Molecular Mechanisms of the 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase Producing Trichoderma asperellum MAP1 in Enhancing Wheat Tolerance to Waterlogging Stress

Abstract

Waterlogging stress (WS) induces ethylene (ET) and polyamine (spermine, putrescine, and spermidine) production in plants, but their reprogramming is a decisive element for determining the fate of the plant upon waterlogging-induced stress. WS can be challenged by exploring symbiotic microbes that improve the plant's ability to grow better and resist WS. The present study deals with identification and application of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing fungal endophyte Trichoderma asperellum (strain MAP1), isolated from the roots of Canna indica L., on wheat growth under WS. MAP1 positively affected wheat growth by secreting phytohormones/secondary metabolites, strengthening the plant's antioxidant system and influencing the physiology through polyamine production and modulating gene expression. MAP1 inoculation promoted yield in comparison to non-endophyte inoculated waterlogged seedlings. Exogenously applied ethephon (ET synthesis inducer) and 1-aminocyclopropane carboxylic acid (ACC; ET precursor) showed a reduction in growth, compared to MAP1-inoculated waterlogged seedlings, while amino-oxyacetic acid (AOA; ET inhibitor) application reversed the negative effect imposed by ET and ACC, upon waterlogging treatment. A significant reduction in plant growth rate, chlorophyll content, and stomatal conductance was noticed, while H₂O₂, MDA production, and electrolyte leakage were increased in non-inoculated waterlogged seedlings. Moreover, in comparison to non-inoculated waterlogged wheat seedlings, MAP1-inoculated waterlogged wheat exhibited antioxidant-enzyme activities. In agreement with the physiological results, genes associated with the free polyamine (PA) biosynthesis were highly induced and PA content was abundant in MAP1-inoculated seedlings. Furthermore, ET biosynthesis/signaling gene expression was reduced upon MAP1 inoculation under WS. Briefly, MAP1 mitigated the adverse effect of WS in wheat, by reprogramming the PAs and ET biosynthesis, which leads to optimal stomatal conductance, increased photosynthesis, and membrane stability as well as reduced ET-induced leaf senescence.

Keywords: ACC deaminase enzyme; Trichoderma asperellum; biofertilizer; endophytic fungus; ethylene; polyamines; waterlogging stress; wheat.

FIGURE 1

Characterization of MAP1 isolate. (A) IAA, proline, phenols, flavonoid content, and root colonization. (B) ACC deaminase activity. (C) %DPPH, free radical scavenging activity. (D) ACC deaminase gene expression of MAP1. Quantitative data represent means ± SD of three independent experiments and at least six technical replicates.

FIGURE 2

eto1-MAP1 co-culturing assay. (A) Phenotypic analysis showing comparison of MAP1-inoculated, etiolated seedlings of Col-0 accession and eto1 mutant (Col-0 background) with non-inoculated seedlings. (B) Growth kinetics of Col-0 and eto1 mutant seedlings. Quantitative data represent means ± SD of three independent experiments and at least 10 technical replicates each. The asterisks indicate a significant difference compared to untreated control (^***p≤ 0.005).

FIGURE 3

Effect of the fungal strain MAP1 on wheat plant growth. (A) Schematic representation of the wheat seedling bioassay experimental setup. Uniformly germinated wheat seedlings were transferred to the soil pots pre-mixed with MAP1 biomass. Seven-day-old wheat seedlings were inoculated with MAP1 culture filtrate. Fifteen-day-old seedlings were irrigated (8 h before onset of flooding treatment) with ACC, ethephon, and AOA. Fifteen-day-old plants were exposed to waterlogging stress for 5 days continuously and were subjected to a recovery period of further 5 days. Note the arrows indicating the time point of experimental activities. (B) Phenotypic analysis of wheat seedlings, with and without MAP1 inoculation exposed to WS and chemical supplementation. (C) Fresh weight of wheat seedlings compared to control. Quantitative data represent means ± SD of three independent experiments and at least 10 technical replicates each. The asterisks indicate a significant difference compared to untreated control (^***p≤ 0.005).

FIGURE 4

Determination of the effect of WS on (A) chlorophyll content, (B) growth rate, and (C) stomatal conductance of wheat plants with and without MAP1 inoculation. Quantitative data represent means ± SD of three independent experiments and at least six technical replicates each. The asterisks indicate a significant difference compared to untreated control (^***p≤ 0.002).

FIGURE 5

Effect of WS on (A) H₂O₂ production in wheat plants with and without MAP1 inoculation, exposed to WS. Quantitative data represent means ± SD of three independent experiments and at least 10 technical replicates each. (B) DAB staining using 1 cm-long segments of the 3rd leaf from 25-day-old seedlings with and without MAP1 inoculation. Different letters indicate statistically significant differences between treatments (p ≤ 0.05, DMRT).

FIGURE 6

Effect of WS on (A) MDA content and (B) leaf electrolyte leakage in wheat plants with and without MAP1 inoculation, exposed to WS. Quantitative data represent means ± SD of three independent experiments and at least six technical replicates each. Different letters indicate statistically significant differences between treatments (p ≤ 0.002, DMRT).

FIGURE 7

Effect of WS on antioxidant enzyme activity (A) SOD (left-axis), reduced glutathione (GSH) contents (right-axis) (B) CAT (left-axis), POD (right-axis) in wheat plants with and without MAP1 inoculation. Quantitative data represent means ± SD of three independent experiments and at least 10 technical replicates each. The asterisks indicate a significant difference compared to untreated control (^***p≤ 0.005).

FIGURE 8

Effect of WS on free polyamines and ACC content. (A) Spermidine (left axis) and ACC content (right axis). (B) Spermine (left axis) and putrescine (right axis) in wheat plants with and without MAP1 inoculation. Quantitative data represent means ± SD of three independent experiments and at least 10 technical replicates each. The asterisks indicate a significant difference compared to untreated control (^***p≤ 0.05).

FIGURE 9

Expression profiling of polyamine biosynthesis genes, ET biosynthesis and signaling genes, photosynthetic activity-related genes, stress-reducing genes, and membrane transporter genes (root and stomatal) by qRT-PCR. Quantitative data represent means ± SD of three independent experiments and at least four technical replicates each. The asterisks indicate a significant change in gene expression compared to untreated control (^∗p≤ 0.05; ^∗∗p≤ 0.01).

FIGURE 10

Model of T. asperellum MAP1 action during waterlogging-induced stress.