Journal of Chemical Ecology Vol. 7, No. 2, 1981
EXAMINATION OF NORTH AMERICAN BISON SALIVA FOR POTENTIAL PLANT GROWTH REGULATORS
J . K . D E T L I N G , l C.W. R O S S , 2 M . H . W A L M S L E Y , 3 D . W . H I L B E R T , 1 C.A. B O N I L L A , 4 a n d M.I. D Y E R 1 ~Natural Resource Ecology Laboratory, 2Department of Botany and Plant Pathology, Department of Zoology and Entomology, and 4Department of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado
(Received April 18, 1980; revised June 5, 1980) Abstraet--A series of laboratory bioassays were utilized to test for the
presence of potential plant growth factors in saliva from a large native ungulate, the North American bison (Bison bison L.). Whole saliva enhanced Avena coleoptile growth at high pH, whether alone or in combination with indoleaceticacid (IAA). However, this enhancement was a result of salts in the saliva (primarily NaHC03) rather than of other compounds acting hormonally, enhancing IAA activity, or inhibiting IAA oxidase activity as possibly occurs with some insect salivas. Additionally, the absence of detectable cytokininsin the saliva was indicated by its failure to enhance cucumber cotyledon expansion. This suggests that biochemical control of plant growth by salivary compounds following grazing is probably not an important component of this ruminant's interactions with its food plants, as has been suggested for some herbivores. Key Words--Saliva, bison, Bison bison, Avena sativa, oats, Cucumus sativus, cucumber, Avena coleoptile, indoleacetic acid, plant growth regulator, herbivore. INTRODUCTION M u c h o f the recent r e s e a r c h on b i o c h e m i c a l i n t e r a c t i o n s b e t w e e n p l a n t s a n d h e r b i v o r e s has d e a l t with the effects o f s e c o n d a r y p l a n t c h e m i c a l s on h e r b i v o r e s (Levin, 1976; Cates a n d R h o a d e s , 1977; R o s e n t h a l a n d J a n z e n , 1979). A n o t h e r b i o c h e m i c a l a s p e c t o f p l a n t - h e r b i v o r e i n t e r a c t i o n s c o n c e r n s the w a y in which h e r b i v o r e s m a y affect g r o w t h o f their h o s t p l a n t s b y injecting p l a n t g r o w t h - r e g u l a t i n g c o m p o u n d s into p l a n t tissues d u r i n g feeding. F o r 239 0098-0331/81/0300-0239503.00/0 9 1981 Plenum Publishing Corporation
240
DETLING ET AL.
example, there have been a number of reports of naturally occurring plant growth regulators, such as cytokinins (Englebrecht et al., 1969; Engelbrecht, 1971), auxins (Miles and Lloyd, 1967; Miles, 1968a), or compounds which interact with them (Hori, 1974; 1975; 1976), in relatively high concentrations in the salivary systems of a variety of herbivorous insects. Miles' (1968b) literature review suggests that certain of these compounds might account for some of the toxic reactions of plants following insect feeding. Although their potential role in plant-herbivore interactions has received little attention, there is also evidence that a number of compounds of animal origin, such as steroid and protein gonadatropin-like compounds, have plant growthregulating properties (Leshem, 1967; Leshem and Lunenfeld, 1968; Leshem et al., 1969; Stowe and Hudson, 1969; Geuns, 1978; Loeys and Geuns, 1978). Little research regarding potential effects of saliva from vertebrate herbivores on plant growth has been conducted, and the few available results are variable. Results from a preliminary experiment (Reardon et al., 1972) indicated that bovine saliva, when applied to the cut leaf surfaces of defoliated sideoats grama (Bouteloua curtipendula) plants, enhanced forage and root yield relative to that of similarly clipped control plants. However, in later experiments, bovine saliva did not significantly affect growth of this species (Reardon et al., 1974) except when plants were grown in sand with very low fertility (Reardon and Merrill, 1978). Nevertheless, because thiamine, a reported component of bovine saliva (Reardon et aL, 1972), apparently enhanced plant growth under some conditions, these investigators suggested that it was an active compound in this plant--animal association and that the grazing animal was necessary to maintain production of native rangelands (Reardon and Merrill, 1978). By contrast, Johnston and Bailey (1972) found that bovine saliva had no effect on growth or tillering of two species of Festuca. Similarly, we (Detling et al., 1980) recently reported that a single application of saliva from another ruminant, the North American bison (Bison bison L.), had no measurable effect on metabolism or growth of blue grama (Bouteloua gracilis), regardless of intensity of defoliation or plant nitrogen status. In spite of the apparent lack of effect of bison saliva on blue grama (Detling et al., 1980), it might be argued that one or more plant growth regulators are actually present, but that they are effective at different concentrations or frequencies of application, at different phenological stages of plant development, or in different plant species. In this paper, we report the results of a series of experiments to determine whether saliva from this native North American herbivore contains any of a variety of plant growth factors capable of influencing oat coleoptile elongation or cucumber cotyledon expansion.
