Salt stress-accelerated proteasomal degradation of LBD11 suppresses ROS-mediated meristem development and root growth in Arabidopsis

Abstract

Roots absorb water and nutrients from the soil, support the plant’s aboveground organs, and detect environmental changes, making them crucial targets for improving crop productivity. Particularly sensitive to soil salinity, a major abiotic stress, roots face significant challenges that threaten global agriculture. In response to salt stress, plants suppress root meristem size, thereby reducing root growth. However, the mechanisms underlying this growth restriction remain unclear. Here, we investigate the role of reactive oxygen species (ROS) in this process and reveal that LATERAL ORGAN BOUNDARIES DOMAIN 11 (LBD11) plays a central role in ROS-mediated regulation of meristem size and the salt stress-induced inhibition of root growth. Under normal conditions, LBD11 controls the expression of key ROS metabolic genes, maintaining ROS homeostasis within root developmental zones to control meristem size and overall root growth. Upon sensing salt stress, LBD11 undergoes rapid proteasome-mediated degradation, leading to decreased distribution of O₂^⋅−, which in turn curtails meristem size and limits root length. Our findings highlight an unexplored plant adaptation strategy, where the growth-promoting LBD11/ROS pathway is downregulated to finely regulate root growth under challenging conditions. We propose a strategy for developing crops with heightened resilience and increased yields in salt-affected environments.

Plants inhibit meristem size and root growth under salt stress. However, the molecular mechanisms underlying this active growth inhibition remain unclear. This study provides evidence demonstrating that LBD11-modulated ROS distribution plays a crucial role in mediating the repression of meristem size and root growth, facilitating plant adaptation to high salinity.

Introduction

Roots are essential for plant growth and development, absorbing water and minerals from the soil and providing structural support (Tajima, 2021). They also detect changes in the surrounding soil and dynamically adjust their growth to optimize resource access and acquisition (Van Zelm et al., 2020; Jia et al., 2022). Understanding the developmental plasticity of roots is critical for securing crop production.

High soil salinity limits water availability due to sodium accumulation, which increases the soil’s osmotic pressure, disrupts nutrient uptake, and induces ion toxicity, thereby inhibiting growth and causing yield losses (Van Zelm et al., 2020). Unlike halophytes, which can grow and reproduce in very highly saline soils (Flowers et al., 2015), most crop plants are salt-sensitive glycophytes (Julkowska and Testerink, 2015). Optimizing crop salt tolerance could significantly increase yields on salinized lands. Studies suggest that mitigating stress-triggered growth reduction is beneficial for plant survival under adverse conditions (Skirycz et al., 2011; Zhang et al., 2020). This active growth inhibition is distinct from the passive growth repression caused by salt-mediated disturbances in growth-promoting metabolic and physiological processes. For example, DELLA proteins negatively regulate plant growth and development (Xue et al., 2022), are stabilized under salt stress, leading to growth inhibition and enhanced plant survival (Achard et al., 2006). Nevertheless, the mechanisms by which salt-activated signaling responses modulate plant growth remain poorly understood.

As the primary sensors of salinity, roots rapidly reduce growth, reallocating resources to adapt to high salt environments (Julkowska and Testerink, 2015; Van Zelm et al., 2020). This adaptive growth response primarily relies on meristem cell activity (West et al., 2004; Liu et al., 2015). Under optimal conditions, the balance between meristem cell proliferation and differentiation is tightly regulated by coordinating hormonal networks, molecular factors, and environmental cues (Vanstraelen and Benková, 2012; Motte et al., 2019). Salt stress rapidly inhibits cell division, leading to reduced meristem size and diminished root growth (West et al., 2004; Liu et al., 2015; Jiang et al., 2016; Scintu et al., 2023; Wang et al., 2023), due to decreased auxin signaling, (Liu et al., 2015; Jiang et al., 2016), reduced sucrose concentration in roots (Wang et al., 2023), and increased cytokinin activity, which triggers meristem cell differentiation (Scintu et al., 2023). Nevertheless, the molecular mechanisms behind salt-induced meristem inhibition have yet to be elucidated.

Reactive oxygen species (ROS) are crucial regulators of meristem cell maintenance (Tsukagoshi et al., 2010; Zeng et al., 2017; Yamada et al., 2020; Dang et al., 2023). Superoxide (O₂^⋅−) primarily accumulates at the root tips in the meristem zone to promote cell proliferation, whereas hydrogen peroxide (H₂O₂) is more abundant in the elongation and maturation zones, controlling cell differentiation. The transition from an O₂^⋅− to an H₂O₂ gradient marks the shift from cell proliferation to elongation, dictating meristem size (Tsukagoshi et al., 2010; Tsukagoshi, 2016; Yamada et al., 2020). The balance of ROS within this transition zone is crucial for meristem maintenance and root growth. Upon salt exposure, rapid ROS waves coupled with Ca²⁺ influx alters downstream signaling that slows root growth (Julkowska and Testerink, 2015; Van Zelm et al., 2020). During the first 24 hours of salt stress, dynamic changes in RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) gene expression play a central role in the ROS production networks during the initial response to salt stress (Xie et al., 2011). However, how ROS gradients change in response to salt stress and how the diverse roles of ROS are interconnected to flexibly modulate stem cell fate and plant growth under adverse conditions remain elusive.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a plant-specific family of transcription factors (TFs), play crucial roles in various aspects of plant development (Xu et al., 2016). In roots, JAGGED LATERAL ORGANS (JLO/LBD30) regulates the expression of auxin-inducible genes such as PLETHORA (PLT) and PIN-FORMED, which are crucial for maintaining meristem activity (Rast and Simon, 2012; Rast-Somssich et al., 2017). Other LBD members, such as LBD16, LBD18, and LBD29, are essential for lateral root formation (Lee et al., 2009; Feng et al., 2012). LBD proteins integrate developmental signals with environmental cues to modulate the plasticity of root architecture, enabling plants to adapt to environmental challenges. For example, Medicago MtLBD1 mediates the inhibition of primary root growth under salt stress (Ariel et al., 2010a; 2010b). However, the molecular mechanisms by which LBD proteins integrate developmental programs and environmental stimuli to regulate root growth remain unclear.

We reveal that LBD11 acts as a key regulator of ROS homeostasis in the vascular cambium (Dang et al., 2023), serving as a molecular switch that rapidly inhibits meristem development and root growth upon sensing high salinity. Our study uncovers a novel mechanism that integrates redox homeostasis, stem cell maintenance, and root development in response to environmental variables.

Results

Correlation between ROS distribution and meristem size under salt stress

To explore the relationship between ROS distribution and meristem size during early salt stress responses, we conducted a time-course experiment examining O₂^⋅− distribution in the root tips of wild-type (WT) Arabidopsis thaliana plants subjected to NaCl treatments. Using nitroblue tetrazolium (NBT), a specific O₂^⋅− indicator (Tsukagoshi et al., 2010), we stained the roots at regular intervals from 0 to 24 hours after NaCl treatments. In untreated plants, NBT staining was concentrated mainly in the meristem region (Figure 1A), consistent with previous reports (Tsukagoshi et al., 2010; Yamada et al., 2020). After 24 hours of NaCl challenge, treated plants exhibited decreased O₂^⋅− distribution, whereas O₂^⋅− levels increased in untreated plants over the same period (Figure 1A). Given that O₂^⋅− promotes cell proliferation (Tsukagoshi et al., 2010), this reduction correlated with a decrease in meristem size in NaCl-treated plants compared to controls (Figure 1B).

Figure 1.

Salt stress reduces O₂^⋅− levels and inhibits meristem size.

(A) O₂^⋅− distribution in the root tip of WT plants decreases over 0–24 hours of 100 mM NaCl treatment, as determined by NBT staining (dark purple). Black arrowheads mark the junction between the meristem and elongation zones. Data are presented as mean ± SEM. n, number of biologically independent samples at each time point.

(B) Meristem cell number decreases over 0–24 hours of 100 mM NaCl treatment. White and blue arrowheads denote the meristem zone. Data are presented as mean ± SEM. n, number of biologically independent samples at each time point.

