For OsGF14C overexpression vector construction, the coding region sequence of OsGF14C was amplified from the blast-resistant line BC10 using the primers in Supplemental Table 1 . The resulting product (843bp) was cloned into pEASY-T1 (TransGen) vector and verified by sequencing. The entry clone for overexpressing plants was then inserted into PHQSN (modified from pCAMBIA1390) which harbors a CaMV35S promoter. The positive plasmid was electroporated into Agrobacterium tumefaciens EHA105 and then introduced into calli of the cultivar Lijiangxintuanheigu (LTH) via Agrobacterium-mediated genetic transformation.

Total RNA was extracted with Trizol reagent (Invitrogen) and purified with NucleoSpin RNA Clean-up (MACHEREYNAGEL) according to the manufacturers’ instructions. RNA quality and quantity were assessed by formaldehyde denaturing agarose gel electrophoresis and spectrophotometry (Nanodrop-1000), respectively. The purified total RNA was reverse-transcribed using the Primescript™ RT reagent kit (Takara) to generate cDNA and real-time PCR was carried out using SYBR ExTaq™ (Takara). Actin gene was chosen as a reference gene. Gene expression was quantified by the comparative CT method. Experiments were performed in triplicate, and the results were presented by their means ± standard derivation (SD). Gene-specific primers used were listed in Supplemental Table 1 .

We got OsGF14C over expression plants by vector construction and rice transformation. The relative expression level of OsGF14C in every line was measured by quantitative real-time PCR. The seeds of T₀, T₁, and T₂ segregating progeny were used for salinity treatment. After incubation at 49°C for 96 h to break dormancy, seeds were soaked in water for 36 h and then incubated at 30°C for 48 h for pre-germination. The seeds that germinated uniformly were sown in cultural boxes with kimura nutrient mixture and grown in the tissue culture incubator for ~10 d. The kimura nutrient mixture was changed into new kimura nutrient mixture containing 120 mmol/L Nacl. After 7 d treatment, the culture solution was change back to normal kimura nutrient mixture without Nacl. After ~10 d recovery, the survival rate was calculated. The treatment was repeated three times.

Electrolyte leakage was measured using the method reported in our previous study (Dong et al., 2019). Briefly, at 0 h, 2 d, 3 d, 4 d before and after Nacl treatment, 0.1 g of leaf material was collected and washed several times in ultrapure water and then immersed in 20 mL ultrapure water for 24 h. The electrical conductivity of the resulting solution was measured and defined as R1. This solution was placed in boiling water for 20-30 min. After cooling, the electrical conductivity was measured and defined as R2. Relative electrolyte leakage (%) = (R1/R2)×100%. The experiment were repeated three times.

We got OsGF14C over expression plants by vector construction and rice transformation. Wild-type LTH plants and 15 lines of T₁ segregating progeny germinated from T₀ transgenic seeds were grown in soil in greenhouse. The relative OsGF14C transcription level was measured by quantitative real-time PCR. M. oryzae isolate GD08-T13 inoculum was used for blast resistance evaluation as described in our previous study (Dong et al., 2021). Briefly, when the plants grow to tillering stage, the leaves of the plants were inoculated with GD08-T13 using the punch method. The inoculated plants were sprayed with water for 2-3 min every 2 h to maintain the humidity. After 10 days inoculation, the lesion size of each leaf was measured. The correlation between the disease area and relative OsGF14C transcription was analysed in excel. The treatment was repeated three times.

We amplified the protein coding region of OsGF14C from BC10 using the primers in Supplemental Table 1 and cloned it into the pUC18 vector with the coding sequence for enhanced green fluorescent protein (eGFP) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter provided by Yang et al. (2014). Dehulled rice seeds of LTH were surface-sterilized using 1.5% sodium hypochlorite for 40 min, then germinated and cultured on half-strength Murashige and Skoog medium in the tissue culture room for ~10 d. Seedlings were grown hydroponically under natural light for 3 d. The protoplast isolation and transformation were performed following the method of Yang et al. (2014). After 24 h incubation at 28°C without light, the rice protoplasts were observed and photographed under a laser confocal microscopy (Zeiss LSM710, Germany).

