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South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
Osmotic priming effects on emergence of Physalis angulata and the influence of abiotic stresses on physalin content
Manuela Oliveira de Souza a *, Cíntia Luiza Mascarenhas de Souza a, Claudinéia Regina Pelacania,
Marcio Soares b, José Luiz Mazzeib, Ivone Maria Ribeiro b,
Concei^ao Pereira Rodrigues b, Therezinha Coelho Barbosa Tomassinib
a State University ofFeira de Santana (UEFS), BR 116, Km 03, CEP 44031-460, Feira de Santana, BA, Brazil
b Natural Product Chemistry Laboratory, PN2, Farmanguinhos-Fiocruz, Sizenando Nabuco 100, CEP 21041-250, Rio de Janeiro, RJ, Brazil
ARTICLE INFO
ABSTRACT
Article history: Received 26 February 2013 Received in revised form 29 July 2013 Accepted 30 July 2013 Available online 25 August 2013
Edited by P Berjak
Keywords: Biomass Growth Saline stress Water restriction
Physalis angulata is a medicinal plant with valuable pharmacological activities. The physiology of stress in this plant can play an important role in the induction or maximization of the production of physalin F, B, D and G, bioactive secondary metabolites described as immunosuppressive, anti-malarial and anti-leishmanial agents. P. angulata was cultivated from seeds which had been previously primed in PEG 6000 solution and non-primed seeds. After 45 days, the plants were exposed to water restriction and saline stress in the field for 13 days. Seedling emergence and growth after stress treatment were assessed. Seco-steroids were quantified in leaves and stems by HPLC/PDA. The emergence rate was 14% higher in primed seeds. The types of irrigation proved to have a significant influence on the number of leaves and fruits, plant height and stem diameter, irrespective of whether the seeds were primed or not. The biomass of the fruits, stems and roots was also decreased by water restriction and saline stress. Physalin content in ethanol extracts increased in leaves, mainly after saline stress and from primed seeds. Despite the biomass reduction caused by the treatments, stress application led to an increase in the production of bioactive metabolites.
© 2013 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction
Secondary metabolites have a limited and restricted taxonomic distribution in plants and their biosynthesis can be modulated by environmental conditions (Torssell, 1997; Kutchan, 2001).
Although recognized as potentially harmful, the induction of plant stress has been practiced to modulate the production of metabolites which are involved in essential physiological processes (Flowers, 2004; Sairam and Tyagi, 2004). Likewise, the controlled induction of plant stress can increase the content of pharmacologically active substances (Abreu and Mazzafera, 2005; Capanoglu, 2010).
Abiotic stresses such as osmotic stress, water restriction and salinity are among the factors that can lead to a variation in the content of secondary metabolites (Harborne and Williams, 2000; Taiz and Zeiger, 2010). Chemical products of several biotic or abiotic sources as well as physical factors are elicitors and may trigger a response in live organisms, resulting in an accumulation of products (Zhao et al., 2001; Yu et al., 2002). Water restriction and saline stress can cause increases in the content of metabolites in plants (Krishna, 2003; Kagale et al.,
Abbreviations: NP, non-primed; P, primed; PEG, polyethylene glycol; HPLC, high performance liquid chromatography.
* Corresponding author. Tel.: +55 71 3181 6109, +55 71 9330 0122 (Mobile). E-mail address: oliva.manuela@gmail.com (M.O. de Souza).
0254-6299/$ - see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.! 016/j.sajb.2013.07.025
2007) and there are studies that have applied sodium chloride (NaCl) as an elicitor to induce secondary metabolites (Abrol et al., 2012). Provision of 50 mM NaCl proved effective in inducing the in vitro production of secondary metabolites in Swertia chirata (Abrol et al., 2012). The bio-synthetic and metabolic pathways followed by each species are very specific (Jahangir et al., 2009) and some of the generated products participate in the secondary plant metabolism and play a role in protecting or minimizing stress that a plant may undergo. Brassica oleracea increases its glucosinolate content when it is subjected to saline stress, indicating the involvement of these metabolites in the response to stress (Lopez-Berenguer et al., 2008).
Germination and the early seedling development are the growth phases which are most influenced by the abiotic stresses. Therefore, pre-germination treatments involving invigoration, priming effectiveness and physiological seed conditioning are applied to improve the seed quality and seedling establishment of cultivated plants. Seeds are soaked in water or in an aqueous solution for enough time to establish an osmotic equilibrium with the medium (Heydecker and Coolbear, 1977; Roberts and Ellis, 1989). This method causes high and synchronized rates of seed germination (Heydecker et al., 1973; Iqbal and Ashraf, 2007; Varier et al., 2010) and emergence (Bradford, 1986; Flors etal., 2007).