(C) After 24 hours of NaCl treatment, O₂^⋅− distribution decreases, and the transition zone shifts closer to the quiescent center (QC), from approximately 250 μm from the QC in controls to approximately 200 μm in treated plants. The positions of the transition points of the O₂^⋅− and H₂O₂ gradients coincide with the corresponding transition zones in both control and NaCl-treated conditions. The position of the transition zone is defined as the distance from the QC to the meristem-elongation boundary in WT plants. At 4 days after imbibition (dai), WT plants were treated with 100 mM NaCl. DHE (red) and BES-H₂O₂-Ac (green) fluorescent indicators were used for the simultaneous detection of O₂^⋅− and H₂O₂ in the same root. Blue arrowheads and vertical dashed lines indicate the transition zone, whereas orange arrowheads and vertical dashed lines mark the transition points of the O₂^⋅− and H₂O₂ gradients.

(D) The DHE/BES-H₂O₂-Ac fluorescence ratio in the transition zone remains around 0.92 after 24 hours of NaCl treatment comparable to that of untreated plants. At 4-dai, WT plants were treated with 100 mM NaCl. Vertical dashed lines represent the transition points of O₂^⋅− and H₂O₂ gradients, with the black arrow highlighting the shift in the transition point after NaCl treatment.

(E) Primary root length decreases after 5 days of NaCl treatment compared to controls. Percentage values indicate the reduction in root growth rate between untreated and NaCl-treated plants.

(F) Relative LBD11 transcript levels over 0–24 hours of 100 mM NaCl treatment in 7-dai WT plants. RD29A expression serves as a marker for salt stress activation. Data are presented as mean ± SEM (n = 3).

In (A), (B) and (E), 5-dai WT plants were used for treatments. In (C) and (E), bar graphs display the mean of individual data points. Error bars, ±SEM. n, the number of biologically independent samples. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-tailed Student’s t-test). Scale bars, 100 μm in (A)–(C), and 10 mm in (E). All experiments were independently repeated at least three times with consistent results. See also Supplemental Figures 1, 2 and 3.

Meristem size is dictated by the transition of O₂^⋅− and H₂O₂ gradients between the meristem and the elongation zone (Tsukagoshi, 2016; Zhou et al., 2020). To examine changes in these gradients after 24 hours of NaCl treatment, we used dihydroethidium (DHE) and BES-H₂O₂-Ac fluorescent indicators to simultaneously detect O₂^⋅− and H₂O₂ distribution, respectively (Owusu-Ansah et al., 2008; Tsukagoshi et al., 2010). In control plants, DHE fluorescence, indicative of O₂^⋅− levels, was observed in the meristem zone, while BES-H₂O₂-Ac fluorescence, reflecting H₂O₂ distribution, was predominantly concentrated in the elongation zone (Figure 1C) (Tsukagoshi et al., 2010; Yamada et al., 2020). In the transition zone (approximately 250 μm from the quiescent center, QC) (Figure 1C and Supplemental Figure 1A), the ratio of DHE to BES-H₂O₂-Ac (DHE/BES-H₂O₂-Ac) fluorescence was approximately 0.92 (Supplemental Figure 1B), marking the relative transition point of O₂^⋅− and H₂O₂ gradients (Figure 1C). After NaCl treatment, DHE fluorescence decreased, indicating reduced O₂^⋅− levels in the meristem and transition zones, while the BES-H₂O₂-Ac fluorescence pattern remained largely unchanged compared to the controls (Figure 1C; Supplemental Figure 1C). NaCl treatment also caused the transition zone to shift closer to the QC (approximately 200 μm from QC) (Figure 1C). These changes coincided with a reduction in meristem size compared to untreated plants (Figure 1B). Notably, despite this positional shift, the DHE/BES-H₂O₂-Ac fluorescence ratio in the transition zone remained at around 0.92 (Figure 1D), which suggests that this specific ratio is crucial for defining the transition from cell division to differentiation. Given that meristem size determines the rate of root growth (Beemster and Baskin, 1998), we then assessed primary root elongation under prolonged salt treatment. After 5 days of NaCl treatment, root length was approximately 19.5% shorter than that of untreated controls (Figure 1E). These findings indicate a link between decreased O₂^⋅− distribution and the inhibition of meristem size and root growth in response to salt stress.

Superoxide dismutases (SODs), which are categorized into three types based on their metal cofactor (iron [FSDs], copper [CSDs], or manganese), convert O₂^⋅− to H₂O₂ (Alscher et al., 2002). We investigated the transcriptional expression of SOD genes in WT plants exposed to NaCl over a 24-hour period. Notably, only CSD2, FSD2, and FSD3 showed slight upregulation after 3 hours of NaCl treatment, while the expression of other SOD genes remained unchanged in treated plants compared to the controls (Supplemental Figure 2A). However, a significant reduction in O₂^⋅− levels and inhibition of meristem size were observed within 3 hours of NaCl treatment (Figures 1A and 1B), suggesting the involvement of other factors that modulate redox status and meristem size during the early salt stress responses.

LBD11 acts as a potential transducer that integrates redox status and meristem responses to salt stress

To identify transducers that orchestrate the integration of redox status with the salt-induced repression of meristem size, we conducted a comprehensive analysis of genes that showed transcriptional changes (up- or downregulation of ≥2-fold) in response to both NaCl (Wang et al., 2021) and H₂O₂ (Chen et al., 2021) treatments of 2 hours in Arabidopsis roots (Supplemental Figure 2B). Our objective was to pinpoint key transcriptional regulators that modulate ROS dynamics, driving meristem responses to high salt conditions. Among the 542 genes that were upregulated upon NaCl treatment and downregulated upon H₂O₂ treatment, 69 encoded TFs (Supplemental Figures 2C and 2D; Supplemental Table 1), including LBD11 (Supplemental Table 1), a central player in maintaining ROS homeostasis for stable vascular cambium activity (Dang et al., 2023).

To gain insights into the potential role of LBD11 in modulating ROS levels and regulating meristem size under salt stress, single-cell transcriptomic analysis was used to examine the spatial distribution of LBD11 in root developmental zones (Shahan et al., 2022). The analysis revealed high LBD11 expression in the vascular tissues of the elongation and maturation zones, with lower expression in the meristem (Supplemental Figure 3). Additionally, LBD11-regulated ROS metabolic genes—PEROXIDASE 71 (PRX71), RBOHD, and RBOHF (Dang et al., 2023)—exhibited expression patterns similar to those of LBD11 (Supplemental Figure 3), suggesting a role for LBD11 in controlling the balance of ROS distribution within the transition zone to regulate meristem size and root growth. LBD11 transcript levels varied during different phases of salt stress response (Van Zelm et al., 2020), with a marked elevation during the stress sensing and quiescent phase (0–9 hours after NaCl treatment), followed by a decline as plants entered the growth recovery phase (>9 hours of NaCl treatment) (Figure 1F). Collectively, these results suggest that LBD11 may regulate ROS distribution through PRX71, RBOHD, and RBOHF within the transition zone to modulate meristem size in response to salt stress.

