[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
Alfalfa (Medicago sativa L.) is a perennial leguminous plant recognised for its high nutritional value, substantial yield, broad geographical distribution, and adaptability to diverse environmental conditions. Despite its moderate salinity tolerance, alfalfa yield declines significantly in high-salinity regions [1]. Therefore, enhancing alfalfa yields in mildly saline soils is crucial for soil reclamation and the advancement of animal husbandry.

High salt concentrations induce ionic toxicity and osmotic stress [2,3]. Excess sodium ions (Na+) stimulate the accumulation of reactive oxygen species (ROS), leading to oxidative stress in plants. Plants have developed defence mechanisms to regulate ion balance and manage ionic and oxidative stress. Plasma membrane proteins are crucial in sensing and transmitting high-salt signals, such as the calcium-binding protein SOS3, Na+/H+ transporter SOS1, and HKT1 in the SOS pathway [4,5]. In addition, studies have shown that plasma membrane proteins PIP2 [6], H+-ATPase [7,8], and calmodulin (CaM) [9] play important roles in the salt stress response.

RCI2 (Rare Cold-Inducible Protein 2) is a plasma membrane protein that is significantly induced by salt, low temperature, and drought. Arabidopsis plants overexpressing AtRCI2 exhibit enhanced resistance to high salinity, demonstrating less growth inhibition and reduced Na+ accumulation [10]. Furthermore, increased exogenous Na+ levels resulted in reduced potassium (K+) content in the roots and shoots of rci2a mutants [11]. These findings suggested that RCI2 plays a crucial role in the

regulation of cation homeostasis. In addition to maintaining plasma membrane stability, RCI2 may influence membrane fluidity under salt stress. Studies have indicated that membrane fluidity in wild-type Arabidopsis under high Na⁺ or K⁺ conditions is significantly greater than in rci2a mutants, but lower than that in transgenic plants expressing 35S: MpRCI2 [12]. Moreover, RCI2 has been proposed to stabilise the plasma membrane and prevent damage during cold stress [13,14].

Overall, the significance of the RCI2 protein in mitigating salt, drought, and cold stress is evidenced by RCI2 gene overexpression in transgenic plants [15–17]. Recent discoveries of RCI2 class genes across various plant species have revealed their functional specificity [18–20]. Understanding the specific functions of RCI2 proteins requires an examination of their unique structural features. RCI2 proteins possess two transmembrane domains (TMD) and a hydrophilic C-terminal region. Arabidopsis RCI2 genes can be classified into two groups based on the presence of a hydrophilic C-terminus following the second TMD: tail-type (AtRCI2D/E/F/G) and tailless-type (AtRCI2A/B/C/H) [21]. The role of C-terminal in complementation of NaCl sensitivity in pmp3 yeast mutants is well-documented, tailless RCI2 proteins can complement Δpmp3 yeast tolerance to NaCl, while tailed RCI2 proteins failed to do so [19]. AtRCI2D (75 aa, tail-type) does not complement Δpmp3 yeast, whereas its deletion isoform AtRCI2D-D1 (57 aa, tailless-type) succeeds [21]. Despite these insights, the functional differences between tailed and tailless RCI2 proteins under stress conditions in plants remain unclear.

Long et al. [22] identified five RCI2 genes from Medicago truncatula and cloned MsRCI2A from Medicago sativa. Their pmp3 yeast complementation and green fluorescence protein (GFP) fusion experiments revealed that MtRCI2A-2C can complement yeast pmp3 mutants and localise in the cell membrane, whereas MtRCI2D and MtRCI2E fail to complement Δpmp3 and are localised in both the cell membrane and intracellular membranes, including the endoplasmic reticulum (ER) [23]. Previous studies that transformed five MsRCI2 genes into alfalfa showed differential salt and alkali tolerance among transgenic plants. Additionally, differences in the transcriptional regulation of ion channel-related genes (H⁺-ATPase, SOS1, NHX1, etc.) by MsRCI2 were observed. However, the molecular mechanisms underlying the functional specificity of tailed and tailless RCI2 genes remain unclear [24,25]. Thus, this study aims to systematically compare the functional differences between MsRCI2B (54 aa, tailless) and MsRCI2E (76 aa, tailed) in response to salt stress. We will investigate their roles in salt tolerance in transgenic alfalfa through analyses of gene expression, protein localization, and protein interactions. The objectives of this study were to elucidate the molecular mechanisms governing salt stress tolerance in alfalfa and identify candidate genes for improving alfalfa cultivars.

Germinated seeds of Alfalfa (Medicago sativa "Longmu 806") were cultivated in plastic pots filled with a 1:1 (v/v) mixture of vermiculite and perlite. The greenhouse conditions were maintained at 24 ± 2 °C with a 16-h light and 8-h dark cycle and a relative humidity of 55 %. During the initial growth phase, both the control and abiotic stress treatment groups received a 1/5 strength Hoagland nutrient solution. After approximately one month of cultivation, alfalfa lines exhibiting similar growth status were selected for further experimentation.

The seedlings were subjected to treatments with salt (200 mmol/L NaCl), alkali (200 mmol/L NaHCO₃, pH 8.5), salicylic acid (SA, 100 μmol/L), and hydrogen peroxide (H₂O₂, 100 μmol/L) for durations of 0, 1, 3, and 6 h, after which leaf samples were collected.

Given the low natural pollination rate of alfalfa, seed propagation under laboratory conditions poses challenges. Therefore, this study employed asexual propagation techniques to reproduce both wild-type (WT) and transgenic lines (OE#RCI2). The plants were irrigated with a 200 mmol/L NaCl solution at a dose of 200 mL per pot once for salt treatment.

