Dufulin activates OsDUF6 protein against salt stress in rice plant

Dufulin activates OsDUF6 protein against salt stress in rice plant | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dufulin activates OsDUF6 protein against salt stress in rice plant Guangming Ma, Yong zhang, Xiangyang Y. Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3852076/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2024 Read the published version in BMC Plant Biology → Version 1 posted 8 You are reading this latest preprint version Abstract Background Dufulin is a chemical immune activator in rice plant. Soil salinity is one of the main environmental stresses in rice production. When plants are exposed to salt stress, a range of cellular equilibria will be disrupted. Previous studies have shown that Dufulin has a positive effect on salt tolerance in rice. Results In this study, we studied the mechanism of Dufulin in response to salt stress. Based on the transcriptome analysis of Dufulin in the process of salt tolerance in rice, we selected the OsDUF6 protein located on the cell membrane and studied its molecular function by overexpression of OsDUF6 . The results showed that the salt-induced decreases in root, stem, and leaf length and increased leaf yellowing rate and Na + concentration in the wild-type plant were improved in the overexpressed lines, and increased the enzyme activity of the SOD, POD, CAT and PAL. OsDUF6 played a positive role in Na + transport by comparing the growth of the salt-sensitive yeast mutant complemented with OsDUF6 . In addition, RT-qPCR analysis confirmed that the overexpression of OsDUF6 significantly changed the expression level of genes related to growth and stress tolerance. Conclusions Combined with previously published data, our results supported that OsDUF6 is an important functional factor of Dufulin to promote salt stress resistance in rice and plays a role in promoting salt tolerance in rice. Rice OsDUF6 Salt stress Functional analysis Overexpression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction One of the major abiotic stresses encountered by food crops during growth is increased soil salinity. 1–3 More than 20% of the world’s agricultural land is affected by high salinity, a figure which is expected to rise in the future. 4 When food crops are subjected to salt stress, the plants respond at the morphological, cellular, and molecular levels. 5 When encountering salt stress, the homeostasis between reactive oxygen species (ROS) production and scavenging will be broken, resulting in excessive accumulation of ROS, which will destroy the original biological functions of the plant to a certain extent and hinder the normal physiological processes of the plant. 6,7 Plant morphological changes under high salt conditions included impaired root establishment, occurrence of leaf rolling, yellowing, decreased thousand-seed weight, and decreased number of spikelets per ear, the latter ultimately resulting in decreased harvest index and grain yield. 8,9 Rice ( Oryza sativa ) is the main food crop for much of the world’s population and is also one of the most important staple foods in daily life. 10 With climatic changes observed in recent years, rice needs to respond to biological and abiotic stresses. The use of pesticides and fungicides has reduced the importance of biological stresses in rice production. However, rice also needs to respond to several abiotic stresses. Among these, salt stress is an important focus of current research, and it has been found that rice is more sensitive to drought and salt stress than other grains, such as barley, wheat, and rye, 11,12 making rice an urgent target for studies into mechanisms of salt tolerance. At present, people pay more attention to food health, while the quality of rice as a staple food is also a key target. 13,14 Moreover, how to improve the quality of rice while increasing its yield, achieving the best of both worlds, is the result that more people want. Despite this, it has become a major public issue to improve rice production by promoting rice tolerance to salt stress. 15,16 Based on the transcriptomic data from rice plants exposed to salt stress after being sprayed with the plant antiviral agent Dufulin, which has been shown to induce salt stress tolerance, we found that OsDUF6 exhibited significantly changed expression among the differentially expressed genes (DEGs). 17 Studying salt stress after spraying with a disease-resistance inducer, such as Dufulin, is in line with the current environment of how to tolerate abiotic stress after preventing biotic stress. Analysis of the molecular function of OsDUF6 will allow us to confirm whether a gene conferring tolerance to salt stress, the expression of which changes in response to treatment with a chemical, Dufulin, which induces salt tolerance, is an important gene with respect to response to abiotic stresses. Therefore, analysis of the response of the OsDUF6 ( LOC_Os12g33300 ) gene to abiotic stress in rice plants and a study of its molecular function can enrich our understanding of the rice salt stress signal network. The current study investigated the molecular function of OsDUF6 and its positive impact on salt tolerance in rice after overexpression, based on an in-depth exploration of transcriptomic data with respect to response to a chemical promoting salt tolerance in rice. Such research could provide novel insights into the molecular function of inducers of salt tolerance, and identify new salt tolerance genes for exploitation in molecular breeding of rice for salt tolerance. 2. Materials and methods 2.1 Materials Dufulin (93%), an active ingredient, was synthesized in the State Key Laboratory of Green Pesticides, Guizhou University. The treatment concentration was 500 mM (Ma et al., 2022). The seeds of rice materials used in the experiment were Nipponbare ( Oryza༎Sativa L༎spp༎japonica ), and the seeds of tobacco materials were Nicotiana benthamiana . 2.2 Sources and cultivation of transgenic overexpressed rice The pBWA (V) HS-OsDUF6 plasmid was constructed, and 1 µL plasmid was added to 50 µL strain EHA105 Agrobacterium competent cells for Agrobacterium tumefaciens transformation. Then, Agrobacterium infection co-culture, callus screening. The transformation-positive rice callus was inoculated onto the differentiation medium and cultured at 25–27°C for 15–20 d. After the differentiation of 2–5 cm buds, the rice callus was transferred onto the rooting medium and cultured at 30°C for 7–10 d. The transgenic rice seedlings( Nipponbare ) were then transferred to plastic pots containing a soil/perlite mixture and grown in a growth chamber at 26°C and 72% relative humidity for 16 h/8 h (day/night). The rice plants cultured after using the CTAB method to extract rice genomic DNA, and then PCR was used to detect positive plants. 2.3 Salt stress treatment of plant material Full and intact seeds of homozygous overexpressed and wild-type rice( Nipponbare ) of similar size were selected and germinated for 3–5 d at the same time. After germination, they were transplanted into plastic pots of a soil/perlite mixture in the greenhouse [26°C and 72% relative humidity for 16 h/8 h (day/night)]. After 15 d growth, wild-type and transgenic rice plants were sprayed with 200 mM NaCl to induce salt stress and were photographed and sampled after a further sixth days. 2.4 Physiological index The phenotypes of transgenic and wild-type rice( Nipponbare ) after six days of salt stress were observed, and root, stem, and leaf lengths were measured to evaluate any growth differences. Then, each plant was dried to obtain dry weights. The sodium (Na +) concentrations of rice plants were measured by flame spectrometry. 2.5 Rice sample collection and total RNA extraction The roots, stems, and leaves of the overexpressed transgenic and wild-type rice( Nipponbare ) were collected after six days of salt stress. They were snap-frozen in liquid nitrogen and stored at − 80°C. Total RNA was extracted from the preserved sample tissues of the overexpressed lines and the corresponding wild-type variety, following the instructions of the manufacturer of the RNA prep Pure Plant Kit (Tiangen, Beijing, China). The RNA concentration was measured with a NanoPhotometer (IMPLEN, Munich, Germany), and the standard DNA concentration of the subsequent experiment was selected for preservation. 2.6 Bioinformatic analysis of the OsDUF6 gene The sequences of the OsDUF6 protein and its homologous protein sequences were obtained from the NCBI. In the ExPASy2 website ( http://au.expasy.org/tools/protparam.html ), physical and chemical parameters were predicted for preliminary OsDUF6 proteins [ 18 ]. Based on this preliminary information, the subcellular localization ( http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ ), the protein secondary structure ( http://bioinf.cs.ucl.ac.uk/psipred/ ), interaction protein database ( https://cn.string-db.org/ ), tertiary structure model ( https://www.uniprot.org/ ), and transmembrane signal peptide ( https://www.dtu.dk/english ) ) of OsDUF6 were predicted and analyzed online [ 19 – 24 ]. Based on the OsDUF6 gene sequence, a search was carried out for homologous sequences among multiple species in NCBI; any sequence identified was downloaded and MEGA software was used to carry out multiple sequence alignment, and the neighborhood connection method to was used to construct a phylogenetic tree [ 25 ]. 2.7 Determination of enzyme activity of OE and WT after salt stress Following the manufacturer's instructions, the appropriate enzyme assay kits (Jiangbin, Nanjing, China) were used to assay plant tissue homogenate (1: 9 tissue weight: buffer [phosphate buffer (0.1 mol/L pH 7–7.4)] volume for the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) [ 17 ]. 2.8 RT-qPCR analysis The total RNA, extracted as described above, was converted into cDNA using the StarScript III All-in-one RT Mix with gDNA Kit (Genstar, Beijing China), and diluted with ddH 2 O. In the 20 µL reaction volume, 2 × RealStar Fast SYBR qPCR Mix (Genstar, China) was used for premixing, and DEPC-treated H 2 O was used to adjust the volume. The reaction was carried out in the LightCycler 96 RT-qPCR assay system (Roche, Basel, Switzerland) and the reaction was completed according to the dissolution curve automatically set by the instrument. The relative gene expression rate between the overexpression lines and the wild-type lines was compared using the 2 −△△Ct method [ 26 ], and three biological replicates were conducted for each sample. All quantitative primers are listed in Table S1 and were synthesized by Beijing Tsingke Biology (Beijing, China). 2.9 General subcellular localization experiment The coding sequence of OsDUF6 ( LOC_Os12g33300 ) was cloned into a pCam35S-GFP vector. After the plasmid obtained was used to transform Agrobacterium strain GV3101 in its receptive state, the bacteria were cultured in a 28°C incubator for 2–3 d. After screening a single colony to confirm its transformation-positive nature, the culture was multiplied in liquid medium. After centrifugation, the bacterial pellet was resuspended and, after dark incubation for 3 h, was inoculated into leaves of 2-week-old Nicotiana benthamiana plants of similar growth form. Three days later, the injected leaves were cut into 2 mm × 2 mm squares and placed on slides, with the fluorescence from the green fluorescent protein (GFP) being observed under a confocal laser scanning microscope(ZEISS, Oberkochen, Germany). 