Y. Sreenivasulu and Dilip Amritphale*

School of Studies in Botany, Vikram University, Ujjain 456 010, India

Relative efficacy of various dormancy breaking chemicals is known to relate to their physical/chemical properties. Acetaldehyde required a much shorter treatment time to stimulate the germination of secondarily dormant cucumber (Cucumis sativus L.) seeds than ethanol and other less lipophilic compounds. T₅₀ (exposure time required for 50% germination) was inversely correlated to the K_o/w (octanol–water partition coefficients) of the chemicals applied. Lipid bilayer fluidity in microsomal membranes of chemically-treated seeds was greater than in untreated control seeds imbibed in water for an equal time period. There was a significantly positive correlation between T₅₀ and fluorescence anisotropy of the outer lipid monolayer in seed microsomal membranes, which indicated that membrane fluidity could be a significant event during the process of chemically-induced germination of secondarily dormant cucumber seeds.

Secondary seed dormancy in cucumber is known to be released by acetone, ethanol and a few other organic compounds with a concomitant change in the permeability of the cell membranes¹². The aim of the present investigation was to determine (i) whether the dormancy-breaking activity of certain nonhormonal chemicals in cucumber is a function of their lipophilicity, and (ii) whether a change in fluidity, if any, in seed cellular membranes occurs during the chemical-induced transition from dormancy to germination in secondarily dormant cucumber seeds.

*For correspondence.

Seeds of cucumber (Cucumis sativus L. cv. Poinsett 76) were purchased from Indo-American Exports, Bangalore, India. Dormancy was induced by exposing nondormant seeds (germination ~ 95% at 20°C in darkness) to intermittent far-red light (7.5 m mols m^–2 s^–1) (ref. 13) while imbibing in – 1.8 MPa polyethylene glycol solution in darkness at 20 ±  1°C. Secondarily dormant seeds thus obtained (germination ~ 10% at 20°C in darkness; viability ~ 95% in tetrazolium tests¹⁴ were immersed for various durations in acetaldehyde, n-propanol, acetone, ethanol and methanol in glass-stoppered flasks at 10°C. Besides offering a reasonable K_o/w range (octanol–water partition coefficients)¹⁵, the selected chemicals showed no visible toxic effects on germination and subsequent seedling growth, at least for the treatment duration and temperature tested. After the treatment, the seeds were air-dried for 24 h and then used in germination and other studies.

All germination tests were conducted with 25 seeds per replicate and 4 replicates per treatment. Seeds were allowed to imbibe on water-saturated filter paper circles for 3 days at 20 ±  1°C in darkness. The criterion for germination was the emergence of the radicle through the testa. The germination experiments were repeated at least twice. The average values for percent germination were arcsin transformed before statistical analysis.

Microsomal membranes were isolated from 18 h water-imbibed dormant (control)/chemically-treated seeds following Hodges and Mills¹⁶ and Wheeler and Boss¹⁷. This period was selected because splitting of the perisperm–endosperm envelope, the first visible germination event in cucumber seed, began between 18 and 24 h of dark imbibition in water at 20°C in the chemically-treated seeds. Changes in the structure and functions of membranes, if any, after this time period could be an admixture of germination/seedling biochemistry not highly relevant to the transition from dormancy to germination. Decoated seeds (testa removed) were homogenized in 50 mM Tris-MES (pH 7.2) containing 0.25 mM sucrose, 3 mM ethylenediamine tetra-acetic acid (EDTA), 2.5 mM dithiothreitol (DTT), 2 mM phenylmethylsulphonyl fluoride (PMSF) and 0.6% insoluble polyvinylpyrrolidone (PVP) in a prechilled pestle-mortar. The homogenate was filtered through a nylon mesh and centrifuged at 10,000 g for 10 min at 0°C in a refrigerated centrifuge with a fixed angle rotor (Kubota 6900, Japan). The supernatant was recentrifuged at 36,000 g for 45 min at 0°C and the resulting pellet (microsomal fraction) was suspended in a medium containing 5 mM potassium phosphate (pH 7.8), 0.25 M sucrose and 50 mM EDTA. The absorbance of the membrane suspension was adjusted to 0.05 absorbance unit at the corresponding excitation peaks each for 1,6-diphenyl 1,3,5-hexatriene (DPH), and its derivatives DPH-propionic acid (DPH-PA) and trimethylammonium-DPH (TMA-DPH) to standardize light scattering¹⁸.

