Cell wall reconstruction and DNA damage repair play a key role in the improved salt tolerance effects of He-Ne laser irradiation in tall fescue seedlings

Figures & data

Table 1. Treatment procedure of different groups of tall fescue seedlings.

Treatment	Light	NaCl	Hydroxyurea	He–Ne laser illumination	Dark culture
	(PPFD 100 μmol m^−2 s^−1)	(150 mM)	(10 mM)	(632.8 nm, 5 mW mm^−2)	(25 °C)
CK	14 h d^−1	–	–	–	10 h d^−1
H2	14 h d^−1	–	–	2 min d^−1 (7 d)	10 h d^−1
H4	14 h d^−1	–	–	4 min d^−1 (7 d)	10 h d^−1
H6	14 h d^−1	–	–	6 min d^−1 (7 d)	10 h d^−1
N	14 h d^−1	10 d	–	–	10 h d^−1
N+H2	14 h d^−1	10 d	–	2 min d^−1 (7 d)	10 h d^−1
N+H4	14 h d^−1	10 d	–	4 min d^−1 (7 d)	10 h d^−1
N+H6	14 h d^−1	10 d	–	6 min d^−1 (7 d)	10 h d^−1
N+H+U	14 h d^−1	10 d	7 d	2, 4, 6, 8, 10 min d^−1, respectively (7 d)	10 h d^−1

Notes: CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress; N+H+U: combined treatment with He–Ne laser (1, 2, 4, 6, 8, 10 m d⁻¹) and hydroxyurea (10 mM) prior to salt stress.

Fig. 1. The procedure of the preparation of plant cell wall materials (a) and sequential extraction of cell wall polysaccharide (b).

Table 2. Effects of He–Ne laser irradiation on plant growth and development.

Treatment	Fresh weight (g)	Leaf length (mm)	Leaf width (mm)	Leaf area index	Plant height (cm)	Root length (cm)
CK	0.28 ± 0.02b	102 ± 2.3b	4.3 ± 0.8b	2.3 ± 0.6b	14.2 ± 1.2b	13.9 ± 2.6b
H2	0.35 ± 0.05a	125 ± 1.8a	6.2 ± 0.3a	4.1 ± 0.8a	16.7 ± 2.5a	21.9 ± 3.3a
H4	0.29 ± 0.03b	105 ± 3.5b	4.5 ± 0.4b	2.5 ± 0.9b	15.7 ± 1.1b	14.3 ± 2.5b
H6	0.27 ± 0.01b	102 ± 3.1b	4.3 ± 0.6b	2.6 ± 0.4b	14.9 ± 3.4b	14.3 ± 2.1b
N	0.11 ± 0.02c	42 ± 1.5d	1.9 ± 0.2c	0.8 ± 0.09c	5.3 ± 1.8e	4.6 ± 1.0 d
N+H2	0.14 ± 0.06c	45 ± 2.6d	2.1 ± 0.5c	1.1 ± 0.1c	6.1 ± 2.7e	5.6 ± 2.7d
N+H4	0.26 ± 0.05b	75 ± 2.8c	4.2 ± 0.7b	2.2 ± 0.3b	12.8 ± 3.8c	11.4 ± 3.5b
N+H6	0.13 ± 0.03c	54 ± 3.2d	2.4 ± 0.4c	1.8 ± 0.6b	10.4 ± 1.8d	10.2 ± 1.4c

Notes: Data are presented as the means ± SD from five independent experiments (n = 5). Different letters followed with bars indicate significant differences at p < 0.05 according to Duncan’s multiple range test. CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress.

Fig. 2. ROS level (a, H₂O₂ relative content; b, the generation rate of O₂^•−) and MDA contents (c) in tall fescue seedlings under He–Ne laser irradiation and salt stress.

Notes: Data are presented as the means ± SD from five independent experiments (n = 5). Different letters followed with bars indicate significant differences at p < 0.05 according to Duncan’s multiple range test. CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress.

Fig. 3. Effects of salt stress and He–Ne laser irradiation on cell viability with trypan blue staining assay kit in tall fescue seedlings (a–d, i–l: root tissues; e–h, m–p: leaf tissues).

Notes: CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress. 10×, bar = 500 μm.

Fig. 4. The agarose gel electrophoresis analysis and the completed genomic DNA relative contents determination in leaves and roots of tall fescue seedlings (a,c, leaf tissues; b,d, root tissues).

Notes: The results of agarose gel electrophoresis analysis (a, leaf tissues; b, root tissues). M: DNA marker; 1: controls without any stress treatment; 2, 3, 4: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; 5: salt stress (150 mM) for 10 d; 6, 7, 8: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress, respectively. The completed genomic DNA relative contents (c, leaf tissues; d, root tissues) according to signal strength of DNA on the agarose gel. Data are presented as the means ± SD from five independent experiments (n = 5). Different letters followed with bars indicate significant differences at p < 0.05 according to Duncan’s multiple range test. CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress.

