Temperature and GA3 as Modulating Factors in the Biosynthesis of Alkaloids during Imbibition and Early Development of Annona x atemoya Mabb. cv. ‘Gefner’ Seedlings
by
, , , , and
Gustavo Cabral da Silva
1
,
Ivan de-la-Cruz-Chacón
2,
Ana Beatriz Marques Honório
1,
Bruna Cavinatti Martin
1,
Marília Caixeta Sousa
1,
Felipe Girotto Campos
1,
Carmen Sílvia Fernandes Boaro
1 and
Gisela Ferreira
1,* 1
Department of Biodiversity and Biostatistics, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, SP, Brazil
2
Laboratorio de Fisiología y Química Vegetal, Instituto de Ciencias Biológicas, Universidad de Ciencias y Artes de Chiapas (UNICACH), Tuxtla Gutiérrez 29039, CHIS, Mexico
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(9), 766; https://doi.org/10.3390/horticulturae8090766
Submission received: 30 July 2022
/
Revised: 20 August 2022
/
Accepted: 22 August 2022
/
Published: 26 August 2022
(This article belongs to the Collection Seed Dormancy and Germination of Horticultural Plants)
Abstract
:Alkaloids are products of the specialized metabolism of plants and temperature is a factor capable of modulating their biosynthesis. Species of the Annonaceae family biosynthesize alkaloids and present dormancy in their seeds, which can be overcome with the use of gibberellins. Therefore, the aim of this work was to evaluate whether temperature variations and the use of gibberellin in seeds affect the production of alkaloids during germination and early development of Annona x atemoya Mabb. cv. ‘Gefner’ seedlings. Results showed that the temperature of 30 °C associated with imbibition in water caused an increase in the production of total alkaloids and liriodenine and that the use of gibberellin decreased production. In addition, it was possible to identify the presence of nine other alkaloids with organ-specific distribution. The presence of none of them was induced by the effect of temperature or gibberellic acid. Therefore, it could be concluded that temperature variation and the use of GA3 alter the biosynthesis of alkaloids, with high temperature causing increased concentration, but the use of GA3 reducing production.
1. Introduction
Alkaloids are specialized metabolites containing nitrogen. The structural diversity of alkaloids is as wide as the range of their biological activities, such as antimicrobial, cytotoxic, antitumor, antiprotozoal, and antiviral actions [1,2,3,4,5,6]. Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites. These alkaloids are typically isolated from plants of the order of Ranunculales, including Papaveraceae, Ranunculaceae, Berberidaceae, Menispermaceae, Magnoliaceae, and Annonaceae families [3].
The Annonaceae family has a great diversity of alkaloids, with reports of about 934 alkaloids, the most abundant being BIA-type alkaloids [7]. BIAs frequently found in annonaceous species are anonaine, asimilobine, isoboldine, isocoridine, liriodenine, stephalagine, nuciferine, atherospermidine, reticuline, laurotenine, lanuginosine, discretine, and xylopine [7,8,9,10,11]. Liriodenine alkaloid is perhaps the specialized metabolite most widely distributed in the Annonaceae, and this oxoaporphine is found in at least 86 genera and 240 Annonaceae species [3]. It is a molecule with biological activities, such as antitumor, antibacterial, antifungal, and antimalarial [3,7,12]. This BIA has been suggested as a defense molecule against phytopathogens in seedlings of the genus Annona [12,13].
The Atemoya (Annona x atemoya Mabb.) is an interspecific annonaceous hybrid between Annona cherimola Mill. and Annona squamosa L. that stands out due to the production and commercialization of fruits. Its phytochemical investigation led to seven benzylisoquinoline alkaloids, including two aporphine (anonaine and asimilobine), three oxoaporphine (lanuginosine, liriodenine and lysicamine) and two proaporphine (pronuciferine and stepharine) [11,14].
Previous phytochemical studies with Annona macroprophyllata (≡Annona diversifolia), Annona cacans, and Annona muricata have shown that BIAs are present in different plant organs during the early seedling development [15,16,17,18], supporting the idea of a specific organ location and dependence on plant development [18]. Some of these alkaloids showed antimicrobial activity during early development [13]. The production of alkaloids with antimicrobial activities from the beginning of the germination process is a defense mechanism of the plant against biotic stress, being a strategy to guarantee its establishment in the environment and perpetuation of the species [15,19].
The alkaloids, biosynthesized from the specialized metabolism, represent a chemical interface between plants and the surrounding environment. Thus, their biosynthesis is often affected by environmental conditions, such as temperature, which can influence the composition and concentration of molecules [20,21,22,23,24]. However, the temperature affects not only the response of the specialized metabolism of plants, but also affects water absorption and the chemical reactions that regulate the metabolism involved in the germination process [25]. In addition, hormones such as gibberellins, which act in the development of the plant and in overcoming seed dormancy, can also affect the production of alkaloids [16].
Overcome dormancy occurs by altering the relationship between endogenous abscisic acid (ABA) and gibberellin (GA). The increase in GA (germination promoter) reduces the ABA (germination inhibitor) content, which results in increased degradation of the reserves, resulting in complete germination with primary root protrusion [26]. Several authors have reported the use of gibberellins (GAs) to overcome the dormancy of Annona seeds [26,27,28,29], as well as temperature variations altering the germination process [30,31,32]. However, the effect of temperature variation associated with the use of exogenous gibberellins in the production of alkaloids during the germination process and in the initial development of seedlings is a topic that deserves to be explored.