Full-length amplification primers for MsRCI2B and MsRCI2E were designed based on the nucleic acid sequences of MtRCI2B (Medtr7g111350) and MtRCI2E (Medtr4g130660). These primers are detailed in Supplementary Table S1. Phylogenetic analysis of MsRCI2A/B/C/D/E/F, yeast PMP3 (DB661484), wheat WPI6 (AB030210), rice OsLti6a/b (AY607689, AY607690), Arabidopsis AtRCI2A/B/C/D/E/F/G/H (AT3G05880, AT3G05890, AT1G57550, AT2G24040, AT4G30650, AT4G30660, AT4G28088, AT2G38905), and Camelina sativa RCI2A/E (JQ809231, JQ809232) was performed using MEGA software. Multiple sequence comparisons of RCI2 genes were conducted using DNAMAN version 8.0 (Lynnon Biosoft) with default parameters. Phylogenetic trees were constructed using MEGA 6.2 (http://www.megasoftware.net/) applying the Neighbor-Joining method. All DNA sequences were translated into amino acid sequences for analysis.

Total RNA was extracted from the leaves, stems, and roots of 1-month-old wild-type and transgenic alfalfa lines. Plants were treated with salt, alkali, SA, and H₂O₂ solutions, and roots or leaves were collected. The Plant RNAprep Pure Kit (Kangwei, China) was used to extract total RNA according to the protocol. The extracted RNA was analyzed using a 2.0 % agarose gel and quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, USA). The first strand cDNA was immediately synthesized using the reverse transcription kit (Vazyme, China). Gene-specific primers, designed using PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), are listed in Supplementary Table S1. RT-qPCR was performed in a 96-well (10 μL) format using Trans Start Top Green qPCR SuperMix (Vazyme Biotech, Nanjing, China), with GAPDH as the internal reference gene. The relative expression level was calculated using the 2^-ΔΔCT method with three biological replicates per sample to ensure statistical significance.

For double enzyme cleavage of p2300 mCherry, the BamHI and KpnI cleavage sites were selected. Following the one-step cloning kit protocol from Vazyme was ligated to a linear vector and transferred to E. coli DH5α. Correct clones were identified, and the resulting constructs were introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterial cells were suspended in infiltration buffer containing 10 mmol/L MgCl₂, 10 mmol/L MES, and 150 μmol/L acetosyringone (pH 5.7). After infiltration into 4-week-old Nicotiana benthamiana leaves, mCherry fluorescence signals were observed and measured after 48 h using a confocal laser scanning microscope. The intensity correlation analysis function of ImageJ will determine the membrane-to-cytoplasm ratio, ensuring consistent fluorescence acquisition parameters, as detailed in Schneider et al. [26].

Yeast two-hybrid (Y2H) assays were conducted to examine the interaction between MsRCI2 and other membrane protein in yeast cells using DUALmembrane system. The SfiI site was selected as the single-enzyme cleavage site of pBT3-N vector, while the SmaI site was used for the pPR3-N vector. MsRCI2 and the membrane proteins were ligated with a linear vector according to the one-step cloning kit from Vazyme (Beijing, China), and transfected into DH5α. The resulting constructs were introduced into the yeast strain Y2HGold via co-transformation. The transformants were cultured on a selective synthetic dropout (SD) medium lacking leucine (Leu) and tryptophan (Trp) (SD/-Leu/-Trp). The yeast colonies were re-streaked onto dropout medium (SD/-Leu/-Trp/-His) containing 3 mmol/L 3-AT and grown for 5–7 days at room

temperature.

Bimolecular fluorescence complementation (BiFC) assay was performed to observe the physical interaction between MsRCI2 and membrane proteins in viable plant cells. The coding sequences of MsRCI2 and membrane proteins were individually inserted into the C-terminal of nYFP and cYFP. The single construct vector were introduced into Agrobacterium tumefaciens strain GV3101. Two positive clones of GV3101 were mixed in equal amounts, and the agrobacterial cells were suspended in an infiltration buffer. The suspensions were infiltrated into 4-week-old Nicotiana benthamiana leaves. After 48 h, YFP fluorescence signals in the tobacco leaves were observed and measured under a confocal laser scanning microscope.

GST-pull down assay was conducted to observe the interaction between MsRCI2E and MsRCI2B in vitro. The vector pET1932 and pGEX-4T were digested by EcoRV and SmaI, respectively. The coding sequences of MsRCI2B/E was ligated to a linear vector by one-step cloning. The recombinant GST-MsRCI2E and His-MsRCI2B was induced to express by Isopropylthiogalactoside (IPTG) in BL21 (DE3) E. coli. The cells were sonicated and centrifuged to remove impurities, and the supernatants were collected as total proteins. The MsRCI2B/2E protein fused with His tag or GST was purified using tag affinity purification methods with Ni-NTA agarose beads or glutathione-Sepharose 4B beads, respectively. Equal amounts of GST-MsRCI2E and His-MsRCI2B proteins were incubated with the respective beads in binding buffer (20 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, and 5% glycerol) at 4°C for 2 h. The beads were then pelleted and washed for five times with binding buffer before being boiled in 1× SDS loading buffer for 5 min. The proteins were fractionated using SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was immunoblotted with antibodies specific to the anti-His or GST epitopes.

The coding sequence of MsRCI2s-GFP/mCherry or another gene driven by the CaMV 35S promoter was cloned into the pMDC123 binary expression vector (35S::Gene-GFP/Cherry). To construct a dual-gene co-expression vector, one gene fragment from 35S::Gene-GFP/Cherry was amplified and ligated into another gene expression vector using one-step cloning approach. The resulting constructs were introduced into the Agrobacterium rhizogenes LBA4404 strain for transformation into the Longmu 806 cultivar using the cotyledonary node transformation system. Selection of resistant plants was carried out using 1.0 mg/L glufosinate-ammonium [24,25].

