Phytotoxicity and Distribution of Sorgoleone in Grain Sorghum Germplasm

INTRODUCTION

Recently, a more phytotoxic inhibitor was discovered within the root exudates of grain sorghum (Sorghum bicolor L. Moench). Netzley, and Butler (1986) isolated sorgoleone {2- hydroxy- 5- methoxy- 3- [(8'Z, ll'Z)- 8' ,1l' ,14' - pentadecatriene]- p- benzoquinon} from hydrophobic root exudates of sorghum. Sorgoleone, the major p-benzoquinone, and three other structurally related minor p-benzoquinones together constitute 90% or more of the root exudates. These p-benzoquinones are also contact allergens with effects similar to those of poison ivy (Netzly et al., 1988). Sorgoleone has been shown to be a potent inhibitor of chlorophyll formation in Lemna minor L. and also inhibits the growth of some broadleaf and grass weeds (Einhellig and Souza, 1992). Sorgoleone appears to act by blocking mitochondrial electron transport as evidenced by inhibition of state III and state IV respiration in mitochondria isolated from etiolated seedlings of soybean [Glycine max (L) Merr.] and corn (Zeo mays L) (Rasmussen et al., 1992). Recent studies using soybean leaf disks and isolated pea chloroplasts have indicated that sorgoleone is an effective inhibitor of C02-dependent oxygen evolution (Einhellig et al., 1993). However, the site at which sorgoleone acts to inhibit photosynthetic oxygen evolution is not known precisely. An important method of measuring interference with photosynthetic electron transport by a herbicide or other inhibitor involves measuring chlorophyll fluorescence (Devine et al., 1993). If a herbicide compound inhibits transfer of electrons from the primary electron acceptor Q-a to the secondary electron acceptor (QB), the excitation energy builds up in photosystem II and leads to increased fluorescence emission, which can be conveniently measured with a fluorometer.

This research was conducted with an objective to investigate the effects of sorgoleone on seed germination, seedling growth in selected crop and weed species, and inhibition of photosynthetic electron transport in resistant and susceptible cell cultures of potato (Solanum tuberosum L.) and common groundsel (Senecio vulgaris L.). A second objective was to evaluate sorgoleone production within a diverse collection of grain sorghum germplasm.

MATERIALS AND METHODS

Weed and Crop Germination Bioassays. Sorgoleone activity was bioassayed using selected weed and vegetable crops. Growth inhibition as measured by radicle elongation was evaluated in velvetleaf (Abutilon theophrasti Medic.), foxtail millet [Setaria italica (L.) Beauv.], curly cress (Lepidium sativum L.), barnyardgrass [Echinochloa crus-galli.(L.) Beauv.], lettuce (Lactuca sativa L.), and tomato (Lycopersicon esculentum L.). Twenty seeds of each weed/crop were germinated in 4.5 cm Petri dishes containing appropriate sorgoleone test solution. Sorgoleone was formulated at concentrations of 0, 125, 250, 500, and 1000 uM by initially dissolving in methanol and then mixing with water. Methanol concentration in the test solution did not exceed 4%. A 1000 uL aliquot of the appropriate test solution was added to individual Petri dishes lined with Whatman No.1 filter paper. Control dishes received 1000 uL of methanol-water solution. After seeds were placed on moistened filter paper, Petri dishes were transferred to a container and incubated in a seed incubator at 29°C for 3 days in the dark. At the end of the incubation period, radicle length was measured. Treatments were replicated four times, and the experiment was repeated.

Bioassay of Weed Growth in Hydroponics. Sorgoleone activity was bioassayed in hydroponic culture with four weed species. Seeds of velvetleaf (A. theophrasti Medic.), ivyleaf morning-glory [Ipomea hederacea (L.) Jacq.], large crabgrass tdigitaria sanguinalis (L.) Scop.], and barnyardgrass [E. crusgalli (L.) Beauv.] were grown in the greenhouse in plastic containers with vermiculite as growth medium. When seedlings were approximately 3 weeks old, they were washed and transferred to beakers containing half-strength Hoagland's solution (Hoagland and Arnon, 1950). After 3 days of acclimation, uniformly grown seedlings were placed in flasks containing fresh nutrient solution and various concentrations of sorgoleone including 0, 10, 50, 100, and 200,uM. Because of the difficulty of getting sorgoleone into aqueous solution due to its hydrophobicity, a concentrated stock solution was prepared in acetone. This stock was added to nutrient medium to obtain appropriate concentrations of sorgoleone. Acetone concentration in the medium was below 2.5%, and controls were formulated with and without acetone. Each treatment was replicated two or three times with five seedlings per replicate. The nutrient solution was aerated, and plants were maintained under fluorescent lighting for 10 days. Shoot and root dry weights of plants were recorded after drying for approximately 120 h at 47°C. Mean dry weights were calculated, and GR50 values were estimated for roots and shoots of each weed by regression analysis of sorgoleone concentration versus root or shoot dry weights.