Physalis angulata L. (family Solanaceae) is a plant used as a commercial herbal medicine and it is found worldwide in tropical and
subtropical areas. It has been widely used in traditional treatment of malaria, asthma, hepatitis, sleeping sickness, dermatitis, rheumatism, earache, and fever (Chiang et al., 1992b; Lin et al., 1992; Caceres et al., 1995; Freiburghaus et al., 1996; Ankrah et al., 2003; Choi and Hwang, 2003). Extracts of whole P. angulata induced cellular death and apopto-sis in human oral cancer cell lines (Lee et al., 2009), roots have shown anti-inflammatory and immunomodulatory properties (Bastos et al., 2008) and the stems have demonstrated anti-neoplastic activity and cell proliferation inhibition of J774 (mouse monocytic cell line), SP 2/0 (mouse myeloma), Ehrlich carcinoma (sarcoma induced by methylcholanthrene), P3653 (mouse plasmocytoma), Neuro-2a (mouse neuroblastoma), MK2 (monkey epithelial cells) and BW yeasts (Ribeiro et al., 2002).
Pharmacological activities have been related to 13,14-seco-16,24-cyclo-steroids, and mainly to the isolated physalins F, B, D and G (Fig. 1) (Chiang et al., 1992a,b; Tomassini et al., 2000; Soares et al., 2003). Physalins F and D showed anti-malarial activity (Sa et al., 2011). Physalins F and B showed both in vitro and in vivo anti-leishmanial activities and inhibited dermal leishmaniasis (Guimaraes et al., 2009). A study with the crude extract from aerial parts of P. angulata showed the possible effect of physalin D on the growth inhibition of Mycobacterium tuberculosis (Januario et al., 2002). Physalins F, B and G inhibited the in vitro proliferation of splenocyte cultures, prevented in vivo the rejection in allogeneic transplantation (Soares et al., 2006), inhibited macrophage activation and protected against lipopolysaccharide-induced death (Soares et al., 2003).
Despite many studies on the pharmacological action of the physalins from P. angulata, there is a lack of understanding about the effects of oscillations in environmental conditions on the production and on the regulation of physalin synthesis. Therefore, the present study aims to evaluate the effect of osmotic seed priming on growth and development of P. angulata plants, as well as, on the physalin content when the mature plants have been exposed to water restriction and saline stress.
2. Material and methods
2.1. Chemicals
Analytical grade ethanol (EtOH) was applied to physalin extraction. Trifluoroacetic acid, methanol and acetonitrile (HPLC/UV grade) were purchased from Tedia (Fairfield, USA) and used throughout the chro-matographic analysis, together with ultra purified water (resistivity of 18.1 Mft-cm) from NanoPure Diamond system (Barnstead/Thermolyne, Dubuque, IA, USA). Reference materials of physalins F, B, D and G (96-98% purity by HPLC) were isolated from dry stems of identified P. angulata plants collected in Belem (Para State, Brazil) and were purified and characterized according to procedures described elsewhere (Soares et al., 2003, 2006).
22. P. angulata seeds
P. angulata seeds were collected from mature fruits of cultivated plants in a greenhouse at the Horto Florestal Station at the Universidade
F 5p,6p-epoxy
D 5a-OH, 6p-OH
Fig. 1. Chemical structures of the bioactive physalins isolated from Physalis angulata.
Estadual de Feira de Santana, Bahia, Brazil (38°55'30.59" W; 12°16' 17.07" S). The seeds were dried for 24 h at 20 °C and 11% relative humidity. The seeds were kept in a desiccator containing super-saturated potassium chloride solution, until there were no differences between fresh and dry weights in batches. After drying, the seeds (moisture content 7.2%) were separated in lots of 500 units and further stored in the dark at 4 °C in polypropylene tubes until use.
2.3. Osmotic priming and emergence
Samples of 1000 seeds of P. angulata were immersed in a polyethylene glycol (PEG 6000) solution at osmotic potential of —1.2 MPa. This was previously established as priming potential for the species (Souza et al., 2011). The immersion was performed in 25 ml-polypropylene tubes for 10 days at 35 °C using a germination chamber coupled with an aeration system. After the pre-germination period, the seeds were taken from the solution, washed and dried at room temperature until a constant weight was reached.
2.4. Experiments in the greenhouse
Ten primed (P) or ten non-primed (NP) seeds were sown in 15 kg substrate (1:1 mixture of soil and washed sand) in each of 80 polyethylene containers. All the pots were kept in an agricultural greenhouse with 40% of the radiation incident on the greenhouse at the Experimental Horto Florestal Station in September 2009 and the substrate was watered daily. The emergence rate (%) was evaluated twice (10 and 20 days after sowing). After the last evaluation was conducted, following thinning, one seedling - the most vigorous - was retained per pot.
At 45 days of cultivation, the plants from non-primed and primed seeds were separated into 3 groups and each was exposed to one of the irrigation treatments: (1) with daily watering — maintaining maximum moisture capacity (100% irrigation) (control group), (2) irrigated with 50% of the daily requirement and (3) irrigated with 0.9% NaCl solution with an electrical conductivity of 10 dS m—1. The respective volumes were 316, 158 and 316 ml. The amounts of water and saline solution used were calculated based on the capacity of the substrate to retain water and this was evaluated after 24 h. The plants were kept under these conditions for 13 days until withering points of the aerial parts could be seen in both stress treatments.
