Embed Size (px)
Citation preview
Field nodules of Alnus incana ssp. rugosa and Myrica gale exhibit pronounced acetylene-induced declines in nitrogenase activity
Christa R. Schwintzer and John D. Tjepkema
Abstract: The time course of acetylene reduction was examined in field nodules of speckled alder (Alnus incana ssp. rugosa (Du Roi) Claus.) and sweet gale (Myrica gale L.) with an open flow-through system. When detached speckled alder nodules were measured in the laboratory, there was an initial peak rate of nitrogenase activity between 2 and 3 min followed by pronounced declines to 50% of the peak rate (early summer) and 66% (late summer) at 9 min, after which there was little further change. Speckled alder nodules measured in the field while still attached to the plant also had a peak rate between 2 and 3 min. Most sweet gale nodules had a peak rate at 2-3 min and a sharp decline to 27% at 21 min followed by a partial recovery to 49% at 60 min. The time courses of field nodules of speckled alder and sweet gale were comparable with those of intact, growth chamber grown seedlings. The initial peak rate is the most accurate measure of nitrogenase activity and the only reliable way to measure this is with an open, flow-through system. We describe a simple, inexpensive, flow-through system for use in the field.
Key words: acetylene-induced decline, Alnus incarza ssp. rugosa, actinorhizal plants, Myrica gale, nitrogen fixation, nitrogenase activity.
RCsumC : Les auteurs ont examine le deroulement de la reduction de 1'acCtylkne chez des nodules recueillis aux champs, d'aulne rugeux (Alnus incana ssp. rugosa (Du Roi) Claus.) et de rnyrique baurnier (Myrica gale L.), en utilisant un systkrne d'Ccoulement ouvert. Lorsqu'on rnesure I'activitC de nodules d'aulne rugeux au laboratoire, on observe un pic initial d'activitC nitrogknasique aprks 2-3 min, suivi d'une chfite rnarquCe de 50% par rapport au taux du pic (debut de 1'CtC) et 66% (fin de I'automne) h 9 min, aprks quoi il y a peu de changernents. Les rnesures affectuCes sur le terrain sur des nodules d'aulne rugeux encore attaches i la plante montrent Cgalernent un pic aprks 2-3 rnin et une chiite marquee h 21 % aprks 21 min, suivi d'un retour partiel i 49% aprks 60 min. Les dCroulements du processus chez les nodules de l'alne rugeux et du myrique baumier aux champs sont cornparables h ceux de plants intacts cultivCs en chambre de croissance. Le taux du pic initial est la rnesure la plus precise de l'activitk nitrogknasique et le seul rnoyen fiable pour la rnesurer avec un systkme d'ecoulement ouvert. Les auteurs dkcrivent un systkrne h Ccoulernent ouvert, simple et peu onereux, utilisable sur le terrain.
Mots cle's : declin produit par l'acCtylene, Alr~us incana ssp. riigosa, plantes actinorhiziennes, Myrica gale. fixation de I'azote, activitC de la nitrogCnase. [Traduit par la rCdaction]
Introduction little or no recoverv in others 1Rosendahl and Huss-Dane11
The reduction of acetylene to ethylene is widely used to measure nitrogenase activity because it has high sensitivity, is fast and inexpensive, and allows repeated assays of the same material. Unfortunately, the rate of acetylene reduction often varies as a function of time, with an initial peak fol- lowed by a decline. This phenomenon has been termed the acetylene-induced decline. In legume nodules, there is typi- cally little or no recovery from this decline (Minchin et al. 1983; Witty et al. 1986), whereas in seedlings of actinorhizal plants there is a variable recovery, with the recovered rate closely approaching the initial rate in some instances and
I Received December 2, 1996.
C.R. Schwintzer' and J.D. Tjepkema. Department of Plant Biology and Pathology, 5722 Deering Hall, University of Maine, Orono, ME 04469-5722, U.S.A.
' Author to whom all correspondence should be addressed.
