Cell-specific expression of tryptophan decarboxylase and 10-hydroxygeraniol oxidoreductase, key genes involved in camptothecin biosynthesis in Camptotheca acuminata Decne (Nyssaceae)

Abstract

Background: Camptotheca acuminata is a major natural source of the terpenoid indole alkaloid camptothecin (CPT). At present, little is known about the cellular distribution of the biosynthesis of CPT, which would be useful knowledge for developing new strategies and technologies for improving alkaloid production.

Results: The pattern of CPT accumulation was compared with the expression pattern of some genes involved in CPT biosynthesis in C. acuminata [i.e., Ca-TDC1 and Ca-TDC2 (encoding for tryptophan decarboxylase) and Ca-HGO (encoding for 10-hydroxygeraniol oxidoreductase)]. Both CPT accumulation and gene expression were investigated in plants at different degrees of development and in plantlets subjected to drought-stress. In all organs, CPT accumulation was detected in epidermal idioblasts, in some glandular trichomes, and in groups of idioblast cells localized in parenchyma tissues. Drought-stress caused an increase in CPT accumulation and in the number of glandular trichomes containing CPT, whereas no increase in epidermal or parenchymatous idioblasts was observed. In the leaf, Ca-TDC1 expression was detected in some epidermal cells and in groups of mesophyll cells but not in glandular trichomes; in the stem, it was observed in parenchyma cells of the vascular tissue; in the root, no expression was detected. Ca-TDC2 expression was observed exclusively in leaves of plantlets subjected to drought-stress, in the same sites described for Ca-TDC1. In the leaf, Ca-HGO was detected in all chlorenchyma cells; in the stem, it was observed in the same sites described for Ca-TDC1; in the root, no expression was detected.

Conclusions: The finding that the sites of CPT accumulation are not consistently the same as those in which the studied genes are expressed demonstrates an organ-to-organ and cell-to-cell translocation of CPT or its precursors.

Figure 1

Biosynthesis of camptothecin. Tryptophan decarboxylase (TDC); geraniol 10-hydroxylase (G10H); NADPH:cytochrome P450 reductase (CPR); secologanine synthase (SLS); strictosidine synthase (STR). Double arrows indicate the involvement of multiple enzymatic steps.

Figure 2

CPT concentration in the shoot apex and in the first 4 leaves of plantlets and mature plants of Camptotheca acuminata, and the effect of drought-stress (D-S) on CPT production. Each value represents the means of three independent determinations; the vertical lines and different letters above the bars indicate standard errors (SE) and statistically significant differences (P ≤ 0.05) between concentrations.

Figure 3

Optical micrographs of fresh leaf cross sections of Camptotheca acuminata 3-month-old plantlets observed under UV-light. (A) Epidermal cells accumulating CPT (arrows) localized on the abaxial side of the leaf midrib; (B) accumulation of CPT in a glandular trichome (white arrow) and in some epidermal cells (yellow arrows) surrounding it; (C) group of segregator idioblasts (white arrow) containing CPT crystals localized in the leaf mesophyll, at midrib level; in the section, glandular trichomes (light-blue arrows) and non-glandular trichomes (yellow arrows) can be observed; (D) CPT accumulation in a group of segregator idioblasts localized in the spongy parenchyma adjacent to the abaxial epidermis. Abaxial Epidermis (AbE); Adaxial Epidermis (AdE); Palisade Parenchyma (PP); Spongy Parenchyma (SP). (A) bar = 5 μm; (B, C) bar = 10 μm; (D) bar = 50 μm.

Figure 4

Ca-TDC1 expression in the leaf and stem of 3-month-old Camptotheca acuminata plantlets. Fresh cross sections of the leaf (A) and primary stem (D) stained with 0.1% toluidine blue to show the anatomical structure. Paraffin-embedded cross sections of leaves (B and C) and primary body of the stem (E and F), treated with Ca-TDC 1 antisense (B and E) and sense (C and F) digoxigenin-labelled probes. The square in the bottom right corner of D indicates a vascular bundle, that is, the part of the stem section shown in E and F. The hybridization signals in the leaf treated with antisense probe (B) are present in some spongy parenchyma cells (arrow), in parenchymatic subepidermal cells (double arrows), and in two epidermal cells (arrowheads). In the stem treated with antisense probe (E), hybridization signals are present in parenchyma cells associated with vascular bundles (arrow). No hybridization signals are present in the sections of leaf (C) and stem (F) treated with sense probe. Abaxial Epidermis (AbE); Adaxial Epidermis (AdE); Palisade Parenchyma (PP); Spongy Parenchyma (SP). (A, B, C, E, F) bar = 50 μm; (D) bar = 100 μm.

Figure 5

Ca-TDC2 expression in the leaf Camptotheca acuminata plantlets subjected to drought-stress. Paraffin-embedded cross sections of leaves treated with Ca-TDC 2 antisense (A) and sense (B) digoxigenin-labelled probes. The hybridization signals are present in some palisade parenchyma cells (arrows). Abaxial Epidermis (AbE); Adaxial Epidermis (AdE); Palisade Parenchyma (PP); Spongy Parenchyma (SP). (A, B) bar = 50 μm; (D) bar = 100 μm.

Figure 6

Ca-HGO expression in the leaf and stem of 3-month-old Camptotheca acuminata plantlets. Paraffin-embedded cross sections of leaves (A and B) and primary body of the stem (C and D), treated with Ca-HGO antisense (A and C) and sense (B and D) digoxigenin-labelled probes. The hybridization signals in the leaf treated with antisense probe (A) are present in all mesophyll cells. In the stem treated with antisense probe (C), hybridization signals are present in parenchyma cells associated with vascular bundles (black arrows). No hybridization signals are present in the sections of leaf (B) and stem (D) treated with sense probe. Abaxial Epidermis (AbE); Adaxial Epidermis (AdE); Palisade Parenchyma (PP); Spongy Parenchyma (SP). Bar = 50 μm.

Figure 7

Diagram displaying the expression of Ca-TDC1, Ca-TDC2, and Ca-HGO genes, and CPT accumulation in different cells, tissues, and organs. Root (A), stem (B), and leaf (C). Epidermal Idioblast (EI); Glandular Trichomes (GT); Group of Idioblast Cells (GIC).