Acetogenins and alkaloids during the initial development of Annona muricata L. (Annonaceae)

1 Introduction

Seed germination and seedling formation are marked by the metabolic activation that triggers the set of gradual and progressive changes in size, structure, and function. The plants invest the resources, endowed by the mother plant, in the form of reserve material, to accomplish the mechanisms that allow them to develop and establish themselves in the ecosystem [1], [2], [3]. Among this mechanisms are the division, expansion, and cellular differentiation that result from the differential expression of the genetic material. When the plant becomes an autotrophic organism, the metabolic costs are supported by photosynthesis [1].

During this initial process, plants are exposed to multiple biotic and abiotic factors that lead to death, making it a critical phase of their life. At this stage, so vulnerable to predation and environmental changes, plants activate mechanisms for their defense and survival [4], which may be physical (such as the development of pubescences or thorns) or chemical (such as the production of specialized metabolites). These are dependent on the increase of the biomass, on the reserve material stored in the seeds and, when the plant grows, on the photosynthesis.

In contrast to the morphological, physiological, and genetic aspects of germination and seedling development, knowledge about metabolites specialized in defense or protection is less documented. However, its biosynthesis is one of the most important strategies for its survival, and it has been analyzed especially in studies on herbivory [5]. Research on metabolites specialized in early development has reported around 200 molecules, including alkaloids, cyanogenic glycosides, anthocyanins, flavonoids, and simple phenols [6].

The presence and organ-specific distribution of specialized metabolites during these stages is variable among species. This distribution can be part of different strategies such as the defense of roots against insects, microorganisms, and parasites of the soil [7], or of leaves and stems against herbivores and pathogens, or as protection against UV radiation [8], among others.

Among plants, the Annonaceae family is characterized by having a specialized active metabolism, with privileged biosynthetic routes for the production of alkaloids and “annonaceous acetogenin” molecules. Both are toxic substances and have diverse biological activities [9], [10], [11], [12], [13], [14]. A few species have reported the presence of more than 934 benzylisoquinoline alkaloids [15], [16], [17] and more than 540 acetogenins [18]. These metabolites have been isolated from different organs and tissues of adult plants, and as far as it has been possible to deduce, the two metabolic pathways are expressed during all the stages of life, but the diversity of its metabolites is possible to find in different organs and moments. As for the alkaloids, organ-specific variation during early development has only been described in two studies, referring to the oxoaporfin alkaloid liriodenine. De-la-Cruz and González Esquinca reported [19] that the variation of liriodenin in Annona diversifolia is associated with the degree of development and differentiation of tissues, so this alkaloid was first detected in the endosperm of the seeds at 5 and 10 days of imbibition, later in the radicle and the hypocotyl, reaching its maximum concentration in six-leaved seedlings (2827.1 nmol g⁻¹ dry matter), but was not found in cotyledons and leaves. These data allow us to infer and prove that the alkaloid forms part of the antifungal defense system on Rhizopus stolonifer (Ehrenb: Fr.) and Aspergillus glaucus L. fungi isolated from the same seeds and which disappear just as the radicle emerges [20].

From Annona muricata L., a popular species in traditional medicine and widely studied, have been isolated 99 acetogeninas [21] and 10 alkaloids of fruits, leaves, roots, and seeds [15], [16], [22], [23], [24]. With the exception of the acetogenins rolliniastatin-2 and laherradurin [25], the moment in which the biosynthesis of these compounds is initiated and its location or variation in the plant is unknown. Understanding the presence and distribution of alkaloids and acetogenins during the early stages of the plant development contributes to the understanding of the dynamics of specialized metabolism, as well as it provides some elements to guide research on the defense mechanisms in which these specialized metabolites participate. Therefore, this study had as an objective to determine the distribution pattern and its dependence to stages of early development of four acetogenins and five benzylisoquinolinic alkaloids, constitutive compounds of A. muricata.

2 Materials and methods

2.1 Stages of growth evaluated

Two phenological stages were analyzed: root emergence (phases 1, 2, and 5 cm of radicle) and the formation of seedlings (leaf primordium phases (Fp), two leaves and six leaves). A total of six development phases were analyzed, which, in order to establish analogies with other phenological works, were quadrated to stages 0 and 1 according to the BBCH scale [26] (Table 1).

Table 1:

Morphophysiological characteristics of germination and seedling development of A. muricata.

