Multi-Omics Analysis Of The Integrated Transcriptome-Metabolome-Rhizosphere Microbiome Regulatory Model During The Development To Senescence Of Rhododendron

The flower is an important reproductive organ of the plant and also influences and determine its ornamental value.Flower development and senescence is a fundamental developmental process that involves complex regulation at multiple levels.Therefore,studying the regulation of flower development and senescence from a multi-omics perspective is of irreplaceable importance to the economic and ornamental value of flowers.With a wide variety of species and gorgeous flowers,Rhododendron is one of the ten most famous traditional flowers in China."Guizhou Baili Rhododendron" is a national 5A level ecological tourism demonstration area and is by far the largest natural Rhododendron forest belt in the world,with high scientific research,ornamental and ecological value.The revenue of Baili Rhododendron has become a key pillar of local economic income.Therefore,the study of the transcriptome-metabolome-rhizosphere microbiome regulation model of Rhododendron development and senescence provides a scientific theory for plant flowering regulation,and is also important for prolonging the ornamental period of Rhododendron to promote the economic income of Baili Rhododendron.With Rhododendron delavayi Franch and Rhododendron hybridum as materials,this project,from the perspective of "transcriptome-secondary metabolites-rhizosphere microbial community",using bioinformatics technology,this paper revealed the molecular regulation mechanism in the process of Rhododendron development and senescence,and expounded the regulation mode of secondary metabolites-rhizosphere microbial community in the process of Rhododendron development and senescence,provided new scientific basis for regulating and extending the flowering period of Rhododendron.The main results of this study are as follows:1.The petals of Rhododendron delavayi from six stages of development to senescence(the time of bud stage,pollen dehiscence stage,pollination stage,after pollination stage,senescence stage and fading stage)were used as material to determine the transcriptome and metabolome of the petals in each period using high-throughput sequencing and chromatography-mass spectrometry tandem techniques.The analysis showed that there were 37,031 differential genes in Rhododendron delavayi from development to senescence,including 1,057 differential transcription factors.The highest number of differential genes was found in the senescence and fading stages,indicating that a large number of genes are involved in the senescence process of Rhododendron.Using time series(STEM)and co-expression network(WGCNA)for modular analysis of differentially expressed genes,it was found that 11 time series gene clusters were significantly correlated with flower development and senescence,and46 co-expression network modules were constructed for differentially expressed genes during flower development and senescence.A total of 973 metabolites were detected during the floral development and senescence process of Rhododendron.Using STEM and WGCNA for modular analysis of metabolites,it was found that 7 time series metabolite clusters were significantly correlated with floral development and senescence,while a total of 5 co-expression network modules were constructed for metabolites in flower development and senescence.2.Transcription factors play an important role in plant senescence.That 1,057 differential transcription factors belong to 64 TF families,and STEM analysis revealed that transcription factors from 4 clusters were significantly correlated with the floral development and senescence of Rhododendron;Based on literature analysis,it was found that transcription factors such as EIN,NAC,ERF,WRKY,ABF,and MYB in the four clusters are all involved in the regulation of plant leaf or flower senescence.Further analysis revealed a significant positive correlation between Rd WRKY75 and the ethylene biosynthesis gene Rd ACS1,as well as a significant positive correlation with the ethylene biosynthesis precursor SAdenosyl-L-Methionine.Both of them were significantly upregulated during senescence.We therefore hypothesized that Rd WRKY75 plays an important role in ethylene-mediated senescence in Rhododendron by regulating the expression of Rd ACS1,which affects ethylene accumulation.Rd WRKY42 was significantly positively correlated with the senescencerelated gene Rd YLS9 and the salicylic acid signal transduction related gene PR1,both of which were significantly upregulated during senescence.We speculated that Rd WRKY42 may play a positive regulatory role in Rhododendron senescence by directly regulating