Overexpression of OsDUF846.2 enhances the sensitivity of rice to salt and heat stresses

Abstract

Soil salinity and heat stress are major abiotic stress factors restricting rice growth, development, and yield potential. DUFs (Domains of Unknown Function) are proteins with structurally conserved but functionally undefined domains, widely present across organisms. The DUF846 family has been demonstrated to participate in plant growth and development as well as trans-Golgi network sorting and secretion. However, their functional roles in abiotic stress tolerance responses in rice remain poorly understood. In this study, we focused on OsDUF846.2, a member of the rice DUF846 family. Our investigation revealed that OsDUF846.2 responds to both salt and heat stress in rice. Following salt and heat stress treatments, OsDUF846.2 overexpression lines exhibited more severe damage, lower survival rates, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) accumulation, reduced antioxidant enzyme activities, and decreased proline and soluble sugar contents compared to wild type (WT). Transcriptome analysis indicated that OsDUF846.2 may enhance the sensitivity of rice to salt stress and heat stress by regulating salt stress-related pathways such as cytoskeletal stability and antioxidant defense system, and heat stress-related pathways such as protein homeostasis maintenance and RNA metabolism. These findings indicate that OsDUF846.2 negatively regulates the response of rice to salt stress and heat stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07426-5.

Introduction

Rice (Oryza sativa L.), one of the world’s three major cereal crops, is highly sensitive to climate change. It is highly susceptible to various abiotic stresses, including drought, salinity, extreme temperatures, and heavy metal contamination [1], Salinity stress poses a severe threat to plant growth, development, and crop productivity [2], mainly by affecting three aspects: osmotic stress, ionic stress, and oxidative stress. Moderate salinity levels (4–8 dS/m) can decrease the yield of major crops such as maize, rice, soybean, and potatoes by more than 50% [3]. Similarly, the effects of heat stress on rice are increasingly emphasized. Under heat stress, rice experiences reduced photosynthetic efficiency, increased respiration rates, and impaired pollen development and pollen tube growth, ultimately resulting in significant declines in yield and grain quality [4]. Studies have shown that for every 1℃ rise in global average temperature, rice yields are reduced by an average of 3.2% [5]. Therefore, elucidating the response mechanisms of rice to these abiotic stresses is crucial for developing stress-resistant varieties and enhancing crop productivity.

Reactive oxygen species (ROS), including superoxide anion radicals (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and singlet oxygen (¹O₂), act as signaling molecules and play critical roles in various biological processes. Under abiotic stress, ROS levels in plants deviate from normal ranges, leading to redox imbalances that impair or even disrupt specific cellular functions. Low ROS levels trigger signaling pathways that alter normal plant metabolism, while excessive ROS accumulation induces oxidative damage to cells [6, 7]. Plants primarily counteract oxidative stress through endogenous defense systems, which include enzymatic antioxidants (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; glutathione peroxidase, GPX; glutathione S-transferase, GST; and peroxiredoxin, PRX) and non-enzymatic antioxidants (ascorbic acid, AsA; glutathione; flavonoids; carotenoids; and non-protein amino acids) [8–10]. The GhbZIP53-GhWRKY68-GhSODs module positively regulates salt tolerance by increasing SOD activity in cotton plants [11]. Mutation of OsQHB enhances salt tolerance in rice seedlings by increasing ROS-scavenging enzyme activity and reducing ROS and malondialdehyde (MDA) accumulation under salt stress [12]. OsEDS1, which confers heat stress tolerance, promotes H₂O₂ scavenging by stimulating catalase activity through the OsEDS1-catalase association [13].

DUFs protein families are characterized by conserved amino acid sequences and structurally uncharacterized protein domains [14]. Studies indicate that DUF families play significant roles in regulating plant growth, development, and responses to biotic and abiotic stresses [15]. For instance, overexpression of PdDUF266A in poplar increases cellulose content and enhances biomass yield [16]. DUF538 proteins influence plant growth and development while acting as stress-responsive regulators, often modulating phosphoinositide signaling, trichome development, endoplasmic reticulum-related stress responses, and potentially functioning as hydrolases in plants [17]. The DUF1644-containing OsSIDP366 gene may act as a regulator of PBs/SGs, positively regulating drought and salt stress responses in rice [18]. Although extensive studies have demonstrated the critical roles of DUF domains in regulating plant growth, development, and responses to biotic and abiotic stresses, research specifically focusing on the DUF846 family remains limited.

The DUF846 family (Pfam ID: PF05832) is widely distributed across eukaryotes and particularly prevalent in plants, especially in crops. Notably, it comprises two members in rice (Oryza sativa L.), three in Arabidopsis thaliana (Arabidopsis thaliana (L.) Heynh.), three in wheat (Triticum aestivum L.), one in barley (Hordeum vulgare L.), one in cucumber (Cucumis sativus L.), three in Soybean (Glycine max (L.) Merr.), one in Tomato (Solanum lycopersicum L.), six in White poplar (Populus tomentosa Carrière) and four in Maize (Zea mays L.).

Among which the Arabidopsis (Arabidopsis thaliana) ECHIDNA protein contains a DUF846 structural domain, which is a key mediator of the secretion function of the trans-Golgi network (TGN), and is involved in the secretion of various substances, for example, ECHIDNA interacts with green fluorescent seed 9 (gfs9) in Arabidopsis, which plays a key role in flavonoid accumulation and seed coat pigmentation by regulating the vesicular transport pathway and vesicle development [19]. Additionally, ECHIDNA has been found to be involved in the secretion of the cell wall components such as hemicellulose and pectin, the growth hormone transporter protein AUX1, and waxes [20–22]. Arabidopsis ech mutants have been shown to be defective in root and hypocotyl elongation, indicating their involvement in mediating cell expansion and elongation [23]. Furthermore, impaired TGN transporter in ech mutants leads to altered anther secretion, affecting downy layer and pollen wall development, as well as anther cleavage and pollen tube formation, thereby reducing male fertility [24]. In poplar (Populus spp.), the ECHIDNA gene was identified from the expression profile of the vascular cambium and is associated with secondary xylem formation [25]. Although there are many reports on the involvement of the ECHIDNA gene in plant growth and development, as well as in TGN sorting and secretion, little is known about their role in abiotic stress responses in rice.

In our present study, we found that the expression of OsDUF846.2 changes in response to treatment with various abiotic stress, is an important gene with respect to improving response to abiotic stresses. Analysis of the molecular function of OsDUF846 will allow us to confirm whether a gene conferring tolerance to salt stress and heat stress. Therefore, these studies can identify new salt-tolerance and heat-tolerance genes for exploitation in molecular breeding, and enrich our understanding of the rice salt and heat stress signaling network, including exploration of the DUF family.

