Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway
- Michigan State University, United States
Figures
Figure 1 with 6 supplements
Acylsugars in solanaceae.
(A) An example acylsugar from tomato. The nomenclature of acylsugars and the ASAT enzymes responsible for acylation of specific positions are described. Carbon numbering is shown in red. Sl refers …
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Figure 1—source data 1
Species sampled at the NYBG and analysis of their trichome types.
Tissues sampled are described.
- https://doi.org/10.7554/eLife.28468.010
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Figure 1—source data 2
Values used to generate the figures in Figure 1C–E and Figure 1—figure supplement 2.
Values indicate normalized peak areas where Normalized peak area = [(Acylsugar peak area)/(Area of internal standard)]/Dry weight of tissue.
- https://doi.org/10.7554/eLife.28468.011
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Figure 1—source data 3
Raw data and parameter files used to analyze Solanaceae species samples.
zip file includes dry weights of all samples, QuanLynx method files and the output files used to make Figure 1C,D,E and Figure 1—figure supplement 2.
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Figure 1—source data 4
mzmine 2 parameter file and peak lists.
These files were used to process RAW mass spectrometric files for the calculation of Shannon Entropy (Figure 1G, Figure 1—figure supplement 5).
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Figure 1—source data 5
Data used to make Figure 1H.
Sheet 5A shows the numerical counts of peaks shared between samples while Sheet 5B shows the percentage values centered on the row sample, as described in the legend.
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Figure 1—figure supplement 1
Tomato ASATs are members of the BAHD enzyme family.
(A) SlASATs shown in a phylogenetic tree with all other BAHDs in cultivated tomato genome. The ‘Solyc’ in the gene names has been shortened to ‘S’. BAHD is an acronym for four acyltransferase …
Figure 1—figure supplement 2
Normalized and integrated acylsugar peak areas in different species.
(A–J) Peak area of each acylsugar was normalized using the internal standard peak area and dry weight, and were summed across different isomers of the same acylsugar. Colors represent % normalized …
Figure 1—figure supplement 3
The complexity of acylsugar phenotype across multiple acylsugar producing species.
Undiluted trichome extracts collected from plants at the NYBG were run on a 110 min gradient on a C18 column as described in the Methods (Figure 1—figure supplement 6). Peaks consistent with …
Figure 1—figure supplement 4
Positive mode CID mass spectra of select representative acylsugars from five species.
(A–E) Each spectrum was generated at elevated collision energy to generate fragment ions. Selected fragment ions obtained at the same retention time as the assigned pseudomolecular ion of the …
Figure 1—figure supplement 5
Peak and tissue specialization
(A) As shown in Figure 1G, the peaks are largely limited to single samples. Hence, the peak specificity (Si) is at the theoretical maximum. (B) A plot of tissue specialization vs Shannon Entropy …
Figure 1—figure supplement 6
Description of the LC gradients used in this study.
First column includes the method run time and the column used.
Figure 2 with 8 supplements
In vitro validation of Salpiglossis ASAT candidates.
(A) NMR derived structures of three Salpiglossis acylsugars. NMR resonances used to interpret the first three structures are described in Figure 2—source data 1. We verified the plant under study as …
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Figure 2—source data 1
NMR chemical shifts for four acylsugars purified from Salpiglossis plants.
This data was used to infer Figure 2A.
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Figure 2—figure supplement 1
Phylogenetic positions of Salpiglossis, Hyoscyamus and Salpiglossis candidate enzymes.
(A,B) Phylogeny based on the ndhF (A) and trnLF spacer (B) sequence amplified from Salpiglossis and Hyoscyamus DNA (this study) and other sequences downloaded from NCBI. Accession numbers of NCBI …
Figure 2—figure supplement 2
Trichome preferentially expressed BAHD enzymes.
Additional information about the transcripts is provided.
Figure 2—figure supplement 3
Confirmation of differential expression results from RNA-seq using semi-quantitative RT-PCR.
(A, B) Verification of trichome high expression of certain transcripts using semi-quantitative RT-PCR in S.quitoense (A) and Salpiglossis (B). The comparisons between Trichome (T) and Shaved stem (S)…
Figure 2—figure supplement 4
SsASAT1 reactions with different acyl CoA substrates.
Shown are extracted ion chromatograms of SsASAT1 reactions using sucrose as the substrate and various acyl CoAs as donors. No product was found with acetyl CoA as donor. LC/MS analyses were …
Figure 2—figure supplement 5
Comparative analyses of LC/MS retention times of enzyme reaction products.
(A,B) Comparison between S1:5(5) produced by SsASAT1, PaASAT1 and SlASAT1 on the C18 (A) and the BEH amide (B) column. The dashed line connect the peaks aligned based on their retention times, and …
Figure 2—figure supplement 6
SsASAT2 does not acylate sucrose.
