Floral and insect-induced volatile formation in Arabidopsis lyrata ssp. petraea, a perennial, outcrossing relative of A. thaliana

Abstract

Volatile organic compounds have been reported to serve some important roles in plant communication with other organisms, but little is known about the biological functions of most of these substances. To gain insight into this problem, we have compared differences in floral and vegetative volatiles between two closely related plant species with different life histories. The self-pollinating annual, Arabidopsis thaliana, and its relative, the outcrossing perennial, Arabidopsis lyrata, have markedly divergent life cycles and breeding systems. We show that these differences are in part reflected in the formation of distinct volatile mixtures in flowers and foliage. Volatiles emitted from flowers of a German A. lyrata ssp. petraea population are dominated by benzenoid compounds in contrast to the previously described sesquiterpene-dominated emissions of A. thaliana flowers. Flowers of A. lyrata ssp. petraea release benzenoid volatiles in a diurnal rhythm with highest emission rates at midday coinciding with observed visitations of pollinating insects. Insect feeding on leaves of A. lyrata ssp. petraea causes a variable release of the volatiles methyl salicylate, C11- and C16-homoterpenes, nerolidol, plus the sesquiterpene (E)-beta-caryophyllene, which in A. thaliana is emitted exclusively from flowers. An insect-induced gene (AlCarS) with high sequence similarity to the florally expressed (E)-beta-caryophyllene synthase (AtTPS21) from A. thaliana was identified from individuals of a German A. lyrata ssp. petraea population. Recombinant AlCarS converts the sesquiterpene precursor, farnesyl diphosphate, into (E)-beta-caryophyllene with alpha-humulene and alpha-copaene as minor products indicating its close functional relationship to the A. thaliana AtTPS21. Differential regulation of these genes in flowers and foliage is consistent with the different functions of volatiles in the two Arabidopsis species.

Fig. 1

Volatile emission from flowers of A. lyrata ssp. petraea. GC–MS chromatograms of volatiles collected from flowers of two individual plants 4A2 (a) and 2B1 (b) of a German A. lyrata ssp. petraea population (Plech). Volatiles were collected from six (a) or eight (b) detached flowers for 2 h (between 11 a.m. and 1 p.m.) in a closed-loop stripping system as described under “Materials and methods”. 1 benzaldehyde, 2 phenylacetaldehyde, 3 acetophenone, 4 nonanal, 5 decanal, S internal standard 1-bromodecane. Nonanal and decanal are background volatile contaminants. The chemical structures of benzaldehyde and phenylacetaldehyde are shown. c Emission rates of benzaldehyde and phenylacetaldehyde from different individuals of the Plech population. Calculations are based on volatile collections from three separate sets of flowers per plant

Fig. 2

Organ-specific emission of floral volatiles. GC–MS chromatograms of volatiles emitted from a single intact flower (a), the detached petals of a single flower (b), and a single flower from which petals were removed (c). Volatiles were trapped in 1-ml glass vials by solid-phase microextraction (SPME) as described under “Materials and methods”. Flowers were from a Plech plant with an “4A2”-type volatile profile (see Fig. 1a, c)

Fig. 3

Diurnal changes of floral and vegetative volatile emissions from A. lyrata ssp. petraea. High resolution volatile measurement recorded by PTR-MS for a single intact flowering plant (Plech). Volatile emission was monitored continuously over a time course of 48 h under a 13 h light/11 h dark rhythm as described in “Materials and methods”. The protonated masses of benzaldehyde (107), phenylacetaldehyde (121), and sesquiterpene olefins including cyclosativene (205) are recorded. The scale for mass 205 is shown on the right

Fig. 4

Insect-induced volatile emissions from leaves of A. lyrata ssp. petraea. 20–30 P. xylostella larvae were placed on rosette leaves of individual non-flowering plants (population Neutras, Germany) and removed 24 h later. Volatiles were collected from control plants (upper panel) and insect-damaged plants (lower panel) of two selected lines, NT20 (a) and NT51 (b) as described under “Materials and methods”. GC–MS chromatograms are shown only for volatiles collected on day 2 since emission rates were highest on this day. 1 DMNT, 2 methyl salicylate, 3 (+)-cyclosativene, 4 (E)-β-caryophyllene, 5 α-humulene, 6 nerolidol, 7 TMTT, S nonyl acetate standard. Unlabeled peaks represent background volatiles or compounds whose identity could not be precisely determined by library comparison

Fig. 5

Herbivore-induced transcription of an A. thaliana (E)-β-caryophyllene synthase gene homologue in A. lyrata ssp. petraea rosette leaves. Semi-quantitative RT-PCR analysis of transcript levels of an A. lyrata ssp. petraea gene (AlCarS) with high sequence similarity to the A. thaliana (E)-β-caryophyllene synthase (At5g23960/AtTPS21). Induced transcripts were observed only in rosette leaves of plants that emitted (E)-β-caryophyllene after 20 h of feeding by P. xylostella. Control reactions were performed with RNA from undamaged clonal plants of the same line. Reactions with primers for actin-8 were performed to judge equality of the cDNA template concentration

Fig. 6

Protein sequence comparison of AlCarS with A. thaliana AtTPS21. Amino acid sequences of AlCarS and AtTPS21 are 91% identical. Identical amino acids are shaded in black. Dashes indicate gaps inserted for optimal alignment. The DDXXD motif that is highly conserved among class I terpene synthases and obligatory for catalytic activity is underlined

Fig. 7

Formation of (E)-β-caryophyllene by recombinant AlCarS enzyme. AlCarS was expressed in E. coli, extracted, and incubated with the substrate all-trans-FPP. The resulting terpene products were analyzed by GC–MS. The major product was identified as (E)-β-caryophyllene (1). Other products were α-humulene (2) and α-copaene (3). The mass spectrum of the (E)-β-caryophyllene product is shown in comparison to that of an authentic (E)-β-caryophyllene standard (inset)