Figure 1.  Effect of acetaldehyde (a), n-propanol (b), acetone (c), ethanol (d), and methanol (e) on germination (expressed as % G) of secondarily dormant cucumber seeds.

Fifty microlitres of 2 mM DPH, DPH-PA and TMA-DPH each in dimethylsulphoxide were added in 25 ml vigorously stirring suspension medium. Twenty five

Table 1.  Fluorescence anisotropy of DPH-, DPH-PA-, and TMA-DPH-labelled microsomal
membranes from 18 h water-imbibed dormant (control) and chemically-treated seeds

T₅₀ (min) K₀/w

Figure 2.  Relationship between K_o/w (octanol–water partition coefficients) and T₅₀ (time required to stimulate 50% germination); acetaldehyde (A), n-propanol (B), acetone (C), ethanol (D), and methanol (E).

microlitres for each probe were added separately to 5.0 ml membrane suspension. Labelling was performed at 37°C for 30 min. Fluorescence polarization measurements were carried out with LS 30 luminescence spectrometer (Perkin-Elmer, UK). The samples at 20°C were excited with vertically polarized light at 350 nm (DPH) 354 nm (DPH-PA) and 355 nm (TMA-DPH) and, vertically and horizontally polarized light emissions were read at 452 nm (DPH) and 430 nm (DPH-PA and TMA-DPH). Anisotropy values were calculated from the degree of polarization in accordance with Shinitzky and Barenholz¹⁹. Acetaldehyde required relatively much shorter exposure time to evoke germination level equal to other chemicals (Figure 1). Among other chemicals, a 20 min exposure to acetone and ethanol induced much higher germination than an equal exposure to n-propanol and methanol. Treating seeds more than 30 min with n-propanol and methanol, but not with acetone and ethanol, reduced percent germination possibly on account of toxicity (data not given). Because the maximum germination inducible with n-propanol was relatively lower than that obtained with other chemicals, T₅₀ (exposure time required for 50% germination) for each chemical was determined from a regression equation (Figure 1) and was plotted against K_o/w (an index of lipophilicity) for each chemical for lipophilicity – activity comparisons (Figure 2). Acetaldehyde, which had the highest lipophilicity among the chemicals tested, required lowest exposure time to induce 50% germination, whereas methanol having the lowest lipophilicity showed highest T₅₀. Cohn³, who used primarily dormant red rice seeds as a model system, showed the efficacy of dormancy-breaking compounds applied in aqueous phase to be an inverse function of their lipophilicity. In the present work with secondarily dormant cucumber seeds also a negative correlation was found between K_o/w and dormancy-breaking activity of the tested chemicals applied in nonaqueous phase.

Besides alcohols, several dormancy-breaking compounds such as ethyl ether, chloroform, acetone and others are known to affect the membranes^4,10,11. The fluorescence polarization has proven to be one of the most sensitive, reproducible and convenient means of probing the fluidity and organization of membranes²⁰. Comparison of fluorescence anisotropy, as monitored by DPH, which is known to partition uniformly in the lipid bilayer thus giving an overall reflection of the membrane status^18,19, indicated an increase in the fluidity of membranes with all the chemicals except methanol (Table 1). This suggested that the lipid bilayer in microsomal membranes in dormant seeds was relatively more rigid than those from treated seeds. However, because the membranes isolated from the dry dormant (control) and chemically-treated seeds showed little difference in fluorescence anisotropy values (data not shown), presumably the change occurred later in the germination.

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Received 18 April 1998; revised accepted 21 September 1998.

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