Effect of chilling acclimation on germination and seedlings response to cold in different seed coat colored wheat (Triticum aestivum L.)

Abstract

Background: Flavonoids can protect plants against extreme temperatures and ROS due to their antioxidant activities. We found that deep-purple seed coat color was controlled by two gene interaction (12:3:1) from the cross between yellow and deep-purple seed coat colored inbreds. F_2:3 seeds were grouped in 3 by seed coat color and germinated under chilling (4 °C) and non-acclimated conditions (18 °C) for a week, followed by normal conditions (18 °C) for three weeks and a subsequent chilling stress (4 °C) induction. We analyzed mean daily germination in each group. Additionally, to study the acclimation in relationship to the different seed coat colors on the germination ability and seedling performances under the cold temperatures, we measured the chlorophyll content, ROS scavenging activity, and expression levels of genes involved in ROS scavenging, flavonoid biosynthetic pathway, and cold response in seedlings.

Results: The results of seed color segregation between yellow and deep purple suggested a two-gene model. In the germination study, normal environmental conditions induced the germination of yellow-seed, while under chilling conditions, the germination ratio of deep purple-seed was higher than that of yellow-colored seeds. We also found that the darker seed coat colors were highly responsive to cold acclimation based on the ROS scavenging enzymes activity and gene expression of ROS scavenging enzymes, flavonoid biosynthetic pathway and cold responsive genes.

Conclusions: We suggest that deep purple colored seed might be in a state of innate pre-acquired stress response state under normal conditions to counteract stresses in a more effective way. Whereas, after the acclimation, another stress should enhance the cold genes expression response, which might result in a more efficient chilling stress response in deep purple seed seedlings. Low temperature has a large impact on the yield of crops. Thus, understanding the benefit of seed coat color response to chilling stress and the identification of limiting factors are useful for developing breeding strategies in order to improve the yield of wheat under chilling stress.

Keywords: Cold tolerance; Flavonoids; Low temperature; Purple wheat; ROS.

Fig. 1

Experimental design and crosses of RILs for color segregation. a Schematic representation of experimental design and treatments. NA: non-acclimated, chilling acclimated (CA), BCS: before chilling stress, ACS: after chilling stress; (b) crosses for the generation of the seed coat color segregation and seed coat color segregation groups based on our genetic analysis. Seed coat color segregation, bulked groups from average scores making each groups: yellow seeds from scores from 1 to 5; medium purple seeds, scores > 5 to 7, and deep purple seeds, scores > 7 to 9

Fig. 2

Phenolics and anthocyanins quantification in seeds. a Free phenolics, (b) total phenolics, and (c) anthocyanin quantification in yellow (Ye)-, medium purple (MP)- and deep purple (DP)- seed groups. Data are means ± SEM of three replicates. Significant differences, evaluated by t-test, are marked by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 when compared to Ye-seed

Fig. 3

Germination percentage under non-acclimated and chilling acclimated seeds. Germination percentage per day in yellow (Ye-seed), medium purple (MP-seed), and deep purple seed groups (DP-seed) under non-acclimated (NA) and chilling-acclimated (CA) conditions. Data are presented as means ± SEM of three replicates. Different letters indicate significant differences in each coordinate value between groups (p ≤ 0.05).”A” means no significant difference between Ye-, MP-, and DP- seed under non-acclimated and DP-seed groups under CA conditions

Fig. 4

Heatmap showing the effect of the second chilling stress in gene expression. ROS (reactive oxygen species) scavenging genes; flavonoid, anthocyanins, and transcription factors regulating the flavonoid biosynthesis pathway; and cold response genes were measured in yellow- (Ye-), medium purple- (MP-), and deep purple- (DP-) seed groups of seedlings germinated under non-acclimated (NA) (18 °C) and chilling acclimated (CA) (4 °C) conditions before chilling stress (BCS) (18 °C) and after chilling stress (ACS) (4 °C for 6 h). Ye BCS and Ye ACS sample were used for comparison within groups (NA and CA). Numbers represent the fold changes. Relative color values are based of the subtraction of the row median, divided by row standard deviation. Data are presented as means ± SD of three biological replicates