LBD11 controls ROS balance for meristem maintenance and root development

To explore the regulatory role of LBD11 in modulating redox homeostasis and governing meristem size, we examined the expression pattern of LBD11 using the LBD11_pro:GUS transcriptional reporter line. GUS staining, which indicates LBD11 promoter activity, was observed in the vascular tissues extending from the transition to elongation zones (Figure 2A), consistent with results from single-cell transcriptomic analysis (Supplemental Figure 3). Next, we assessed the distribution of O₂^⋅− and H₂O₂ in the developmental zones of roots in WT, the lbd11 mutant, and two independent LBD11-overexpressing lines (35S_pro:LBD11-HA) using DHE and BES-H₂O₂-Ac indicators. The lbd11 mutant exhibited reduced DHE fluorescence, indicating decreased O₂^⋅− levels, while LBD11-overexpressing lines displayed increased DHE fluorescence, suggesting elevated O₂^⋅− distribution, compared to WT plants (Figure 2B). In contrast, the BES-H₂O₂-Ac fluorescence pattern, which reflects H₂O₂ distribution, was similar across WT and mutant lines (Supplemental Figure 4A). The changes in O₂^⋅− distribution in lbd11 and LBD11-overexpressing plants were associated with shifts in the transition zone: closer to QC in the lbd11 mutant (approximately 200 μm from QC) and further from the QC in LBD11-overexpressing plants (approximately 300 μm from QC) compared to the WT (approximately 250 μm from QC) (Figure 2B; Supplemental Figure 4A). Notably, despite these positional shifts, the DHE/BES-H₂O₂-Ac fluorescence ratio in the transition zone remained consistent at approximately 0.92 across WT, lbd11 mutant, and LBD11-overexpressing plants (Figure 2C), underscoring this ratio as a cue to cease cell division. NBT staining further validated these changes, showing an approximately a 25% reduction in O₂^⋅− levels in the lbd11 mutant and approximately a 35% increase in LBD11-overexpressing plants relative to the WT (Supplemental Figure 4B). Consistent with the reduced O₂^⋅− distribution, the lbd11 mutant had a smaller meristem size and shorter primary root length, while the LBD11-overexpressing lines exhibited elevated O₂^⋅− levels, increased meristem sizes, and longer root lengths compared to the WT (Figures 2D and 2E). We confirmed the functional significance of LBD11 in modulating ROS-mediated meristem size using two LBD11 complementation lines (LBD11_pro:LBD11-GFP/lbd11), which showed comparable O₂^⋅− levels, meristem sizes, and primary root lengths to WT plants (Supplemental Figures 4C–4E).

Figure 2.

LBD11 controls ROS homeostasis for meristem maintenance and root growth.

(A)LBD11 promoter activity is detected in the vascular tissues of the root transition and elongation zones of the LBD11_pro:GUS reporter line. EZ, elongation zone, MZ, meristem zone; TZ, transition zone. Analysis was conducted on 8-dai plants.

(B) O₂^⋅− distribution decreases in the lbd11 mutant but increases in LBD11-overexpressing plants compared to the WT, as determined by simultaneous DHE and BES-H₂O₂-Ac staining. Blue arrowheads and dashed lines mark the transition zone, while orange arrowheads and dashed lines indicate the transition points of the O₂^⋅− and H₂O₂ gradients.

(C) Ratio of DHE/BES-H₂O₂-Ac fluorescence in the transition zone remains consistent at approximately 0.92 across the WT, lbd11 mutant and LBD11-overexpressing plants. This ratio indicates the transition points of the O₂^⋅− and H₂O₂ gradients, represented by vertical dashed lines.

(D) Meristem cell number decreases in the lbd11 mutant but increases in LBD11-overexpressing plants compared to the WT. White and blue arrowheads indicate the meristem size.

(E) Average primary root length in the WT, lbd11 mutant, and LBD11-overexpressing plants over 12 days. The image shows plants at 10 dai. Each dot represents the root length of an individual plant, with mean values indicated by red horizontal lines.

(F) O₂^⋅− levels in root tips of LBD11_pro:FSD2-GFP and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants are lower than in WT and LBD11-overexpressing plants, respectively, while they remain similar in the lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants, as revealed by NBT staining. Analysis was conducted on 6-dai plants (see also Supplemental Figure 5B).

(G) Meristem cell numbers are comparable between the lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants, while they decrease in LBD11_pro:FSD2-GFP and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants compared to WT and LBD11-overexpressing plants, respectively (see also Supplemental Figure 5C).

(H) Primary roots are shorter in LBD11_pro:FSD2-GFP and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants compared to WT and LBD11-overexpressing plants, respectively, while the lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants exhibit similar root lengths. Analysis was conducted on 10-dai plants. Data are displayed as box and whisker plots with individual data points. Horizontal lines indicate the median, and whiskers represent the maximum and minimum values (see also Supplemental Figure 5D).

In (B) and (C), 5-dai WT plants were used for analysis. In (D), (F), and (G), bar graphs show mean values with individual data points. Error bars are ±SEM. n indicates the number of biologically independent samples. ∗p < 0.05, ∗∗∗∗p < 0.0001 (two-tailed Student’s t-test). Different letters indicate statistically significant differences at p < 0.05 (determined by one-way ANOVA followed by Tukey’s multiple comparisons test). Scale bars = 50 μm in (A), 100 μm in (B) and (D), and 10 mm in (E). All experiments were conducted independently at least three times with consistent results. See also Supplemental Figures 4 and 5.

To further elucidate the role of LBD11 in modulating O₂^⋅− distribution and its impact on meristem size, we measured these parameters in the WT, lbd11 mutant, and LBD11-overexpressing lines using diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase activity (Tsukagoshi et al., 2010). A 24-hour DPI treatment significantly reduced O₂^⋅− levels and decreased meristem size in both WT and LBD11-overexpressing plants. In contrast, these characteristics remained consistent between DPI-treated and untreated lbd11 mutant plants (Supplemental Figures 4F and 4G). To substantiate the role of LBD11-controlled O₂^⋅− distribution in meristem maintenance genetically, we investigated meristem features in WT, the lbd11 mutant, and LBD11-overexpressing plants harboring the LBD11_pro:FSD2-GFP transgene (Dang et al., 2023), which scavenges O₂^⋅− from cells in the transition and elongation zones (Supplemental Figure 5A). This scavenging activity significantly reduced the O₂^⋅− levels in WT and LBD11-overexpressing plants, while the O₂^⋅− distribution in lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants remained comparable (Figure 2F; Supplemental Figure 5B). Corresponding to the decreased O₂^⋅− levels, both LBD11_pro:FSD2-GFP/WT and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA transgenic plants exhibited smaller meristem sizes and shorter primary root lengths compared to their respective controls (Figures 2G and 2H; Supplemental Figures 5C and 5D). However, meristem size and root length did not differ significantly between the lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants (Figures 2G and 2H; Supplemental Figures 5C and 5D). Previous studies reported that UPBEAT1 (UPB1) negatively regulates peroxidase genes in the elongation zone to decrease O₂^⋅− levels, thereby maintaining ROS homeostasis within the root developmental zones (Tsukagoshi et al., 2010). Despite their contrasting roles in ROS regulation, LBD11 appears to modulate redox homeostasis independently of UPB1 signaling, as UPB1 transcriptional levels remained unchanged in the lbd11 mutant relative to the WT (Supplemental Figure 5E). Additionally, the peroxidase genes regulated by LBD11 differ from those controlled by UPB1 (Tsukagoshi et al., 2010; Dang et al., 2023) (Supplemental Figure 5F). Collectively, these results strongly suggest that LBD11 modulates redox homeostasis in root development zones to regulate meristem size and primary root growth.