The evidence pointed out leads us to the objective of investigating whether temperature and the use of GA3 modulate the synthesis of benzylisoquinoline alkaloids in Annona x atemoya Mabb. cv. ‘Gefner’ from seed imbibition to early seedling development.
2. Materials and Methods
2.1. Plant Material
Annona x atemoya Mabb. cv. ‘Gefner’ seeds were collected (May 2019) from ripe fruits in a commercial orchard in the municipality of Itapetininga, São Paulo, Brazil, and transported to the Plant Biology Sector, Biostatistics, Plant Biology, Parasitology and Zoology Department, São Paulo State University (UNESP), Botucatu, freezer at (4–5 °C) for 30 days, when the experiment was started.
2.2. Experimental Design and Experiment Implementation
The experimental design was completely randomized, in a 3 × 2 factorial scheme (temperatures × water and GA3) with 4 replicates of 25 seeds per treatment. Treatments consisted of seeds imbibed in distilled water and gibberellic acid (GA3) (ProGibb® 400, North Chicago, IL, USA) at a concentration of 500 mg L−1, for 36 h [27] associated with different temperatures (20 °C, 30 °C, and 20–30 °C (alternating, 8 and 16 h, respectively) with 16 h light and 8 h dark photoperiod).
From a total of 4720 atemoya seeds, 100 were used (4 replicates of 25 seeds) before the imbibition (dry seeds), to do the alkaloid extraction (seed coat and endosperm). After the imbibed process for 36 h under different treatment conditions, the seeds (4620) were divided into 3 groups. In the first group (100 seeds/treatment = 600 seeds), seed coat and endosperm were submitted to the alkaloid extraction method. In the second group, (570 seeds/treatment = 3420 seeds) the seeds were kept in germinators under controlled conditions (previously described) until the formation of seedlings with 5 centimeters of roots, separated from the formed structures (seed coat, endosperm, root, hypocotyl and cotyledons), which were subjected to the alkaloid extraction method. Seeds in the third group (100 seeds/treatment = 600 seeds) were submitted to the germination test.
2.3. Germination Test
For the germination test, 4 replicates of 25 seeds per treatment were used, with seeds imbibed in distilled water and GA3 at different temperatures (20 °C, 30 °C, and 20–30 °C (alternating, 8 and 16 h, respectively)). After 36 h of imbibition, the seeds were placed in germination paper rolls (Germitest = germination test) moistened with 2.5 times the paper mass and kept in germinators under the conditions established in the treatments. The following variables were evaluated: germination percentage, speed, and average germination time.
2.4. Extraction and Quantification of Total Alkaloids and Liriodenine
Seed and seedling structures obtained by the different imbibition and germination conditions of each treatment were dried, ground, and weighed equally in four replicates per structure of each treatment. Samples were carbonated with saturated anhydrous sodium carbonate solution (Na2CO3) and allowed to dry completely in oven at 30 °C. Alkaloids were extracted with chloroform (CHCl3) under stirring for 2 h and then filtered. The chloroformic phase was extracted with 1 M hydrochloric acid solution (HCl) and then alkalized with Na2CO3 until reaching pH 9.5. Once again, it was re-extracted with CHCl3 and allowed to evaporate at room temperature in the dark until further analyses [15]. After complete CHCl3 evaporation, samples were resuspended with the same solvent and evaluated in spectrophotometer with ultraviolet light source and the total alkaloid content was determined by spectrophotometry at 254 nm using liriodenine as standard in the preparation of the standard curve (y = 0.0881x − 0.0112, R2 = 0.9949) [15].
After determining the total alkaloid content, the profile was evaluated and the quantification of liriodenine for each treatment was performed using ultra high performance liquid chromatography (UHPLC - Thermo Fisher-Scientific®, Waltham, MA, USA) and Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software (Catalog number: CHROMELEON7, Waltham, MA, USA), with gradient pump and UV-Vis detector using C18 reverse phase column (150 × 4.6 mm and 5 μm particle diameter). The mobile phase was water (pH 3.5 with acetic acid) and methanol in a 30:70 isocratic model, with a flow rate of 1 mL/min, keeping the column temperature at 30 °C. Detection was performed in UV at 254 nm. For liriodenine quantification, calibration curves were performed by analyzing stock solution series (y = 0.3595x − 0.0011; R2 = 0.9989 for samples with up to 10 μg of liriodenine in the extract and y = 0.3658x + 1.142; R2 = 0.9992 for samples with more than 10 μg of liriodenine in the extract) [15]. The identification of asimilobine alkaloids, discretine, lanuginosine, laurotetanine, liriodenine, N-methyl-laurotetanine, norglaucine, oxoglaucine, reticuline, xylopine, and xylopinine was performed in comparison with the standards provided by Emmanoel Vilaça Costa and Jackson Roberto Guedes da Silva Almeida.
2.5. Statistical Analysis
The total alkaloids and liriodenine data were submitted to analysis of variance (Two-Way ANOVA) using the SigmaPlot software (Version 12.5, Chicago, IL, USA) and the means were compared by the Tukey test at 5% (p < 0.05) [33].