Extracted ion chromatogram of masses corresponding to S1:4(4), S1:5(5) and S1:6(6). No peaks corresponding to these products are seen. Additional reactions with individual CoAs -- aiC6 CoA and nC12 …
Figure 2—figure supplement 7
SsASAT3 acylates at the R3 position.
An indirect assessment of SsASAT3 positional specificity using Petunia ASAT4, which acylates at the pyranose R6 position (A) The chromatogram shows that Petunia ASAT4, which is known to acylate at …
Figure 2—figure supplement 8
SsASAT5 putative secondary activities.
(A) SsASAT5 can transfer C2 to S3:15 (5,5,5) isolated from Solanum pennellii-derived Backcross Inbred Line BIL6180 (unpublished data; Ofner et al., 2016), which has the three acyl chains on the R2, …
Figure 3 with 2 supplements
In planta validation of SsASAT1 and SsASAT2 candidates.
(A) Two representative plants with the phytoene desaturase gene silenced using VIGS shown with 18% reflectance gray card. SsPDS-1 and SsPDS-2 have two different regions of the SsPDS transcript …
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Figure 3—source data 1
Raw and processed values obtained through qPCR.
Data was used to make Figure 3B.
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Figure 3—source data 2
Normalized peak areas calculated for VIGS experiments.
Individual worksheets describe data obtained from independent experiments. This data was used to make Figures 3D, F, 4B and D.
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Figure 3—figure supplement 1
Results of knockdown of SsASAT1, SsASAT2 and SsASAT5 transcripts in a distinct replication of VIGS experiments.
(A) SsASAT1 knockdown shows reduction of acylsugar levels. (B) SsASAT2 and SsASAT5 knockdown causes substantial changes in Salpiglossis acylsugar profiles. The accumulating acylsugars in these two …
Figure 3—figure supplement 2
Individual acylsugar levels in SsASAT2 VIGS replicate 1.
The boxplots denote the summed normalized acylsugar peak areas, obtained using the LC/MS extracted ion chromatograms, across all plants in the experiment. Blue and red asterisks indicate significant …
Figure 4 with 3 supplements
VIGS phenotypes of SsASAT3 and SsASAT5 knockdown plants.
In each sub-figure, the left hand panel shows a representative chromatographic phenotype while the right hand panel shows distributions of the aggregated peak areas of all plants of the tested …
Figure 4—figure supplement 1
SsASAT3 knockdown boxplots for levels of individual acylsugars.
Statistical significance of the difference between TRV2-LIC and ASAT3-2 was tested using Kolmogorov-Smirnov test. ** represents p<0.01 and * represents p<0.05. Boxes where the acylsugars had …
Figure 4—figure supplement 2
Positive mode fragmentation patterns of novel acylsugars found in SsASAT3 VIGS knockdown plants.
In (A,B,C), mass spectra obtained at elevated collision energies of three different novel acylsucrose peaks are shown. We make the assumption that fragment ions and the most abundant pseudomolecular …
Figure 4—figure supplement 3
Hypothesized routes of metabolite flow in VIGS knockdown plants.
(A) SsASAT3 knockdown and (B) SsASAT5 knockdown. The perturbed pathway steps are denoted by a blurred arrow and two red lines. All acylation positions are hypothesized based on in vitro data and …
Figure 5
Model for the Salpiglossis acylsugar biosynthetic pathway.
The question marks indicate unidentified enzymes. The blue colored acyl chains are positioned on the sucrose molecule based on results of positive mode fragmentation characteristics, co-elution …
Figure 6 with 4 supplements
The origins of acylsugar biosynthesis.
(A) Gene tree showing the characterized ASATs (blue squares) and other BAHDs in Clade III of the BAHD family of enzymes (D'Auria, 2006). Only Lamiid species are included in this tree, given …
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Figure 6—source data 1
Results of BLAST against 1kp database.
The ‘Sequences’ sheet shows the Petunia sequences used for TBLASTN against the Asterids database of 1kp (Nadakuduti et al., 2017). Other sheets show the tabular output of TBLASTN using individual ASATs as the query. The best matching non-Solanaceae sequences were used for making the phylogenetic trees shown in Figure 6 and Figure 6—figure supplement 3A.
- https://doi.org/10.7554/eLife.28468.041
Figure 6—figure supplement 1
The phylogenetic context of tomato ASATs.
Results of protein BLAST (BLASTP) between SlASAT sequences and proteomes of each species in the Phytozome database. The % identity (% IDT) values for the top BLASTP hits in each species are shown.
Figure 6—figure supplement 2
Robustness of the phylogenetic relationships.
(A) A maximum likelihood tree obtained using the best model (JTT + G + I + F with five rate categories) as per maximum likelihood based search of multiple models. Sites with <70% coverage were …
Figure 6—figure supplement 3
Additional related sequences do not affect our inferences regarding ASAT clade emergence.
