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Sunflower Allelochemicals Mode of action in germinating Mustard Seeds.

Renata Bogatek1, Agnieszka Gniazdowska1, Joanna Stępień1, Ewa Kupidłowska2

1Department of Plant Physiology, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland, www.sggw.waw.pl Email bogatek@delta.sggw.waw.pl
2
Department of Plant Anatomy and Cytology, Institute of Plant Experimental Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland, www.biol.uw.edu.pl

Abstract

Commonly observed actions of allelochemicals are inhibition of seed germination and abnormalities of seedling development. Catabolism of storage reserves and energy production are most important factors ensuring undisturbed seed germination.

The inhibiting effect of sunflower allelochemicals on seed germination appears to be mediated through a disruption of cellular metabolism rather than through organelle damage. Reserve mobilisation seems to be blocked or delayed during allelopatic stress. Disorder in respiration rate (O2 uptake) leads to a limitation on the availability of metabolic energy (ATP level) and in consequence decreases seed germination and seedlings growth.

Media summary

Sunflower allelochemicals block catabolism of storage reserves and energy production during germination of mustard seeds.

Key Words

Reserve mobilisation, respiration, ATP/ADP level, isocitrate lyase, oil/protein bodies

Introduction

Sunflower can actively influence the growth of surrounding plants due to its high allelopathic potential. More than 200 natural allelopathic compounds from different cultivars of sunflower were isolated (Macias et al. 2002 and references therein). Most of the known allelochemicals, inhibit or delay seed germination. This effect was reported for seeds of many species (Inderjit and Duke 2003; Weston and Duke 2003).

Germination consist of several different phases: imbibition, catabolic and finally anabolic phase resulting in radicle protrusion. Therefore radicle emergence is considered as completion of germination and the starting point for seedling growth. During catabolic phase storage materials are digested supporting substrates for biosynthetic processes and respiration. Storage proteins in maturing seeds are accumulated mostly in insoluble forms as protein bodies, which are digested by peptidases during seed germination to soluble peptides, degraded then to aminoacids (Bewley and Black 1994). It was reported, that in seeds of Brassicaceae breakdown of storage proteins starts earlier and proceeds faster than mobilization of storage oils (Murphy et al. 1989). Triglycerides present in oleosomes are β-oxidated to fatty acids which are converted to acetyl-CoA and than to sucrose through the glyoxylate cycle and gluconeogenesis. Isocitrate lyase (ICL) is the key enzyme of the glyoxylate cycle, thus it’s activity may by crucial for providing substrate (succinate) for mitochondrial respiration.

The aim of this study was to investigate the effect of the allelochemicals from sunflower leaf on germination of mustard seeds. Reserves (proteins and lipids) mobilization processes as well as respiration during seed germination were studied both at the biochemical and ultrastructural level.

Material and methods

Plant material and growth conditions

Allelopathic extract from sunflower (Helianthus annuus) cv. Ogrodowy (10 % (w/v)) was prepared as described by Bogatek et al. (2005).

Mustard seeds (Sinapis alba L.) were germinated in 9 cm Petri dishes (50 seeds per dish) on a filter paper moistened with extract from sunflower leaf or distilled water (control) at 20 C in darkness. Seed viability was estimated using the tetrazolium test. Germination and seed viability were determined after 0.75 (18 hours), 2, 4 and 8 days in 5 repetitions.

Oxygen uptake

Oxygen consumption by intact seeds was measured with a Hansatech gas-phase oxygen electrode unit in a 20 ml container at room temperature in darkness. Results are expressed as nmol O2/g FW/s and correspond to the means of the values SD from 3 independent repetitions.

Adenosine phosphate assays

Adenosine phosphates were extracted from seeds according to Olempska-Beer and Bautz-Freeze (1984). ATP and ADP contents of the extracts were measured using the bioluminescence method, as described by Saglio et al. (1979). Results are the means of the values SD from 3 independent repetitions.

Enzyme determination

Isocitrate lyase activity (threo-Ds-isocitrate glyoxylate lyase, EC 4.1.3.1, ICL) was determined according to Bogatek et al. (1989). Results are expressed as nmol/mg prot/h and correspond to the means of the values SD from 5 independent repetitions.

Proteins were determined by the method of Bradford (1976) using bovine serum albumin (BSA) as a standard.

Microscopic observations

Small block of tissues from cotyledon, radicle and the basal part of the hypocotyl, were fixed according to Karnovsky (1965) after 0, 18, 48 hours of imbibition in water or in extract from sunflower leaf. After 24 h postfixation in 1% osmium tetroxide samples were dehydrated through a graded alcohol series and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate (Reynolds 1963) and examined with a JEOL 1200X electron microscope.

