Soil salinization is one of the major limiting factors for crop growth and production (Wei et al., 2021). Saline soils are widely distributed globally in 100 countries on 6 continents, with a total area of 1.173 × 10⁷ km² (Hassani et al., 2021). Rice (Oryza sativa L.), a major food crop in Asia, is a salt-sensitive crop. Particularly, it is sensitive to salt stress during the seedling and reproductive stages (Ganie et al., 2019). Generally, damage to rice due to salt stress mainly occurs via osmotic stress, membrane lipid peroxidation, and ion toxicity (Lee et al., 2013; Shen et al., 2015). Salt stress increases Na⁺ content in rice cells and reduces K⁺ uptake under ion competition, resulting in increased Na⁺/K⁺ and disruption of ion balance eventually leading to cell death under the dual action of osmotic stress and ion poisoning (Feng et al., 2019).

Maintaining the dynamic balance of Na⁺ and K⁺ is an important means for plants to cope with salt stress, mainly through long-distance transport of ions in the xylem and phloem and accumulation and compartmentalization of ions (Liu et al., 2022). Ion-transport proteins are the key factors in the maintenance of Na⁺ and K⁺ homeostasis in plants under salt stress (Jabnoune et al., 2009). In rice, several transporter protein families, mainly HKT, HAK, AKT, NHX, and SOS, regulate ion homeostasis. The high-affinity K⁺ transporter (HKT) family in rice has two subfamilies. A member of the subfamily I, namely, OsHKT1;5 (SKC1), transports Na⁺ from rice shoot to root via xylem unloading to reduce Na⁺ accumulation in the shoot under salt stress (Ren et al., 2005). A member of the subfamily II, namely, OsHKT2;1 (OsHKT1), maintains cytoplasmic Na⁺/K⁺ homeostasis by selectively transporting Na⁺ or Na⁺ and K⁺ under the regulation of the clock component OsPRR73 (Jabnoune et al., 2009; Wei et al., 2021). OsHAK21 (qSE3), a member of the HAK/KUP/KT transporter family, regulates salt tolerance at the germination and seedling establishment stages of rice by regulating K⁺ and Na⁺ uptake, increasing abscisic acid (ABA) biosynthesis, and activating ABA signaling responses (He et al., 2019). Shaker K⁺ channel protein, namely, OsAKT2, plays an important role in K⁺ loading and redistribution from the phloem and maintains cellular Na⁺/K⁺ homeostasis by regulating the K⁺ redistribution process (Tian et al., 2021). OsNHX1, a vesicular membrane NHX-type antiporter, reverses cation transport into the vesicles by ion compartmentalization under salt stress to maintain cellular ion homeostasis and osmotic potential (Fukuda et al., 2004; Liu et al., 2010; Fukuda et al., 2011; Ma et al., 2016; Bassil et al., 2018; Wu et al., 2018). Rice plasma membrane Na⁺/H⁺ exchange protein, namely, salt overly sensitive 1 (OsSOS1), which is activated by the protein kinase complex OsCIPK24-OsCBL4, maintains salt tolerance in rice in a specific manner by Na⁺ efflux (Martínez-Atienza et al., 2007; El Mahi et al., 2019).