Semi-quantitative RT-PCR and quantitative real-time RT-PCR were performed to detect the mRNA expression levels of MsRCI2 in transgenic alfalfa. Gene-specific primers were designed using Primer 5.0 software (Table S1), and the reaction specificity was assessed by examining the melting curves of RT-PCR products. The relative expression level was calculated using the 2^-ΔΔCt method, with three biological replicates.

Each experiment was performed in triplicate, yielding consistent results. Different letter indicates significant differences according to Duncan’s multiple range test at p < 0.05 using One-way AMOVA.

(A)
Wild-type (WT) and transgenic plants were subjected to a 200 mmol L⁻¹ NaCl solution. The relative chlorophyll content in the leaves was measured using a chlorophyll analyzer (TYS-B) at 11 a.m. on the sixth day of salt stress, under full sunlight. Relative conductivity, proline (Pro), soluble sugar, and malondialdehyde (MDA) contents were measured to complement the phenotypic analysis and evaluate the salt tolerance of the transgenic plants. Relative conductivity was determined using the vacuum infiltration method. MDA, Pro, and soluble sugar contents were analyzed using physiological kit (Suzhou Keming Biotechnology Co., Ltd., China), with measurements conducted on a UV–visible spectrophotometer. Antioxidant enzymes activities were also evaluated following salt treatment. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were measured using the physiological kits, while hydrogen peroxide (H₂O₂) and oxygen free radical (OFR) contents were detected and quantified using a UV–visible spectrophotometer. Plant materials were then subjected to in diaminobenzidine (DAB), nitroblue tetrazolium (NBT) staining solution, placed in a vacuum for 30 min, and stained overnight. The next day, stained leaves were transferred to a decolorizing solution (ethanol: acetic acid: glycerol = 3:1:1) and boiled in water bath until all chlorophyll residues were removed.

(B)
To prepare the standard solution, 1.907 g of analytical grade KCl and 0.6355 g of NaCl were weighed and dissolved in deionized water to a final volume of 1 L. Leaves and roots of WT and transgenic alfalfa were initially dried at 105°C, followed by further drying in an oven at 80°C until a constant weight was achieved. A 0.05 g sample was weighed from each dried plant material. The nitration solution was prepared by mixing 5 mL of concentrated nitric acid, 1 mL of 60% trichloroacetic acid, and 0.5 mL of concentrated sulfuric acid. This solution was added to the sample, which was subsequently extracted in a constant-temperature water bath at 90°C. The resulting solution was filtered and analyzed for Na⁺ and K⁺ content using flame atomic absorption spectrometry.

employed for each sample to ensure statistical significance.

(C) BiFC assay illustrating the interaction of MsRCI2B with MsRCI2E. MsRCI2B and MsRCI2E were cloned into vectors containing YFPN or YFPC sequences. The resulting plasmids were co-expressed in tobacco leaves for 16 h, and YFP signals were observed using a confocal microscope.

All experiments in this study were performed in triplicate, and the average of the three measurements was calculated for analysis. GraphPad Prism 9.0 was used for graphical analysis, while one-way ANOVA was employed for significance testing (p < 0.001 represented by ***, p < 0.01 represented by **, p < 0.05 represented by *).

To explore the differences in abiotic stress responses between tailless MsRCI2B and tailed MsRCI2E, we analyzed their gene expression under treatments with 100 μmol/L SA, 200 mmol/L NaCl, 200 mmol/L NaHCO₃, and 100 μmol/L H₂O₂ (Fig. 1A). After NaCl treatment, the expression of MsRCI2B/E peaked after 1 h of treatment. For the NaHCO₃ treatment, both MsRCI2B/E initially decreased but subsequently increased, reaching peak levels at 12 h. In contrast, exogenous SA treatment resulted in distinct expression patterns, with MsRCI2B showing significant upregulation, peaking at 1 h and then decreasing.

an additional 20 hydrophilic amino acid C-terminal tails following the second TMD, therefore, MsRCI2E was classified as tailed-RCI2. Phylogenetic analysis (Fig. S1B) categorized these proteins into two distinct groups based on their branching patterns. MsRCI2A-C from alfalfa and AtRCI2A-B from Arabidopsis were grouped into one branch, while MsRCI2D-F and AtRCI2C-H formed another branch.

MsRCI2B and MsRCI2E were amplified using cDNA as a template, resulting in 165 bp and 231 bp fragments, respectively. Compared to other members of the RCI2 gene family, including MtRCI2s and AtRCI2, both MsRCI2B and MsRCI2E retain the characteristic features of PMP3 proteins, such as two putative transmembrane domains (TMD), as indicated by arrows in Fig. S1A. The amino acid sequence similarity between MsRCI2B and MsRCI2E was 55.76%, with MsRCI2E containing an additional 20 hydrophilic amino acid C-terminal tails following the second TMD, therefore, MsRCI2E was classified as tailed-RCI2. Phylogenetic analysis (Fig. S1B) categorized these proteins into two distinct groups based on their branching patterns. MsRCI2A-C from alfalfa and AtRCI2A-B from Arabidopsis were grouped into one branch, while MsRCI2D-F and AtRCI2C-H formed another branch.

The expression levels of MsRCI2B/E did not change significantly after H2O2 treatment.

