The aerial epidermis is a major site of quinolizidine alkaloid biosynthesis in narrow-leafed lupin

Abstract

Lupins are promising protein crops that accumulate toxic quinolizidine alkaloids (QAs) in the seeds, complicating their end-use. QAs are synthesized in green organs (leaves, stems, and pods) and a subset of them is transported to the seeds during fruit development. The exact sites of biosynthesis and accumulation remain unknown; however, mesophyll cells have been proposed as sources, and epidermal cells as sinks. We investigated the exact sites of QA biosynthesis and accumulation in biosynthetic organs of narrow-leafed lupin (Lupinus angustifolius) using mass spectrometry-based imaging (MSI), laser-capture microdissection coupled to RNA-Seq, and precursor feeding studies coupled to LC-MS and MSI. We found that the QAs that accumulate in seeds ('core' QAs) were evenly distributed across tissues; however, their esterified versions accumulated primarily in the epidermis. Surprisingly, RNA-Seq revealed strong biosynthetic gene expression in the epidermis, which was confirmed in leaves by quantitative real-time polymerase chain reaction. Finally, feeding studies using a stably labeled precursor showed that the lower leaf epidermis is highly biosynthetic. Our results indicate that the epidermis is a major site of QA biosynthesis in narrow-leafed lupin, challenging the current assumptions. Our work has direct implications for the elucidation of the QA biosynthesis pathway and the long-distance transport network from source to seed.

Keywords: LCM; Lupinus angustifolius; MALDI‐MSI; laser‐capture microdissection; lupin alkaloids; plant specialized metabolites.

Fig. 1

Transverse distribution of quinolizidine alkaloids (QAs) in a leaf of narrow‐leafed lupin (Lupinus angustifolius) as determined by matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI). (a) Bright‐field microscopy image, with the red box denoting the area that was further analyzed by high‐res MALDI‐MSI (bar, 1 mm). ad, leaf adaxial (upper) surface; ab, leaf abaxial (lower) surface; midvein, central midvein. (b–i) Individual MALDI‐MS images of eight QAs at a spatial resolution of 5 μm (bar, 0.4 mm). Each image was obtained by selecting the exact mass of the protonated QA (±5 ppm, Supporting Information Table S1) and normalizing by the total ion current. The color scale represents signal intensity.

Fig. 2

Transverse distribution of quinolizidine alkaloids (QAs) in a stem of narrow‐leafed lupin (Lupinus angustifolius) as determined by matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI). (a) Bright‐field microscopy image, with the red box denoting the area that was further analyzed by high‐res MALDI‐MSI (bar, 1 mm). ep, epidermis; par, parenchyma; phl, phloem; xyl, xylem; pith, inner pith. (b–i) Individual MALDI‐MS images of eight QAs at spatial resolution of 5 μm (bar, 0.4 mm). Each image was obtained by selecting the exact mass of the protonated QA (±5 ppm, Supporting Information Table S1) and normalizing by the total ion current. The color scale represents signal intensity.

Fig. 3

Transverse distribution of quinolizidine alkaloids (QAs) in a developing pod of narrow‐leafed lupin (Lupinus angustifolius), including an enclosed seed, as determined by matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI). The pod was harvested at c. 30 DAA. (a) NLL pod with dotted line illustrating the analyzed transverse plane. (b) Bright‐field microscopy image with red box denoting the area that was further analyzed by high‐res MALDI‐MSI (bar, 1 mm). vs, ventral suture; pod, pod wall; sc, seed coat; em, seed embryo. (c–j) Individual MALDI‐MS images of QAs at 20 μm spatial resolution (bar, 1 mm). Each image was obtained by selecting the exact mass of the protonated QA (± 5 ppm, Supporting Information Table S1) and normalizing by the total ion current. The color scale represents signal intensity.

Fig. 4

Expression of quinolizidine alkaloid (QA) biosynthetic genes lysine decarboxylase (LDC) and copper amine oxidase (CAO) in organs of narrow‐leafed lupin (Lupinus angustifolius) as determined by laser‐capture microdissection coupled to RNA‐Seq. (a–c) Heat maps of average expression of LDC (Luan_Oskar_PB12_103708) in leaf (a), stem (b), and developing pod (c) as shown on the Lupin eFP Browser. The scale on the left represents TMM‐normalized transcripts per million (TPM) values. The corresponding heat maps for CAO (Luan_Oskar_PB23_106386) are similar and are not depicted here. (d–f) Graphical representation of LDC and CAO expression in leaf (d), stem (e), and developing pod (f). Box plots represent the mean (line within box) as well as minimum and maximum values (whiskers) of two biological replicates (stem epidermis has one replicate). Asterisks indicate a significant difference as determined by differential gene expression analysis (DESeq2; Wald test): *, P _adj < 0.05; **, P _adj < 0.01.

