Combining transcriptomics and biochemical analysis to understand the accumulation changes of polysaccharides in different harvest periods of Gastrodia elata

Gastrodia elata polysaccharides (GEP), recognized for their diverse biological activities, are significant bioactive components of the orchid Gastrodia elata Blume. However, the dynamics of GEP accumulation across different developmental stages and the underlying molecular mechanisms remain to be elucidated. We quantified the sugar content of G. elata at three distinct harvest periods and found that prolonged growth duration led to an initial decrease followed by an increase in the total reducing sugar content, as well as in the levels of fructose, glucose, and GEP. Conversely, the sucrose content displayed an inverse trend, increasing initially before subsequently decreasing. The KEGG enrichment analysis primarily identified pathways associated with carbohydrate metabolism. We reconstructed the GEP biosynthesis pathway and generated a gene expression heatmap to trace the metabolic conversion from sucrose to uridine diphosphate glucose (UDP-Glc). The expression patterns of most genes in this pathway were closely correlated with the fluctuations in GEP content. This expression pattern was further validated by real-time quantitative PCR (RT-qPCR). The HXK1 and PGM1 genes were identified as potential key regulators, likely encoding rate-limiting enzyme genes in the GEP biosynthetic pathway. The in vitro enzyme-linked reaction confirmed the function of the PGM1 enzyme. Enzymatic characterization of the recombinant PGM1 enzyme and determined its optimal temperature and pH to be 35 °C and 8.0, respectively. The kinetic parameters of the recombinant PGM1 were determined, with K m of 0.4545 mM and K cat was 15.47 min −1 . Molecular docking studies identified key amino acid residues that may influence the catalytic activity of the PGM1 enzyme. This study provided fundamental data to inform the harvesting and breeding of G. elata and offers critical insights into the biosynthetic mechanisms of GEP. • Analysis of the accumulation and changes of sugar substances in G. elata. • The biosynthesis pathway and gene expression heat map of GEP were constructed. • The correlation analysis revealed that PGM1 and HXK1 are the key genes. • Investigated the enzymatic function and enzymatic properties of PGM1. • Molecular docking further revealed the catalytic mechanism of the PGM1 enzyme.