We investigated the subcellular localization of tailless MsRCI2B and tailed MsRCI2E using 35S:mCherry fusion fluorescent proteins, including MsRCI2Em (a C-terminal tail-deleted variant of MsRCI2E). As shown in Fig. 1B, the MsRCI2B-mCherry fusion protein was exclusively localised to the plasma membrane (PM), whereas the MsRCI2E-mCherry fusion protein was observed in both the PM and intracellular compartments. The MsRCI2Em-mCherry fusion protein was predominantly localised in the PM. Analysis using ImageJ software (Fig. 1C) revealed that the membrane-to-cytoplasm ratios of MsRCI2B and MsRCI2Em were significantly higher than that of MsRCI2E (p < 0.001). These results indicate that tailless RCI2 proteins were primarily localised to the plasma membrane. In contrast, tailed RCI2 proteins were found in the internal membranes. This observation aligns with previous localization studies of CsRCI2F [27].

We cloned six RCI2 genes (MsRCI2A-F) from alfalfa and validated their interactions using a DUAL membrane yeast two-hybrid (Y2H) system (Fig. S2). Only yeast cells harbouring the RCI2A/B/D-pBT3-N and RCI2E-pPR3-N pairs grew on SD/-Leu-Trp-His containing 3 mM 3-AT for high stringency, even at a 1000-fold dilution. This suggests that MsRCI2E interacts heterologously with MsRCI2A/B and MsRCI2D to form heterodimers but does not form homodimers. We further explored the RCI2B–RCI2Em interaction; however, yeast cells harbouring RCI2B-pBT3-N and RCI2Em-pPR3-N did not grow on SD/-Leu-Trp-His medium (Fig. 2A), indicating that deletion of the C-terminal tail of MsRCI2E prevented its interaction with MsRCI2B. To further validate this interaction, we expressed and purified GST, GST-MsRCI2E, and His-MsRCI2B recombinant proteins from the E. coli system for in vitro GST pull-down assays. Biochemical data showed that GST-MsRCI2E, but not GST, specifically pulled His-MsRCI2B down (Fig. 2B). This indicated that MsRCI2B physically interacts with MsRCI2E in vitro.

MsRCI2B is localised in the plasma membrane (PM), whereas MsRCI2E is localised within the cell. Bimolecular fluorescence complementation (BiFC) was used to study their interactions and localization. When the MsRCI2E-YFPC and MsRCI2B-YFPN fusion proteins were transiently co-expressed in tobacco leaf cells, yellow fluorescence was observed in the plasma membrane (Fig. 2C), indicating that the interaction between MsRCI2B and MsRCI2E was concentrated in the PM. Moreover, after salt stress treatment for 3 h, the two proteins interacted with each other and their localization remained unchanged.

To assess the salt and alkali tolerance of individual RCI2 proteins and their interactions, MsRCI2B and MsRCI2E were separately transferred into yeast cells and co-transferred as MsRCI2B–RCI2E. Yeast growth was monitored on YPDA solid medium with varying NaCl concentrations (0, 100, 150, and 200 mmol/L) and pH levels (7, 7.5, 8, and 8.5) (Fig. S3). These results indicate that MsRCI2B and the MsRCI2B–RCI2E co-transfer groups exhibited greater salt and alkali tolerance than the MsRCI2E group.

To further investigate the effects of MsRCI2E and MsRCI2B on alfalfa growth and salt tolerance, we generated transgenic alfalfa lines overexpressing MsRCI2E, MsRCI2B, and MsRCI2Em (OE#RCI2E, OE#RCI2B, and OE#RCI2Em) (Fig. S4A). Additionally, we obtained transgenic alfalfa co-transformed with both MsRCI2B and RCI2E genes (OE#RCI2B-2E) to assess the synergistic effects on salt tolerance. Following selection with glyphosate, PCR verification (using the bar gene), and qPCR (using the MsRCI2 genes), we isolated 5–7 independent transgenic lines (Fig. S4BC). Root fluorescence assays were conducted on RCI2-overexpressing lines with the fusion of fluorescent protein genes (RCI2B-Cherry, RCI2E-GFP) (Fig. S4D). Transgenic alfalfa co-expressing RCI2E and RCI2B (OE#RCI2B-2E 1#, 2#, and 12#) exhibited significantly higher expression levels of both genes than non-transgenic controls. Colocalization of Cherry and GFP fluorescence signals in the roots indicated successful coexpression.

For growth and salt tolerance analyses, we subjected RCI2-overexpressing alfalfa lines, including those coexpressing RCI2B and RCI2E, and non-transgenic control alfalfa (WT) to salt stress. Under control conditions, notable differences were observed in plant height, electrical conductivity, chlorophyll content, and branching number between some transgenic lines and WT (p < 0.05 or p < 0.001), particularly in the OE#RCI2E line (Fig. 3, Fig. S5). After 10 days of treatment with 200 mmol/L NaCl, WT growth was severely inhibited, exhibiting leaf wilting and near death. In contrast, transgenic alfalfa maintained better growth with fewer yellowed leaves (Fig. 3A). Differences in salt tolerance among transgenic lines were evident: OE#RCI2E had significantly higher plant height (p < 0.001), OE#RCI2E and OE#RCI2B-2E also exhibited higher chlorophyll content. Although the most salt-tolerant transgenic line remains uncertain, these results confirm that RCI2B, RCI2E, and the C-terminal deletion of RCI2E (RCI2Em) play distinct roles in alfalfa salt tolerance.

The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as the proline, soluble sugar, and malondialdehyde (MDA) concentrations, were measured in all transgenic alfalfa lines (OE#) and wildtype lines (WT) under salt treatment (Fig. 3 and Fig. S5). Following salt treatment, both proline and soluble sugars increased significantly in all lines, with transgenic lines exhibiting markedly higher levels than the WT lines (p < 0.001) (Fig. S5DE). In contrast, SOD and POD activities were significantly higher in transgenic lines under untreated conditions. After salt treatment, SOD, POD, and CAT activities increased significantly in all transgenic lines and were markedly higher than those in the WT plants (Fig. 3C). However, the MDA levels were lower in the transgenic lines than in the WT lines (Fig. 3D).

