Introduction

Plants are sessile organisms and are therefore constantly affected by various abiotic stresses. In particular, salinity affects 7% of the land surface of world; it is estimated that more than 50% of all arable lands will be saline by 2050 (Wang et al. 2003). Hence, salinity is one of the most severe stress for plant growth and crop productivity, and therefore, improvement in salt tolerance for economically important crops is an important objective. Salinity stress causes an excessive increase in the Na+ concentration in plant cells resulting from the competition between Na+ and K+ for the same binding sites in cellular metabolic processes, such as enzymatic reactions, protein synthesis, and ribosome function (Marschner 1995). Na+ interferes with K+ uptake and consequently disrupts ion homeostasis. It is important to maintain the low concentration of Na+ in the cytosol for proper functioning of many cytosolic enzymes and physiological processes in living cells (Duggleby and Dennis 1973). Besides ionic imbalance, salinity increases reactive oxygen species (ROS) production, which causes oxidative damage to nucleic acids, proteins, carbohydrates, and lipids in plants (You and Chan 2015). Plants have developed an efficient enzymatic and non-enzymatic antioxidative system to protect themselves against oxidative damage. The ROS scavenging by antioxidant enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) has been proven to be a crucial mechanisms of plants for stress tolerance (You and Chan 2015). To cope with unfavorable growth conditions such as soil salinization, plants have developed unique molecular, biochemical, and physiological defense mechanisms that are controlled by multiple genes. So far, a number of salt stress-induced genes and genes improving salt tolerance and ion homeostasis have been characterized in a range of plants. Plasma membrane (PM) is the first part to respond against abiotic stresses; therefore, it would be important to examine the response of proteins located in the PM to salt stress.

Rare-cold-inducible 2 (RCI2) gene is a PM protein (PMP3) that is associated with responses to various abiotic stresses such as low temperature, drought, osmotic stress, and high salinity (Rocha 2016). RCI2 encodes small and highly hydrophobic integral membrane proteins bearing two conserved transmembrane domains (TMD) (Chang-Qing et al. 2008). RCI2s have been identified in a wide variety of plant species and are altered by salt stress (Rocha 2016). Further, the functional roles of RCI2s in salt tolerance were shown through complementation studies of yeast pmp3 mutants exhibiting salt sensitivity. A number of plant RCI2 genes exhibit salt tolerance in the pmp3 mutant; however, some RCI2 do not complement the salt sensitivity (Kim et al. 2021; Rocha 2016). In contrast to studies in yeast, there are fewer studies describing the salt stress-responsive functions of RCI2 in plants. The overexpression of MePMP3-2 in cassava and AlTMP1 and AlTMP2 in Aeluropus contributes to salt tolerance in rice and tobacco, respectively (Ben Romdhane et al. 2017, 2018; Yu et al. 2016). In addition, AtRCI2A, ZmPMP3-1, TaRCI, and MsRCI2A also enhance salt tolerance in different plant species (Mitsuya et al. 2006; Fu et al. 2012; Khurana et al. 2015; Long et al. 2015). Alternatively, the overexpression of MpRCI recovers the salt tolerance in the AtRCI2A knockout mutant that exhibits salt sensitivity (Liu et al. 2012). It was proposed that the genes could confer salt tolerance by maintaining ion homeostasis, increasing membrane integrity, improving plant water status, and decreasing oxidative damage in cells. Although previous studies have clearly indicated the possible functional roles of RCI2s in salt adaptation or tolerance in plants, relatively few knowledge on the roles of RCI2 in crop plants exists.

Camelina sativa L. is an economically important oilseed crop for biofuel production; it can be grown on marginal lands that are not well suited for food crops and has the potential for low cost and high value oil. Eight RCI2s (CsRCI2A to CsRCI2H) are present in the Camelina genome. These genes are subdivided into two groups: Group I (CsRCI2A-C and CsRCI2H) belongs to the yeast PMP3 family and contains only two TMDs, whereas Group II (CsRCI2D to CsRCI2G) contains an extra C-terminal hydrophilic tail. In our previous study, we found that salt stress-induced two different genes of Camelina RCI2 including CsRCI2A and CsRCI2E had different roles in salt response; CsRCI2A enhances salt tolerance in yeast pmp3 mutants but CsRCI2E did not (Kim et al. 2016). Furthermore, we have recently shown that one of Group I gene, CsRCI2H, enhances seed germination and seedling growth under salt stress in Camelina (Kim et al. 2020). It is interesting to determine the functions of Group II gene under salt stress. Although previous study have found that Group II genes including CsRCI2D, CsRCI2E, and CsRCI2G are induced by salt stress, it is uncertain salt stress-responsive roles of Group II genes. Here, we showed that CsRCI2D has a positive effect on seed germination and postgermination seedling growth of Camelina under salt stress, which is achieved by decreasing the accumulation of Na+ and ROS in plant cells.

