rice lines susceptible and resistant to BPH stress with the application of distilled water, cPTIO, and SNP. The negative control involved no stress, whereas the positive control was treated with distilled water spray only,and followed by BPH stress. Similarly, cPTIO and SNP were sprayed through foliar applicationon the rice lines, and the mortality rate was observed after 7 and 14 days (Figure 4A and B). The survival percentage was significantly lower in the CNDH-susceptible rice lines for the positive control group treated with distilled water and the groups treated with 100 and 200 µM cPTIO compared to the SNP treatment group (Figure 4A). However, in the CNDH-resistant rice lines, we found no significant difference between the positive control and SNP-treated lines, but the 100 and 200 µM cPTIO-treated plants showed a decrease in their survival rate (Figure 4A). We found that treatment with 100 and 200 µM cPTIO significantly decreased the survival percentage of the rice seedlings; however, treatment with 100 and 200 µM SNP foliar applications significantly enhanced the survival percentage and decreased the mortality rate in both susceptible and resistant CNDH rice lines (Figure 4B).
3.5 No-associated gene expression under BPH stress
We observed the relative expression of genes associated with NO synthesis at different time points (0, 6, 12, and 24 h) under BPH stress. In this study, we used 14-day-old seedlings after BPH infestation. We observed the differential expression of OsNOA1, OsNR, and OsEF-1α in both susceptible and resistant rice lines at different time points. We observed high OsNOA1 expression at 6 h in the BPH-susceptible rice line CNDH 14-2. Similarly, high induction was observed in the resistant rice lines CNDH 78 and CNDH 87 after 12 and 24 h, respectively (Figure 5A). However, OsNR activity was gradually induced in the CNDH 48-3 rice line, and the highest induction was observed after 24 h compared to that in the other CNDH lines and their respective control groups (Figure 5B). Regarding the OsEF-α1 gene, we observed significant upregulation as the time interval increased. However, its maximum expression was observed after 6 h in the CNDH-78 resistant rice line compared with the other lines and their control groups (Figure 5C).
3.6 Determination of NO content under BPH infestation
The results showed distinct patterns of NO levels among the rice lines (Figure 6A). The susceptible rice lines CNDH 14-2 and CNDH 48-3 exhibited a gradual increase in NO content over time and reached the highest level of relative absorbance after 24 h of BPH stress compared with their respective control groups (0 h). This suggests that BPH infestation triggered a notable elevation in NO levels in the susceptible rice lines. In contrast, the resistant rice lines displayed different responses. They exhibited the highest relative absorbance of NO at earlier time points, specifically 6, 12, and 24 h after infestation. These findings indicate that the resistant rice lines mounted a rapid and robust defense against BPH stress, as shown by their earlier and sustained increased NO levels, whereas the susceptible rice lines exhibited a robust defense only after 24 h of BPH stress (Figure 6A). We further confirmed the NO signal by detecting green fluorescence after 24 h of BPH stress and compared it with that of the negative control groups. It was observed that BPH feeding significantly inhibited the internal NO levels in the susceptible rice lines CNDH 14-2 and CNDH 48-3 compared to the positive control and 100 µM cPTIO treatment under BPH stress conditions (Figure 6B). In contrast, the resistant rice lines CNDH 78 and CNDH 87 showed high fluorescence activity under BPH stress compared to the susceptible rice lines. However, 100 µM SNP application robustly increased the internal NO levels and showed high fluorescence in both the susceptible and resistant rice lines under BPH stress. This observation suggests that SNP application might alleviate the effects of BPH feeding (Figure 6B).
NO signaling is known to be part of the plant’s response to microbial and insect attacks. In this study, we aimed to investigate whether the application of SNP enhanced NO, which plays a role in the wounding response specific to phloem damage caused by insects 22. It has been suggested that NO may be released by the phloem, possibly through an enzymatic process. Moreover, in the plant apoplasts, NO can be produced nonenzymatically through the oxidation of nitrite under acidic conditions 25. In the current study, we investigated the feeding effect of BPH on susceptible and resistant rice lines (Fig. 1A). After 24 h of BPH stress, susceptible rice lines showed a higher relative absorbance compared to the resistant rice lines (Fig. 6A). This suggests that the susceptible rice lines might release NO content at a slower rate than the resistant rice lines. Understanding these differences in NO release may contribute to enhancing BPH resistance in rice crops. Further research is required to explore the underlying molecular and physiological mechanisms involved in this response.