Root growth repression under salt stress is dependent on LBD11

Drawing on the transcriptional responsiveness of LBD11 to salt stress (Figure 1F) and its established role in modulating ROS homeostasis for meristem maintenance (Figure 2), we investigated how LBD11 integrates redox status and meristem size under salt stress conditions. We subjected WT, lbd11 mutant, and LBD11-overexpressing plants to NaCl treatment and analyzed ROS distribution between the meristem and elongation zones using simultaneous DHE and BES-H₂O₂-Ac staining. While BES-H₂O₂-Ac fluorescence patterns, which indicate H₂O₂ distribution, remained largely unchanged across all conditions after 24 hours of NaCl treatment (Supplemental Figure 6A), the WT and LBD11-overexpressing plants exhibited decreased DHE fluorescence, reflecting lower O₂^⋅− levels than those of untreated controls (Figure 3A; Supplemental Figure 6A). Conversely, the lbd11 mutant displayed similar DHE fluorescence under both NaCl-treated and untreated conditions, suggesting minimal changes in O₂^⋅− levels (Figure 3A; Supplemental Figure 6A). This reduction in O₂^⋅− distribution in WT and LBD11-overexpressing plants under salt stress coincided with a shift in the transition zone closer to the QC, while the position of the transition zone in the lbd11 mutant remained relatively unchanged (Figure 3B). Despite these positional shifts, the DHE/BES-H₂O₂-Ac fluorescence ratio in the transition zone remained consistent at approximately 0.92 across all genotypes (Figure 3C), indicating that this ratio marks the transition point of O₂^⋅− and H₂O₂ gradients and defines the location of the transition zone under both control and salt stress conditions (Figures 3B and 3D). NBT staining corroborated these observations, showing a marked reduction in O₂^⋅− levels in WT and LBD11-overexpressing plants after 24 hours of NaCl treatment, while O₂^⋅− levels in the lbd11 mutant remained unchanged (Supplemental Figure 6B). Consistent with changes in ROS distribution, meristem size was significantly reduced in NaCl-treated WT and LBD11-overexpressing plants compared to their untreated controls, whereas NaCl treatment did not decrease meristem size in the lbd11 mutant (Figure 3E). Subsequently, prolonged salt stress (5 days) strongly inhibited primary root length in NaCl-treated WT (by 19.4%) and LBD11-overexpressing plants (by 25.5% and 25.4%, respectively), while the lbd11 mutant showed only a minor reduction in root growth (by approximately 11.6%) (Figure 3F). These results underscore the crucial role of LBD11 in coordinating redox status and meristem size under salt stress conditions.

Figure 3.

LBD11 coordinates redox status and meristem size in response to salt stress.

(A) NaCl treatment for 24 hours reduces O₂^⋅− distribution in WT and LBD11-overexpressing plants compared to untreated controls, while O₂^⋅− distribution remains unchanged in the lbd11 mutant, as revealed by simultaneous DHE and BES-H₂O₂-Ac staining. Blue arrowheads and dashed lines indicate the transition zone, while orange arrowheads and dashed lines mark the transition points of the O₂^⋅− and H₂O₂ gradients.

(B) After 24 hours of NaCl treatment, the transition zone and transition points of O₂^⋅− and H₂O₂ gradients shift closer to the QC in WT and LBD11-overexpressing plants, while these remain mostly unchanged in the lbd11 mutant.

(C) Ratio of DHE/BES-H₂O₂-Ac fluorescence in the transition zone remains consistent at approximately 0.92 across NaCl-treated and untreated WT, lbd11 mutant and LBD11-overexpressing plants. This ratio marks the transition points of the O₂^⋅− and H₂O₂ gradients as indicated in (B), represented by vertical dashed lines.

(D) Correlation analysis shows that the transition points of the O₂^⋅− and H₂O₂ gradients are within the transition zone in both NaCl-treated and untreated WT, lbd11 mutant, and LBD11-overexpressing plants.

(E) NaCl treatment decreases meristem cell count in WT and LBD11-overexpressing plants compared to controls, while the lbd11 mutant shows insensitivity to NaCl treatment. 5-dai plants were subjected to a 24-hour, 100 m-M NaCl treatment. White and blue arrows indicate the meristem zone.

(F) Primary root length is less affected in the lbd11 mutant but is more severely decreased in LBD11-overexpressing lines compared to WT plants, 5 days after growing on 100 mM NaCl. Percentage values indicate the rate of root growth reduction of untreated and NaCl-treated plants.

In (A)–(D), 4-dai plants were treated with 100 mM NaCl for 24 hours. In (B), (E), and (F), bar graphs show mean values with individual data points. Error bars are ±SEM. n indicates the number of biologically independent samples. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-tailed Student’s t-test). Different letters indicate statistically significant differences at p < 0.05 (determined by one-way ANOVA followed by Tukey’s multiple comparisons test). Scale bars, 100 μm in (A) and (E), and 10 mm in (F). All experiments were conducted three times with similar results. See also Supplemental Figures 6, 7, and 8.

To further substantiate the functional relevance of LBD11/ROS orchestration in salt-triggered growth repression, we examined O₂^⋅− distribution, meristem size, and root growth in LBD11 complementation lines under salt stress. After a 24-hour NaCl treatment, the lbd11 mutants showed no change in O₂^⋅− levels or meristem sizes, whereas WT and LBD11 complementation lines had decreased O₂^⋅− distribution and meristem sizes compared to untreated controls (Supplemental Figures 7A and 7B). After 5 days of NaCl treatment, primary root length was significantly reduced in WT and LBD11 complementation lines (by approximately 17.4%, 17%, and 16.3%, respectively), compared to a 10.8% reduction in lbd11 mutants (Supplemental Figure 7C). Collectively, these findings demonstrate that cellular responses driving the repression of meristem size and root growth under salt stress are dependent on LBD11.

Furthermore, differentially expressed genes in LBD11-overexpressing plants were not enriched in Gene Ontology categories related to salt stress signaling (Dang et al., 2023). The expression of key salt stress–responsive genes (Huang et al., 2018) was not significantly different across WT, lbd11 mutant, and LBD11-overexpressing lines (Supplemental Figure 8), suggesting that LBD11 primarily regulates plant growth responses to salt rather than modulating the levels of salt-induced transcripts.

Cellular responses to salt stress trigger rapid proteasomal degradation of LBD11

Although the transcript levels of LBD11 were upregulated by NaCl treatment (Figure 1F), the concurrent suppression of LBD11-dependent O₂^⋅− levels and meristem size during salt stress (Figure 3; Supplemental Figure 7) suggests that the LBD11 protein undergoes degradation. We analyzed LBD11 protein accumulation at different phases of the salt stress response (Van Zelm et al., 2020), noting rapid degradation during the sensing and quiescent phases, followed by increased abundance during the growth recovery phase (Figure 4A). To investigate the cellular mechanism behind NaCl-mediated LBD11 degradation, we tested the effects of several proteolysis inhibitors. The salt-triggered degradation of LBD11 was inhibited by the proteasome inhibitor MG132, but not by the protease inhibitors pepstatin A and PMSF, suggesting that LBD11 degradation is specifically mediated by proteasome activity (Figures 4B and 4C). Using transient Arabidopsis leaf mesophyll protoplasts co-transfected with LBD11 (35S_pro:LBD11-HA) and ubiquitin-related constructs (35S_pro:FLAG-UBQ10, 35S_pro:FLAG-SUMO1, 35S_pro:FLAG-SUMO3, and 35S_pro:FLAG-SUMO5), we demonstrated that the proteasomal degradation of LBD11 is ubiquitin (Ub) dependent (Zhang et al., 2015b) (Supplemental Figure 9A). This Ub-mediated LBD11 proteasomal degradation was consistent even under optimal developmental conditions (Supplemental Figure 9B). In protoplasts co-expressing LBD11-HA and FLAG-UBQ10, we detected a change in LBD11 ubiquitination levels under salt stress. Following a 1-hour treatment with NaCl, we observed reductions in LBD11 protein abundance and associated Ub molecules via immunoprecipitation (Figure 4D). However, co-treatment with NaCl and MG132 resulted in a marked increase in LBD11-bound Ub molecules compared to the control (Figure 4D), confirming that salt stress mediates LBD11 degradation through the Ub–proteasome system (UPS). Since ubiquitination is the conjugation of Ub molecules and the lysine (K) residues of target proteins (Tracz and Bialek, 2021), we searched for the ubiquitination sites on LBD11 by mutating individual K residues to arginine (R) and assessing the stability of these mutants under salt stress in the protoplast system. K83R and K150R mutants had reduced degradation (with approximately 53.1% and 39.8% of the mutant LBD11 proteins degraded, respectively) compared to WT LBD11 and other lysine mutants (>60% degradation) (Figure 4E). A subsequent ubiquitination analysis showed that ubiquitination levels of NaCl-treated K83R and K150R mutant proteins were comparable to their respective controls, whereas the ubiquitination levels of WT LBD11 and other lysine mutants increased substantially after NaCl treatment (Figure 4F). These findings suggest that salt stress enhances the UPS-mediated degradation of LBD11 through ubiquitination at K83 and predominantly at K150. Collectively, these results illustrate that early salt stress signaling represses LBD11, inhibiting meristem development.

Figure 4.

Salt accelerates LBD11 degradation through the UPS.