Results

Characterization of CsRCI2D

Eight RCI2 genes in Camelina are divided into two groups depending on the existence of C-terminal tail. CsRCI2D harbors two TMDs (TMD1 and TMD2) and C-terminal tail region, which is typical structural characteristics of group II genes (Fig. S1), but group I genes do not possess C-terminal tail as yeast PMP3.

(ScPMP3). CsRCI2D consists of 75 amino acid residues and shows highly significant homology with AtRCI2D (96.1%) (Fig. S1b). Previously, we investigated other CsRCI2 protein localized in the plasma membrane (Kim et al. 2020). The YFP-CsRCI2D fusion protein which is transiently expressed in tobacco leaf cells, was localized in not only the PM but also the intracellular membranes such as the unidentified punctate structures as indicated by the arrows (Fig. S2). This result imply that CsRCI2D may be endocytosed in plant cells. To further confirm the subcellular localization of CsRCI2D, western blot analysis was performed using antibodies of endomembrane marker proteins such as PM H+-ATPase (PM), V-ATPase (tonoplast), and BIP (ER) from fourteen membrane fractions which are separated by sucrose density gradient centrifugation (Fig. 1). CsRCI2D expression was observed at fractions 7 to 14 with strong intensity at 10 to 11. However, the expression of CsRCI2D shifted to fraction 7 to 9 under NaCl stress. This result indicated that localization of CsRCI2D shifted to lower density of membrane from PM by NaCl exposure. Although all endomembrane marker proteins showed different expression pattern each other, the expressed fractions of all endomembrane marker proteins were partially overlapped with CsRCI2D. These results suggest that CsRCI2D is associated to membrane trafficking such as internalization of PM proteins to another organelle.

Fig. 1
figure 1

Subcellular localization of CsRCI2D in Camelina. Western analysis of membrane proteins from Camelina. Fourteen linear membrane fractions by sucrose gradient were isolated and used for western analysis using antibodies of CsRCI2D, PM H+-ATPase, V-ATPase, and binding immunoglobulin protein (BIP)

Full size image

Functional Complementation of CsRCI2D in Yeast Mutant

CsRCI2D was significantly increased by threefold after 150 mM NaCl treatment, suggesting that CsRCI2D may play a crucial role in salt stress response (Fig. 2a). To predict the functional roles of CsRCI2D in salt tolerance, we determined whether CsRCI2D complements the salt sensitivity of yeast pmp3 mutant (△pmp3). △pmp3 cells and △pmp3 cells expressing CsRCI2D displayed salt sensitivity under 0.5 M and 1 M NaCl (Fig. 2b). Previous findings revealed that the C-terminal tail in CsRCI2 disrupts salt tolerance in △pmp3 (Kim et al. 2016). When the C-terminal tail-truncated CsRCI2D (CsRCI2D△58) was expressed in △pmp3, CsRCI2D△58 could complement the salt sensitivity of △pmp3, which is similar growth with the yeast BY4741 (WT) (Fig. 2b).

