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Allelopathic effect of Solanum lycocarpum leaf extract on protein synthesis in sesame seedlings

Camila A. Jerônimo1, Fabian Borghetti1 and Cezar Martins de Sá2

1 Departamento de Botanica, Universidade de Brasilia, 70910-970, Brasilia, Brasil. Email: fborghet@unb.br
2
Departamento de Biologia Celular Universidade de Brasilia, 70910-970, Brasilia, Brasil. Email: sasa@unb.br

Abstract

Solanum lycocarpum is a shrub of the Cerrado vegetation in central Brazil. Aqueous extracts of its leaves at 1% significantly reduced root growth and inhibited root hair and lateral root differentiation in sesame (Sesamum indicum) seedlings. In order to identify proteins involved in these morphological changes, we have investigated the protein synthesis during sesame growth in water and in leaf aqueous extracts. One-day old sesame seedlings were incubated at 30oC under a 12h photoperiod (white light), either in 1% (w/v) aqueous leaf extract or in distilled water. Protein homogenates were prepared from root and shoot parts (except cotyledons) after 48 hours of incubation. Six hours before protein extraction 35S-methionine was applied on the roots. The proteins were solubilized in a Laemmli buffer (Tris 80mM, SDS 2%, mercaptoethanol 2%, pH 6.8), centrifuged and the supernatant stored at -18oC. The protein samples were run in a SDS-PAGE gel electrophoresis at 50 mA. After, the gels were dried and exposed to kodak t-mat films for seven days. The comparison of fluorograms of the control (water) and treated (leaf extract) seedlings showed a few differences. A polypeptide of about 50 kDa was induced in the roots, and a polypeptide of 45 kDa was induced in the shoot parts of the treated seedlings. On the other hand, a polypeptide of about 66 kDa almost disappeared from the fluorograms of the treated roots. These preliminary results suggest that the morphological changes observed in treated sesame seedlings seem to be related to a few changes in protein turnover.

Media Summary

Aqueous leaf extract of Solanum lycocarpum at 1% inhibited root growth and differentiation, and induced changes in protein turnover of sesame seedlings.

Keywords

Allelopathy, Cerrado, sesame, Solanum lycocarpum, Protein

Introduction

Several allelopathic studies have attempted to investigate the effects of either plant extracts or isolated chemicals on plant growth and metabolism. By this approach it has been shown that phytotoxic terpenoids can inhibit enzyme activity (as cineole), disrupt plasma membrane (dehydrozaluzanin C) (Duke and Oliva 2004), and phenolic compounds as cinnamic and benzoic acids can also act on enzyme and phytohormone activity and on mineral uptake (Einhellig 2004), among other examples (Macias et al. 2004). On the other hand, a few studies have been conducted to investigate the effects of plant extracts on protein synthesis. Romero-Romero et al. (2002) showed that aqueous extracts of four native shrubs of the mexican desert (Sicyos deppei, Accacia sedillense, Sebastiania adenophora and Lantana camara) reduced root growth and induced an overall increase in protein synthesis in roots of Zea mays, Phaseolus vulgaris, Cucurbita pepo and Lycopersicon esculentum. Allelochemicals produced by leaves of Callicarpa acuminata were tested on root growth, protein synthesis and enzyme activity of seedlings of P. vulgaris, L. esculentum and Z. mays (Cruz-Ortega et al., 2002). In particular, the synthesis of a protein (99% similarity with subunits of α-amilase inhibitor) was induced in roots of P. vulgaris and the synthesis of a polypeptide (69-95% similarity to glutathione-S-transferase) was promoted in roots of L. esculentum (Cruz-Ortega et al., 2002).

Solanum lycocarpum St. Hil. (Solanaceae) is a shrub native of the Cerrado vegetation in the Central Brazil. This species is very common in disturbed lands, as pasture and fragmented areas. Single fruits may reach weight of 400-600 g and contains around 400 seeds. Previous studies have shown that seeds (Borghetti, Pessoa 1997), leaves (Oliveira et al. 2004a) and fruits (Oliveira et al. 2004b) of S. lycocarpum have allelopathic properties, inhibiting both seed germination and seedling growth of some target species as Sesamum indicum (sesame). In particular, aqueous leaf extracts were found to inhibit root growth and root hair differentiation and to impair geotropic curvature of sesame seedlings at concentration as low as 1% (figure 1). In order to identify proteins involved in these morphological changes, this paper aims at investigating the effects of S. lycocarpum leaf extracts on root and shoot growth and on protein synthesis of sesame seedlings.