1988; Tjepkema e< al. 1988; ~ o n z and Schwintzer 1989; Silvester and Harris 1989; Tjepkema and Murry 1989; Silvester and Winship 1990). The extent of both the decline and the recovery depends on the plant species, plant age, and the conditions under which the seedlings were grown and assayed (Tjepkema et al. 1988; Tjepkema and Schwintzer 1992; Schwintzer and Tjepkema 1994; Harris and Silvester 1994).
The nodules of actinorhizal plants are perennial, coralloid structures consisting of multiple nodule lobes each of which is a modified lateral root e err^ and Sunell 1990). A nodule, sometimes termed a nodule cluster, may arise from a single infection site or multiple infection sites and usually functions for several years in the field (Akkermans and van Dijk 1976; Schwintzer et al. 1982). In the north temperate zone, nitro- genase activity in nodules of Alnus ssp. and sweet gale (Myrica gale L.) appears after bud break in spring, reaches maximum activity in early or middle summer after full expan- sion of the leaves, and disappears about the time of leaf fall in autumn (Schwintzer et al. 1982, and references therein).
Can. J . Bot. 75: 1415- 1423 (1997) O 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Can. J. Bot. Vol. 75, 1997
The time course of acetylene reduction has not yet been measured in field nodules in actinorhizal plants. In seedlings of Coriaria arborea Lindsay, the decline decreases with seedling age until it is absent in 12-month-old seedlings (Harris and Silvester 1994). This suggests that the decline might be much more pronounced in seedlings than in field nodules. In seedlings, acetylene-reduction time courses have been well studied in sweet gale (Tjepkema and Schwintzer 1992; Schwintzer and Tjepkema 1994) but have not yet been examined in speckled alder (Alnus incana ssp. rugosa (Du Roi) Claus.).
In the present work we investigated the time course of acetylene reduction in field nodules of speckled alder and sweet gale using detached roots assayed in the laboratory. We also measured the time course in sveckled alder nodules in situ in the field and determined the time course for nodules of speckled alder seedlings grown under controlled conditions. Lastly, we measured the effect of moderate disturbance (detopping, removing the roots below the nodulated zone, and shaking off the growth medium) on nitrogenase activity in seedlings. Our goals were to (i) determine if field nodules exhibit an acetylene-induced decline and if so, its extent and whether it varies with the time of the season; (ii) compare the decline in field nodules, one or more years old, with that in seedlings; and (iii) develop improved methods for measuring nitrogenase activity in field nodules of actinorhizal plants.
We used an open, flow-through system because this allows detailed resolution of the time course and accurate determi- nation of the initial peak rate of nitrogenase activity. The initial peak rate is the most reliable measure of nitrogenase activity in legume nodules that have a decline and in actinor- hizal nodules (Minchin et al. 1983, 1994; Schwintzer and Tjepkema 1994). The only practical way to measure the initial peak rate is with an open, flow-through system (Minchin et al. 1994). This is especially true in actinorhizal plants where the peak rate often persists for less than 2 min (Tjepkema et al. 1988; Schwintzer and Tjepkema 1994).
Materials and methods
Field nodules
Field sites Speckled alder nodules were examined in a well-established stand of tall shrubs in Orono, Maine, located approximately 5 min from our laboratory. The stand grew on a poorly drained soil rich in silt and clay and consisted primarily of vigorous speckled alder shrubs but also included a few individuals of Salix spp.
Sweet gale nodules were collected in Caribou Bog, Old Town, Maine, approximately 20 min from our laboratory. The site was in an open, weakly minerotrophic peatland dominated by low shrubs including vigorous clumps of sweet gale, Chatnaedaphne calyculata (L.) Moench, and Rhododendrorl canadense (L.) Torr. The ground surface under the sweet gale shrubs was covered with either Sphagrlum spp. or litter.