Fig. 2
figure 2

Expression pattern of CsRCI2D and complementation of Δpmp3 mutant under salt stress. a One-week-old Camelina seedlings were treated with 150 mM NaCl for 3, 12, and 24 h, and the transcript level of CsRCI2D was quantified by real-time RT-PCR. Values are mean ± SE of three independent experiments, and significant differences are indicated by asterisks (P < 0.05). b WT (BY4741) and Δpmp3 mutant cells harboring the CsRCI2D, C terminal tail truncated CsRCI2D (CsRCI2D△58), and pYES-DEST52 were grown on SG agar plates with 0.5 and 1 M NaCl at 30 °C. TM: transmembrane domain; the numbers indicate total amino acids

Full size image

CsRCI2D Improves Germination and Root Growth under Salt Stress

To determine whether the CsRCI2D or CsRCI2D△58 affects plant salt tolerance, CsRCI2D- or CsRCI2D△58-overexpressing transgenic Camelina plants (T3) were generated, and their responses to salt stress were assessed. Under normal growth conditions, both transgenic Camelina showed no significant difference in germination, plant growth, and development compared to that in WT (data not shown). Under 200 mM NaCl, while CsRCI2D-overexpressing transgenic seeds displayed 100% germination at 36 h, the WT seeds showed no germination (Fig. 3a). In the case of 300 mM NaCl, none of the WT seeds germinated at 48 h, whereas 100% of CsRCI2D-overexpressing transgenic seeds germinated (Fig. 3b). After germination, the roots of CsRCI2D-overexpressing transgenic plants grew more rapidly than those of WT under salt stress (Fig. 3c). Interestingly, CsRCI2D△58-overexpressing Camelina displayed similar germination rates and postgermination seedling growth with CsRCI2D-overexpressing Camelina under salt stress (Fig. 3).

Fig. 3
figure 3

Effects of salt stress on seed germination and seedling growth of CsRCI2D- and CsRCI2D△58-overexpressing transgenic Camelina. The seed germination rates of WT, CsRCI2D- and CsRCI2D△58-overexpressing transgenic Camelina were scored on the MS plate containing 200 (a) or 300 mM NaCl (b) and photographs were taken on 48 h. c Germinated seeds were further grown vertically for 5 days under 200 mM NaCl. Mean values ± SE were obtained from three independent experiments (n = 15). Significant differences between wild type and transgenic plants are indicated by *P < 0.05 and **P < 0.01 using the Student’s t test

Full size image

Effects of Salt Stress on Ion Content in CsRCI2D-Overexpressing Camelina

To ascertain whether the salt-tolerant phenotype was associated with the accumulation of Na+, K+, or Ca2+ upon salt stress, ion content was analyzed (Table 1). Under normal growth condition, similar contents of Na+, K+, or Ca2+ in the roots and shoots were investigated between WT and transgenic plants. After NaCl treatment, Na+ content greatly increased in WT and transgenic plants. Na+ was more accumulated in the roots than shoots in both WT and transgenic plants upon salt stress. Na+ content in the roots and shoots of transgenic plants was obviously lower than that in WT. Under normal condition, K+ content in the roots of WT was slightly higher than transgenic plants but similar K+ contents were investigated in the shoots of WT and transgenic plants. K+ content in transgenic plants was similar in the roots but was higher in the shoots compared to that in WT under salt stress (Table 1). In contrast, less Ca2+ contents were observed in salt-treated plants than in control plants. Ca2+ content between WT and transgenic plants were similar in both the roots and shoots under salt stress (Table 1).

Table 1 Effects of salt stress on the Na+, K+, and Ca2+ content in the roots and shoots of WT and the CsRCI2D-expressing Camelina
Full size table

Transcription of Potassium Transporter Genes

Retention of high K+ ion in plant cell is important for salt tolerance in plant. To investigate the reason of different K+ contents between WT and transgenic Camelina under salt stress, transcript levels of K+ transporter genes such as CsKEA1 (K+ efflux antiporter 1), CsHKT1 (high affinity K+ transporter 1), CsAKT1 (K+ transporter 1), and CsKAT1 (K+ channel in Arabidopsis thaliana 1) were measured by quantitative RT-PCR (Fig. 4). Transcript levels of all genes displayed similar expression in both WT and transgenic plants under normal condition. Transcript levels of CsKEA1 and CsHKT1 in both WT and transgenic plants were significantly decreased by salt treatment. Under salt stress, the expression of CsAKT1 in transgenic plants was higher than WT. Notably, CsKAT1 in transgenic plants was significantly up-regulated under salt stress compared to that in WT (Fig. 4).

