Identification and characterization of two P450 enzymes from Citrus sinensis involved in TMTT and DMNT biosyntheses and Asian citrus psyllid defense

Abstract

The homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are the major herbivore-induced plant volatiles that help in defense directly by acting as repellants and indirectly by recruiting insects’ natural enemies. In this study, DMNT and TMTT were confirmed to be emitted from citrus (Citrus sinensis) leaves infested with Asian citrus psyllid (Diaphorina citri Kuwayama; ACP), and two cytochrome P450 (CYP) genes (CsCYP82L1 and CsCYP82L2) were newly identified and characterized. Understanding the functions of these genes in citrus defense will help plan strategies to manage huanglongbing caused by Candidatus Liberibacter asiaticus (CLas) and spread by ACP. Quantitative real-time PCR (qPCR) analysis showed that CsCYP82L1 and CsCYP82L2 were significantly upregulated in citrus leaves after ACP infestation. Yeast recombinant expression and enzyme assays indicated that CsCYP82L1 and CsCYP82L2 convert (E)-nerolidol to DMNT and (E,E)-geranyllinalool to TMTT. However, citrus calluses stably overexpressing CsCYP82L1 generated only DMNT, whereas those overexpressing CsCYP82L2 produced DMNT and TMTT. Furthermore, ACPs preferred wild-type lemon (Citrus limon) over the CsCYP82L1-overexpressing line in dual-choice feeding assays and mineral oil over TMTT or DMNT in behavioral bioassays. Finally, yeast one-hybrid, electrophoretic mobility shift, and dual luciferase assays demonstrated that CsERF017, an AP2/ERF transcription factor, directly bound to the CCGAC motif and activated CsCYP82L1. Moreover, the transient overexpression of CsERF017 in lemon leaves upregulated CsCYP82L1 in the absence and presence of ACP infestation. These results provide novel insights into homoterpene biosynthesis in C. sinensis and demonstrate the effect of homoterpenes on ACP behavior, laying a foundation to genetically manipulate homoterpene biosynthesis for application in huanglongbing and ACP control.

Acknowledgements

This study was funded by the Guangdong Provincial Key Realm R&D Program (Grant No. 2020B0202090005) and the Scientific Innovation Strategy-construction of High-Level Academy of Agriculture Science (Grant No. R2020PY-JG002). We thank TopEdit (www.topeditsci.com) for the English language editing of our manuscript.

Author contributions

X.S. and X.Z. designed the experiments; X.S. performed the experiments and analyzed the data; C.H. and G.Y. supervised the study; X.Z. and X.S. wrote and revised the drafts. The manuscript has been read and approved by all authors.

Data availability

All data generated during the study are provided in the published article and the supplementary files.

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Hu L, Wu Y, Wu D. et al. The coiled-coil and nucleotide binding domains of BROWN PLANTHOPPER RESISTANCE14 function in signaling and resistance against planthopper in rice. Plant Cell. 2017; 29:3157-85

[2] Richter A, Schaff C, Zhang ZW. et al. Characterization of biosynthetic pathways for the production of the volatile homoterpenes DMNT and TMTT in Zea mays. Plant Cell. 2016; 28:2651-65

[3] Zeng L, Liao Y, Li J. et al. α-Farnesene and ocimene induce metabolite changes by volatile signaling in neighboring tea (Camellia sinensis) plants. Plant Sci. 2017; 264:29-36

[4] McCormick AC, Unsicker SB, Gershenzon J. The specificity of herbivore-induced plant volatiles in attracting herbivore enemies. Trends Plant Sci. 2012; 17:303-10

[5] Mas F, Vereijssen J, Suckling DM. Influence of the pathogen Candidatus Liberibacter solanacearum on tomato host plant volatiles and psyllid vector settlement. J Chem Ecol. 2014; 40:1197-202

[6] Unsicker SB, Kunert G, Gershenzon J. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Curr Opin Plant Biol. 2009; 12:479-85

[7] Arimura G, Ozawa R, Shimoda T. et al. Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature. 2000; 406:512-5

[8] Su JW, Zeng JP, Qin XW. et al. Effect of needle damage on the release rate of Masson pine (Pinus massoniana) volatiles. J Plant Res. 2009; 122:193-200

[9] Lee S, Badieyan S, Bevan DR. et al. Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis. Proc Natl Acad Sci USA. 2010; 107:21205-10

[10] Azuma H, Toyota M, Asakawa Y. Intraspecific variation of floral scent chemistry in Magnolia kobus DC. (Magnoliaceae). J Plant Res. 2001; 114:411-22

[11] Van Poecke RMP, Posthumus MA, Dicke M. Herbivore-induced volatile production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: chemical, behavioral, and gene-expression analysis. J Chem Ecol. 2001; 27:1911-28

[12] Liu DF, Huang XZ, Jing WX. et al. Identification and functional analysis of two P 450 enzymes of Gossypium hirsutum involved in DMNT and TMTT biosynthesis. Plant Biotechnol J. 2018; 16:581-90

[13] Oluwafemi S, Bruce TJA, Pickett JA. et al. Behavioral responses of the leafhopper, Cicadulina storeyi China, a major vector of maize streak virus, to volatile cues from intact and leafhopper-damaged maize. J Chem Ecol. 2011; 37:40-8

[14] Sobhy IS, Woodcock CM, Powers SJ. et al. cis-Jasmone elicits aphid-induced stress signalling in potatoes. J Chem Ecol. 2017; 43:39-52

[15] Jing TT, Du WK, Gao T. et al. Herbivore-induced DMNT catalyzed by CYP82D47 plays an important role in the induction of JA-dependent herbivore resistance of neighboring tea plants. Plant Cell Environ. 2021; 44:1178-91

