Heterografted chrysanthemums enhance salt stress tolerance by integrating reactive oxygen species, soluble sugar, and proline

Abstract

Chrysanthemum, one of the most important commercial ornamental crops, is susceptible to salinity, which limits its cultivation and application in coastal and inland saline areas. Grafting is widely used to improve the salt tolerance of horticultural crops, but the mechanisms of grafted chrysanthemum responses to salt stress remain unclear. In this study, we showed that heterografted chrysanthemums with Artemisia annua as rootstock exhibited increased salt tolerance compared with self-grafted and self-rooted chrysanthemums. Under high salt stress, the roots of heterografted chrysanthemums enrich Na⁺, resulting in a reduction of Na⁺ toxicity in the scion, with only a small amount of Na⁺ being transported to the leaves. On the other hand, the roots of heterografted chrysanthemums alleviated high Na⁺ stress via enhanced catalase enzyme activity, downregulation of the expression of reactive oxygen species (ROS) accumulation-related genes, massive accumulation of soluble sugars and proline, and upregulation of the expression of heat shock protein-related genes to enhance salt tolerance. In addition, the leaves of heterografted chrysanthemums respond to low Na⁺ stress by increasing peroxidase enzyme activity and soluble sugar and proline contents, to maintain a healthy state. However, self-grafted and self-rooted plants could not integrate ROS, soluble sugars, and proline in response to salt stress, and thus exhibited a salt-sensitive phenotype. Our research reveals the mechanisms underlying the increased salt tolerance of heterografted chrysanthemums and makes it possible to have large-scale cultivation of chrysanthemums in saline areas.

Figure 1

HG chrysanthemum plants display significantly enhanced salt tolerance compared with SR and SG chrysanthemum plants. a Phenotypes. b Leaf area injured per plant. The data in (a) and (b) were obtained after 8 days of salt treatment. The data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment and are means of three replicates ± standard deviations. Means marked with different lower-case letters differed significantly at P < .05

Figure 2

Roots of HG plants enriched Na⁺ to reduce the Na⁺ content of leaves. a Relative electrical conductivity. b MDA content. c Na⁺ content. d K⁺ content. e Na⁺/K⁺. Plants were treated as described in Fig. 1. Roots and leaves were sampled after 8 days of salt stress. Data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment. Data are means of three replicates (± standard deviations), and means marked with different lower-case letters differed significantly at P < .05.

Figure 3

Statistical and KEGG enrichment analyses of DEGs. a Statistical analysis of upregulated and downregulated DEGs. b Venn diagrams of DEGs in the leaf. c Venn diagrams of DEGs in the root. d KEGG enrichment analysis of DEGs in SRT versus SRC of the leaf. e KEGG enrichment analysis of DEGs in HGT versus HGC of the root. Data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment.

Figure 4

DEGs related to ROS and Ca²⁺. a Heat map of leaf DEGs related to ROS and Ca²⁺. b Heat map of root DEGs related to ROS and Ca²⁺. c Regulated pathway analysis of DEGs related to ROS and Ca²⁺ in the leaves of SR, SG, and HG plants. d Regulated pathway analysis of DEGs related to ROS and Ca²⁺ in the roots of SR, SG, and HG plants. The three columns of the heat map represent SR, SG, and HG plants from left to right. e Verification of the expression of four DEGs from (a) by qRT–PCR. f POD activity in leaves and roots of SR, SG, and HG plants. g CAT activity in leaves and roots of SR, SG, and HG plants. Roots and leaves were sampled after 0, 1, 4, and 8 days of salt stress. Data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment. Data are means of three replicates (± standard deviations), and means marked with different lower-case letters differed significantly at P < .05.

Figure 5

DEGs involved in starch and sucrose metabolism. a Heat map of leaf DEGs involved in starch and sucrose metabolism. b Heat map of root DEGs involved in starch and sucrose metabolism. c Verification of the expression of four DEGs in (a) by qRT–PCR. d Soluble sugar content in leaves and roots of SR, SG, and HG plants. Data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment. Data are means of three replicates (± standard deviations), and means marked with different lower-case letters differed significantly at P < .05.

Figure 6

DEGs involved in proline and arginine metabolism. a Heat map of leaf DEGs involved in proline and arginine metabolism. b Heat map of root DEGs involved in proline and arginine metabolism. c Verification of the expression of three DEGs in (a) by qRT–PCR. Left vertical axis represents fragments per kilobase per million mapped fragments (FPKM) from RNA sequencing; right vertical axis represents relative gene expression level from qRT–PCR. R-values are the relative coefficients between qRT–PCR and RNA sequencing. d Proline content in leaves and roots of SR, SG, and HG plants. Data were obtained under 0 mM NaCI (C) or 120 mM NaCI (T) treatment. Data are means of three replicates (± standard deviations), and means marked with different lower-case letters differed significantly at P < .05.

Figure 7

A model for mechanisms underlying the enhanced salt tolerance of HG chrysanthemum plants.