Proposed model of acyl sugar synthesis in S. pennellii (A) and N. benthamiana (B) assuming the same intracellular locations generally observed for primary metabolic pathways of BCAA synthesis and catabolism. Solid arrows and broken arrows depict major and minor metabolic routes, respectively, in acyl sugar formation deduced from chain composition. Proposed two-carbon elongation by plastidial FAS of BCFAs from a 2MeC3:0 precursor is shown for S. pennellii (A). Plausible operation of the iso and anteiso branches of the α-keto acid elongation pathway is shown for branched-chain elongation in N. benthamiana (B). EC numbers are as follows: TD (EC 4.2.1.16), ALS (EC 2.2.1.6), IPMS (EC 2.3.3.13), IPMD (EC 1.1.1.85), IPDS (EC 4.2.1.33), BCKD (EC 2.7.11.4), and KAS (EC 2.3.1.41). ER, Endoplasmic reticulum.

Glandular trichomes in N. benthamiana and S. pennellii. A, Scanning electron microscopy of a N. benthamiana stem shows one large swollen-stalk trichome (lt) and small trichomes capped with single (s), twin (t), or multiple-celled (m) secretory heads. Bar = 200 μm. B, Light microscopy of acyl sugar-secreting type IV trichomes of S. pennellii, indicating a glandular head (h). Bar = 200 μm. C to E, Common glandular head types of small secretory trichomes in N. benthamiana. Bars = 20 μm. F, S. pennellii type VI glandular trichome visualized with scanning electron microscopy. Bar = 100 μm. G, Trichomes harvested from leaves of N. benthamiana by brushing, including large type (lt). Bar = 500 μm. H, Trichomes similarly harvested from S. pennellii leaves showing glandular heads (h) and broken stalks of type IV trichomes (st). Bar = 100 μm.

Functional classification of differentially expressed genes in the trichomes of N. benthamiana and S. pennellii. Functional assignment is shown for the products of genes that are preferentially expressed in leaf trichomes or total underlying leaf in N. benthamiana (A) and S. pennellii (B). These genes showed at least a 2-fold difference in expression. The y axis lists categories of putative gene function, and the x axis shows the proportion of preferentially trichome-expressed genes in a given functional category.

Acyl sugar and wax alkane production of N. benthamiana and S. pennellii plants impaired in BCAA breakdown. Analysis of BCKD gene expression (A) and function by gene knockdown using VIGS (B–F). A, Semiquantitative RT-PCR analysis of the expression of genes encoding components of the BCKD complex in different tissues of N. benthamiana and S. pennellii. Tri (st), Stem trichomes; Tri (lf), leaf trichomes; Epi (st), stem epidermis; Flwr, flower; −Contr, no cDNA PCR control. cDNA levels were normalized according to actin transcript levels. The gene encoding HR7, highly expressed in trichomes and epidermis of tomato and tobacco, served as an additional control. B, Major acyl sugars in young N. benthamiana leaf exudates after BCKD E1-β silencing, separating methanol extracts by LC-MS. Control plants were treated with a TRV2 vector containing no insert. Acyl sugars are defined by their molecular mass (Supplemental Table S1) and their approximate abundance measured by comparison with the internal standard Suc monolaurate. Error bars indicate se (n = 8). Significant differences are as follows: P < 0.01 (molecular weight 594–762), P < 0.05 (molecular weight 804). FW, Fresh weight. C, Composition of FAMES derived from N. benthamiana total acyl sugars separated in B. Error bars indicate se (n = 8; P < 0.01). D, Wax alkane composition of a young N. benthamiana leaf after BCKD E1-β silencing, separated by gas chromatography-mass spectrometry. Error bars indicate se (n = 4). n-C₂₉, Nonacosane; n-C₃₁, hentriacontane; n-C₃₃, tritriacontane; n-C₃₂, dotriacontane; i-C₂₉, 2-methyloctacosane; i-C₃₁, 2-methyltriacontane; i-C₃₃, 2-methyldotriacontane; ai-C₃₀, 3-methylnonacosane; ai-C₃₂, 3-methylhentriacontane; ai-C₃₄, 3-methyltritriacontane. E, Composition of FAMES derived from S. pennellii acyl sugars extracted in methanol from young expanded leaves. Error bars indicate se (n = 12). F, Wax alkane composition in S. pennellii. Error bars indicate se (n = 6). In D to F, asterisks denote significant differences (* P < 0.05, ** P < 0.01) obtained using Student's t test.

Role of fatty acid synthesis in acyl sugar side chain production in S. pennellii trichomes. Acyl-CoAs were measured in wild-type trichomes (A and B), and the effects of cerulenin inhibition (C) and KAS I gene silencing (D–F) were determined. A and B, Acyl-CoA profiles of S. pennellii leaf and stem trichome extracts compared with extracts from total underlying leaf and stem tissues. Acyl-CoA chains below C10:0 were separated with a porous graphite carbon column (A). C10:0 and over were separated with a C18 column (B). Internal standard was 0.2 pmol mg⁻¹ fresh weight (FW) heptadecanoyl-CoA (17:0). tri, Trichome. C, Effect of cerulenin on acyl sugar fatty acid composition; values represent the abundance of FAMES derived from leaf acyl sugars extracted with methanol. The inset shows the sum of acyl sugar-derived FAMES. con, Controls; cer 100, 100 μm cerulenin. Error bars indicate se (n = 10). Asterisks denote significant differences (* P < 0.05, ** P < 0.01) using Student's t test. D, Morphology of a S. pennellii plant in which KAS IA and KAS IB were down-regulated using VIGS, compared with plants treated with a TRV2 control strain or in which the PDS gene was silenced. Bars = 8 cm. E, Composition of acyl sugar-derived FAMES from young expanded leaves in which KAS I genes were down-regulated using VIGS. Control plants were treated with a TRV2 vector containing no insert. Error bars indicate se (n = 8). Significant differences relative to the TRV2 control are as follows: * P < 0.05, ** P < 0.01. F, Acyl-CoA HPLC profiles of trichomes in plants in which KAS IA or KAS IB was down-regulated using VIGS. EXSTD, Mixture of different fatty acid standards; the internal standard used in this experiment was C17:0 at 0.2 pmol mg⁻¹ fresh weight of plant material.