3. Results and Discussion
In general, it was observed that temperature was characterized as a factor of variation in alkaloid concentration (total alkaloids and liriodenine) in the early development stages (seed imbibition and early seedling). Furthermore, although the use of GA3 in the imbibition solution has favored the overcoming of seed dormancy (which was expected), it reduced the biosynthesis of alkaloids during the early stage of seedling development.
In newly collected (dry) Annona x atemoya Mabb. cv. ‘Gefner’ seeds, total alkaloids were detected in higher concentration in the endosperm (11.401 µg g−1) than in the seed coat (2.057 µg g−1), and liriodenine was found in endosperm (0.618 µg g−1), and not found in the seed coat, which indicates that the storage of these molecules occurred before dispersion, during the seed formation process (seed organogenesis), thus differing from results published with species of the same genus, such as Annona macroprophyllata (≡Annona diversifolia), A. purpurea, A. lutescens, and A. muricata, where the biosynthesis of alkaloids begins at germination or when the seedling becomes photosynthetically active [3].
In the same way as total alkaloids, liriodenine was detected in the endosperm of newly collected Annona x atemoya seeds and imbibed seeds in GA3 or H2O for 36 h at different temperatures (20 °C, 30 °C and 20–30 °C) (Table 1). Studies carried out with Annona cacans [16] corroborate our results that liriodenine was found at a higher concentration in the endosperm, when compared to the seed coat.
During the 36 h of imbibition, a period in which there is activation of the germinal metabolism [27,30,34], it was found that the temperature and the use of GA3 altered the biosynthesis of alkaloids in seeds. Seeds kept in H2O at 30 °C had the highest concentration of total alkaloids (22.777 µg g−1), not differing from seeds treated with GA3 and 20 °C, which was the treatment that promoted the highest liriodenine concentration in the endosperm during this period (Table 1).
In addition to the changes in alkaloid biosynthesis promoted by the treatments, it was possible to identify the presence of 10 alkaloids (asimilobine, discretine, lanuginosine, laurotetanine, liriodenine, N-methyl-laurotetanine, norglaucine, oxoglaucine, reticuline, xylopine, and xylopinine, in early seedling development (Table 2 and Table 3).
Liriodenine was the only alkaloid found in all treatment conditions in seed and seedling structures of Annona x atemoya cv. ‘Gefner’, being also reported in studies with species of the same genus as Annona cacans (seedling), Annona macroprophyllata (≡Annona diversifolia) (seedling), Annona crassiflora (young plants), and Annona emarginata (adult) [8,9,12,16].
Alkaloids such as lanuginosine and N-methyl-laurotetanine were absent in the structures of seeds imbibed for 36 h, being identified from the seedling structures, regardless of temperature and imbibition condition. While the alkaloid xylopinine remained present only in the endosperm of the seeds and was absent in the seed coat, however, during the initial development of the seedling it was possible to observe its presence in all structures and treatments. The other alkaloids were present in at least one or more structures of seeds or seedlings, and the presence or absence associated with the temperature or imbibition condition was not evident.
The application of gibberellins is common in seeds for species of the Annonaceae family to overcome dormancy, as it alters the hormonal balance between Abscisic Acid (ABA) and Gibberellins (GAs) and promotes the biosynthesis of hydrolytic enzymes that degrade seed reserves, making energy available for embryo development and subsequent germination [26,28], which was observed in other studies with atemoya seeds [27,35] and this experiment, with significant dormancy-overcoming of seeds treated with GA3. Temperature variation also affects, in general, the germination of Annona seeds, as alternating temperatures stimulate the highest germination percentage [25,30,31], including in atemoya seeds [32,35]. However, for atemoya seeds, it was observed in this experiment that temperature variation is no longer significant with the use of GA3 (Table 4).
The results obtained during imbibition reinforce the importance of GA3 to overcome dormancy in Annona seeds (Table 4) and its use increased the concentration of alkaloids in the endosperm during imbibition (when associated with a temperature of 20 °C, similar to seeds imbibed in water at a temperature of 30 °C) (Table 1). However, it caused a reduction in the biosynthesis of alkaloids during seedling development (Table 5). These observations show that the stimulus provided by GA3 directs the energy process to overcoming dormancy and promoting germination and not towards specialized metabolism, justifying the lower biosynthesis of alkaloids with the use of this regulator at different temperatures during imbibition.
When seeds were imbibed in H2O, the germination process was slower (slower speed, longer average time, lower germination percentage and, therefore, higher dormancy) compared to seeds that received GA3 (Table 4). However, in seeds imbibed in water, an increase in specialized metabolism was observed with the biosynthesis of alkaloids during imbibition and after germination, with an increase in the concentration of total alkaloids in all structures (seed coat, endosperm, root, hypocotyl, and cotyledons), with the only exception for temperatures of 20–30 °C (in the endosperm) and 20 °C (in the hypocotyl) (Table 5).
In seedling structures, it was also possible to observe the presence of liriodenine, with the highest values found in the root and endosperm, 127.831 µg g−1 (H2O, 30 °C) and 38.163 µg g−1 (GA3, 20–30 °C), respectively, but also found in structures such as hypocotyl and cotyledons, which suggests mobility of these alkaloids or biosynthesis in different locations. In addition, liriodenine was detected in the coat of seeds that were still attached to the seedling (Table 6). Studies carried out in Annona macroprophyllata (≡Annona diversifolia) [15] did not detect the presence of liriodenine in the seed coat (kept attached to seedlings) and seedling cotyledons; being found only in root, hypocotyl, and endosperm structures.