2.5. Growth analysis
After the treatments described above the following growth parameters were assessed: number of leaves and fruits, plant height and stem diameter. For the measurement of plant height a millimeter ruler was used to measure from the soil surface to the apex of the highest leaf stretched vertically. The stem diameter (mm) was measured at the height at the soil surface with the aid of a digital caliper (Cosa 111-101EB). After evaluation of growth, leaves, stems, flowers and roots from plants from the same pre-germination and stress treatment were separated and placed in forced-air circulation oven to dry, at 40° ± 5 °C, for 8 days and the final dry masses were determined. Samples of dry leaves as well as dry stems were combined and ground before the quantitative determination of the physalin.
2.6. Quantitative determination of physalins in leaves and stems
Fifty grams of the ground material (from leaves or stems) was extracted three times with 200 ml ethanol. The extracts of both plant organs from the various treatments were filtered through filter paper (J-Prolab, 200 |am thickness, 14 |am pore size) and concentrated in a rotary evaporator R-124 (BÜCHI Labortechnik, Switzerland) at 175 mbar and 40 °C, until semi-solid residues were obtained. The residues were dried in a desiccator until they achieved a constant weight. Three 10-mg aliquots of each sample were independently
Table 1
Emergence rate (%, mean and standard deviation) of non-primed (NP) or primed (P) seeds of Physalis angulata.
Days after sowing Pre-germination treatment
10 64 ± 11 78 ± 11*
20 79 ± 13 89 ± 8*
* Data are different (p < 0.05) within the same periods of observation.
weighed (±0.01 mg), diluted with 400 |al methanol, sonicated for 2 min, and filtered through centrifugal filter devices (Durapore, PVDF membrane, 0.2 |am) for physalin content determination by high performance liquid chromatography (HPLC).
The HPLC system (Shimadzu Co., Japan) LC-10AVP consisted of two LC-10AD pumps, a DGU-12A degasser, the SIL-10AD autosampler, CT0-10A column oven and a SPD-M10A photodiode array detector (PDA) with scanning at 200-800 nm with 1 nm resolution. The data analysis was performed using CLASS-VP v.6.13 SP2 software (Shimadzu Co.). The column used was a Hibar 250 x 4 mm with a reversed phase LiChrospher 100 RP18 5 |am (Merck, Darmstadt) connected to a Supelcosil C18 guard-column (2 cm). Gradient elution was carried out at 30 °C with 0.05% trifluoroacetic acid in ultrapure water (phase A) and acetonitrile (phase B). The steps of the gradient program at a flow rate of 1 ml min-1 were as follows: 3%B for 0 to 3 min, 3-10%B for 3-6 min, 10%B for 6-9 min, 10-18%B for 9-12 min, 18%B for 1215 min, 18-21%B for 15-18 min, 21%B for 18-21 min, 21-35%B for 21-45 min, 35%B for 45-55 min, 35-80%B for 55-70 min, 80%B for 70-75 min, returning to 3%B in 75-78 min and holding for 7 min before the following injection (20 |i). The chromatograms were monitored by UV detection at wavelengths of 225 and 310 nm. Reference materials of physalins D, G, F, and B at 1 mg/ml in methanol were injected for assignment by similarity to UV spectra and to the retention times which were 38.5, 39.7, 55.5, and 66.8 min, respectively. The physalin content was measured based on a standard curve (at 225 nm) using physalin D at five concentrations (10, 20, 40, 500, and 1000 ^g ml-1 in methanol). These reference solutions were injected in triplicate (20 |al) to determine the calibration curve and to check linearity and validity by ANOVA test.
2.7. Experimental design and statistical analysis
The experiments in the greenhouse were designed as a randomized block with factorial arrangement 2 x 3 for the analysis of the effects of seed priming and of irrigation conditions, respectively. Each irrigation treatment consisted of seven pots with one plant per pot in four replicates.
The data underwent analysis of variance (ANOVA) and Tukey's studentized range HSD test (p < 0.05) using the statistical program SISVAR (Ferreira, 2011).
Hierarchical cluster analysis of the multivariate data about growth and dry mass of the plants and physalin content in their respective extracts was performed. Euclidean distances were used to calculate the similarity matrix for which dendrograms were constructed using Action statistical software.
3. Results and discussion
3.1. Emergence and growth of P. angulata
Ten days after sowing, the primed seeds showed a significantly (p < 0.05) higher emergence rate than the untreated seeds (Table 1), with 14% higher seedling germination rates. This behavior was maintained at the second observation (20 days after sowing), demonstrating that seed priming is efficient in improving the emergence rate and seedling growth of P. angulata.
Using this technique, the desired benefits of seedling establishment of P. angulata can be achieved ensuring a good yield of plant material per planting area. These results are similar to those found in other works that have asserted the importance of osmotic priming in the early stages of plant development (Patane et al., 2009; Dantas et al., 2010).