(A) LBD11 undergoes rapid degradation upon NaCl treatment. Total protein was extracted from LBD11-overexpressing plants treated with 100 mM NaCl over 0–24 hours, followed by Western blot analysis using an anti-HA antibody.

(B and C) Salt-mediated degradation of LBD11 is inhibited by the 26S proteasome inhibitor MG132, but not by the protease inhibitors pepstatin A and PMSF. 7-dai LBD11-overexpressing plants were treated with 100 mM NaCl alone or in combination with 100 μM MG132, 100 μM Pepstatin A, or 1 mM PMSF for 1 hour (B) or for indicated times (C).

(D) Ubiquitination and proteasomal degradation of LBD11 are enhanced upon NaCl treatment. Leaf mesophyll protoplasts were co-transfected with 35S_pro:LBD11-HA and 35S_pro:FLAG-UBQ10, and then treated with 300 mM NaCl with and without 50 μM MG132 for 1 hour.

(E) The LBD11 mutants K83R and K150R exhibit reduced degradation in response to salt compared to WT LBD11 and other K mutants. Protoplast cells were transfected with LBD11-HA and K-mutated LBD11-HA variants, then treated with 300 mM NaCl for 1 hour.

(F) Under salt stress conditions, K83R and K150R LBD11 variants show decreased ubiquitination levels compared to WT LBD11 and other K mutants. Protoplast cells were co-transfected with LBD11-HA or K-mutated LBD11-HA variants and 35S_pro:FLAG-UBQ10, followed by treatment with 300 mM NaCl and 50 μM MG132 for 1 hour.

In (A)–(C), 7-dai plants were used for analysis. The numbers represent LBD11 protein levels relative to the mock control, which is set as 1. Total protein was isolated and subjected to Western blot analysis using an anti-HA antibody. Detection of a constitutively expressed tubulin serves as the protein loading control. In (D) and (F), cells were lysed and proteins were analyzed by Western blot (Input) or IP using an anti-HA antibody followed by Western blotting. In (D)–(F), CBB-stained rubisco large subunit (RbcL) confirms equal protein loading. All experiments were conducted three times with consistent results. See also Supplemental Figure 9.

LBD11-controlled ROS metabolic enzymes mediate root meristem responses to salt stress

LBD11acts as a transcriptional regulator for PRX71, RBOHD, and RBOHF, which generate cambium-specific ROS for vascular cambium activity (Dang et al., 2023). These genes are predominantly expressed in the vascular tissues of the elongation and maturation zones in roots (Supplemental Figure 3 and Supplemental Figure 10A), mirroring the expression pattern of LBD11 (Figure 2A; Supplemental Figure 3). In the lbd11 mutant, promoter activity of PRX71, RBOHD, and RBOHF is suppressed (Supplemental Figure 10A), indicating that LBD11 transcriptionally regulates these ROS metabolic genes within the root developmental zones. To explore the functional implications of these ROS metabolic enzymes on meristem size, we examined O₂^⋅− distribution in the root tip of their respective loss-of-function mutants using NBT staining (Dang et al., 2023). The results showed that compared to the WT, O₂^⋅− levels were reduced in the prx71-1 (by approximately 15%), rbohd (by approximately 24%), and rbohf (by approximately 20%) single mutants; meristems were smaller, and primary roots were shorter (Supplemental Figure 10B, 10C, and 10D). The lbd11 prx71-1 and lbd11 rbohf double mutants had meristem sizes and primary root lengths similar to those of the prx71-1 and rbohf single mutants, respectively (Supplemental Figures 10E and 10F). These results underscore LBD11’s role in modulating key ROS metabolic genes to ensure optimal ROS levels in developmental zones for meristem maintenance and root growth.

Having established the roles of PRX71, RBOHD, and RBOHF in root growth regulation, we next investigated their importance in root growth responses under salt stress. We examined O₂^⋅− levels and meristem features in WT, prx71-1, rbohd, and rbohf single mutants following a 24-hour NaCl treatment. Salt stress substantially repressed O₂^⋅− distribution and reduced meristem size in the WT (Figures 5A and 5B). In contrast, O₂^⋅− levels and meristem sizes in the NaCl-treated prx71-1, rbohd, and rbohf single mutants were comparable to those in the untreated controls (Figures 5A and 5B). After 5 days of NaCl treatment, root growth inhibition in prx71-1, rbohd, and rbohf single mutants was less severe (showing reductions of approximately 12.1%, 10.1%, and 11.2%, respectively) compared to the WT (approximately 19.5% reduction) (Figure 5C). Notably, 24-hour NaCl treatment did not reduce the meristem size in the lbd11 prx71-1 and lbd11 rbohf double mutants (Figure 5D). After 5 days of NaCl treatment, the lbd11 prx71-1 and lbd11 rbohf plants showed root growth inhibition rates comparable to those of the prx71-1 and rbohf single mutants, respectively (Figure 5E). Collectively, these results demonstrate that PRX71, RBOHD, and RBOHF act in concert with LBD11 to mediate the cellular responses that regulate meristem size under salt stress.

Figure 5.

Abolition of LBD11-regulated ROS metabolic genes mitigates salt-induced growth inhibition.

(A) O₂^⋅− levels in the root tip of WT plants decrease after 24 hours of treatment with 100 mM NaCl compared to the control. In contrast, O₂^⋅− levels remain similar between untreated and NaCl-treated prx71-1, rbohd, and rbohf mutants, and are lower than in the WT. O₂^⋅− distribution was detected using NBT staining. Black arrowheads mark the transition zone. Bar graphs show means ± SEM.

(B) Meristem cell numbers in the WT decrease after 24 hours of 100 mM NaCl treatment compared to untreated plants, whereas they remain similar between untreated and NaCl-treated prx71-1, rbohd and rbohf mutants. White and blue arrowheads indicate the meristem zone.

(C) Primary root length was measured in the WT, prx71-1, rbohd, and rbohf mutants after 5 days of 100 mM NaCl treatment. Percentage values indicate the reduction rate of root growth between untreated and NaCl-treated counterparts.

(D) A 24-hour, 100-mM NaCl treatment decreases meristem cell number in the WT compared to untreated controls, whereas the lbd11, prx71-1, and rbohf single mutants, as well as lbd11 prx71-1 and lbd11 rbohf double mutants, display meristem cell numbers comparable to their respective untreated plants. White and blue arrowheads mark the meristem zone. Bar graphs show means ± SEM.

(E) Measurement of primary root length in the WT, lbd11, prx71-1, and rbohf single mutants, as well as lbd11 prx71-1 and lbd11 rbohf double mutants, after 5 days of growth on 100 mM NaCl compared to their respective untreated controls. Bar graphs show means ± SEM. Percentage values indicate the reduction rate of root growth between untreated and NaCl-treated plants.

In (A)–(E), 5-dai plants were treated with 100 mM NaCl. In (B) and (C), data are displayed as box and whisker plots with individual points. The center horizontal line represents the median and whiskers extend to the minimum and maximum values. n refers to the number of biologically independent samples.

Different letters indicate statistically significant differences at p < 0.05 (determined by one-way ANOVA followed by Tukey’s multiple comparisons test). Scale bars, 100 μm in (A), (B), and (D), and 10 mm in (C) and (E). All experiments were conducted three times with consistent results. See also Supplemental Figure 10.