The samples were ground in liquid nitrogen to powder. 1 g of sample was dissolved in 5 mL of water, and placed in an ice water bath for ultrasonic extraction for 1 h. After centrifugation at 4°C, 12000 rpm for 5 min, 1 mL supernatant extract was mixed with 1 mL aqueous anthalenediamine solution (6 g/L), derivatization reaction was performed for 8 h at room temperature and dark conditions, filtered at 0.22 um, and then conducted chromatograph analysis. Chromatographic conditions are as follows: chromatographic column: Kromasil; Anti-phase chromatography column (150 mm × 4.6 mm, 5 um); Mobile phase: 0.1%(V/V) acid aqueous solution (A) and methyl alcohol (B); gradient elution program: 0~5 min: B= 30%; 5~10 min: B up from 30% gradient to 90%; 10~15 min: B= 90%; 15~16 min: B gradually decreased from 90% to 30%; 16~20 min: B= 30%; strength: 1.0 ml/min. Detection wavelength: 318nm; Column temperature: 30°C; Sample volume: 10uL; Retention time: 10.3min. Methylglyoxal content= Detection concentration*Sample extraction volume (dilute volume)/the weight of sample.

To investigate the biological functions of OsGF14C in salinity and blast resistance in rice, we first analyzed the expression patterns of OsGF14C under salinity stress and blast infection. The experiments were conducted at 0h, 12h, 24h and 48h after stress treatments. The results indicated that the expression levels of OsGF14C significantly increased at 24h and 48h after salinity treatment, while significantly decreased at 24h after blast inoculation ( Figure 1 ).

To confirm the functions of OsGF14C in salinity and blast resistance in rice, we produced the OsGF14C overexpressing plants (OsGF14C-OX) using the variety Lijiangxintuanheigu (LTH) as material, which is susceptible to blast. Fifteen independent transgenic lines were generated and the OX plants showed no differences in morphological phenotypes compared to wild type plants.

Four independent homozygous lines (OX-4, OX-9, OX-12 and OX-15) with different high transcription levels of OsGF14C were selected for evaluation of salinity tolerance ( Figure 2A ). The results showed that the relative electrolyte leakages, which is an indicator of membrane injury under salinity stress, of all the OsGF14C-OX lines were significantly lower than the wild-type plants on the fourth day after high salinity stress treatment ( Figure 2B ). After salinity stress treatment for seven days and then recovery for ten days, the survival rates of the OsGF14C-OX lines were significantly higher than their wild-type plants ( Figures 2C, D ). We also measured the transcription level of OsGF14C in the fifteen OX lines and conducted blast resistance evaluation using M. oryzae isolate GD08-T13 by measuring the infected lesion areas ( Figures 3A–C ). Correlation analysis demonstrated a positive correlation between the expression level of OsGF14C and infected leaf area (R^{2 =} 0.643; Figure 3D ), suggesting that increased OsGF14C expression may reduce the blast resistance in rice.

In order to gain further insights into the function of OsGF14C in rice response to stresses, the temporal and spatial expression patterns of OsGF14C in root, stem and leaf at different stages were analyzed. As shown in Figure 4A , the expression of OsGF14C can be detected in all rice tissues examined in the present study and relative higher expression level was observed in leaf at seedling stage.

To investigate the sub-cellular localization of OsGF14C, the coding domain sequence of OsGF14C was fused with the green fluorescence protein (eGFP) under the control of the cauliflower mosaic virus 35S promoter. The OsGF14C::eGFP fusion vector and the control vector (empty eGFP vector) were transiently expressed in rice protoplasts. The laser confocal microscope showed that the green fluorescent signal in the protoplasts expressing eGFP control vector was distributed both in the nucleus and the cytoplasm, whereas the green fluorescent signal was distributed in the cytoplasm of the rice protoplasts transfected with the OsGF14C::eGFP vector ( Figure 4B ). The results demonstrated OsGF14C was localized in cytoplasm.