Some studies have reported that seeds hydrate slowly during osmotic priming, allowing a longer time for the repair of macromolecules and for a more favorable metabolic balance at the beginning of germination. Increases in enzymatic and metabolic activities, DNA synthesis, ATP production, and membrane damage repairs allow tissue formation in a more direct way, reducing the risks of damage to the embryo axis. These characteristics are inherent to osmotic priming and seem to be related to the increase in the seed vitality during the subsequent germination step (Khan, 1992).
Table 2
Growth characteristics of Physalis angulata originated from non-primed (NP) and primed (P) seeds and in different types of irrigation.
100% water
50% water
NaCl aq.
100% water
50% water
NaCl aq.
Number of leaves Number of fruits Plant height (cm) Stem diameter (cm)
155 ± 15 29 ± 4 62.0 ± 2.3 13.0 ± 0.6
123 ± 8a 20 ± 7a 52.0 ± 9.1 11.6 ± 0.8a
123 ± 5a 20 ± 6a 57.0 ± 10.2 11.7 ± 0.6a
140 ± 13 25 ± 3 67.4 ± 5.5 12.3 ± 1.6
114 ± 6a 20 ± 3 51.6 ± 9.6a 11.3 ± 0.7
134 ± 5 25 ± 5 54.6 ± 8.4a 11.2 ± 0.6
a Significantly different values (p < 0.05) compared to the plants watered daily (100% irrigation) are indicated. There was no significant difference (p > 0.05) between non-primed (NP) and primed (P) seeds.
Table 3
Dry mass (g plant-1) of organs of the Physalis angulata plants grown from non-primed (NP) and primed (P) seeds and in different types of irrigation.
NP P
100% water 50% water NaCl aq. 100% water 50% water NaCl aq.
Leaves 3.75 ± 0.3 2.95 ± 0.3a 3.72 ± 0.2 3.37 ± 0.4 2.83 ± 0.3a 3.40 ± 0.1
Flowers 0.06 ± 0.1 0.05 ± 0.1 0.04 ± 0.1 0.05 ± 0.1 0.06 ± 0.1 0.05 ± 0.1
Fruits 1.85 ± 0.4 1.15 ± 0.3a 1.28 ± 0.5a 1.97 ± 0.5 1.15 ± 0.2a 1.41 ± 0.3a
Stems 4.67 ± 0.4 4.20 ± 0.6 3.97 ± 0.6a 4.98 ± 0.8 3.42 ± 0.6a 3.55 ± 0.5a
Roots 3.23 ± 0.3 2.27 ± 0.5a 2.51 ± 0.2a 3.29 ± 0.5 2.14 ± 0.5a 2.67 ± 0.4a
a Significantly different (p < 0.05) values from plants watered daily (100% irrigation) are indicated. There was no significant difference (p > 0.05) in dry mass whether plants were grown from non-primed (NP) or primed (P) seeds.
Fig. 2. Dendrogram of the effects on elongation growth of the plant and on dry mass of the organs of Physalis angulata after water restriction (a) and saline stress (b) (from Tables 2 and 3).
The restricted irrigation of P. angulata and the saline solution decreased the number of leaves and fruits, the plant height and the stem diameter (Table 2), with only one exception (number of fruits of plants grown from primed seeds and after exposure to saline stress), and the reduction was significant (p < 0.05) in some conditions. Seed priming did not affect (p > 0.05) the parameters analyzed (Table 2).
The occurrence of fluid and saline stresses during the initial phase of the development can promote a reduction in growth, the number of branches and leaves, a decrease in the photosynthetic surface and consequently, a lower production of flowers and fruits (Karamanos et al., 1982; Boutra and Sanders, 2001). The yields of cultivated plants in soil under fluid or saline stresses will depend on the adaptive mechanisms that enable them to keep up growth and high photosynthetic activity. This is reflected in desirable production levels even under prolonged stress conditions. When fewer fruits, seeds and leaves are produced, the economic value of such plants is reduced (DaMatta, 2007).
According to Koyro (2000), the reduction in the number of leaves is the most pronounced morphological change observed during salt stress. Leaf expansion can decrease significantly as the plants are exposed to high salinity irrigation and, this mechanism is dependent on the capacity of the plant to avoid excessive concentration of ions in transpiring tissues and, as consequence the plant produces new leaves at a much faster rate (Miranda et al., 2010).
The lowest growth rates, that were induced in plants exposed to water restriction or saline stress were significant (p < 0.05) among the plants from the primed seeds, unlike plants from non-primed ones (Table 2). This behavior could be a disadvantage of osmotic priming on plant growth, however, plants from seeds with and without the pre-germination treatment did not grow differently (p > 0.05). The reduction in plant height may be associated to a decline in cellular
expansion and growth, due to the low turgor pressure in addition to foliar senescence (Jaleel et al., 2008).
3.2. Dry mass production by the organs of the plant
The pre-germination treatment (osmotic priming) had no significant effect (p > 0.05) on the dry mass of each organ of the plant (Table 3). Thus the results in Tables 2 and 3 for plants from non-primed or primed seeds were grouped, divided by values found in the respective control group and compared in a cluster analysis (Fig. 2) to express the quantitative decrease or increase by the irrigation treatments.