Materials and methods

Bioinformatic analysis

Obtain the sequences of the OsDUF846.2 (LOC_Os05g06700) from NCBI and perform a cis-acting element analysis of the promoter sequence upstream of the start codon on a 1500 bp sequence region by using PlantCare (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Ensemble Plants (https://plants.ensembl.org/index.html) obtained all DUF846 protein sequences of rice, Arabidopsis (Arabidopsis thaliana (L.) Heynh.), wheat (Triticum aestivum L.), barley (Hordeumvulgare L.), cucumber (Cucumis sativus L.), soybean (Glycine max (L.) Merr.), tomato (Solanum lycopersicum L.), hairy poplar (Populus trichocarpa Torr. & Gray) and maize (Zea mays L.). The phylogenetic tree analysis was conducted using Mega, the motif analysis was performed on the MEME Suite (https://meme-suite.org/meme/tools/meme), and the image processing was carried out by TBtools.

Plants construction and growth condition

The T3-generation overexpression (OE) lines used in this study, with Nip (Oryza sativa L. subsp. Japonica cv. Nipponbare) as the genetic background, were provided by Wuhan Tianwen Biotechnology Co., Ltd. Seeds were soaked in water at 37℃ for 1 d, followed by sterilization with 1% NaClO solution for 0.5 h. After thorough rinsing, the seeds were transferred to germination trays and placed in a growth chamber. Uniformly growing seedlings were cultivated in a growth chamber using a modified Yosida medium [26] with alternating photoperiods of 28 °C/16 h (8000 lx light) and 22 °C/8 h (0 lx darkness) until reaching the three-leaf stage (approximately 2 weeks old). The modified nutrient solution formulation is provided in Supplementary Table 3.

GUS staining assay

The sequences of the OsDUF846.2 and that of the OsDUF846.2 protein and its homologs were obtained from the NCBI. The promoter sequence upstream of the start codon on a 1500 bp sequence region of OsDUF846.2 was cloned into pCAMBIA1305 vector and obtained proOsDUF846.2::GUS plants through Agrobacterium tumefaciens mediated genetic transformation by infection of non-transgenic plant [27]. Positive transgenic plants were screened using PCR and overexpression lines were assayed for expression using RT-qPCR. Tissue samples from heading-stage proOsDUF846.2::GUS plants and 5-day-old proOsDUF846.2::GUS seedlings treated with 50 µmol/L ABA, 20% PEG, 180 mmol/L NaCl, 42℃, or 4℃ for 10–12 h were collected. Samples were incubated in GUS staining solution (50 mmol/L NaPO₄ (pH 7.2), 5 mmol/L K₃Fe(CN)₆, 5 mmol/L K₄Fe(CN)₆, 0.1% (w/w) Triton-100 and 1 mmol/L X-Gluc) in the dark for 12 h. Subsequently, tissues were destained with 75% ethanol. GUS staining patterns were documented using a ZEISS stereomicroscope.

All of the primer sequences used for the vector construction and detection are listed in Supplemental Table 1.

Subcellular localization

The OsDUF846.2 full coding sequence (CDS), removed the stop condon, was cloned into the pCMBIA1300-35 S-GFP vector, followed by recombination to construct 35 S: OsDUF846.2-GFP, which was transformed into Agrobacterium tumefaciens EHA105 and stored at −80℃. The 35 S: OsDUF846.2-GFP fusion construct and an ER-localized marker were transiently co-expressed in Nicotiana benthamiana leaves via Agrobacterium tumefaciens strain EHA105-mediated transformation. After dark incubation for 48 h, fluorescence signals were visualized using an Olympus FV3000 confocal laser scanning microscope.

Stress treatments in OsDUF846.2 overexpression seedlings

The 14-day-old wild-type seedlings were subjected to 24 h stress treatments at 20% PEG, 180 mmol/L NaCl, 50 µmol/L ABA, 4℃ and 42℃, respectively. Samples were collected at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h after treatment initiation, followed by performing expression level analysis to explore the response to abiotic stress.

Seeds of WT and OE lines were sterilized and germinated in control (standard nutrient solution) or salt stress conditions (120 mmol/L NaCl-supplemented nutrient solution), germination rates were recorded every 24 h over a 7-day period. Three independent biological replicates were conducted, with 50 seeds per line evaluated in each replicate.

Shoot height of 3-day-old seedlings of WT and OE lines were measured after control conditions or salt stress (120mmol/L NaCl-supplemented nutrient solution) treatment for 5 days. Three independent biological replicates were conducted, with 16 seedlings per line evaluated in each replicate.

The three-leaf stage seedlings of WT and OE lines (approximately 14 d in standard nutrient solution) were treated with 180 mmol/L NaCl-supplemented nutrient solutions for 4 d, allowed by recovery for 10 d under normal growth conditions. Survival rates were quantified post-recovery. Three independent biological replicates were conducted, with 16 seedlings per line evaluated in each replicate. Three independent biological replicates were conducted, with each replicate comprising three pots. For each pot, 16 seedlings per line were evaluated.

Shoot height of 3-day-old seedlings of WT and OE lines were measured after control conditions (28℃/22℃ day/night cycle) or heat-stress (38℃/25℃ day/night cycle) treatment under a 16-h light/8-h dark photoperiod for 7 days. Three independent biological replicates were conducted, with 16 seedlings per line evaluated in each replicate.

The three-leaf stage seedlings of WT and OE lines (approximately 14 d in standard nutrient solution) were subjected to 45℃ for 33 h, allowed by recovery for 10 d under normal growth conditions. Survival rates were quantified post-recovery. Three independent biological replicates were conducted, with 16 seedlings per line evaluated in each replicate. Three independent biological replicates were conducted, with each replicate comprising three pots. For each pot, 16 seedlings per line were evaluated.

Seedlings of WT and OE lines were cultured in control conditions and 0.1 µmol/L GA₃-supplemented hydroponic solution for 7 days after germination, and their shoot height was measured. Three independent biological replicates were conducted, with each replicate comprising three pots. For each pot, 10 seedlings per line were evaluated.

Measurement of the physiological and biochemical indicator

Aboveground tissue samples were collected from three-leaf-stage seedlings of WT and OE lines before and after treatment with 180 mmol/L NaCl solution for 2 days, as well as before and after treatment with 45 °C (heat stress) for 1 day. All experiments and analyses were performed using samples collected from the same batch. Three technical replicates were completed for each sample.

Frozen aerial tissue (0.2 g) was ground to powder in liquid nitrogen and homogenized in 3 mL of 100 mmol/L phosphate buffer (pH 7.8). The homogenate was centrifuged at 10,000 × g and 4℃ for 10 min. Enzyme activities of SOD (EC 1.15.1.1) and MDA content, were subsequently measured as described in the previous study [28, 29]. Proline content in the supernatant was determined using the acid-ninhydrin method, with proline concentration calculated based on a standard curve prepared with L-proline [30].

Soluble sugar content assay

The soluble sugar content was measured with modifications adapted from previous methods [31], three technical replicates were completed for each sample. Aerial tissue samples (0.1 g) were ground to powder in liquid nitrogen, mixed with 1.5 mL of distilled water, and incubated in a boiling water bath for 20 min. After cooling, the homogenate was filtered and diluted to 40 mL with distilled water. A 1 mL aliquot of the extract was mixed with 5 mL of anthrone reagent (prepared in 80% concentrated sulfuric acid), boiled for 10 min, cooled, and absorbance was measured at 620 nm.