SALIVA AS PLANT GROWTH REGULATOR
241
METHODS AND MATERIALS
All experiments utilized saliva collected between 10:00 AM and noon from two 7-year-old esophageally fistulated bison (a female and a castrated male) maintained at the Pawnee Site, USDA Central Plains Experiments Range near Nunn, Colorado. Saliva samples, usually 1 liter from each animal, were kept on ice during transport (1 hr) to Colorado State University where they were centrifuged at 12,000 g for 1.5 hr at 4~ to remove major microbial and particulate contaminants. Supernatants were lyophilized, and the resulting residues were weighed and kept frozen until immediately prior to their use in an experiment. Results of an earlier experiment indicated that amylase activity, and therefore likely activity of other complex organic molecules, was not affected by these collection, handling, and storage procedures (Detling et al., 1980). Prior to growth studies, residues were dissolved in 10 mM Tris HC1, pH 8.6, to give various concentrations of solids relative to their concentration in whole saliva. Maintenance of this high pH during all experiments was necessitated by the strong buffering capacity and high pH (8.6-8.8) of the saliva. Of the 11.38 g/liter of soluble salts in the saliva sample we analyzed, Na +, and K § (determined by atomic absorption) accounted for 3.03 and 0.24 g/liter, respectively, and HCO3- (determined titrimetrically) accounted for 7.17 g/liter. The first group of experiments, patterned after those of Hori (1974, 1975, 1976), were designed to determine whether plant growth factors capable of enhancing oat coleoptile elongation are present in bison saliva. Oat (Avena sativa L. cv. Victory) seedlings were grown in vermiculite for 92 hr at 25~C in darkness. After excision of the apical 3 mm of each coleoptile, a subapical section of 7 mm was removed for growth studies. All cutting was done in dim red light. Sections were rinsed twice with distilled water during 4 hr of preincubation darkness. Initial lengths of 20-25 sections then were measured with a dissecting microscope and ocular micrometer, and all subsequent length changes were determined relative to these initial lengths. Each experimental unit consisted of 10 sections incubated at 25~C for 24 hr in 5.5 ml of 10 mM Tris HCI, with or without saliva and IAA, in 9-cm plastic Petri dishes. The second g r o u p of experiments utilized a cucumber cotyledon expansion assay (Narain and Laloraya, 1974; Green and Muir, 1978) to test for the presence of eytokinins in the saliva. Cotyledons from 3 to 4-day old, dark-grown cucumbers (Cucumus sativus L. cv. Marketer) were utilized in all cases. Fresh weights of 20 cotyledons were determined immediately following their excision, and subsequent weight changes were determined by comparison with these initial weights. Cotyledons were incubated in darkness for 3
1.5
20
No IAA
A
40
1-I
IAA
60
I -I IAA
80
I00 0 Salivg
Relative
I
~o
2~
'2L.,-,
B
85
Concentrotion
io
,t,
t00 0 (%)
20
\
• ~o I
40
60
\NoHCO3 +10 m g , I -
80
]AA
tOO
FIG. 1. (A) Promotion of coleoptile elongation by bison saliva at various concentrations of IAA. The concentration of lyophilized solids representing the 100% relative saliva concentration was 14 mg/ml. Each value is the mean of l0 coleoptile sections. (B) Promotion of coleoptile elongation by the 0 to 500-dalton fraction of bison saliva with and without 1 rag/liter IAA. Two liters of saliva yielded 12 g of solids in the 0 to 500-dalton fraction following fractionation by ultrafiltration. Thus, the 100% relative saliva concentration here was 6 mg/ml of incubation solution. Each value is the mean + SE of l0 sections. (C) Similarity of saliva and NaHCO~ effects on coleoptile elongation at 0 and l0 mg/liter IAA. Each value is the mean from two experiments, each with 10 coleoptile sections per treatment. There were 17 mg of lyophilized solids per ml incubation medium in the 100% saliva concentration. The NaHCO3 concentrations used were calculated relative to the HCO3- concentrations in the whole saliva used; thus, a 100% NaHCO3 concentration here was 12.5 mg NaHCO3/ml incubation medium (148 mM).