Chlorophyll fluorescence can accurately reflect the process of light energy uptake and conversion by plants and effect of salt stress on the photosynthetic system of plants. In rice leaves, salt stress disrupts the balance of production and scavenging of reactive oxygen species (ROS) on the thylakoid membrane. This causes oxidative reactions that damage the thylakoid membrane, reduce the regulation of excitation energy partitioning between photosystems PSI and II, reduce photosynthetic efficiency, and alter fluorescence properties (Faseela et al., 2020; He et al., 2020). Theerawitaya et al. explored the physiological characteristics of rice varieties Pokkali (salt tolerant) and IR29 (salt sensitive) under salt stress and observed that the maximal fluorescence (Fm), maximum quantum efficiency of PSII (Fv/Fm), and net photosynthetic rate (Pn) were significantly higher in Pokkali than in IR29 after salt stress (Theerawitaya et al., 2020). Moreover, Singh et al. confirmed that Fv/Fm of salt-sensitive rice varieties decreased significantly with increased duration of salt stress, whereas no significant changes were observed in salt-tolerant varieties (Singh and Sarkar, 2014). He et al. reported that OsHCF222, a high chlorophyll fluorescent protein localized in the endoplasmic reticulum and chloroplasts, is a key factor regulating chlorophyll fluorescence properties of rice. This protein forms a complex by binding to its interacting protein OsABCI7, which regulates cellular ROS homeostasis to maintain the stability of the thylakoid membrane (He et al., 2020). Zhu et al. reported that the differential expression of OsHCF222 and OsABCI7 directly affected the changes in chlorophyll fluorescence parameters of japonica rice germplasm with different salt tolerances and may be involved in the regulation of salt tolerance in rice seedlings (Zhu et al., 2022).

In recent years, many studies have reported the regulation of salt tolerance in rice by ion homeostasis and chlorophyll fluorescence, focusing on the phenotype or gene expression. However, only few studies have comprehensively evaluated salt tolerance in rice by combined analysis of phenotype, haplotype, and gene expression. It is largely unclear how these three aspects interact and regulate each other, and the key reasons for differential responses to salt stress by salt-tolerant and salt-sensitive accessions are not studied. In this study, we aimed to provide foundational insights into these issues. Morphological indices and physiology indices associated with ion homeostasis and chlorophyll fluorescence were examined for 12 japonica rice germplasm accessions with different salt tolerances. The expression characteristics and haplotypes of ion-transporter proteins and chlorophyll fluorescence-related genes were analyzed. This study provided an important reference for studying molecular mechanisms of salt tolerance in rice.

The energy produced by photosynthesis is a key factor supporting crop growth. Photosynthetic pigments, photosystems, electron transport systems, and CO₂ reduction pathways involved in the photosynthesis process are affected by salt stress (Ashraf and Harris, 2013). Chloroplasts support rice growth by converting light energy into chemical energy, and excess energy is dissipated as heat (NPQ) or emitted as chlorophyll fluorescence (Maxwell and Johnson, 2000). These three processes compete with each other, and under stress, more energy is converted to heat and chlorophyll fluorescence with the reduction in photosynthesis. Assessment of chlorophyll fluorescence parameters can effectively indicate the rate of photochemical reactions and extent of heat dissipation (Tsai et al., 2019). Wang et al. reported that the permeability and function of the thylakoid membrane in chloroplasts were significantly impaired when large amounts of salt ions accumulated in rice cells over a long period (Wang et al., 2009), and the activity of their photosystem and chlorophyll fluorescence were significantly reduced (Faseela et al., 2020). Fm in rice leaves significantly reduced under salt stress compared with the normal treatment (Theerawitaya et al., 2020; Zhu et al., 2022), and Fv/Fm decreased significantly in salt-sensitive varieties (Sarkar et al., 2013; Singh and Sarkar, 2014). In the present study, Fm and Fv/Fm of STA and SSA exhibited significant decreases with increasing duration of salt stress ( Table 2 ). Y, ETR, and NPQ of SSA were significantly lower than those on the CK group at 3 days after salt stress and exhibited a highly significant decrease at day 6 of salt stress. Fm, Fv/Fm, Y, ETR, and NPQ of STA at 6 days after salt stress were highly significantly higher than those of SSA ( Figure 2 ), indicating that the electron transfer rate and photochemical reaction rate of STA were higher and the PSII complex was less damaged under salt stress. NPQ reflects the level of excess energy converted into heat dissipation. It was reported that NPQ of rice varieties with different salt tolerances increased significantly under salt stress (Moradi and Ismail, 2007), whereas another study reported increased NPQ in Pokkali (salt-tolerant indica rice) and decreased NPQ in IR-29 (salt-sensitive indica rice) (Lee et al., 2013). In our study, NPQ of SSA was significantly lower than that of the CK group at days 3 and 6 after salt stress. However, NPQ of STA was lower than that of the CK group at day 3, but it significantly improved at day 6 compared with that at day 3 ( Figure 2 ). It may be due to lower inhibition of light energy conversion efficiency in STA at the early stage of salt stress, and only limited excess energy was produced for heat dissipation; therefore, NPQ was lower at day 3 after salt stress. With the prolongation of duration of stress, the photosystem conversion efficiency gradually decreased to reduce the damage to the thylakoid membrane from excess energy. In STA, a thermal dissipation mechanism was initiated, which converted the excess energy into thermal energy for dissipation, resulting in an increase in NPQ and a protective effect on the thylakoid membrane.