This increase in alkaloid concentration during the early seedling development seems to be related to the defense capacity of plants in order to guarantee their establishment in the environment [13]. Thus, in addition to the different alkaloid concentrations obtained in seed structures at 36 h of imbibition, changes were also observed in the total alkaloid concentrations in seedling structures as a function of treatments (Table 5).
Among seedling structures, roots were those with the highest concentration of total alkaloids (342.344 µg g−1) when seeds were imbibed in H2O and kept at 30 °C, indicating that it is the structure with the highest biosynthesis of these molecules in atemoya seedlings during early development. Similarly, roots were also the structure with the highest alkaloid production in Annona muricata L. [18], Annona lutescens [17], and Annona macroprophyllata (≡Annona diversifolia) [15]. On the other hand, there was the effect of lower temperature (20 °C) on the increase of alkaloid concentrations in cotyledons, endosperm, and seed coat, still kept attached to developing seedlings.
The changes that occurred in alkaloid concentrations in different structures as a function of temperature, from the imbibition process to the early seedling development, provide information that contributes to a greater understanding of how this species adapts to environmental temperature variations. Although Christie, Alfenito, and Walbot [20] and Wallaart et al. [24] observed that low temperatures significantly influence the levels of specialized metabolites, which was also observed in this experiment in some seedling structures at 20° C, the authors did not work with alkaloids.
4. Conclusions
Temperature and GA3 cause alterations in the biosynthesis of total alkaloids and liriodenine from the beginning of the germination process to the early Annona x atemoya Mabb. cv. ‘Gefner’ seedling development. It could also be concluded that the use of the GA3 regulator to break dormancy reduces the biosynthesis of alkaloids and liriodenine in the early development stages, while high temperature (30 °C) stimulates the biosynthesis of alkaloids in seedling roots.
Liriodenine, lanuginosine, laurotetanine, N-methyl-laurotetanine, xylopine, and xylopinine alkaloids have a more extensive presence in time and space; Liriodenine, lanuginosine, and laurotetanine are molecules that are formed during embryogenesis, while N-methyl-laurotetanine, xylopine, and xylopinine are formed during seed germination.
Author Contributions
Conceptualization, G.C.d.S. and G.F.; data curation, G.C.d.S., A.B.M.H., B.C.M., M.C.S., F.G.C. and G.F.; formal analysis, G.C.d.S., F.G.C. and C.S.F.B.; investigation, G.C.d.S., I.d.-l.-C.-C., A.B.M.H., B.C.M., M.C.S., F.G.C., C.S.F.B. and G.F.; methodology, G.C.d.S., I.d.-l.-C.-C., A.B.M.H., B.C.M., M.C.S., F.G.C., C.S.F.B. and G.F.; resources, I.d.-l.-C.-C., C.S.F.B. and G.F.; supervision, C.S.F.B. and G.F.; validation, G.F.; writing—original draft, G.C.d.S., C.S.F.B. and G.F.; writing—review and editing, G.C.d.S., I.d.-l.-C.-C., C.S.F.B. and G.F. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)-Finance Code 001 and CAPESPrint, Unesp.
Institutional Review Board Statement
Not applicable.
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest.
References
- De-la-Cruz-Chacón, I.; López-Fernández, N.Y.; Riley-Saldaña, C.A.; Castro-Moreno, M.; González-Esquinca, A.R. Antifungal Activity in Vitro of Sapranthus microcarpus (Annonaceae) against Phytopathogens. Acta Bot. Mex. 2019, 126, 1–9. [Google Scholar] [CrossRef]
- dos Santos, R.C.; Chagas, E.A.; de Melo Filho, A.A.; Takahashi, J.A.; Montero, I.F.; Chagas, P.C.; Ribeiro, P.R.E.; de Melo, C.G.R. Bioactive Extracts from Annona hypoglauca. Chem. Eng. Trans. 2018, 64, 289–294. [Google Scholar] [CrossRef]
- González-Esquinca, A.R.; De-La-Cruz-Chacón, I.; Castro-Moreno, M.; Orozco-Castillo, J.A.; Riley-Saldaña, C.A. Alkaloids and Acetogenins in Annonaceae Development: Biological Considerations. Rev. Bras. De Frutic. 2014, 36, 1–16. [Google Scholar] [CrossRef]