2.10 Confirmation of yeast functional complementation We used a salt-sensitive Saccharomyces cerevisiae mutant Axk3k that lacks the main plasma membrane Na + transporter [ 27 ]. The full-length sequence of OsDUF6 was cloned into the PYES2 yeast expression vector. First, the Axt3k strain, stored at − 80°C, was activated, and then, according to the manufacturer's instructions, the yeast transformation kit (Coolaber, Beijing, China) was used to convert it into the receptive state. Then, the OsDUF6 plasmid was introduced into the prepared receptive Axt3k by the PEG/LiAc method, and the transformed yeast, after centrifugation, was spread onto SD/-Ura solid medium, and the plate cultured in an inverted orientation, in a 30°C incubator for 3–4 d to achieve yeast colonies of the appropriate size. After a single colony was selected and confirmed to be transformation-positive, the transformed yeast was suspended in ddH 2 O and diluted with ddH 2 O to form the concentration gradient of 10-fold serial dilutions. Approximately 3 µL of the yeast suspension was applied onto the Arg phosphate (AP) solid medium plate containing 0, 25, 50, 75, or 100 mM NaCl, incubated upside down at 30°C to grow for 3–4 d, and the yeast growth was observed and recorded. 2.11 Data analysis All data were analyzed by ANOVA to identify statistically significant ( P < 0.05 and P < 0.01)) differences in measured parameters between the wild-type and overexpression lines. Three independent biological replicates were used for each measurement. Experimental data are expressed as mean ± standard error. Graphs were generated using Prism software (v 9.0) (GraphPad Software, Boston, MA, USA). 3. Results 3.1 Functional prediction and analysis of the OsDUF6 protein The molecular weight and isoelectric point (pI) of OsDUF6 were 41.46 kDa and 8.87, respectively, and the number of amino acids encoded was 373. Through the prediction of its transmembrane domain and signal peptide, the results showed that the deduced protein had ten transmembrane domains, so that it was a transmembrane protein, but lacked a signal peptide (Fig. 1 A,B). The subcellular localization of the protein was predicted online and indicated to be in the cell membrane (Fig. S1 ). The secondary structure of OsDUF6 was predicted by PSIPRED. It can be seen from the prediction results that helix and coil constituted almost its entire secondary structure, with strand encoding only two amino acids, so that the secondary structure is relatively stable (Fig. S2). Then, on the basis of the secondary structure, we predicted the tertiary structure of OsDUF6, with the results showing that the AlphaFold produced a very high per-residue confidence score (pLDDT) > 90, so that this tertiary structure could be applied to experimental research to a certain extent (Fig. S3). By comparing and screening with the STRING database, based on the rice varieties studied in this experiment, the Oryza sativa japonica Group was selected to predict the interaction proteins of OsDUF6 in the database. In Fig. 1 C, A0A0P0YAX6 is our target protein, and the intricate lines represent the interaction network of OsDUF6; in this interaction network, it is clearly divided into two parts. However, according to the meaning of the line representation, we found that the prediction results are based on the mined text, and the proteins interacting with OsDUF6 are not based on accurate data or experimental results, which will require us to determine the actual interacting proteins based on experimental results (Fig. 1 C). The homologous sequence of the OsDUF6 protein sequence was studied using NCBI technology. Then, MEGA6.0 software was used to achieve multiple sequence comparisons, and the amino acid sequence obtained after comparison was used to construct an evolutionary phylogenetic tree. The results showed that OsDUF6 had a high degree of genetic relationship with the WAT1-related protein At5g64700 ( Oryza sativa japonica Group ) and hydroxy protein OsJ36299 ( Oryza sativa japonica Group), but it had a distant genetic relationship with the hydroxy protein BS78-09G001100 ( Paspalum vaginatum ) and the hydroxy protein EJB05-22345 partial ( Eragrostis curvula ) (Fig. 2 A,B). 3.2 OsDUF6 encodes a membrane-localized protein In order to detect the general subcellular localization of OsDUF6 protein, the coding sequence (CDS) region of OsDUF6 was cloned and the expression vector of the OsDUF6-YFP (yellow fluorescent protein) fusion protein was constructed and was used to transform Agrobacterium , which was cultured, incubated, and injected into tobacco. Finally, the subcellular localization was preliminarily determined by laser scanning confocal microscopy to detect the fluorescence signal. As shown in Fig. 3 , the fluorescence of the 35S:: OsDUF6-YFP construct was detected in the cell membrane. Comparison of the prediction results and the subcellular localization results showed that they were similar, so that the OsDUF6 gene was determined to encode a membrane-localized protein. 3.3 Functional analysis of OsDUF6 in yeast mutants under NaCl stress In order to test the function of OsDUF6 in salt tolerance, OsDUF6 was cloned into yeast expression vector pYES2, and then transformed into S. cerevisiae mutant Axt3k (ena1–4:: HIS3,nha1:: LEU2, nhx1:: KanMX), which is very sensitive to Na + stress. For each transformed yeast strain, colony growth responded indirectly to a gradient of NaCl concentrations on AP medium. The results showed that the yeast mutant carrying the OsDUF6 gene still grew at the highest NaCl concentration on the AP medium, whereas the control mutant group barely grew (Fig. 4 ). This growth of the complemented yeast mutant confirmed the functional salt tolerance of the OsDUF6 gene. 3.4 Overexpression of OsDUF6 promotes salt tolerance in rice In order to clarify the function of OsDUF6 transgenic rice in terms of salt stress response, seeds of the transgenic rice lines and its corresponding wild-type plant were grown in a greenhouse, and 200 mM NaCl was added at the soil matrix at a specified time for salt stress treatment. Under normal, non-stress conditions, there was no significant phenotypic difference between transgenic lines and the wild-type strain. After sixth days of salt treatment, according to phenotypic observations, all rice plants (transgenic lines and wild type) were damaged, though to varying extents (Fig. 5 ). The area of yellowed tissue of leaves of transgenic plants was smaller than that of the wild-type plants; furthermore, the length of the roots, leaves, and stems of both genotypes were reduced under salt stress (compared with the non-stressed control), but the decrease in transgenic plant size was much smaller than that of the wild-type plants (Fig. 6 B,C). 3.5 Overexpression of OsDUF6 reduced the concentration of Na + concentration in transgenic rice plants under salt stress Na + homeostasis is an important characteristic of plant salt tolerance. Therefore, we determined the concentration of Na + in the roots and leaves of wild-type and overexpressed lines. Compared with the Na + concentration of the wild-type plants, the accumulation of Na + in the overexpression lines was less, which indicates that the stress tolerant mechanism was due, at least in part, to the transgenic plants being more efficient at transporting Na + into/out of the plant cells (Fig. 6 A). 3.6 Overexpression of OsDUF6 increases the activity of antioxidant enzymes under salt stress In order to detect the level of ROS scavenging in overexpressed lines and wild-type plants, we measured the activities of the main direct antioxidant enzymes SOD, CAT, and POD, and an indirect antioxidant enzyme, PAL, in the overexpressed lines and wild-type lines. After assay, it was found that, in response to salt treatment, the increase in SOD, PAL, CAT, and POD activities in overexpression lines were higher than those in the wild-type lines (Fig. 7 ), with the response of the three direct antioxidant enzymes being much higher than that of the indirect antioxidant enzyme, PAL. These results suggest that OsDUF6 overexpression lines can increase the activity of four antioxidant enzymes SOD, PAL, CAT and POD when rice is exposed to salt stress, so that the accumulation of ROS in rice under salt stress is reduced to a certain extent, compared with wild-type lines, thereby alleviating a series of adverse oxidative stress effects caused by ROS accumulation in rice and promoting rice tolerance to salt stress. 3.7 Overexpression of OsDUF6 changed the expression of related genes under salt stress Our previous research had shown that, when rice was subjected to salt stress after spraying with Dufulin, OsDUF6 expression in the rice transcriptome was found to be upregulated, with the relative expression of some other genes also changing dynamically [ 17 ]. Therefore, we selected a number of genes putatively related to growth and stress tolerance and the expression of these genes were presented as a heatmap, based on the previous transcriptome data (Fig. 8 A). Based on these data, we conducted RT-qPCR to confirm the changes in expression level of these genes in the leaves of transgenic and wild-type plants. The results confirmed that the dynamic changes in expression of these genes were consistent with those from the previous transcriptome data and were associated with increased plant salt tolerance (Fig. 8 B). Therefore, these results indicate that overexpression of OsDUF6 changed the expression level of these genes to a certain extent, which means that OsDUF6 expression is closely related to that of these genes. In summary, according to the results of RT-qPCR, in the presence of salt stress, overexpression of OsDUF6 has a positive effect on rice growth and stress-related genes via an effect on Na + transport, which further explains its role in promoting salt stress tolerance in rice. 4. Discussion Salinity is a major abiotic stress factor that seriously affects crop growth and yield, causing significant economic losses and threatening global food security [ 28 , 29 ]. At present, 23.02 billion hectares of farmland worldwide can be used for food production. However, due to inappropriate crop irrigation methods (leading to salinization) and excessive fertilizer usage, as well as natural effects, such as sea level rise, salt intrusion into coastal areas, some 2% of farmland is affected by salt, and the proportion of impacts on agricultural development is also increasing year by year [ 30 ]. Rice is a very important cereal crop that feeds half of the world's population and is particularly sensitive to salt stress [ 31 ]. There have been many studies on salt-tolerant transgenic rice, most of which were based on research into homologous species and gene families to identify the genes that change the salt response of rice [ 32 – 34 ]. Prior to the current study, the OsDUF6 gene had no