Fig. 5

Visualization of chloroplasts in epidermal cells of narrow‐leafed lupin (Lupinus angustifolius). In each panel, Chl autofluorescence (red) is overlaid on the respective bright‐field microscopy image (grey). Black bars represent a distance of 40 μm. (a) Leaf abaxial epidermis. (b) Leaf adaxial epidermis. (c) Stem epidermis. (d) Developing pod.

Fig. 6

Accumulation of isotopically labeled compounds in tissue fractions of narrow‐leafed lupin (Lupinus angustifolius) leaves upon feeding with labeled L‐lysine. Whole leaves were fed via the cut petiole, and leaves were dissected at four time points after the start of the feeding period (6, 10, 18, and 24 h). The respective tissue fractions (abaxial epidermis, mesophyll, and leaf without abaxial epidermis) were analyzed by LC‐MS. Data points and error bars represent the mean and SD of 3 biological replicates. (a) Accumulation of the fed, labeled l‐lysine in the tissue fractions across time points. Single asterisks represent a significant difference between abaxial epidermis and the leaf without abaxial epidermis, and double asterisks represent significant differences between abaxial epidermis and both the leaf without abaxial epidermis and the mesophyll (P _adj < 0.05 on Tukey's test). ns, not significant. (b) Accumulation of the QA lupanine in the tissue fractions across time points. Significant differences are represented as described for (a). (c) Accumulation of ammodendrine (likely side product of the early QA pathway) in the tissue fractions across time points. Significant differences are represented as described for a, with the exception of the 10 h time point, for which a non‐parametric statistical test was used (P _adj < 0.05 on Dunn's test).

Fig. 7

Distribution of isotopically labeled lupanine in transverse sections of narrow‐leafed lupin (Lupinus angustifolius) leaves at 6 h (left column) and 10 h (right column) after continuous feeding with isotopically labeled l‐lysine. Tissue sections were analyzed by high‐res matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI) at 5 μm spatial resolution. Compounds were visualized by selecting the respective m/z ratios of their protonated forms (±5 ppm, Supporting Information Table S1) and normalizing by the total ion current. (a, b) Bright‐field microscopy images with red boxes representing the areas that were further analyzed (bar, 1 mm). (c, d) MALDI‐MSI visualization of labeled l‐lysine. The color scale (right) represents signal intensity (bar, 0.4 mm). (e, f) MALDI‐MSI visualization of unlabeled lupanine (same color scale; bar, 0.4 mm). (g, h) MALDI‐MSI visualization of labeled lupanine (same color scale; bar, 0.4 mm). (i, j) Merged and recolored MALDI‐MS images of unlabeled lupanine (blue) and labeled lupanine (green) (bar, 0.4 mm).

Fig. 8

Spatial model for quinolizidine alkaloid (QA) biosynthesis and transport in narrow‐leafed lupin (NLL, Lupinus angustifolius). The aerial epidermis is a major site of QA biosynthesis, at least until the formation of lupanine (1) (pathway sub‐cellular localization is yet to be defined). The esterified QAs accumulate in the epidermis, likely within the vacuole (2) (Mende & Wink, 1987). The core QAs (e.g. lupanine) must move from the epidermis to the phloem‐associated cells for long‐distance transport to the seeds. It is yet to be shown whether this is via a symplastic or apoplastic route, or both (3a). The apoplastic phloem loading mechanism depicted (3b) is generally found in Fabaceae species (although not yet confirmed for NLL) (Bourquin et al., ; Wimmers & Turgeon, 1991). After long‐distance transport through the phloem, the QAs likely enter the seed coat cells through symplastic connections with the vasculature (4) (van Dongen et al., ; Tegeder, 2014). Finally, an apoplastic transport step is required for the QAs to accumulate in embryo cells (5) (Tegeder, 2014). Dotted black arrows represent symplastic transport, solid black arrows represent apoplastic transport, blue arrows represent transport into the vacuole, and brown arrows represent QA biosynthesis steps. This figure was created in BioRender (BioRender.com/a33n741).