Results and discussion

Almost all (97 %) control mustard seeds germinated within 4 days (Table 1). The prolonged culture in water resulted in growth of etiolated seedlings. Allelochemicals from sunflower leaf completely inhibited germination of mustard seeds (Table 1). Even after 8 days of imbibition no radicles pierced the seed envelope nor was seedling growth observed. During the whole experiment (till the 8th day) treated seeds remained at the developmental stage complementary to that detected for control seeds after 0.75 day. Therefore seeds after 0.75 days of imbibition in water were regarded as the control. Moreover seed imbibition during first 18 hours of sunflower extracts treatment showed no statistically significant differences compared to seeds germinated in water (data not shown), indicating that alterations in seed germination are due to toxicity of allelopathic compounds not only to water stress (Oracz et al. 2004). Although sunflower allelochemicals inhibited seed germination, their influence on seed viability was less pronounced. During the first four days of germination in the presence of sunflower allelochemicals most seeds remained viable (Table 1). The extended treatment was lethal leading to the death of all seeds after two weeks (Bogatek et al. 2005). Inhibition of wild mustard seed germination in the presence of leaf extracts from four different sunflower cultivars was previously observed by Leather (1983).

Table 1. Mustard seed germination and viability in the presence of 10 % (w/v) sunflower leaf extract. SD was less than 2 %.

 

Germination (%)

Viability (%)

 

0.75 days

2 days

4 days

8 days

0.75 days

2 days

4 days

8 days

Control

22

78

97

98

98

97

95

97

Allelopathy

0

1

2

4

95

92

84

65

As in other fat-storing species, during mustard seed germination, the glyoxylate cycle plays a key role in catabolism of triacylgricerides present in storage tissue. Conversion of isocitrate into succinate catalyzed by ICL provides substrates for biosynthetic processes and mitochondrial respiration. ICL activity increased in control seeds (Figure 1) and in young seedlings cultured in water (data not shown). ICL activity in mustard seeds germinating in the presence of sunflower allelochemicals increased similarly as in the control during the early catabolic phase. It declined to low levels during the next 3 days (Figure 1), indicating inhibition in further lipid mobilization. Similar results were detected during canola (Brassica napus) seeds germinating in the presence of ferulic and p-coumaric acids (Baleroni et al. 2000), or sunflower seeds germination affected by alkaloids from Datura stramonium (Levitt et al. 1984). Maffei et al. (1999) noticed decreased ICL activity and the disappearance of ICL protein in extracts of cucumber (Cucumis sativus) seedlings incubated with 3,4-dimethoxybenzoic and vanilic acids. Decrease in ICL activity in allelopathy stressed seeds were in agreement with ultrastructural observation. The cup-shaped oil bodies, arising due to partial digestion of the lipid contents (Bergfeld et al.1978; Huang 1992) were visible both in control and treated embryos (Figure 2 A, D) at the beginning of germination; but afterwards, the oil bodies disappeared only in the control, whereas their number remained significant in the treated material (Figure 2 C, F).

Figure 1. Activity of isocitrate lyase in control mustard seeds germinated in water (□) or in the presence of sunflower leaf allelochemicals (■).

Total endopeptidase activity measured in the crude extract both in control and allelopathy stressed mustard seed was low and increased only a little during the first 18 hours of imbibition. Longer allelopathy treatment did not influence endopeptidase activity, which remained at steady state levels (data not shown).

Degradation of protein bodies, which appeared as a loss of their dense matrix was visible only in the control (Figure 2 B). Small vacuoles developed as a result of storage protein digestion, fused to form in the cells a large vacuolar system (Figure 2C). In the cells of treated embryos, the structure of protein bodies was unchanged in the culture period (Figure 2 E, F).

Figure 2. Ultrastructural changes in storage organelles of radicle cells during germination, A-C control, D-E treated with sunflower allelochemicals. 36 h of culture: A and D cup-shaped oil bodies, B digested and E undigested protein bodies. 48 h of culture: C fusion of vacuoles following storage protein digestion, F numerous, undigested protein and oil bodies.

Respiration rate in control seeds represented the typical characteristic during seed germination (Figure 3 A). It increased markedly when the growth phase had started (for control seedlings cultured in water; data not shown). In the presence of sunflower allelochemicals oxygen uptake by seeds remained at a low level (similar to that detected for dry seeds) during the whole period of the experiment. An insignificant increase was observed only during the early catabolic phase. Low oxygen uptake by mustard seeds treated by sunflower leaf extract correlated well with decreased ATP/ADP levels (Figure 3 B). Low energy production may be due not only to low respiration rate but also to increased partitioning of alternative oxidase in mitochondrial the electron transport chain as it was reported in many plants under allelopathic stress (Peňuelas et al. 1996; Cruz–Ortega et al. 1988; Massardo et al. 1994).

A

B

Figure 3. Oxygen uptake by mustard seeds (A) and ATP/ADP ratio (B) in mustard seeds during germination in water (□) or in the presence of sunflower leaf allelochemicals (■).

Conclusions

Both biochemical and cytological observations indicate that mobilization of storage proteins is almost completely suppressed, while degradation of lipid bodies is initiated slightly only during the early phase of germination in mustard seeds treated with sunflower allelochemicals. The delay or inhibition of reserve mobilization, a process which usually takes place rapidly during seed germination, can lead to an insufficient supply of respiratory substrates, resulting in low respiration rate and in consequence ATP deficiency in seeds exposed to allelochemicals. Thus the presented data suggest that the inhibiting effect of sunflower allelochemicals on mustard seed germination appears to be mediated through a disruption of normal cellular metabolism rather than through organelle damage.

Acknowledgments

The authors express their gratitude to Dr D. Vinell from laboratory Physiologie Vegetale Appliquee, Universite P. & M. Curie, France headed by Prof. F. Corbineau for ATP and ADP determination.

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