Physiological and Biochemical Mechanisms and Cytology of Cold Tolerance in Brassica napus

Abstract

Cold damage has negatively impacted the yield, growth and quality of the edible cooking oil in Northern China and Brassica napus L.(rapeseed) planting areas decreased because of cold damage. In the present study we analyzed two Brassica napus cultivars of 16NTS309 (highly resistant to cold damage) and Tianyou2238 (cold sensitive) from Gansu Province, China using physiological, biochemical and cytological methods to investigate the plant's response to cold stress. The results showed that cold stress caused seedling dehydration, and the contents of malondialdehyde (MDA), relative electrolyte leakage and O₂ ^- and H₂O₂ were increased in Tianyou2238 than 16NTS309 under cold stress at 4°C for 48 h, as well as the proline, soluble protein and soluble sugars markedly accumulated, and antioxidant enzymes of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were higher in 16NTS309 compared with in Tianyou2238, which play key roles in prevention of cell damage. After exposure to cold stress, the accumulation of the blue formazan precipitate and reddish brown precipitate indicated that O₂ ^- and H₂O₂, respectively, were produced in the root, stem, and leaf were higher than under non-cold conditions. Contents of O₂ ^- and H₂O₂ in cultivar Tianyou2238 were higher than 16NTS309, this is consistent with the phenotypic result. To understand the specific distribution of O₂ ^- in the sub-cellular, we found that in both cultivars O₂ ^- signals were distributed mainly in cambium tissue, meristematic cells, mesophyll cytoplasm, and surrounding the cell walls of root, stem, leaves, and leaf vein by morphoanatomical analysis, but the quantities varied. Cold stress also triggered obvious ultrastructural alterations in leaf mesophyll of Tianyou2238 including the damage of membrane system, destruction of chloroplast and swelling of mitochondria. This study are useful to provide new insights about the physiological and biochemical mechanisms and cytology associated with the response of B. napus to cold stress for use in breeding cold-resistant varieties.

Keywords: Brassica napus; biochemical index; cold stress; cytology; physiological index; super-oxide anion (O2−).

Figure 1

The chromosomes were counter stained with the DAPI solution. B. napus cultivars of (A) 16NTS309 and (B) Tianyou2238.

Figure 2

Phenotypic symptoms of cultivars 16NTS309 (A, C, E) and Tianyou2238 (B, D, F) in response to cold stress. Morphological characteristics of B. napus in December in the natural environment (A, B), representative phenotypes of the plants under non-cold (C, D), and cold stress conditions (E, F).

Figure 3

The activity of catalase (CAT) (A), peroxidase (POD) (B), superoxide dismutase (SOD) (C), proline (D), protein (E), soluble sugars (F), malondialdehyde (MDA) (G), relative electrolyte leakage (H), O₂ ⁻ (I), and H₂O₂ (J) content accumulation in leaves of 16NTS309 and Tianyou2238 under cold stress at 4°C for 48h. Overwintering rates was recorded in natural environment. Average overwintering rstes (K) in plants of 16NTS309 and Tianyou2238 under harsh winter conditions. The majuscules indicate a significant difference (P<0.05) for the data of the cold stress treated samples compared with non-cold stressed samples. The different small letters indicate significant differences at p < 0.05. The mean values were calculated from three biological replicates. Error bars denote standard error of the mean.

Figure 4

The O₂ ⁻ (A-D), H₂O₂ (E-H) levels in seedlings stained with Nitrotetrazolium blue chloride (NBT) and 3, 3ʹ-Diaminobenzidine (DAB) of cultivars 16NTS309 and Tianyou2238 under non-cold (A, C, E, G) and cold stress (B, D, F, H) conditions. B. napus cultivars 16NTS309 (A, B, E, F) and Tianyou2238 (C, D, G, H).

Figure 5

B. napus cultivars 16NTS309 and Tianyou2238 under non-cold (A, B, E, F) and cold stress (C, D, G, H) conditions for 48 h. The deepest root samples with blue formazan precipitate areas in 16NTS309 (A–D) and Tianyou2238 (E–H) were sectioned for morphoanatomical analysis. ep, epidermis; ca, cambium; sp, secondary phloem; sx, secondary xylem. Scale bars = 50 µm and 100 µm.

Figure 6

B. napus cultivars 16NTS309 and Tianyou2238 under non-cold (A, B, E, F) and cold stress (C, D, G, H) conditions for 48 h. The deepest stem samples with blue formazan precipitate areas in 16NTS309 (A–D) and Tianyou2238 (E–H) were sectioned for morphoanatomical analysis. vb, vascular bundle; co, cortex; ep, epidermis. Scale bars = 50 µm and 100 µm.

Figure 7

B. napus cultivars 16NTS309 and Tianyou2238 under non-cold (A, C) and cold stress (B, D) conditions for 48 h. The deepest leaf samples with blue formazan precipitate areas in 16NTS309 (A, B) and Tianyou2238 (C, D) were sectioned for morphoanatomical analysis. po, pore; ep, epidermis; sp, spongy parenchyma; pt, palisade tissue; vb, vascular bundle; ep, epidermis. Scale bars = 100 µm.

Figure 8

B. napus cultivars 16NTS309 and Tianyou2238 under non-cold (A, C) and cold stress (B, D) conditions for 48 h. The deepest leaf vein samples with blue formazan precipitate areas in 16NTS309 (A, B) and Tianyou2238 (C, D) were sectioned for morphoanatomical analysis. ep, epidermis; xy, xylem; vb, vascular bundle. Scale bars = 100 µm.

Figure 9

Transmission electron micrographs of leaf ultrastructure from cultivar 16NTS309 under non-cold (A–D) and cold stress (E–H) conditions for 48 h. Mi, mitochondria; Ch, chloroplast; GL, granum lamellae; OG, osmiophilic globule; CW, cytoderm.Scale bars = 1 µm and 500 nm.

Figure 10

Transmission electron micrographs of leaf ultrastructure from cultivar Tianyou2238 under non-cold (A–E) and cold stress (F–I) conditions for 48 h. Mi, mitochondria; Ch, chloroplast; GL, granum lamellae; S, starch granules; OG, osmiophilic globule; CW, cytoderm. Scale bars = 2 µm and 500 nm.

Figure 11

Plant callus system for leaf segments of B. napus cultivar Tianyou2238 (cold sensitive). To characterize callus growth in response to cold stress, calluses were grown in 200-ml flasks containing 50 ml of liquid MS medium. After four weeks incubation, the formed calluses (a) and plants (b) were divided into groups: one was maintained at 25°C as the non-cold control and the other was subjected to cold stress at 4°C.

Figure 12

The deepest leaf (A), callus (B, C), root (D–F) samples with blue formazan precipitate areas from the callus of cultivar Tianyou2238 were sectioned for morphoanatomical analyses. ep, epidermis; mc, mesophyll cell; mn, meristematic nodule; me, meristem; pc, parenchymal cell. Scale bars = 50 µm and 100 µm.

Figure 13

Mechanism of reactive oxygen species (ROS) diffusion in cytoplasmic and other cells. Chloroplasts, mitochondria, cell wall peroxidases, and plasma membrane NADPH oxidases play important roles in ROS production. ROS as highly dynamic signalling molecules that ROS propagate throughout the different tissues and cells, and between different organelles and cells over long distances. When the plant defence system collapses, excessive accumulation of ROS from multiple sources results in ROS bursts that damage other surrounding cells and severely damage cellular structures.