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). 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. This work was supported by the Hohhot Key R&D Project (2023-JBGS-S-1); the National Center of Pratacultural Technology Innovation (Under Preparation) Special Fund for Innovation Platform Construction (CCPTZX2024GJ06); the Shandong Provincial Natural Science Foundation, China (ZR2023QC212, ZR202210270038); the National Natural Science Foundation of China (No. 32401476, No. 32441003); and the Taishan Scholar Program of Shandong, and Qingdao New Energy Shandong Laboratory of Key Projects Programs (Grant: QNESL KPP202302). The authors declare that they have no conflict of interest. C.F., W.L., R.Y., Y.S. and Y.Z. designed research; R.Y., Y.S., Y.Z., C.B., Y.L. and J.S. performed experiments; C.F., W.L., R.Y., Y.S., Y.Z., Z.W., F.Y. and X.W. analysed the data; C.F., W.L., R.Y., Y.S. and Y.Z. wrote the manuscript. The data that supports the findings of this study are available in the supplementary material of this article. Figures S1-S6 Supplementary Figures. Tables S1-S3 Supplementary Tables. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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Claims 20
Claim · model-written summary
MtNAC33 expression was significantly induced under mannitol-simulated drought and NaCl-induced salt stress.
- Taxon
- Medicago truncatula
Medicago truncatulae1 - Treatment
- mannitol-simulated drought and NaCl-induced salt stresse0
- Gene
- MtNAC33e1
- Outcome
- MtNAC33 expressione0
- Direction
- increaseinferrede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“its expression was analysed under mannitol-simulated drought and NaCl-induced salt stress. Results confirmed significant induction of MtNAC33 expression in these stress conditions”
e1“The Medicago truncatula NAC transcription factor MtNAC33 (Medtr3g096140)”
Claim · model-written summary
MtNAC33 overexpression in Arabidopsis enhanced drought resistance relative to wild-type plants.
- Taxon
- Arabidopsis thaliana
Arabidopsis thalianae2 - Treatment
- drought stress (10 days without watering)e3
- Gene
- MtNAC33e2
- Outcome
- drought resistancee3
- Direction
- enhancede3
- Comparator
- wild-type plantse3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“To assess its functional role, MtNAC33 was overexpressed in Arabidopsis thaliana”
e3“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)”
Claim · model-written summary
MtNAC33 overexpressing Arabidopsis showed no significant difference from wild-type plants under salt stress.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae2
- Treatment
- salt stresse3
- Gene
- MtNAC33e2
- Outcome
- differencese3
- Direction
- no significant differencese3
- Comparator
- wild-type plantse3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e2“To assess its functional role, MtNAC33 was overexpressed in Arabidopsis thaliana”
e3“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)”
Claim · model-written summary
MtNAC33 overexpression delayed flowering in alfalfa.
