Overexpression of MtNAC33 enhances biomass yield and drought tolerance in alfalfa

Abstract

Alfalfa (Medicago sativa L.), a highly valuable perennial forage legume, is extensively cultivated worldwide (Russelle, 2001). As global warming exacerbates evaporation rates, severe drought conditions, characterized by mud cracking, have increasingly affected alfalfa cultivation regions. Drought stress can decrease stomatal conductance, impair photosynthesis activity and induce reactive oxygen species (ROS) accumulation in Alfalfa plants. Therefore, it reduces alfalfa growth and accelerates flowering, leading to significant declines in biomass yield and forage quality. Previous studies have shown that the plant-specific NAC (NAM, ATAF1,2 and CUC2) transcription factors play crucial roles in plant response to diverse environmental stresses. For example, NACs are involved in cold response of tomato, salt tolerance of soybean and disease resistance of Arabidopsis. Recent studies have also highlighted that OsNAC120 and OsNAC016 regulated the balance between plant growth and drought tolerance by promoting gibberellin (GA) biosynthesis, brassinosteroid (BR) signalling and repressing abscisic acid (ABA)-mediated drought responses (Wu et al., 2022; Xie et al., 2024). These insights provide a framework for developing crop varieties with improved biomass yield under drought conditions.

The Medicago truncatula NAC transcription factor MtNAC33 (Medtr3g096140), one member of the NAC2 subfamily, clusters phylogenetically with Arabidopsis NAC082 and NAC103 (Figure S1). Previous studies revealed that MtNAC33 is induced by mannitol and NaCl treatments in Medicago seedlings (Ling et al., 2017), but its biological functions remain largely unexplored. To elucidate the role of MtNAC33 in drought tolerance, its expression was analysed under mannitol-simulated drought and NaCl-induced salt stress. Results confirmed significant induction of MtNAC33 expression in these stress conditions (Figure 1a; Figure S2). To assess its functional role, MtNAC33 was overexpressed in Arabidopsis thaliana. Two transgenic lines, MtNAC33OE-A and MtNAC33OE-C, with the highest MtNAC33 expression levels, were selected for analysis. Under drought stress (10 days without watering), MtNAC33OE plants exhibited enhanced drought resistance (Figure S3) but showed no significant differences with wild-type plants under salt stress (Figure S4).

Figure 1

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Overexpression of MtNAC33 improves biomass yield and drought tolerance in alfalfa. (a) Quantitative real-time PCR (qRT-qPCR) analysis of MtNAC33 expression levels in M. truncatula (R108) shoots and roots at 0, 3, 6, 12 and 24 h after treatment with 300 mM mannitol. Left Y-axis means MtNAC33 expression levels in shoot, right Y-axis means MtNAC33 expression levels in root. MtActin was used as the internal control. Values are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); ns, no significance, **P < 0.01, ****P < 0.0001. (b) qRT-PCR analysis of MtNAC33 expression levels in transgenic alfalfa lines. MsActin was used as the internal control. Values are means ± SE (n = 3). Different letters indicate significant difference determined by one-way ANOVA (P < 0.05, Tukey's multiple-range test). (c) Leaf characterization of control and MtNAC33OE alfalfa plants. (d) Ratio of leaf width to length. Values are means ± SE (n = 10). One-way ANOVA (Dunnett's multiple-range test); ***P < 0.001, ****P < 0.0001. (e) Leaf-to-stem weight ratio and (f) whole dry weight per plant in control and MtNAC33OE alfalfa plants. Values in are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); *P < 0.05, **P < 0.01. (g) Starch, (h) soluble protein and (i) soluble carbohydrate contents in control and MtNAC33OE alfalfa plants. Values in are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); **P < 0.01, ***P < 0.001. (j) Net photosynthetic rate in control and MtNAC33OE alfalfa plants. Values in are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); *P < 0.05, **P < 0.01. (k, l) Phenotype of control and MtNAC33OE transgenic alfalfa under drought stress and (m, n) following re-watering. After 25 days of growth under optimal moisture conditions post-cutting, drought treatment was imposed by withholding water for 20 days. Following 15 days of drought, plants were re-watered and grown under normal conditions for 1 week. Bar = 10 cm. (o–q) Delayed fluorescence of chlorophyll in leaves after 24 h (o), 48 h (p), and 72 h (q) detached from plants. Values represent total chlorophyll fluorescence density. (r–u) Stomatal morphology in leaves of control (r, t) and MtNAC33OE alfalfa plants (s, u) following drought treatment. (v) Percentage of closed stomata in control and MtNAC33OE leaves under drought conditions and (w) stomatal aperture after ABA treatment. Values in are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); *P < 0.05, **P < 0.01, ***P < 0.001. (x) MDA and (y) proline contents in MtNAC33OE and wild-type alfalfa under drought stress. Values in are means ± SE (n = 3). One-way ANOVA (Dunnett's multiple-range test); **P < 0.01, ***P < 0.001. (z) Heatmap of transcriptomic data showing differentially expressed genes regulating photosynthetic rate and leaf development under normal conditions. (aa) Differentially expressed genes involved in stomatal closure and (ab) ROS pathway regulation under drought conditions. Orange font, gene number coding Phot1 and GGR proteins.