In additional, the levels of H2O2 and oxygen free radicals (OFR) were measured, and diaminobenzidine (DAB), nitroblue tetrazolium (NBT) staining was performed (Fig. 3D, Fig. S5FG). Under untreated conditions, OE#RCI2B and OE#RCI2B-2E lines exhibited lower H2O2 levels than WT, while the OE#RCI2E line had higher OFR levels. After salt treatment, the H2O2 and OFR levels were significantly lower in all transgenic lines than in the WT, with DAB, and NBT staining, corroborating the changes observed in the H2O2 and OFR levels.

Although all transgenic lines demonstrated enhanced antioxidant capacity, variations were observed between them. Specifically, the OE#RCI2E line exhibited higher conductivity, H2O2 and OFR levels, and lower CAT enzyme activity than the OE#RCI2B and OE#RCI2Em lines. Thus, the presence of the C-terminal tail of MsRCI2E appears to reduce the gene's ROS-scavenging capacity, while interactions between RCI2B and RCI2E proteins may complement the function of the RCI2E gene.

To elucidate the mechanisms underlying salt tolerance in transgenic alfalfa, we comprehensively analyzed Na+, K+, and Na+/K+ ratios in the roots and leaves of both the transgenic and WT lines (Fig. 4AB). Under control conditions, K+ content varied significantly, with the OE#RCI2Em line exhibiting markedly lower K+ levels than WT (p < 0.001). A consistent pattern emerged after exposure to salt stress for 10 days: K+ content declined, Na+ content increased, and Na+/K+ ratios increased. All transgenic lines, except OE#RCI2E, notably exhibited significantly lower Na+ content and Na+/K+ ratios than the WT. Most lines did not exhibit

significant differences in root except OE#RCI2E-2B, which consistently displayed higher K+ content than other lines (p < 0.01). Furthermore, OE#RCI2B lines exhibited significantly elevated Na+/K+ ratios after stress (p < 0.001), indicating a unique response to salt stress in the root.

To gain insights into the molecular mechanisms underlying these changes in ion homeostasis, we analyzed the expression levels of the key ion transporter genes MsSOS1, MsAKT1, and MsNHX1 (Fig. 4CD). MsSOS1, a plasma membrane Na+/H+ antiporter, consistently upregulated upon salt treatment, with OE#RCI2B and OE#RCI2Em lines exhibiting significantly higher MsSOS1 expression than WT and other transgenic lines (p < 0.001). MsAKT1, a K+ transport channel, responds variably to salt stress in leaves and roots. In leaves, OE#RCI2B and OE#RCI2B-2E lines displayed the highest MsAKT1 expression, which correlated with lower Na+/K+ ratios, suggesting efficient K+ uptake and transport. In roots, however, MsAKT1 expression was highest in OE#RCI2B and OE#RCI2Em, with OE#RCI2B-2E showing the lowest. MsNHX1, a vacuole Na+/H+ antiporter, was most pronouncedly upregulated in OE#RCI2Em lines (p < 0.001). Overexpression of RCI2 genes influences plasma membrane ion transporters and affects ion homeostasis under salt stress. Notably, MsRCI2E overexpression partially balanced ion homeostasis, albeit less effectively than MsRCI2B. The truncated RCI2Em variant demonstrated superior regulation, whereas the interaction between RCI2B and RCI2E appeared to compensate for the functional deficiencies in RCI2E.

We also assessed the expression of MsRCI2A-2F in each transgenic alfalfa line (Fig. S6). Overexpression of MsRCI2B or MsRCI2Em genes upregulates MsRCI2A, MsRCI2D, and MsRCI2F genes, whereas OE#RCI2E lines exhibited a significant decrease in MsRCI2B expression. After salt stress treatment, MsRCI2A, MsRCI2D, and MsRCI2F gene expression increased in OE#RCI2B and OE#RCI2Em lines. These findings suggest complex regulatory interactions among RCI2 genes, each fulfilling a distinct function without redundancy.

Regarding the interaction of the RCI2 protein, Kim et al. [27]

Fig. 5. Interaction of MsRCI2B/E with MsPIP1;4 and the function of protein interaction in salt tolerance.
(A) BiFC assay demonstrating the interaction between MsRCI2E and PIP1;4. The plasmid was co-expressed in tobacco leaves for 16 h, and fluorescence signals were observed before and after treatment with 50 mmol/L NaCl for 6 h.
(B) Yeast two-hybrid assays showing the interaction of PIP1;4 with MsRCI2E/Em.
(C) Comparison of fluorescence signal intensity inside and outside the membrane.
(D, E) H₂O₂ content and NBT staining of WT and transgenic lines (OE#) under salt stress.
(F, G) OFR content and DAB staining of WT and transgenic lines (OE#) under salt stress. Each experiment was conducted in triplicate, with at least three technical replicates. Statistical comparisons were performed using Tukey's test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

reported an association between CsRCI2E and CsRCI2F with CsPIP2;1. To further explore this association, we employed a yeast two-hybrid approach to screen for plasma membrane proteins (PMPs) that specifically interact with MsRCI2B or MsRCI2E. Our findings revealed that MsCaM1 interacts with MsRCI2B, whereas MsPIP1;4 and MsHVP1 interact with MsRCI2E. These interactions were subsequently validated using the BiFC assay (Figs. 5A, 6A, S7), indicating that the interacting proteins were not solely confined to the plasma membrane but were also present within the cytoplasm. Notably, the MsPIP1;4-MsRCI2E interaction produced a yellow fluorescent ring proximal to the plasma membrane, suggesting a vesicular structure accompanied by additional fluorescence within the cell. Another, yeast two-hybrid experiments revealed that the interaction between MsRCI2Em and MsHVP1 was lost (Fig. 6B), while its interaction with MsPIP1;4 were notably weakened (Fig. 5B).