LBD11-dependent redox status determines growth responses to salt stress

LBD11-overexpressing plants exhibited elevated O₂^⋅− levels in the root tip and increased meristem size (Figure 2); however, salt stress caused greater inhibition to their root growth than it did for the WT (Figure 3). In contrast, the lbd11, prx71-1, rbohd, and rbohf mutants, which had lower levels of O₂^⋅− and reduced meristem size, experienced less root growth inhibition under salt stress compared to the WT (Figures 2, 3, and 5; Supplemental Figure 10). Previous studies have shown that increased FSD activity and reduced O₂^⋅− content are associated with salt tolerance (Bose et al., 2014; Kaur et al., 2016). Therefore, we postulated that mitigating plant sensitivity to salt-induced root growth inhibition could be achieved by eliminating O₂^⋅− in the root developmental zones through FSD activity. To test this hypothesis, we investigated O₂^⋅− levels in the root tip, meristem size, and primary root length of WT, lbd11 mutant, LBD11-overexpressing plants, and LBD11_pro:FSD2-GFP transgenic lines under salt stress (Figures 2F–2H; Supplemental Figures 5A–5D). After 24 hours of NaCl treatment, O₂^⋅− levels and meristem size were substantially reduced in the WT and LBD11-overexpressing plants, whereas these features were insignificantly altered in LBD11_pro:FSD2-GFP/WT plants and only slightly decreased in LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants (Figures 6A–6D). In lbd11 mutants and LBD11_pro:FSD2-GFP/lbd11 transgenic plants, O₂^⋅− levels and meristem size were comparable between NaCl-treated and untreated conditions (Figures 6A–6D). Consequently, after a prolonged NaCl treatment for 5 days, LBD11_pro:FSD2-GFP/WT and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA transgenic plants exhibited significantly less reduction in root growth than the WT and LBD11-overexpressing plants, respectively (Figure 6E). In contrast, root growth inhibition was similar in the lbd11 mutant and LBD11_pro:FSD2-GFP/lbd11 plants (Figure 6E). These findings suggest that maintaining low O₂^⋅− levels in the root tip is beneficial for sustaining root growth during salt stress, supporting the downregulation of the LBD11/ROS-dependent system to improve plant adaptation to this challenging environmental condition.

Figure 6.

Reduced O₂^⋅− distribution diminishes the inhibitory effects of salt on root growth.

(A and C) Distribution of O₂^⋅− in the root tip, as revealed by NBT staining, was assessed in WT LBD11_pro:FSD2-GFP/WT, lbd11 mutant, LBD11_pro:FSD2-GFP/lbd11, LBD11-overexpressing, and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants after a 24-hour treatment with 100 mM NaCl. Black arrowheads mark the transition zone.

(B and D) Meristem cell numbers were quantified in WT, LBD11_pro:FSD2-GFP/WT, lbd11 mutant, LBD11_pro:FSD2-GFP/lbd11, LBD11-overexpressing, and LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA plants following 24 hours of NaCl exposure. White and blue arrowheads indicate the meristem zone.

(E) Primary root length was measured in the WT, lbd11 mutant, and LBD11-overexpressing plants, each harboring the LBD11_pro:FSD2-GFP construct, compared to their respective controls after 5 days of 100 mM NaCl treatment. Percentage values indicate the reduction rate of root growth between untreated and NaCl-treated counterparts.

In (A)–(E), 5-dai plants were treated with 100 mM NaCl. In (C)–(E), bar graphs show means ± SEM. n refers to the number of biologically independent samples. Different letters denote statistically significant differences at p < 0.05 (determined by one-way ANOVA followed by Tukey’s multiple comparisons test). Scale bars, 100 μm in (A) and (B), 10 mm in (E).

Discussion

Understanding how plants navigate complex environments for their survival and reproduction is not only a fundamental area of scientific inquiry but also vital for sustainable agriculture and food security. Many studies have focused on plant defense mechanisms against stress-induced cellular damage; however, our understanding of how plants actively adjust their growth to thrive in adverse conditions remains limited (Julkowska and Testerink, 2015; Van Zelm et al., 2020; Zhang et al., 2020). In this study, we demonstrate the pivotal roles of the LBD11/ROS regulatory system in modulating meristem size and driving significant growth inhibition in response to salt stress (Figure 7).

Figure 7.

Proposed model of LBD11-regulated ROS homeostasis in modulating meristem size and root growth under stress conditions.

Under optimal growth conditions, LBD11 regulates the transcription of key ROS metabolic genes, such as PRX71, RBOHD, and RBOHF, to promote O₂^⋅− production in the transition zone. This activity enhances meristem size and supports robust root growth. Concurrently, LBD11 undergoes continuous degradation via the UPS to maintain ROS homeostasis, ensuring proper meristem function. In response to salt stress, UPS-mediated degradation of LBD11 accelerates, leading to a significant reduction in O₂^⋅− levels. This altered ROS distribution restricts meristem size and root growth, enabling the plant to adapt to stressful conditions. EZ, elongation zone; MZ, meristem zone; TZ, transition zone.

The dynamic interplay between O₂^⋅− and H₂O₂ orchestrates the transition from the cell proliferation to differentiation along the root tip, thereby determining meristem size (Tsukagoshi et al., 2010; Yamada et al., 2020). As meristem cells enter the transition zone, they retain their proliferative activity until reaching the elongation zone, which underscores the importance of ROS balance in this transitional region (Tsukagoshi et al., 2010). LBD11 regulates PRX71 and RBOHD/F expression to drive O₂^⋅− production, maintaining ROS homeostasis and ensuring proper meristem size. The disruption of LBD11, PRX71, and RBOHD/F in the lbd11, prx71-1, rbohd, and rbohf mutants led to decreased O₂^⋅− levels and reduced meristem size (Figure 2; Supplemental Figure 10). Conversely, LBD11 overexpression resulted in elevated O₂^⋅− distribution in the transition zone and increased meristem size (Figure 2). These findings strongly suggest that stable LBD11 levels are critical for maintaining redox homeostasis within root developmental zones. Consistent with this, LBD11 is continuously degraded via the UPS to maintain a balanced ROS level (Supplemental Figure 9B). Moreover, H₂O₂ has been shown to downregulate LBD11 (Dang et al., 2023) (Supplemental Figure 1C), suggesting a feedback loop where H₂O₂ accumulation in the elongation zone curbs excessive LBD11 expression, thereby maintaining redox homeostasis. Future investigations will explore the cellular components responsible for UPS-mediated LBD11 degradation, offering deeper insights into the molecular mechanisms by which plants coordinate ROS homeostasis for stem cell maintenance.

Ub-mediated protein degradation plays a crucial role in plant growth and development (Christians et al., 2012; Liu et al., 2013; Hu et al., 2014) and responses to environmental stresses (Zhang et al., 2015a, 2015b, 2019). Although salt stress initially enhances LBD11 transcription during the quiescent phase (Figure 1F), LBD11 protein is quickly degraded via the UPS (Figure 4A). This degradation leads to reduced meristem size, consistent with the observed arrest of plant growth during the quiescent phase (Julkowska and Testerink, 2015; Van Zelm et al., 2020). Notably, LBD11 protein levels increase during the recovery phase following salt stress, surpassing those of the untreated control, which facilitates plant growth recovery (Figure 4A). Despite the partial recovery during the growth recovery phase, prolonged exposure to salt stress induces constitutive ROS production and accumulation of sodium ions in the shoot, which damages plant cells, reduces photosynthesis, and ultimately impairs plant growth (Julkowska and Testerink, 2015; Mittler et al., 2022).

Salt stress induces LBD11 transcription (Figure 1F) while simultaneously promoting LBD11 protein degradation during the quiescent phase (Figure 4A). Although the molecular mechanisms underlying salt-mediated transcriptional changes and degradation of LBD11 remain unclear, the dynamics of abscisic acid (ABA) signaling transcripts and ABA levels during the quiescent and recovery phases (Geng et al., 2013) suggest a possible role of ABA in these processes. ABA, a crucial regulator of salt stress responses (Cutler et al., 2010), promotes growth at low concentrations and inhibits growth at high levels (Finkelstein and Rock, 2002). During the quiescent phase, ABA-associated transcriptional networks are upregulated (Geng et al., 2013), which coincides with elevated LBD11 transcript levels (Figure 1F). Interestingly, ABA levels peak during the quiescent phase and decline during the recovery phase (Geng et al., 2013), showing an inverse relationship with LBD11 protein abundance (Figure 4A). Understanding the dynamic interplay between ABA and LBD11 levels during the early and late stages of salt stress response could provide important insights into how plants rapidly adjust their growth to withstand not only high salt conditions but also other environmental stresses.