- Levorato Vinche, A.D.; de-La-Cruz-Chacón, I.; González-Esquinca, A.R.; de Fátima da Silva, J.; Ferreira, G.; dos Santos, D.C.; Garces, H.G.; de Oliveira, D.V.M.; Marçon, C.; de Souza Cavalcante, R.; et al. Antifungal Activity of Liriodenine on Agents of Systemic Mycoses, with Emphasis on the Genus Paracoccidioides. J. Venom. Anim. Toxins Incl. Trop. Dis. 2020, 26, e20200023. [Google Scholar] [CrossRef] [PubMed]
- Rinaldi, M.V.N.; Díaz, I.E.C.; Suffredini, I.B.; Moreno, P.R.H. Alkaloids and Biological Activity of Beribá (Annona hypoglauca). Rev. Bras. De Farmacogn. 2017, 27, 77–83. [Google Scholar] [CrossRef]
- Tamfu, A.N.; Ceylan, O.; Fru, G.C.; Ozturk, M.; Duru, M.E.; Shaheen, F. Antibiofilm, antiquorum sensing and antioxidant activity of secondary metabolites from seeds of Annona senegalensis, Persoon. Microb. Pathog. 2020, 144, 104191. [Google Scholar] [CrossRef]
- Lúcio, A.S.S.C.; Almeida, J.R.G.D.S.; da-Cunha, E.V.L.; Tavares, J.F.; Barbosa Filho, J.M. Alkaloids of the Annonaceae: Occurrence and a Compilation of Their Biological Activities. Alkaloids Chem. Biol. 2015, 74, 233–409. [Google Scholar]
- Honório, A.B.M.; De-La-cruz-chacón, I.; Martínez-Vázquez, M.; da Silva, M.R.; Campos, F.G.; Martin, B.C.; da Silva, G.C.; Fernandes Boaro, C.S.; Ferreira, G. Impact of Drought and Flooding on Alkaloid Production in Annona crassiflora Mart. Horticulturae 2021, 7, 414. [Google Scholar] [CrossRef]
- Ovile Mimi, C.; De-la-Cruz-Chacón, I.; Caixeta Sousa, M.; Aparecida Ribeiro Vieira, M.; Ortiz Mayo Marques, M.; Ferreira, G.; Silvia Fernandes Boaro, C. Chemophenetics as a Tool for Distinguishing Morphotypes of Annona emarginata (Schltdl.) H. Rainer. Chem. Biodivers. 2021, 18, 1–14. [Google Scholar] [CrossRef]
- Corrêa, P.L.C.; De-la-Cruz-Chacón, I.; Sousa, M.C.; Vieira, M.A.R.; Campos, F.G.; Marques, M.O.M.; Boaro, C.S.F.; Ferreira, G. Effect of Nitrogen Sources on Photosynthesis and Biosynthesis of Alkaloids and Leaf Volatile Compounds in Annona sylvatica A. St.-Hil. J. Soil Sci. Plant Nutr. 2022, 22, 956–970. [Google Scholar] [CrossRef]
- Mannino, G.; Gentile, C.; Porcu, A.; Agliassa, C.; Caradonna, F.; Bertea, C.M. Chemical Profile and Biological Activity of Cherimoya (Annona cherimola Mill.) and Atemoya (Annona atemoya) Leaves. Molecules 2020, 25, 2612. [Google Scholar] [CrossRef] [PubMed]
- De-La-Cruz Chacón, I.; Riley-Saldaña, C.A.; González-Esquinca, A.R. Secondary Metabolites during Early Development in Plants. Phytochem. Rev. 2013, 12, 47–64. [Google Scholar] [CrossRef]
- de la Cruz-Chacón, I.; González-Esquinca, A.R.; Fefer, P.G.; Garcia, L.F.J. Liriodenine, Early Antimicrobial Defence in Annona diversifolia. Z. Für Nat. Sect. C J. Biosci. 2011, 66, 377–384. [Google Scholar] [CrossRef]
- Rabêlo, S.V.; Costa, E.V.; Barison, A.; Dutra, L.M.; Nunes, X.P.; Tomaz, J.C.; Oliveira, G.G.; Lopes, N.P.; Santos, M.D.F.C.; Almeida, J.R.G.d.S. Alkaloids Isolated from the Leaves of Atemoya (Annona cherimola × Annona squamosa). Braz. J. Pharmacogn. 2015, 25, 419–421. [Google Scholar] [CrossRef]
- De La Cruz Chacón, I.; González-Esquinca, A.R. Liriodenine Alkaloid in Annona diversifolia during Early Development. Nat. Prod. Res. 2012, 26, 42–49. [Google Scholar] [CrossRef]
- Sousa, M.C.; Bronzatto, A.C.; González-Esquinca, A.R.; Campos, F.G.; Dalanhol, S.J.; Boaro, C.S.F.; Martins, A.L.; da Silva Almeida, J.R.G.; Costa, E.V.; De-la-Cruz-Chacón, I.; et al. The Production of Alkaloids in Annona cacans Seedlings Is Affected by the Application of GA4+7 + 6-Benzyladenine. Biochem. Syst. Ecol. 2019, 84, 47–51. [Google Scholar] [CrossRef]
- Castro-Moreno, M.; Tinoco-Ojangurén, C.L.; Cruz-Ortega, M.D.R.; González-Esquinca, A.R. Influence of Seasonal Variation on the Phenology and Liriodenine Content of Annona lutescens (Annonaceae). J. Plant Res. 2013, 126, 529–537. [Google Scholar] [CrossRef]