Hits obtained using various BLAST strategies (A,B) and using Petunia Clade III BAHDs (C) are shown in a phylogenetic context with ASATs and other relevant homologs. The novel hits that were not …
Figure 6—figure supplement 4
LC/MS profiles of leaf surface metabolites from 11 species from the Gentianales, Lamiales, Boraginales orders collected from the living collection at the MSU Botanical Gardens.
Positive controls S. lycopersicum M82 and Salpiglossis sinuata are also included. Samples were run on a C18 column as described in the Methods. These profiles show that peaks with m/z ratios …
Figure 7 with 1 supplement
The evolution of acylsugar biosynthesis in Solanaceae.
(A) ASAT activities overlaid on the Solanaceae phylogenetic relationships. Each colored square represents a single ASAT, starting from ASAT1 and moving sequentially down the pathway to ASAT5 (left …
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Figure 7—source data 1
Results of BLAST searches performed using ASAT sequences as queries and multiple databases as subjects.
BLASTP or TBLASTN was performed, and only the top hit of a query is shown. Results were used to infer Figure 7A.
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Figure 7—source data 2
Syntenic blocks between pairs of species identified by MCScanX.
Grey and red colors indicate non-tandem and tandem-duplicated genes, respectively, as identified by MCScanX. Comparisons between tomato and Solanum phureja are based on results of a manual analysis performed using the GBrowse utility on the SpudDB database.
- https://doi.org/10.7554/eLife.28468.045
Figure 7—figure supplement 1
Some aASAT2 orthologs cannot catalyze sucrose to mono-acylsucrose conversion.
(A) Neighbor-joining tree made using Salpiglossis and tomato ASATs and their best hits in S. nigrum (Sn) and H. niger (Hn) transcriptome database. Tree was made using default parameters in MEGA6 for …
Figure 8
Proposed model for evolution of acylsugar biosynthesis over 100 million years.
Events in plant evolution are noted on the left and the acylsugar biosynthetic pathway developments are noted on the right of the timeline. Tri-acylsugars produced by Salpiglossis and tomato …
Tables
Table 1
RNA-seq data statistics
https://doi.org/10.7554/eLife.28468.015| Item | S. nigrum | S. quitoense | H. niger | S. sinuata |
|---|---|---|---|---|
| Original read pairs | 81,314,841 | 85,374,110 | 86,161,659 | 80,302,734 |
| Filtered read pairs (% original) | 73346531 (90.2%) | 76734781 (89.9%) | 76819022 (89.2%) | 71129160 (88.6%) |
| Normalized read pairs (% normalized) | 17301238 (23.6%) | 15350023 (20.0%) | 20779972 (27.1%) | 17905057 (25.2%) |
| Total transcript isoforms | 160,583 | 124,958 | 189,711 | 149,136 |
| Longest isoforms (% total) | 78,020 (49%) | 72,426 (58%) | 96,379 (51%) | 77,970 (52.3%) |
| With > 10 reads | 32,105 | 32,044 | 38,252 | 32,798 |
| With predicted peptide > 50aa | 23,224 | 22,289 | 26,262 | 23,570 |
| Differentially expressed*,† (%>10 reads) | 10,386 (32%) | 12,194 (38%) | 9007 (24%) | 7091 (22%) |
| Trichome high† (% differentially expressed) | 2292 (22.1%) | 3547 (29.1%) | 3321 (36.8%) | 1888 (26.6%) |
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* Differentially expressed genes at p<0.05 (corrected for multiple testing); 2 p<0.05, fold change >2
† Some differentially expressed transcripts were confirmed by RT-PCR, as shown in Figure 2—figure supplement 3.
Additional files
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Supplementary file 1
GO categories enriched among trichome-high genes in different Solanaceae species.
The enrichment columns show whether the GO is enriched in trichome high transcripts in this species, based on Fisher Exact Test 1: yes, 0: no. In the ‘# of genes’ column, 0 = GO category not enriched among trichome high genes in this species.
- https://doi.org/10.7554/eLife.28468.047
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Supplementary file 2
Sequences identified in this study.
The transcript identifier as per the assembler Trinity nomenclature is noted in the part after the bar (|). Green and yellow highlighted sequences are the two regions targeted for VIGS in Salpiglossis.
- https://doi.org/10.7554/eLife.28468.048
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Supplementary file 3
Primer sequences used in this study.
- https://doi.org/10.7554/eLife.28468.049
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Supplementary file 4
Experimental conditions for VIGS experiments.
- https://doi.org/10.7554/eLife.28468.050
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Supplementary file 5
MEGA6 aligned file in the *.
mas format. These sequences were used to make Figure 6A.
- https://doi.org/10.7554/eLife.28468.051