known function. In this present study, based on the transcriptome analysis of rice sprayed with Dufulin and then subjected to salt stress [ 17 ], we studied the function of OsDUF6 gene, transgenic plants were generated to overexpress the OsDUF6 gene, and their molecular functions were studied through a series of indicators. Research has shown that a high concentration of Na + enters the body of plants under salt stress, and thence induce the accelerated accumulation of ROS [ 35 , 36 ]. When a high concentration of ROS is generated, it will cause oxidative damage to the membrane; in serious cases, it can disrupt cellular metabolism, leading to the inhibition of normal growth and development of the plants [ 37 , 38 ]. Through subcellular protein localization test and software prediction, it was preliminarily determined that the OsDUF6 protein is located on the cell membrane (Fig. 3 and Fig. S1 ). Therefore, overexpression of OsDUF6 will directly and positively affect the salt tolerance of rice by mediating Na + transport through the membrane, a finding of great significance in the study of salt resistance of rice. When plants are exposed to salt stress, ROS will be generated, which will cause plants to be unable to clear them in time, resulting in their accumulation in cells and in oxidative stress damage to particular components of the cells [ 39 , 40 ]. Studies have also demonstrated that ROS accumulation depends largely on the balance between ROS production and concurrent ROS clearance by scavenging or quenching [ 41 , 42 ]. ROS-scavenging enzymes play an important role in this process, so that the activities of ROS-scavenging enzymes, such as SOD, CAT, and POD, affect the ROS levels in stressed plants[ 43 , 44 ]. Three research groups had previously reported that the overexpression of genes encoding human leukocyte antigen-B associated transcript 1 ( OsBAT1 ), late-embryogenesis-abundant (LEA) proteins (e.g., OsLEA1a ), and plant ferritoxin-like protein ( PFLP ) in rice could enhance salt stress tolerance [ 45 – 47 ]. These genes can each promote the production of ROS- scavenging enzymes, which can keep the dynamic balance of ROS from being disrupted during salt stress. Therefore, the improvement of salt tolerance exhibited by transgenic lines can be related to an increase in the ROS-scavenging capacity during salt stress. In order to further clarify the effect of OsDUF6 induction on salt stress in rice, we compared the accumulation level and activity of ROS scavenging enzyme in transgenic lines and wild plants. It was found that the activities of the ROS-scavenging enzymes CAT, POD, PAL, and SOD increased significantly in transgenic lines (Fig. 7 ), compared with the corresponding wild-type line. In conclusion, the increased ROS scavenging enzyme activity in the OsDUF6- overexpressed lines protects the membrane system, mediates the transport and prevents the accumulation of Na+, and hence alleviates the osmotic and oxidative stress damage, promoting the ability of the transgenic plants to tolerate salt stress. In addition, in order to identify the gene function of OsDUF6 , OsDUF6 was introduced into yeast mutant Axt3k , and characterization of the complemented line confirmed its role in maintaining monovalent cation homeostasis and salt stress tolerance, by restoring the Na + transport and tolerance of the Axt3k mutant. It was confirmed that the molecular function of OsDUF6 included increased tolerance to salt stress. 5. Conclusions On the basis of previous studies, after heatmap analysis, we confirmed the expression of ten genes associated with growth and stress tolerance through RT-qPCR, and the results were similar to those from transcriptomics. It has been proved that overexpression of OsDUF6 can make these genes change dynamically when tolerating salt stress, thus promoting the ability to tolerate salt stress. Therefore, this study explains that, when rice is exposed to salt stress, the overexpression of OsDUF6 can enhance the expression of genes to improve the salt tolerance of rice, providing a research process and a factor for further exploring the ability of rice to tolerate salt stress at the molecular function level of genes. However, the specific pathways through which OsDUF6 regulates or interacts with genes which play a role in rice tolerating salt stress are unclear and deserve further investigation. Abbreviations OE Overexpression SOD Superoxide dismutase PAL Phenylalanine ammonolyase CAT Catalase POD Peroxidase ROS Reactive oxygen species DEGs Differentially expressed genes RT-qPCR Real time quantitative polymerase chain reaction NCBI National Center for Biotechnology Information AP Arg phosphate CDS Coding sequence CTAB Cetyltrimethylammonium Bromide PCR Polymerase chain reaction Declarations Author contributions XL and GM designed the experiment. GM and YZ added writing content. All authors have read and agreed to the published version of the manuscript. Funding This work was supported in part by the National Key Research and Development Program of China (Grant No. 2021YFD1700101) and the National Natural Science Foundation of China (Grant No. 31960546 and 32172461), Talents of Guizhou Science and Technology Cooperation Platform [Grant No. (2021)5623], Guizhou Science and Technology Cooperation Foundation [Grant No. ZK(2021)140], and a Unique Feature Project of Guizhou Provincial Education Department [Grant No. KY(2021)056]. Availability of Data and Materials All of the datasets are included within the article an5d its additional files. Ethics Approval and Consent to Participate Not applicable. Consent for Publication Applicable. Competing interests The authors declare no competing interests. Author details Corresponding Author XiangYang Li- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China; E-mail : [email protected] Authors GuangMing Ma- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China. Yong Zhang- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China. References Farooq M, Hussain M, Wakeel A, Siddique KH. M. Salt stress in maize: effects, resistance mechanisms, and management. A review. Agron Sustain Dev. 2015;35:461–81. Ali A, Yun DJ. Salt stress tolerance; what do we learn from halophytes? J Plant Biol. 2017;60(5):431–9. Hussain S, Zhang JH, Zhong C, Zhu LF, Cao XC, Yu SM, Jin QY. Effects of salt stress on rice growth, development characteristics, and the regulating ways: A review. J Integr Agric. 2017;16(11):2357–74. Jadamba C, Kang K, Paek NC, Lee SI, Yoo SC. Overexpression of Rice Expansin7 (Osexpa7) Confers Enhanced Tolerance to Salt Stress in Rice. Int J Mol Sci. 2020;21(2):454. Ashraf M, Akram NA. Improving salinity tolerance of plants through conventional breeding and genetic engineering: An analytical comparison. Biotechnol Adv. 2009;27(6):744–52. Akladious SA, Mohamed HI. Ameliorative effects of calcium nitrate and humic acid on the growth, yield component and biochemical attribute of pepper (Capsicum annuum) plants grown under salt stress. Sci Hortic. 2018;236:244–50. Kohli SK, Khanna K, Bhardwaj R, Abd Allah EF, Ahmad P, Corpas FJ. Assessment of Subcellular ROS and NO Metabolism in Higher Plants: Multifunctional Signaling Molecules. Antioxid (Basel Switzerland). 2019;8(12):641. Razzaq A, Ali A, Safdar LB, Zafar MM, Rui Y, Shakeel A, Shaukat A, Ashraf M, Gong W, Yuan Y. Salt stress induces physiochemical alterations in rice grain composition and quality. J Food Sci. 2020;85(1):14–20. van Zelm E, Zhang Y, Testerink C. Salt Tolerance Mechanisms of Plants. Annu Rev Plant Biol. 2020;71:403–33. Reddy INBL, Kim BK, Yoon IS, Kim KH, Kwon TR. Salt tolerance in rice: focus on mechanisms and approaches. Rice Sci. 2017;24(3):123–44. Huang L, Zhang F, Zhang F, Wang W, Zhou Y, Fu B, Li Z. Comparative transcriptome sequencing of tolerant rice introgression line and its parents in response to drought stress. BMC Genom. 2014;15(1):1026. Jia M, Luo N, Meng X, Song X, Jing Y, Kou L, Liu G, Huang X, Wang Y, Li J, Wang B, Yu H. OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice. J Genet Genomics. 2022;49(8):766–75. Fukagawa NK, Ziska LH, Rice. Importance for Global Nutrition. J Nutr Sci Vitaminol. 2019;65(Supplement):2–S3. Wang Z, Zhou Y, Ren XY, Wei K, Fan XL, Huang LC, Zhao DS, Zhang L, Zhang CQ, Liu QQ, Li QF. Co-Overexpression of Two Key Source Genes, OsBMY4 and OsISA3, Improves Multiple Key Traits of Rice Seeds. J Agric Food Chem. 2023;71(1):615–25. Frukh A, Siddiqi TO, Khan MIR, Ahmad A. Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress. Plant Physiol Biochem. 2020;146:55–70. Gupta A, Shaw BP. Biochemical and molecular characterisations of salt tolerance components in rice varieties tolerant and sensitive to NaCl: the relevance of Na + exclusion in salt tolerance in the species. Funct. Plant Biol. 2020;48(1):72–87. Ma G, Zhang Y, Li X. Dufulin enhances salt resistance of rice. Pestic Biochem Physiol. 2022;188:105252. Walker JM, editor. The proteomics protocols handbook. Humana press. New Jersey: Pasaic County; 2005. Chou KC, Shen HB. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE, 2010, 5(6), e11335. Nielsen H. Predicting Secretory Proteins with SignalP. Methods in molecular biology. (Clifton N J). 2017;1611:59–73. Buchan DWA, Jones DT. The PSIPRED Protein Analysis Workbench: 20 years on. Nucleic Acids Res. 2019;47(W1):W402–7. Hallgren J, Tsirigos KD, Pedersen MD, Almagro Armenteros JJ, Marcatili P, Nielsen H, Winther O. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. BioRxiv. 2022, 2022–04. Coudert E, Gehant S, Castro E, Pozzato M, Baratin D, Neto T, Sigrist CJA, Redaschi N, Bridge A. UniProt Consortium. Annotation of biologically relevant ligands in UniProtKB using ChEBI. Bioinf (Oxford England). 2023;39(1):btac793. Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, Gable AL, Fang T, Doncheva NT, Pyysalo S, Bork P, Jensen LJ, von Mering C. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638–46. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.). 2001, 25(4), 402–408. Kong M, Luo M, Li J, Feng Z, Zhang Y, Song W, Zhang R, Wang R, Wang Y, Zhao J, Tao Y, Zhao Y. Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance. Genomics. 2021;113(4):1940–51. Zhang Y, Xiao Y, Zhang Y, Dong Y, Liu Y, Liu L, Wan S, He J, Yu Y. Accumulation of Galactinol and ABA Is Involved in Exogenous EBR-Induced Drought Tolerance in Tea Plants. J Agric Food Chem. 2022;70(41):13391–403. Zhang Q, Liu Y, Jiang Y, Li A, Cheng B, Wu J. OsASR6 Enhances Salt Stress Tolerance in Rice. Int J Mol Sci. 2022;23(16):9340. Kamran M, Parveen A, Ahmar S, Malik Z, Hussain S, Chattha MS, Saleem MH, Adil M, Heidari P, Chen JT. An Overview of Hazardous Impacts of Soil Salinity in Crops, Tolerance Mechanisms, and Amelioration through Selenium Supplementation. Int J Mol Sci. 2019;21(1):148. Ma X, Feng F, Wei H, Mei H, Xu K, Chen S, Li T, Liang X, Liu H, Luo L. Genome-Wide Association Study for Plant Height and Grain Yield in Rice under Contrasting Moisture Regimes. Front. Plant Sci. 2016;7:1801. Singh V, Singh AP, Bhadoria J, Giri J, Singh J, T V V, Sharma PC. Differential expression of salt-responsive genes to salinity stress in salt-tolerant and salt-sensitive rice (Oryza sativa L.) at seedling stage. Protoplasma. 