0
0.5
._~ g ~, i.o
~
20 t
2.5
r~ ~4 >
Z
Z7
to to
243
SALIVA AS PLANT G R O W T H REGULATOR
days in Petri dishes containing 3 ml of a solution of 20 mM KC1, 10 mM Tris HCI (pH 8.4), and reconstituted bison saliva at concentrations of 0-40% of that of whole saliva. Four Petri dishes, each containing 10 cotyledons, were used for each treatment. After the 3-day incubation period, fresh weight of cotyledons was determined by weighing in groups of 5. Sensitivity of the cotyledons was confirmed with an additional treatment involving the addition of 0.017 mM zeatin.
RESULTS AND DISCUSSION Although coleoptile elongation was relatively small at pH 8.6, bison saliva did enhance elongation, the optimum concentration being 10-20% that of whole saliva (Figure 1A). Elongation was further enhanced by the addition of IAA, the optimum concentration of which was about 10 rag/liter (57 # M). Addition of 2% (w/v) sucrose to saliva with and without l mg/liter IAA yielded growth curves (data not shown) similar to those without sucrose in Figure 1A, suggesting that saliva does not act primarily as an energy source. To determine the active salivary component(s), we first tested thiamine because of its reported growth regulation of sideoats grama (Reardon et al., 1972, 1974; Reardon and Merrill, 1978). At 1 mg/liter, alone or with various concentrations of saliva, this vitamin did not promote elongation (data not shown). We then separated reconstituted saliva by ultrafiltration into
---oNo.co,
// / EE 075 _
050
//
0.251 /
/ /
/
i!o
,~
IAA Concentration (mg.I -~)
FIG. 2. Stimulation of coleoptile elongation by sodium and potassium salts (31 mM) at various I A A concentrations. Each salt value represents the mean from three or four experiments, each with 10 sections per treatment; no salt values are means from seven experiments.
244
DETLING ET AL.
fractions of 10,000 daltons. Only the < 500-dalton fraction was consistently active. When solids in this fraction were varied relative to their abundance in whole saliva, dose-response curves with and without 1 mg/liter IAA (Figure IB) were similar to those produced by unfractionated saliva (Figure 1A). Because of the large amounts of NaHCO3 in saliva from both cattle (Johnston and Bailey, 1972) and bison, we investigated its effects with and without IAA. Data in Figure IC indicate that NaHCO3, when varied according to its content in saliva, caused doseresponse curves with overall shapes almost identical to those caused by whole saliva and the
EXAMINATION OF NORTH AMERICAN BISON SALIVA FOR POTENTIAL PLANT GROWTH REGULATORS
J . K . D E T L I N G , l C.W. R O S S , 2 M . H . W A L M S L E Y , 3 D . W . H I L B E R T , 1 C.A. B O N I L L A , 4 a n d M.I. D Y E R 1 ~Natural Resource Ecology Laboratory, 2Department of Botany and Plant Pathology, Department of Zoology and Entomology, and 4Department of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado
(Received April 18, 1980; revised June 5, 1980) Abstraet--A series of laboratory bioassays were utilized to test for the
presence of potential plant growth factors in saliva from a large native ungulate, the North American bison (Bison bison L.). Whole saliva enhanced Avena coleoptile growth at high pH, whether alone or in combination with indoleaceticacid (IAA). However, this enhancement was a result of salts in the saliva (primarily NaHC03) rather than of other compounds acting hormonally, enhancing IAA activity, or inhibiting IAA oxidase activity as possibly occurs with some insect salivas. Additionally, the absence of detectable cytokininsin the saliva was indicated by its failure to enhance cucumber cotyledon expansion. This suggests that biochemical control of plant growth by salivary compounds following grazing is probably not an important component of this ruminant's interactions with its food plants, as has been suggested for some herbivores. Key Words--Saliva, bison, Bison bison, Avena sativa, oats, Cucumus sativus, cucumber, Avena coleoptile, indoleacetic acid, plant growth regulator, herbivore. INTRODUCTION M u c h o f the recent r e s e a r c h on b i o c h e m i c a l i n t e r a c t i o n s b e t w e e n p l a n t s a n d h e r b i v o r e s has d e a l t with the effects o f s e c o n d a r y p l a n t c h e m i c a l s on h e r b i v o r e s (Levin, 1976; Cates a n d R h o a d e s , 1977; R o s e n t h a l a n d J a n z e n , 1979). A n o t h e r b i o c h e m i c a l a s p e c t o f p l a n t - h e r b i v o r e i n t e r a c t i o n s c o n c e r n s the w a y in which h e r b i v o r e s m a y affect g r o w t h o f their h o s t p l a n t s b y injecting p l a n t g r o w t h - r e g u l a t i n g c o m p o u n d s into p l a n t tissues d u r i n g feeding. F o r 239 0098-0331/81/0300-0239503.00/0 9 1981 Plenum Publishing Corporation
240
DETLING ET AL.