Electrolyte leakage rate measurement

Aerial tissue samples (0.1 g) were cut into segments, rinsed with deionized water, and incubated in 50 mL of deionized water at 25℃ with shaking at 200 r/min for 5 h. The initial electrolyte leakage (R1) was determined using a conductivity meter. Samples were then boiled for 30 min, cooled to room temperature, and the total electrolyte leakage (R2) was measured. The relative electrolyte leakage rate was calculated as: Relative electrolyte leakage rate (%) = (R1/R2) × 100 [32]. Three technical replicates were completed for each sample.

Staining

As previously described [33], nitroblue tetrazolium (NBT) and 3’-diaminobenzidine (DAB) were subsequently measured after salt stress and heat stress, respectively. Using Evans Blue as described [34], leaves were immersed in 0.25% Evans Blue solution in the dark for 24 h. After decolorizing with absolute ethanol, observe them under a ZEISS stereomicroscope.

Agronomic trait measurements

Six plants per row were randomly selected from field-grown rice plants to measure plant height and tiller number. For each line, the main panicle was evaluated to determine the seed-setting rate. A total of 500 grains per line were randomly selected and analyzed for grain length and width using an automatic rice seed analyzer (JLM-Mini1600, Shanghai, China). Grain weight was measured with an electronic balance (NJNONITALAB DNA233B, Nanjing, China). All measurements were performed in three independent biological replicates.

RNA extraction and RT-qPCR analysis

Total RNA was extracted using the TRIzol reagent method. For reverse transcription, 2 µg of total RNA was reverse-transcribed into cDNA using the HiFi Script All-in-one RT Master Mix for qPCR (CWBIO, China), and the cDNA was stored at −20℃ for subsequent analysis. RT-qPCR was performed with Super Star Universal SYBR Master Mix (CWBIO, China). The rice Ubiquitin gene (LOC_Os01g22490) was used as an internal reference to normalize gene expression levels. Obtain the standardized relative transcription levels by the 2^−ΔΔCT method [35]. Primer sequences for RT-qPCR are provided in Supplementary Table S2.

Transcriptome sequencing and RNA-seq analysis

14-day-old seedlings of WT and OE lines under control conditions, 180 mmol/L NaCl for 8 h, or 45℃ for 6 h were collected, six biological replicates per group, flash-frozen in liquid nitrogen, and stored at −80℃. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions. RSEM [36] was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DEGseq [35]. DEGs with |log2FC|≧1 and FDR < 0.001 (DEGseq) were considered to be significantly different expressed genes. GO (Gene Ontology) functional enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were carried out on the online platform of Majorbio Cloud Platform (www.majorbio.com) by Goatools and Python scipy software, respectively. The online platform of Majorbio Cloud Platform also was used to construct volcano plots and Venn diagram.

Statistical analysis

Data are showed as the mean c standard error of three or more independent replicates. Data were analyzed using Student’s t-test, One-way ANOVA or Two-way ANOVA. Significant differences were denoted as P < 0.05 (*) and P < 0.01 (**). GraphPad Prism was used to derive all significance text and graphical representation. Three technical replicates were completed for each sample.

Result

Bioinformatics analysis

Based on its classification as the second member of the DUF846 family, it comprises two members in rice, the gene was designated OsDUF846.2. Then using the PlantCARE online tool, a 1,520 bp promoter region upstream of the OsDUF846.2 start codon was analyzed. Multiple abiotic stress-related cis-acting regulatory elements were identified (Fig. 1A), including the CGTCA-motif (JA-responsiveness), GARE-motif (GA-responsive element), LTR (low-temperature-responsive element), MYB (drought-inducible element), W-box (WRKY transcription factor-binding site). These results indicate that the gene is inducible or regulated at the transcriptional initiation level by jasmonic acid (JA), gibberellin (GA), low temperature, drought and so on.

Fig. 1.

Promoter element analysis and protein sequence analysis. (A) The promoter elements analysis of OsDUF846.2. (B) A phylogenetic analysis on the DUF846 protein family of rice, Arabidopsis thalian, maize and Populus tomentosa. Red Box: OsDUF846.2

To investigate the evolutionary conservation of the OsDUF846.2 protein, we analyzed DUF846 family members from Rice (Oryza sativa L.), Arabidopsis (Arabidopsis thaliana (L.) Heynh.), Wheat (Triticum aestivum L.), Barley (Hordeum vulgare L.), Cucumber (Cucumis sativus L.), Soybean (Glycine max (L.) Merr.), Tomato (Solanum lycopersicum L.), White poplar (Populus tomentosa Carrière), and Maize (Zea mays L.). Phylogenetic analysis revealed strong sequence conservation among these orthologs (Fig. 1B).

Expression pattern of OsDUF846.2 and subcellular localization

To investigate the tissue-specific expression of OsDUF846.2, GUS histochemical staining was performed on various tissues of proOsDUF846.2::GUS transgenic plants at the booting stage. Staining was observed in stem, leaf, sheath, ligule, stem internode, glume, and anther, with varying intensities (Fig. 2A). RT-qPCR analysis further confirmed its transcriptional activity across tissues, with the highest expression levels detected in spikelet (Fig. 2B), consistent with the GUS staining results. These findings indicated that OsDUF846.2 was ubiquitously expressed in rice tissues.

Fig. 2.

OsDUF846.2 expression pattern analysis. (A) GUS staining results of different tissue sites for stem, leaf, sheath, ligule, stem internode, glume, and anther. (B) Relative expression levels of different tissue sites. (C) GUS staining of roots of proOsDUF846.2::GUS transgenic plants under abiotic stresses. Student’s t-test was used to calculate the P values (n = 3), *P < 0.05, and **P < 0.01. The error bars represent the mean ± standard error (n = 3). Scale bars: 2 mm. (D) The expression levels of the OsDUF846.2 gene during 24 h of treatment with 120mmol/L and 180 mmol/L NaCl, 20% PEG, 4℃, 38℃, and 42℃, respectively. The error bars represent the mean ± standard error, One-way ANOVA test was used to calculate the P values (n = 3), *P < 0.05, and **P < 0.01

To assess its responsiveness to abiotic stress, 5-day-old proOsDUF846.2::GUS seedlings were treated with 20% PEG, 180 mmol/L NaCl, 42℃, or 4℃, respectively, and GUS staining was carried out 12 h later. Under control conditions, strong GUS staining was observed in roots. However, abiotic stress treatments induced distinct changes in staining patterns. Compared with the control, the staining in the root tissue was deepened after treatment, especially under 20% PEG, 4℃, and 42℃ treatments (Fig. 2C). These results indicated that OsDUF846.2 responded to multiple abiotic stresses.

Furthermore, we used RT-qPCR to detect the changes in the expression levels of the OsDUF846.2 gene during 24 h of treatment with 120mmol/L and 180 mmol/L NaCl, 20% PEG, 4℃, 38℃, and 42℃ (Fig. 2D). We found its transcription level in expression of the gene under various conditions w.r.t. to the control sample at each time point was changed to varying degrees, from the minimum 0.03-fold under 20% (w/v) PEG6000 treatment to the maximum 12.48-fold under 180 mmol/L NaCl treatment. Besides, its highest level appeared at different stress time points when treated with 20% PEG, 4℃ and 42℃ treatment, which was 2.27-, 3.02- and 3.36-fold, respectively. And, its lowest level appeared at different stress time points when treated with, 120mmol/L and 38℃ treatment, which was 0.13- and 0.06-fold, respectively. These results showed that OsDUF846.2 may respond to multiple abiotic stresses, particularly to the salt treatment exhibiting the highest fold change in expression levels.

