The Regulation Mechanism Of Polyamine Metabolism In Response To Citrus Huanglongbing Infection

Citrus is the largest cultivated area and the highest yield fruit in China and also an important industry for poverty alleviation and rural revitalization.Citrus Huanglongbing(HLB)is caused by Candidatus Liberibacter asiaticus(CLas)and transmitted by citrus psyllid.Due to the lack of effective radical cure method,citrus yield and fruit quality were seriously affected,resulting in huge economic losses.Many studies have showed that HLB infection could trigger many metabolic alterations in citrus,mainly involving amino acids,organic acids,hormones,phenols,and flavonoids.However,the role of polyamine metabolic pathway has not been well studied.In the early stage,the research group through multiomics combined analysis found that the polyamine synthesis pathway was significantly enriched and the relative expression of gene was up-regulated after citrus infection with HLB.Therefore,conducting studies on the regulation of polyamine metabolism will contribute to our understanding of HLB pathology and provide a theoretical basis for the scientific and effective management of HLB in citrus.In this study,we used Newhall navel orange(Citrus sinensis(L)Osb.)leaves as experimental materials.According to the results of detection whether infected with HLB by polymerase chain reaction(PCR)and determination of CLas content by quantitative real-time PCR(q RT-PCR).We divided into three groups: healthy control group(H),asymptomatic group(ASY)and symptomatic group(SYM).Firstly,high performance liquid chromatography(HPLC)and ultra high performance liquid chromatography tandem quadrupole time of flight mass spectrometry(UPLC-QTOF-MS)were used to qualitatively and quantitatively to analyze 9polyamine compounds in polyamine metabolic pathway of combination with reference standards.The relative expression of 14 related genes in polyamine metabolic pathway were analyzed by q RT-PCR,and the activities of 4 key enzymes were detected.In order to evaluate the relationship between polyamine metabolites and citrus HLB tolerance,we selected six citrus subfamily plants with different HLB tolerance to compare the content of polyamine metabolites.The main results were as follows:1.HPLC and UPLC-QTOF-MS methods for the determination of 9 polyamine compounds in citrus leaves were established.The recovery were 93.06～110.92%,intra day precision(RSD)and intra day precision(RSD)were within 8.65% and8.66%,respectively,indicating that methods have good repeatability and high accuracy.Methods were applied to the determination of polyamine compounds in H,ASY and SYM leaves.The results showed that the content range was 0.10～62.00μg/g FW.Compared with H group,the content of arginine,ornithine,γ-aminobutyric acid and putrescine remarkably increased after infection with HLB,and reached the highest in SYM group.Among them,the content of arginine and ornithine increased dramatically,from 0.33 and 0.13 μg/g FW increased to 59.86 and 2.58 μg/g FW,respectively.However,the levels of spermidine and spermine showed a downward trend after infected by HLB,from 2.87 and 1.58 μg/g FW decreased to 0.57 and 1.31μg/g FW,respectively.In addition,the content of agmatine,1,3-diaminopropane and methionine was the highest in ASY group,61.51,5.59 and 4.13 μg/g FW,respectively.2.qRT-PCR was used to evaluate the expression of 14 key genes in polyamine metabolic pathway.The results showed that the relative expression of amino cyclopropanecarboxylate oxidase(ACO),arginase(ARG),ornithine decarboxylase(ODC),argininosuccinate lyase(ASL),diamine oxidase(DAO)and argininosuccinate synthase(ASS)up-regulated with the increase of CLas in Newhall navel orange leaves,and reached the highest in SYM group.In addition,the relative expression of agmatine deiminase(ADI),arginine decarboxylase(ADC),methionine adenosyltransferase(MAT)and spermidine synthase(SPDS)reached the highest in ASY group.However,the relative expression of 1-aminocyclopropane-1-carboxylate synthase(ACS),polyamine oxidase(PAO),s-adenosylmethionine decarboxylase(SAMDC)and spermine synthase(SPMS)down-regulated with the increase of CLas.In addition,the enzyme activities of four key enzymes(ADC,ODC,ADI,and ARG)in polyamine metabolic pathway were also detected.The enzyme activities of ODC and ARG increased significantly with the development of HLB symptoms,and reached the highest level in SYM group.However,the enzyme activities of ADC and ADI were the highest in ASY group.3.The biosynthetic pathway of polyamines in plants is initiated mainly with arginine through two different routes.The first route is arginine generates agmatine through ADC,and agmatine then generates putrescine through ADI;the second route is that arginine uses ARG to generate ornithine,which is decarboxylated by ODC to directly generate putrescine.The results showed that the agmatine content,enzymatic activity and relative expression of ADC and ADI in the first route reached the highest levels in the ASY group.However,in the second route,ornithine content,relative expression of ARG and ODC,and enzymatic activity reached the highest levels in SYM leaves.It is hypothesized that in the first route plays an important role in the ASY period,while in the SYM period the second route is the main pathway.4.The content of 9 polyamine compounds in six citrus subfamilies plants with different HLB tolerance ranged from 0.10～105.00 μg/g FW,and there were significant differences in polyamines content among different plants.The content of1,3-diaminopropane and spermine were the highest in Eureka lemon(C.limon(L.)Burm.f.),the content of putrescine,spermine,arginine,and ornithine were the highest in Kamuning(Murraya exotica L.),and the content of methionine andg-aminobutyric acid were the highest in Wampee(Clausena lansium(Lour.)Skeels).The total content of 9 polyamine compounds in six citrus subfamilies plants with different HLB tolerance were as follows: Kamuning > Wampee > Eureka lemon >Shatian pomelo(C.maxima(Burm.)Merr.cv.Shatian Yu.)> Newhall navel orange > Ponkan(reticulata Blanco cv.Ponkan).The total content of polyamine compounds in tolerant group(Kamuning)was the highest,in the susceptible group(Ponkan)was the lowest.Thus,the polyamines content was positively correlated with HLB tolerance,and polyamines may become tolerance markers in citrus with different HLB resistance.This study revealed the regulatory mechanism of the polyamine metabolic pathway in the response to HLB of Newhall navel orange,clarified the relationship between citrus subfamily plants with different HLB tolerance and polyamine compounds,provided a theoretical basis for breeding for resistance in citrus and also for HLB prevention and control and development of resistant varieties.