It is possible that the BPH induces the non-enzymatic generation of NO in rice by the salivary sheath that is formed by the deposition of solid saliva during the probing activity of the BPH. It has previously been reported that in the resistant cultivars, there is a notably higher frequency of BPH probing and a greater number of salivary sheaths observed in the phloem compared to the susceptible cultivars 6,27. Contrary to our initial expectations, our study revealed higher probing activity of BPH in the susceptible rice lines compared to the resistant rice lines. Additionally, we observed higher feeding rates under distilled water and cPTIO treatment compared to SNP applications. This indicates that SNP might enhance NO production, thereby leading to a reduced feeding effect in comparison to cPTIO (Fig. 2B-F).
This study suggests that seedling mortality is not primarily caused by the act of probing and ingesting by the BPH. Instead, it is likely linked to the loss of phloem sap or water. The feeding symptoms resemble those seen during drought stress 6. In addition, changes in the stomatal apertures and root structure, which are early morphological responses to both drought and BPH feeding, result in a reduction of the plant shoot height. Plants adjust their growth patterns as an important adaptive mechanism to minimize damage when water becomes limited 28. In a study on NO function, there are several substances that can either stimulate or scavenge NO production. One commonly used NO-releasing compound or NO donor in many laboratories is SNP 29,30, and 2-(4-Carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) is a commonly used NO-specific scavenger in numerous studies 31. Our study revealed that exogenous SNP enhanced internal NO production under BPH stress, whereas cPTIO reduced the levels of internal NO (Fig. 6B). Previous studies provide compelling evidence revealing the role of exogenously applied NO in enhancing water deficit tolerance in both detached wheat leaves and wheat seedlings that were subjected to drought stress conditions. A recent study reported that the application of SNP alleviated drought stress by enhancing internal NO levels, whereas the application of cPTIO reversed this effect 32,33.
In our study, 100 µM cPTIO treatment led to increased RWC and EL in both the susceptible and resistant rice lines. However, the application of SNP during BPH stress reversed these effects, thus indicating its protective role in rice plants (Fig. 3A and B). Previous studies have shown that NO promotes root expansion in plants such as rice 34 and cucumber 35 compared to untreated plants. A previous study has also shown that SNP application with NH4+ and cPTIO application with NO3− resulted in auxin levels in the roots similar to those observed under NO3− and NH4+ supply, respectively. In contrast with the wild-type, the OSpinb mutant showed lower auxin levels, along with a reduction in the lateral roots and shorter seminal roots 36. Another previous study suggests that SNP and indole-3-butyric acid (IBA) induce lateral root formation in rice during the seedling stage 37. It has been reported that SNP and multiwalled carbon nanotubes (MWCNTs) significantly enhanced the RWC, chlorophyll content, growth metrics, and photosynthesis parameters 38. In our study, we found that 100 µM SNP stimulated root growth compared to the positive control and significantly enhanced root growth in the resistant and susceptible rice lines (Fig. 2E). Shoot growth under 100 µM SNP application dramatically increased compared to the positive control and cPTIO-treated plants and showed almost similar or higher response compared to the negative control (Fig. 2F).