Na⁺, a mineral element required for rice growth, can be promoted at low Na⁺ concentrations, particularly under K⁺-deficient conditions (Horie et al., 2007). However, high concentrations of Na⁺ can have direct toxic effects on cells, e.g., inhibition of multiple metabolic processes by competing with K⁺, which was required for a variety of biochemical reactions and protein synthesis (Belgaroui et al., 2018). On this basis, Na⁺ produces sustained osmotic and oxidative stresses that damage the integrity of cell membranes and cell walls to inhibit rice growth (Ismail et al., 2014; Voxeur and Höfte, 2016; Zhu, 2016; Yang and Guo, 2018a, YangGuo, 2018b; Zhao et al., 2021). To adapt to saline soil, plants have evolved complex mechanisms to maintain Na⁺ homeostasis, which is mainly controlled by ion-transport proteins (Zhu, 2016; Yang and Guo, 2018a). OsHKT1;5, isolated from the salt-tolerant indica rice variety (Nona Bokra), plays an important role in regulating ion homeostasis in indica rice (Ren et al., 2005; Hauser and Horie, 2010; Alnayef et al., 2020). In our study, the expression of OsHKT1;5 was significantly higher in the roots of Huangluo than in those of Shanfuliya ( Figure 5A ), whereas the Na⁺ content was significantly lower in the roots of Huangluo than in those of Shanfuliya at 6 days after salt stress ( Supplementary Figure 1 ). This indicated that OsHKT1;5 plays an important role in regulating Na⁺ homeostasis in japonica rice. OsHKT2;1, a versatile protein of HKT subgroup II, regulates ion transportation. OsHKT2;1 cotransports Na⁺ and K⁺ under physiological conditions, separately cotransports Na⁺ under high Na⁺ concentration, and takes up Na⁺ to maintain normal rice growth under K⁺-deficient conditions (Jabnoune et al., 2009; Hauser and Horie, 2010; Suzuki et al., 2016; Hartley et al., 2019). However, we observed that the expression of OsHKT2;1 was significantly higher in Huangluo than in Shanfuliya; additionally, Theerawitaya et al. reported that Pokkali exhibited a significant increase in OsHKT2;1 expression after salt stress, which was approximately 2.5-fold higher than that under normal growth conditions (Theerawitaya et al., 2020). This may be because of K⁺ deficiency due to high Na⁺ concentrations and the uptake of Na⁺ by OsHKT2;1 to maintain normal growth. Although OsAKT2 expressed in the shoot phloem loaded and transported K⁺ to the roots for redistribution to alleviate rice salt stress (Tian et al., 2021), our results revealed that the expression of OsAKT2 was suppressed. This may be a protective mechanism against K⁺ loss in the shoots, causing excessive Na⁺/K⁺ in shoot. In addition, we observed that both OsNHX1 and OsHKT2;1 were expressed in the vascular sheaths (Horie et al., 2007; Fukuda et al., 2011), with high synergism ( Figures 5B–E ), speculating that a coexpression network may exist between them; however, so far, no reliable basis is obtained for this inference.

