Comparative transcriptome analysis reveals the potential mechanism of seed germination promoted by trametenolic acid in Gastrodia elata Blume.

Gastrodia elata Blume (GEB) is a potential medicinal and edible plant with several active components and pharmacological activity that has a high application value in medicine and the food business. However, in natural conditions, GEB seed has a very low germination rate and depends on two specific fungi, germinal and nutritive fungi, to complete the germination process and growth. Armillaria mellea, while acting as a nutrient supplier, actually inhibits the germination of GEB seeds. Mycena strains, as the main germinating fungi, can facilitate germination but cannot support the subsequent growth and development of GEB. It requires symbiotic interactions with Mycena and Armillaria mellea to obtain nutrients for its complex life cycle. Our previous studies have shown that trametenolic acid (TA) can effectively promote seed germination of GEB. The aim of this study was to use transcriptome sequencing to further understand the potential mechanism of seed germination triggered by TA in GEB, in order to lay the groundwork for developing a new germination-growth system for GEB with Armillaria mellea. The untreated symbiotic group (Group A) did not germinate in the seed germination test. The high-dose TA-treated symbiotic group (Group B), the low-dose TA-treated symbiotic group (Group C), and the non-symbiotic untreated germination group (Group A) had germination rates of 85.01, 61.18 and 27.39%, respectively. This indicates that TA treatment can induce symbiosis with Armillaria mellea in GEB seeds and significantly increase germination rates. Transcriptome sequencing (RNA-seq) of Groups A, B, and C identified 86,843 annotated genes. There were more down-regulated genes than up-regulated genes, with 3912, 2518, and 814 differentially expressed genes (DEGs) between B and A, C and A, and B and C, respectively. The DEGs were mainly involved in DNA transcription factors, cell wall actions, plant-pathogen interactions, phenylpropanoid biosynthesis, phytohormone signal transduction, and starch-sucrose metabolism pathways. Six genes were confirmed using qRT-PCR: Down-regulated genes in the lignin biosynthesis pathway include MYB4 and 4CL, while GA20ox1 in the gibberellin biosynthesis pathway was also down-regulated. Up-regulated genes in the plant-pathogen interaction pathway are AIB and WRKY51, with MYB44 in the lignin biosynthesis pathway showing up-regulation. The transcriptomics results supported these expression patterns. Lignin, GA, and abscisic acid (ABA) levels were analyzed in GEB protocorms to understand how TA promotes germination. Results showed that groups B and C had lower lignin and ABA levels, but higher GA levels compared to group A. The study revealed that certain genes play a crucial role in promoting GEB seed germination through TA, by regulating gene expression to alter lignin content and hormone levels, breaking seed dormancy, facilitating seed-fungus interactions, and promoting symbiotic relationships with Armillaria mellea. TA modulates the expression of genes involved in lignin biosynthesis and hormone signaling, leading to an increase in GA content and a decrease in ABA and lignin content. This helps seeds break dormancy and promote germination. Additionally, TA can enhance GEB's defense response against fungi by regulating plant-pathogen interaction genes. It also improves the interactions between GEB and Armillaria mellea, overcoming the technical challenges associated with using Armillaria mellea as a germinating fungus. This establishes a new symbiotic germination-growth system between Armillaria mellea and GEB, laying the foundation for further research on the molecular mechanisms of GEB seed germination.