It has been reported that susceptible rice cultivars in Asia experience substantial yield losses because of BPH infestations 39. A recent study 40 found a connection between a protein called OsMAPK20-5 and NO production in N. lugens-infested rice plants, which sheds light on its potential role in enhancing plant defense against pests. In the current study, the positive control and treatment with 100 µM cPTIO significantly decreased the survival percentage in the BPH-susceptible rice lines, whereas 100 µM SNP treatment enhanced the survival rate of rice seedlings under BPH stress (Fig. 4A and B). A previous study suggests that the NOS pathway is considered a primary mechanism to synthesize NO in wounded plant tissue 22. The expression levels of the OsNOA1 gene were significantly upregulated in the susceptible rice line CNDH14-2 after 6 h of BPH stress. However, it was also induced after 24 h in both the susceptible rice line CNDH 48 − 3 and the resistant lines, CNDH-78 and CNDH-87, under BPH stress conditions (Fig. 5A). Because it is unclear whether the expression of OsNOA1 leads to increased NOS activity, the activity of NO was also measured. We observed a high prevalence of NO after 6 and 12 h of BPH stress in both the susceptible and resistant rice lines (Fig. 6A). Therefore, it might be possible that rice under BPH stress activates the NOS pathway and releases a high concentration of NO for defense against BPH infestation. It has been reported that OsNR activity remains unaffected by BPH infestation in the cultivar DV85, whereas a downregulation was observed in the cultivar TN1 2. In the current study, we observed a significant downregulation of OsNR in the susceptible rice line CNDH 14 − 2. However, with the progression in infestation time, OsNR was significantly upregulated in the susceptible rice line, CNDH 48 − 3, and the resistant rice line, CNDH 87 (Fig. 5B).
Some studies have shown that the process of photosynthesis in leaf blades is hindered when plants are subjected to BPH infestation stress, and plants infested by BPH exhibit a lower concentration of nitrogen in their leaves compared to unaffected plants 41. It has been reported that the presence of over 30 BPH nymphs per tiller significantly diminishes the plants ability to absorb nitrogen from the soil through their roots, which indicates a substantial reduction in nitrogen uptake under these conditions 42. However, the investigations conducted in our studies did not determine the specific factor responsible for the decline in leaf nitrogen concentration and the reduced nitrogen absorption by the plants. However, based on our research findings, the reduced expression of OsNR could be attributed to the decrease in leaf photosynthesis induced by BPH infestation. This decline in photosynthesis limits the root system’s activity because of the hindrance in assimilate translocation from the leaves to the roots, particularly in the susceptible rice line, CNDH 14 − 2 (Fig. 5A). Our results suggest that the production of NO in response to BPH feeding might not solely rely on the activity of the NR pathways. Instead, it appears to be partially dependent on the NOS pathway, thus highlighting the complexity of the plants response to BPH infestation (Fig. 5A, 6A and B).
In the current study, we investigated the impact of SNP and cPTIO potassium salt on rice plants under BPH stress, and revealed that, compared to the negative control, the positive control and 100 µM cPTIO-treated groups had decreased growth and RWC and an increased EL and death rate. Gene expression analysis showed distinct patterns in the resistant and susceptible rice lines. Resistant lines exhibited increased NO content, while susceptible lines showed elevated NO levels after 24 h of stress. Considering the importance of rice as a worldwide staple food, the application of 100 µM SNP enhanced internal NO content, improved agronomic traits, and protected the rice from BPH effects.
Conflict of Interest
The authors declare no conflict of interests.
Author Contributions
M-F planned, designed, and conducted the experimental study, performed data analysis, and wrote the findings, R-J, S-A, D-D-Z, Z-K, assisted in collecting experimental data, J-R-P provided the rice lines and contributed to providing experimental resources. K-K-M evaluated and edited the final version of the manuscript.
Funding
No Funding
Acknowledgments
This work was supported by a grant from the Securing National Standard Breeding Big Data and Building Deep Data (Project No. RS-2023-00230677).
Data Availability Statement
The data support the findings of this study are available upon request from the authors, with the understanding that the data will be used solely for the purpose of scientific research and not for commercial or other non-research purpose. Restriction may apply to the availability of these data, which were used under license for this study.
Ethics approval and consent to participate.
Not applicable.
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Table 1. List of primers used in the study.
|
Genes |
Forward Primer |
Reverse Primer |
|
OsNOA1 |
5ʹ-AGAGAAGTTGGAGTTACATTGAC-3ʹ |
5ʹ-ACTCTCAAATGCAGTTCACCAC-3ʹ |
|
OsNR |
5ʹ-GCAGAAGTTGGAGTTACATTGAC-3ʹ |
5ʹ-GCGGTTGTCCTTGTAATGGT-3ʹ |
|
OsEf-1α |
5ʹ-AGACCACCAAGTACTACTGCAC-3ʹ ׳ |
5ʹ-CCACCAATCTTGTACACATCC-3ʹ |
No competing interests reported.