Fig.4
figure 4

Expression pattern of K+ transporter and channel genes in CsRCI2D-overexpressing Camelina. One-week-old WT and CsRCI2D-overexpressing plants were treated with 150 mM NaCl for 12 h and the transcript levels of the genes were determined by real-time RT-PCR. Values are mean ± SE of three independent experiments. The same letter indicates no significant difference at P < 0.05

Full size image

CsRCI2D Decreases H2O2 and MDA under Salt Stress

In general, salt stress increases reactive oxygen species (ROS) at the cellular level disturbing the cellular metabolism (You and Chan 2015). Salt stress markedly increased the accumulation of H2O2 in both WT and transgenic plants, but the H2O2 levels in transgenic plants are much lower than those in WT (Fig. 5a). A similar pattern as that for H2O2 was observed for MDA, which is a reliable biomarker of oxidative stress. The MDA in both WT and transgenic plants was significantly increased by salt stress, but it was much lower in transgenic plants than in the WT (Fig. 5b).

Fig. 5
figure 5

Effects of salt stress on H2O2 and MDA in CsRCI2D-overexpressing Camelina. Ten-day-old WT and CsRCI2D-expressing plants were treated with 150 mM NaCl for 12 h and the levels of H2O2 (a) and MDA (b) were measured. Values are mean ± SE of three independent experiments. The same letter indicates no significant difference at P < 0.05

Full size image

Responses of Antioxidant Enzyme Genes under Salt Stress

To investigate the possible mechanism of salt tolerance in CsRCI2D-overexpressing plants, we determined the transcript levels of antioxidant genes responsible for ROS scavenging (Fig. 6). Under normal condition, two–threefold higher transcript levels of all genes, CsCuSOD1, CsCuSOD2, CsCuSOD3, CsMnSOD1, CsFeSOD1, CsFeSOD2, CsCAT1, CsCAT2, CsCAT3, CsAPX1, and CsGR2, were found in transgenic plants compared to WT. However, salt stress significantly induced the transcription of all genes in WT. In particular, CsCuSOD1 CsCuSOD3, CsMnSOD1, CsFeSOD1, CsCAT1, and CsAPX1 transcripts increased by approximately three–eightfold compared to the WT. In the case of transgenic plants, only the CsFeSOD1 transcript was further increased about sixfold by salt stress.

Fig. 6
figure 6

Effects of salt stress on the expression of antioxidant-related genes in CsRCI2D-overexpressing Camelina. Ten-day-old WT and CsRCI2D-overexpressing plants were treated with 150 mM NaCl for 12 h and the transcript levels of the genes were determined by real-time RT-PCR. Values are mean ± SE of three independent experiments. The same letter indicates no significant difference at P < 0.05

Full size image

Discussion

The present results indicate that CsRCI2D contributes positively to seed germination and postgermination seedling growth of Camelina under high salinity stress. We have recently found that CsRCI2H, a different group (no tail type) of gene, plays a role in germination and seedling growth in the presence of salt (Kim et al. 2020). Although this report implies that Group I gene is involved in the salt tolerance in plant, it is unknown the roles of Group II gene (tail type) in Camelina under salt stress. The aim of the present study is to determine the functional roles of CsRCI2D in plant salt stress responses. In general, many plant RCI2 proteins are mainly located in the PM but rarely endoplasmic reticulum (ER), Golgi apparatus, and intracellular membranes (Rocha 2016). In addition, internalization of RCI2B (LTI6b) in to the vacuolar lumen was observed in Arabidopsis when exposed to salt stress (Ueda et al. 2016). YFP-CsRCI2D fusion protein was observed in unidentified punctate structure at intracellular membrane (Fig. S2), which is the same finding as with Arabidopsis RCI2s (AtRCI2C, E, F) (Medina et al. 2007). Moreover, sucrose density gradient and western analysis showed that membrane fraction of CsRCI2D was shifted to lower density fractions when Camelina was exposed to NaCl stress. This result clearly showed localization of CsRCI2D can travel to other membrane system (Fig. 1). Recently, it was reported that several RCI2s can travel to other organelles such as early endosome and vacuole (Kim et al. 2021). It would be of interest to further verify the subcellular redistribution of CsRCI2D under various abiotic stresses.