Water restriction decreased the biomass of leaves, fruits, stems and roots in the plant significantly (p < 0.05) (Table 3). No effect on the dry mass of flowers was observed, certainly due to the low weight measurement sensitivity. A reduction in biomass is expected because decreases in the water absorption by the roots inhibit meristematic activity and cell elongation (Chartzoulakis and Klapaki, 2000), as has been observed in seedlings of Beta vulgaris L. (Sadeghian and Yavari, 2004). Water restriction also results in a reduction in growth and development of the plants (Chartzoulakis and Klapaki, 2000; Sadeghian and Yavari, 2004). The cluster analysis of the multivariate data found in Tables 2 and 3 illustrates the strong similarities of the effects of the water restriction (Fig. 2A) on all the measured aspects relative to elongation characteristics of P. angulata: number and mass of leaves, diameter and mass of stems and plant height.
In plants exposed to salinity the reduction in root volume could be a favorable feature as it limits their ability to accumulate toxic ions in the aerial parts or in the whole plant (Munns, 2002; Alarcon et al., 2006; Miranda et al., 2010). However in the present study exposure to salinity
Content of physalins (mg g-1) in dried ethanol extracts of Physalis angulata organs of plants grown from non-primed (NP) and primed (P) seeds and subjected to the different irrigation regimes.
NP P
100% water 50% water NaCl aq. 100% water 50% water NaCl aq.
Leaves
Physalin F 13.2 ± 3.4 29.4 ± 15.0 22.1 ± 3.1 20.3 ± 3.6 18.2 ± 1.3 38.1 ± 2.4a,b
Physalin B 16.6 ± 4.6 37.1 ± 19.3 25.4 ± 3.4 26.2 ± 4.4 31.4 ± 1.9 51.3 ± 3.2a,b
Physalin D 11.0 ± 1.4 20.8 ± 6.5a 9.3 ± 1.3 11.3 ± 2.2 10.7 ± 0.5b 17.8 ± 2.3a,b
Physalin G 2.3 ± 0.4 7.0 ± 2.8a 3.5 ± 0.5 3.0 ± 1.0 5.6 ± 0.4a 6.4 ± 1.0a,b
Physalin F 43.2 ± 3.0 20.6 ± 2.7a 7.6 ± 1.1a 25.7 ±11.4 11.5 ± 1.0b 17.2 ± 2.6b
Physalin B 45.2 ± 3.1 24.2 ± 3.5a 7.7 ± 1.2a 29.1 ± 12.7 22.8 ± 1.4 16.0 ± 2.2b
Physalin D 42.6 ± 3.3 18.7 ± 1.0a 3.5 ± 0.5a 17.4 ± 7.7b 8.9 ± 1.4b 8.1 ± 1.2b
Physalin G 5.0 ± 0.4 6.0 ± 0.4 0.6 ± 0.1a 4.3 ± 2.6 3.5 ± 1.1b 1.7 ± 0.3b
Significantly (p < 0.05) different values are indicated: arelative to those obtained for samples from plants watered daily (100% irrigation) or boriginating from non-primed (NP) seeds, respectively.
Fig. 3. Dendrogram of the growth characteristics and physalin content determined in plant organs of Physalis angulata after water restriction (a) and saline stress (b).
had negative consequences on P. angulata, with a decrease in the biomass of fruits, stems and roots due to salt stress (Table 3). The effects of saline solution on the dry mass cannot have been a consequence of the decrease in growth characteristics because the similarities in intensity among the respective parameters for each plant organ (of the mass and the respective number or diameter) were not identified by the cluster analysis (Fig. 2B). The best observed similarities among the parameters were found between the plant height and stem diameter and between the plant height and mass of leaves.
The main factors responsible for inhibiting plant growth by salt stress are the decrease in the osmotic potential of the soil, ionic toxicity and nutrient imbalances. As a result plants tend to reduce stomatal activity, preventing water loss through perspiration which therefore leads to decreases in plant growth and photosynthetic rates (Flowers, 2004).
3.3. Quantification ofphysalins in extracts of leaves and stems of P. angulata
Leaves and stems of the plants exposed to 13 days of water restriction and saline stress showed differences in their physalin content (Table 4).
It is interesting to observe that both water restriction and salt stress induced significant increases in the physalin content in leaves and decreases in stems (Table 4).
Leaves showed higher levels of physalins F, B, D and G (untreated seeds) or only B and G (primed seeds) in the plants exposed to water restriction than in the control group. Despite the seed type (non-primed or primed) the decreasing effect of water restriction on the physalin content in stems was observed for physalins F, B and D.
Salt stress induced the two most intense effects (p < 0.05) on physalin content which were dependent on the pre-germinative treatment: (i) increase (p < 0.05) in leaves of plants from primed seeds, reaching higher values than those found with untreated seeds, and (ii) decrease in the stems of plants from non-primed seeds.
