In the present study, the accumulation pattern of CPT in C. acuminata was compared with the expression pattern of Ca-TDC1, Ca-TDC2, and Ca-HGO genes, which are involved in TIA biosynthesis. Both the accumulation of CPT and the expression of Ca-TDC and Ca-HGO genes at the cellular level were investigated in samples collected from plants at different stages of development and subjected to drought-stress, since it is well known that the biosynthesis, transport, and accumulation of plant alkaloids are strongly associated with development and with biotic and abiotic environmental stimuli [6, 24, 30, 31].
The first step of this experiment was to determine whether drought-stress increases CPT production. In a study on the relationship between drought-stress and CPT production in C. acuminata , only plants whose seeds came from certain geographic locations showed increased CPT production in response to drought-stress. In our plants, chemical analyses confirmed that drought-stress induced a significant increase in CPT production. Other studies have shown that CPT production in C. acuminata is also enhanced by other types of adverse growing conditions, such as heavy shade , heat shock , pruning , and nutritional stress [14, 34]. These results support the hypothesis that CPT plays a role in the chemical defence of the plant. Pathogenic and herbivorous attacks can result in the loss of cells, tissues, or entire organs, which are replaced with more difficulty in plants with retarded growth; for this reason, these plants require greater defences than the same species grown under favourable environmental conditions. Although the hypothesis that CPT is involved in chemical defence has not been directly proven , it is supported by indirect evidence, such as the lack of damage caused by insects and pathogens in C. acuminata plantations in the USA . It is also supported by our finding that the number of accumulation sites decreased with plant age, as did the CPT content, which is consistent with the results of other studies [17, 18, 37]. Moreover, the role of other alkaloids in chemical defence has been proven for other plant species [6, 38–41].
The second step of this experiment was to determine whether the quantity of CPT was associated with the accumulation pattern at the cellular level. In all of the samples, fluorescent microscope analyses showed that CPT accumulation occurred in the same cellular sites, in particular, in the GTs (in the leaf and young stem), in some EIs (in the leaf, stem, and root), and in the GICs (in the parenchymatic tissues of the leaf, stem, and root). CPT accumulation was not observed in all of the GTs, which could be explained in two ways: i) only some of the GTs are able to produce and/or accumulate CPT; or ii) all of the GTs are able to produce and/or accumulate CPT, but some of them do it constitutionally, whereas others do so exclusively when induced by specific stimuli. The latter hypothesis is supported by the finding that the percentage of CPT accumulating GTs was much higher in the plantlets subjected to drought-stress, compared to same-age unstressed plantlets.
To identify the sites of the early stages of CPT biosynthesis at the cellular level and determine whether these sites are the same as those of CPT accumulation, the cell-specific localization of Ca-TDC and Ca-HGO expression was investigated. In several species, alkaloid biosynthesis occurs in cells, tissues and organs that are different from those where accumulation takes place. For example, in Solanaceae species, the tropane alkaloids are first synthesised in the root and then transported, through the vascular tissue, to the bud and leaf, which are the main sites of accumulation [24, 30, 42]. One way of investigating the compartmentalisation of alkaloid biosynthesis is to localize the expression of genes involved in their biosynthetic pathway. In C. roseus, RNA in situ hybridization combined with immunocytolocalization techniques has demonstrated that the genes involved in the early stages of vindoline biosynthesis (TDC and STR1) are expressed in the epidermis of the stem, leaf, and flower bud, and in the apical meristem of the root tip, whereas the genes involved in the terminal stages (D4H and DAT) are expressed in the laticifer and idioblast cells of the leaf, stem and flower bud . These results demonstrate that vindoline biosynthesis involves the participation of different cell types and that it requires the intercellular translocation of the pathway intermediates.
Several studies carried out on C. acuminata  and C. roseus [43, 22] have shown that an increase in TIA biosynthesis is accompanied by an increase in TDC activity; thus these enzymes seem to play a leading role in the regulatory control of the TIA biosynthetic pathway. In our study, the hybridization signals obtained with Ca-TDC1 and Ca-TDC2 probes were very intense and circumscribed to single cells or small groups of cells; in the surrounding tissues, no hybridization signals were observed, not even weak signals. Since TDC enzymes are involved in the biosynthesis of not only TIAs but also other metabolites (e.g., proteins and, in some species, IAA), it was surprising that in our study Ca-TDC expression was limited to specific cells. It is possible that these genes are expressed in the majority of cells but that the expression levels are too low to be detected by in situ hybridization, possibly because of the strong dilution factor of the probes used.