According to the previous studies, the defense-related genes play important roles in blast resistance in rice, and the accumulation of toxic ions, such as Na⁺ is the main reason for ion toxicity in rice under salinity stresses (Yan et al., 2022). To elucidate the regulatory mechanisms underlying OsGF14C in response to salinity and blast stresses in rice, we detected the expression changes of defense-related genes and Na⁺ transport-related genes in transgenic and wild type plants.

The results indicated that the expression of the lipoxygenase gene LOX2 was significantly induced in OsGF14C-OX lines compared to the wild type plants. In contrast, the expression levels of three pathogenesis-related genes (PR1, PR5, PR10a) and a chitinase gene (CHT1, also named PR-3 chitinase 1), were significantly lower in OsGF14C-OX lines than that in wild type plants ( Figure 5 ).

We also evaluated the expression pattern of the HKT, NHX and SOS1 genes, which are the important player in regulating the ion homeostasis in rice under salinity stress. Our results showed that the expression levels of the three HKT genes (HKT1, HKT4, HKT6) were significantly lower in OsGF14C-OX lines than that in wild type plants both before and after salinity stress treatment ( Figure 6 ). However, there was no significant difference in the expression levels of NHX1, NHX5, and SOS1 between the OsGF14C-OX lines and the wild type plants ( Supplemental Figure 1 ).

Plant GF14 proteins usually interact with a broad range of proteins to exert their functions. In the previous study, Zhang et al. (2017) reported 29 (exclude OsGF14C itself) client proteins of OsGF14C in rice root by affinity chromatography. Among the 29 client protein genes, 22 of them were found to be expressed in our OsGF14C-OX RNA-seq results. Among these 22 genes, only 6 of them had significant expression changes in OsGF14C-OX plants compared to wild-type plants ( Table S2 ). In order to confirm these results, we further evaluated the expression levels of these gene by qRT-PCR. The results demonstrated that the expression levels of two other OsGF14 genes (GF14E and GF14F) and a sucrose synthase gene SUS1 were significantly lower in OsGF14C-OX lines than that in the wild type plants. In contrast, the expression levels of two self-defense mechanism related genes (GLYI-11 and GSTF2), as well as a cell structure and growth-related gene TUBC3 were significantly higher in OsGF14C-OX lines than that in the wild type plants ( Figure 7 ).

It has been reported that GF (14-3-3) played very important roles in plant growth and development, as well as in response to various stresses. There are eight GF genes in rice, named GF14A-GF14H. The other studies and our previous studies have confirmed that GF14B and GF14E functioned in regulating disease resistance and drought tolerance in rice (Manosalva et al., 2011; Liu et al., 2016a; Liu et al., 2016b). Although it was reported that OsGF14C responded differently in various biotic and abiotic stresses in rice (Chen et al., 2006), the molecular functions of OsGF14C involved in biotic and abiotic stresses resistances in rice remain to be elucidated. In present study, the sub-cellular localization of OsGF14C in rice protoplasts revealed that OsGF14C accumulated only in the cytoplasm, which is consistent with the observations in rice callus and onion epidermal cells ( Figure 4B ). Differently, GF14B, GF14E and GF14F were localized in both nuclei and cytoplasm (Chen et al., 2006). Furthermore, the temporal and spatial expression analysis indicated that OsGF14C exhibited higher expression levels in all the tissues examined in the present study at the seedling stage compared to those at the ripening stage ( Figure 4A ). The different sub-cellular localization, temporal and spatial expression patterns imply that OsGF14C might have different functions in rice. Our transgenic experiments further confirmed this speculation.