Because salt stress induced transcript of CsRCI2D (Fig. 2a), it is likely that CsRCI2D may have important functional roles in salt stress response. The roles of salt-induced plant RCI2s including AtRCI2D (Medina et al. 2007; Rocha 2016) has been studied in yeast system. Previous reports found that the salt sensitivity of yeast △pmp3 mutant lacking PMP3 was restored following the ectopic expression of a number of RCI2 genes from Arabidopsis, rice, and maize. The current results revealed that CsRCI2D could not complement the △pmp3 yeast; however, the deletion of the C-terminal tail of CsRCI2D complemented salt sensitivity (Fig. 2b). These observations, which show that the C-terminal tail modulates the function of CsRCI2D in salt tolerance, are consistent with previously reported results for other RCI2s. CsRCI2A and CsRCI2H which have no C-terminal tail and tail-deleted CsRCI2E enhanced salt tolerance in the △pmp3 (Kim et al. 2016, 2020). Furthermore, the C-terminal tail containing alfalfa RCI2s, MtRCI2D and MtRCI2E, could not complement the △pmp3 mutant, whereas no tail genes (MsRCI2A and MtRCI2A–C) complemented the salt sensitivity (Long et al. 2015). It was suggested that two TMDs in the no tail type proteins were likely to be involved in regulation of membrane potential and consequently maintained the intracellular ion homeostasis. Therefore, it appears that the C-terminal tail in CsRCI2D may interfere regulation of membrane potential and thus directly do not function to recover salt sensitivity when expressed in the yeast. In contrast to yeast, CsRCI2D contributes to salt tolerance in Camelina. CsRCI2D overexpression accelerated seed germination and root growth in Camelina compared to WT upon high salinity (Fig. 3). Interestingly, CsRCI2D△58-overexpressing transgenic Camelina displayed similar salt tolerance during germination and seedling growth with CsRCI2D-overexpressing Camelina (Fig. 3), implying that C-terminal tail does not improve salt tolerance in Camelina. Previous studies have shown that overexpression of AtRCI2A and CsRCI2H which harbor only TMD domain without C-terminal tail enhances salt tolerance of transgenic plants (Mitsuya et al. 2006; Kim et al. 2015). Collectively, it seems that TMD but C-terminal tail may have crucial role in plant salt tolerance. Further study is needed to determine the exact manner of TMD or C-terminal tail by which different RCI2 protein functions in plants.

When plants are exposed to high salinity, increasing the content of Na+ in the cells damages plants (Marschner 1995). Excessive Na+ causes ion deficiency and ion imbalance by competing with other ions, such as Ca2+ and K+ in plants. Therefore, maintaining an elevated K+/Na+ ratio is an important mechanism to decrease the negative effects of Na+ toxicity (Giri et al. 2007). A previous study showed that the overexpression of CsRCI2H, AlTMP2 and AtRCI2A decreased the accumulation of Na+ and resultantly enhanced salt resistance in Camelina, tobacco, and Arabidopsis (Mitsuya et al. 2006; Ben-Romdhane et al. 2018; Kim et al. 2020). On the contrary, rci2a mutant that is the disruption of AtRCI2A led to an increase in Na+ accumulation and displayed salt sensitivity (Mitsuya et al. 2005). Although K+ in both root and shoot of WT showed higher or similar level compared to transgenic plants under normal condition, CsRCI2D transgenic plants inhibited the accumulation of Na+ in both root and shoot and significantly increased K+ in the shoot compared to WT under salt stress. Therefore, it is apparent that the salt tolerance of CsRCI2D transgenic plants is closely related to maintenance of higher K+/Na+ ratios in roots and shoots compared to those in WT (Table 1). Maintenance of high K+ concentration by K+ transporters in plant cells is important for salt tolerance. It appears that high CsKAT1 in transgenic plants under salt stress could confer K+ accumulation in the transgenic plant cells (Fig. 4).