An effective antioxidant system is necessary for preventing oxidative stress while preserving optimal levels of ROS for cellular signaling during salt stress (Bose et al., 2014; Kaur et al., 2016; Yang and Guo, 2018). Halophytes, with intrinsically high SOD activity—particularly for FSDs— are more resilient to soil salinity compared to glycophytes (Taji et al., 2004; Bose et al., 2014). In rice, salt-resistant cultivars display lower O₂^⋅− contents than salt-sensitive cultivars (Kaur et al., 2016), underscoring the importance of maintaining low O₂^⋅− levels for plant survival during salt stress. Consistent with these findings, our study demonstrated that the lbd11, prx71-1, rbohd, and rbohf mutants, which are characterized by lower O₂^⋅− levels in developmental zones, experience less growth inhibition under salt stress compared to WT plants (Figures 2, 3, and 5). Additionally, by driving FSD2 expression with an LBD11 promoter to decrease O₂^⋅− distribution in the transition and elongation zones, we mitigated the inhibitory effect of salt on root growth (Figure 6). Our results provide insights into the direct connection between ROS metabolism and plant growth under adverse conditions (Miller et al., 2010), highlighting promising avenues for improving plant resilience in dynamically changing habitats.

The manner by which salt-perturbed ROS distribution affects growth inhibition is not fully understood. Auxin is crucial for cell division in the meristem (Di Mambro et al., 2017). Salt stress reduces the expression of auxin transporter genes, including AUXIN/INDOLE-3-ACETIC ACID and PIN-FORMED, leading to decreased auxin levels in the root meristem and inhibition of meristem cell division (Liu et al., 2015; Jiang et al., 2016). This response is associated with a disruption of redox balance, suggesting a connection between redox signaling and auxin distribution in the salt-mediated repression of meristem cell proliferation (Jiang et al., 2016). PLETHORA2 (PLT2), a master regulator of root stem cells (Blilou et al., 2005), is critical for maintaining meristem cell activity during salt stress (Hao et al., 2023). Notably, both PLT2 transcript levels and PLT2 stability are modulated by meristematic O₂^⋅− levels (Yamada et al., 2020). Therefore, we postulate that salt stress inhibits meristem cell proliferation by reducing local O₂^⋅− production, thereby repressing auxin levels and auxin-modulated cellular components. The failure to elicit these responses in the lbd11 mutant, however, confers benefits for plant growth fitness, facilitating survival under salt stress.

Our study establishes a direct connection between the modulation of ROS homeostasis by LBD11 and the active inhibition of plant growth in response to salt stress. While it is known that salt stress perception rapidly induces ROS waves to trigger systemic signaling (Van Zelm et al., 2020), our findings specifically highlight the reduction of local O₂^⋅− levels as a key mechanism for inhibiting root growth. This underscores the importance of spatial ROS regulation in directing plant growth responses to salt. Future research should aim to unravel the molecular mechanisms that govern the degradation and recovery of LBD11 during early salt stress responses. Additionally, we seek to decode the downstream signaling controlled by the LBD11/ROS pathway and elucidate the higher-order cellular and tissue regulation of ROS distribution. These investigations will deepen our understanding of how plants dynamically modulate their growth and stress responses, adapting to ever-changing environments to ensure successful reproduction.

Methods

Plant materials and growth conditions

A. thaliana Columbia (Col-0) was the WT background for all mutants and transgenic lines used in this study. The lbd11 mutant (GABI-426C05) was obtained from the ABRC at Ohio State University. The prx71-1, rbohd and rbohf mutants were obtained from Arnaud et al. (2017). Transgenic lines, including 35S_pro:LBD11-HA, LBD11_pro:GUS, LBD11_pro:LBD11-GFP/lbd11, LBD11_pro:FSD2-GFP/WT, LBD11_pro:FSD2-GFP/lbd11, LBD11_pro:FSD2-GFP/35S_pro:LBD11-HA, PRX71_pro:GUS, RBOHD_pro:GUS, RBOHF_pro:GUS, PRX71_pro:GUS/lbd11, RBOHD_pro:GUS/lbd11, and RBOHF_pro:GUS/lbd11, as well as the lbd11 prx71-1 and lbd11 rbohf double mutants, were generated as described in Dang et al. (2023).

Seeds were surface-sterilized for 7 minutes with 70% (v/v) ethanol and 0.05% Triton X-100 (Sigma), followed by two rinses with 100% ethanol for 1 minute each. Seeds were plated on ½ Murashige and Skoog salt mixture including vitamins (½ MS) medium (Duchefa), supplemented with 1% (w/v) sucrose and 1% (w/v) agar (Sigma), at pH 5.6–5.7. After 3 days of stratification at 4°C in the dark, plates were transferred to a growth chamber under a 16-hour light/8-hour dark photoperiod at 22°C.

To investigate the responsiveness of LBD11, SOD genes, and ROS metabolic genes to salt stress, 7 days after imbibition (dai), Col-0 seedlings were acclimated in ½ MS liquid media and then supplemented with 100 mM NaCl. Plants were harvested at indicated time points, snap-frozen in liquid nitrogen, and total RNA was isolated. Similarly, to assess the expression of salt stress marker genes, 7-dai seedlings were challenged with 100 mM NaCl in ½ MS liquid media for 1 hour.

Plasmid construction

To generate the 35S_pro:LBD11-HA construct for transient expression in leaf mesophyll protoplasts, the genomic DNA sequence of LBD11 (1537 bp) without the stop codon was PCR-amplified from Col-0 genomic DNA. The PCR products were digested with BglII and StuI restriction enzymes and inserted downstream of the 35SC4PPDK promoter in the HBT95 expression vector fused with two HA epitope tags (Sheen, 1996). The 35S_pro:FLAG-UBQ10, 35S_pro:FLAG-SUMO1, 35S_pro:FLAG-SUMO3, and 35S_pro:FLAG-SUMO5 plasmids were generated by PCR amplification of UBQ10 (1374 bp), SUMO1 (300 bp), SUMO3 (333 bp), and SUMO5 (324 bp) from total cDNA and cloned into the HBT95 expression vector with two FLAG epitope tags at their N-terminus. K-mutated LBD11 constructs were produced by site-directed mutagenesis on the 35S_pro:LBD11-HA construct (HBT95 vector) using complementary mutant primer pairs extending 10–15 nucleotides on either side of the modification sites. Following PCR amplification (initial denaturation at 95°C for 5 minutes; 20 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 7 minutes; final extension at 72°C for 15 minutes), the methylated template DNA in the PCR products was digested with DpnI restriction enzyme. Primers used for cloning are listed in Supplemental Table 2.

Measurement of meristem cell number and root length

Meristem cell number was determined following a previously described method (Dello Ioio et al., 2007). Briefly, seeds were grown vertically on ½ MS medium. At 6 dai, plants with meristems reaching a final size of approximately 30 cells were selected for counting. Cortex cells were counted from the QC to the first elongated cell to determine meristem size (Dello Ioio et al., 2007).

To measure meristem cell number under salt stress or DPI treatment, 5-dai seedlings were transferred to treatment media consisting of ½ MS medium supplemented with 0.5% (w/v) sucrose and either 100 mM NaCl (Samchun) or 100 μM DPI (Sigma) for 24 hours. Seedlings were stained with 5 μg/mL propidium iodide for approximately 1 minute, and root tips were visualized using a confocal microscope (LSM800, Zeiss) at wavelengths of 600–650 nm.

To observe root growth, seedlings were grown vertically on ½ MS medium, and primary root lengths were measured on the indicated days. To investigate root growth under NaCl treatment, 5-dai plants grown vertically on ½ MS medium with 0.5% (w/v) sucrose were transferred to the same medium supplemented with 100 mM NaCl for an additional 5 days.

Detection of ROS signals

NBT (Alfa Aesar) staining was performed as previously described (Dunand et al., 2007). Briefly, seedlings were stained in NBT solution (2 mM NBT, 20 mM phosphate buffer, pH 6.1) for 2 minutes and rinsed with distilled water. NBT signals were observed and imaged at a 10× objective using an Axioplan 2 microscope (Carl Zeiss). Total NBT staining intensities were calculated from the QC to the transition zone using the Photoshop histogram function (0, white; 255, black).