- Riley-Saldaña, C.A.; Cruz-Ortega, M.D.R.; Martínez Vázquez, M.; De-La-Cruz-Chacón, I.; Castro-Moreno, M.; González-Esquinca, A.R. Acetogenins and Alkaloids during the Initial Development of Annona muricata L. (Annonaceae). Z. Für Nat. Sect. C J. Biosci. 2017, 72, 497–506. [Google Scholar] [CrossRef]
- Riley-saldaña, C.A.; Cruz-ortega, M.R. Do Colletotrichum gloeosporioides and Rhizopus stolonifer Induce Alkaloidal and Antifungal Responses in Annona muricata Seedlings? Z. Für Nat. Sect. C J. Biosci. 2022, 77, 1–7. [Google Scholar] [CrossRef]
- Christie, P.J.; Alfenito, M.R.; Walbot, V. Impact of Low-Temperature Stress on General Phenylpropanoid and Anthocyanin Pathways: Enhancement of Transcript Abundance and Anthocyanin Pigmentation in Maize Seedlings. Planta 1994, 194, 541–549. [Google Scholar] [CrossRef]
- Elgorashi, E.E.; Drewes, S.E.; Van Staden, J. Organ-to-Organ and Seasonal Variation in Alkaloids from Crinum macowanii. Fitoterapia 2002, 73, 490–495. [Google Scholar] [CrossRef]
- Gobbo-Neto, L.; Lopes, N.P. Plantas Medicinais: Fatores de Influência No Conteúdo de Metabólitos Secundários. Quim. Nova 2007, 30, 374–381. [Google Scholar] [CrossRef]
- Roca-Pérez, L.; Boluda, R.; Gavidia, I.; Pérez-Bermúdez, P. Seasonal Cardenolide Production and Dop5βr Gene Expression in Natural Populations of Digitalis obscura. Phytochemistry 2004, 65, 1869–1878. [Google Scholar] [CrossRef] [PubMed]
- Wallaart, T.E.; Pras, N.; Beekman, A.C.; Quax, W.J. Seasonal Variation of Artemisinin and Its Biosynthetic Precursors in Plants of Artemisia annua of Different Geographical Origin: Proof for the Existence of Chemotypes. Planta Med. 2000, 66, 57–62. [Google Scholar] [CrossRef]
- Zucareli, V.; Ferreira, G.; Silvério, E.R.V.; Amaro, A.C.E. Luz e Temperatura Na Germinação de Sementes de Annona squamosa L. Rev. Bras. De Biociências 2007, 5, 840–842. [Google Scholar]
- Ferreira, G.; De-La-cruz-chacón, I.; González-Esquinca, A.R. Changes in Hormonal Balance as Key to Reserve Degradation after Dormancy Overcoming in Annona macroprophyllata and Annona purpurea Seeds. Rev. Bras. Frutic. 2020, 42, 1–11. [Google Scholar] [CrossRef]
- de Oliveira, M.C.; Ferreira, G.; Guimarães, V.F.; Dias, G.B. Germinação de Sementes de Atemoia (Annona cherimola Mill. × A. squamosa L.) CV “Gefner” Submetidas a Tratamentos Com Ácido Giberélico (GA3) e Ethephon. Rev. Bras. Frutic. 2010, 32, 27. [Google Scholar] [CrossRef]
- Ferreira, G.; De-La-Cruz-Chacón, I.; González-Esquinca, A.R. Superação Da Dormência de Sementes de Annona macroprophyllata e Annona purpurea Com o Uso de Reguladores Vegetais. Rev. Bras. Frutic. 2016, 38, 1–10. [Google Scholar] [CrossRef]
- Rego, C.H.Q.; Cardoso, F.B.; Cotrim, M.F.; Cândido, A.C.D.S.; Alves, C.Z. Ácido Giberélico Auxilia Na Superação Da Dormência Fisiológica E Expressão De Vigor Das Sementes De Graviola. J. Neotrop. Agric. 2018, 5, 83–86. [Google Scholar] [CrossRef]
- Costa, P.N.; Bueno, S.S.C.; Ferreira, G. Germination Phases of Annona emarginata (SCHLTDL.) H. Rainer Seeds under Different Temperatures. Rev. Bras. Frutic. 2011, 33, 253–260. [Google Scholar] [CrossRef]
- Oliveira, I.V.d.M.; Andrade, R.A.D.; Martins, A.B.G. Influência Da Temperatura Na Germinação de Sementes de Annona montana. Rev. Bras. Frutic. 2005, 27, 344–345. [Google Scholar] [CrossRef]
- Gomes, R.L.; Passos, J.R.d.S.; Gimenez, J.I.; Sousa, M.C.; De-Pieri-Oliveira, M.d.F.; Mimi, C.O.; Ferreira, G. Optimum Sample Size in the Germination of Atemoya Seeds (Annona × atemoya Mabb.). J. Agric. Sci. 2019, 11, 239. [Google Scholar] [CrossRef]
- Costa, J.R. Técnicas Experimentais Aplicadas Às Ciências Agrárias. Embrapa Agrobiol. 2003, 54, 1–54. [Google Scholar]
- Stenzel, N.M.C.; Murata, I.M.; Neves, C.S.V.J. Superação Da Dormência Em Sementes de Atemóia e Fruta-Do-Conde. Rev. Bras. Frutic. 2003, 25, 305–308. [Google Scholar] [CrossRef]
- Braga, J.F.; Ferreira, G.; Pinho, S.Z.d.; Braga, L.F.; Sousa, M.P. Germination of Atemoya (Annona cherimola Mill. × A. squamosa L.) Cv. Gefner Seeds Subjected to Treatments with Plant Growth Regulators. Int. J. Sci. Nat. 2010, 1, 120–126. [Google Scholar]
Table 1.
