The down-regulation of MsWOX13-2 promotes enhanced waterlogging resilience in alfalfa

Abstract

Soil waterlogging events are predicted to escalate globally as a result of climate change, threatening the sustainability of alfalfa (Medicago sativa L.) and livestock production in the future. WUSCHEL-related homeobox (WOX) transcription factors are known to play a role in numerous developmental processes and abiotic stress responses; however, their function in waterlogging resilience has not been investigated as of yet. In the present study, we functionally characterized the alfalfa MsWOX13-2 gene, which we found to be differentially expressed in response to waterlogging. Although the RNAi-mediated silencing of MsWOX13-2 in alfalfa did not affect growth or morphology under normally watered conditions, MsWOX13-2 RNAi plants exhibited higher chlorophyll retention and maximum quantum efficiency of photosystem II, as well as greater survivability, compared to empty vector genotypes under waterlogging. Subsequent analyses indicated that MsWOX13-2 RNAi leaves accumulated less H₂O₂ and displayed a greater increase in superoxide dismutase activity under waterlogging, resulting in reduced oxidative damage, which may have contributed to the enhanced waterlogging tolerance in these genotypes. RNA-Seq analysis confirmed alterations in the transcript levels of genes related to antioxidants, as well as those involved in photosynthesis, anaerobic fermentation, phytohormone-related pathways, and transcriptional regulation in the leaves of WOX13-2 RNAi genotypes compared to wild type following waterlogging stress. Bi-allelic mutation of MsWOX13-2 in alfalfa using CRISPR/Cas9 confirmed its function in waterlogging response. Overall, our findings suggest that MsWOX13-2 acts as a negative regulator of waterlogging response in alfalfa, providing a novel candidate for downstream breeding endeavors in this important species.

Keywords: Medicago sativa; MsWOX13‐2; alfalfa; crop improvement; forage; waterlogging tolerance.

Figure 1

Gene expression patterns of MsWOX13‐2. (A) Tissue‐specific expression of MsWOX13‐2 in alfalfa. (B) Transcriptional response of MsWOX13‐2 to waterlogging stress in alfalfa. Blocks represent the mean of 4–8 biological replicates and bars indicate standard errors. Lower case letters indicate statistically significant differences between groups within a treatment as determined by the Kruskal–Wallis test followed by Dunn's multiple comparison test with ‘Bonferroni’ adjustment (P < 0.05). * and N.S. denote significant differences at P ≤ 0.05 or no significant difference, respectively, between normally watered and waterlogged conditions as determined by two‐tailed t‐tests (assuming unequal variance).

Figure 2

The RNAi‐mediated silencing of MsWOX13‐2 leads to improved waterlogging tolerance in alfalfa. (A) Representative images of plants grown under normally watered conditions, as well as those subjected to waterlogging for 7 and 14 days. (B) Survival rate of empty vector and MsWOX13‐2 RNAi genotypes under normally watered and waterlogged conditions. Blocks represent mean values of survival rates for two experimental sets. Green dots represent data points from set 1 and black dots represent data points from set 2. In each experimental set, 18 biological replicates of three independent empty vector genotypes (pooled), and 5–7 biological replicates of RNAi‐203 and RNAi‐205 genotypes, respectively, were utilized for assessment.

Figure 3

Morphological evaluation of empty vector and MsWOX13‐2 RNAi genotypes under normally watered (NW) and waterlogged (WL) conditions. (A) Plant height, (B) number of branches, (C) shoot fresh weight, (D) shoot dry weight, (E) root length, and (F) root dry weight of empty vector and MsWOX13‐2 RNAi genotypes measured under both NW and WL conditions. (G) Representative images of empty vector and MsWOX13‐2 RNAi roots under NW conditions and after 14 days of waterlogging. Blocks represent mean values of 18–24 biological replicates of three independent empty vector genotypes (pooled), and 7–8 biological replicates of each MsWOX13‐2 RNAi genotype, respectively. Bars indicate standard errors. Lower case letters denote statistically significant differences between groups as determined by two‐way anova followed by Tukey's HSD test (P ≤ 0.05).

Figure 4

Photosynthesis‐related characteristics in empty vector and MsWOX13‐2 RNAi genotypes under normally watered (NW) and waterlogged conditions. (A) Chlorophyll content, (B) transpiration rate (E), (C) light‐saturated photosynthetic rate (A _sat), (D) stomatal conductance to water vapor (g _sw), (E) quantum yield of photosystem II (Φ _PSII), (F) electron transfer rate at photosystem II (PSII), and (G) maximum quantum efficiency of PSII (F _v/F _m) in empty vector and MsWOX13‐2 RNAi genotypes under NW conditions and after waterlogging for 7 and 14 days. Blocks represent mean values of 12–18 biological replicates of three independent empty vector genotypes (pooled), and 4–8 biological replicates of each MsWOX13‐2 RNAi genotype, respectively. Bars indicate standard errors. Lower case letters denote statistically significant differences between groups as determined by two‐way anova followed by Tukey's HSD test (P ≤ 0.05).

Figure 5

Antioxidant and osmolyte characteristics in the leaves of empty vector and MsWOX13‐2 RNAi genotypes under normally watered (NW) and waterlogged (WL) conditions. (A) Representative images of leaflets from empty vector and MsWOX13‐2 RNAi genotypes following DAB staining. (B) Malondialdehyde levels, (C) superoxide dismutase activity, (D) ascorbate peroxidase activity, (E) and catalase activity in the leaves of alfalfa genotypes under WL and NW conditions. (F) Representative images of trifoliate leaves from empty vector and MsWOX13‐2 RNAi genotypes 14 days after the initiation of waterlogging, (G) anthocyanin index, (H) flavonol index, (I) proline levels, and (J) soluble carbohydrate levels in the leaves of alfalfa genotypes grown under NW and WL conditions. Blocks represent the mean values of 9–18 biological replicates of three independent empty vector genotypes (pooled), and 3–8 biological replicates of each MsWOX13‐2 RNAi genotype, respectively. Bars indicate standard errors. Lower case letters denote statistically significant differences between groups as determined by two‐way anova followed by Tukey's HSD test (P ≤ 0.05). EV, empty vector genotypes.

Figure 6

Transcriptional changes in various metabolic pathways in MsWOX13‐2 RNAi genotypes compared to wild‐type. (A) Transcriptional changes under normally watered conditions, and (B) transcriptional changes after waterlogging for 14 days. Boxes depict the extent of log₂ fold changes in significantly up‐regulated (green) or down‐regulated (red) differentially expressed genes in each pathway. Myo‐ino, myo‐inositol metabolism; Raf, raffinose metabolism; Tre, trehalose metabolism.

Figure 7

Proposed model illustrating the role of MsWOX13‐2 in alfalfa under waterlogging stress. The transcriptional suppression of MsWOX13‐2 under waterlogging results in decreased levels of ROS, increased antioxidant activity, and altered transcript levels of genes associated with antioxidants, photosynthesis, fermentation, phytohormones, and transcription factors. This, in turn, leads to higher chlorophyll content and improved maximum quantum efficiency of photosystem II (F _v/F _m) in alfalfa leaves, supporting sustained growth and enhanced survival under stress conditions. The dotted arrow indicates a putative indirect linkage.