senescence genes and influencing salicylic acid signaling transduction.3.Combined transcriptome and metabolome analysis revealed that 22 differential gene modules were significantly correlated with 5 metabolite modules.After KEGG enrichment of differential genes and metabolites,it was found that all modules were significantly enriched to 11 metabolic pathways,including Glutathione metabolism and Biosynthesis of cofactors.Both glutathione peroxidase(GPX)and γ-glutamyl transpeptidase(GGT)were significantly upregulated during the flower senescence stage of Rhododendron,and it was hypothesized that these two enzymes may delay the flower senescence of Rhododendron by scavenging reactive oxygen species(ROS)free radicals and maintaining antioxidant levels in petal cells.4.Plant hormones have been revealed as important signals that initiate and regulate the senescence process.Analysis of transcriptome combined with metabolomic data revealed that three signal transduction pathways,jasmonic acid,salicylic acid and abscisic acid,may be synergistically involved in the regulation of the flower development and senescence of Rhododendron.Firstly,the content of jasmonic acid was highest at the bud stage,and then continued to increase after a significant decline at the pollen dehiscence stage,and jasmonic acid was significantly positively correlated with the expression changes of Rd COI1 and Rd MYC2,suggesting that jasmonic acid may play a positive regulatory role in the early stage of Rhododendron development through signal transduction pathway.The content of salicylic acid significantly increases during the senescence period,and is positively correlated with downstream Rd NPR1,Rd TGA,and Rd PR1 expression changes,suggesting that salicylic acid may play a promoting role in the flower senescence process of Rhododendron through signal transduction pathway.Meanwhile,abscisic acid also increased significantly during senescence,and the changes in abscisic acid content were significantly positively correlated with changes in Rd PYL and Rd SNRK2 expression,suggesting that abscisic acid may also play a role in promoting senescence in Rhododendron through signal transduction pathway.5.CircRNAs act as miRNA sponges and play regulatory roles through circ RNA-miRNA-m RNA networks.In this study,ten differentially expressed circ RNAs that may bind to miRNAs were identified and seven circ RNA-miRNA-m RNA networks were constructed;The molecular mechanism that circ RNAs play a regulatory role in flower development and senescence by mediating jasmonic acid signaling pathway was proposed.6.Using root system and rhizosphere soil from five stages of the development to senescence process(the time of bud stage,initial blooming stage,full-bloom stage,senescence stage and fading stage)of Rhododendron hybridum as materials,a full length microbial diversity sequencing was conducted on rhizosphere soil samples,and root exudates were identified.It was found that during the development to senescence process of Rhododendron,the rhizosphere bacterial community and root metabolites underwent significant changes,especially during the fullbloom and fading stages.Network analysis revealed that eight OTU modules consisting of Acidocella aluminiidurans,Tepidisphaera mucosa and Bdellovibrio bacteriovorus were significantly correlated with four metabolite clusters consisting of Quercetin-5-O-glucuronide,Isorhamnetin-3-O-(6’’-malonyl)glucoside,and Vitexilactone during the flower development and senescence of Rhododendron,indicating that the rhizosphere bacterial community is closely associated with metabolites during the development of Rhododendron.At the same time,analysis was conducted on the differential floral metabolites during the development and senescence process of Rhododendron Rhododendron delavayi and the differential root secretion metabolites during the development and senescence process of Rhododendron hybridum.It was found that during the development and senescence process of Rhododendron,11 metabolites,including Eugenol,Caffeic acid,8-hydroxyquinoline,Myricetin,Procyanidin A2,trans-Cinnamic Acid and Salicylic acid,underwent the same significant changes in both flowers and roots.A cross-domain network analysis of 11 metabolites and rhizosphere microorganisms revealed that 11 metabolites were significantly positively or negatively correlated with bacteria such as Flavitalea antarctica,Hypsibius dujardini and Rhizomicrobium electricum,and that the key bacterium Pseudomonas was associated with plant growth and development;this suggests that these root exudates and rhizosphere bacteria may be involved in flower development and senescence through interactions during the development to senescence of Rhododendron.