2018;255(6):1667–81. Kim JY, Lee SJ, Min WK, Cha S, Song JT, Seo H. S. COP1 controls salt stress tolerance by modulating sucrose content. Plant Signal Behav. 2022;17(1):2096784. Shakri T, Che-Othman MH, Md Isa N, Sukiran NL, Zainal Z. Morpho-Physiological and Stress-Related Gene Expression of Rice Varieties in Response to Salinity Stress at Early Vegetative Stage. Agriculture. 2022;12:638. Ahanger MA, Aziz U, Alsahli AA, Alyemeni MN, Ahmad P. Influence of Exogenous Salicylic Acid and Nitric Oxide on Growth, Photosynthesis, and Ascorbate-Glutathione Cycle in Salt Stressed Vigna angularis. Biomolecules. 2019;10(1):42. Parvin K, Nahar K, Hasanuzzaman M, Bhuyan MHMB, Mohsin SM, Fujita M. Exogenous vanillic acid enhances salt tolerance of tomato: Insight into plant antioxidant defense and glyoxalase systems. Plant Physiol Biochem. 2020;150:109–20. Ahmad P, Tripathi DK, Deshmukh R, Singh VP, Corpas FJ. Revisiting the role of ROS and RNS in plants under changing environment. Environ Exp Bot. 2019;161:1–3. Decros G, Baldet P, Beauvoit B, Stevens R, Flandin A, Colombié S, Gibon Y, Pétriacq P. Get the Balance Right: ROS Homeostasis and Redox Signalling in Fruit. Front Plant Sci. 2019;10:1091. Mittova V, Guy M, Tal M, Volokita M. Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii. J Exp Bot. 2004;55(399):1105–13. Ellouzi H, Oueslati S, Hessini K, Rabhi M, Abdelly C. Seed-priming with H2O2 alleviates subsequent salt stress by preventing ROS production and amplifying antioxidant defense in cauliflower seeds and seedlings. Sci Hortic. 2021;288:110360. Yang Y, Guo Y. Unraveling salt stress signaling in plants. J Integr Plant Biol. 2018;60(9):796–804. Liu Y, Yang T, Lin Z, Gu B, Xing C, Zhao L, Dong H, Gao J, Xie Z, Zhang S, Huang X. A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation. Plant Biotechnol J. 2019;17(9):1770–87. Li C, Sun X, Chang C, Jia D, Wei Z, Li C, Ma F. Dopamine alleviates salt-induced stress in Malus hupehensis. Physiol Plant. 2015;153(4):584–602. Yang Y, Guo Y. Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol. 2018;217(2):523–39. Tuteja N, Tarique M, Trivedi DK, Sahoo RK, Tuteja R. Stress-induced Oryza sativa BAT1 dual helicase exhibits unique bipolar translocation. Protoplasma. 2015;252(6):1563–74. Huang HE, Ho MH, Chang H, Chao HY, Ger MJ. Overexpression of plant ferredoxin-like protein promotes salinity tolerance in rice (Oryza sativa). Plant Physiol Biochem. 2020;155:136–46. Wang Z, Zhang Q, Qin J, Xiao G, Zhu S, Hu T. OsLEA1a overexpression enhances tolerance to diverse abiotic stresses by inhibiting cell membrane damage and enhancing ROS scavenging capacity in transgenic rice. Funct Plant Biol. 2021;48(9):860–70. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 19 Feb, 2024 Reviews received at journal 31 Jan, 2024 Reviewers agreed at journal 24 Jan, 2024 Reviewers agreed at journal 23 Jan, 2024 Reviewers invited by journal 17 Jan, 2024 Editor assigned by journal 16 Jan, 2024 Submission checks completed at journal 16 Jan, 2024 First submitted to journal 10 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3852076","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267349137,"identity":"77f3586b-23c4-4852-ae9f-d3d3678de3f8","order_by":0,"name":"Guangming Ma","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Guangming","middleName":"","lastName":"Ma","suffix":""},{"id":267349138,"identity":"3f46642b-50c3-45d3-a939-bc15686359a5","order_by":1,"name":"Yong zhang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"zhang","suffix":""},{"id":267349139,"identity":"c7da78be-1e4d-4800-ac43-03288e4196ae","order_by":2,"name":"Xiangyang Y. Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYFAC5oYDCRUQpgSRWhgbDjw4Q6oWxodtpGgxuJHYeCBxXq29wQHmg7d5GOzyCGqRnJHYcCBx23FmgwNsydY8DMnFBLXwS4C1HGMzOMBjJs3DcCCxgZAWNrCWOcd4DA7wfyNOC8SWhhoJoC1sxGmR7HkIjJdjBwwkD7MZW84xSCasxeB48uGPP2rq7PmONz+88abCjrAWBoEEEHkYmArAJhBUDwT8B0BkHTFKR8EoGAWjYKQCANurP0YeSgfQAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Xiangyang","middleName":"Y.","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-01-11 02:59:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3852076/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3852076/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-04921-z","type":"published","date":"2024-03-26T15:01:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49776146,"identity":"7223c86d-33ae-4fe5-8e4c-16fb65a4ae1c","added_by":"auto","created_at":"2024-01-17 20:55:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254886,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional prediction and analysis. (A) Prediction of OsDUF6 protein transmembrane region; (B) Secretory signal peptide; (C) Prediction of OsDUF6 interacting protein regulatory network.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/21c0adf34e37ba899d137ac0.png"},{"id":49776143,"identity":"718047b6-8f43-4c29-9575-77749903e423","added_by":"auto","created_at":"2024-01-17 20:55:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3597715,"visible":true,"origin":"","legend":"\u003cp\u003eHomology sequence alignment and phylogenetic tree analysis of \u003cem\u003eOsDUF6\u003c/em\u003e. (A, Homology sequence alignment; B, Phylogenetic tree analysis)\u003c/p\u003e","description":"","filename":"floatimage410.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/10016fdfbafbdac6993d7aa6.jpeg"},{"id":49776144,"identity":"17083729-cfa0-43d9-be99-259abf66751d","added_by":"auto","created_at":"2024-01-17 20:55:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":532995,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral subcellular localization experiment of OsDUF6 protein\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/513ad0b86672fcbeb492d2db.jpeg"},{"id":49776145,"identity":"69c63d8b-9a93-4ed4-99ec-61f14643f24a","added_by":"auto","created_at":"2024-01-17 20:55:39","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":300545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOsDUF6\u003c/em\u003e gene promotes the growth of yeast mutant Axt3k under salt stress.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/5da02f7a6fdda967e7c973a7.jpeg"},{"id":49776151,"identity":"5ac3639d-5479-49a2-bc98-423f4ea1fc54","added_by":"auto","created_at":"2024-01-17 20:55:39","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":844214,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth characteristics of rice after salt stress. (left, wild-type; right, OE).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/2f0380708ea4a5b408fe5df5.jpeg"},{"id":49776304,"identity":"64d04a09-f020-4ac8-b3c9-a465b1bfb02c","added_by":"auto","created_at":"2024-01-17 21:03:39","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":358699,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological indicators. (A) Leaf yellow rate; (B) Na\u003csup\u003e+\u003c/sup\u003e content; (C) Length of root, stem and leaf. [mean values displayed in each bar followed by different letters significantly differ according to Student’s t-test (*P \u0026lt; 0.05, **P \u0026lt; 0.01). Vertical bars indicate SD (n = 3)]\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/1fc85e08f295c1298f16a256.jpeg"},{"id":49776305,"identity":"630a50c8-b8be-4c60-92bf-c27c37fedaf8","added_by":"auto","created_at":"2024-01-17 21:03:39","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":187909,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of rice growth-related enzymes under salt stress. [mean values displayed in each bar followed by different letters significantly differ according to Student’s t-test (*P \u0026lt; 0.05, **P \u0026lt; 0.01). Vertical bars indicate SD (n = 3)]\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/198d71c12e4373d8624a4dc8.jpeg"},{"id":49776148,"identity":"6dd27249-22bb-4abe-b599-53cdc3b68122","added_by":"auto","created_at":"2024-01-17 20:55:39","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":298006,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs associated with salt stress treatment.\u003cstrong\u003e \u003c/strong\u003e(A) Cluster heat map of DEGs. Red indicates up-regulated genes, and blue indicates down-regulated genes. Ten selected gene names were marked. (DFL, Dufulin-treated group; CK, control group); (B) RT-qPCR validation. Ten genes related to growth and stress resistance were verified. [mean values displayed in each bar followed by different letters significantly differ according to Student’s t-test (*P \u0026lt; 0.05, **P \u0026lt; 0.01). Vertical bars indicate SD (n = 3)]\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/133a34d8c208e283c3b2e432.jpeg"},{"id":53869978,"identity":"07b91e2d-6bf2-48dc-a6eb-12d3db0f1eb7","added_by":"auto","created_at":"2024-04-01 15:12:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1450684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/c8090ad3-fda9-4114-8236-7b93ba7193e2.pdf"},{"id":49776149,"identity":"60d50943-20f6-479e-814a-7a9aae7f83ba","added_by":"auto","created_at":"2024-01-17 20:55:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":845957,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3852076/v1/0d3d6e412a11b502fe47c21f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dufulin activates OsDUF6 protein against salt stress in rice plant","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOne of the major abiotic stresses encountered by food crops during growth is increased soil salinity.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e More than 20% of the world\u0026rsquo;s agricultural land is affected by high salinity, a figure which is expected to rise in the future.\u003csup\u003e4\u003c/sup\u003e When food crops are subjected to salt stress, the plants respond at the morphological, cellular, and molecular levels.\u003csup\u003e5\u003c/sup\u003e When encountering salt stress, the homeostasis between reactive oxygen species (ROS) production and scavenging will be broken, resulting in excessive accumulation of ROS, which will destroy the original biological functions of the plant to a certain extent and hinder the normal physiological processes of the plant.\u003csup\u003e6,7\u003c/sup\u003e Plant morphological changes under high salt conditions included impaired root establishment, occurrence of leaf rolling, yellowing, decreased thousand-seed weight, and decreased number of spikelets per ear, the latter ultimately resulting in decreased harvest index and grain yield.\u003csup\u003e8,9\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e) is the main food crop for much of the world\u0026rsquo;s population and is also one of the most important staple foods in daily life.\u003csup\u003e10\u003c/sup\u003e With climatic changes observed in recent years, rice needs to respond to biological and abiotic stresses. The use of pesticides and fungicides has reduced the importance of biological stresses in rice production. However, rice also needs to respond to several abiotic stresses. Among these, salt stress is an important focus of current research, and it has been found that rice is more sensitive to drought and salt stress than other grains, such as barley, wheat, and rye,\u003csup\u003e11,12\u003c/sup\u003e making rice an urgent target for studies into mechanisms of salt tolerance. At present, people pay more attention to food health, while the quality of rice as a staple food is also a key target.