Characterization of Trichome-Expressed BAHD Acyltransferases in Petunia axillaris Reveals Distinct Acylsugar Assembly Mechanisms within the Solanaceae

Abstract

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato (Solanum lycopersicum), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3', and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petuniaaxillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris, tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris, PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3' position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3' position in tomato, is absent in P. axillaris Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis.

Figure 1.

Acylsucroses from S. lycopersicum and P. axillaris. A, Structures of characteristic tetra-acylsucroses from S. lycopersicum. Details of the length of the acyl chains typically found at each position in the most abundant metabolites are provided together with the identity of the ASAT enzymes responsible for acylsucrose assembly and the order in which they catalyze acylation reactions. Data represented are summarized from previous reports (Schilmiller et al., 2012, 2015; Ghosh et al., 2014; Fan et al., 2016). B, Structures of characteristic acylsucroses from P. axillaris. Details of the length of the acyl chains typically found at each position are derived from NMR-based structural characterization (Liu et al., 2017). The order of assembly of P. axillaris acylsucroses and the identification of the corresponding ASAT enzymes is the subject of this study.

Figure 2.

Expression analysis of putative P. axillaris ASATs. The expression of candidate ASATs was determined in whole petioles, shaved petioles with trichomes removed, and trichomes of P. axillaris. Three biological and three technical replicates for each sample were analyzed. Data are presented as mean expression values ± se relative to those observed in shaved stems. Values labeled with different letters are significantly different (least square means, P < 0.05).

Figure 3.

Phylogenetic analysis of putative ASATs. An unrooted phylogenetic tree of known S. lycopersicum (tomato) ASATs together with related amino acid sequences from S. lycopersicum, Solanum tuberosum (potato), Capsicum annuum (pepper), and P. axillaris was constructed using the maximum likelihood method from a multiple sequence alignment of deduced full-length amino acid sequences. The tree was constructed using MEGA version 5, and bootstrap values of 50 or greater are shown from 2,000 replicates. Known tomato ASATs are shown in red, and putative trichome preferentially expressed ASATs from P. axillaris are in blue. Subsequent functional characterization of the putative P. axillaris ASATs reported herein led to the identification of four enzymes involved in acylsugar assembly, PaxASAT1 to PaxASAT4. The positions on the Suc molecule where the addition of acyl chains occurs following ASAT-mediated catalysis are provided in parentheses.

Figure 4.

The P. axillaris acylsugar profile is altered by the silencing of Pax21699 and Pax31629. A, Comparison of the acylsugar profile of P. axillaris expressing TRV2 empty vector, TRV2::Pax21699, or TRV2::Pax31629 constructs. Negative ion mode total ion current liquid chromatography-mass spectrometry (LC-MS) chromatograms are shown, indicating both reduced [e.g. S(m)5:26 (m,5,5,5,8)] and increased [e.g. S3:18 (5,5,8), S(m)4:21 (m,5,5,8), S4:19 (4,5,5,5), and S4:20 (5,5,5,5)] abundance of individual acylsugars in TRV2::Pax21699- and TRV2::Pax31629-silenced lines. The peak corresponding to a 5 µm telmisartan internal standard is labeled IS. Details of individual acylsugars are provided in Supplemental Table S2 and Supplemental Figure S5. B and C, Quantification of acylsugar levels in TRV2::Pax21699- and TRV2::Pax31629-silenced lines. The abundances of individual and total (insets) acylsugars in TRV2::Pax21699- and TRV2::Pax31629-silenced lines relative to those in TRV2 empty vector controls are shown. Data are presented as peak area normalized to dry weight and internal standard peak area and represent means ± se of 12 biological replicates. Asterisks denote significant differences (**, P < 0.01; ***, P < 0.001) as determined by Student's t tests.

Figure 5.

P. axillaris acylsugar levels are reduced in Pax36474- and Pax39119-silenced lines. The abundances of individual and total (insets) acylsugars in TRV2::Pax36474-silenced (A) and TRV2::Pax39119-silenced (B) lines relative to those in TRV2 empty vector controls are shown. Data are presented as peak area normalized to dry weight and internal standard peak area and represent means ± se of 12 biological replicates. Asterisks denote significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) as determined by Student's t tests.

Figure 6.

ASAT1 activity is not conserved in P. axillaris and S. lycopersicum. A, In vitro formation of S1:5 by recombinant S. lycopersicum ASAT1 (SlASAT1) and P. axillaris ASAT1 (PaxASAT1) using Suc as the acyl acceptor and either iC5-CoA or aiC5-CoA as the acyl donor. Negative ion mode LC-MS extracted ion chromatograms for the reaction product mass-to-charge ratio (m/z) 461.1 (S1:5; [M+Cl]⁻) are shown. The vertical dashed line highlights a retention time shift of S1:5 products formed by SlASAT1 and PaxASAT1, suggesting that different S1:5 isomers are formed by these enzymes. B, Structures of the S1:5 reaction products formed by SlASAT1 and PaxASAT1 using iC5-CoA and aiC5-CoA, respectively. Data for S1:5(iC5^R4) formed by SlASAT1 are based on prior structural characterization (Schilmiller et al., 2015; Fan et al., 2016), while the structure of S1:5(aiC5^R2) was determined by NMR (Supplemental Table S4).

Figure 7.

Identification of ASAT2, ASAT3, and ASAT4 activities of P. axillaris. A, In vitro formation of S2:10 by recombinant P.  axillaris ASAT2 (PaxASAT2) using S1:5 (aiC5^R2) as the acyl acceptor and aiC5-CoA as the acyl donor. A negative ion mode LC-MS extracted ion chromatogram for the reaction product m/z 555.2 is shown. B, In vitro formation of S3:17 by recombinant P. axillaris ASAT3 (PaxASAT3) using S2:10 (aiC5^R2, aiC5^R4) as the acyl acceptor and iC7-CoA as the acyl donor. A negative ion mode LC-MS extracted ion chromatogram for the reaction product m/z 667.3 is shown. C, In vitro formation of S4:22 by recombinant P. axillaris ASAT4 (PaxASAT4) using S3:17 (aiC5^R2, iC7^R3, aiC5^R4) as the acyl acceptor and aiC5-CoA as the acyl donor. A negative ion mode LC-MS extracted ion chromatogram for the reaction product m/z 751.4 is shown. D to F, Tandem mass spectra of the acylsucroses formed by PaxASAT2, PaxASAT3, and PaxASAT4. The fragmentation of specific reaction products of m/z 555.2, 667.3, and 751.4 presented in A to C is shown. Fragmentation in negative mode reveals the length of the acyl chains esterified to the Suc core due to the loss of acyl groups as ketenes.

Figure 8.

Comparison of tetra-acylsucrose assembly in tomato and P. axillaris. A, Structure of a generic tetra-acylsucrose from tomato. B, Structure of a generic acylsucrose from P. axillaris. The four steps required for the formation of tetra-acylsucroses in each species are shown. Orthologous enzymes predicted by sequence analysis are indicated by the same color. Note that the predicted orthologous relationships between SlASAT1 and PaxASAT2 and between SlASAT2 and PaxASAT3 are not conserved at the biochemical level, and this, together with the presence-absence variation of other ASATs between tomato and petunia, results in differential acylsucrose assembly in these two species.