Sequence polymorphism analysis helps us to understand the mechanisms of adaptation to salt stress in different rice germplasms (Oomen et al., 2012). He et al. identified 14 SNPs and 2 indels in the OsHAK21 promoter and coding region, where four SNPs located in the coding region caused amino acid substitution. Haplotype analysis revealed that 2036T SNP located in the promoter was significantly positively correlated with seed germination under salt stress, and Hap3 containing 2036T SNP was a typical salt-tolerant haplotype (He et al., 2019). In our study, all japonica rice germplasm accessions exhibited the 2036C SNP in OsHAK21 promoter. Interestingly, we observed one SSR sequence in its promoter, and the STS of germplasm accessions gradually decreased with the increase of AGA trinucleotide repeat motifs ( Figure 6B ). The expression of OsHAK21 was significantly higher in Huangluo (Hap2) than in Shanfuliya (Hap4), indicating that the SSR sequence may affect the salt tolerance of rice seedlings by regulating the expression of OsHAK21. We observed that an ABRE cis-acting element located in this SSR region is associated with response to ABA, and OsHAK21 is involved in the ABA synthesis, ABA signaling pathway-related gene expression, and inhibition of ROS accumulation to regulate salt tolerance in rice (cf. PlantCARE database) (Lescot et al., 2002; He et al., 2019). Similarly, this occurs in the OsNHX1 promoter. We detected an indel site at OsNHX1 promoter (−822 bp); Huangluo (Hap2) was A, Shanfuliya (Hap1) was a single-base deletion, and Hap3 was AA. Hap2 had significantly higher STS than the other two haplotypes, and the OsNHX1 expression of Huangluo was significantly higher than that of Shanfuliya. This indicated that with the insertion or deletion of A, this locus may regulate salt tolerance in rice by affecting the expression of OsNHX1 ( Figure 6C ). In rice shoots, K⁺ can be loaded into the phloem via OsAKT2 and other transporter proteins and transported downward, with approximately 80% returning to rice roots, which helps rice roots maintain Na⁺/K⁺ homeostasis and normal functions (Tian et al., 2021). Haplotype analysis revealed that Hap2 and Hap4 were salt tolerant, and Hap3 was salt sensitive. Only one SNP (−1866 bp) was detected in the promoter between Hap2 and Hap3 ( Figure 6D ), which suggested that the SNP may be a key locus affecting OsAKT2 expression under salt stress in rice seedlings. Meanwhile, one SNP was detected in OsAKT2 coding region, resulting in an amino acid substitution (glutamate to glycine, E-G; Supplementary Figure 2A ); however, this locus did not have significant effect on salt tolerance in rice at the seedling stage. The SNP presented in the exon of OsABCI7 caused the substitution of valine to alanine (V-A; Supplementary Figure 2B ), which was not located in the critical domain of OsABCI7 (He et al., 2020). This significantly affected salt tolerance in rice seedlings ( Figure 6E ). Multigene combinatorial haplotype analysis suggested that the polymorphic loci significantly associated with salt tolerance located at OsHAK21 (−1231 bp), OsNHX1(−822 bp), OsAKT2 (−1866 bp), and OsABCI7 (+1605 bp) can be used to develop molecular markers for the molecular identification of salt-tolerant rice varieties.

