Transcriptomic and metabolomic analyses provide insight into the volatile compounds of citrus leaves and flowers

Abstract

Background: Previous reports have mainly focused on the volatiles in citrus fruits, and there have been few reports about the volatiles in citrus leaves and flowers. However, citrus leaves and flowers are also rich in volatile compounds with unique aromas. Here, to investigate the volatiles in citrus leaves and flowers, volatile profiling was performed on leaves from 62 germplasms and flowers from 25 germplasms.

Results: In total, 196 and 82 volatile compounds were identified from leaves of 62 citrus germplasms and flowers of 25 citrus germplasms, respectively. The dominant volatile terpenoids were more diverse in citrus leaves than in peels. A total of 34 volatile terpenoids were commonly detected in the leaves of at least 20 germplasms, among which 31 were overaccumulated in the leaves of wild or semiwild germplasms. This result was consistent with the high expression levels of five genes and one key gene of the mevalonate and 2-C-methyl-D-erythritol-4-phosphate (MEP) biosynthetic pathways, respectively, as well as the low expression levels of geranylgeranyl diphosphate synthase of the MEP pathway, relative to the levels in cultivars. Fully open flowers showed increased levels of four terpene alcohols and a decrease in sabinene content compared with balloon-stage flowers, especially in sweet orange. A monoterpene synthase gene was identified and functionally characterized as a sabinene synthase in vitro.

Conclusions: Collectively, our results suggest that 31 important terpenoids are abundant in wild or semiwild citrus germplasms, possibly because of a negative effect of domestication on the volatiles in citrus leaves. The sweet smell of fully open flowers may be attributed to increased levels of four terpene alcohols. In addition, a sabinene synthase gene was identified by combined transcriptomic and metabolomic analyses.

Keywords: Citrus; STPS; Terpenoid biosynthesis; Volatile profile; Wild or semiwild germplasms.

Fig. 1

Total volatile content in citrus leaves. The line above shows the 14 germplasms with the highest total volatile levels among all 62 citrus germplasms. LSM: loose-skin mandarin; PA: papeda; CI: citron; SO: sour orange; IC: C. ichangensis; SW: sweet orange; PU: pummelo; TRI: Poncirus trifoliata

Fig. 2

CPCoA of citrus germplasms based on volatile profiles of leaves. LSM: loose-skin mandarin; PA: papeda; CI: citron; SO: sour orange; IC: C. ichangensis; SW: sweet orange; PU: pummelo; TRI: Poncirus trifoliata

Fig. 3

Differential gene expression patterns of two terpenoid biosynthetic pathways in leaves of cultivated citrus species and wild or semiwild germplasms. a: biosynthetic pathway of terpenoids in plants. AACT: acetoacetyl-CoA thiolase; HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR: HMG-CoA reductase; MVK: mevalonate kinase; PMK: phosphomevalonate kinase; IDI: isopentenyl diphosphate isomerase; DXS:1-deoxy-d-xylulose 5-phosphate synthase; DXR: 1-deoxy-d-xylulose 5-phosphate reductoisomerase; IPP: isopentenyl diphosphate; FPPS: farnesyl pyrophosphate synthase; GPPS: geranyl pyrophosphate synthase; GGPPS: geranylgeranyl diphosphate synthase. b: differentially expressed genes in MVA and MEP pathways; c: differences in accumulation of monoterpenes and sesquiterpenes in the leaves of wild or semiwild species and cultivars

Fig. 4

RT-qPCR analysis of the transcript levels of four genes in the MVA pathway and two genes in the MEP pathway in cultivar and wild or semiwild citrus germplasms. Transcript levels are expressed relative to the expression level of the gene encoding the Actin extension protein. C: cultivar citrus germplasms; W: wild or semiwild citrus germplasms

Fig. 5

Monoterpene compounds produced by recombinant STPS in vitro. pET-28a(+) + GPP: pET-28a(+) empty vector added with GPP; STPS + GPP: STPS protein added with GPP. Standards: six authentic standards were used for compound identification. P1: α-pinene, P2: sabinene, P3: β-myrcene, P4: D-limonene, P5: trans-β-ocimene, P6: linalool