To further investigate its potential functions in alfalfa responses to drought stress, MtNAC33 was overexpressed in the widely cultivated Medicago sativa cultivar Zhongmu No. 1 using an Agrobacterium-mediated ultrasonic-assisted leaf disc transformation protocol (Zhao et al., 2024). Two transgenic lines, MtNAC33OE-6 and MtNAC33OE-17, with the highest MtNAC33 expression levels, were selected for further studies (Figure 1b). Compared to control plants, MtNAC33OE alfalfa exhibited delayed flowering (Figure S5), increased leaf width-to-length ratio (Figure 1c,d), higher leaf-to-stem weight ratio—a key indicator of forage quality (Figure 1e)—and a 31%–43% increase in dry matter biomass (Figure 1f). Additionally, MtNAC33OE plants accumulated significantly higher levels of starch, soluble protein and soluble carbohydrates (Figure 1g–i), alongside an enhanced net photosynthetic rate (Figure 1j).

Clonal MtNAC33OE and wild-type alfalfa plants, propagated via shoot cuttings, were subjected to drought stress analysis. After 20 days of drought, control plants exhibited severe wilting and droopy leaves (Figure 1k), whereas MtNAC33OE plants remained green and healthy (Figure 1l). Following rehydration, control plants failed to recover (Figure 1m), while MtNAC33OE plants showed almost recovery (Figure 1n). A delayed chlorophyll fluorescence assay revealed higher Fv/Fm ratios in detached leaves of MtNAC33OE plants compared to controls after 24, 48 and 72 h of dehydration (Figure 1o–q). Given that stomatal closure is a critical response to dehydration, the stomatal conductance of abaxial surfaces of 72 h-detached leaf was assessed using scanning electron microscopy (SEM). SEM analysis revealed a greater percentage of closed stomata in MtNAC33OE leaves under drought conditions compared to controls (Figure 1r–v; Figure S6). Notably, ABA treatment induced stronger stomatal closure in MtNAC33OE leaves (Figure 1w). Additionally, MtNAC33OE plants exhibited reduced malondialdehyde (MDA) and proline levels under drought stress (Figure 1x,y), further supporting their enhanced drought tolerance.

To investigate the global impact of MtNAC33 overexpression, transcriptomic analysis was performed on MtNAC33OE and control plants. A total of 26 162 differentially expressed genes (DEGs) were identified, including 14 372 up-regulated and 11 790 down-regulated genes in MtNAC33OE plants under normal conditions. Genes related to auxin biosynthesis and signalling pathways were significantly altered (Figure 1z; Table S1), while photosynthesis-related genes, such as Phototropin-1 (Phot1) and Geranylgeranyl Reductase (GGR), were up-regulated, consistent with observations of enhanced leaf size, photosynthetic rate and biomass yield. Under drought conditions, genes involved in stomatal closure (CLE25, KAT1, PIP2;1, CPK3, CPK4) and ROS regulation (BCB, PER34, AOX1D, SOD) were significantly up-regulated in MtNAC33OE plants (Figure 1aa,ab; Table S2). These findings provide insights into the mechanisms of photosynthetic rate promotion, stomatal regulation and ROS clearance in MtNAC33OE plants, warranting further investigation.

In conclusion, this study identified MtNAC33 as a key transcription factor in Medicago with significant potential for enhancing biomass yield and drought tolerance in forage legumes.