Accumulation of calycosin and its 7-O-β-d-glucoside and related gene expression in seedlings of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao induced by low temperature stress

The changes in calycosin and calycosin-7-O-β-d-glucoside content as well as the expression of genes involved in their biosynthesis were monitored in roots, stems and leaves of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao seedlings during 10 days of low temperature treatment. The concentrations of calycosin and its 7-O-β-d-glucoside in the different tissues were analyzed using high-performance liquid chromatography. Higher glycoside contents were observed at 2°C than that at 16°C in all the tested tissues, however, the aglycone was scarcely detected in both leaves and stems at either 16 or 2°C. cDNA fragments encoding four structural genes from the calycosin pathway, namely chalcone synthase, isoflavone synthase, isoflavone 3'-hydroxylase and UDP-glucose: calycosin-7-O-glucosyltransferase were isolated from A. membranaceus var. mongholicus seedlings by polymerase chain reaction (PCR) and sequenced. Real-time quantitative reverse transcript PCR demonstrated that in leaves and stems, five genes (including phenylalanine ammonia lyase), exhibited clear differences in their accumulation pattern in response to a low temperature stress, which was consistent with the increased content of calycosin-7-O-β-d-glucoside. In the roots, transcription of the five genes was down-regulated at 2°C, but the contents of calycosin and its glucosides were higher than that at 16°C. These findings indicate that low temperature stress could induce accumulation of calycosin and its glucosides in different tissues of the seedlings of A. membranaceus var. mongholicus but the mechanisms regulating the accumulation were different.