The Molecular Mechanism Of Endogenous Hormones Regulate Horned Gall Development

Rhus chinensis is an important economic tree that has long been used in folk medicine in Asia,and can especially produce horned galls caused by fluid-feeding aphid parasite.The horned gall enriches gallotannin that can be widely used for medical applications and food industry and agriculture.Tannin is the main component in gallnut,and its productions,such as tannic acid and gallic acid,are widely used in medicine,printing and dyeing,petroleum exploration,extraction of rare metal,food engineering,etc.Gall development is controlled by gradual increasing of aphid population,which is a extremely complicated and precise physiological processes.However,the molecular mechanism of gall development has not been disclosed.Plant hormones play a crucial role in the signaling cascade system.But,the majority of studies on the regulation of endogenous hormones in gall development focus on the simple determination.In the study,based on the temporal and spatial variation of endogenous hormone during gall development,transcriptome sequencing,digital gene expression,gene cloning and gene expression analysis were combined.The molecular mechanism of tree response and gall formation was revealed,which make a foundation for mining potential operation means of hormone regulation or genetic control the gall development.The main results are as follows:(1)The change rules of endogenous hormones in the process of gall developmentThe concentration of 7 endogenous hormones including auxin(IAA),gibberellin(GA),cytokinin(CTK),abscisic acid(ABA),jasmonic acid(JA),salicylic acid(SA)and ethylene(ETH)at different developmental stages(days from gall initiation of 30,85,100,115,125,140,155 and 170d)and different tissues(including Gall,GL,LW and CL)were assayed.Seven kinds of endogenous hormone in different developmental stages and tissues and their interactions have significant differences.Of which,the concentration of IAA,CTK,JA,SA,ABA and ETH increased in the second stage(85d),and the concentration of CTK,ABA,JA and ETH increased in the fifth stage(125d),and the concentration of IAA,CTK,SA and ETH increased in the final stage(170d),which indicated that both promoting and inhibiting hormones regulate the gall development in different stages,and that trees produce an obvious hormone response after aphid attack.Generally,except GA,the hormone concentration of the samples in gall tree is higher than that in the non-gall tree.The most of hormones(IAA,CTK,ABA,JA,SA and ETH)that elevated during the gall development showed the similar trend.Among four kinds of samples,only GA showed the opposite trend,indicating that GA may play a unique role during gall development.(2)The relationship between the of hormone change and gall developmentThe gall volume and aphid population showed a significantly positive correlation,and aphid population and GA concentration displayed a significantly positively correlation.However,GA concentration is negatively correlated with CTK,ABA and ETH.Principal component analysis indicated that GA clustered into an independent group among all hormones.In addition,GA concentration is positively correlated with gall volume.These data showed that GA is the key to regulation of gall development.In order to explain the effects of environment on hormone,three samples from gall tree(Gall,GL,tree and LW)were divided by the control leaf that was from non-gall tree galls(CL).The standardized data showed that when the tree was attacked by aphids,the overall GA level in gall tree samples decreased,of which,the decrease of GA level in gall was less compared with GL and LW.Furthermore,exogenous hormone was applicated.Compared with control,IAA and CTK did not have a obvious effect on gall development.From 30 to 85 d,ABA promoted the gall development.But after 85 d,ABA inhibited the gall development.Also,the application of ABA inhibitor—sodium tungstate caused the leaves wither and seedling death,showing that the role of ABA is very complicated: it promote gall development in the early stage and inhibit gall growth in late stage.We speculate that the reason of seedlings death is due to lack of the protective function of ABA inducing water stress.Compared with other treatments,GA significantly promote the gall development.Therefore we propose a model: the host plants would like to suppress the gall development by decrease of endogenous GA concentration.However,aphids antagonize this behavior using different means,so as to promote the gall development.