Our results showed that the expression of OsGF14C was induced by salinity stress while suppressed by blast infection ( Figure 1 ). The relative electrolyte leakages, which is an indicator of salinity resistance, of the OsGF14C-OX lines were significantly lower than that of the wild-type plants after salinity stress treatment ( Figure 2B ). In consistence with these results, after salinity treatment for seven days and then recovery for ten days, the survival rates of the OsGF14C-OX lines were significantly higher than that of their wild type plants ( Figures 2C, D ). On the contrary, for the blast disease resistance, the infected lesion areas of OsGF14C-OX lines were larger than the wild type plants and the expression levels of OsGF14C was positively correlated with infected leaf areas in OsGF14C-OX lines ( Figure 3 ), indicating negative effect of OsGF14C in regulating blast resistance in rice. Taken together, our results suggest that overexpression of OsGF14C can improve salinity tolerance but reduce blast resistance in rice.

It is well known that PR genes play important roles in plant defense responses (Kaur et al., 2017). Our previous study and other studies also suggest that the PR genes are involved in blast resistance in rice (Tezuka et al., 2019; Dong et al., 2021). CHT1, which is also named as pathogenesis related (PR)-3 chitinase 1, is another typical defense response gene. Previous studies showed that CHT1 played important roles in disease resistances, such as sheath blight disease and blast disease in rice (Hao et al., 2012). To investigate whether OsGF14C mediated blast resistance is associated with PR genes, we analyzed the expression of the PR genes in the present study. The results showed that the expression levels of PR1, PR5, PR10a and CHT1 in OsGF14C overexpressing lines were all significantly lower than that in wild type plants, suggesting that OsGF14C negatively regulates blast resistance in rice by suppression of PR genes.

In plants, GF proteins are known to be involved in regulating a large number of biological processes via their interaction with numerous client proteins in a phosphorylation-dependent manner. Recently, 87 client proteins were identified through affinity chromatography assay using four GF proteins in rice, including the GF14C (Zhang et al., 2017). Twenty-nine (exclude OsGF14C itself) out of the 87 client proteins can interact with OsGF14C. Among these 29 client proteins, the expression levels of OsGF14E and OsGF14F, were significantly lower in OsGF14C-OX plants than that in wild type plants. In our previous study, we have functionally confirmed that OsGF14E and OsGF14F could positively regulate blast resistance in rice (Liu et al., 2016b; Ma et al., 2022). These results suggest that OsGF14C might negatively regulate blast resistance by suppressing the expression of OsGF14E and OsGF14F. However, further studies are needed to elucidate whether OsGF14C regulates blast resistance by an indirect way or by both indirect and direct ways.

GFP (Glyoxalase family protein, also named GLY or GLO) and GSTF2 (Glutathione S-transferase) are another two client proteins of OsGF14C reported by Zhang et al. (2017). GLYI catalyzes methylglyoxal (MG) metabolism in the presence of GSH and produces an intermediate compound S-lactoylglutathione. According to previous studies, MG is a potent cytotoxic compound produced from glycolysis. The rate of glycolysis increases under stresses, and lead to MG accumulation in plants. MG accumulation is toxic to cells as it inhibits cell proliferation and results in a number of adverse effects, such as increasing the degradation of proteins and inactivating the antioxidant defense system (Hossain et al., 2009). Previous studies showed that overexpression of GLY I+II together in tobacco led to enhancement of salinity and zinc tolerance (Singla-Pareek et al., 2003; Singla-Pareek et al., 2006). Heterologous expression of OsGLYI-11.2 in Escherichia coli and tobacco also resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lowering the Na⁽⁺⁾/K⁽⁺⁾ ratio and maintenance of reduced glutathione levels (Mustafiz et al., 2014). In the present study, the expression levels of GLYI-11 and GSTF2, and the contents of MG were significantly lower in OsGF14C-OX lines than that in wild type plants ( Figures 7 , 8 ). Taken together, these results suggest that the positive role of OsGF14C in regulating salinity tolerance might be partially attributed to its interactions with GLYI-11 and GSTF2, and leading to the reduction of MG.

