基于全长转录组和代谢组联合分析揭示湿地松胚性愈伤褐变的发生机制

[Objective]The browning of embryogenic callus is closely related to genotype.By utilizing anti-browning and easy-browning embryonic clones,this study aims to elucidate the molecular mechanisms underlying the browning of embryogenic callus,providing a theoretical basis for selecting anti-browning resources in pine trees and optimizing somatic embryogenesis protocols.[Methods]The study focused on anti-browning and easy-browning embryogenic clones derived from the same half-sibling family of slash pine(Pinus elliottii).Embryogenic callus from both clones were sampled on 0,14,and 28 days after subculture(DAS).Full-length transcriptome and second-generation transcriptome sequencing analyses were conducted,and liquid chromatography-tandem mass spectrometry(LC-MS/MS)was employed for targeted metabolomic analysis of flavonoids and/or phenolic compounds.Through multi-omics association analysis and co-expression analysis,important genes and regulatory networks related to the embryogenic callus browning were identified.[Results]Expression profile analysis based on 63,334 full-length transcripts obtained from three generations of sequencing(with an annotation rate of 97.27％)indicated that while the anti-browning clone exhibited rapid growth,only 17 differentially expressed genes(DEGs)were found between 0 and 14 DAS.In contrast,there were over 13,710 DEGs between the two clones.The DEGs between the two clones exhibited divergent expression trends,with a key differential time point at 14 DAS.Genes that were upregulated earlier on 14 DAS in the easy-browning embryogenic callus were similar to those that did not upregulate until 28 DAS in the anti-browning callus.These genes were associated with Gene Ontology(GO)terms and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways related to flavonoid biosynthesis,stress responses,and phenylpropanoid biosynthesis.Exceeding the subculture period of 14 days,a significant number of genes showed downregulation in the embryogenic callus of both clones,associated with GO terms and KEGG pathways related to methylation,chromatin modification,and RNA modification.Further exploration through Weighted Correlation Network Analysis(WGCNA)revealed 14 co-expression modules,with genes in the lightcyan module significantly enriched in GO terms and KEGG pathways related to phenylpropanoid and flavonoid biosynthesis.A transcriptional regulatory network for browning was constructed based on this module,identifying 9 transcription factors,including WRKY6 and ERF4,along with 31 key genes.Targeted metabolomic analysis identified 61 flavonoid/phenolic compounds,most of which were found at higher levels in the easy-browning callus.Multi-omics association analysis indicated a significant accumulation of 4-hydroxycinnamic acid and naringenin in the easy-browning callus on 14 DAS,consistent with the expression patterns of related genes.High expressions of PePAL,PeCYP73A4,PeCHS,and PeCHI promoted the accumulation of these substances,while transcription factors PeWRKY6,PeERF4,and PeNAC098 may have upregulated these genes,collectively contributing to browning.[Conclusion]The anti-browning clones were able to maintain relative stability in internal gene expression during the 14-day subculture period,with significant changes occurring only after exceeding the subculture period.Conversely,the easy-browning clones exhibited many genes that underwent premature expression changes,leading to an early accumulation of phenolic compounds.Key genes such as PeWRKY6 and PeCYP73A4 were associated with the excessive accumulation of 4-hydroxycinnamic acid and naringenin in embryogenic callus,triggering browning.