Table 1. Inhibition of Growth As Measured by Radicle Length of Selected Plant Species in the Presence of Various Concentrations of Sorgoleone (a)

Screening of Sorghum Germplasm for Sorgoleone Production. A diverse collection of grain sorghum germplasm (Table 3) was screened for sorgoleone production with 5-day-old seedling roots. Germination percentage and root fresh weight were recorded for each germplasm, and total quantity of sorgoleone produced was determined. The extraction procedure for sorgoleone is as outlined above. The purity of the sorgoleone extract was also assessed using reversed phase HPLC as described above.

Cell Cultures. An atrazine-susceptible genotype of Potato (PO-WT) was established in liquid suspension culture, made photoautotropic (LaRosa et al., 1984), and used for the selection of an atrazine-resistant (PO-VAR) cell line (Smeda et al., 1993). For additional comparison, atrazine-susceptible (CG-VAR) and resistant (CG-VAR) common groundsel plants were used to establish photoautotrophic liquid suspension cultures (yerkes and Weller, 1993). Both WT and VAR photoautotrophic potato and common groundsel cell suspension cultures were maintained on carbohydrate-free media under conditions of 2% C02 and 200 umol M-2 S-1 photosynthetic photon flux density. All experiments were conducted with cells in mid-exponential phase of growth and suspended in their respective growth media (Smeda et al., 1993; Yerkes and Weller, 1993).

Measuring Photosynthetic Electron Transport. Photosynthetic electron transport was measured with a Clark-type oxygen electrode (Hansatech D2/2 system) in the absence and presence of sorgoleone at concentrations ranging from 0.01 to 100 uM. For each measurement, 1 mL of cell suspension (30 mg mL-1) was added to the sample cuvette. Temperature of the samples was maintained at 29°C with a circulating water bath. After a steady rate of respiration was attained, sorgoleone was added under dim light and allowed to absorb for 3 min. A light intensity of 225 umol M-2 S-1 photosynthetic photon flux density was used to drive electron transport, and oxygen evolution was measured polarographically.

Chlorophyll Fluorescence. Cell suspensions (30 mg mL-1) of WT and VAR potato and common groundsel were incubated with sorgoleone at concentrations ranging from 0 to 100 uM for 30 min and harvested by vacuum filtration on Whatman No. 4 filter paper. Two hundred milligrams of cells was packed into a 35 uL cuvette and dark-adapted for 15 min before chlorophyll fluorescence measurement. A portable fluorescence meter (Morgan CF-1000) was used for measurement of chlorophyll fluorescence. At the end of dark adaptation, cells were exposed for 10 s to an actinic light of 200 umol m-2 s-2 photosynthetic photon flux density. Fv/Fm (ratio that reflects photochemical efficiency), T1/2 (half-time to fluorescence maximum), and quenching capacity were measured and expressed as percent of control.

RESULTS AND DISCUSSION

Table 2. Effect of Sorgoleone on the Growtb of 15-Day-Old Weed Seedlings under Hydroponic Culture

The bioassay in hydroponic culture showed that sorgoleone could remain in solution over a 10 day period when dissolved in a suitable solvent and sonicated before and after addition to the nutrient medium. Sorgoleone inhibited the growth of large crabgrass, velvetleaf, and barnyardgrass, and the inhibition was concentration dependent (Table 2). Among the species tested, large crabgrass was most sensitive, with a GR50 of 10 uM for shoot growth. However, there was no apparent effect of sorgoleone on root growth of large crabgrass. Studies by Einhellig and Souza (1992) have demonstrated similar response of large crabgrass shoot growth to sorgoleone in aqueous culture, with a 10 uM concentration reducing more than 50% of shoot growth. Reduction in root growth compared to control was observed in their studies, but no significant differences were found between 10, 50, and 100 uM. Differential tolerance to sorgoleone was observed among weed species, with ivyleaf morningglory showing the most tolerance as evidenced by little effect of sorgoleone on either shoot or root growth over the range of concentrations tested (Table 2). Sorgoleone caused chlorosis in sensitive tissues, with chlorotic shoot tissue observed particularly in large crabgrass and barnyardgrass. The effect of sorgoleone on tissue chlorosis is an inclination that sorgoleone may inhibit chlorophyll biosynthesis. Research on its mode of action has indicated that sorgoleone is an inhibitor Of C02-dependent 02 evolution (Einhellig et al., 1993). In our experiments, living root systems were often discolored by the presence of sorgoleone, but root weights were generally less affected by sorgoleone presence than shoot weights. Compared to other natural products with herbicidal activity, sorgoleone is very active at concentrations of 10-200 uM, similar to many synthetic herbicides.