MsPIP1;4, a plasma membrane aquaporin, is most similar to Arabidopsis PIP1 (At4g00430) [28]. Although PIP1 and PIP2 have similar amino acid sequences and molecular weights, their channel activities and functions differ [29,30]. To investigate the effect of MsPIP1;4 and MsRCI2E specific interaction on salt tolerance in alfalfa, salt stress treatments were performed on tobacco with transient expression based on BiFC. After 3 h of salt stress, interacting proteins were enriched in the stomata guard cell. Following 6 h of salt stress, yellow fluorescence was enhanced in the cytoplasm (Fig. 5AC).

Additionally, we obtained several alfalfa lines: overexpressing the MsPIP1;4 gene (OE#PIP1;4), co-expressing MsPIP1;4 and MsRCI2E (OE#RCI2E-PIP). These transgenic lines and WT plants were subjected to salt stress. Before and after salt stress treatment, the OE#PIP1;4 lines exhibited lower levels of H2O2 in the leaves. After salt treatment, the levels of OFR and MDA were the lowest among all lines (Fig. 5DF, S9A).

The results of DAB and NBT staining corroborated the measurements of H2O2 and OFR (Fig. 5EG). However, in the co-transformed lines (OE#RCI2E-PIP), there were no significant differences in H2O2 and OFR levels compared to the WT. Moreover, the activities of antioxidant enzymes SOD, POD, and CAT showed no significant differences between the OE#PIP1;4 and OE#RCI2E-PIP lines (Fig. S9B). The expression of the H2O2 production-related gene, MsANN1, did not differ significantly between the two lines (Fig. S9C). Given that neither H2O2 production nor ROS-scavenging enzyme activity differed significantly, the variation in H2O2 levels in the leaves may be related to its efflux. Based on the localization results of the interacting proteins MsPIP1;4 and MsRCI2E, it is inferred that the interaction between MsRCI2E and MsPIP1;4 is closely associated with the intracellular trafficking of PIP1;4 proteins. This interaction likely reduces PIP1;4 proteins on the plasma membrane and a diminished ability to export H2O2.

H+-PPases (proton-pumping pyrophosphatases, HVP) are a class of membrane-bound pyrophosphatases that generate energy by hydrolysing inorganic pyrophosphate (PPi) and transporting protons across the cell membrane [31]. More studies have indicated that H+-PPase plays a crucial role in plant resistance to abiotic stress [32]. In the OE#RCI2E transgenic lines, the expression of the MsHVP1 gene was significantly upregulated, and the enzyme activity of H+-PPase was also significantly increased compared to WT plants (Fig. 6C). Thus, MsRCI2E positively regulated the function of H+-PPase under salt stress. Similarly, we obtained different transgenic lines and preliminarily determined the role of the interaction between MsRCI2E and HVP1 in the plant response to salt stress through differences in salt tolerance among transgenic lines. After salt stress treatment, the OE#RCI2E and OE#RCI2E-HVP lines exhibited higher levels of K+ compared to WT (p < 0.01, p < 0.001), along with elevated Na+ levels and Na+/K+ ratios (Fig. 6D). This indicated that the interaction between MsRCI2E and HVP1 enhanced the ion balance under salt stress and reduced Na+ influx. Additionally, the expression levels of ion channel-related genes such as NHX1, PM-H+-ATPase, SOS1, and AKT1 were significantly higher in the OE#RCI2E-HVP line compared to the OE#RCI2E line and markedly higher than in WT (Fig. 6E). These findings suggest that the interaction between MsRCI2E and HVP1 plays a synergistic role in enhancing salt tolerance in alfalfa.

In summary, the interactions of MsRCI2E with MsPIP1;4 or HVP1 exerted distinct effects on the salt tolerance of alfalfa, highlighting the role of RCI2s in salt stress responses through interactions with membrane proteins.

The special function of the C-terminal tail of the MsRCI2E protein

In alfalfa, there are six RCI2 genes that can be classified into tailless-type RCI2 (MsRCI2A-D, 54 amino acids) and tailed-type RCI2 (MsRCI2E, 76 amino acids) (Fig. S1). In Arabidopsis, tailless AtRCI2 (A-C) and AtRCI2H are found in the plasma membrane (PM), whereas tailed AtRCI2D is predominantly located in the endoplasmic reticulum (ER) [21]. Similarly, in Camelina sativa, tailless CsRCI2A is localised in the PM, whereas tailed CsRCI2D is found in both the membrane and cytosol, including the ER and endomembrane vesicles [19,33]. In alfalfa, tailless MsRCI2D [24] and MsRCI2B are localised to the PM, whereas tailed MsRCI2E is present in both the membrane and the cytosol (Fig. 1).

Renard et al. demonstrated that deletion of the C-terminal tail of SNA2 (mSNA2) shifts its localization from the PM to the ER, suggesting that the C-terminal tail may serve as a destination sequence that directs the protein to its correct location [34]. To investigate this, we truncated the C-terminus of MsRCI2E (MsRCI2Em). Analysis using mCherry fusion proteins revealed that MsRCI2Em, similar to the tailless MsRCI2B, was exclusively localised to the PM (Fig. 1). This finding indicated that the C-terminal tail significantly influenced the intracellular localization of RCI2 proteins.