Concentration of total alkaloids (µg g−1 dry mass) and liriodenine (µg g−1 dry mass) in the structures (seed coat and endosperm) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (solution with H2O or GA3) at different temperatures (20 °C, 30 °C and 20–30 °C).
Table 1.
Concentration of total alkaloids (µg g−1 dry mass) and liriodenine (µg g−1 dry mass) in the structures (seed coat and endosperm) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (solution with H2O or GA3) at different temperatures (20 °C, 30 °C and 20–30 °C).
| Total Alkaloid Concentration | Liriodenine Concentration | ||||
|---|---|---|---|---|---|
| Temperature | H2O | GA3 | H2O | GA3 | |
| Seed coat | 20 °C | 0.779 Aa * | 1.771 Aa | ND | ND |
| 30 °C | 1.553 Aa | 1.346 Aa | ND | ND | |
| 20–30 °C | 1.481 Aa | 1.833 Aa | ND | ND | |
| Endosperm | 20 °C | 13.857 BCa | 19.272 Aa | 0.000 Ab | 0.661 Aa |
| 30 °C | 22.777 Aba | 14.205 Ab | 0.199 Aa | 0.239 ABa | |
| 20–30 °C | 8.665 Ca | 11.248 Aa | 0.000 Aa | 0.000 Ba | |
(*) Means followed by the same letter, uppercase in the column (temperatures in each imbibition solution), and lowercase in the row (imbibition solutions in each temperature), for each structure, do not differ from each other by Tukey’s test at 5% probability. ND: not detected.
Table 2.
Presence (x) or absence ( ) of alkaloids in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originating from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
Table 2.
Presence (x) or absence ( ) of alkaloids in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originating from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
| Seed Imbibition H2O | Seed Imbibition GA3 | |||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Alkaloids | Seed Coat | Endosperm | Hypocotyl | Cotyledon | Root | Sead Coat | Endosperm | Hypocotyl | Cotyledon | Root | ||||||||||||||||||||
| 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | |
| Asimilobine | x | x | x | x | x | x | x | x | x | x | ||||||||||||||||||||
| Discretine | ||||||||||||||||||||||||||||||
| Lanuginosine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | |
| Laurotetanine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | ||||||||
| Liriodenine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x |
| N-metil-Lauroteanine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | ||||||||||||
| Norglaucine | x | |||||||||||||||||||||||||||||
| Oxoglaucine | x | x | x | x | x | x | ||||||||||||||||||||||||
| Reticuline | x | |||||||||||||||||||||||||||||
| Xylopine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | |||||||
| Xylopinine | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | |||||||
Table 3.
Presence (x) or absence ( ) of alkaloids in the seed structures (seed coat and endosperm) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (solution with H2O or GA3) at different temperatures (20 °C, 30 °C, and 20–30 °C) and dry seeds.
Table 3.
Presence (x) or absence ( ) of alkaloids in the seed structures (seed coat and endosperm) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (solution with H2O or GA3) at different temperatures (20 °C, 30 °C, and 20–30 °C) and dry seeds.
| Alkaloids | Seed Dry | Seed Imbibition H2O | Seed Imbibition GA3 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Endosperm | Seed Coat | Endosperm | Seed Coat | Endosperm | Seed Coat | |||||||||
| 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | 20 | 30 | 20–30 | |||
| Asimilobine | x | x | x | x | x | x | x | x | x | x | ||||
| Discretine | ||||||||||||||
| Lanuginosine | x | x | ||||||||||||
| Laurotetanine | x | x | x | x | x | |||||||||
| Liriodenine | x | x | x | x | x | x | x | x | x | x | x | x | ||
| N-metil-Lauroteanine | ||||||||||||||
| Norglaucine | ||||||||||||||
| Oxoglaucine | x | x | x | |||||||||||
| Reticuline | ||||||||||||||
| Xylopine | x | |||||||||||||
| Xylopinine | x | x | x | x | x | x | ||||||||
Table 4.
Germinability (Germination percentage-% G; Percentage of dormant seeds-% D; Percentage of dead seeds-% M; Germination speed index-GSI; Mean germination time-MGT) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during germination.
Table 4.
Germinability (Germination percentage-% G; Percentage of dormant seeds-% D; Percentage of dead seeds-% M; Germination speed index-GSI; Mean germination time-MGT) of Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during germination.
| Temperature | H2O | GA3 | |
|---|---|---|---|
| % G | 20 °C | 15 Cb * | 71 Aa |
| 30 °C | 42 Bb | 71 Aa | |
| 20–30 °C | 55 ABa | 62 Aa | |
| % D | 20 °C | 59 Aa | 10 Ab |
| 30 °C | 35 Ba | 0 Ab | |
| 20–30 °C | 25 Ba | 0 Ab | |
| % M | 20 °C | 26 Aa | 19 Aa |
| 30 °C | 23 Aa | 29 Aa | |
| 20–30 °C | 20 Aa | 38 Aa | |
| GSI | 20 °C | 0.209 Cb | 0.805 Ca |
| 30 °C | 1.063 Bb | 2.155 ABa | |
| 20–30 °C | 1.618 ABa | 1.676 Ba | |
| MGT | 20 °C | 18.75 Ab | 25.685 Aa |
| 30 °C | 13.219 Ba | 9.268 Bb | |
| 20–30 °C | 10.504 Ba | 10.202 Ba |
(*) Means followed by the same letter, uppercase in the column (temperatures in each imbibition solution), and lowercase in the row (imbibition solutions in each temperature), for each structure, do not differ from each other by Tukey’s test at 5% probability.