[16] Jagoueix S, Bove J, Garnier M. The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria. J Bacteriol. 1994; 44:379-86

[17] Fancelli M, Borges M, Laumann RA. et al. Attractiveness of host plant volatile extracts to the Asian citrus psyllid, Diaphorina citri, is reduced by terpenoids from the non-host cashew. J Chem Ecol. 2018; 44:397-405

[18] Tholl D, Sohrabi R, Huh J. et al. The biochemistry of homoterpenes—common constituents of floral and herbivore-induced plant volatile bouquets. Phytochemistry. 2011; 72:1635-46

[19] Sun XL, Yu T, Bin ML. et al. Transcriptomic analysis reveals the defense mechanisms of citrus infested with Diaphorina citri. Hortic Plant J. 2023; 9:450-62

[20] Xu Q, Chen LL, Ruan XA. et al. The draft genome of sweet orange (Citrus sinensis). Nat Genet. 2013; 45:59-66

[21] Howe GA, Jander G. Plant immunity to insect herbivores. Annu Rev Plant Biol. 2008; 59:41-66

[22] Wu JQ, Baldwin IT. New insights into plant responses to the attack from insect herbivores. Annu Rev Genet. 2010; 44:1-24

[23] Bruessow F, Gouhier-Darimont C, Buchala A. et al. Insect eggs suppress plant defence against chewing herbivores. Plant J. 2010; 62:876-85

[24] Howe GA, Major IT, Koo AJ. Modularity in jasmonate signaling for multistress resilience. Annu Rev Plant Biol. 2018; 69:387-415

[25] Irmisch S, McCormick AC, Günther J. et al. Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar. Plant J. 2014; 80:1095-107

[26] Khan ZR, Midega CAO, Bruce TJA. et al. Exploiting phytochemicals for developing a ’push-pull’ crop protection strategy for cereal farmers in Africa. J Exp Bot. 2010; 61:4185-96

[27] Hegde M, Oliveira JN, Da Costa JG. et al. Identification of semiochemicals released by cotton, Gossypium hirsutum, upon infestation by the cotton aphid, Aphis gossypii. J Chem Ecol. 2011; 37:741-50

[28] Kappers IF, Asaph A, Herpen TWJM. et al. Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis. Science. 2005; 309:2070-2

[29] Moraes MCB, Laumann RA, Pareja M. et al. Attraction of the stink bug egg parasitoid Telenomus podisi to defence signals from soybean activated by treatment with cis-jasmone. Entomol Exp Appl. 2009; 131:178-88

[30] Li FQ, Li W, Lin YJ. et al. Expression of lima bean terpene synthases in rice enhances recruitment of a beneficial enemy of a major rice pest. Plant Cell Environ. 2018; 41:111-20

[31] Ling SQ, Rizvi SAH, Xiong T. et al. Volatile signals from guava plants prime defense signaling and increase jasmonate-dependent herbivore resistance in neighboring citrus plants. Front Plant Sci. 2022; 13:833562

[32] Pu QY, Liang J, Shen QQ. et al. A wheat β-patchoulene synthase confers resistance against herbivory in transgenic Arabidopsis. Genes. 2019; 10:441

[33] Sun Y, Huang XZ, Ning YS. et al. TPS46, a rice terpene synthase conferring natural resistance to bird cherry-oat aphid, Rhopalosiphum padi (Linnaeus). Front Plant Sci. 2017; 8:110

[34] Alquézar B, Volpe HXL, Magnani RF. et al. Engineered orange ectopically expressing the Arabidopsis β-caryophyllene synthase is not attractive to Diaphorina citri, the vector of the bacterial pathogen associated to huanglongbing. Front Plant Sci. 2021; 12:641457

[35] Li SY, Wang H, Li FQ. et al. The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles. Plant J. 2015; 84:296-308

[36] Gong ZH, Luo YQ, Zhang WF. et al. A SlMYB75-centred transcriptional cascade regulates trichome formation and sesquiterpene accumulation in tomato. J Exp Bot. 2021; 72:3806-20

[37] Rupasinghe S, Schuler MA. Homology modeling of plant cytochrome P450s. Phytochem Rev. 2006; 5:473-505

[38] Xu J, Wang XY, Guo WZ. The cytochrome P450 superfamily: key players in plant development and defense. J Integr Agric. 2015; 14:1673-86

[39] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C_T method. Nat Protoc. 2008; 3:1101-8

[40] Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007; 2:1565-72

[41] Li DD, Shi W, Deng XX. Agrobacterium -mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Rep. 2002; 21:153-6

[42] Murashige T, Tucker DPH. Growth factors requirements of citrus tissue. In: Chapman HD,ed. Proceedings of the 1st International Citrus Symposium. vol. III. Riverside, CA: Riverside Color Press; 1969, 1155-61

[43] Tan B, Li D, Xu S. et al. Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in citrus. Tree Genet Genomes. 2009; 5:529-37

[44] Aharoni A, Giri AP, Deuerlein S. et al. Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell. 2003; 15:2866-84

[45] Williams L, Blackmer JL, Rodriguez-Saona C. et al. Plant volatiles influence electrophysiological and behavioral responses of Lygus hesperus. J Chem Ecol. 2010; 36:467-78

[46] Bruce TJA, Matthes MC, Chamberlain K. et al. cis-Jasmone induces Arabidopsis genes that affect the chemical ecology of multitrophic interactions with aphids and their parasitoids. Proc Natl Acad Sci USA. 2008; 105:4553-8

[47] Figueiredo JFL, Römer P, Lahaye T. et al. Agrobacterium-mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay. Plant Cell Rep. 2011; 30:1339-45