Laboratory measurements of acetylene reduction The rate of acetylene reduction was measured in an open, flow- through system consisting of a gas reservoir, a peristaltic pump, a cuvette containing one or more nodules attached to a segment of root, and tubing venting waste gas to the outdoors. The cuvette (20 mL) was made from a section of the barrel of a 30-mL plastic syringe and was immersed in a temperature-controlled water bath
to regulate temperature. Gas entered the cuvette via tubing attached to the needle hub and exited through tubing inserted into a neoprene stopper that sealed the open end of the barrel. A gas flow rate of 68 mL . min-' was used.
Gas mixtures containing 10% acetylene were made in saran bags (ANSPEC, Ann Arbor, Mich.) by adding acetylene to air along with sufficient 0, to keep the PO, at atmospheric value and humid- ified by adding small amounts of water to the bags. Acetylene was generated by adding CaC, to excess water. Gas was pumped from the saran bags through the cuvette with the peristaltic pump via connections made with PVC (Tygon) tubing (2.4 mm i.d. and 0.8 mm wall thickness). Gas exiting the cuvette was sampled with 3-mL syringes. The samples were stored in the syringes for 1 to 15 min before analysis for ethylene and acetylene with a gas chro- matograph equipped with a sampling loop and a flame ionization detector. The gas chromatograph was calibrated for ethylene using 1000 p L . L-I standards (+2% accuracy) supplied by Alltech (Deerfield, Ill.).
Experimental procedures Nodules were carefully excavated in the field and detached from the parent plant together with a 20-mm segment of the proximal sub- tending root, wrapped in a damp paper towel and returned to the laboratory in an insulated box partially filled with the substrate in which the roots were growing to minimize temperature changes. In the laboratory, the nodules and their attached root segments were placed in a cuvette that was submersed in a water bath. The temper- ature of the water bath was set prior to departure for the field based on the previous day's soil temperatures and was usually within 1 " C of the ambient soil temperature when the nodule was collected. The maximum difference between ambient soil and water bath temperature was 2°C. Ambient soil temperatures ranged from 16.8 to 21.9"C. The nodules were preincubated for about 10 min in humidified air pumped through the cuvette at 68 mL . min-'. After acetylene was introduced, the initial gas samples were taken at 1, 2, 3, 4 , 6, and 9 min. The time between detachment of the nodule-bearing root segment in the field and exposure to acetylene was 29-46 min for speckled alder and 38 -49 min for sweet gale. In an earlier study with sweet gale, nodule clusters attached to 20-50 mm long root segments maintained a constant rate of acety- lene reduction for 6 h (Schwintzer 1979). When the acetylene reduction assay was completed, the nodules were separated from the roots, and living nodule lobes were dissected out, dried to constant weight, and weighed.
Field measurelnents of acetylene reduction Field measurements were made in situ with the nodules still attached to the plant. The method was similar to that used in the laboratory with the following modifications. The peristaltic pump was a light- weight (567 g) minipump (Variable Flow Tubing Pump, VWR Scientific, Bridgeport, N.J.) powered by a motorcycle battery. The cuvette (5 mL) was made from a portion of the barrel of a 30-mL plastic syringe with slots (6 mm wide and 15 mm deep) cut into opposite sides of the open end. The slots allowed the root that bore the nodule to pass into and out of the cuvette. The spaces in the slots around the root were sealed with Kromopan (LASCOD, Sesto Fiorentino, Italy), an alginate-based compound used in mak- ing dental impressions. As before, gas entered via tubing attached to the needle hub and exited via tubing inserted into a neoprene stopper closing the open end. After the cuvette was installed around the nodule, it was covered with a thin sheet of plastic and then moss and leafy branches to insulate it from temperature changes. The gas bags and gas lines were kept shaded. Temperatures in the cuvette ranged from 15.3 to 22.7"C but did not vary by more than 0.6"C in the course of the measurement of an individual nodule. Gas samples were stored in syringes for 110- 135 min prior to analysis
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Schwintzer and Tjepkerna
for ethylene and acetylene in the laboratory. The syringes were tested for leakage and lost only 2% of their C,H, in 60 min.