In general, various stresses including high salinity induces ROS resulting in oxidative damage in plants. The present results showed that salt stress enhanced H2O2 accumulation in both WT and CsRCI2D-overexpressing plants, but transgenic plants accumulated much less H2O2 (Fig. 5a). Low H2O2 in CsRCI2D-overexpressing plants is correlated with low content of MDA which is indicator of oxidative damage (Fig. 5b), implying that low H2O2 could alleviate cellular membrane damage from oxidative stress. It is supposed that efficient ROS scavenging might occur in transgenic plants. Previous studies have found that salt stress upregulated expression of genes encoding APX, CAT, SOD, and GR in rice and chickpea (Ahmad et al. 2016; Rossatto et al. 2017). Notably, O2− scavenging enzyme genes (CsCuSODs, CsMnSOD, and CsFeSOD) and H2O2 scavenging enzyme genes (CsCATs, CsAPX, and CsGR) in WT were considerably increased by salt stress (Fig. 6). It is considered that the higher induction of antioxidant genes in WT is necessary for scavenge of excess ROS which is much higher level than transgenic plants under salt stress. Interestingly, CsRCI2D overexpression increased all antioxidant genes under normal growth condition, suggesting that possession of excellent ability of ROS scavenging in transgenic plants could confer salt tolerance through immediate ROS scavenging after salt treatment. It would be of interest to investigate whether the high expression of antioxidant genes in CsRCI2D transgenic plants is related in the other stress tolerance.

In conclusion, the results indicate that CsRCI2D enhanced plant salt tolerance during seed germination and postgermination seedling growth of Camelina through low accumulation of Na+ and ROS. It would be interesting to determine how Na accumulation is regulated during salt stress. Moreover, it would be important to determine the roles of CsRCI2D against other stress such as cold or drought, which will provide clues to understand the mechanistic roles of CsRCI2D in abiotic stress responses. As the current knowledge on the roles of CsRCI2 in plant stress is limited, it is imperative to evaluate the functions of many uncharacterized CsRCI2.

Materials and Methods

Plant Materials and Salt Stress Treatment

Camelina seedlings were grown hydroponically (Hoagland’s solution) for 6 weeks in a growth chamber at 25 °C, under a 16 h light/8 h dark photoperiod. Nutrient solution was renewed once a week. Tissue samples including roots, leaves, stems, flowers, and siliques were obtained, and the transcripts of CsRCI2D were determined using RT-PCR. To examine the effect of salt stress on CsRCI2D expression, 1-week-old Camelina seedlings were treated with 150 mM NaCl for 0, 3, 12, 24 h, and 48 h, and the transcript level of CsRCI2D was determined using real-time RT-PCR with the primers, as summarized in Table S1.

Analysis of Subcellular Localization of CsRCI2D

The coding sequence without the stop codon of CsRCI2D was amplified and then cloned into the pFAST vector to generate the YFP-CsRCI2D fusion gene under the control of the CaMV 35S promoter. Subsequently, tobacco leaves were infiltrated with Agrobacterium expressing the YFP-CsRCI2D fusion protein, and the YFP fluorescence signals were visualized under a laser scanning confocal microscope (Leica, Jena, Germany).

Membrane Preparation and Western Blot Analysis

Seven-day-old Camelina seedlings were grown on hydroponics. The seedlings were treated with or without 150 mM NaCl for 24 h. Whole plant samples were rinsed twice using distilled water before homogenization. The samples were ground using ice-cold buffer containing 50 mM 3-(N-Morpholino)propane sulfonic acid (MOPs)-BTP (pH 7.5), 330 mM sucrose, 5 mM EDTA, 5 mM dithiothreitol, 0.5 mM phenylmethanesulphonylfluoride, 0.2% (w/v) bovine serum albumin, and 0.5% PVP-40. The homogenates filtrated by four layer cheese cloth were centrifuged at 10,000g for 15 min at 4 °C. The supernatants were centrifuged at 80,000g for 45 min at 4 °C. The pellet was resuspended in buffer (5 mM MOPs-BTP, 330 mM sucrose, 5 mM KCl, 0.1 mM EDTA, and 1 mM DTT) and loaded on eight phase (15, 20, 25, 30, 35, 40, 45, and 50%) discontinuous sucrose density gradient containing 5 mM MOPs-BTP (pH 7.5) and 5 mM KCl. The sucrose gradients were centrifuged at 100,000g for 3 h at 4 °C and fourteen membrane fractions were collected. The membrane proteins (2 µg) were separated by 12–16% SDS-PAGE and the gels were subsequently used for Western blotting. Proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore). The membrane was incubated with rabbit IGg antibodies of CsRCI2D, PM H+-ATPase, V-ATPase, and binding immunoglobulin protein (BIP). HRP-conjugated anti-rabbit IgG (Santacruz) was used as the secondary antibody.