- Taxon
- Medicago sativa
Medicago sativae7 - Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- floweringe4
- Direction
- delayede4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33 overexpression increased the leaf width-to-length ratio of alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- leaf width-to-length ratioe4
- Direction
- increasede4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33 overexpression gave a higher leaf-to-stem weight ratio in alfalfa, a key indicator of forage quality.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- leaf-to-stem weight ratioe4
- Direction
- highere4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33 overexpression increased dry matter biomass of alfalfa by 31%–43%.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- dry matter biomasse4
- Direction
- increasee4
- Value
- 31%–43%e4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33OE alfalfa accumulated significantly higher starch, soluble protein and soluble carbohydrates than control plants.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- starch, soluble protein and soluble carbohydratese5
- Direction
- highere5
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e5“MtNAC33OE plants accumulated significantly higher levels of starch, soluble protein and soluble carbohydrates (Figure 1g–i), alongside an enhanced net photosynthetic rate (Figure 1j)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33 overexpression enhanced the net photosynthetic rate of alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- MtNAC33OEe4
- Gene
- MtNAC33e4
- Outcome
- net photosynthetic ratee5
- Direction
- enhancede5
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e4“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)”
e5“MtNAC33OE plants accumulated significantly higher levels of starch, soluble protein and soluble carbohydrates (Figure 1g–i), alongside an enhanced net photosynthetic rate (Figure 1j)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
After 20 days of drought, MtNAC33OE alfalfa showed less wilting than control plants.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- 20 days of droughte6
- Outcome
- wilting and droopy leavese6
- Direction
- decreaseinferrede6
- Comparator
- control plantse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“After 20 days of drought, control plants exhibited severe wilting and droopy leaves (Figure 1k), whereas MtNAC33OE plants remained green and healthy (Figure 1l)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
After rehydration, MtNAC33OE alfalfa almost recovered while control plants failed to recover.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- rehydratione6
- Outcome
- recoverye6
- Direction
- almost recoverye6
- Comparator
- control plantse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“Following rehydration, control plants failed to recover (Figure 1m), while MtNAC33OE plants showed almost recovery (Figure 1n)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33OE alfalfa had higher Fv/Fm ratios than controls after 24, 48 and 72 h of dehydration.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- 24, 48 and 72 h of dehydratione6
- Outcome
- Fv/Fm ratiose6
- Direction
- highere6
- Comparator
- controlse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“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”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33OE alfalfa had a greater percentage of closed stomata than controls under drought.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- drought conditionse6
- Outcome
- percentage of closed stomatae6
- Direction
- greatere6
- Comparator
- controlse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“SEM analysis revealed a greater percentage of closed stomata in MtNAC33OE leaves under drought conditions compared to controls”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
ABA treatment induced stronger stomatal closure in MtNAC33OE alfalfa leaves.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- ABA treatmente6
- Outcome
- stomatal closuree6
- Direction
- strongere6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“Notably, ABA treatment induced stronger stomatal closure in MtNAC33OE leaves (Figure 1w)”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33OE alfalfa had reduced malondialdehyde (MDA) and proline levels under drought stress.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- drought stresse6
- Outcome
- malondialdehyde (MDA) and proline levelse6
- Direction
- reducede6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“MtNAC33OE plants exhibited reduced malondialdehyde (MDA) and proline levels under drought stress”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
MtNAC33OE alfalfa had 26 162 differentially expressed genes compared with control plants.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Outcome
- differentially expressed genese6
- Value
- 26 162e6
- Qualifier
- 14 372 up-regulated and 11 790 down-regulatede6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“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”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
Genes related to auxin biosynthesis and signalling pathways were significantly altered in MtNAC33OE alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Outcome
- auxin biosynthesis and signalling pathwayse6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“Genes related to auxin biosynthesis and signalling pathways were significantly altered”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
Photosynthesis-related genes Phot1 and GGR were up-regulated in MtNAC33OE alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Gene
- Phototropin-1 (Phot1) and Geranylgeranyl Reductase (GGR)e6
- Outcome
- photosynthesis-related genese6
- Direction
- up-regulatede6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“photosynthesis-related genes, such as Phototropin-1 (Phot1) and Geranylgeranyl Reductase (GGR), were up-regulated”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
Genes for stomatal closure (CLE25, KAT1, PIP2;1, CPK3, CPK4) were significantly up-regulated under drought in MtNAC33OE alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- drought conditionse6
- Gene
- CLE25, KAT1, PIP2;1, CPK3, CPK4e6
- Outcome
- genes involved in stomatal closuree6
- Direction
- up-regulatede6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“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”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
Claim · model-written summary
Genes for ROS regulation (BCB, PER34, AOX1D, SOD) were significantly up-regulated under drought in MtNAC33OE alfalfa.
- Taxon
- Medicago sativaMedicago sativae7
- Material
- Zhongmu No. 1e7
- Treatment
- drought conditionse6
- Gene
- BCB, PER34, AOX1D, SODe6
- Outcome
- ROS regulatione6
- Direction
- up-regulatede6
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e6“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”
e7“in the widely cultivated Medicago sativa cultivar Zhongmu No. 1”
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