\u003csup\u003e13,14\u003c/sup\u003e Moreover, how to improve the quality of rice while increasing its yield, achieving the best of both worlds, is the result that more people want. Despite this, it has become a major public issue to improve rice production by promoting rice tolerance to salt stress.\u003csup\u003e15,16\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBased on the transcriptomic data from rice plants exposed to salt stress after being sprayed with the plant antiviral agent Dufulin, which has been shown to induce salt stress tolerance, we found that \u003cem\u003eOsDUF6\u003c/em\u003e exhibited significantly changed expression among the differentially expressed genes (DEGs).\u003csup\u003e17\u003c/sup\u003e Studying salt stress after spraying with a disease-resistance inducer, such as Dufulin, is in line with the current environment of how to tolerate abiotic stress after preventing biotic stress. Analysis of the molecular function of \u003cem\u003eOsDUF6\u003c/em\u003e will allow us to confirm whether a gene conferring tolerance to salt stress, the expression of which changes in response to treatment with a chemical, Dufulin, which induces salt tolerance, is an important gene with respect to response to abiotic stresses. Therefore, analysis of the response of the \u003cem\u003eOsDUF6\u003c/em\u003e (\u003cem\u003eLOC_Os12g33300\u003c/em\u003e) gene to abiotic stress in rice plants and a study of its molecular function can enrich our understanding of the rice salt stress signal network.\u003c/p\u003e \u003cp\u003eThe current study investigated the molecular function of \u003cem\u003eOsDUF6\u003c/em\u003e and its positive impact on salt tolerance in rice after overexpression, based on an in-depth exploration of transcriptomic data with respect to response to a chemical promoting salt tolerance in rice. Such research could provide novel insights into the molecular function of inducers of salt tolerance, and identify new salt tolerance genes for exploitation in molecular breeding of rice for salt tolerance.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eDufulin (93%), an active ingredient, was synthesized in the State Key Laboratory of Green Pesticides, Guizhou University. The treatment concentration was 500 mM (Ma et al., 2022). The seeds of rice materials used in the experiment were \u003cem\u003eNipponbare\u003c/em\u003e(\u003cem\u003eOryza༎Sativa L༎spp༎japonica\u003c/em\u003e), and the seeds of tobacco materials were \u003cem\u003eNicotiana benthamiana\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sources and cultivation of transgenic overexpressed rice\u003c/h2\u003e \u003cp\u003eThe pBWA (V) HS-OsDUF6 plasmid was constructed, and 1 \u0026micro;L plasmid was added to 50 \u0026micro;L strain EHA105 \u003cem\u003eAgrobacterium\u003c/em\u003e competent cells for \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e transformation. Then, \u003cem\u003eAgrobacterium\u003c/em\u003e infection co-culture, callus screening. The transformation-positive rice callus was inoculated onto the differentiation medium and cultured at 25\u0026ndash;27\u0026deg;C for 15\u0026ndash;20 d. After the differentiation of 2\u0026ndash;5 cm buds, the rice callus was transferred onto the rooting medium and cultured at 30\u0026deg;C for 7\u0026ndash;10 d. The transgenic rice seedlings(\u003cem\u003eNipponbare\u003c/em\u003e) were then transferred to plastic pots containing a soil/perlite mixture and grown in a growth chamber at 26\u0026deg;C and 72% relative humidity for 16 h/8 h (day/night). The rice plants cultured after using the CTAB method to extract rice genomic DNA, and then PCR was used to detect positive plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Salt stress treatment of plant material\u003c/h2\u003e \u003cp\u003eFull and intact seeds of homozygous overexpressed and wild-type rice(\u003cem\u003eNipponbare\u003c/em\u003e) of similar size were selected and germinated for 3\u0026ndash;5 d at the same time. After germination, they were transplanted into plastic pots of a soil/perlite mixture in the greenhouse [26\u0026deg;C and 72% relative humidity for 16 h/8 h (day/night)]. After 15 d growth, wild-type and transgenic rice plants were sprayed with 200 mM NaCl to induce salt stress and were photographed and sampled after a further sixth days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Physiological index\u003c/h2\u003e \u003cp\u003eThe phenotypes of transgenic and wild-type rice(\u003cem\u003eNipponbare\u003c/em\u003e) after six days of salt stress were observed, and root, stem, and leaf lengths were measured to evaluate any growth differences. Then, each plant was dried to obtain dry weights. The sodium (Na\u003csup\u003e+)\u003c/sup\u003e concentrations of rice plants were measured by flame spectrometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Rice sample collection and total RNA extraction\u003c/h2\u003e \u003cp\u003eThe roots, stems, and leaves of the overexpressed transgenic and wild-type rice(\u003cem\u003eNipponbare\u003c/em\u003e) were collected after six days of salt stress. They were snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA was extracted from the preserved sample tissues of the overexpressed lines and the corresponding wild-type variety, following the instructions of the manufacturer of the RNA prep Pure Plant Kit (Tiangen, Beijing, China). The RNA concentration was measured with a NanoPhotometer (IMPLEN, Munich, Germany), and the standard DNA concentration of the subsequent experiment was selected for preservation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Bioinformatic analysis of the OsDUF6 gene\u003c/h2\u003e \u003cp\u003eThe sequences of the OsDUF6 protein and its homologous protein sequences were obtained from the NCBI. In the ExPASy2 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://au.expasy.org/tools/protparam.html\u003c/span\u003e\u003cspan address=\"http://au.expasy.org/tools/protparam.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), physical and chemical parameters were predicted for preliminary OsDUF6 proteins [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Based on this preliminary information, the subcellular localization (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the protein secondary structure (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"http://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), interaction protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), tertiary structure model (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and transmembrane signal peptide (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.dtu.dk/english\u003c/span\u003e\u003cspan address=\"https://www.dtu.dk/english\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) ) of OsDUF6 were predicted and analyzed online [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on the \u003cem\u003eOsDUF6\u003c/em\u003e gene sequence, a search was carried out for homologous sequences among multiple species in NCBI; any sequence identified was downloaded and MEGA software was used to carry out multiple sequence alignment, and the neighborhood connection method to was used to construct a phylogenetic tree [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Determination of enzyme activity of OE and WT after salt stress\u003c/h2\u003e \u003cp\u003eFollowing the manufacturer's instructions, the appropriate enzyme assay kits (Jiangbin, Nanjing, China) were used to assay plant tissue homogenate (1: 9 tissue weight: buffer [phosphate buffer (0.1 mol/L pH 7\u0026ndash;7.4)] volume for the activity of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 RT-qPCR analysis\u003c/h2\u003e \u003cp\u003eThe total RNA, extracted as described above, was converted into cDNA using the StarScript III All-in-one RT Mix with gDNA Kit (Genstar, Beijing China), and diluted with ddH\u003csub\u003e2\u003c/sub\u003eO. In the 20 \u0026micro;L reaction volume, 2 \u0026times; RealStar Fast SYBR qPCR Mix (Genstar, China) was used for premixing, and DEPC-treated H\u003csub\u003e2\u003c/sub\u003eO was used to adjust the volume. The reaction was carried out in the LightCycler 96 RT-qPCR assay system (Roche, Basel, Switzerland) and the reaction was completed according to the dissolution curve automatically set by the instrument. The relative gene expression rate between the overexpression lines and the wild-type lines was compared using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and three biological replicates were conducted for each sample. All quantitative primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and were synthesized by Beijing Tsingke Biology (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 General subcellular localization experiment\u003c/h2\u003e \u003cp\u003eThe coding sequence of \u003cem\u003eOsDUF6\u003c/em\u003e (\u003cem\u003eLOC_Os12g33300\u003c/em\u003e) was cloned into a pCam35S-GFP vector. After the plasmid obtained was used to transform \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101 in its receptive state, the bacteria were cultured in a 28\u0026deg;C incubator for 2\u0026ndash;3 d. After screening a single colony to confirm its transformation-positive nature, the culture was multiplied in liquid medium. After centrifugation, the bacterial pellet was resuspended and, after dark incubation for 3 h, was inoculated into leaves of 2-week-old \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants of similar growth form. Three days later, the injected leaves were cut into 2 mm \u0026times; 2 mm squares and placed on slides, with the fluorescence from the green fluorescent protein (GFP) being observed under a confocal laser scanning microscope(ZEISS, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Confirmation of yeast functional complementation\u003c/h2\u003e \u003cp\u003eWe used a salt-sensitive \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e mutant \u003cem\u003eAxk3k\u003c/em\u003e that lacks the main plasma membrane Na\u003csup\u003e+\u003c/sup\u003e transporter [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The full-length sequence of \u003cem\u003eOsDUF6\u003c/em\u003e was cloned into the PYES2 yeast expression vector. First, the \u003cem\u003eAxt3k\u003c/em\u003e strain, stored at \u0026minus;\u0026thinsp;80\u0026deg;C, was activated, and then, according to the manufacturer's instructions, the yeast transformation kit (Coolaber, Beijing, China) was used to convert it into the receptive state. Then, the \u003cem\u003eOsDUF6\u003c/em\u003e plasmid was introduced into the prepared receptive \u003cem\u003eAxt3k\u003c/em\u003e by the PEG/LiAc method, and the transformed yeast, after centrifugation, was spread onto SD/-Ura solid medium, and the plate cultured in an inverted orientation, in a 30\u0026deg;C incubator for 3\u0026ndash;4 d to achieve yeast colonies of the appropriate size. After a single colony was selected and confirmed to be transformation-positive, the transformed yeast was suspended in ddH\u003csub\u003e2\u003c/sub\u003eO and