Complementation Analysis of Yeast pmp3 Mutant

ORF of CsRCI2D without stop codon and C terminal tail truncated CsRCI2D (CsRCI2D△58) were amplified by PCR using the corresponding primers (Table S1). These fragments were ligated with the yeast expression vector pYES-DEST52 (Invitrogen) and were transformed into a S. cerevisae mutant strain YDR276c lacking for pmp3 (△pmp3) which exhibits high sensitivity to NaCl. Further, BY4741 (WT) and the transformed △pmp3 cells were cultured (OD600 0.3–0.4) and tenfold dilution series were spotted onto SG agar plates supplemented with NaCl (0, 0.5, and 1 M).

Generation of CsRCI2D and CsRCI2D△58-Overexpressing Transgenic Camelina

A gateway cloning system was used to construct the overexpression vector. The ORF of CsRCI2D and CsRCI2D△58 harboring Gateway acceptor sites (5′ attR1-attR2 3′) were amplified with gene-specific primers and inserted into the pDONR zeocin vector. Subsequently, the vectors were introduced to a pCB302-3 vector which has the CaMV 35S promoter through LR reaction (Invitrogen). Siliques from flowering plants were removed and the plants were used for A. tumefaciens (GV3101)-mediated vacuum infiltration (Lu and Kang 2008). The harvested seeds were planted on 50 µg mL−1 glufosinate ammonium selection medium for the identification of the transgenic plants. After further selection of the transgenic lines employing a 3:1 segregation ratio in T1 generation, the T3 generation of the homozygous lines were utilized for a subsequent analysis.

Phenotypic Analysis

The WT and transgenic Camelina seeds were harvested at the same time and were used for the germination test and seedling growth assay. The surface-sterilized seeds were sown on Hoagland medium supplemented with 200 mM or 300 mM NaCl, and the germination rate was recorded for 4 days. The germinated seeds were incubated vertically for estimating seedling growth.

Determination of Na+, K+, and Ca2+

The WT and transgenic seeds were inoculated in the Hoagland medium. After 5 days, the seedlings were transferred to the net floated on the surface of Hoagland’s solution in a plastic box. After 3 weeks, the plants were treated with 150 mM NaCl for 12 h. The plants were washed thoroughly with distilled water and then dried at 60 °C. Dried plant materials were used for the determination of Na+, K+, and Ca2+ content by ICP-OES (Kim et al. 2019).

Determination of H2O2 and MDA Content

To detect H2O2, and MDA content, 10-day-old WT and transgenic Camelina plants were treated with 150 mM NaCl for 12 h. H2O2 was determined by measuring absorbance at 410 nm according to Wu et al. (2009). Lipid peroxidation (MDA) was determined using the thiobarbituric acid (TBA)-based colorimetric method (Heath and Packer 1968).

RNA Extraction and Real-Time RT-PCR

The WT and transgenic plants were grown hydroponically for 10 days and treated with 150 mM NaCl for 12 h. Total RNA was extracted from the tissues using TRIZOL reagent, treated with RQ1 DNase (Promega, Madison, WI, USA), and then further purified using an RNeasy Clean-up kit (Qiagen,Valencia, CA, USA). Two hundred nanogram of RNA was amplified using a one-step real-time RT-PCR kit (Qiagen) with the primers for antioxidant enzyme genes (CsCuSOD1, CsCuSOD2, CsCuSOD3, CsFeSOD1, CsMnSOD1, CsCAT1, CsCAT2, CsCAT3, CsAPX1, and CsGR) and K+ transporters (CsKEA1, CsHKT1, CsKAT1, CsAKT1) listed in Table S1. Real-time RT-PCR was conducted using a Rotor-Gene Q real-time thermal cycling system (Qiagen) and QuantiTect SYBR Green RT-PCR kit (Qiagen). CsActin was used as the internal control gene. All experiments were repeated three times with different batches of RNA samples.

Statistical Analysis

Two-way ANOVA was used for all data except for germination rate, followed by Tukey’s post hoc test. Different letters indicate significant differences between genotypes/treatments.