Sampling and data analysis Almost all speckled alder nodules and all sweet gale nodules were not visible prior to excavation. In a given hole, the first nodule 2 1 year old that was encountered was selected. For the laboratory measurements, a few speckled alder nodules were excluded because they were attached to roots that were too large to fit into the cuvette. For the field measurements of speckled alder, some nodules were excluded because they were attached to roots that did not fit into the cuvette because they were too thick, branched, or crooked. As a result of these limitations, the nodules sampled were representative of the nodule population but did not constitute a random sample.
Means were compared by ANOVA followed by a Tukey HSD multiple comparisons test where appropriate. Comparisons of means within the acetylene reduction time courses in Figs. 1-4 were com- parisons of relative rates of acetylene reduction (i.e., percentages).
Time courses for nodules of growth chamber grown seedlings
Plant growth Speckled alder seedlings were grown in a growth chamber (16 h light at 22°C : 8 h dark at 20°C, 475 pmol . m-? . s- ' PPF, cool white fluorescent and incandescent lamps) either hydroponically or in vermiculite from seeds collected in Orono, Maine. Hydroponi- cally grown seedlings were inoculated with Frankia strain HFPArI3 and grown without aeration in one-quarter strength modified N-free Hoagland's solution in wide-mouth, 250-mL (nominal size) plas- tic bottles (125 mm tall). The composition of modified N-free Hoagland's solution, at full strength, was (mg . L-') CaSO, . 2H20, 344; K,SO,, 435; MgSO, . 7H20, 492; Ca(H,PO,), . H20, 126; and FeEDTA, 42; with 1.0 mL. L- ' trace elements as formulated by Monz and Schwintzer (1989). The fluid level was kept below the region of the nodules, with a gas space of about 125 mL and a liquid volume of 150 mL. The plants were used for experiments approximately 1 I weeks after inoculation.
Vermiculite-grown seedlings were inoculated with crushed speckled alder nodules and grown in cone-shaped plastic tubes (210 mm tall; diameter at the top 40 mm) with a volume of 160 mL. The seedlings were watered daily using one-quarter strength modi- fied N-free Hoagland's solution for 2 days and distilled water on the third. The plants were used approximately 9 weeks after inoculation.
Measurements of acetylene reduction Measurements were made on intact plants using methods similar to those used for laboratory measurements of field nodules with the following modifications. In hydroponically grown seedlings, acety- lene reduction was measured in the growth chamber in the bottles in which the plants grew. Six weeks before the measurements, the upper, nodulated portion of the root system was surrounded by a cuvette made of a section of the barrel of a 30-mL syringe (65 mm tall). The upper end of the cuvette was sealed by a rubber stopper with a hole through which the stem of the plant passed. Just prior to the measurement, the stem was sealed into the stopper with plasticine. Inlet tubing entered the cuvette through the stopper and ended 45 mm below the stopper. Exit tubing was attached flush with the lower surface of the stopper. The lower end of the cuvette was sealed by immersion into the nutrient solution. The cuvette volume was approximately 20 mL and the flow rate was 65 mL . min-'. The temperature in the bottles during measurements was 26°C.
In vermiculite-grown seedlings, acetylene reduction was measured in the laboratory in the tubes in which the plants grew. Inlet tubing was sealed to the lower, narrow end of the tube, and the wide upper end was sealed with a rubber stopper with two holes. The stem of
the plant passed through one hole and was sealed in place with Kromopan. Outlet tubing, flush with the bottom of the stopper, passed through the other hole. The cuvette was immersed in a 26°C water bath. The gas volume in the cuvette was approximately 100 mL, and the flow rate was 290 mL . min-I.
Effect of disturbance Seedlings of red alder (Ainus rubra Bong.) and sweet gale were produced from seed, inoculated, and grown in a growth chamber in vermiculite in cone-shaped tubes using methods similar to those for growth chamber grown seedlings. Red alder seeds were obtained from F.W. Schumacher, Inc. (Sandwich, Mass.) and sweet gale seeds were collected in Caribou Bog, Old Town, Maine. Red alder seedlings were inoculated with crushed nodule inoculum as for speckled alder, and sweet gale seedlings were inoculated with Frankia strain LLR161101.
