β-Aminobutyric acid promotes stress tolerance, physiological adjustments, as well as broad epigenetic changes at DNA and RNA nucleobases in field elms (Ulmus minor)

Abstract

Background: β-Aminobutyric acid (BABA) has been successfully used to prime stress resistance in numerous plant species; however, its effectiveness in forest trees has been poorly explored thus far. This study aimed to investigate the influence of BABA on morphological, physiological, and epigenetic parameters in field elms under various growth conditions. Epigenetic changes were assessed in both DNA and RNA through the use of reversed-phase ultra-performance liquid chromatography (UPLC) coupled with sensitive mass spectrometry.

Results: The presented results confirm the influence of BABA on the development, physiology, and stress tolerance in field elms. However, the most important findings are related to the broad epigenetic changes promoted by this amino acid, which involve both DNA and RNA. Our findings confirm, for the first time, that BABA influences not only well-known epigenetic markers in plants, such as 5-methylcytosine, but also several other non-canonical nucleobases, such as 5-hydroxymethyluracil, 5-formylcytosine, 5-hydroxymethylcytosine, N6-methyladenine, uracil (in DNA) and thymine (in RNA). The significant effect on the levels of N6-methyladenine, the main bacterial epigenetic marker, is particularly noteworthy. In this case, the question arises as to whether this effect is due to epigenetic changes in the microbiome, the plant genome, or both.

Conclusions: The plant phenotype is the result of complex interactions between the plant's DNA, the microbiome, and the environment. We propose that different types of epigenetic changes in the plant and microbiome may play important roles in the largely unknown memory process that enables plants to adapt faster to changing environmental conditions.

Keywords: Drought stress; Epigenetics; Forest trees; Holoepigenome; Hologenome; Non-canonical nucleobases; Plant memory; Priming; Stress tolerance; β-Aminobutyric acid.

Fig. 1

Influence of BABA on plant growth. Treatments were carried out by drenching and spraying four-month-old elm greenhouse seedlings with different BABA concentrations. Plants were kept under normal watering (NW) or drought stress (DS) conditions during 8 weeks. Plant height increase (PHI) during stress tests was measured. Box plots present median, upper and lower quartiles; whiskers show minimum and maximum values, n = 30. Statistical data analyses: Kruskal Wallis test and multiple comparisons of Nemenyi. Comparisons were made between treatment groups and the corresponding watering control group, 0 (NW) or 0 (DS). Statistically significant differences (p < 0.05) are shown with different letters above groups. P-values respect to controls are shown: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Influence of BABA on leaf pigments concentrations and NBI index. Treatments were carried out by drenching and spraying four-month-old elm greenhouse seedlings with different BABA concentrations. Plants were kept under normal watering (NW) or drought stress (DS) conditions during 8 weeks. Results are based on measurements of the epidermal UV absorbance of chlorophyll (a), anthocyanins (b) and NBI (c) with a Dualex device. Uppermost leaves were measured at the end of the experiments. Box plots present median, upper and lower quartiles; whiskers show minimum and maximum values, n = 30. Statistical tests: Statistical data analyses: Kruskal Wallis test and multiple comparisons of Nemenyi. Comparisons were made between treatments groups and the corresponding watering control group, 0 (NW) or 0 (DS). Statistically significant differences (p < 0.05) are shown with different letters above groups. P-values respect to controls are shown: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Influence of BABA on the maximum quantum yield of PSII photochemistry (Fv/Fm). Treatments were carried out by drenching and spraying four-month-old elm greenhouse seedlings with different BABA concentrations. Plants were kept under normal watering (NW) or drought stress (DS) conditions during 8 weeks. Fv/Fm was measured at the youngest fully developed leaves after stress tests with a PAM fluorometer. Box plots present median, upper and lower quartiles; whiskers show minimum and maximum values. n = 5–12, ANOVA/Tukey. Statistically significant differences (p < 0.05) are shown with different letters above groups. P-values respect to controls are shown: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Epigenomic changes promoted by BABA. Treatments were carried out by drenching and spraying two-month-old elm greenhouse seedlings with 0.5 mM BABA. Plants were watered regularly and no drought stress was applied. The concentrations of several non-canonical deoxynucleosides, N6-mdA (a), 5-mdC (b), 5-hmdU (c), 5-hmdC (d), 5-fdC (e), dU (f), and 8-oxodG (g), were measured in DNA isolated from the youngest fully developed leaves in control (0 mM BABA) and treated elm seedlings (BABA 0.5 mM, 1 or 7 days after treatment). Box plots present median, upper and lower quartiles; whiskers show minimum and maximum values, n = 24. Statistical tests: a parametric (one-way ANOVA and multiple comparisons of Tukey) or a non-parametric (Kruskal-Wallis test and multiple comparisons of Nemenyi) test were used. Statistically significant differences (p < 0.05) are shown with different letters above groups. P-values respect to control are shown: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Epitranscriptomic changes promoted by BABA. Treatments were carried out by drenching and spraying two-month-old elm greenhouse seedlings with 0.5 mM BABA. Plants were watered regularly and no drought stress was applied. The concentration of two non-canonical ribonucleosides, 5-mrC (a) and rT (b), were measured in RNA isolated from the youngest fully developed leaves in control (0 mM BABA) and treated elm seedlings (BABA 0.5 mM, 1 or 7 days after treatment). Box plots present median, upper and lower quartiles; whiskers show minimum and maximum values. n = 10–20. Statistical tests: a parametric (one-way ANOVA and multiple comparisons of Tukey) or a non-parametric (Kruskal-Wallis test and multiple comparisons of Nemenyi) test were used. Statistically significant differences (p < 0.05) are shown with different letters above groups. P-values respect to controls are shown: *p < 0.05, **p < 0.01, ***p < 0.001