To investigate the subcellular localization of the OsDUF846.2-GFP fusion protein, we conducted a co-localization study using the endoplasmic reticulum (ER)-localized Marker-RFP in epidermal cells of tobacco plants. The results demonstrated that the fluorescence signals of OsDUF846.2-GFP and the ER marker showed significant overlap (Fig. 3), indicating that the OsDUF846.2-GFP fusion protein was localized to the endoplasmic reticulum.

Fig. 3.

Subcellular localization of the OsDUF846.2-GFP fusion protein in tobacco. Scale bars: 20 μm

Overexpression of OsDUF846.2 reduces salt tolerance in rice

Based on the analysis of GUS staining, relative transcript levels and pre-test results under different abiotic stresses, the present study hypothesized that the OsDUF846.2 overexpression lines might respond to salt stress. Therefore, in the background of Nipponbare, the OE lines were generated to study its biological function. Seven independent OsDUF846.2-overexpressing lines were confirmed by RT-qPCR, and three lines (OE1, OE8, OE10) with distinct expression levels (low, moderate, and high) for subsequent experiments. (Figure S1). In order to study the effect of OsDUF846.2 overexpression on the salt tolerance of rice, seeds of WT and overexpression lines were treated in a NaCl solution containing 120 mmol/L (simulated moderate salt stress). The results showed that the overexpression lines had a much slower germination rate and a lower final germination rate than WT (Fig. 4A). To ensure normal seedling growth, only moderate salt stress (120 mmol/L NaCl) was simulated in the growth phenotyping experiments. Therefore, 2-day-old overexpression lines and WT seedlings were incubated in 0 and 120 mmol/L NaCl solutions for 5 d, and seedling heights were measured. The results showed that in the control group, the seedling height of OsDUF846.2 overexpression seedlings was significantly lower than that of WT. Under 120 mmol/L NaCl treatment, seedling height of overexpression lines was significantly more inhibited than WT (Fig. 4B, C).

Fig. 4.

Tolerance analysis of OsDUF846.2 overexpression lines under salt stress. (A) Germination rate of WT and OsDUF846.2 overexpression lines under normal condition and 120 mmol/L NaCl treatment. (B, C) Growth of seedlings of WT and OE lines under control conditions and salt stress. Two-way ANOVA test were used to calculate the P values (n = 3), **P < 0.01. Scale bars: 3 cm. (D, E) The survival rates of WT and OE lines under salt stress. The error bars represent the mean ± standard error, One-way ANOVA test were used to calculate the P values (n = 3), **P < 0.01. Scale bars: 3 cm

Severe salt stress (180 mmol/L NaCl) was simulated specifically for the statistical analysis of seedling survival rates. 14-day-old WT and OsDUF846.2 overexpression seedlings were placed in culture medium containing 180 mmol/L NaCl for 4 d until the leaves were chlorotic. Subsequently, they were placed in normal nutrient solution for 7 d, and the survival rate was counted. After salt stress treatment, the OsDUF846.2 overexpression lines showed more severe wilting compared with the WT, indicating more severe salt damage. After recovery, the survival rate of the overexpression lines was significantly lower than that of the WT (Fig. 4D, E). In conclusion, overexpression of OsDUF846.2 enhances the sensitivity of rice seedlings to salt stress.

Overexpression of OsDUF846.2 affects the accumulation of osmoregulatory substances and ROS levels under salt stress

To investigate the extent of damage to the cell membrane of the overexpression line under salt stress, the leaves of WT and OsDUF846.2 overexpression lines were stained with Evans blue before and after treatment with 180 mmol/L NaCl. Changes in the MDA content as well as relative electrolyte leakage rates were also measured. After salt stress treatment, the leaves of the overexpression line showed deeper staining than those of WT (Fig. 5A). Additionally, the MDA content and relative electrolyte leakage rate were significantly higher in the overexpression line compared to WT (Fig. 5B, C).

Fig. 5.

Analysis of cell membrane damage, osmotic substance synthesis, and accumulation and scavenging of ROS in WT and OE lines under salt stress. (A) Evans blue staining. (B) MDA content. (C) Relative electrolyte leakage. (D) proline content. (E) Soluble sugar content. (F). NBT staining. (G) O₂₋ content. (H) DAB staining. (I) SOD activity. The error bars represent the mean ± standard error, Two-way ANOVA test was used to calculate the P values (n = 3), **P < 0.01. Scale bars: 4 mm

To understand the role of OsDUF846.2 in osmoregulation, the accumulation of proline and soluble sugar were measured in the overexpression lines under salt stress. After 180 mmol/L NaCl treatment, the proline content and soluble sugar content of the overexpression line were significantly lower than those of WT (Fig. 5D, E).

Hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) are important components of ROS. To investigate the accumulation of ROS in OsDUF846.2 overexpression seedlings under salt stress, in this study, we stained the leaves of each strain under normal conditions and after treatment with 180 mmol/L NaCl using DAB and NBT. After salt treatment, more blue and tan deposits appeared on the surface of the leaves of the overexpression lines (Fig. 5F, H), indicating that the leaves of the overexpression lines accumulated more O₂⁻ and H₂O₂ than the WT. Further determination of the O₂⁻ content revealed that the O₂⁻ content of the leaves of the overexpression lines was significantly higher than that of the WT after salt treatment (Fig. 5G). Additionally, SOD activity in the overexpression lines were lower than that in the WT after salt stress (Fig. 5I).

The aforementioned results demonstrated that the expression of OsDUF846.2 in rice caused a more significant impairment of cell membranes under salt stress conditions. Concurrently, the expression of OsDUF846.2 affected the osmoregulatory process and ROS accumulation in rice under salt stress, leading to enhanced salt sensitivity.

Overexpression of OsDUF846.2 reduces heat tolerance in rice

In this study, we found that OsDUF846.2 gene responded to heat stress to some extent in addition to salt stress. To ensure normal seedling growth, only moderate heat stress (<40 °C) was simulated in the growth phenotyping experiments. To investigate the growth of overexpressed seedlings under heat stress, overexpression lines and WT seeds were germinated for 3 d and incubated at 38 °C for 7 d before counting the height of seedlings. In the control group, the seedling height of OsDUF846.2 overexpression lines was significantly lower than that of WT. Under the 38℃ treatment, the height of all seedlings was suppressed, but the overexpression line showed a significantly greater suppression compared to WT (Fig. 6A, B). In this study, we also simulated severe heat stress (45 °C) specifically to examine the survival rate of overexpressed seedlings under heat stress. 14-day-old seedlings of each strain were treated in an incubator at 45 °C for 33 h. The overexpressed lines showed more severe wilting and damage and had a significantly lower survival rate than the WT after heat stress treatment compared with the WT (Fig. 6C, D). In conclusion, overexpression of OsDUF846.2 increased the sensitivity of rice seedlings to heat stress.