Acetylene reduction was measured as described above for ver- miculite grown seedlings except that for the untreated controls, samples were taken only at 1, 1.5, 2 , 2.5, and 3 min, and the gas was then switched to moist air. After 30 min the gas was again switched to acetylene, and samples were taken at 1, 1.5, 2, 2.5, 3, 4, and 5 min.
For the disturbed plants, each seedling was removed from the growth tube after the initial exposure to acetylene, and the shoot was cut off above the rubber stopper just above the Kromopan seal. Next the roots were separated starting from the bottom of the root system, as much vermiculite as possible was shaken off, and roots below the nodulated segment of the root system were cut off. To compensate for the volume lost from the root ball, 40 mL of glass beads that had been brought to water bath temperature were added to the bottom of the tube. Finally, the plant was replaced in its tube, moist air was passed over the root system, and the root system was again exposed to acetylene 30 min after the end of the first exposure to acetylene.
Results
Field nodules Speckled alder nodules that were collected in the field in early (June 24 - July 5 1994) or late summer (August 8 - 18 1994) and measured in the laboratory, had similar acetylene- reduction time courses (Fig. I). These had peak rates 2- 3 min after introduction of acetylene, and the peaks were followed by pronounced declines to 50% of the peak rate (early summer) and 66% (late summer) at 9 min. kfter this there was little further change. Mean values for the two curves did not differ significantly at the peak (2 min; P = 0.09), end of the decline (9 min; P = 0.26), or after 40 min (P = 0.17). The early- and late-summer nodules had similar biomasses but differed strongly in specific nitrogenase activity with the early summer nodules being approximately three times as active as the late-summer nodules (Table 1).
Speckled alder nodules that were measured in situ in the field in late summer (August 8-31 1994) had acetylene- reduction time courses with peak rates between 2 and 3 min followed by a modest decline to 90% at 6 min and a gradual, further decline to 76% at 40 min (Fig. 2). Mean values for this curve were not significantly different from those for the late-summer nodules measured in the laboratory at the peak (2 min; P = 0.32) and 40 min (P = 0.08) but were signifi- cantly higher (90 vs. 64%) at 12 min (P = 0.003). The biomasses and nitrogenase activities of the nodules measured in situ were similar to those of the late-summer nodules measured in the laboratory (Table 1).
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
1418 Can. J. Bot. Vol. 75, 1997
Fig. 1. Relative rates of acetylene reduction as a function of time after acetylene addition in speckled alder nodules collected in the field in Orono, Maine, in early summer (0) (June 24 - July 5, 1994) or late summer (0) (Aug. 8-29, 1994) and measured in the laboratory. See Table 1 for more information. Values are mean f SE, n = 9 (early summer) or 11 (late summer).
T i m e a f t e r a d d i t i o n o f a c e t y l e n e ( rn in)
Table 1. Dry mass and specific activity of speckled alder and sweet gale nodules collected in the field in Maine in 1994.
Plant Measurement Nodule dry mass Specific activity"
Dates location N (8) (pmol C,H, . min-' . g-')
Speckled alder June 24 - July 5 Laboratory 9 0.126f 0 . 0 3 4 ~ 1.24f 0 . 2 7 ~ Speckled alder Aug. 12-29 Laboratory 11 0.093f0.021~ 0.35f0.066 Speckled alder Aug. 8-31 Field 8 0.091 f 0 . 0 1 9 ~ 0.50f0. 13b Sweet gale, ty picalh July 7 - 12 Laboratory 7 0.051 f 0.01 la 1.20f 0 . 2 5 ~ Sweet gale, delayedr July 7 - 14 Laboratory 4 0.034f 0 . 0 1 5 ~ 0.45 f0.04b
Note: Values are mean SE. Values followed by the same letters are not significantly different (P 5 0.05) within species and within columns.
