Functional identification of CCR1 gene in apple (Malus halliana) demonstrates that it enhances saline-alkali stress tolerance

Abstract

Abstract Background Lignin is a complex aromatic polymer that plays an important biological role in maintaining plant structure and defending plants against biotic and abiotic stresses. Cinnamoyl-CoA reductase (CCR) is a key enzyme involved in the lignin synthesis-specific pathway and regulates lignin biosynthesis and accumulation. Methods Based on transcriptome data, MhCCR1, which was significantly induced by saline-alkali stress, was cloned from Malus halliana. The physicochemical properties, evolutionary relationships and cis-acting elements were analyzed. We obtained transgenic materials by heterologous transformation of MhCCR1 into Arabidopsis thaliana and tobacco, as well as homologous transformation into apple callus, and characterized their saline-alkali stress resistance by a series of physiological and biochemical experiments. And yeast two-hybridization technique was applied to screen and validate the interacting proteins. Results We found that overexpression of MhCCR1 enhanced the tolerance of A. thaliana, tobacco and apple calli under saline-alkali stress, and caused a variety of physiological and biochemical changes. As compared to the wild type, the transgenic plants showed better growth, higher lignin, chlorophyll and proline contents, lower conductivity and MDA content, and significant increase in antioxidant enzyme activities (SOD, POD, CAT) in the transgenic lines under stress condition. In addition, expression of saline-alkali stress-related genes in overexpressed A. thaliana were also higher than in WT, including the antioxidant genes, the Na+ transporter genes, and the H+-ATPase genes, while expression of the K+ transporter genes displayed opposite changes. Meanwhile, the expression levels of genes related to lignin synthesis, AtPAL1, AtCOMT, AtC4H, At4CL1, and AtCCOAOMT, were also significantly up-regulated. At last, the Y2H experiment confirmed the interaction between MhCCR1 and MhMYB4, MhMYB1R1, MhHXK, and MhbZIP23 proteins. Conclusions These results suggest that MhCCR1 may play a positive regulatory role in saline-alkali tolerance of transgenic lines by regulating the lignin content, osmoregulatory substances, chlorophyll content, antioxidant enzyme activities, and genes related to saline-alkali stress, thus providing excellent resistance genes for the stress-responsive regulatory network of apples, and providing a theoretical basis for the cultivation of saline and alkali-resistant apple varieties. Graphical Abstract

Keywords

• Lignin synthesis
• CCR1
• Saline-alkali stress
• Malus halliana
• Y2H