example, there have been a number of reports of naturally occurring plant growth regulators, such as cytokinins (Englebrecht et al., 1969; Engelbrecht, 1971), auxins (Miles and Lloyd, 1967; Miles, 1968a), or compounds which interact with them (Hori, 1974; 1975; 1976), in relatively high concentrations in the salivary systems of a variety of herbivorous insects. Miles' (1968b) literature review suggests that certain of these compounds might account for some of the toxic reactions of plants following insect feeding. Although their potential role in plant-herbivore interactions has received little attention, there is also evidence that a number of compounds of animal origin, such as steroid and protein gonadatropin-like compounds, have plant growthregulating properties (Leshem, 1967; Leshem and Lunenfeld, 1968; Leshem et al., 1969; Stowe and Hudson, 1969; Geuns, 1978; Loeys and Geuns, 1978). Little research regarding potential effects of saliva from vertebrate herbivores on plant growth has been conducted, and the few available results are variable. Results from a preliminary experiment (Reardon et al., 1972) indicated that bovine saliva, when applied to the cut leaf surfaces of defoliated sideoats grama (Bouteloua curtipendula) plants, enhanced forage and root yield relative to that of similarly clipped control plants. However, in later experiments, bovine saliva did not significantly affect growth of this species (Reardon et al., 1974) except when plants were grown in sand with very low fertility (Reardon and Merrill, 1978). Nevertheless, because thiamine, a reported component of bovine saliva (Reardon et aL, 1972), apparently enhanced plant growth under some conditions, these investigators suggested that it was an active compound in this plant--animal association and that the grazing animal was necessary to maintain production of native rangelands (Reardon and Merrill, 1978). By contrast, Johnston and Bailey (1972) found that bovine saliva had no effect on growth or tillering of two species of Festuca. Similarly, we (Detling et al., 1980) recently reported that a single application of saliva from another ruminant, the North American bison (Bison bison L.), had no measurable effect on metabolism or growth of blue grama (Bouteloua gracilis), regardless of intensity of defoliation or plant nitrogen status. In spite of the apparent lack of effect of bison saliva on blue grama (Detling et al., 1980), it might be argued that one or more plant growth regulators are actually present, but that they are effective at different concentrations or frequencies of application, at different phenological stages of plant development, or in different plant species. In this paper, we report the results of a series of experiments to determine whether saliva from this native North American herbivore contains any of a variety of plant growth factors capable of influencing oat coleoptile elongation or cucumber cotyledon expansion.
SALIVA AS PLANT GROWTH REGULATOR
241
METHODS AND MATERIALS
All experiments utilized saliva collected between 10:00 AM and noon from two 7-year-old esophageally fistulated bison (a female and a castrated male) maintained at the Pawnee Site, USDA Central Plains Experiments Range near Nunn, Colorado. Saliva samples, usually 1 liter from each animal, were kept on ice during transport (1 hr) to Colorado State University where they were centrifuged at 12,000 g for 1.5 hr at 4~ to remove major microbial and particulate contaminants. Supernatants were lyophilized, and the resulting residues were weighed and kept frozen until immediately prior to their use in an experiment. Results of an earlier experiment indicated that amylase activity, and therefore likely activity of other complex organic molecules, was not affected by these collection, handling, and storage procedures (Detling et al., 1980). Prior to growth studies, residues were dissolved in 10 mM Tris HC1, pH 8.6, to give various concentrations of solids relative to their concentration in whole saliva. Maintenance of this high pH during all experiments was necessitated by the strong buffering capacity and high pH (8.6-8.8) of the saliva. Of the 11.38 g/liter of soluble salts in the saliva sample we analyzed, Na +, and K § (determined by atomic absorption) accounted for 3.03 and 0.24 g/liter, respectively, and HCO3- (determined titrimetrically) accounted for 7.17 g/liter. The first group of experiments, patterned after those of Hori (1974, 1975, 1976), were designed to determine whether plant growth factors capable of enhancing oat coleoptile elongation are present in bison saliva. Oat (Avena sativa L. cv. Victory) seedlings were grown in vermiculite for 92 hr at 25~C in darkness. After excision of the apical 3 mm of each coleoptile, a subapical section of 7 mm was