In all of the samples, Ca-TDC1 transcripts were detected in the leaf and stem. In these organs, some of the cellular sites with Ca-TDC1 showed a similar localization with respect to CPT accumulation, that is, the epidermal and parenchymatic tissues. No Ca-TDC1 transcripts were observed in the GTs, but interestingly, hybridization signals were sometimes detected in the EIs surrounding them, which are the same cellular sites in which CPT accumulation was sometimes observed. These data suggest that CPT might be biosynthesised in these EIs and then transported to the GTs, which serve as sinks for CPT, even if they are not capable of biosynthesising this alkaloid.
In none of the analysed samples was Ca-TDC1 expression detected in the root, although CPT does accumulate in this organ. Previous results demonstrated that no CPT was produced by roots regenerated in vitro from leaf explants; by contrast, roots originating from micro-cuttings (with axillary buds) accumulated CPT, though at a low concentration . López-Meyer and Nessler  detected Ca-TDC1 expression in all parts of one-year-old C. acuminata plants, including the root, although in this organ the expression level was very low. It is possible that this gene was also expressed in our plants but that the amount of the transcripts was too low and delocalised to be detected by in situ RNA hybridization. Lu et al.  and Lu and McKnight  cloned and characterized, respectively, the α-subunit of anthranilate synthase (ASA) and the β-subunit of tryptophan synthase (TSB) from C. acuminata, enzymes involved in the indole pathway. They demonstrated that both ASA and TSB enzymes were expressed in the root of C. acuminata at very low levels compared to the other parts of the plant. Although the root is a site of CPT accumulation, the above-mentioned results suggest that this organ is not a site of CPT biosynthesis, at least for the early stages of the biosynthetic pathway. This is in contrast with the opinion of other authors [11, 28] who have hypothesized that this alkaloid may be completely synthesized in the root and then transferred to the shoot organs, such as occurs for tropane alkaloids and nicotine [24, 30, 42]. In another CPT-producing plant, Ophyorrhiza pumila, the highest TDC expression was detected in the root, which is the main site of CPT accumulation, and no expression was detected in the leaf, in which CPT accumulation is very low .
The Ca-TDC2 transcripts were observed exclusively in the leaf of plantlets subjected to drought-stress, and these samples Ca-TDC1 transcripts were also detected. López-Meyer and Nessler  did not observe Ca-TDC2 expression in unstressed plantlets at any point in their development; they induced the expression of this gene by eliciting C. acuminata leaf disks with yeast extract and methyl jasmonate, which did not affect Ca-TDC1 expression. Based on these results, the authors hypothesized that Ca-TDC2 is a part of an inducible defence system, whereas Ca-TDC1 is part of a developmentally regulated defence system.
The expression of Ca-TDC2 was detected both in the leaf and stem, in some EIs and ICs, as found for Ca-TDC1, yet the number of these cellular sites per section was higher than those in the sections treated with the Ca-TDC1 probe. In stressed plants, in addition to an increase in CPT, there was an increase in the number of cells with CPT accumulation. This suggests that C. acuminata possesses, in both the leaf and stem, specialised cells whose capacity to biosynthesize and accumulate CPT is activated exclusively in response to stress.
Ca-HGO gene was expressed in the leaf and stem but not in the root. In the stem, Ca-HGO transcript was observed in the same sites as Ca-TDC 1 and 2 expression. In the leaf, Ca-HGO expression was detected in chlorenchyma cells, yet differently from that which was found for Ca-TDC 1 and 2, it extended to the entire mesophyll and was not restricted to specific groups of cells.
The different localization of Ca-HGO and Ca-TDC transcripts reflects a different localization of iridoid and indole biosynthetic pathways, from which derived CPT intermediates (secologanin and tryptamine). The compartmentation of biosynthetic pathways implies that there is a cell-to-cell transport of these intermediates, and that they accumulate in cells where the late stages of CPT biosynthesis occur. Multi-cellular compartmentation has been demonstrated for other alkaloid-producing species , such as Atropa belladonna, Hyoscyamus niger, Papaver somniferum, Thalictrum flavum, and Chatharanthus roseus. In C. roseus, which has been the most widely studied species in terms of indole alkaloid biosynthesis, the early iridoid pathway occurs in adaxial phloem parenchyma cells of aerial organs, whereas the late stage of both the iridoid pathway and indole pathway occurs in epidermal cells .