Screening of sorghum germplasm for sorgoleone production indicated that considerable variability exists among genotypes with regard to the amount of sorgoleone produced (Table 3). RTx433 produced as little as 0.67 mg/g of root fresh weight, whereas B Redlan and IS 1318C produced 17.8 and 14.2 mg of sorgoleone, respectively. Most other genotypes ranged between 1.5 and 10 mg/g of root fresh weight. In a different collection of germplasm, Hess et al. (1992) observed that all genotypes of Sorghum produced equal amounts of sorgoleone regardless of whether they were resistant or susceptible to striga (Striga asiatica). Furthermore, they indicated that sorgoleone production is quite sensitive to environmental conditions, particularly to the amount of moisture on the filter paper. It appears, from our studies performed under controlled environmental conditions, that production and secretion of sorgoleone may also be dependent on inherent genetic differences among Sorghum genotypes, apart from environmental factors. On average, sorgoleone accounted for 85-90% of the root exudate composition of the germplasm tested, although the actual amount ranged between 76 and 99%. It is interesting to observe that Sorghum species vary not only in the amount of sorgoleone produced but also in its purity, which may suggest differential allelopathic interference among genotypes.

Table 3. Sorgoleone Production by Various Sorghum Genotypes (a)

Oxygen evolution, a measure of whole chain photosynthetic electron transport, decreased in both atrazine- resistant (VAR) and -susceptible (WT) cell cultures of potato and common groundsel with increasing concentrations of sorgoleone (Figure 1). In potato cell cultures, 1uM sorgoleone effectively reduced oxygen evolution by 50% compared to the untreated control. Furthermore, responses of both WT and VAR cells were identical over the range of sorgoleone concentrations investigated. In contrast, WT common groundsel was more sensitive to inhibition by sorgoleone as compared to VAR, although the pattern of oxygen evolution followed a similar decreasing trend in both as a function of concentration. Less than 1uM sorgoleone reduced oxygen evolution by 50% in WT common groundsel cells compared to nearly 2 uM sorgoleone required by the VAR cells to obtain a similar level of inhibition. Interestingly, the response of sorgoleone is very similar to the observed response for diuron in inhibiting oxygen evolution of WT and VAR common groundsel cells (Yerkes and Weller, 1995). Also, in studies with spinach thylakoids, sorgoleone strongly inhibited oxygen evolution, and the pattern of inhibition was strikingly similar to that of diuron (Nimbal et al., unpublished data). Evidence for the inhibition of photosynthetic oxygen evolution by sorgoleone has also been reported in Glycine max leaf disks and intact chloroplasts of Pisum sativum (Einhellig et al., 1993). Atrazine, another well-known inhibitor of photosynthesis, in contrast, exhibited differential response by inhibiting oxygen evolution in WT, but not in the VAR common groundsel cells (Yerkes and Weller, 1995). These studies suggest that both sorgoleone and diuron may act at the same site to inhibit photosynthetic electron transport. Atrazine resistance in potato VAR cells is due to a serine to threonine substitution at position 264 of the photosystem II reaction center polypeptide, D1 (Smeda et al., 1993). On the other hand, in common groundsel VAR cells and most other triazine-resistant higher plants characterized, resistance is due to a serine to glycine substitution at the same position of the D1 polypeptide.

Chlorophyll fluorescence has been regarded as an important technique in determining inhibition of photosynthetic electron transport. Dark-adapted, photoautotrophic cells, when exposed to photosynthesis-saturating light, exhibit a typical rise in fluorescence followed by a rapid exponential decay in the absence of any PS II inhibiting herbicide or inhibitor. The Presence of such an inhibitor/herbicide can markedly slow the fluorescence decay, indicating that the reoxydation OfQ-A by QB is inhibited. When we measured the flash-induced chlorophyll fluorescence in the presence of sorgoleone, the Fv/Fm was found to be similar for WT and VAR cell types of both potato and common groundsel (Figure 2). This indicates that sorgoleone caused fluorescence in the VAR cells almost to the same extent as in WT cells. This is not unexpected because photosynthetic electron transport, as measured by oxygen evolution, was affected similarly in both WT and VAR cell types. Increasing sorgoleone concentrations progressively reduced the elapsed time to reach fluorescence maximum (Figure 3). T1/2 was reduced similarly in WT and VAR cells of common groundsel, but the effect was less for VAR potato cells compared to WT potato cells. The reason for this differential response is not clear, although VAR potato cells exhibited inherently low T1/2 time compared to WT potato cells.

ABBREVIATIONS USED

LITERATURE CITED