| Phellodendron chinense Schneid is a special species of Phellodendron Rupr in Rutaceae.It is a traditional Chinese herbal medicine.Its stem bark and root bark contain a large number of medicinal components such as berberine and phellodendrine.It has the functions of clearing away heat and dampness,detoxifying and treating sores,purging fire and removing steam.Besides it could treat senile hypertension,cerebral hemorrhage,coronary heart disease,angina pectoris,expand coronary vessels,increase coronary flow and so on.Plant growth and active ingredient accumulation are regulated by drought stress,and the mechanism by which drought stress regulates the growth and alkaloid accumulation of seedlings of Phellodendron chinense Schneid is not clear.In this study,we selected the seedlings of Phellodendron chinense Schneid as experimental materials,this paper explored the effects of drought stress on the growth,physiological characteristics and content of medicinal components of Phellodendron chinense Schneid seedlings,and clarified the effects of drought stress in the biosynthesis of berberine and phellodendrine.We analyzed the expression pattern of DEGs by transcriptomic techniques to elucidate the molecular mechanism of response to drought stress in seedlings of Phellodendron chinense Schneid.At the same time,the effects of different levels of nitrogen application(0 N,2 N,4 N and 8 N)on the growth,resistance characteristics and berberine accumulation of Phellodendron chinense Schneid seedlings under drought stress were studied to clarify the physiological function of exogenous nitrogen under drought stress.It provides a theoretical basis for the follow-up scientific planting,improving the quality and effect of Phellodendron amurense.The main results are as follows:1.Compared with the control,moderate drought stress(MD)and severe drought stress(SD)significantly reduced the shoot fresh weight,total fresh weight,chlorophyll a content,total chlorophyll content,carotenoid content,Pn,Tr Gs,Fv/Fm and ΦPSⅡ;increased the content of chlorophyll b,Ci,soluble sugar and soluble protein in leaves.It significantly increased the content of malondialdehyde(MDA)and peroxide in leaves,significantly increased the activities of SOD and CAT,and significantly reduced POD and APX activities.At the same time,the content of NH4+-N,the activities of NR and GS and the expression of Pc(S)-NR and Pc(S)-GS were significantly increased in roots and leaves.The results showed that drought stress inhibited the growth of Phellodendron chinense Schneid seedlings,promoted intracellular nitrogen accumulation and translocation uptake,and increased the activity of oxidase to improve its drought resistance.2.MD group and SD group significantly increased the contents of berberine and phellodendrine in root bark and stem bark of Phellodendron chinense Schneid seedlings,increased the expression level of Pc(S)-THBO in roots.and increased their expression levels in stems and leaves respectively.It was indicated that drought stress promoted the synthesis and accumulation of berberine in Phellodendron chinense Schneid by increasing the expression level of Pc(S)-THBO.3.The analysis results of transcriptome sequencing data based on Illumina novaseq 6000 platform showed that a total of 54325 unigenes were obtained,and the assembled N50 was 1837bp,with an average length of 1057bp.The assembled unigenes were annotated to eight databases.8056、22776、17936、15851、19284、16293、21850 and 27924 unigenes were obtained in functional annotations respectively.In three comparison groups(NC vs MD&SD,NC vs MD,NC vs SD),1117,1416 and 1995 DEGs were identified in KEGG database respectively.It was mainly involved in plant hormone signal transduction,MAPKplant signal pathway,isoquinoline biosynthesis,phenylpropane metabolism and other metabolic pathways.The key genes in ethylene signal pathway and alkaloid synthesis pathway were up-regulated.It was showed that Phellodendron chinense Schneid responds to drought stress through ethylene signal pathway and indicated that drought stress promotes the accumulation of phellodendrine.The differential gene expressions of Pc(S)-ACS,Pc(S)ETR2,Pc(S)-CTR1,Pc(S)-EBF2,Pc(S)-ERF1,Pc(S)-PP2C6,Pc(S)-PP2C77,Pc(S)-DELLA,Pc(S)-TyrAT,Pc(S)-DDC,Pc(S)-BBE8,Pc(S)-BBE13 were randomly selected for qRT-PCR verification.The detection results were consistent with the trend of transcriptome sequencing results,indicated that transcriptome data was reliable.4.2 N and 4 N significantly decreased MDA content and GS activity of Phellodendron chinense Schneid seedlings in MD and SD groups,increased soluble protein content,POD and CAT activity,NH4+-N and NO3--N contents,NR activity.Pn,Tr,Gs,Fv/Fm and ΦPSⅡ,qL and NPQ,increased the shoot fresh weight,root fresh weight and total fresh weight,and alleviated the inhibitory effect of drought stress on the growth of Phellodendron chinense Schneid seedlings.At the same time,2 N and 4 N significantly increased the contents of berberine and phellodendrine in the stem bark and root bark of Phellodendron chinense Schneid seedlings in MD and SD groups,respectively.The results showed that the appropriate concentration of exogenous nitrogen significantly improved the drought resistance of Phellodendron chinense Schneid seedlings under drought stress,and promoted the synthesis and accumulation of berberine and phellodendrine.Based on the analysis results of growth physiological indexes and transcriptome data of Phellodendron chinense Schneid seedlings treated by drought stress,it was clear that drought stress(MD and SD)inhibited the growth of Phellodendron chinense Schneid seedlings and promoted the synthesis and accumulation of phellodendrine.The regulation model of drought stress through promoting ethylene synthesis and signal passingreducing photosynthetic efficiency-inhibiting growth was constructed to clarify the molecular mechanism of leaves of Phellodendron chinense Schneid seedlings in response to drought stress.At the same time,the appropriate concentration of exogenous nitrogen(2 N and 4 N)alleviated the inhibitory effect of drought stress on the growth of Phellodendron chinense Schneid seedlings,enhanced drought resistance,and promoted the accumulation of alkaloids.It provides data support and theoretical basis for the improvement of quality and efficiency of Phellodendron chinense Schneid. |
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