Table 5.
Concentration of total alkaloids (µg g−1 dry mass) in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originating from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
Table 5.
Concentration of total alkaloids (µg g−1 dry mass) in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originating from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
| Temperature | H2O | GA3 | |
|---|---|---|---|
| Seed coat | 20 °C | 27.291 Aa * | 0.818 Ab |
| 30 °C | 5.169 BCa | 0.375 Aa | |
| 20–30 °C | 4.035 Ca | 2.17 Aa | |
| Endosperm | 20 °C | 168.218 Aa | 13.609 Bb |
| 30 °C | 46.133 BCa | 28.24 Ba | |
| 20–30 °C | 34.151 Cb | 83.5 Aa | |
| Root | 20 °C | 147.039 Ca | 44.782 Bb |
| 30 °C | 342.344 Aa | 119.647 Ab | |
| 20–30 °C | 173.474 BCa | 88.002 ABb | |
| Hypocotyl | 20 °C | 20.947 Cb | 33.22 Aa |
| 30 °C | 41.469 Ba | 21.755 Ab | |
| 20–30 °C | 51.603 ABa | 27.48 Ab | |
| Cotyledon | 20 °C | 122.053 Aa | 8.582 Bb |
| 30 °C | 84.985 Aa | 56.581 Bb | |
| 20–30 °C | 129.832 Aa | 41.967 Bb |
(*) Means followed by the same letter, uppercase in the column (temperatures in each imbibition solution), and lowercase in the row (imbibition solutions in each temperature), for each structure, do not differ from each other by Tukey’s test at 5% probability.
Table 6.
Liriodenine concentration (µg g−1 dry mass) in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originated from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
Table 6.
Liriodenine concentration (µg g−1 dry mass) in seedling structures (seed coat, endosperm, root, hypocotyl, and cotyledon) originated from Annona x atemoya Mabb. cv. ‘Gefner’ seeds immersed for 36 h (with H2O or GA3) at temperatures of 20 °C, 30 °C, and 20–30 °C and kept at the same temperatures during early development.
| Temperature | H2O | GA3 | |
|---|---|---|---|
| Seed coat | 20 °C | 0.000 Ba * | 0.144 Ca |
| 30 °C | 1.286 Aa | 0.956 ABa | |
| 20–30 °C | 0.359 Ba | 0.819 Ba | |
| Endosperm | 20 °C | 6.921 Aa | 2.112 Ba |
| 30 °C | 18.656 Aa | 13.116 Ba | |
| 20–30 °C | 7.011 Ab | 38.163 Aa | |
| Root | 20 °C | 27.453 Ba | 13.210 Aa |
| 30 °C | 127.831 Aa | 26.912 Ab | |
| 20–30 °C | 46.304 Ba | 22.658 Aa | |
| Hypocotyl | 20 °C | 20.328 Aa | 3.755 Bb |
| 30 °C | 6.868 Aa | 3.133 Ba | |
| 20–30 °C | 7.429 Aa | 2.591 Ba | |
| Cotyledon | 20 °C | 0.000 Bb | 25.084 Aa |
| 30 °C | 16.483 Ca | 21.890 ABa | |
| 20–30 °C | 30.932 Aa | 13.348 CBb |
(*) Means followed by the same letter, uppercase in the column (temperatures in each imbibition solution), and lowercase in the row (imbibition solutions in each temperature), for each structure, do not differ from each other by Tukey’s test at 5% probability.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
da Silva, G.C.; de-la-Cruz-Chacón, I.; Honório, A.B.M.; Martin, B.C.; Sousa, M.C.; Campos, F.G.; Boaro, C.S.F.; Ferreira, G. Temperature and GA3 as Modulating Factors in the Biosynthesis of Alkaloids during Imbibition and Early Development of Annona x atemoya Mabb. cv. ‘Gefner’ Seedlings. Horticulturae 2022, 8, 766. https://doi.org/10.3390/horticulturae8090766
AMA Style
da Silva GC, de-la-Cruz-Chacón I, Honório ABM, Martin BC, Sousa MC, Campos FG, Boaro CSF, Ferreira G. Temperature and GA3 as Modulating Factors in the Biosynthesis of Alkaloids during Imbibition and Early Development of Annona x atemoya Mabb. cv. ‘Gefner’ Seedlings. Horticulturae. 2022; 8(9):766. https://doi.org/10.3390/horticulturae8090766
Chicago/Turabian Styleda Silva, Gustavo Cabral, Ivan de-la-Cruz-Chacón, Ana Beatriz Marques Honório, Bruna Cavinatti Martin, Marília Caixeta Sousa, Felipe Girotto Campos, Carmen Sílvia Fernandes Boaro, and Gisela Ferreira. 2022. "Temperature and GA3 as Modulating Factors in the Biosynthesis of Alkaloids during Imbibition and Early Development of Annona x atemoya Mabb. cv. ‘Gefner’ Seedlings" Horticulturae 8, no. 9: 766. https://doi.org/10.3390/horticulturae8090766
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