Figure 1: Effects of aqueous leaf extracts of Solanum lycocarpum on the growth of Sesamum indicum seedlings. From right to left, 0, 1, 2, 3 and 4% leaf extract concentration (w/v). The seedlings were grown for five days at 30oC, photoperiod of 12 h (white light). White bar = 1 cm.

Methods

Sesamum indicum (Pedaliaceae) has been used as the target species because their seeds present a fast germination (Carvalho et al. 2001) and their seedlings grow uniformly at 30°C. Seeds of sesame were provided by EMBRAPA (Brazilian Agricultural Research Corporation). To prepare the extracts, mature leaves of S. lycocarpum were harvested from naturally growing population in the campus of the Universidade de Brasilia. The leaves were oven-dried at 50°C for 24 h, grounded with a pistil and a mortar, and dissolved in distilled water to obtain a 5% stock solution. After 24 h of incubation at 4°C, the stock solution was filtered and diluted to prepare 1% and 3% test solutions (w/v). The test solutions were prepared just before their use in the experiments. One-day old sesame seedlings were disposed in Petri dishes layered with one filter-paper and the respective test solution. Distilled water was used as control. The experiments were conducted at 30°C, and a photoperiod of 12 h (white light). After two and five days of incubation, roots and shoots of the treated seedlings were measured and compared to the control (Kruskal Wallis`Test, p=0.05; Biostat 2.0).

Protein homogenates were prepared from root and shoot parts (except cotyledons) after 48 hours of incubation both in water and in the extract at 1%. Six hours before protein extraction 35S-methionine (about 30 µCi/seedling) was applied on the root hairs. The proteins were dissolved in a modified Laemmli buffer (Tris 80mM, SDS 2%, β-mercaptoethanol 2%, pH 6.8), centrifuged at 10000 rpm/10 min and the supernatant stored at -18°C. The protein samples (20 μg per lane) were run in a SDS-PAGE gel electrophoresis at 50 mA. After, the gels were stained in coomassie blue, dried and exposed to kodak t-mat films for up to seven days.

Results

Leaf extracts of S. lycocarpum significantly reduced root growth, and inhibited root hair and lateral root differentiation in sesame seedlings after two and five days of incubation (table 1).

Table 1. Effect of aqueous leaf extracts of Solanum lycocarpum on the growth of shoots and roots and on root differentiation of Sesamum indicum. N=30/treatment. The seedlings were grown for two or five days at 30oC under white light at two extract concentration (w/v). Different letters within columns indicate significant differences in the average length (Kruskal Wallis, p=0.05). Standard deviation are in parenthesis. +++ relative quantity in respect to the control.

Treatment

Shoot length (cm)

Root length (cm)

Lateral roots

Root hairs

 

Two days

Five days

Two days

Five days

   

Water

0.77 (0.20) a

1.72 (0.41) a

1.88 (0.49) a

5.42 (1.60) a

++++

+++++

1% leaf extract

0.57 (0.22) b

1.41 (0.42) a

0.68 (0.27) b

1.55 (0.71) b

+++

+++

3% leaf extract

0.52(0.22) b

0.73 (0.48) b

0.37 (0.25) b

0.25 (0.31) c

++

+

The roots were more affected than the shoots. After five days the root growth was reduced by about 80% at extract at 1%, but the shoot growth was not significantly affected up to 3% concentration (table 1).

Despite the effects of the extracts on shoot growth, no abnormality was observed in the shoot morphology of the treated seedlings. The cotyledons expanded, and the hypocotyls greened similarly to the control seedlings. Therefore, it is not known whether the reduction of the shoot growth was a direct effect of the extracts on the shoots or simply a consequence of extract effects on root metabolism. These results are similar to that found by Oliveira et al. (2004a), and indicate that for sesame the roots are more susceptible to the leaf extracts than the shoots.One-dimensional gel electrophoresis of the protein samples obtained from root and shoot parts of sesame showed almost no difference when comparing control versus treated seedlings. Protein patterns of roots and shoots were very similar (figure 2-A).