removed for growth studies. All cutting was done in dim red light. Sections were rinsed twice with distilled water during 4 hr of preincubation darkness. Initial lengths of 20-25 sections then were measured with a dissecting microscope and ocular micrometer, and all subsequent length changes were determined relative to these initial lengths. Each experimental unit consisted of 10 sections incubated at 25~C for 24 hr in 5.5 ml of 10 mM Tris HCI, with or without saliva and IAA, in 9-cm plastic Petri dishes. The second g r o u p of experiments utilized a cucumber cotyledon expansion assay (Narain and Laloraya, 1974; Green and Muir, 1978) to test for the presence of eytokinins in the saliva. Cotyledons from 3 to 4-day old, dark-grown cucumbers (Cucumus sativus L. cv. Marketer) were utilized in all cases. Fresh weights of 20 cotyledons were determined immediately following their excision, and subsequent weight changes were determined by comparison with these initial weights. Cotyledons were incubated in darkness for 3
1.5
20
No IAA
A
40
1-I
IAA
60
I -I IAA
80
I00 0 Salivg
Relative
I
~o
2~
'2L.,-,
B
85
Concentrotion
io
,t,
t00 0 (%)
20
\
• ~o I
40
60
\NoHCO3 +10 m g , I -
80
]AA
tOO
FIG. 1. (A) Promotion of coleoptile elongation by bison saliva at various concentrations of IAA. The concentration of lyophilized solids representing the 100% relative saliva concentration was 14 mg/ml. Each value is the mean of l0 coleoptile sections. (B) Promotion of coleoptile elongation by the 0 to 500-dalton fraction of bison saliva with and without 1 rag/liter IAA. Two liters of saliva yielded 12 g of solids in the 0 to 500-dalton fraction following fractionation by ultrafiltration. Thus, the 100% relative saliva concentration here was 6 mg/ml of incubation solution. Each value is the mean + SE of l0 sections. (C) Similarity of saliva and NaHCO~ effects on coleoptile elongation at 0 and l0 mg/liter IAA. Each value is the mean from two experiments, each with 10 coleoptile sections per treatment. There were 17 mg of lyophilized solids per ml incubation medium in the 100% saliva concentration. The NaHCO3 concentrations used were calculated relative to the HCO3- concentrations in the whole saliva used; thus, a 100% NaHCO3 concentration here was 12.5 mg NaHCO3/ml incubation medium (148 mM).
0
0.5
._~ g ~, i.o
~
20 t
2.5
r~ ~4 >
Z
Z7
to to
243
SALIVA AS PLANT G R O W T H REGULATOR
days in Petri dishes containing 3 ml of a solution of 20 mM KC1, 10 mM Tris HCI (pH 8.4), and reconstituted bison saliva at concentrations of 0-40% of that of whole saliva. Four Petri dishes, each containing 10 cotyledons, were used for each treatment. After the 3-day incubation period, fresh weight of cotyledons was determined by weighing in groups of 5. Sensitivity of the cotyledons was confirmed with an additional treatment involving the addition of 0.017 mM zeatin.
RESULTS AND DISCUSSION Although coleoptile elongation was relatively small at pH 8.6, bison saliva did enhance elongation, the optimum concentration being 10-20% that of whole saliva (Figure 1A). Elongation was further enhanced by the addition of IAA, the optimum concentration of which was about 10 rag/liter (57 # M). Addition of 2% (w/v) sucrose to saliva with and without l mg/liter IAA yielded growth curves (data not shown) similar to those without sucrose in Figure 1A, suggesting that saliva does not act primarily as an energy source. To determine the active salivary component(s), we first tested thiamine because of its reported growth regulation of sideoats grama (Reardon et al., 1972, 1974; Reardon and Merrill, 1978). At 1 mg/liter, alone or with various concentrations of saliva, this vitamin did not promote elongation (data not shown). We then separated reconstituted saliva by ultrafiltration into
---oNo.co,
// / EE 075 _
050
//
0.251 /
/ /
/
i!o
,~
IAA Concentration (mg.I -~)
FIG. 2. Stimulation of coleoptile elongation by sodium and potassium salts (31 mM) at various I A A concentrations. Each salt value represents the mean from three or four experiments, each with 10 sections per treatment; no salt values are means from seven experiments.
244
DETLING ET AL.
fractions of 10,000 daltons. Only the < 500-dalton fraction was consistently active. When solids in this fraction were varied relative to their abundance in whole saliva, dose-response curves with and without 1 mg/liter IAA (Figure IB) were similar to those produced by unfractionated saliva (Figure 1A). Because of the large amounts of NaHCO3 in saliva from both cattle (Johnston and Bailey, 1972) and bison, we investigated its effects with and without IAA. Data in Figure IC indicate that NaHCO3, when varied according to its content in saliva, caused doseresponse curves with overall shapes almost identical to those caused by whole saliva and the
