Acetoin Promotes Plant Growth and Alleviates Saline Stress by Activating Metabolic Pathways in Lettuce Seedlings

Abstract

Acetoin is a volatile organic compound, which is a class of metabolites produced by plant growth-promoting rhizobacteria. The mechanisms underlying plant growth promotion by acetoin and its potential to induce saline stress tolerance in plants are poorly understood. Lettuce (Lactuca sativa L. var. ramosa Hort.) seedlings in hydronics and pots under non-saline or saline conditions were foliar-sprayed with 10 mL of 0 or 1 mg·mL^-1 acetoin at 7 and 14 d after transplantation and harvested 7 d after the second spray. Shoots and roots of hydroponic lettuce seedlings were harvested at 6 and 24 h after treatment for RNA sequencing. Seedlings sprayed with acetoin showed more vigorous growth, with higher shoot and root biomass than those of the controls, in both hydronic and pot modes. The transcriptomic analysis revealed acetoin application resulted in 177 differentially expressed genes (39 upregulated and 138 downregulated) in shoots and 397 differentially expressed genes (112 upregulated and 285 downregulated) in roots. These DEGs, mainly involved in plant hormone signal transduction and the mitogen-activated protein kinase, have the potential to trigger plants' responses to various environmental stimuli, including stress and developmental signals. Under saline conditions, acetoin-treated plants showed increased net leaf photosynthesis and activities of several defense enzymes, indicating that acetoin enhances both fundamental growth and the plant's stress defenses, especially against salinity. In summary, acetoin appears to act through a complex interplay of genetic and biochemical mechanisms, influencing key signaling pathways and physiological processes that lead to improved growth and stress tolerance in lettuce seedlings.

Keywords: bio-fertilizer; photosynthesis; saline stress; transcriptome; volatile organic compounds.

Figure 1

Effects of acetoin (AT) on phenotypic response of lettuce in the hydroponic experiment. (A) Phenotype of plants, (B) shoot dry weight and root dry weight, (C) number of leaves, and (D) value of SPAD under CK (non-AT control) and AT treatments. Different letters indicate significant differences (p < 0.05) according to an independent sample t-test.

Figure 2

Numbers of differentially expressed genes associated with biological processes, cellular components, and molecular functions found using Gene Ontology [13] enrichment analysis. (A) GO analysis of DEGs in the shoots of Lettuce. (B) GO analysis of DEGs in the roots of Lettuce. Blue bar represents up-regulated DEGs. Orange bar represents down-regulated DEGs. CK6/24 represents the non-acetoin (AT) control at 6 h (24 h post treatment); AT6/24 represents the AT control at 6 h (24 h post treatment). L and R represent the shoots and roots of lettuce seedlings, respectively.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) classification of differentially expressed genes (DEGs). Results are summarized as five main KEGG A Classes: metabolism, genetic information processing, cellular processes, organismal systems, and environmental information processing. CK6/24 represents the non- acetoin (AT) control at 6 h (24 h post treatment); AT6/24 represents the AT control at 6 h (24 h post treatment). L and R represent the shoots and roots of lettuce seedlings, respectively. Numbers indicate the number of DEGs.

Figure 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) related to plant hormones and signal transduction at 6 and 24 h post acetoin treatment. (A) KEGG pathway analysis of DEGs related to plant hormones and signal transduction at 6 h. (B) and at 24 h. Red and green indicate up- and downregulated genes, respectively and the numbers on the left represent the differential expression of genes. ABA represents abscisic acid; PYR/PYL, PP2C, SnRK2, ABF, ETR, CTR1, EIN2, EIN3, and ERF1/2 represent the genes encoding abscisic acid receptor PYR/PYL family, protein phosphatases 2C, SNF1-related type 2 protein kinase, ABA-response element binding factors, ethylene receptor, serine/threonine-protein kinase CTR1, ethylene-insensitive protein 2, ethylene-insensitive protein 3, and ethylene-responsive transcription factor 1/2, respectively.

