Functional genomics reveals that a compact terpene synthase gene family can account for terpene volatile production in apple

Abstract

Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple 'Royal Gala' expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.

Figure 1.

Floral and vegetative terpene emission in cv Royal Gala. GC-MS analysis is shown for the predominant terpene volatiles trapped from the headspace of cv Royal Gala vegetative and floral tissues. Averaged data for biologically replicated analyses are shown. Terpenes produced at a rate less than 0.5 ng g⁻¹ fresh weight (FW) h⁻¹ are not included. The complete data set is presented in Supplemental Table S1. The bourbonene isomer was not determined, as the retention indices for α- and β-bourbonene are very similar.

Figure 2.

Volatile terpene emission in cv Royal Gala floral tissues. GC-MS analysis is shown for the predominant terpene volatiles trapped from the headspace of cv Royal Gala floral tissues. Stg/Sty/Sta, Combined sample containing stigma, style, and stamen tissue. Averaged data for biologically replicated analyses are shown. Terpenes produced at less than 0.5 ng g⁻¹ fresh weight (FW) h⁻¹ are not included. The complete data set is presented in Supplemental Table S2.

Figure 3.

cv Royal Gala fruit at various stages of development: 30 DAA (A), 60 DAA (B), 90 DAA (C), 120 DAA (D), and 150 DAA (harvest ripe; E). Bars = 1 cm.

Figure 4.

Phylogeny of the apple TPS family. Maximum likelihood analysis is shown for the predicted full-length TPSs from cv Royal Gala (RG) and cv Golden Delicious (GD) apple with selected full-length grape, poplar, pear, and tomato TPSs. Bootstrap values are shown as a percentage of 100 replicates. The scale bar represents 0.5 substitutions per site. Abbreviations for previously identified and/or functionally annotated TPS enzymes, including GenBank accession numbers in parentheses, are as follows: grape VvGwECar1 = (E)-β-caryophyllene synthase (ADR74192), VvCSaFar = (E,E)-α-farnesene synthase (ADR74198), VvPNRLin = (3R)-linalool synthase (ADR74209), VvPNaPin1 = pinene synthase (ADR74202), VvGwbOci = (E)-β-ocimene synthase (ADR74204), VvGwGer = geraniol synthase (ADR74217), VvPNLinNer2 = (3S)-linalool/(E)-nerolidol synthase (ADR74211), and VvCSENerGl = (E)-nerolidol/(E,E)-geranyl linalool synthase (ADR74219); apple MdRGAFS1 = (E,E)-α-farnesene synthase (AAX19772) and MdRGEKS = ent-kaurene synthase (AFG18184); pear PcAFS1 = (E,E)-α-farnesene synthase (AAT70237); tomato SleKS = ent-kaurene synthase (AEP82778) and SlCPS = CDP synthase (BAA84918); poplar PtCPS = CDP synthase (EEE81383).

Figure 5.

In planta analysis of cv Royal Gala sesquiterpene synthase enzyme activity. Full-length sesquiterpene synthase enzymes were cloned into the pHEX2 plant transformation vector and transiently expressed in tobacco leaves. Selected ion (m/z 93) headspace GC-MS profiles are as follows. A, MdAFS-RG1: peak 1, (Z,E)-α-farnesene; peak 2, (E,E)-α-farnesene. B, MdCAR-RG1: peak 3, (E)-β-caryophyllene; peak 4, α-caryophyllene. C, MdGDS-RG1: peak 3, (E)-β-caryophyllene; peak 5, germacrene-D. D, MdNES-RG1: peak 6, (E)-nerolidol. E, GUS: empty vector control. Products were confirmed by comparison with authentic standards and/or GC-MS retention indices.

Figure 6.

In planta analysis of cv Royal Gala monoterpene synthase enzyme activity. Full-length monoterpene synthase enzymes were cloned into the pHEX2 plant transformation vector and transiently expressed in tobacco leaves. Selected ion (m/z 93) headspace GC-MS profiles are as follows. A, MdPIN/CAM-RG1: peak 1, α-pinene; peak 2, camphene; peak 3, β-pinene; peak 4, β-myrcene; peak 5, limonene. B, MdOCS-RG1: peak 6, (E)-β-ocimene. C, MdLIS-RG1: peak 7, linalool. D, GUS: empty vector control. Products were confirmed by comparison with authentic standards and/or GC-MS retention indices.

Figure 7.

TPS gene expression in cv Royal Gala flowers and leaves. Expression profiles are shown for sesquiterpene (A), monoterpene (B), and diterpene (C) synthases in different flower and leaf tissues. ^aMdEKS-RG1 and MdCDS-RG1 have not been functionally verified but are predicted to encode the respective cv Royal Gala ent-kaurene and CDP synthases. YLE, Young leaf; MLE, mature leaf; STP, stipule; FLB, flower bud; FLO, fully opened flower; PED, pedicel; SEP, sepal; PET, petal; ST, stigma, style, and stamen; OVA, ovary. The data were analyzed using the target-reference ratio calculated with the LightCycler 480 software, enabling a comparison of the level of expression of different gene family members compared with the EF1α reference gene considered stable and unchanging in the different tissues during development. The specific primers used for each gene are detailed in Supplemental Table S7. Data are presented as means ± se (n = 4).

Figure 8.

TPS gene expression in cv Royal Gala fruit tissues. Gene expression profiles are shown for sesquiterpene (A), monoterpene (B), and diterpene (C) synthases in different tissues during apple fruit development. Gene expression data analysis was carried out as for Figure 7. Data are presented as means ± se (n = 4).

Figure 9.

TPS gene expression in the fruit of five heritage apple cultivars. Fruit were picked at approximately 150 DAA, and gene expression data analysis was carried out using the same conditions and primers as described in Figure 5. The expression of each TPS gene in cv Royal Gala fruit at 150 DAA from Figure 8 has been included for comparison. Data are presented as means ± se (n = 4).