(3)The screen of differentially expressed gene during gall developmentTo disclose the mechanism of the gall development,transcriptome analysis by Illumina deep sequencing of four tissues are performed.Total of 86.17 million high-quality reads with an average GC content of 43.73% were generated,and assembled into 59,522 unigenes with a mean length of 889 bp.We found that DEGs abundantly enrich in biosynthesis of secondary metabolites,plant-pathogen interaction,and plant hormone signal transduction,and that these DEGs related to three pathways above mentioned highly expressed at galls compared with other leaf tissues.Principle component analysis showed that IAA and ABA signal transduction dominates hormone signal transduction.Furthermore,quantitative real-time PCR were performed to verify the transcriptome data,including genes associated with hormone signal transduction: auxin response factor 5-like isoform 2(ARF),serine/threonine-protein kinase bri1,putative(SPKB),Auxin-responsive protein IAA1(ARP),two-component response regulator ARR12-like(TCRR),F-box protein GID2-like(FPG),serine/threonine-protein kinase SRK2E-like isoform 1(SPK),protein phosphatase 2c(PP2C),ethylene-insensitive protein 2-like(EIP),ethylene receptor(ER),BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 precursor,putative(BIRK),DELLA protein GAI1-like isoform 1(DPGI)and LMYC2,and genes related to secondary metabolism: anthranilate synthase alpha subunit(ASAS),chloroplast biotin carboxylase(CBC)and cytochrome P450(CP),and genes associated with plant-pathogen interaction: Pathogenesis-related protein 1a(PP1A),hypothetical protein PRUPE_ppa002088mg(PPA),and genes involved in sugar metabolism: Cellulose synthase A catalytic subunit(CSCS),glycosyltransferase 8E(G8E)and soluble acid invertase(SAI).These transcripts can be classified three cluster according to expression:(1)down-regulation(ARF,ASAS and SPKB),double curve(ARP,TCRR,FPG,PPIA,BIRK,CBC,PPA,G8 E and CP)and single peak(SPK,PP2 C,EIP,ER,SAI,CSCS,DPGI and LMYC2).The integrated gene expression profile disclosed that phytohormone signal transduction possibly coordinates the primary and secondary metabolism.(4)The clone of the related GA and ABA genes and its effects on gall developmentAbundant enrichment of signal transduction of seven kinds of hormone were identified,which is accorded with our previous study that seven types of hormone concentration show significant difference.However,the PCA result between hormone concentration and signal transduction cannot completely overlap,possibly due to the difference between hormone synthesis and signal transduction.Interestingly,ABA owns high weight in PCA results both hormone concentration and signal transduction,suggesting it play a key role for forming galls.In this study,22 transcripts were related to ABA signal transduction,of which,19 transcripts were anotated as protein phosphatase 2c and the majority of them retain higher expression in galls compared with other tissues.Based on the above results,the transcript encoding protein phosphatase 2C and GAI were cloned and analyzed.We cloned a full length of cDNA with length of 1013bp(RcPP2C)and 977bp(RcGAI).Both these two genes belong to constitutive expression by gene expression analysis.From the initial to terminal stage,the expression of RcPP2 C shows a fluctuation.Combined with exogenous experiments,it might indicates that ABA is involved in stress response and water metabolism during gall development,in which,RcPP2 C gene perform a negative role to regulate of ABA.It is also found that RcGAI down-regulates from initial to terminal stage,and that expression of RcGAI negatively correlates with the GA content,and a significantly negatively correlates with gall volume,which suggests that RcGAI gene might be a negative regulator of GA response to affect gall development.In summary,this study clarifies the overall decrease pattern of GA influenced by aphids and host plants,and reveals that GA promotes the gall formation and development.In addition,we constructed the whole transcriptional data by deep sequencing,and found that the secondary metabolism,plant pathogen interactions and hormone signal transduction pathway significantly enriched during gall development.Furthermore,RcGAI gene associated with GA signaling transduction,RcPP2 C gene related to ABA transduction were screened and cloned,and their gene expression were analyzed.The final results showed that RcGAI gene negatively regulates GA reaction and affect gall development.