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) related to MAPK signaling pathway–plant at 6 and 24 h post acetoin treatment. KEGG pathway analysis of DEGs related to MAPK signaling pathway–plant at 6 (A) and 24 h (B). Red and green indicate up- and downregulated genes, respectively and the numbers on the left represent the differential expression of genes. ABA represents abscisic acid; PYR/PYL, PP2C, SnRK2, MAPKKK17/18, MKK3/9, ETR/ERS, CTR1, EIN3/EIL, ERF1, PDF1.2, CHIB, CALM/CML, RbohD, and OXI1 represent the genes encoding abscisic acid receptor PYR/PYL family, protein phosphatases 2C, SNF1-related type 2 protein kinase, mitogen-activated protein kinase kinase kinase 17/18, mitogen-activated protein kinase 3/9, ethylene receptor, serine/threonine-protein kinase CTR1, ethylene-insensitive protein 3, ethylene-responsive transcription factor 1, defensin-like protein 16, basic endochitinase B, calmodulin/calmodulin-like protein, respiratory burst oxidase homologous protein D, and serine/threonine-protein kinase OXI1, respectively. MPK1/2, MPK7/14, MPK3/6, and MPK8 represents the genes encoding mitogen-activated protein kinase 1/2, 7/14, 3/6, and 8, respectively.

Figure 6

Relative expression levels of 10 differentially expressed genes were measured using qRT-PCR (2^−ΔΔCt). CK represents the non-acetoin (AT) control. According to independent sample t-test, different letters indicate significant differences (p < 0.05). Ls represents lettuce; EBF2, MKK9, CP1, CTR1, CML35/39/41/46, PP2CA, and PYL4 represent the genes encoding the EIN3-binding F-box protein 2, mitogen-activated protein kinase 9, calmodulin, serine/threonine-protein kinase, calmodulin-like protein 35/39/41/46, protein phosphatases 2C, and ABA receptor PYR1-like protein, respectively.

Figure 7

Effects of acetoin (AT) on the growth of lettuce under non-saline and saline conditions in the pot experiment. (A) Phenotype of shoots, (B) value of SPAD, (C) plant height, (D) leaf area, (E) shoot fresh weight, (F) root fresh weight, (G) shoot dry weight, and (H) root dry weight under CK (non-AT control) and AT treatments. According to independent sample t-test, different letters indicate significant differences (p < 0.05).

Figure 8

Effects of acetoin (AT) on the photosynthetic parameters of lettuce seedlings under non-saline and saline conditions in the pot experiment. (A) Net photosynthetic rate (Pn), (B) transpiration rate (Tr), (C) stomatal conductance (gsw), and (D) intercellular CO₂ concentration (Ci) under CK (non-AT and non-NaCl treatment control), CK + AT, NaCl (3 g NaCl per kg of soil), and NaCl + AT treatments. According to independent sample t-test, different letters indicate significant differences (p < 0.05).

Figure 9

Dynamic changes in the defense enzyme activities in leaves of lettuce seedlings under non-saline and saline conditions in the pot experiment. (A) Superoxide dismutase (SOD) activity; (B) malondialdehyde (MDA) activity; and (C) soluble protein content under CK (non-AT and non-NaCl treatment control), CK + AT, NaCl (3 g NaCl per kg of soil), and NaCl + AT treatments. According to independent sample t-test, different letters indicate significant differences (p < 0.05).

Figure 10

Schematic of growth-promotion mechanisms in lettuce influenced by acetoin. Red modules represent upregulated genes or positive promotion. Green modules represent downregulated genes. Yellow modules represents related physiological responses or indirectly regulated genes. Ls represents lettuce; CALM/CML, CTR1, MKK9, EBF1/2, PYR/PYL, MPK8, EIN3/EIL, PP2C, ERF1/2, and SnRK2 represent the gene encoding calmodulin/calmodulin-like protein, serine/threonine-protein kinase CTR1, mitogen-activated protein kinase kinase 9, EIN3-binding F-box protein 1/2, abscisic acid receptor PYR/PYL family, mitogen-activated protein kinase 8, ethylene-insensitive protein 3, protein phosphatases 2C, ethylene-responsive transcription factor 1/2, and SNF1-related type 2 protein kinase, respectively; ROS represents reactive oxygen species.