In Camelina, CsRCI2D proteins lacking the tail complemented Δpmp3 yeast, but adding a C-terminal tail of CsRCI2D to a tailless RCI2 protein hindered its ability to complement NaCl tolerance in Δpmp3 yeast [19]. However, the functions of the C-terminal tail-deleted RCI2-overexpressing plants have not been extensively studied. We cloned MsRCI2E and its C-terminal tail-deletion variant, MsRCI2Em, and generated transgenic alfalfa lines overexpressing the MsRCI2E gene and MsRCI2Em. Salt tolerance evaluation revealed that OE#RCI2Em lines exhibited greater salt tolerance than OE#RCI2E lines, characterised by enhanced ROS scavenging and antioxidant capacity (Fig. 3) and improved ion homeostasis (Fig. 4).

The differing salt tolerance functions of MsRCI2E and MsRCI2Em may result from changes in cellular localization and variations in protein interactions or regulation. Our results revealed that MsRCI2E interacts with MsRCI2B, MsHVP1, and MsPIP1;4. Notably, deletion of the C-terminal tail in MsRCI2E results in the loss of interactions with MsRCI2B and MsHVP1, as well as a weakened interaction with MsPIP1;4, suggesting that the C-terminal tail is crucial for interactions with specific plasma membrane proteins. Additionally, differences in the expression of genes such as MsSOS1, MsNHX1, and MsAKT1 between OE#MsRCI2E and OE#MsRCI2Em lines (Fig. 4) further emphasise the importance of the C-terminal tail in mediating MsRCI2E’s response to salt stress through its regulation of or interaction with membrane proteins.

Functional differences of MsRCI2B and MsRCI2E genes undergoing salt stress

RCI2 is crucial for various abiotic stress responses, particularly for regulating plant tolerance to cold, drought, alkaline, and salt stress [21]. However, within the same species, different RCI2 genes exhibit functional specificity [23]. In this study, both MsRCI2B and MsRCI2E responded to salt stress; however, their expression levels and trends differed significantly. Specifically, MsRCI2B exhibited higher expression levels than MsRCI2E under salt and alkaline stress (Fig. 1A). Additionally, overexpressing the MsRCI2B gene resulted in stronger salt tolerance compared to overexpressing MsRCI2E, as evidenced by higher soluble sugar accumulation, increased SOD and CAT activities, and lower ROS and MDA levels (Fig. 3). Furthermore, MsRCI2B better regulates ion homeostasis under salt stress, as indicated by its lower Na+/K+ ratio (Fig. 4). These differences may be attributed to disparities in gene structure, expression, regulation, and protein function [35].

The regulatory mechanisms of RCI2 expression remain underexplored, and no known transcription factors have been identified for these genes. Previous studies have indicated that overexpression of MsRCI2 genes can regulate the expression of other MsRCI2 genes [24,25]. This suggests a distinct regulatory mechanism between MsRCI2B and MsRCI2E. A yeast two-hybrid assay revealed that MsRCI2E interacts with MsRCI2B and MsRCI2D (Fig. S2). Still, none of the RCI2 proteins formed homodimers, indicating that regulatory relationships may be transcriptional or dependent on protein expression balance.

Several transgenic lines evidence indicate that functional specialisation among RCI2 paralogs is determined by differences in the activities of their cognate proteins. Liu et al. found reduced H+-ATPase activity and gene expression in rci2a mutants compared with wild-type and 35S: MpRCI-rci2a transgenic plants [12]. Our findings similarly showed that overexpressing MsRCI2B and MsRCI2E affected the expression of MsSOS1, MsNHX1, and MsAKT1 genes, with OE#RCI2B lines showing significant upregulation compared to OE#RCI2E lines (Fig. 4). This suggests that the different regulatory patterns of MsRCI2B and MsRCI2E concerning ion transport genes contribute to their distinct functions in salt tolerance.

Two main factors contribute to the functional differences in salt tolerance between MsRCI2B and MsRCI2E. First, the C-terminal tail of MsRCI2E influenced its localization and protein interactions. As previously mentioned, deletion of this tail in MsRCI2E significantly enhanced

Most researchers believe that, because of their small size, RCI2s cannot function as ion transporters on their own [39]. However, this does not preclude the possibility that the two RCI2 oligomers form transporters or interact with other membrane proteins. In this study, we found that MsRCI2B interacts with MsRCI2E but does not interact with itself. Upon interaction, these two proteins localised to the plasma membrane (Fig. 2). However, based on available data, it is difficult to determine whether their interaction leads to dimer formation or the establishment of an ion channel. Indeed, the ionic regulation capability of alfalfa co-transformed with MsRCI2B and MsRCI2E was not superior to that of the OE#MsRCI2B lines.

We compared the salt tolerance of various transgenic alfalfa lines, including those individually transformed with MsRCI2B or MsRCI2E and those co-transformed with both genes. The OE#RCI2B-2E lines exhibited higher levels of proline and soluble sugars and lower ROS compared to the OE#RCI2E lines (Fig. S5), and they showed the lowest Na⁺/K⁺ ratios (Fig. 4A). Additionally, the expression levels of MsAKT1 in the leaves of OE#MsRCI2B-2E lines were significantly higher (Fig. 4CD). These observations suggest that the interaction between MsRCI2B and MsRCI2E may alleviate the negative regulatory effects of the C-terminal tail on plant salt tolerance and enhance alfalfa salt tolerance by modulating the expression of ion homeostasis-related genes.

salt tolerance and improved antioxidant and ion homeostasis. Second, the specificity of interacting proteins is crucial. MsRCI2B interacts with CAM1, whereas MsRCI2E interacts with PIP1;4 and HVP1. These proteins are all involved in regulating ion transport and salt stress signalling [36–38]. The specificity of the protein interactions ultimately determines the functional diversity of RCI2 proteins.