Fig. 5. Apopladder assay and DNA damage analysis under salt stress and He–Ne laser irradiation in leaves and roots of tall fescue seedlings (a, c, leaf tissues; b, d, root tissues).

Notes: The results of agarose gel electrophoresis analysis (a, leaf tissues; b, root tissues). M: 1 kb DNA ladder marker; 1: controls without any stress treatment; 2, 3, 4: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; 5: salt stress (150 mM) for 10 d; 6, 7, 8: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress, respectively. Relative DNA “smear” degree (c, leaf tissues) and DNA “laddering” degree (d, root tissues). Data are presented as the means ± SD from five independent experiments (n = 5). Different letters followed with bars indicate significant differences at p < 0.05 according to Duncan’s multiple range test. CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress.

Table 3. Effects of He–Ne laser irradiation on the monosaccharide composition proportions of cell wall polysaccharide of tall fescue seedlings.

Treatment	Fucose (g kg^−1)	Glucose (g kg^−1)	Mannose (g kg^−1)	Arabinose (g kg^−1)	Galatose (g kg^−1)	Xylose (g kg^−1)	Rhamnose (g kg^−1)
CK	3.4 ± 1.2a	298.4 ± 10.3c	8.5 ± 1.5c	48.4 ± 2.3a	31.7 ± 4.1a	15.3 ± 1.2a	5.6 ± 0.8a
H2	3.6 ± 0.5a	295.6 ± 8.7c	9.2 ± 1.8c	47.9 ± 1.9a	31.9 ± 2.2a	16.3 ± 2.2a	5.9 ± 0.3a
H4	3.5 ± 0.7a	299.6 ± 11.5c	8.8 ± 1.1c	51.3 ± 2.8a	31.3 ± 2.9a	15.4 ± 3.1a	6.1 ± 1.2a
H6	3.3 ± 0.4a	295.9 ± 11.7c	8.9 ± 0.8c	49.9 ± 4.5a	33.6 ± 3.6a	17.8 ± 1.3a	5.7 ± 1.5a
N	1.2 ± 0.4c	456.3 ± 14.9b	43.1 ± 0.9b	21.2 ± 3.6c	10.2 ± 1.5b	14.8 ± 0.9a	5.3 ± 0.7a
N+H2	1.3 ± 0.6c	479.4 ± 15.2b	73.5 ± 0.8a	22.9 ± 1.3c	10.8 ± 1.2b	15.1 ± 1.1a	6.2 ± 0.3a
N+H4	2.8 ± 0.5b	568.8 ± 18.3a	103.7 ± 1.2a	41.8 ± 3.3b	11.7 ± 0.9b	15.9 ± 0.8a	6.1 ± 0.2a
N+H6	1.6 ± 0.7c	483.3 ± 17.2b	85.6 ± 0.4a	25.3 ± 2.4c	11.2 ± 1.8b	14.9 ± 0.6a	5.6 ± 1.4a

Notes: Data are presented as the means ± SD from five independent experiments (n = 5). Different letters followed with bars indicate significant differences at p < 0.05 according to Duncan’s multiple range test. CK: controls without any stress treatment; H2, H4, H6: He–Ne laser illumination alone for 2, 4, and 6 min d⁻¹ under normal conditions, respectively; N: salt stress (150 mM) for 10 d; N+H2, N+H4, N+H6: He–Ne laser illumination for 2, 4, and 6 min d⁻¹ prior to salt stress.

Fig. 6. The FTIR spectra analysis of cell wall polysaccharides (a, e, the chelating pectin fraction; b, f, the alkali-soluble pectin fraction; c, g, hemicelluloses; d, h, celluloses) from leaves (a–d) and roots (e–h) materials of tall fescue seedlings.

Notes: CK: controls without any stress treatment; H: He–Ne laser illumination alone for 4 min d⁻¹ under normal conditions; N: salt stress (150 mM) for 10 d; N+H: He–Ne laser illumination for 4 min d⁻¹ prior to salt stress.

Fig. 7. Schematic of a putative mechanism through which cell wall polysaccharides are protected against physicochemical damages induced by salt stress that result in plant cell wall structure reconstruction.

Notes: Then, cell wall also enhances genomic DNA self-repair capacity, improved DNA injury due to reduced genomic DNA “smear” or “tailing” occurrence and decreased DNA “laddering” phenomena via unknown mechanisms. Thereby, resistance-related genes transcriptional levels are activated, finally improved defense resistance against unfavorable stressors, and enhanced abiotic stress tolerance.