Fig. 6.

Heat stress tolerance analysis of WT and OE lines. (A, B) Growth phenotype of seedlings of WT and OE lines under heat stress. Two-way ANOVA test were used to calculate the P values (n = 3), **P < 0.01. Scale bars: 3 cm. (C, D) Tolerance of OsDUF846.2 overexpression lines to heat stress. The error bars represent the mean ± standard error, One-way ANOVA test were used to calculate the P values (n = 3), **P < 0.01. Scale bars: 3 cm

Overexpression of OsDUF846.2 affects the accumulation of osmoregulatory substances and ROS levels under heat stress

Membrane thermal stability is an important indicator of heat tolerance. Evans Blue staining shows, after heat stress treatment, the leaves of the overexpression lines exhibited deeper staining than WT (Fig. 7A). The MDA content and relative electrolyte leakage rate were also significantly higher in the overexpression lines than in the WT (Fig. 7B, C). These results indicated that the cell membranes of OsDUF846.2 overexpression lines sustained greater damage under heat stress compared to WT.

Fig. 7.

Analysis of cell membrane damage, osmotic substance synthesis, and accumulation and scavenging of ROS in overexpression lines under heat stress. (A) Evans blue staining. (B) MDA content. (C) Relative electrolyte leakage. (D) proline content. (E) Soluble sugar content. (F) NBT staining. (G) O₂₋ content. (H) DAB staining. (I) SOD activity. The error bars represent the mean ± standard error, Two-way ANOVA test was used to calculate the P values (n = 3), *P < 0.05, and **P < 0.01. Scale bars: 4 mm

To understand the role of OsDUF846.2 in osmoregulation, the accumulation of proline and soluble sugar in the overexpression lines under normal conditions and after heat stress treatment was determined in this study. After 45℃ treatment, the proline and soluble sugar contents in the overexpression lines were significantly lower than those in the WT (Fig. 7D, E). These results suggested that OsDUF846.2 modulated rice heat stress tolerance by affecting osmoregulation.

Similar to salt stress, heat stress also leads to the accumulation of ROS. In this study, the leaves of each strain were stained with NBT and DAB under normal conditions and after 45℃ treatment to observe ROS accumulation. After heat stress treatment, more blue and tan deposits were observed on the leaves of the overexpression lines, indicating higher accumulation of O₂⁻ and H₂O₂ compared to WT (Fig. 7F-H). Changes in SOD activity were detected in overexpressed seedlings before and after 45℃ treatment. SOD activity in the overexpression lines significantly lower than that in the WT after heat stress treatment (Fig. 7I). These results suggested that the overexpression strain accumulated more ROS and reduced the ability to scavenge ROS under heat stress.

Analysis of differentially expressed genes in seedlings of OsDUF846.2 overexpression lines

Overexpression of OsDUF846.2 significantly alters the expression of various genes, potentially affecting plant stress responses. To investigate the effect of overexpression of OsDUF846.2 on the gene network, RNA was extracted from above-ground parts of WT, OsDUF846.2-OE1 and OsDUF846.2-OE8 plants at the three-leaf stage, and transcriptome sequencing was performed. Compared to WT, under normal conditions, 1832 differentially expressed genes (DEGs) were detected in the OsDUF846.2-OE1 line, while 8629 DEGs were detected in the OsDUF846.2-OE8 line (Figure S2A, B). Among these, 911 DEGs were co-expressed in both overexpression lines (Fig. 8A). Gene ontology (GO) functional annotation of these 911 DEGs was conducted to better understand the effect of OsDUF846.2 overexpression on stress response. GO enrichment analysis revealed that these DEGs were mainly enriched in the following categories: “extracellular region”, “oxidoreductase activity”, “heme binding” and “apoplast” (Fig. 8B). Further analysis revealed that genes involved in the expansion and construction of the cell wall were enriched in the “extracellular region” category (Figure S2C), while genes associated with gibberellin biosynthetic process were enriched in the “oxidoreductase activity” category (Fig. 10C). Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these DEGs were involved in pathways such as “diterpenoid biosynthesis”, “DNA replication”, “flavone and flavonol biosynthesis”, and “motor proteins” pathways (Fig. 8C). Subsequently, the expression changes of these 911 DEGs under heat and salt stress conditions were investigated. Under salt stress conditions, among the 911 DEGs, 102 genes were significantly up-regulated and 363 genes were significantly down-regulated in WT (Figure S2D), and the expression patterns of the 911 DEGs in OE1 were consistent with those in WT (Fig. 8D). And under heat stress conditions, 167 significantly up-regulated genes and 441 significantly down-regulated genes were identified in WT (Figure S2E). Interestingly, the expression patterns of the 911 DEGs in OE8 exhibited an opposite trend compared to those in WT (Fig. 8E).

Fig. 8.

RNA-seq analysis of WT and OE lines under normal conditions. (A) Venn diagram of DEGs detected in OsDUF846.2-OE1 and OsDUF846.2-OE8 lines under normal conditions. (B) GO enrichment analysis results of DEGs co-expressed in OE lines under normal conditions. (C) KEGG enrichment analysis results of DEGs co-expressed in OE lines under normal conditions. (D) Heatmap of gene expression profiles for DEGs co-expressed in OE lines under normal conditions, under salt stress. (E) Heatmap of gene expression profiles for DEGs co-expressed in OE lines under normal conditions, under heat stress

Fig. 10.

Dwarfing phenotype analysis of OsDUF846.2 overexpression. (A, B) Growth phenotype of WT and OE lines under control conditions and exogenous GA₃ treatment. (C) GO analysis of DEGs, co-expressed in OE lines under normal conditions, enriched in the category of “oxidoreductase activity”. (D) Relative expression levels of OsGA2ox5, OsGA2ox6, OsGA2ox9 and OsGA20ox2 in each line. The error bars represent the mean ± standard error, the student’s t-test was used to calculate the P values (n = 6), *P < 0.05, and **P < 0.01. Scale bars: 3 cm

These results indicated that overexpression of OsDUF846.2 influenced the expression of genes involved in growth and development, environmental adaptation, defense responses, the synthesis and regulation of secondary metabolites, and abiotic stress response.

OsDUF846.2 modulates agronomic traits and growth in rice

In this experiment, the agronomic traits of OsDUF846.2 overexpression lines were determined. At the maturity stage, the overexpression lines exhibited significantly reduced plant height, seed set rate, and main panicle length compared to WT; however, the overexpression lines produced a higher number of effective tillers than WT (Fig. 9A-E). Meanwhile, the grain lengths of three overexpression lines of OsDUF846.2, were lower than that of WT, and the grain widths were also reduced in all overexpression lines (Fig. 9F-H). Furthermore, the thousand-grain weights of the overexpression lines were all lower than that of WT (Fig. 9I). These findings indicated that OsDUF846.2 overexpression negatively impacted rice yield-related traits.

Fig. 9.