Stages	Escale BBCH	Growth phases (days)	Morphophysiological events	Keys
Germination	05	1 cm of radicle (12–14)	Mobilization of substances of endosperm reserves, elongation of radicle	Rd1
	06	2 cm of radicle (15–17)	Mobilization of substances of endosperm reserves, elongation of radicle	Rd2
	07	5 cm of radicle (20–22)	Differentiation of the embryonic axis in radicle and hypocotyl. Mobilization of substances from the endosperm reserves	Rd5
Seedling	11	With foliar primordium (25–28)	Partially open green cotyledons. First pair of true leaves and separated from the apex of the stem. Branched root. Photosynthesis	Fp
	12	With two leaves (30–35)	First pair of leaves in their final size. Independence of the seedling. Photosynthesis	2 Lf
	16	With six leaves (60–64)	Six dark green leaves, developed root system. Vegetative growth. Photosynthesis	6 Lf

1. Stages and phases growth were adjusted to BBCH scale (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie).

In order to obtain the plants in different stages of their development, the germination of 1000 seeds was induced using the technique of “between paper” [27], in rolls of brown paper with 50 seeds, which were placed in plastic containers of 1 L with 150 mL of water. The seeds were washed every third day to prevent the appearance of fungi, and changes of paper and water were also made. Then, 200 seeds were harvested in the root emergence stage, while another 500 seeds were kept in paper rolls until reaching the Fp stage. After reaching this stage, the seeds were sowed in 500-mL extruded polystyrene foam cups, with previously sterilized soil, in an autoclave FE-397 (Felisa, Jalisco, México) until reaching the two- (2 Lf) and six-leaf (6 Lf) phases. The whole experiment was maintained inside a Conviron^® series CMP4030 germination chamber (Conviron, Winnipeg, Canada) with a controlled environment at 28 °C, with a 12-h photoperiod, fluorescence lamps at an intensity of 500 μM m⁻² s⁻¹, and a relative humidity between 65% and 75%. From each stage of development, radicles, roots, stems, and leaves were carefully separated with pruning shears. Each organ was placed in brown paper bags and made to dry at room temperature for 4 days.

3 Results

During the two stages of growth, it was possible to detect the presence of four acetogenins and five alkaloids with variable organ distribution (Figure 1).

Figure 1:

Acetogenins and alkaloids detected in the developmental stages of A. muricata seedlings. Their presence in roots (R), stems (S), and leaves (L) are indicated.

3.1 Acetogenins and alkaloids in roots

In the roots, two acetogenins were detected from the early stages of growth: cis-uvarimicin IV (p=9.104^e−05, χ²=25.955) when the radicle just emerges (Rd1), and mosinone (p=3.918^e−05, χ²=27.839), 25 days later, when the Fp has been reached. The most abundant acetogenin in the development phase of Fp is mosinone, while cis-uvarimicin IV is in the 2 Lf. In this tissue, both molecules persist during the 2 Lf to 6 Lf (Figure 2A).

Figure 2:

Ontogenetic variation and organ-specific distribution of acetogenins and alkaloids during the early development of A. muricata. Roots (A, B); stems (C, D); leaves (E, F). The values represent the average of three replicates, and standard deviations are indicated. The difference between means was considered significant at 95% confidence interval.

Five alkaloids were found (Table 3), namely, anonaine, nornuciferine, and reticuline from early stages, at the beginning of germination, when the radicle reaches 1 cm in length (Rd1). On the other hand, the asimilobine (p=4.192^e−05, χ²=27.686) alkaloid appears when the radicle measures 5 cm (Rd5); and coreximine (3.65^e−05, χ²=27.99) at 30 days, when the seedling has reached the Fp. In the roots these compounds increase in relation to the development of the plant, being more abundant in the 2 Lf seedling, with the exception of asimilobine and anonaine (p=0.000112, χ²=25.507), which remains constant during leaf formation (Figure 2B).

3.4 Accumulation dynamics

Acetogenins and alkaloids in A. muricata occur in different organs and at different times (accumulation dynamics). The richness (number) and abundance of alkaloids increase as the plant develops, with the highest concentration being observed during the 6 Lf. In contrast, there is no similar pattern in acetogenins: while their abundance is the same in the 6 Lf, each molecule has a unique dynamic (Figure 3).

Figure 3:

Comparison of relative abundances (accumulation dynamics) of acetogenins and alkaloids during six phases of initial development of A. muricata. The relative abundances were calculated: ([amount detected at×time/highest amount detected in a particular tissue]×100).

