Exogenous Stilbenes Improved Tolerance of Arabidopsis thaliana to a Shock of Ultraviolet B Radiation

Abstract

Excessive ultraviolet B (UV-B) irradiation is one of the most serious threats leading to severe crop production losses. It is known that secondary metabolite biosynthesis plays an important role in plant defense and forms a protective shield against excessive UV-B irradiation. The contents of stilbenes and other plant phenolics are known to sharply increase after UV-B irradiation, but there is little direct evidence for the involvement of stilbenes and other plant phenolics in plant UV-B protection. This study showed that foliar application of trans-resveratrol (1 and 5 mM) and trans-piceid (5 mM) considerably increased tolerance to a shock of UV-B (10 min at 1800 µW cm^-2 of irradiation intensity) of four-week-old Arabidopsis thaliana plants that are naturally incapable of stilbene production. Application of trans-resveratrol and trans-piceid increased the leaf survival rates by 1-2%. This stilbene-induced improvement in UV-B tolerance was higher than after foliar application of the stilbene precursors, p-coumaric and trans-cinnamic acids (only 1-3%), but less than that after treatment with octocrylene (19-24%), a widely used UV-B absorber. Plant treatment with trans-resveratrol increased expression of antioxidant and stress-inducible genes in A.thaliana plants and decreased expression of DNA repair genes. This study directly demonstrates an important positive role of stilbenes in plant tolerance to excessive UV-B irradiation, and offers a new approach for plant UV-B protection.

Keywords: UV-B irradiation; piceid; plant UV tolerance; resveratrol; stilbenes.

Figure 1

The effect of exogenous stilbenes, stilbene precursors and octocrylene on the leaf survival of Arabidopsis thaliana exposed to ultraviolet B (UV-B) irradiation. The leaf surface of four-week-old A. thaliana rosettes was treated with t-resveratrol (a), t-piceid (b), p-coumaric acid (c), t-cinnamic acid (d), octocrylene (e) and then exposed to UV-B 12 h after the chemical treatments. UV-B (312 nm) was applied for 10 min at a distance of 15 cm above the pots and as described [33]. C: water-treated A. thaliana exposed to the same UV-B irradiation conditions. The leaf survival rates were determined as the number of visibly green leaves seven days after the UV-B exposure. The data are presented as mean ± standard error. The data were obtained from five biological replicates (n = 40). *, ** Significantly different from the control water-treated A. thaliana at p < 0.05 and 0.01, respectively, according to the Student’s t test.

Figure 2

A representative HPLC profile (310 nm) of t-resveratrol and cis-resveratrol standards (a) and stilbenes extracted 2 h (b) and 12 h (c) after ultraviolet B (UV-B)- and t-resveratrol treatments of Arabidopsis thaliana. Trans-resveratrol (1) and cis-resveratrol (2).

Figure 3

The effect of t-resveratrol and UV-B treatments on the expression of selected stress-responsive genes of Arabidopsis thaliana. The expression levels of ABA biosynthesis genes ((a), ABA1; (b), ABA2) and antioxidant genes ((c), CAT1; (d), CSD1; (e), CSD2) were analyzed. The four-week-old A. thaliana rosettes were sprayed with t-resveratrol and then exposed to UV-B 12 h after application of t-resveratrol. RNA was isolated before treatments, 12 h and 24 h after UV-B irradiation. C1 and C2: control A. thaliana not exposed to UV-B irradiation and not treated with t-resveratrol; 0: water-treated A. thaliana plants exposed to UV-B irradiation; 1 and 5: t-resveratrol-treated A. thaliana plants exposed to UV-B irradiation; r.u.–relative units. Means followed by the same letter were not different at p ≤ 0.05 using Student’s t test (three independent experiments).

Figure 4

The effect of t-resveratrol and UV-B treatments on the expression of selected stress-responsive genes of Arabidopsis thaliana. The expression levels of stress-inducible regulatory genes ((a), ABF3; (b), KIN1, (c), RD26; (d), RD29a; (e), RD29b) and ion transporter genes ((f), NHX1; (g), SOS1) was analyzed. The four-week-old A. thaliana rosettes were sprayed with t-resveratrol and then exposed to UV-B 12 h after application of t-resveratrol. RNA was isolated before treatments, 12 h and 24 h after UV-B irradiation. C1 and C2: control A. thaliana not exposed to UV-B irradiation and not treated with t-resveratrol; 0: water-treated A. thaliana plants exposed to UV-B irradiation; 1 and 5: t-resveratrol-treated A. thaliana plants exposed to UV-B irradiation; r.u.–relative units. Means followed by the same letter were not different at p ≤ 0.05 using Student’s t test (three independent experiments).

Figure 5

The effect of t-resveratrol and UV-B treatments on the expression of selected DNA repair genes of Arabidopsis thaliana. The expression levels of DNA demethylases ((a), DME; (b), DML3), DNA repair proteins ((c), Rad4; (d), Rad23), DNA polymerase ((e), Pol), DNA glycosylases ((f), UNG1), and photolyase ((g), UVR2; (h), UVR3) was analyzed. The four-week-old A. thaliana rosettes were sprayed with t-resveratrol and then exposed to UV-B 12 h after application of t-resveratrol. RNA was isolated before treatments, 12 h and 24 h after UV-B irradiation. C1 and C2: control A. thaliana not exposed to UV-B irradiation and not treated with t-resveratrol; 0: water-treated A. thaliana plants exposed to UV-B irradiation; 1 and 5: t-resveratrol-treated A. thaliana plants exposed to UV-B irradiation; r.u.–relative units. Means followed by the same letter were not different at p ≤ 0.05 using Student’s t test (three independent experiments).

Figure 6

A representative HPLC-UV (310 nm) profile of the methanol extracts from Arabidopsis thaliana plants before (a) and 12 h after 5 mM p-coumaric (b). 1: coumaroyl hexoside (isomer 1); 2: glucohirsutin; 3: coumaroyl hexoside (isomer 2); 4: sinapoyl hexoside; 5: robinin; 6: p-coumaric acid; 7: kaempferol-3-O-glucosid-7-O-ramnoside; 8: sinapoyl malate.