"Specific activity values are calculated, using nodule dry mass, for the peak rate of acetylene reduction. "Nodules with typical curves. 'Nodules with curves that have a delayed maximum.
Sweet gale nodules produced two different kinds of time courses. Seven of the 11 nodule samples had time courses with a peak at 2-3 min and had a sharp decline to 27% at 21 rnin followed by a partial recovery to 49% at 60 min (Fig. 3). This curve is typical of time courses reported earlier for hydroponically grown sweet gale seedlings (Tjepkema and Schwintzer 1992; Schwintzer and Tjepkema 1994), and we designated this kind as a "typical" time course. The remaining four nodule samples had time courses in which the highest rate was observed at the end of the measurement period (Fig. 3). We designated this kind as a "delayed maxi- mum" time course. The two types of nodules had similar biomasses, but specific nitrogenase activities were approxi- mately 2.5 times larger in nodules with a typical rather than a delayed maximum time course (Table 1).
with peak rates between 2 and 3 min, followed by a modest decline to 78 % at 12 min, and little further change to 60 rnin (Fig. 4). After this the rate recovered to 93.0 + 2.6% (mean + SE) at 90 rnin and 98.0 Jr 1.5% at 150 min. The shoots had a biomass of 0.50 & 0.08 g dry mass.
When speckled alders were grown in vermiculite, nodules on intact seedlings had acetylene-reduction time courses with peak rates between 1 and 2 rnin followed by a sharp decline to 83.2 + 3.8 % at 4 rnin followed by little further change to 9 rnin and then a gradual decline to 73.2 Jr 3.8% at 25 min. (Fig. 4). The shoots had a biomass of 1.3 1 + 0.19 g dry mass. The curves for the hydroponically and vermiculite grown plants were similar except at 3 and 4 rnin when they differed significantly (P I 0.05). The vermiculite-grown plants had greater shoot biomass (P I 0.05), nodule mass, and specific nitrogenase activity (Table 1) than the hydro-
Nodules of growth-chamber grown seedlings p ~ n i ~ i l l y grown plants. When speckled alders were grown hydroponically, nodules In speckled alder, all time courses showed an initial sharp on intact seedlings had acetylene-reduction time courses peak between 1 and 3 rnin followed by a decline, which in
O 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Schwintzer and Tjepkerna 141 9
Fig. 2. Relative rates of acetylene reduction as a function of time after acetylene addition in speckled alder nodules measured in situ in the field in Orono, Maine, in late summer (Aug. 8-31, 1994). See Table 1 for more information. Values are mean f SE, n = 8.
0 + 0 L 100
T ime a f t e r a d d i t i o n o f ace ty lene ( m i n )
turn was followed by little or no recovery by 40 and 60 min after the addition of acetylene. Thus the time courses for speckled alder were similar for overwintering nodules from the field (Figs. 1 and 2) and nodules on young seedlings grown in a growth chamber (Fig. 4). The mean value at 40 min ranged from a low of 48% in the early summer nodules to a high of 76 % in the nodules measured in situ and in the seedlings grown in hydroponics. With the exception of the early summer values, none of the 40-min values differed significantly from each other (P 1 0.05). The early summer values differed from all others except the late-summer values.
Effect of disturbance When untreated red alder and sweet gale seedlings were exposed to acetylene for 3 min and again 30 min later, the peak rate of acetylene reduction remained the same (Table 2). When the seedlings were subjected to a moderate disturbance (removal of the shoot and lower portion of the root system and shaking to remove part of the vermiculite growth medium), the post-disturbance peak rate was not significantly different (P 10.05) from the predisturbance rate (Table 2).