The metabolic functions of the physalins on the protection of the plants against effects by elicitor agents in adverse environmental conditions are still not known, but the findings in the present work indicate that the application of the elicitor NaCl was associated with an increase in the production of physalins, mainly in the leaves of plants from primed seeds. These findings may be important to enhance production of physalins by applying a combination of cultivation techniques (saline irrigation) with pre-germinative conditioning (osmotic priming), which were shown to be favorable for emergence rates (Table 1) although these conditions affected both growth characteristics (Table 2) and biomass (Table 3).
Variations of the physalin content (Table 4) were compared to data regarding growth characteristics and to dry mass of leaves and stems (Tables 2 and 3) by a cluster analysis (Fig. 3). The results from plants from non-primed or primed seeds were grouped in this analysis. It can be observed that the effects on the physalin content in the leaf and in stem extracts occurred under different conditions than those affecting the biomass or dry mass, respectively. Thus the increase in the content of physalins (Table 4) does not seem to be associated to decreasing biomass. The effects on the biosynthesis of physalins could explain the difference in the content of the substances.
The different effects on the physalin content in leaves and stems are clear in the cluster analysis (Fig. 3A and B), regardless of the irrigation treatment. As can be observed in Table 4 and in Fig. 3, the content of physalins F and B was affected in a similar way. The saline solution was the treatment that increased physalins F and B in the leaves, and significantly (p < 0.05) in the primed seeds. There was more similarity among the effects on physalin D, F and B content than among the physalin G, F and B content, except only in the leaves of plants exposed to water restriction (Fig. 3). However, with few exceptions, the proportionality among the contents of the four physalins was maintained in leaves and stems of the P. angulata, regardless of the pre-germination and irrigation treatments used: B > F > D > G (Fig. 4). The ratios should match the values actually found in the respective plant parts, because they appear not to depend on the selectivity of the extraction solvent (ethanol). In polar solvents, as in this case, physalins have greater
Fig. 4. Box plot graph of the content distribution of each physalin relative to total identified physalins obtained in leaves and stems of Physalis angulata after all irrigation treatments (from Table 4, n = 36).
solubility than other steroids, but the proportionality of the contents (Fig. 4) occurred in the reverse order of the solubility in ethanol (D > G > F > B). Thus, the effects observed are suggested to have been the result of action on the biosynthetic pathway that precedes the synthesis of physalin B which seems to be the precursor of the other type B physalins, as physalins F, D and G in P. angulata (Row et al., 1980; Tomassini et al., 2002).
The importance of P. angulata in therapeutic applications justifies studies aimed at a better understanding of the biochemical processes that regulate the production of physalins. A large number of works have focused on the potential therapeutic properties of P. angulata however, there is a lack of work dealing with the induction of physalin biosynthesis. In this context the present work has addressed these issues investigating how the production of physalins in leaves and stems is influenced by seed priming and irrigation stresses (water restriction or saline stress) on P. angulata plants grown in a greenhouse.
Acknowledgments
This work was supported by the State of Bahia Research Support Foundation (FAPESB), National Council for Scientific and Technological Development (CNPq) and Program for Technological Development in Health Products (PDTIS/Fiocruz).
References
Abreu, I.N., Mazzafera, P., 2005. Effects of water and temperature stress on the content of active constituent of Hypericum brasiliense Choisy. Plant Physiology and Biochemistry 43, 241-248.
Abrol, E., Vyas, D., Koul, S., 2012. Metabolic shift from secondary metabolite production to induction of anti-oxidative enzymes during NaCl stress in Swertia chirata Buch-Ham. Acta Physiologiae Plantarum 34, 541-546. Alarcón, J.J., Morales, M.A., Ferrández, T., Sánchez-Blanco, M.J., 2006. Effects of water and salt stresses on growth, water relations and gas exchange in Rosmarinus officinalis. The Journal of Horticultural Science and Biotechnology 81, 845-853. Ankrah, N.A., Nyarko, A.K., Addo, P.G., Ofosuhene, M., Dzokoto, C., Marley, E., Addae, M.M., Ekuban, FA, 2003. Evaluation of efficacy and safety of a herbal medicine used for the treatment of malaria. Phytotherapy Research 17, 697-701. Bastos, G.N., Silveira, A.J., Salgado, C.G., Pican<;o-Dmiz, D.L., Do Nascimento, J.L., 2008. Physalis angulata extract exerts anti-inflammatory effects in rats by inhibiting different pathways. Journal of Ethnopharmacology 118, 246-251. Boutra, T., Sanders, F.E., 2001. Influence of water stress on grain yield and vegetative growth of two cultivars of bean (Phaseolus vulgaris L.). Journal of Agronomy and Crop Science 187, 251-257. Bradford, K.J., 1986. Manipulation of seed water relations via osmotic priming to improve
germination under stress conditions. Horticultural Science 21,1105-1112. Caceres, A., Menendez, H., Mendez, E., Cohobon, E., Samayoa, B.E., Jauregui, E., Peralta, E., Carrilo, G., 1995. Antigonorrhoeal activity of plants used in Guatemala for the treatment of sexually transmitted diseases. Journal of Ethnopharmacology 48,85-88. Capanoglu, E., 2010. The potential of priming in food production. Trends in Food Science
and Technology 21,399-407. Chartzoulakis, K., Klapaki, G., 2000. Response of two greenhouse pepper hybrids to NaCl
salinity during different growth stages. Scientia Horticulturae 86, 247-260. Chiang, H.C., Jaw, S.M., Chen, P.M., 1992a. Inhibitory effects of physalin B and physalin F
on various human leukemia cells in vitro. Anticancer Research 12,155-162. Chiang, H.C., Jaw, S.M., Chen, C.F., Kan, W.S., 1992b. Antitumor agent, physalin F from
Physalis angulata L. Anticancer Research 12,837-843. Choi, E.M., Hwang, J.K., 2003. Investigations of anti-inflammatory and antinociceptive activities of Piper cubeba, Physalis angulata and Rosa hybrida. Journal of Ethnopharmacology 89,171-175.