4 Discussion

Different morphophysiological events occur during the initial phases of A. muricata development (Table 1). After imbibition and during the metabolism’s activation, the reservoirs are mobilized to start elongation and embryo differentiation, the aperture of the testa, and the emergence of the radicle. At the same time, the specialized metabolism (secondary metabolism) is expressed, a fact which is documented in 99 species of different families [6]. Important metabolic investments are also destined within the Annonaceae, especially for alkaloid and acetogenin biosynthesis, clearly established within this family, as is documented in multiple systematized reports by Cavé et al. [34] and Lucio et al. [17], among others.

The ontogenic relation between the specialized metabolism and the development of the plant in Annonaceae has been documented in A. diversifolia Saff. (synonym of Annona macroprophyllata Donn. Sm), by De la Cruz and González-Esquinca [19], alongside the biosynthesis of liriodenine during the seed’s imbibition. Also, in a paper by Brechú-Franco et al. [35], it was demonstrated that idioblasts (specialized cells that contain alkaloids and acetogenins) are formed during the development of the radicle and the hypocotyl.

In the present study, it was found that the biosynthesis of both alkaloids and acetogenins in A. muricata occurs simultaneously during the initial stages of development. Both types of molecules appear in variable proportions, developmental stages, and organs, signaling their crucial role in the plant development. The documented acetogenins had been found before in the stems, roots, leaves, and seeds of adult specimens [18], [32], [34], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], but it was not until now that their presence was detected in the early stages of development.

Four monotetrahydrofuran acetogenins were detected in the early stages. These include cis-uvarimicin IV, mosinone, muricina B, and cis-annonacin-10-one; all of them related with organ differentiation. cis-uvarimicin IV and mosinone appear in the roots when growth still depends on reserve material, with only one report of its presence in the roots of an adult specimen [49], while muricin B and cis-annonacin-10-one are only present when the plant is photosynthetically active. Both acetogenins can be found also in the leaves and seeds of adult plants [50], a trait that could indicate their dependence on photosynthesis. The presence of these acetogenins in the plant’s photosynthetic phases, indicate a kind of survival function that occurs in the vulnerable stages of development. Also, it was noted that the acetogenins laherradurin and rolliniastatin-2 were present in A. muricata seedlings [25], where it serves a larvicide function. González-Coloma et al. [51] reported that laherradurin is more active than rolliniastatin-2 on Spodoptera littoralis Boisduval, a folivore, with an 86% and 29.7% mortality rate, respectively. The biosynthesis of Annonaceous acetogenins is not known, but with some methods, such as the isolation of precursors, as well as the presence of numerous asymmetric centers and biomimetic hemisyntheses, it is thought that acetogenins originated from a precursor of three carbon atoms and a fatty acid [34]. The latter is very abundant in the seeds of the species of the Annonaceae family.

The insecticidal activity of cis-annonacin-10-one, in A. muricata has only been demonstrated on Oncopeltus fasciatus Dallas and on Spodoptera frugiperda Smith and Abbo [52], [53] or fungicidal properties, as shown by scuamocin, scuamocin G, and scuamostatin, on Phytophthora infestans Mont [54]. These acetogenins have not been found in A. muricata before or in the present analysis; nonetheless, the presence of these molecules must suppose an evolutionary advantage, which enables the plants to adapt to their environment.

The following alkaloids were detected in the early developmental stages of A. muricata: reticuline, coreximine, anonaine, asimilobine, and nornuciferine (Figure 1). These have also been found in the organs of other adult plants. These alkaloids, in a similar manner to the acetogenins, show a spatial-temporary and organ-specific distribution.

Reticuline serves as an intermediary alkaloid within the diversification in the biosynthetic pathway that produces six benzylisoquinoline alkaloid synthesis [55]. It has been found in the roots, stems, and leaves of adult A. muricata specimens [15], [16], [22], [23], [24], although their presence in their seeds are undocumented. In the present study, reticuline was detected in radicle emergence, specifically, in roots of over 1 cm in length (Rd1), which signals an early biosynthesis that depends on the endosperm’s reserved material. However, reticuline is also present all over the seedling, during its early phase, and its biosynthesis depends on photosynthetic activity. In both cases, a considerable energy cost is produced, which is to be expected from an ad hoc biosynthetic machinery, whose presence repeats frequently in these and other specialized metabolites.

