Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

Abstract

Background: Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.

Results: Glandular trichomes of sunflower (Helianthus annuus L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon delta-cadinene as one of the two main products with alpha-muuorlene, beta-caryophyllene, alpha-humulene and alpha-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.

Conclusion: This study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.

Figure 1

Display of the exon-intron structure of HaTPS1a (HaGAS1), HaTPS1b (HaGAS2) and HaTPS2 (HaCS) genes. Grey boxes represent exon sequences, lines show intron sections. Length of nucleotide sequences is shown for exons in the boxes and for introns above the line. DDxxD marks the position of the conserved aspartate-rich region. i2: intron 2; i4: intron 4.

Figure 2

Alignment of the deduced amino acid sequences of sesquiterpene synthase genes. HaTPS1a (HaGAS1), HaTPS1b (HaGAS2), and HaTPS2 (HaCS) indicate sesquiterpene synthase genes isolated from sunflower in this study. LsLTC2, CiGAS, and TEAS are germacrene A synthase from Lactuca sativa, germacrene A synthase from Cichorium intybus, and 5-epi-aristolochene synthase from Nicotiana tabacum, respectively. Black: identical amino acids in all sequences; dark grey and light grey: identical amino acids in 5 or 4 enzymes, respectively.

Figure 3

(a) PAGE showing purification of recombinant HaCS-fusion protein using Ni-NTA affinity chromatography. ni: uninduced control; ind: induced E. coli culture; ub: unbound fraction; w1-2: washing steps 1 to 2 using 20 mM imidazol; w3: washing step 3 using 100 mM imidazol; e1-3: elution steps 1 to 3 using 250 mM imidazol; M: marker. (b) GC-MS analysis (m/z 204) of an in vitro incubation of recombinant HaCS protein with FDP. Negative control: incubation of an E. coli protein extract not expressing HaCS under the same conditions. x: unidentified compounds.

Figure 4

GC-MS analysis of sesquiterpene products of the in vivo expression of HaCS in S. cerevisiae. Diagrams show products obtained by the expression of HaCS and thioredoxin-HaCS in comparison to pESC-Leu2d empty vector (negative control). Peak numbers in each panel correspond to numbers above peaks in the chromatogram. Mass spectra of identified products are shown in the top of each panel with a mass spectrum of an authentic standard below. One of the two main products was identified as δ-cadinene; the fragmentation pattern of peak 7 showed high similarities to γ-cadinene but differed slightly in the retention time. RI: retention indice. I: farnesol.

Figure 5

GC-MS analysis of sesquiterpene products of sunflower germacrene A synthases HaGAS1 and HaGAS2 compared to germacrene A standard produced by LsLTC2 from lettuce. (a) In vitro enzyme-substrate reactions using purified recombinant HaGAS1 or HaGAS2 with the substrate FDP showing β-elemene (peak 1), the cope-rearrangement product of germacrene A. Germacrene A: standard produced by expression of LsLTC2 (L. sativa germacrene A synthase 2, chromatogram not true to scale); negative control: incubation of FDP with total proteins isolated from E. coli without plasmid. (b) In vivo products from gene expression in yeast showing β-elemene (peak 2) and germacrene A (peak 3). Negative control: yeast strain carrying the empty vector. Differences between (a) and (b) were caused by the use of different injection port temperatures.

Figure 6

Expression of sesquiterpene synthase genes in different sunflower tissues. (a) RT-PCR analyses of germacrene A synthases, HaCS, farnesyl diphosphate synthase, and ubiquitin gene expression. Total RNA was extracted from roots (R), stems (S), cotyledons (C), young leaves (YL), old leaves (OL), ray flowers (RF), and capitate glandular trichomes (T). The constitutively expressed gene for ubiquitin was used as cDNA loading control and internal standard. Due to high sequence similarity differentiation between HaGAS1 and HaGAS2 was not possible by PCR. (b) Detection of the expressed genes for HaGAS1 and HaGAS2 in trichomes (left) and roots (right) by selective restriction digestion of full length HaGAS1/2 cDNA (◂). HaGAS1 contains a PauI but no DraI recognition site while HaGAS2 contains a DraI site but no PauI recognition site.DraI specifically cuts the amplicon of HaGAS1 (1680 bp) into a 1406 bp (•) and 274 bp (▪) fragment. PauI specifically cuts the 1680 bp HaGAS2 amplicon into 1105 (*) and 575 bp (+) fragments. ND: undigested control. L: marker.

Figure 7

RT-PCR analysis of HaGAS, HaCS, and FDPS gene expression in different developmental trichome stages. (a) Cross section of a sunflower capitulum showing young florets in the centre and older florets at the margin of the capitulum. (b) Micrographs of florets in differently developed trichome stages, as found in the capitulum. (c) Semi-quantitative RT-PCR experiments for identification of secretory active trichome stages. Ubiquitin was used as internal standard and loading control; FDPS: cDNA amplification of the expressed farnesyl diphosphate synthase gene; HaGAS: amplicons for H. annuus germacrene A synthases. HaCS: amplicons for H. annuus cadinene synthase.

Figure 8

Quantitative real-time RT-PCR analysis. (a) qPCR data for expression levels of HaGAS1/2, HaCS and FPPS in differently developed trichomes. Same samples were used for generation of these data and those of figure 7. The expression level is shown in comparison to the presecretory trichome stage. (b) qPCR analysis of the expression level of the indicated genes in different sunflower tissue. Expression level in presecretory trichome stage was used as reference.