Abstract:

4CL （4-coumarate： coenzyme A ligase） is a key enzyme in the lignin synthesis pathway， which has been shown to play an important role in biological and abiotic stress， mechanical damage resistance and other biological processes. However， the aluminum tolerance of Gm4CL2 has not been reported. In this work， Gm4CL2 of Glycine max cv. Tamba was chosen， and its full-length coding sequence was cloned by RT-PCR. The genetic relationship among Gm4CL2protein sequences was analyzed using multiple sequence alignment and phylogenetic tree analysis. Gm4CL2 was transformed into Arabidopsis thaliana and Medicago sativa by Agrobacterium-mediated floral dip and leaf disk method， respectively. Gene expression levels were detected using qRT-PCR. The results of sequence analysis revealed a 1668 bp Gm4CL2 full-length coding sequence， which encodes 555 amino acids and belongs to class I 4CL in dicotyledons. Real-time PCR results showed that Gm4CL2 expression was specifically induced at 50 μmol·L^-1 AlCl₃ （pH 4.5） in 0-2 cm root tips of G. max cv. Tamba seedlings， and the expression levels of AtMATE， AtSTAR1 and AtSTAR2 were significantly up-regulated in root tips of Arabidopsis overexpressing Gm4CL2 under aluminum treatment （P<0.05）. Under Al³⁺ stress， relative root elongation， activities of SOD （superoxide dismutase） and POD （peroxidase） and citrate secretion were significantly elevated， while Evans blue and chrome azure staining and the content of Al³⁺， ROS （reactive oxygen species） and MDA （malondialdehyde） were significantly lower in the roots of Arabidopsis overexpressing Gm4CL2 than in those of the wild type （P<0.05）. The relative root elongation， citrate secretion and biomass of alfalfa overexpressing Gm4CL2 were significantly higher， while Al³⁺ content and Evans blue staining were significantly lower than those of the wild type （P<0.05）. Cell wall composition analysis showed that pectin， and caffeic and ferulic acid contents in Arabidopsis roots overexpressing Gm4CL2 were significantly lower than those of the wild type under both Al³⁺ and non-Al³⁺ stress （P<0.05）. Under Al³⁺ stress， the content of lignin decreased， but the content of 4-coumaric acid increased in Arabidopsis roots overexpressing Gm4CL2 compared with the wild type （P<0.05）.

Key words: Gm4CL2, aluminum tolerance, citric secretion, Arabidopsis, alfalfa