diluted with ddH\u003csub\u003e2\u003c/sub\u003eO to form the concentration gradient of 10-fold serial dilutions. Approximately 3 \u0026micro;L of the yeast suspension was applied onto the Arg phosphate (AP) solid medium plate containing 0, 25, 50, 75, or 100 mM NaCl, incubated upside down at 30\u0026deg;C to grow for 3\u0026ndash;4 d, and the yeast growth was observed and recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Data analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed by ANOVA to identify statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01)) differences in measured parameters between the wild-type and overexpression lines. Three independent biological replicates were used for each measurement. Experimental data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Graphs were generated using Prism software (v 9.0) (GraphPad Software, Boston, MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Functional prediction and analysis of the OsDUF6 protein\u003c/h2\u003e \u003cp\u003eThe molecular weight and isoelectric point (pI) of OsDUF6 were 41.46 kDa and 8.87, respectively, and the number of amino acids encoded was 373. Through the prediction of its transmembrane domain and signal peptide, the results showed that the deduced protein had ten transmembrane domains, so that it was a transmembrane protein, but lacked a signal peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). The subcellular localization of the protein was predicted online and indicated to be in the cell membrane (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The secondary structure of OsDUF6 was predicted by PSIPRED. It can be seen from the prediction results that helix and coil constituted almost its entire secondary structure, with strand encoding only two amino acids, so that the secondary structure is relatively stable (Fig. S2). Then, on the basis of the secondary structure, we predicted the tertiary structure of OsDUF6, with the results showing that the AlphaFold produced a very high per-residue confidence score (pLDDT)\u0026thinsp;\u0026gt;\u0026thinsp;90, so that this tertiary structure could be applied to experimental research to a certain extent (Fig. S3). By comparing and screening with the STRING database, based on the rice varieties studied in this experiment, the \u003cem\u003eOryza sativa japonica\u003c/em\u003e Group was selected to predict the interaction proteins of OsDUF6 in the database. In Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, A0A0P0YAX6 is our target protein, and the intricate lines represent the interaction network of OsDUF6; in this interaction network, it is clearly divided into two parts. However, according to the meaning of the line representation, we found that the prediction results are based on the mined text, and the proteins interacting with OsDUF6 are not based on accurate data or experimental results, which will require us to determine the actual interacting proteins based on experimental results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe homologous sequence of the OsDUF6 protein sequence was studied using NCBI technology. Then, MEGA6.0 software was used to achieve multiple sequence comparisons, and the amino acid sequence obtained after comparison was used to construct an evolutionary phylogenetic tree. The results showed that OsDUF6 had a high degree of genetic relationship with the WAT1-related protein \u003cem\u003eAt5g64700\u003c/em\u003e (\u003cem\u003eOryza sativa japonica Group\u003c/em\u003e) and hydroxy protein \u003cem\u003eOsJ36299\u003c/em\u003e (\u003cem\u003eOryza sativa japonica\u003c/em\u003e Group), but it had a distant genetic relationship with the hydroxy protein \u003cem\u003eBS78-09G001100\u003c/em\u003e (\u003cem\u003ePaspalum vaginatum\u003c/em\u003e) and the hydroxy protein \u003cem\u003eEJB05-22345\u003c/em\u003e partial (\u003cem\u003eEragrostis curvula\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 OsDUF6 encodes a membrane-localized protein\u003c/h2\u003e \u003cp\u003eIn order to detect the general subcellular localization of OsDUF6 protein, the coding sequence (CDS) region of \u003cem\u003eOsDUF6\u003c/em\u003e was cloned and the expression vector of the \u003cem\u003eOsDUF6-YFP\u003c/em\u003e (yellow fluorescent protein) fusion protein was constructed and was used to transform \u003cem\u003eAgrobacterium\u003c/em\u003e, which was cultured, incubated, and injected into tobacco. Finally, the subcellular localization was preliminarily determined by laser scanning confocal microscopy to detect the fluorescence signal. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the fluorescence of the 35S::\u003cem\u003eOsDUF6-YFP\u003c/em\u003e construct was detected in the cell membrane. Comparison of the prediction results and the subcellular localization results showed that they were similar, so that the \u003cem\u003eOsDUF6\u003c/em\u003e gene was determined to encode a membrane-localized protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Functional analysis of OsDUF6 in yeast mutants under NaCl stress\u003c/h2\u003e \u003cp\u003eIn order to test the function of \u003cem\u003eOsDUF6\u003c/em\u003e in salt tolerance, \u003cem\u003eOsDUF6\u003c/em\u003e was cloned into yeast expression vector pYES2, and then transformed into \u003cem\u003eS. cerevisiae\u003c/em\u003e mutant \u003cem\u003eAxt3k\u003c/em\u003e (ena1\u0026ndash;4:: HIS3,nha1:: LEU2, nhx1:: KanMX), which is very sensitive to Na\u003csup\u003e+\u003c/sup\u003e stress. For each transformed yeast strain, colony growth responded indirectly to a gradient of NaCl concentrations on AP medium. The results showed that the yeast mutant carrying the \u003cem\u003eOsDUF6\u003c/em\u003e gene still grew at the highest NaCl concentration on the AP medium, whereas the control mutant group barely grew (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This growth of the complemented yeast mutant confirmed the functional salt tolerance of the \u003cem\u003eOsDUF6\u003c/em\u003e gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Overexpression of OsDUF6 promotes salt tolerance in rice\u003c/h2\u003e \u003cp\u003eIn order to clarify the function of \u003cem\u003eOsDUF6\u003c/em\u003e transgenic rice in terms of salt stress response, seeds of the transgenic rice lines and its corresponding wild-type plant were grown in a greenhouse, and 200 mM NaCl was added at the soil matrix at a specified time for salt stress treatment. Under normal, non-stress conditions, there was no significant phenotypic difference between transgenic lines and the wild-type strain. After sixth days of salt treatment, according to phenotypic observations, all rice plants (transgenic lines and wild type) were damaged, though to varying extents (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The area of yellowed tissue of leaves of transgenic plants was smaller than that of the wild-type plants; furthermore, the length of the roots, leaves, and stems of both genotypes were reduced under salt stress (compared with the non-stressed control), but the decrease in transgenic plant size was much smaller than that of the wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C).\u003c/p\u003e \u003ch2\u003e3.5 Overexpression of OsDUF6 reduced the concentration of Na\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003econcentration in transgenic rice plants under salt stress\u003c/h2\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e homeostasis is an important characteristic of plant salt tolerance. Therefore, we determined the concentration of Na\u003csup\u003e+\u003c/sup\u003e in the roots and leaves of wild-type and overexpressed lines. Compared with the Na\u003csup\u003e+\u003c/sup\u003e concentration of the wild-type plants, the accumulation of Na\u003csup\u003e+\u003c/sup\u003e in the overexpression lines was less, which indicates that the stress tolerant mechanism was due, at least in part, to the transgenic plants being more efficient at transporting Na\u003csup\u003e+\u003c/sup\u003e into/out of the plant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Overexpression of OsDUF6 increases the activity of antioxidant enzymes under salt stress\u003c/h2\u003e \u003cp\u003eIn order to detect the level of ROS scavenging in overexpressed lines and wild-type plants, we measured the activities of the main direct antioxidant enzymes SOD, CAT, and POD, and an indirect antioxidant enzyme, PAL, in the overexpressed lines and wild-type lines. After assay, it was found that, in response to salt treatment, the increase in SOD, PAL, CAT, and POD activities in overexpression lines were higher than those in the wild-type lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003e), with the response of the three direct antioxidant enzymes being much higher than that of the indirect antioxidant enzyme, PAL. These results suggest that \u003cem\u003eOsDUF6\u003c/em\u003e overexpression lines can increase the activity of four antioxidant enzymes SOD, PAL, CAT and POD when rice is exposed to salt stress, so that the accumulation of ROS in rice under salt stress is reduced to a certain extent, compared with wild-type lines, thereby alleviating a series of adverse oxidative stress effects caused by ROS accumulation in rice and promoting rice tolerance to salt stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Overexpression of OsDUF6 changed the expression of related genes under salt stress\u003c/h2\u003e \u003cp\u003eOur previous research had shown that, when rice was subjected to salt stress after spraying with Dufulin, \u003cem\u003eOsDUF6\u003c/em\u003e expression in the rice transcriptome was found to be upregulated, with the relative expression of some other genes also changing dynamically [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we selected a number of genes putatively related to growth and stress tolerance and the expression of these genes were presented as a heatmap, based on the previous transcriptome data (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Based on these data, we conducted RT-qPCR to confirm the changes in expression level of these genes in the leaves of transgenic and wild-type plants. The results confirmed that the dynamic changes in expression of these genes were consistent with those from the previous transcriptome data and were associated with increased plant salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Therefore, these results indicate that overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e changed the expression level of these genes to a certain extent, which means that \u003cem\u003eOsDUF6\u003c/em\u003e expression is closely related to that of these genes. In summary, according to the results of RT-qPCR, in the presence of salt stress, overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e has a positive effect on rice growth and stress-related genes via an effect on Na\u003csup\u003e+\u003c/sup\u003e transport, which further explains its role in promoting salt stress tolerance in rice.