Anonaine, an aporphynic alkaloid, is the most found alkaloid in plants of the Annonaceae family after liriodenine [17]. There is no evidence of its presence in A. muricata seeds. Thus, anonaine, and its biosynthesis, occurs in roots during the early development stage. Furthermore, it is possible to detect anonaine on leaves when these start to develop, in the same way as liriodenine, produced during imbibition and emergence of the radical, while both are located in the seedlings, leaves, roots, hypocotyls, and stems of A. diversifolia [19]. The fact that anonaine can be found only in the stems of the six-leaved seedlings allow us to infer the lack of transport from the roots, as well as the biosynthetic machinery that might be present in the leaves. Anonaine has important fungicidal properties against dermatophyte fungus and bacteria [56].

Asimilobine, another aporphine, and the third most reported alkaloid in the specimens from the Annonaceae family [17], is found in roots (that have reached 5 cm in length), also in leaves, when the seedling has two and six leaves, and has been reported in fruits also [22], [57].

Additionally, the presence of nornuciferine in the fruits [22], [57], alongside its early detection, enables us to infer that it could be (at least initially) a contribution from the mother plant, in contrast with the other alkaloids.

Finally, coreximine, a protoberberine alkaloid, appears to be dependent on the photosynthetic activity of the plant, as it is only found in the leaves’ development stages [23], [58]. The same result is also found in adult A. muricata specimens. For the biosynthesis of this type of alkaloids, a major number of enzymes seems to be involved than in the biosynthesis of aporphinics [24].

Pathway diversification in the biosynthesis of specialized metabolites and its distribution along the seedling enable us to infer that the plant invests more resources in the protection of life-dependent organs, such as the leaves due to their photosynthetic function, or the roots with their important role in water and nutriment transportation [5]. Therefore, roots and leaves would have a bigger production and accumulation of alkaloids and acetogenins. Implicated within the mechanisms of biosynthetic regulation, it is possible to find genes, enzymes, cells, tissues, and organelles, all of which depend on a process linked to organelle and tissue differentiation. Accordingly, the seedling posits resources from distinct reservoirs for its development and simultaneously for specialized metabolite biosynthesis. Acetogenin and alkaloid variations throughout development is characteristic of the specialized metabolism’s dynamics, modulated by gene expression, which, in turn, is different for every molecule (Figures 7 and 8). As it has been demonstrated in seedlings of Arabidopsis thaliana (L.) Heynh [6], [59], [60], [61], [62]. This species produces anthocyanins after 3–5 days of sprouting; likewise, in Catharanthus roseus (L.) G. Don, catharanthine, tabersonine, cindoline, and vincristine alkaloids accumulate after 7–10 days of sprouting [6], [63], [64], [65].

The neurotoxic activity found in four of the five detected alkaloids, as well the cytotoxic and pesticide activity of the acetogenins [51], enables us to suppose that these compounds are produced and maintained all along the complete development of the plant, as part of the defense systems against distinct pathogen species. Paired with the aforementioned process, at least in the case of alkaloids, there is, during early development, a diversification of the biosynthetic pathway beginning with reticuline [66], its main intermediary, which leads to protoberberine or aporfinoid biosynthesis. Although, to date, the biosynthetic pathway of alkaloids of species of the Annonaceae family is not known, there is no in vivo interconversion between them.

The increment of some alkaloids is related with some phytoregulators, for example, the exogenous addition of gibberellic acid (GA₃) specifically in morphine’s yield [67]. Likewise, IAA’s metabolism is tightly related with the specialized metabolism in some species, as is the case of plants in the order Capparales, which include Arabidopsis, where indolic glucosinolates and other indolic defense compounds (camalexine) are formed, starting from the biosynthetic pathway of indol acetic acid [68]. This suggests a relation between gibberellins’ and auxines’ expression, formed during division, elongation, and differentiation of cells alongside [67] the development of roots and leaves and the expression of the specialized metabolism.

5 Conclusion

During its first steps’ stages of development, A. muricata expresses the biosynthesis of diverse acetogenins and alkaloids. This fact is related among the multiple biosynthetic processes that are linked alongside the formation of tissues and organs. To be sure, the importance of the expression for the plant is manifested the biosynthesis capable of initiating and being preserved from the sprouting until (at least) the seedling stage. In some cases, this synthesis depends on the apparition of leaves or can even be organ specific. Even though the understanding of these processes still appears partial and fragmentary (being related mostly within defense systems), the type of studies, such as the present, can be directed toward the research of the importance the processes have for the plants that produce them.