For simultaneous observation of O₂^⋅− and H₂O₂ in a single root, 5-dai or 4-dai seedlings subjected to 24-hour NaCl treatment were incubated in a staining solution containing 10 μM DHE (Thermo Fisher Scientific), 50 μM BES-H₂O₂-Ac (Wako), and 50 mM PBS (pH 7.4) for 30 minutes in the dark and rinsed with 50 mM PBS (pH 7.4). Root tips were then observed at a 10× objective using a confocal microscope (LSM800, Zeiss) with specific settings: for DHE, excitation at 514 nm with a detection range of 516–576 nm; for BES-H₂O₂-Ac, excitation at 488 nm with a detection range of 500–550 nm. A series of 10 images were acquired at depths from 0 to 45 μm, moving from the outermost to the central region of the sample. These images were orthogonally projected to generate a composite image for analysis. Fluorescence intensities of DHE and BES-H₂O₂-Ac were quantified using ZEN 3.4 Zeiss software (LSM800, Zeiss). The transition point, defined as the point where the ratio of DHE to BES-H₂O₂-Ac fluorescence intensity drops below 0.92, was identified.

Real-time qPCR analysis

Real-time qPCR analysis was performed as previously reported (Dang et al., 2023). Total RNA was isolated from snap-frozen tissues using TRIzol reagent (Invitrogen) as per the manufacturer’s instructions. DNA-free total RNA (1 μg) was used for cDNA synthesis with ImProm-II reverse transcriptase (Promega). Real-time qPCR was performed using gene-specific primers with Actin2 serving as the internal control. The primers used for real-time qPCR are listed in Supplemental Table 2.

Histochemical GUS assay

GUS staining was conducted as previously described (Kondo et al., 2014). Seedlings at 6-dai were fixed with 90% acetone for 30 minutes on ice, washed twice with sodium phosphate buffer (pH 7.2), and then vacuum infiltrated with GUS staining solution (50 mM phosphate buffer pH 7.2, 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 0.05% Triton X-100, and 1 mg/mL X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronidase)) for 15 minutes. The seedlings were incubated overnight at 37°C in the dark. After staining, the seedlings were embedded in Visikol Mount (Visikol) and observed using an Axioplan 2 microscope (Carl Zeiss).

Total protein isolation

We transferred 7-dai 35S_pro:LBD11-HA seedlings to ½ MS liquid media for acclimatization and then supplemented them with 100 mM NaCl, with or without 100 μM proteasome inhibitor MG132 (Alfa Aesar), 100 μM Pestatin A (Sigma), and 1 mM PMSF (Sigma) for 1 hour or as indicated. Harvested seedlings were snap-frozen, ground to a fine powder in liquid nitrogen, and homogenized with protein extraction buffer [150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail (Roche)], followed by centrifugation at 13 000 rpm for 10 min at 4°C. Total protein concentration was measured using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad) following the manufacturer’s protocol.

Immunoblotting analysis

Total protein was analyzed by immunoblot as previously described (Ramon et al., 2019) with modifications. Total protein (8 μg) was separated on 7.5% polyacrylamide SDS-PAGE gel in Tris-Gly running buffer [0.025 M Tris, 0.192 M glycine, 0.1% SDS (w/v), pH 8.5] at 60 V for 30 minutes, then at 160 V until the target proteins reached the bottom of the gel (estimated according to the predicted molecular weight). Proteins were then transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore) in Tris-Gly transfer buffer [0.025 M Tris, 0.192 M glycine, 10% (v/v) methanol] at 70 V for 90 minutes in a cold room. The membrane was blocked with 5% (w/v) skimmed milk (Difco) and incubated overnight at 4°C with horseradish peroxidase (HRP)-conjugated anti-HA antibody (1:1500; Roche) or HRP-conjugated anti-TUB antibody (1:4000; Santa Cruz Biotechnology) in 1% skimmed milk. After washing five times with Tris-buffered saline [50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20], the membrane was exposed to Pierce West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) for 1 minute, and then to film for 30 seconds in the dark room. Constitutively expressed tubulin or the rubisco large subunit stained with Coomassie Brilliant Blue R-250 served as the protein loading control. Protein abundance was quantified by calculating the band intensity of the LBD11 protein relative to that of tubulin using the Photoshop histogram function (Adobe Systems Inc.).

Transient expression in the leaf mesophyll protoplasts

Isolation of Arabidopsis leaf mesophyll protoplasts and transient transfection were performed as previously described (Yoo et al., 2007). For the assessment of protein stability under salt stress conditions, approximately 4 × 10⁴ protoplasts were transfected with a total 30 μg of DNA. Following polyethylene glycol (PEG)-Ca²⁺-mediated transformation, the protoplasts were divided into two groups: one served as the control and the other underwent NaCl treatment. Both groups were incubated at room temperature for 5 hours. The control protoplasts were harvested and snap-frozen in liquid nitrogen, while the NaCl treatment group had 300 mM NaCl added for an additional hour. The protoplasts were lysed with 50 μL of protein extraction buffer, and total protein was analyzed by immunoblotting using an HRP-conjugated anti-HA antibody (1:1500; Roche).

In vivo ubiquitination assay

Ubiquitination was performed as previously described (Zhou et al., 2014) with modifications. Approximately 2 × 10⁵ protoplasts were transfected with 100 μg of 35S_pro:LBD11-HA and 35S_pro:FLAG-UBQ10 plasmid DNA and incubated for 6 hours. The protoplasts were then treated with 300 mM NaCl with or without 50 μM MG132 for an additional hour. The cells were harvested and lysed with 200 μL immunoprecipitation (IP) buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5 mM DTT, and a protease inhibitor cocktail (Roche)]. A 20-μL aliquot was taken for the input control. The remaining lysate was incubated with 1 μg of anti-HA antibody [ChIP grade, cat. no ab9110 (Abcam)] for 2 hours at 4°C under gentle rotation. Then, 10 μL of prewashed (3× in IP buffer) Dynabeads Protein G (Thermo Fisher Scientific) was added to the lysate, and incubation continued for at least 2 hours, up to overnight, under gentle rotation at 4°C. After incubation, the beads were washed four times with IP buffer without the protease inhibitor cocktail and once with ice-cold 50 mM Tris-HCl (pH 7.5). Both IP samples and inputs were subjected to immunoblotting analysis using HRP-conjugated anti-HA antibody (1:1500; Roche), HRP-conjugated anti-FLAG antibody (1:1000; Sigma), and HRP-conjugated anti-TUB antibody (1:4000; Santa Cruz Biotechnology).

Statistical analyses

For statistical difference comparisons among multiple groups, one-way ANOVA followed by Tukey’s multiple comparisons test was performed using GraphPad Prism version 10.0.0 for Windows (GraphPad Software, www.graphpad.com). For pairwise comparisons between two groups, two-tailed Student’s t-tests were conducted. p < 0.05 was considered statistically significant.

Funding

This work was supported by grants to I.H. from the New Breeding Technologies Development Program (project no. PJ016538), Rural Development Administration, Republic of Korea; and from the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT, Republic of Korea (project no. 2020R1A2C3012750). T.V.T.D. was supported by BK21 FOUR through the NRF funded by the Ministry of Education, Republic of Korea. H.S.C. was supported by the Basic Science Research Program through the NRF funded by the Ministry of Education, Republic of Korea (grant nos. S-2023-00276819 and RS-2024-00461525). S.L. was supported by the Basic Science Research Program through the NRF funded by the Ministry of Education, Republic of Korea (grant no. 2019R1I1A1A01055449).

Acknowledgments

We thank all our lab members for reading the manuscript and providing helpful suggestions. The authors declare no competing interests.

Author contributions

T.V.T.D. and I.H. conceived the research and designed the experiments. T.V.T.D. performed the experiments and analyzed the data. H.S.C. analyzed simultaneous detection of ROS. S.L. performed the transcriptomic analyses. T.V.T.D. and I.H. wrote the manuscript.

Published: January 8, 2025