| Iris domestica(L.)Goldblatt et Amberley is a perennial herbaceous plant in the family Iridaceae with a wide distribution range and strong drought adaptability.It is an important medicinal and ornamental plant,and also commonly used in traditional Chinese medicine.According to the 2020 edition of the Chinese Pharmacopoeia,the dried rhizome of I.domestica are used as a medicinal material with therapeutic effects including clearing heat,detoxification,relieving phlegm,and sharpening pharynx.Currently,I.domestica cultivation is geographically widespread with significant differences in precipitation,which may contribute to variations in the quality of the medicinal material.Most studies have demonstrated that water regulation is an important agronomic technique for enhancing medicinal material quality.However,the physiological and ecological responses of I.domestica to drought stress and the molecular ecological regulatory mechanisms underlying its isoflavone synthesis are not yet clear,which impedes the technology Innovation of water control for its cultivation.Exploring suitable water conditions for isoflavone accumulation in I.domestica can offer theoretical support for developing rational water management measures in production,as well as a scientific basis for molecular breeding of I.domestica.This study conducted persistent drought and drought rehydration experiments to investigate the physiological and ecological changes in photosynthesis and osmotic regulation in I.domestica.It also investigated the effects on the accumulation of isoflavone components and the expression levels of related genes involved in isoflavone biosynthesis pathways.In addition,the study elucidated the molecular ecological regulatory mechanisms underlying the response of I.domestica to drought stress by combining transcriptome sequencing technology.The key outcomes of this study are as follows:(1)I.domestica has a key drought threshold point in response to persistent drought,which is moderate drought stress.Under persistent drought,physiological and biochemical indicators such as isoflavones content,photosynthetic parameters,chlorophyll fluorescence parameters,osmoregulation substances,and protective enzyme systems underwent significant changes.For example,the content of rhizomes tectorigenin and iridin increased by 9.0% and 23.0%,respectively,under moderate drought stress.In addition,by using principal component analysis to comprehensively evaluate the response of I.domestica to drought,the results showed that the comprehensive score of various indicators was highest(57.80)under MLW drought stress,indicating that I.domestica had the strongest response to drought and the ability to resist drought was strong at this drought,with soil moisture content of 14.3%.Furthermore,enhancing the activity of protective enzymes(SOD,POD,and CAT enzymes)and accumulating osmoregulation substances(Pro)are important ways for I.domestica to resist drought stress.(2)Rehydration is a double-edged sword,and the synthesis and metabolism of isoflavones and photosynthetic physiology of I.domestica can be partially restored or compensated after rehydration;however,resistance physiological indicators such as protective enzymes and non-enzymatic systems like Pro decreased after rehydration.Under RW1 rehydration,the content of five isoflavone monomers in the rhizome and root of I.domestica increased to varying degrees during the vegetative stage,with the content of irisflorentin in the rhizome increasing by 8.0% and the content of that in the roots increasing by 30.6%.For the reproductive stage rhizomes and roots,their isoflavone content decreased or did not change significantly after rehydration.In addition,the dry and fresh weight of the rhizomes and roots during the vegetative and reproductive stages changed little under D1 drought,but significantly decreased under D2 drought(p < 0.05).Based on the above research results and classification of drought levels,this study suggests that moderate drought and rehydration treatment be applied to the I.domestica during the vegetative stage in actual cultivation to improve the quality of the medicinal herb.At this time,the soil moisture content of I.domestica cultivation should be between 13.44% and 16.8%.In the physiological and biochemical response process of I.domestica to drought and rehydration,RW1 rehydration restored the weakened photosynthesis caused by drought,such as Pn,Tr,Gs to the level of the CK group,and increased the actual photochemical efficiency(YII).At the same time,rehydration effectively reduced the enzyme-catalyzed system(SOD and POD enzymes)and non-enzyme-catalyzed system(Pro)indicators.(3)After m RNA sequencing of rhizomes and roots of I.domestica under moderate drought(MLW)and severe drought stress(MDW),a total of 116,612 genes were obtained.Differential gene analysis,GO and KEGG enrichment analysis were further conducted on these genes,and the results showed that there were 6,904 differentially expressed genes,mainly enriched in plant hormone signal transduction and secondary metabolic pathways(such as flavonoid biosynthesis),and the genes in these pathways may be involved in the drought response process of I.domestica.(4)The ABA signaling pathway plays an important role in drought signaling and isoflavone accumulation in I.domestica.During persistent drought,genes involved in calcium ion receptor proteins(CDPK,Cal and CBL),ABA metabolism(ABA3 and A8’H),ABA receptor gene PYL8,and core genes in ABA signaling(Sn RK2 and PP2C)were all regulated by drought stress.Except for the negative regulatory factor PP2 C,which was significantly downregulated,the expression of other genes was significantly promoted.At the same time,there were tissue-specific differences in drought signal transmission between the rhizome and the roots,with the roots being more sensitive to soil moisture stress,exhibiting higher expression levels,and responding more rapidly to water stress.Similar differences also existed in the key enzyme genes involved in flavonoid synthesis,with the flavonoid synthesis and metabolism genes(such as C4 H,F6H)in the rhizome responding to drought stress and upregulated,while those in the roots were significantly downregulated.Overall,rehydration can weaken the transmission of drought signals in shajing and have a negative effect on the expression of flavonoid metabolism genes.In addition,combined with the results of Chapter 3 and this chapter,this study preliminarily constructed a molecular regulatory network for the accumulation of I.domestica isoflavones(including transcription factor b HLH and β-BGLU31-I gene)with ABA signaling pathway as the core.(5)Using transcriptome data from I.domestica,the transcription factor b HLH15 andβ-glucosidase(β-BGLU31-I)gene were successfully cloned.Through bioinformatics software analysis,the proteins encoded by these two genes belonged to hydrophilic unstable proteins without signal peptides.Laser confocal microscopy showed that proteins encoded by Idb HLH115 and β-BGLU31-I genes were localized in the nucleus. |
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