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSalinity is a major abiotic stress factor that seriously affects crop growth and yield, causing significant economic losses and threatening global food security [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. At present, 23.02\u0026nbsp;billion hectares of farmland worldwide can be used for food production. However, due to inappropriate crop irrigation methods (leading to salinization) and excessive fertilizer usage, as well as natural effects, such as sea level rise, salt intrusion into coastal areas, some 2% of farmland is affected by salt, and the proportion of impacts on agricultural development is also increasing year by year [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Rice is a very important cereal crop that feeds half of the world's population and is particularly sensitive to salt stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. There have been many studies on salt-tolerant transgenic rice, most of which were based on research into homologous species and gene families to identify the genes that change the salt response of rice [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Prior to the current study, the \u003cem\u003eOsDUF6\u003c/em\u003e gene had no known function. In this present study, based on the transcriptome analysis of rice sprayed with Dufulin and then subjected to salt stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we studied the function of \u003cem\u003eOsDUF6\u003c/em\u003e gene, transgenic plants were generated to overexpress the \u003cem\u003eOsDUF6\u003c/em\u003e gene, and their molecular functions were studied through a series of indicators. Research has shown that a high concentration of Na\u003csup\u003e+\u003c/sup\u003e enters the body of plants under salt stress, and thence induce the accelerated accumulation of ROS [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. When a high concentration of ROS is generated, it will cause oxidative damage to the membrane; in serious cases, it can disrupt cellular metabolism, leading to the inhibition of normal growth and development of the plants [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Through subcellular protein localization test and software prediction, it was preliminarily determined that the OsDUF6 protein is located on the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Therefore, overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e will directly and positively affect the salt tolerance of rice by mediating Na\u003csup\u003e+\u003c/sup\u003e transport through the membrane, a finding of great significance in the study of salt resistance of rice.\u003c/p\u003e \u003cp\u003eWhen plants are exposed to salt stress, ROS will be generated, which will cause plants to be unable to clear them in time, resulting in their accumulation in cells and in oxidative stress damage to particular components of the cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Studies have also demonstrated that ROS accumulation depends largely on the balance between ROS production and concurrent ROS clearance by scavenging or quenching [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. ROS-scavenging enzymes play an important role in this process, so that the activities of ROS-scavenging enzymes, such as SOD, CAT, and POD, affect the ROS levels in stressed plants[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThree research groups had previously reported that the overexpression of genes encoding human leukocyte antigen-B associated transcript 1 (\u003cem\u003eOsBAT1\u003c/em\u003e), late-embryogenesis-abundant (LEA) proteins (e.g., \u003cem\u003eOsLEA1a\u003c/em\u003e), and plant ferritoxin-like protein (\u003cem\u003ePFLP\u003c/em\u003e) in rice could enhance salt stress tolerance [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These genes can each promote the production of ROS- scavenging enzymes, which can keep the dynamic balance of ROS from being disrupted during salt stress. Therefore, the improvement of salt tolerance exhibited by transgenic lines can be related to an increase in the ROS-scavenging capacity during salt stress. In order to further clarify the effect of \u003cem\u003eOsDUF6\u003c/em\u003e induction on salt stress in rice, we compared the accumulation level and activity of ROS scavenging enzyme in transgenic lines and wild plants. It was found that the activities of the ROS-scavenging enzymes CAT, POD, PAL, and SOD increased significantly in transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003e), compared with the corresponding wild-type line. In conclusion, the increased ROS scavenging enzyme activity in the \u003cem\u003eOsDUF6-\u003c/em\u003eoverexpressed lines protects the membrane system, mediates the transport and prevents the accumulation of Na+, and hence alleviates the osmotic and oxidative stress damage, promoting the ability of the transgenic plants to tolerate salt stress.\u003c/p\u003e \u003cp\u003eIn addition, in order to identify the gene function of \u003cem\u003eOsDUF6\u003c/em\u003e, \u003cem\u003eOsDUF6\u003c/em\u003e was introduced into yeast mutant \u003cem\u003eAxt3k\u003c/em\u003e, and characterization of the complemented line confirmed its role in maintaining monovalent cation homeostasis and salt stress tolerance, by restoring the Na\u003csup\u003e+\u003c/sup\u003e transport and tolerance of the \u003cem\u003eAxt3k\u003c/em\u003e mutant. It was confirmed that the molecular function of \u003cem\u003eOsDUF6\u003c/em\u003e included increased tolerance to salt stress.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOn the basis of previous studies, after heatmap analysis, we confirmed the expression of ten genes associated with growth and stress tolerance through RT-qPCR, and the results were similar to those from transcriptomics. It has been proved that overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e can make these genes change dynamically when tolerating salt stress, thus promoting the ability to tolerate salt stress. Therefore, this study explains that, when rice is exposed to salt stress, the overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e can enhance the expression of genes to improve the salt tolerance of rice, providing a research process and a factor for further exploring the ability of rice to tolerate salt stress at the molecular function level of genes. However, the specific pathways through which \u003cem\u003eOsDUF6\u003c/em\u003e regulates or interacts with genes which play a role in rice tolerating salt stress are unclear and deserve further investigation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Overexpression\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp;Superoxide dismutase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePAL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phenylalanine ammonolyase\u003c/p\u003e\n\u003cp\u003eCAT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Catalase\u003c/p\u003e\n\u003cp\u003ePOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Peroxidase\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eDEGs \u0026nbsp; \u0026nbsp; \u0026nbsp; Differentially expressed genes\u003c/p\u003e\n\u003cp\u003eRT-qPCR \u0026nbsp; \u0026nbsp;Real time quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eNCBI \u0026nbsp; \u0026nbsp; \u0026nbsp; National Center for Biotechnology Information\u003c/p\u003e\n\u003cp\u003eAP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Arg phosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCDS \u0026nbsp; \u0026nbsp; \u0026nbsp;Coding sequence\u003c/p\u003e\n\u003cp\u003eCTAB \u0026nbsp; \u0026nbsp; \u0026nbsp;Cetyltrimethylammonium Bromide\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; Polymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXL and GM designed the experiment. GM and YZ added writing content. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the National Key Research and Development Program of China (Grant No. 2021YFD1700101) and the National Natural Science Foundation of China (Grant No. 31960546 and 32172461), Talents of Guizhou Science and Technology Cooperation Platform [Grant No. (2021)5623], Guizhou Science and Technology Cooperation Foundation [Grant No. ZK(2021)140], and a Unique Feature Project of Guizhou Provincial Education Department [Grant No. KY(2021)056].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the datasets are included within the article an5d its additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics Approval and Consent to Participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for Publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApplicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiangYang Li- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China; E-mail : [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuangMing Ma- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China.\u003c/p\u003e\n\u003cp\u003eYong Zhang- National Key Laboratory of Green Pesticide, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFarooq M, Hussain M, Wakeel A, Siddique KH. M. Salt stress in maize: effects, resistance mechanisms, and management. A review. Agron Sustain Dev. 2015;35:461\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli A, Yun DJ. Salt stress tolerance; what do we learn from halophytes? J Plant Biol. 2017;60(5):431\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain S, Zhang JH, Zhong C, Zhu LF, Cao XC, Yu SM, Jin QY. Effects of salt stress on rice growth, development characteristics, and the regulating ways: A review. J Integr Agric. 2017;16(11):2357\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJadamba C, Kang K, Paek NC, Lee SI, Yoo SC. Overexpression of Rice Expansin7 (Osexpa7) Confers Enhanced Tolerance to Salt Stress in Rice. Int J Mol Sci. 2020;21(2):454.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshraf M, Akram NA. Improving salinity tolerance of plants through conventional breeding and genetic engineering: An analytical comparison. Biotechnol Adv. 2009;27(6):744\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkladious SA, Mohamed HI. Ameliorative effects of calcium nitrate and humic acid on the growth, yield component and biochemical attribute of pepper (Capsicum annuum) plants grown under salt stress. Sci Hortic. 2018;236:244\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohli SK, Khanna K, Bhardwaj R, Abd Allah EF, Ahmad P, Corpas FJ. Assessment of Subcellular ROS and NO Metabolism in Higher Plants: Multifunctional Signaling Molecules. Antioxid (Basel Switzerland). 2019;8(12):641.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRazzaq A, Ali A, Safdar LB, Zafar MM, Rui Y, Shakeel A, Shaukat A, Ashraf M, Gong W, Yuan Y. Salt stress induces physiochemical alterations in rice grain composition and quality. J Food Sci. 2020;85(1):14\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Zelm E, Zhang Y, Testerink C. Salt Tolerance Mechanisms of Plants. Annu Rev Plant Biol. 2020;71:403\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy INBL, Kim BK, Yoon IS, Kim KH, Kwon TR. Salt tolerance in rice: focus on mechanisms and approaches. Rice Sci. 2017;24(3):123\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang L, Zhang F, Zhang F, Wang W, Zhou Y, Fu B, Li Z. Comparative transcriptome sequencing of tolerant rice introgression line and its parents in response to drought stress. BMC Genom. 