DaMatta, F.M., 2007. Ecophysiology of tropical tree crops: an introduction. Brazilian
Journal of Plant Physiology 19, 239-244. Dantas, I.B., Guimaraes, R.M., Pinho, E.V.R.V., Carvalho, M.L.M., 2010. Osmotic priming methodologies in relation to the physiological performance of rangpur lime seeds (Citrus limonia Osbeck). Revista Brasileira de Sementes 32,141-151. Ferreira, D.F., 2011. Sisvar: a computer statistical analysis system. Ciencia e Agrotecnologia 35,1039-1042.
Flors, V., Paradís, M., García-Andrade, J., Cerezo, M., Bosch, G.C., Pilar, GAP., 2007. A tolerant behaviour in salt-sensitive tomato plants can be mimicked by chemical stimuli. Plant Signaling & Behavior 2,50-57. Flowers, T.J., 2004. Improving crop salt tolerance. Journal of Experimental Botany 55, 307-319.
Freiburghaus, F., Kaminsky, R., Nkunya, M.H., Brun, R., 1996. Evaluation of African medicinal plants for their in vitro trypanocidal activity. Journal of Ethnopharmacology 55, 1-11.
Guimaraes, E.T., Lima, M.S., Santos, L.A., Ribeiro, I.M., Tomassini, T.B., Santos, R.R., Santos, W.L., Soares, M.B., 2009. Activity of physalins purified from Physalis angulata in vitro
and in vivo models of cutaneous leishmaniasis. Journal of Antimicrobial Chemotherapy 64,84-87.
Harborne, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481-504.
Heydecker, W., Coolbear, P., 1977. Seed treatments for improved performance survey and attempted prognosis. Seed Science and Technology 5,353-425.
Heydecker, W., Higgins, J., Gulliver, R.L., 1973. Accelerated germination by osmotic seed treatment. Nature 246, 42-44.
Iqbal, M., Ashraf, M., 2007. Seed treatment with auxins modulates growth and ion partitioning in salt-stressed wheat plants. Journal of Integrative Plant Biology 49, 1003-1015.
Jahangir, M., Abdel-Farida, I.B., Kim, H.K., Choia, Y.H., Verpoorte, R., 2009. Healthy and unhealthy plants: the effect of stress on the metabolism of Brassicaceae. Environmental and Experimental Botany 67,23-33.
Jaleel, CA, Manivann, P., Lakshmanan, G.M.A., Gomathinayagam, M., Panneerselvam, R., 2008. Alterations in morphological parameters and photosynthetic pigment responses of Catharanthus roseus under soil water deficits. Colloids and Surfaces. B, Biointerfaces 61,298-303.
Januario, A.H., Rodrigues-Filho, E., Pietro, R.C.L.R., Kashima, S., Sato, D.N., Franca, S.C., 2002. Antimycobacterial physalins from Physalis angulata L. (Solanaceae). Phytotherapy Research 16,445-448.
Kagale, S., Divi, U.K., Krochko, J.E., Keller, W.A., Krishna, P., 2007. Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses. Planta 225, 353-364.
Karamanos, A.J., Elston, J., Wadsworth, R.M., 1982. Water stress and leaf growth of field beans (Vicia faba L.) in the field: water potentials and laminar expansion. Annals of Botany 49, 815-826.
Khan, A.A., 1992. Preplant physiological seed conditioning. Horticultural Review 13, 131-181.
Koyro, H.W., 2000. Effect of high NaCl-salinity on plant growth, leaf morphology, and ion composition in leaf tissues of Beta vulgaris ssp. marittima. Journal of Applied Botany — Angewandte Botanik 74, 67-73.
Krishna, P., 2003. Brassinosteroid-mediated stress responses. Journal of Plant Growth Regulation 22, 289-297.
Kutchan, T.M., 2001. Ecological arsenal and developmental dispatcher. The paradigm of secondary metabolism. Plant Physiology 125,58-60.