Plants have developed intricate mechanisms for Na⁺ uptake, export and compartmentation to reduce the accumulation of Na⁺ (Wu, 2018). So far, the salt-overly-sensitive (SOS) signaling pathway has been considered an importance pathway in salt resistance (Gong et al., 2020; Zelm et al., 2020). The SOS1 encodes a plasma membrane Na⁺/H⁺ antiporter, which uses the H⁺ gradient to drive Na⁺ efflux and thus reducing cytosolic Na⁺ concentration (Shi et al., 2003). However, in the present study, there was no significant difference in the expression levels of SOS1 between OsGF14C-OX plants and wild type plants, either before or after salinity treatment ( Supplemental Figure 1 ). In addition, the central vacuole is an ideal location for Na⁺ sequestration to reduce the Na⁺ accumulation in the cytoplasm. NHX-type transporters play important roles in Na⁺ translocation from cytoplasm to vacuole through a Na⁺/H⁺ exchanger (Blumwald and Poole, 1985; Gong et al., 2020). Nevertheless, our results showed that the expression levels of NHX1 and NHX5 in OsGF14C-OX lines were similar to that in wild type plants, both with and without salinity treatment ( Supplemental Figure 1 ). In contrast, the expression levels of HKT-type transporters including HKT1, HKT4, and HKT6, which mediate the translocation of Na⁺ from extracellular to cytoplasm in OsGF14C-OX plants were significantly lower than that in wild type plants, both before and after salinity treatment ( Figure 6 ). Taken together, these results suggest that overexpression of OsGF14C may positively regulate salinity tolerance by lowering the expression of HKT-type transporter genes and inhibiting the Na⁺ uptake instead of exclusion or compartmentation.

In natural environment, plants are often subjected to both abiotic (such as drought, salt, cold) and biotic (necrotrophic and biotrophic pathogens) stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize the fitness costs. It is essential to find out the genes which could play roles in different stresses resistance and understand how the various stress response pathways interact with one another. In the present study, overexpression of OsGF14C enhanced the resistance to salinity stress but reduced the resistance to blast in rice. The opposite roles of OsGF14C overexpression played in salinity stress and blast disease are consistent with the previous findings that plants exposed to abiotic stresses such as high salinity and drought often display reduced immune activity (Bostock et al., 2014). It is understandable for this conflict as stress responses are costly, plants likely coordinate these responses to minimize fitness costs.

According to previous studies, LOXs, the key enzymes of JA synthesis, have been reported to involve in various physiological processes, including responses to biotic and abiotic stresses (Mostofa et al., 2015). Chu et al. (2019) showed that the Arabidopsis plants inoculated with Pseudomonas PS01 could survive under 225 mM NaCl, while the non-inoculated plants were dead above 200 mM NaCl. The gene expression analysis of the genes related to stress resistance indicated that only LOX2 was up-regulated in the inoculated plants in comparison to the non-inoculated controls (Chu et al., 2019). Furthermore, Zhang et al. (2018b) found that LOX2 and LOX5 were specifically suppressed by Guy11 infection, which are the key JA synthesis genes hijacked by M. oryzae strain Guy11 to subvert host immunity and facilitate pathogenicity. They also proposed that induced expression of OsLOX2/5 may improve resistance to the blast disease in rice (Zhang et al., 2018b). In the present study, our results showed that the expression levels of LOX2 were significantly higher in the OsGF14C-OX plants than that in wild type plants. Given that LOX genes play important roles in regulating both biotic and abiotic stresses in plants, the increased expressions of LOX2 in OsGF14C-OX plants implied LOX2 may partially lead to the differences responses of transgenetic plants to salinity and blast resistance. However, despite that OsGF14C-OX plants showed increased expression of LOX2, the transgenetic plants were more sensitive to rice blast disease than their wild type plants, which seems to be conflict with previous studies. But we also detected reduced expression of some PR genes in OsGF14C-OX plants, which may be the principal reason for the reduction of blast resistance. This also indicated the complexity between different stress response pathways and the importance for understanding the crosstalk between different stress pathways. According to the results from this study, LOX2 may play important roles in coordinating salinity tolerance and blast resistance in rice. However, it need more studies to confirm if LOX2 is the switch gene and how it regulates the salinity tolerance and blast resistance in rice. It is of great significance to study the regulation mechanisms of LOX2 underlying different stresses in crops.