Engineering saline-alkali-tolerant apple rootstock by knocking down MdGH3 genes in M9-T337

Abstract

Soil salinization and alkalization have become an increasingly severe global issues, significantly limiting both the yield and quality of apples (Malus × domestica). M9-T337 is a widely used apple dwarfing rootstock; however, it is sensitive to saline-alkali stress. Therefore, developing saline-alkali tolerant apple rootstocks is essential. In this study, we utilized RNAi (RNA interference) technology to knock down GH3 genes in the M9-T337 background, aiming to engineer a dwarfing and stress-tolerant apple rootstock. We found that MdGH3 RNAi plants exhibited superior morphology compared to M9-T337 under saline-alkali stress conditions, characterized by more robust root systems, increased plant height, a lower Na⁺/K⁺ ratio, and enhanced photosynthetic and antioxidant capacities. Moreover, when MdGH3 RNAi plants were used as rootstocks, the GL-3/MdGH3 RNAi plants also displayed greater plant height, root vitality, photosynthetic ability, and antioxidant capacity compared to GL-3 grafted onto M9-T337 rootstock. Taken together, our study constructed a saline-alkali-tolerant apple rootstock by knocking down MdGH3 genes.

Keywords: MdGH3 RNAi; Apple; M9-T337; Rootstock; Saline-alkali stress.

Fig. 1

MdGH3 RNAi plants are more tolerant to saline-alkali stress. A Photos of M9-T337 and MdGH3 RNAi transgenic apple plants exposed to the control and saline-alkali stress conditions after 15 days. Scale bars: 10 cm. Plant height (B), root viability (C), ion leakage (D) of plants shown in a. Two-month-old M9-T337 and MdGH3 RNAi transgenic apple plants were divided into two groups. The control group was grown in 1/2-strength Hoagland nutrient solution. The saline-alkali stress condition group was treated with 25 mM NaCl and NaHCO₃ (v/v = 1:1, pH = 8.5 ± 0.2). After 15 days of stress treatment, plants were photographed and used in the subsequent experiments. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 2

MdGH3 RNAi plants confer saline-alkali stress tolerance by reducing the Na⁺/K⁺ ratio in apple leaves and roots. The Na⁺ content (A to B), K⁺ content (C to D) and Na⁺/K⁺ ratio (E to F) in the leaves and roots of M9-T337 and MdGH3 RNAi plants under the control and saline-alkali stress conditions. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 3

MdGH3 RNAi plants show the enhanced photosynthetic capacity under saline-alkali stress conditions. Changes in Pn (A), total chlorophyll content (B), chlorophyll a content (C), chlorophyll b content (D), chlorophyll fluorescence (E), Fv/Fm (F) of M9-T337 and MdGH3 RNAi transgenic apple plants under control and saline-alkali stress conditions. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 4

MdGH3 RNAi plants enhance the antioxidant capacity under saline-alkali stress condition. A to E The O₂⁻ content (A), H₂O₂ content (B), MDA content (C), activities of CAT (D), activities of POD (E) of M9-T337 and MdGH3 RNAi plants. Two-month-old M9-T337 and MdGH3 RNAi transgenic apple plants were divided into two groups. The CK group grew in 1/2-strength Hoagland nutrient solution. The MSA conditions group was added with 25 mM NaCl and NaHCO₃ (v/v = 1:1, pH = 8.5 ± 0.2). After 10 days of the stress treatment, plants were used for the subsequent experiments and analyses. Error bars indicate SD (n = 3). Statistical significance was determined using ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 5

Grafting GL-3 onto MdGH3 RNAi plants enhances the saline-alkali stress tolerance of scions. A Morphology of GL-3 grafted on M9-T337 and MdGH3 RNAi plants. Scale bars: 10 cm. Plant height (B), ion leakage (C) of plants in (A). Two-month-old GL-3/M9-T337 and GL-3/MdGH3 RNAi grafted plants were divided into two groups. The Control group grew in 1/2-strength Hoagland nutrient solution. The saline-alkali stress condition group was added with 25 mM NaCl and NaHCO₃ (v/v = 1:1, pH = 8.5 ± 0.2). After 15 days of the stress treatment, plants were photographed and used in the subsequent experiments. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 6

Grafting GL-3 on the MdGH3 RNAi rootstocks facilitate the photosynthetic capacity of scions under saline-alkali stress condition. The Pn (A), Tr (B), Gs (C), SPAD (D), chlorophyll fluorescence (E), Fv/Fm ratios (F) in GL-3 grafted onto M9-T337 and MdGH3 RNAi plants. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Fig. 7

Grafting GL-3 on the MdGH3 RNAi rootstocks confer plants tolerance to saline-alkali stress by activating antioxidant system. The O₂⁻ content (A), H₂O₂ content (B), activities of CAT (C) and POD (D), MDA content (E) of GL-3 scions grafted onto the M9-T337 and MdGH3 RNAi rootstocks. Error bars indicate SD (n = 3). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test