A: Gel

B: Fluorogram

Figure 2: One-dimensional SDS-PAGE gel electrophoresis (left) and fluorogram (right) of protein homogenates obtained from roots and shoots of Sesamum indicum incubated for two days in water or 1% aqueous extract of leaves of Solanum lycocarpum. Twenty micrograms of proteins were loaded per lane. Molecular mass (kDa) is indicated on the left. The treatments are indicated below the lanes.

The comparison of fluorograms of the control and treated seedlings apparently showed three major differences (figure 2-B). A polypeptide of about 50 kDa was induced in the roots and a polypeptide of about 45 kDa was induced in the shoots of the treated seedlings. On the other hand, a polypeptide of about 66 kDa almost disappeared from the fluorograms of treated roots. Overall, the protein synthesis seemed to be more intense in the roots than in the shoots. As expected, differences can be found when comparing protein patterns of roots and shoots, irrespective whether the seedling was incubated in water or in the leaf extract. We are in the course to run bi-dimensional gel electrophoresis for better comparisons of protein patterns of control and treated seedlings.

Conclusion

The effects of S. lycocarpum leaf extracts on sesame growth and differentiation at concentration as low as 1% point out the allelopathic potential of this species of the Cerrado vegetation. These first results regarding protein turnover during sesame growth suggest that the morphological changes observed in treated sesame seedlings are related to a few changes in protein synthesis. The investigation of allelopathic effects on protein synthesis opens the perspective of a better knowledge of allelochemical action at biochemical level.

Aknowledgements

We thank to Fabio N. Noda and Liliam de Oliveira F. Maçanero for technical assistance and CAPES for providing scholarship to C. A. Jeronimo. Fabian Borghetti also thanks to the FINATEC (Fundação de Empreendimentos Científicos e Tecnológicos / Brasilia) for providing support to attend the meeting.

References

Borghetti F, Pessoa DMA (1997) Autotoxicidade e alelopatia em Sementes de Solanum lycocarpum. St. Hil. (Solanaceae) In ‘Contribuição ao conhecimento ecológico do Cerrado’. (Eds LL Leite, CH Saito) pp 54-58. (EdUnB)

Carvalho PGB, Borghetti F, Buckeridge MS, Morhy L, Ferreira Filho EX (2001) Temperature-dependent germination and endo-b-mannanase activity in sesame seeds. Braz. J. Plant Physiol. 13(2), 139-148.

Cruz-Ortega R, Ayala-Cordero G, Anaya AL (2002) Allelochemical stress produced by the aqueous leachate of Callicarpa acuminata: effects on roots of bean, maize, and tomato. Physiol. Plantarum 116, 20-27

Duke SO, Oliva A (2004) Mode of action of phytotoxic terpenoids. In ‘Allelopathy: Chemistry and mode of action of allelochemicals’. (Eds FA Macias, JCG Galindo, JMG Molinillo, HG Cutler) pp. 201-216. (CRC Press)

Einhellig FA (2004) Mode of allelochemical action of phenolic compounds. In ‘Allelopathy: Chemistry and mode of action of allelochemicals’. (Eds FA Macias, JCG Galindo, JMG Molinillo, HG Cutler) pp. 217-238. (CRC Press)

Macias FA, Galindo JCG, Molinillo JMG, Cutler HG (2004) Allelopathy: Chemistry and mode of action of allelochemicals. CRC Press. 372 p.

Oliveira SCC, Ferreira AG, Borghetti F (2004a) Allelopathic effect of Solanum lycocarpum St. Hil. leaves on the germination and growth of Sesamum indicum L. under different temperatures. Acta Botanica Brasilica 18(3), 401-406.

Oliveira SCC, Ferreira AG, Borghetti F (2004b) Effect of Solanum lycocarpum fruit extract on sesame seed germination and seedling growth. Allelopathy Journal 13(2), 201-210.

Romero-Romero T, Anaya AL, Cruz-Ortega R (2002) Screening for the effects of phytochemical variability on cytoplasmic protein synthesis pattern of crop plants. Journal of Chemical Ecology 28, 601-613.

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