Agronomic trait analysis of OsDUF846.2. (A) Plant height. (B) Seed setting rate. (C) Tiller number. (D) Panicle length of main spike. (E) Spike phenotype. (F) Grain characters. (G) Grain length. (H) Grain width. (I) 1000-grain weight. The error bars represent the mean ± standard error, the student’s t-test was used to calculate the P values (n = 6), *P < 0.05, and **P < 0.01. Scale bars: E: 3 cm, F: 1 cm

Transcriptome sequencing analysis revealed that genes involved in gibberellin biosynthetic process were significantly enriched in the “oxidoreductase activity” category of OsDUF846.2 overexpression lines (Fig. 10C). To investigate whether the dwarfing phenotype of OsDUF846.2 overexpression was due to reduced endogenous gibberellin (GA) content, the effects of exogenous GA₃ on the shoot length of OsDUF846.2 transgenic plants were explored. The seedlings of WT and OE lines were cultured in control conditions and 0.1 µmol/L GA₃-supplemented hydroponic solution for 7 days after germination respectively and the shoot height was measured. Under normal conditions, the shoot height of OE lines seedlings was significantly lower than that of WT. But with 0.1 µmol/L GA₃ treatment, the shoot height of all seedlings increased and no significant difference in shoot height was observed between the WT and OE lines (Fig. 10A, B). The result suggested that the dwarf phenotype of OsDUF846.2 overexpression plants may result from a defect in endogenous gibberellin (GA) content.

And then, the transcript levels of GA biosynthesis and inactivation genes were analyzed in overexpression lines and WT using RT-qPCR. The expression level of the late GA biosynthesis gene OsGA20ox2 was significantly down-regulated in the OE lines compared to WT; meanwhile, the expression of GA inactivation-related genes OsGA2ox5, OsGA2ox6, and OsGA2ox9 were significantly up-regulated in the overexpression lines (Fig. 10D).

Therefore, OsDUF846.2 affecting rice plant height by regulated GA content.

Analysis of differentially expressed genes in OsDUF846.2 overexpressing rice plants under salt stress at seedling stage

To further elucidate the salt stress response mechanism of OsDUF846.2, OsDUF846.2-OE1, which exhibited the most stable phenotype under salt stress, was selected for the reliability of data significance and transcriptome sequencing analysis was performed on WT and OsDUF846.2-OE1 plants under salt stress (180 mmol/L NaCl). Under salt stress conditions, 568 genes were up-regulated while 1756 genes were down-regulated in the OsDUF846.2-OE1 line compared to WT (Fig. 11A). GO analysis of the 568 up-regulated DEGs revealed that biological processes such as “lipid biosynthetic process”, “lipid metabolic process” and “monocarboxylic acid metabolic process” were significantly enriched (Fig. 11B) and KEGG pathway analysis identified predominant enrichment of metabolic pathways, including “fatty acid elongation”, “sesquiterpenoid and triterpenoid biosynthesis” and “biosynthesis of various plant secondary metabolites”, in these DEGs (Fig. 11C). Meanwhile, GO analysis of the 1756 downregulated DEGs revealed enrichment in pathways such as “extracellular region”, “apoplast”, “polymeric cytoskeletal fibers”, “heme-binding”, “microtubule”, and “oxidoreductase activity” (Fig. 11D). KEGG enrichment analysis showed that these DEGs were enriched in pathways including “motor proteins”, “phenylpropanoid biosynthesis”, “cutin, suberine and wax biosynthesis”, “diterpenoid biosynthesis” and “starch and sucrose metabolism” pathways (Fig. 11E). These findings indicated that the genes were involved in pathways affecting cytoskeleton stability, redox balance, energy metabolism, secondary metabolite synthesis and phytohormone metabolism. Under salt stress, overexpression of OsDUF846.2 inhibited cell division and elongation, increased oxidative damage, insufficient energy supply, and reduced synthesis of defensive compounds by regulating these DEGs, thereby impairing salt tolerance and defense capacity in rice.

Fig. 11.

RNA-seq analysis of WT and OE lines under salt stress. (A) Volcanic map of DEGs detected in the OsDUF846.2-OE1 line under salt stress. (B, C) GO and KEGG enrichment analysis of up-regulated DEGs detected in OsDUF846.2-OE1 line under salt stress. (D, E). GO and KEGG enrichment analysis of down-regulated DEGs detected in OsDUF846.2-OE1 line under salt stress. (F) Relative expression levels of OsTUB1, OsTUB6, OsPRX38, OsGL1-6, OsNHX1, OsLEA3, and OsHKT1;4 in each line under salt stress. The error bars represent the mean ± standard error, the student’s t-test was used to calculate the P values (n = 3), *P < 0.05, and **P < 0.01

To confirm the results of RNA-seq analysis, OsTUB1, OsTUB6, OsPRX38 and OsGL1-6 were selected for RT-qPCR analysis. Under salt stress, these genes were down-regulated in the OsDUF846.2 overexpression lines, consistent with the RNA-seq findings (Fig. 11D-G). In addition, the expression of stress-related genes, such as OsNHX1, OsLEA3, and OsHKT1;4, was also detected, and these genes were also down-regulated in the OsDUF846.2 overexpression lines under salt stress (Fig. 11F). Overall, RNA-seq analysis revealed that OsDUF846.2 regulated salt tolerance in rice by modulating cytoskeletal stability, genes related to redox homeostasis, and other salt-response-related genes.

Analysis of differentially expressed genes in OsDUF846.2 overexpressing rice plants under heat stress at seedling stage

To further investigate the heat stress response mechanism of OsDUF846.2, OsDUF846.2-OE8, which exhibited the most stable phenotype under heat stress, was selected for the reliability of data significance and transcriptome sequencing analysis was performed on WT and OsDUF846.2-OE8 plants under heat stress (45℃). Under heat stress conditions, 4546 genes were up-regulated, while 3122 genes were down-regulated in the OE8 line compared to WT (Fig. 12A). GO analysis of the 4546 up-regulated DEGs revealed that cellular component such as “cellular anatomical entity”, “cellular_component” and “plasma membrane” were significantly enriched (Fig. 12B) and KEGG pathway analysis identified predominant enrichment in pathways, including “DNA replication”, “biosynthesis of various plant secondary metabolites” and “carotenoid biosynthesis”, in these DEGs (Fig. 12C). Meanwhile, GO analysis of the 3122 down-regulated DEGs showed enrichment in pathways such as “protein folding”, “RNA metabolic processes”, “response to temperature stimulus”, “response to heat”, “RNA processing” and “intracellular organelle” (Fig. 12D). KEGG enrichment analysis further revealed that these DEGs were enriched in pathways including “protein processing in the endoplasmic reticulum”, “ribosome biogenesis in eukaryotes”, “RNA polymerase”, “nucleotide excision repair”, “carotenoid biosynthesis” and “glutathione metabolism” (Fig. 12E). These results indicated that these genes were involved in pathways affecting protein folding, DNA repair, RNA metabolism, antioxidant capacity and intracellular organelle function. Under heat stress, overexpression of OsDUF846.2 affected rice heat stress tolerance by regulating the expression of these DEGs, which related to protein misfolding, aberrant RNA metabolism, decreased DNA repair, decreased antioxidant capacity, impaired organelle function, and metabolic and signaling disorders.