Considering all the results, we summarized the regulatory patterns of chlorophyll fluorescence and ion-transporter protein genes regulating salt tolerance in different rice germplasm accessions under salt stress ( Figure 7 ). When the rice seedlings were subjected to high salt stress, Huangluo (salt-tolerant germplasm) had higher Na⁺ content at day 3 of salt stress than Shanfuliya (salt-sensitive germplasm) ( Supplementary Figure 1 ). This may be because OsHKT2;1 and OsHAK21 are expressed in the root epidermis and translocated more Na⁺ in Huangluo. With high Na⁺ entering the cells, the expression of OsNHX1 was rapidly activated on the vesicle membrane within the first 6 h of salt stress in Huangluo (approximately 4 times the expression of OsNHX1 in Shanfuliya at 3 h) ( Figure 5E ). V-ATP was used up to translocate H⁺ into the cytoplasm, generating an electrochemical potential gradient to translocate Na⁺ into the vesicle, completing ion regionalization to avoid damage to the cell from high Na⁺ content (Wang et al., 2015a, Wang et al., 2019; Goyal et al., 2016). However, OsSOS1, located on the cell membrane, also translocated Na⁺ to the apoplast (Martínez-Atienza et al., 2007; El Mahi et al., 2019). In contrast, the relative expression of OsNHX1 and OsSOS1 in Shanfuliya was low, and only limited Na⁺ was regionalized and translocated out of the cell, with high concentrations of Na⁺ retained in the cytoplasm leading to ion toxicity (Apse et al., 1999; Liu et al., 2010). OsHKT1;5 and OsSOS1, which are expressed in the xylem parenchyma, regulated the content of Na⁺ and transported it to shoot through acting antagonistically (El Mahi et al., 2019). The Na⁺ content in the shoot was higher in Huangluo than in Shanfuliya at 3 days of salt stress ( Supplementary Figure 1 ). It is possible that the high expression of OsSOS1 expelled the excess Na⁺ into the xylem. In Huangluo, OsHKT1;5 transported Na⁺ to the xylem parenchyma to counteract some of the function of OsSOS1 and caused membranes depolarization of xylem parenchyma cells, opening K⁺ channels and releasing K⁺ into the xylem vessels (Ren et al., 2005; Hauser and Horie, 2010; El Mahi et al., 2019; Alnayef et al., 2020). Such an antagonistic mechanism not only avoids the transport of large amount of Na⁺ to the shoot but also uses the high concentration of K⁺ in the shoot to neutralize some of the Na⁺ content to reduce the damage to the roots due to high Na⁺ concentration. Therefore, the Na⁺/K⁺ ratio was significantly lower in the shoot of Huangluo than in that of Shanfuliya ( Supplementary Figure 1C ). As the expression of OsAKT2 was higher in Shanfuliya than in Huangluo ( Figure 5C ), with the shoot K⁺ transported to the roots, the root Na⁺/K⁺ ratio was lower and shoot Na⁺/K⁺ ratio was higher in Shanfuliya than in Huangluo at 3 days of salt stress ( Supplementary Figure 1C ). Concurrently, OsABCI7 and OsHCF222 on rice chloroplast thylakoid membrane responded to salt stress with lower expression in Shanfuliya, which could only form a smaller amount of OsABCI7-OsHCF222 protein complex. Therefore, it was unable tocould not scavenge a lot of ROS through the APX pathway, resulting in an excessive accumulation of ROS, damaging the thylakoid membrane, and affecting normal photosynthesis; this was manifested as a decrease in chlorophyll fluorescence parameters (Fm, Fv/Fm, Y, NPQ, and ETR), RSPAD, and STS (He et al., 2020; Zhu et al., 2022). Huangluo could produce more OsABCI7-OsHCF222 protein complex to maintain ROS homeostasis in the chloroplasts with the assistance of the APX pathway.

In summary, Huangluo was better than Shanfuliya in terms of maintenance of intracellular Na⁺ and K⁺ and ROS homeostasis by coordinating multiple ion-transport proteins (El Mahi et al., 2019). Thus, the cells of Huangluo were relatively intact after 6 days of salt stress and could maintain normal function, exhibiting greater salt tolerance. However, the salt tolerance of Shanfuliya was lower, with most of the cells dying after 6 days of salt stress ( Figure 4A ). Therefore, in Shanfuliya, the damage due to salt was more severe, and eventually, the plants died.

The differential response of salt stress in japonica rice germplasm with different salt tolerances mainly resulted from the disparity in chlorophyll fluorescence and ion-transporter protein gene expressions. And some genes’ differential expressions may due to the genotypic variations in promoter regions. We detected four key variations, including an SNP in the coding region of OsABCI7 and three polymorphic sites in the promoter of OsHAK21, OsNHX1, and OsAKT2, which may play key roles by affecting the response of these genes to salt stress.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

LT supervised and designed the experiments. JS guided the research and wrote the manuscript. JS, HY, CQ, CZ, TB, HD, SM, NW, CL, YZ, TM and PL performed the experiments and data analysis. All authors have read and approved the final manuscript. All authors contributed to the article and approved the submitted version.