RCI2 proteins play an important role in maintaining the ion balance, primarily through interactions with other proteins. Using MsRCI2B and MsRCI2E as bait in a yeast two-hybrid assay, we identified MsCaM1, which interacts with MsRCI2B; MsHVP1, which interacts with MsRCI2E; and MsPIP1;4, which interacts only with MsRCI2E. MsCaM1 is analogous to Arabidopsis CaM7 (At3g43810) [40]. Calmodulin (CaM) is ubiquitous in eukaryotes and is a highly conserved Ca²⁺ binding protein that plays multiple regulatory roles in response to various stimuli, including salt, cold, and drought stresses [41,42]. Therefore, the role of the MsRCI2B in regulating ion homeostasis is closely linked to MsCaM1.

H⁺-PPases (proton-pumping pyrophosphatases, HVP) are a class of membrane-bound pyrophosphatases. In Arabidopsis, overexpression of AVP1 leads to increased salt tolerance, primarily due to enhanced vacuole uptake of Na⁺, which mitigates the toxic effects of Na⁺ [43]. Our study found that the C-terminal tail of MsRCI2E is essential for its interaction with MsHVP1. Co-transformation of RCI2E and HVP1 enhanced ion homeostasis regulation and reflected lower Na⁺/K⁺ ratios. This co-transformation also positively regulated the expression of key genes, such as MsSOS1, MsHKT1, MsNHX1, and MsH⁺-ATPase (Fig. 6), suggesting that MsRCI2E collaboratively regulates ion homeostasis through its interaction with MsHVP1.

Interestingly, MsRCI2E's interaction between MsPIP1;4 was localised near the plasma membrane, within vesicles, and in the cytoplasm, after salt treatment, interaction proteins trafficking into cytoplasm. While the C-terminal tail of RCI2E is not essential for this interaction, its absence weakens binding. Co-transformation with MsPIP1;4 and MsRCI2E enhanced antioxidant enzyme activity; however, the H₂O₂ content in the leaves did not significantly differ from that in the wild-type (Fig. 5D). Research indicates that PIP2 facilitates water permeability. In contrast, PIP1's water transport capability remains controversial [44]. PIPs also exhibit broad-channel activity for various small molecules, including glycerol, CO₂, and H₂O₂ [45,46]. Zhang et al. found that OsPIP2 enhances H₂O₂ permeability under alkaline stress [47]. Furthermore, studies show that AtPIP2 transports H₂O₂ into the cytoplasm via endocytosis under hyperosmotic stress [48]. The diversity of the PIP protein function may explain the unique findings of the present study. In this study, we revealed that under normal conditions, MsPIP1;4 is predominantly localised in vesicle-like structures near the plasma membrane. However, following salt stress treatment, MsPIP1;4 was re-transported to the cytoplasm and guard cells of the stomata (Fig. S8). This dynamic shift in localization closely resembles the behavior of Arabidopsis PIP2;1, as previously reported [49]. In addition, MsPIP1;4 facilitates H₂O₂ efflux, and before and after salt stress treatment, the OE#PIP1;4 lines showed lower H₂O₂ levels in leaves. The interaction between MsRCI2E and MsPIP1;4 assists the intracellular trafficking of MsPIP1;4, leading to reduced PIP1;4 protein levels in the plasma membrane and diminished H₂O₂ efflux. Despite the higher antioxidant enzyme activity, intracellular H₂O₂ accumulation remains notable. H₂O₂ acts as both a reactive oxygen species and a signalling molecule. Thus, the MsRCI2E-MsPIP1;4 interaction under salt stress may not be negative. Since

MsRCI2B also interacts with MsRCI2E, we speculate that interactions among these three proteins may maintain a balance, ensuring PIP1;4 homeostasis at the plasma membrane and regulating intracellular H2O2 levels.

In summary, MsRCI2B is localised to the plasma membrane, where it interacts with proteins such as calmodulin (CaM) and MsRCI2E. In contrast, MsRCI2E is present in both the plasma membrane and cytoplasm, interacting with the aquaporin PIP1;4 and the vacuolar proton pump HVP1. Under salt stress, increased Na+ influx raises cytosolic Ca2+ levels, activating CaM to regulate ion homeostasis. Meanwhile, MsRCI2E enhances HVP1 activity, promoting Na+ sequestration into vacuoles and reducing cytoplasmic ion toxicity. Additionally, MsRCI2E assists PIP1;4 trafficking from plasma membrane to cytoplasm, limiting H2O2 and water efflux, which helps maintain ROS balance and prevent water loss. The interaction between MsRCI2B and MsRCI2E may further modulate MsRCI2E’s function, enhancing plant salt stress tolerance (Fig. 7).

Depeng Zhang: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. Zhongbao Shen: Resources, Project administration, Funding acquisition, Conceptualization. Pin He: Visualization, Methodology, Investigation, Formal analysis. Jianli Wang: Methodology, Investigation, Formal analysis. Donghuan Li: Visualization, Methodology, Investigation, Formal analysis. Jing Meng: Investigation, Formal analysis. Dongmei Zhang: Investigation, Formal analysis. Jia You: Visualization, Formal analysis. Yaqin Luo: Visualization, Formal analysis. Xinsheng Wang: Methodology, Formal analysis. Xu Zhuang: Investigation. Linlin Mu: Investigation. Shichao Zhang: Formal analysis. Weibo Han: Project administration, Funding acquisition, Conceptualization. Hua Cai: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This research was financially supported by the National Natural Science Foundation of China (32071866), and the Science and Technology Innovation 2030-Major Project (2022ZD040120401).

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.140093.

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