Fig. 12.

RNA-seq analysis of WT and OE lines under heat stress. (A) Volcanic map of DEGs detected in the OsDUF846.2-OE8 line under heat stress. (B, C) GO and KEGG enrichment analysis of up-regulated DEGs detected in OsDUF846.2-OE1 line under heat stress. (D, E). GO and KEGG enrichment analysis of down-regulated DEGs detected in OsDUF846.2-OE1 line under heat stress. (F) Relative expression of OsHSP17.0, OsHSP24.1, OsHSP74.8, Oshsp82A, and OsXrn3 in each line under heat stress. The error bars represent the mean ± standard error, the student’s t-test was used to calculate the P values (n = 3), *P < 0.05, and **P < 0.01

To confirm the results of RNA-seq analysis, OsHSP17.0, OsHSP24.1, OsHSP74.8, Oshsp82A and OsXrn3 were selected for RT-qPCR. Under heat stress, these genes were down-regulated in the OsDUF846.2 overexpression lines, which was consistent with the RNA-seq results (Fig. 12F). Overall, the RNA-seq analysis showed that OsDUF846.2 regulated heat tolerance in rice by affecting genes related to protein folding and DNA repair.

Discussion

Rice, as a stationary plant species, can only respond to environmental biotic and abiotic stresses by regulating its internal mechanisms. Crop yields are reduced by abiotic stresses, with soil salinization being a major factor [37]. In addition, with the rise in global temperatures due to industrialization and economic development, the frequency of heat stress events has increased, posing a major challenge to rice production [38]. Understanding the mechanisms by which rice responds to these abiotic stresses is therefore essential for the development of resistant varieties and the enhancement of crop yields.

In recent years, the role of DUF family genes in salt stress and heat stress has been identified in many different plant species. Silencing of GhDUF4228-67 reduced salt tolerance in cotton [39]. Overexpression of GmCBSDUF3, which contains an unknown functional domain (DUF21), increased tolerance to drought and salt stress in Arabidopsis.34 Overexpression of the wheat DUF581 family gene, TaSRHP, in Arabidopsis enhances plant tolerance to salt stress [40]. High temperature-induced expression of wheat DUF860 family gene TaWTF1 overexpressed in Arabidopsis significantly increased plant survival under heat stress [41]. There are many reports on the involvement of DUF846 family members in plant growth and development as well as TGN sorting and secretion functions [19–25, 42], but there is little information on their role in response to abiotic stress responses in rice. In this study, we found that overexpression of OsDUF846.2 enhanced rice sensitivity to salt stress and heat stress and affected rice growth and development.

Our results revealed that OsDUF846.2 overexpression affected osmoregulation in rice under salt stress and heat stress. Both salt stress and heat stress lead to osmotic imbalance in plants, and proline and soluble sugars play important roles in osmoregulation by maintaining cellular osmotic balance and protecting cellular structures [43, 44]. Heat-tolerant tomato improves its heat tolerance by increasing the content of soluble sugars [45]. Increase in proline content in Arabidopsis had a significant effect on the heat tolerance of the plant [46]. In this study, we found that the proline and soluble sugar contents in OsDUF846.2 overexpression lines were significantly lower than those in non-transgenic under salt and heat stresses. Meanwhile, transcriptome enrichment analysis showed that genes related to “starch and sucrose metabolism” were down-regulated in the overexpression line of OsDUF846.2 under salt stress, such as alginate-6-phosphate phosphatase OsTPP2 and OsTPP10, which are important genes involved in alginate synthesis. Alginate enhances the antioxidant defense system and protects plants from salinity-induced oxidative damage [47]. The genes related to “arginine and proline metabolism” were down-regulated in the OsDUF846.2 overexpression line under heat stress, and among them, OsP5CR and OsP5CS2 are important genes for proline synthesis. Therefore, the results showed that OsDUF846.2 significantly impaired the osmotic regulation of cells in vivo by suppressing the expression of genes related to alginate synthesis and proline synthesis, resulting in cellular water loss and growth inhibition, and thus enhanced the sensitivity of rice to salt and heat stresses.

Meanwhile, OsDUF846.2 overexpression affects salt and heat tolerance in rice by regulating ROS homeostasis, and excessive salt and high temperatures impair the form and function of the plasma membrane, leading to an increase in its fluidity and permeability, affecting membrane integrity and enhancing organic and inorganic ion leakage from the cell [48]. MDA is a product of lipid peroxidation, and increased levels of which are usually associated with elevated levels of oxidative stress [49]. The significant increase in MDA content and ion leakage rate in OsDUF846.2 overexpression lines under salt and heat stress indicated that the integrity of their cell membranes was severely disrupted. The accumulation of reactive oxygen species and the reduction of SOD activity in leaves further confirmed that the overexpression lines suffered more severe oxidative damage under salt stress and heat stress. In addition, transcriptome enrichment analysis showed that genes related to “oxidoreductase activity” and “glutathione metabolism” were down-regulated in OsDUF846.2 overexpression lines under salt stress, including peroxidase (POD), ascorbate peroxidase (OsAPX7), glutathione S-transferase (GSTL1) and glutathione reductase (GR3). It was shown that SOD, APX and GR activities were increased under salt stress in leaves of maize salt tolerant genotype BR5033 [50]. Overexpression of ascorbate peroxidase OsAPXa or OsAPXb in rice increased tolerance to salt stress. Constitutive expression of OsGSTU4 in Arabidopsis also increased tolerance to salt stress [51]. The genes related to the pathways “phenylpropanoid biosynthesis” and “cutin suberine and wax biosynthesis” were down-regulated, and phenylacetone and suberin are important secondary metabolites in plants as potent antioxidants for scavenging harmful ROS accumulated under salt stress conditions [52]. The genes related to “glutathione metabolism” were down-regulated in OsDUF846.2 overexpression lines under heat stress, including OsGSTU5, OsRGST1, OsAPX1, and OsAPx3, which play an important role in the protection of plants against high temperature stress. APX reduces its toxicity by converting H₂O₂ to H₂O [53]. High expression of antioxidant enzymes in grapes and tomatoes reduced ROS damage to plants under high temperature conditions [54]. It has been shown that carotenoids capture light energy, scavenge oxygen radicals, and enhance plant tolerance to heat and light stress in plants [55, 56]. The genes of the “carotenoid biosynthesis” are down-regulated under heat stress, including 9-cis-epoxycarotenoid dioxygenases (OsNCED1, OsNCED3, OsNCED4) and β-carotene hydroxylase (OsBCH), which are involved in ABA and carotenoid synthesis, respectively, and play a role in maintaining ROS homeostasis [57]. Therefore, overexpression of OsDUF846.2 may increase the susceptibility of rice to salt and heat stress by affecting enzymatic antioxidant activity and antioxidant synthesis under salt and heat stress, which leads to the accumulation of ROS triggering oxidative damage.