2014;15(1):1026.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia M, Luo N, Meng X, Song X, Jing Y, Kou L, Liu G, Huang X, Wang Y, Li J, Wang B, Yu H. OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice. J Genet Genomics. 2022;49(8):766\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukagawa NK, Ziska LH, Rice. Importance for Global Nutrition. J Nutr Sci Vitaminol. 2019;65(Supplement):2\u0026ndash;S3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Zhou Y, Ren XY, Wei K, Fan XL, Huang LC, Zhao DS, Zhang L, Zhang CQ, Liu QQ, Li QF. Co-Overexpression of Two Key Source Genes, OsBMY4 and OsISA3, Improves Multiple Key Traits of Rice Seeds. J Agric Food Chem. 2023;71(1):615\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrukh A, Siddiqi TO, Khan MIR, Ahmad A. Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress. Plant Physiol Biochem. 2020;146:55\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta A, Shaw BP. Biochemical and molecular characterisations of salt tolerance components in rice varieties tolerant and sensitive to NaCl: the relevance of Na\u0026thinsp;+\u0026thinsp;exclusion in salt tolerance in the species. Funct. Plant Biol. 2020;48(1):72\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa G, Zhang Y, Li X. Dufulin enhances salt resistance of rice. Pestic Biochem Physiol. 2022;188:105252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalker JM, editor. The proteomics protocols handbook. Humana press. New Jersey: Pasaic County; 2005.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou KC, Shen HB. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE, 2010, 5(6), e11335.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNielsen H. Predicting Secretory Proteins with SignalP. Methods in molecular biology. (Clifton N J). 2017;1611:59\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchan DWA, Jones DT. The PSIPRED Protein Analysis Workbench: 20 years on. Nucleic Acids Res. 2019;47(W1):W402\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHallgren J, Tsirigos KD, Pedersen MD, Almagro Armenteros JJ, Marcatili P, Nielsen H, Winther O. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. BioRxiv. 2022, 2022\u0026ndash;04.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoudert E, Gehant S, Castro E, Pozzato M, Baratin D, Neto T, Sigrist CJA, Redaschi N, Bridge A. UniProt Consortium. Annotation of biologically relevant ligands in UniProtKB using ChEBI. Bioinf (Oxford England). 2023;39(1):btac793.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, Gable AL, Fang T, Doncheva NT, Pyysalo S, Bork P, Jensen LJ, von Mering C. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51(D1):D638\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.). 2001, 25(4), 402\u0026ndash;408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong M, Luo M, Li J, Feng Z, Zhang Y, Song W, Zhang R, Wang R, Wang Y, Zhao J, Tao Y, Zhao Y. Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance. Genomics. 2021;113(4):1940\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Xiao Y, Zhang Y, Dong Y, Liu Y, Liu L, Wan S, He J, Yu Y. Accumulation of Galactinol and ABA Is Involved in Exogenous EBR-Induced Drought Tolerance in Tea Plants. J Agric Food Chem. 2022;70(41):13391\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Liu Y, Jiang Y, Li A, Cheng B, Wu J. OsASR6 Enhances Salt Stress Tolerance in Rice. Int J Mol Sci. 2022;23(16):9340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamran M, Parveen A, Ahmar S, Malik Z, Hussain S, Chattha MS, Saleem MH, Adil M, Heidari P, Chen JT. An Overview of Hazardous Impacts of Soil Salinity in Crops, Tolerance Mechanisms, and Amelioration through Selenium Supplementation. Int J Mol Sci. 2019;21(1):148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa X, Feng F, Wei H, Mei H, Xu K, Chen S, Li T, Liang X, Liu H, Luo L. Genome-Wide Association Study for Plant Height and Grain Yield in Rice under Contrasting Moisture Regimes. Front. Plant Sci. 2016;7:1801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh V, Singh AP, Bhadoria J, Giri J, Singh J, T V V, Sharma PC. Differential expression of salt-responsive genes to salinity stress in salt-tolerant and salt-sensitive rice (Oryza sativa L.) at seedling stage. Protoplasma. 2018;255(6):1667\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JY, Lee SJ, Min WK, Cha S, Song JT, Seo H. S. COP1 controls salt stress tolerance by modulating sucrose content. Plant Signal Behav. 2022;17(1):2096784.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShakri T, Che-Othman MH, Md Isa N, Sukiran NL, Zainal Z. Morpho-Physiological and Stress-Related Gene Expression of Rice Varieties in Response to Salinity Stress at Early Vegetative Stage. Agriculture. 2022;12:638.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhanger MA, Aziz U, Alsahli AA, Alyemeni MN, Ahmad P. Influence of Exogenous Salicylic Acid and Nitric Oxide on Growth, Photosynthesis, and Ascorbate-Glutathione Cycle in Salt Stressed Vigna angularis. Biomolecules. 2019;10(1):42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParvin K, Nahar K, Hasanuzzaman M, Bhuyan MHMB, Mohsin SM, Fujita M. Exogenous vanillic acid enhances salt tolerance of tomato: Insight into plant antioxidant defense and glyoxalase systems. Plant Physiol Biochem. 2020;150:109\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad P, Tripathi DK, Deshmukh R, Singh VP, Corpas FJ. Revisiting the role of ROS and RNS in plants under changing environment. Environ Exp Bot. 2019;161:1\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDecros G, Baldet P, Beauvoit B, Stevens R, Flandin A, Colombi\u0026eacute; S, Gibon Y, P\u0026eacute;triacq P. Get the Balance Right: ROS Homeostasis and Redox Signalling in Fruit. Front Plant Sci. 2019;10:1091.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMittova V, Guy M, Tal M, Volokita M. Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii. J Exp Bot. 2004;55(399):1105\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllouzi H, Oueslati S, Hessini K, Rabhi M, Abdelly C. Seed-priming with H2O2 alleviates subsequent salt stress by preventing ROS production and amplifying antioxidant defense in cauliflower seeds and seedlings. Sci Hortic. 2021;288:110360.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Guo Y. Unraveling salt stress signaling in plants. J Integr Plant Biol. 2018;60(9):796\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Yang T, Lin Z, Gu B, Xing C, Zhao L, Dong H, Gao J, Xie Z, Zhang S, Huang X. A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation. Plant Biotechnol J. 2019;17(9):1770\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Sun X, Chang C, Jia D, Wei Z, Li C, Ma F. Dopamine alleviates salt-induced stress in Malus hupehensis. Physiol Plant. 2015;153(4):584\u0026ndash;602.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Guo Y. Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytol. 2018;217(2):523\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuteja N, Tarique M, Trivedi DK, Sahoo RK, Tuteja R. Stress-induced Oryza sativa BAT1 dual helicase exhibits unique bipolar translocation. Protoplasma. 2015;252(6):1563\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang HE, Ho MH, Chang H, Chao HY, Ger MJ. Overexpression of plant ferredoxin-like protein promotes salinity tolerance in rice (Oryza sativa). Plant Physiol Biochem. 2020;155:136\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Zhang Q, Qin J, Xiao G, Zhu S, Hu T. OsLEA1a overexpression enhances tolerance to diverse abiotic stresses by inhibiting cell membrane damage and enhancing ROS scavenging capacity in transgenic rice. Funct Plant Biol. 2021;48(9):860\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rice, OsDUF6, Salt stress, Functional analysis, Overexpression","lastPublishedDoi":"10.21203/rs.3.rs-3852076/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3852076/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDufulin is a chemical immune activator in rice plant. Soil salinity is one of the main environmental stresses in rice production. When plants are exposed to salt stress, a range of cellular equilibria will be disrupted. Previous studies have shown that Dufulin has a positive effect on salt tolerance in rice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we studied the mechanism of Dufulin in response to salt stress. Based on the transcriptome analysis of Dufulin in the process of salt tolerance in rice, we selected the OsDUF6 protein located on the cell membrane and studied its molecular function by overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e. The results showed that the salt-induced decreases in root, stem, and leaf length and increased leaf yellowing rate and Na\u003csup\u003e+\u003c/sup\u003e concentration in the wild-type plant were improved in the overexpressed lines, and increased the enzyme activity of the SOD, POD, CAT and PAL. \u003cem\u003eOsDUF6\u003c/em\u003e played a positive role in Na\u0026thinsp;+\u0026thinsp;transport by comparing the growth of the salt-sensitive yeast mutant complemented with \u003cem\u003eOsDUF6\u003c/em\u003e. In addition, RT-qPCR analysis confirmed that the overexpression of \u003cem\u003eOsDUF6\u003c/em\u003e significantly changed the expression level of genes related to growth and stress tolerance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCombined with previously published data, our results supported that \u003cem\u003eOsDUF6\u003c/em\u003e is an important functional factor of Dufulin to promote salt stress resistance in rice and plays a role in promoting salt tolerance in rice.\u003c/p\u003e","manuscriptTitle":"Dufulin activates OsDUF6 protein against salt stress in rice plant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 20:55:34","doi":"10.21203/rs.3.rs-3852076/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-19T21:50:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-31T15:31:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4af559ae-ce83-4ed4-ac03-dab65ea88f04","date":"2024-01-24T12:24:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49469c04-b858-468c-b548-ecb890eb1239","date":"2024-01-23T05:56:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-17T12:07:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-16T08:24:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-16T08:24:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-01-11T02:49:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f72f18f-5681-44fe-bad3-1ecedbe4b22c","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-01T15:10:05+00:00","versionOfRecord":{"articleIdentity":"rs-3852076","link":"https://doi.org/10.1186/s12870-024-04921-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-03-26 15:01:07","publishedOnDateReadable":"March 26th, 2024"},"versionCreatedAt":"2024-01-17 20:55:34","video":"","vorDoi":"10.1186/s12870-024-04921-z","vorDoiUrl":"https://doi.org/10.1186/s12870-024-04921-z","workflowStages":[]},"version":"v1","identity":"rs-3852076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3852076","identity":"rs-3852076","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}