Lee, H.Z., Liu, W.Z., Hsieh, W.T., Tang, F.Y., Chung, J.G., Leung, H.W.C., 2009. Oxidative stress involvement in Physalis angulata-induced apoptosis in human oral cancer cells. Food and Chemical Toxicology 47, 561-570.
Lin, Y.S., Chiang, H.C., Kan, W.S., Hone, E., Shih, S.J., Won, M.H., 1992. Immunomodulatory activity of various fractions derived from Physalis angulata L. extract. The American Journal of Chinese Medicine 20, 233-243.
Lopez-Berenguer, C., Martinez-Ballesta, M.C., Garcia-Viguera, C., Carvajal, M., 2008. Leaf water balance mediated by aquaporins under salt stress and associated glucosinolate synthesis in broccoli. Plant Science 174, 321-328.
Miranda, D., Fischer, G., Ulrichs, C., 2010. Growth of cape gooseberry (Physalis peruviana L.) plants affected by salinity. Journal of Applied Botany and Food Quality 83,175-181.
Munns, R., 2002. Comparative physiology of salt and water stress. Plant, Cell & Environment 25, 239-250.
Patane, C., Cavallaro, V., Cosentino, S.L., 2009. Germination and radicle growth in unprimed and primed seeds of sweet sorghum as affected by reduced water potential in NaCl at different temperatures. Industrial Crops and Products 30, 1 -8.
Ribeiro, I.M., Silva, M.T.G., Soares, R.D.A., Stutz, C.M., Bozza, M., Tomassini, T.C.B., 2002. Physalis angulata L. antineoplastic activity, in vitro, evaluation from it's stems and fruit capsules. Revista Brasileira de Farmacognosia 12, 21-22.
Roberts, E.H., Ellis, R.H., 1989. Water and seed survival. Annals of Botany 63, 39-52.
Row, L.R., Reddy, K.S., Sarma, N.S., Matsuura, T., Nakashima, R., 1980. New physalins from Physalis angulata and Physalis lancifolia. Structure and reactions of physalins D, I, G and K. Phytochemistry 19,1175-1181.
Sa, M.S., Menezes, M.N., Krettli, A.U., Ribeiro, I.M., Tomassini, T.C.B., Santos, R.R., Azevedo Jr., W.F., Soares, M.B.P., 2011. Antimalarial activity of physalins B, D, F, and G. Journal of Natural Products 74, 2269-2272.
Sadeghian, S.Y., Yavari, N., 2004. Effect of water deficit stress on germination and early seedling growth in sugar beet. Journal of Agronomy and Crop Science 190, 138-144.
Sairam, RK, Tyagi, A., 2004. Physiology and molecular biology of salinity stress tolerance in plants. Current Science 86, 407-421.
Soares, M.B.P., Bellintani, M.C., Ribeiro, I.M., Tomassini, T.C.B., Santos, R.R., 2003. Inhibition of macrophage activation and lipopolysaccaride-induced death by seco-steroids purified from Physalis angulata L. European Journal of Pharmacology 459,107-111.
Soares, M.B.P., Brustolim, D., Santos, L.A., Bellintani, M.C., Paiva, F.P., Ribeiro, Y.M., Tomassini, T.C.B., Santos, R.R., 2006. Physalins B, F and G, seco-steroids purified from Physalis angulata L., inhibit lymphocyte function and allogeneic transplant rejection. International Immunopharmacology 6, 408-414.
Souza, M.0.de, Souza, C.L.M., Pelacani, C.R., 2011. Germination of osmoprimed and non-osmoprimed seeds and initial growth of Physalis angulata (Solanaceae) in saline environments. Acta Botanica Brasilica 25,105-112.
Taiz, L., Zeiger, E., 2010. Plant Physiology, Fifth ed. Sinauer Associates Publishers, Sunderland, Massachusetts.
Tomassini, T.C.B., Barbi, N.S., Ribeiro, I.M., Xavier, D.C.D., 2000. Genus Physalis — a revision of withasteroids. Quimica Nova 23,47-57.
Tomassini, T.C.B., Santos, R.R., Soares, M.B.P., Xavier, D.C.D., Barbi, N.S., Ribeiro, I.M., Soares, R.O.A., Fernandez-Ferreira, E.P., 2002. Process for isolating physalins from plants and pharmaceutical composition containing physalins. U.S. Patent Application U.S. 2002/0103386 A1.
Torssell, K.B.G., 1997. Chemical ecology. In: Torssell, K.B.G. (Ed.), Natural Product Chemistry, a Mechanistic Biosynthetic and Ecological Approach. Pharmaceutical Press, Swedish, pp. 42-79. Varier, A., VarI, A.K., Dadlani, M., 2010. The subcellular basis of seed priming. Current Science 99, 450-456.
Yu, L.J., Lan, W., Qin, W.M., Xu, H.B., 2002. High stable production of taxol in elicited
synchronous cultures of Taxus chinensis cells. Process Biochemistry 38,207-210. Zhao, J., Fujita, K., Yamada, J., Sakai, K., 2001. Improved p-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate. Applied Microbiology and Biotechnology 55, 301-305.