In this study, further analysis of the transcriptome data under salt stress revealed that GO enrichment analysis showed that the down-regulated genes were enriched in the categories of “polymeric cytoskeletal fiber” and “microtubule” and the up-regulated genes were enriched in the categories of “lipid biosynthetic process” and “lipid metabolic process”. The stability of the cytoskeleton lipid metabolic is essential for maintaining cellular morphology and function [58, 59], and the plant cytoskeleton comprises the systemic polymers between actin filaments and microtubules [60, 61], Sustained overexpression of the Arabidopsis TUB9 gene in rice transgenic plants enhanced tolerance to salt stress [62]. Decreased transcript levels of related genes in OsDUF846.2 overexpression lines under salt stress may cause alterations in cytoskeletal stability, leading to disruption of cellular structure and function. KEGG enrichment analysis of down-regulated DEGs showed that the differentially expressed genes were enriched for “Cutin, suberine and wax biosynthesis”. Cutin and suberin are the polymer matrices for lipophilic cell wall barriers. These barriers play roles in protecting plants from biotic and abiotic stresses and in controlling plant morphology [63]. In addition, pathways such as “diterpenoid biosynthesis” and “starch and sucrose metabolism” were also enriched. What’s more, for up-regulated DEGs, KEGG pathway analysis identified predominant enrichment of “biosynthesis of various plant secondary metabolites”. These results suggest that OsDUF846.2 may affect the sensitivity of rice to salt stress by regulating cell structure, energy supply and secondary metabolism of plants.

Further analysis of transcriptomic data under heat stress revealed that GO analysis showed that DEGs down-regulated by OsDUF846.2 overexpression under heat stress were enriched for “protein folding”, “response to temperature stimuli”, “response to heat”, and studies have shown that molecular chaperones of HSPs can stabilize, regenerate, or degrade unfolded proteins, and play a crucial role in helping plant cells to cope with stress conditions, especially heat stress [64]. Overexpression of OsHSP1 increases heat tolerance in Arabidopsis [65], OsHSP101enables long-term acquired heat tolerance in Arabidopsis by forming a positive feedback loop with HSA32 [66]. In addition, pathways such as “RNA metabolism” and “RNA processing” have been enriched. Recent studies using RNA metabolism-related mutants have revealed that RNA processing, RNA decay and RNA stability play an important role in regulating gene expression at a post-transcriptional level in response to abiotic stresses [67]. In addition, KEGG enrichment analysis of down-regulated DEGs has revealed that the “eukaryotic ribosome biogenesis” and “RNA polymerase” pathways are enriched. Among them, key genes such as ribosome assembly factor (OsBMS1) and RNA polymerase subunits (OsRPA49, OsRPC4) were significantly down-regulated, resulting in blocked ribosome maturation [68], abnormal rRNA processing and reduced transcription efficiency. These results suggest that overexpression of OsDUF846.2 under heat stress enhances the sensitivity of rice to heat stress mainly through the down-regulation of related genes enriched in the pathways of protein homeostasis maintenance, RNA metabolism and ribosomal genesis. What’s more, the up-regulated DEGs, however, showed a comparable enrichment profile with those up-regulated DEGs induced by salt stress, predominantly clustering in cellular component-associated categories including “cellular anatomical entity”, “cellular_component” and “plasma membrane” by GO analysis and enriched in “biosynthesis of various plant secondary metabolites” by KEGG analysis. This result further suggests that OsDUF846.2 may affect plant tolerance to abiotic stress by regulating cellular structural components and secondary metabolism of plants.

In addition, OsDUF846.2 is involved in influencing the growth and development of rice. In this study, we constructed proOsDUF846.2::GUS plants and analyzed them by GUS histochemical staining, and found that its expression activity was significantly higher than that of other tissues in leaves and young spikes, suggesting that it may regulate the morphogenesis and reproductive development of the plant through spatiotemporal-specific expression patterns. The agronomic traits of OsDUF846.2 overexpression lines at maturity showed that the height of overexpression lines was significantly reduced, the number of tillers was increased, the length of spikes was shortened, the length and width of grains were reduced, and the fruiting rate was significantly reduced. Transcriptome enrichment analysis showed that many genes involved in catalyzing GA synthesis or metabolism, such as gibberellin 2 oxidase genes OsGA2ox5 and OsGA2ox9, in the OsDUF846.2 overexpression lines. Studies have shown that GA defects lead to plant dwarfism, while GA-deficient mutants produce defective anthers, leading to male sterility [69]. Meanwhile, the down-regulation of GA synthesis genes and up-regulation of catabolism genes in the overexpression strain were found by RT-qPCR, suggesting that OsDUF846.2 may be involved in the regulation of GA synthesis and metabolism. And the result of phenotypic assays under exogenous GA treatment suggested that the dwarf phenotype of OsDUF846.2 overexpression plants may result from a defect in endogenous gibberellin (GA) content. Therefore it was hypothesized that this gene might regulate rice growth and development by interfering with GA synthesis and metabolism.

Based on the above conclusions, future research will focus on: (i) investigating the alterations in cellular structural components and secondary metabolic pathways of OsDUF846.2 under salt stress; (ii) elucidating the molecular mechanisms by which OsDUF846.2 regulates the aforementioned pathways under heat stress; and (iii) exploring the impact of endogenous GA deficiency on plant hormone signaling pathways. These studies aim to achieve a deeper understanding of the molecular mechanisms underlying OsDUF846.2 function.

Conclusions

This study, through phenotypic experiments, physiological and biochemical assays, and transcriptomic analysis, preliminarily proposes the following hypotheses: (1) OsDUF846.2 may influence plant tolerance to abiotic stress by regulating cellular structural components and secondary metabolism; (2) under heat stress, OsDUF846.2 overexpression primarily affects rice sensitivity to heat stress by modulating pathways related to protein homeostasis maintenance, RNA metabolism, and ribosome biogenesis; (3) OsDUF846.2-overexpressing plants may potentially affects spider development through modulation of endogenous gibberellin (GA) content.

These studies enrich our understanding of the rice salt and heat stress signaling network, including exploration of the DUF family.

Abbreviations

DUF

domains of unknown function

ROS

reactive oxygen species

GFP

green fluorescent protein

GUS

β-glucuronidase

NBT

Nitrotetrazolium Blue Chloride

DAB

3’-diaminobenzidine

SOD

superoxide dismutase

MDA

malondialdehyde

DEGs

differentially expressed genes

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

Authors’ contributions

L.L. conceived and designed the experiments; J.Z. (Jiali Zhu) and Z.W. performed the experiments, and wrote the article; H.C., M.C., X.Z., C.M., J.K. and J.Y. analyzed the data, produce the figures; R.C, J.Z. (Jun Zhu) and J.Z. (Jianqing Zhu) provided support and experimental guidance for this study; X.J. and X.Y. provided assistance in the management of the experiment. All authors read and approved the final manuscript.

Funding

This work was supported by the Sichuan Science and Technology Program (2022ZDZX0012).

Data availability

The datasets generated and analysed during the current study are available in the Genome Sequence Archive (GSA) repository, [CRA027684]. And all other data generated or analyzed during this study are included in the manuscript or the NCBI database accession numbers are provided in the manuscript [and its Supplementary Table 1].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.