Discussion
Time courses All field nodules of speckled alder and sweet gale had sub- stantial acetylene-induced declines in nitrogenase activity (Figs. 1-3). Consequently, the initial peak rate of nitrogenase activity must be measured to obtain a reliable measure of nitrogenase activity. This is because in legume nodules that have an acetylene-induced decline (Minchin et al. 1983, 1994), and in seedlings of sweet gale (Schwintzer and Tjepkema 1994), it has been shown that the initial peak rate of acety- lene reduction is the most reliable measure of nitrogenase activity and that other measurements underestimate nitro- genase activity. The only practical way of measuring the
initial peak in actinorhizal plants is with an open, flow- through system with a flow rate (mL . min-') of at least three times the cuvette volume (mL). Lower flow rates and closed systems do not allow for sufficient mixing of the gas phase to resolve the peak rate. We have described a simple flow-through system that can readily be taken to remote areas. In this study, we used it to measure nitrogenase activity in situ. While these measurements are too time consuming for use in routine field measurements, the sys- tem could easilv be modified to measure detached nodules by using a cuvette similar to the one we used for laboratory measurements.
In field-collected nodules of s~eckled alder. similar time courses were obtained in early summer when specific nitro- genase activity was relatively high and in late summer when it was only about one third as high (Fig. 1, Table 1). This pattern suggests that, although there is strong seasonal varia- tion in specific nitrogenase activity, there may be little if any seasonal variation in acetylene-reduction time courses. This conclusion is preliminary because only one species was measured on only two dates. However, those dates include the time of the approximate peak of nitrogenase activity in early summer when shoot growth is active and a period in late summer when nitrogenase activity is much lower and shoot growth is largely complete (Schwintzer et al. 1982, and references therein).
Acetvlene-reduction -time courses of actinorhizae form two groups with respect to recovery to stable rates follow- ing the decline: those that show such a recovery and those that do not. Recovery to stable rates has been found in Alnus rubra, Myrica, and Casuarina (Tjepkema et al. 1988; Tjepkema and Murry 1989), but there is little if any recovery in Coriaria and Datisca (Tjepkema et al. 1988; Silvester and Harris 1989). The time courses for speckled alder in this study appear to belong to the second group because they show little or no recovery by 60 min. The time course
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
1 4 2 0 Can. J . Bot. Vol. 7 5 , 1 9 9 7
Fig. 3. Relative rates of acetylene reduction as a function of time after acetylene addition in sweet gale nodules collected in the field in Old Town, Maine, in midsummer (July 7- 14, 1994) and measured in the laboratory. See Table 1 for more information. Values are mean f SE, n = 7 (typical curves) or 4 (delayed maximum curves).
T y p i c a l c u r v e s
: rr D e l a y e d m a x i m u m c u r v e s
I I I I I I I
T i m e a f t e r a d d i t i o n o f a c e t y l e n e ( rn in)
for the closely related European gray alder (Alnus incana ssp. incana (L.) Moench.) also shows no recovery by 70 rnin (Rosendahl and Huss-Dane11 1988). However, the significance of the difference between red alder, which shows a recovery, and speckled alder is not clear because longer time courses may reveal recovery of activity in speckled alder. We found a recovery to 98% of the initial peak rate in hydroponically grown seedlings of speckled alder at 150 rnin, whereas very little recovery occurred by 60 min (see Results). Whether the other groups of speckled alder nodules and the gray alder nodules (Rosendahl and Huss-Dane11 1988) would have recovered if given more time in acetylene is not known because they were exposed for no more than 70 min.
In sweet gale, field nodules with typical time courses of acetylene reduction (Fig. 3) were similar to time courses reported earlier for hydroponically grown seedlings (Tjepkema et al. 1988; Tjepkema and Schwintzer 1992; Schwintzer and Tjepkema 1994). Thus, as in speckled alder, these field nodules did not differ substantially from nodules of growth chamber grown seedlings. In contrast, the nodules with a delayed maximum had time courses that differed strongly
from those found in seedlings. These delayed-maximum time courses may have been influenced by recovery from oxygen shock. The peat in which the sweet gale nodules grew was very moist and became increasingly water saturated with increasing depth in the rooting zone. In the wetter regions of peat, oxygen concentrations are extremely low, and nodules that develop in this environment adapt to low p02 (Silvester et al. 1988). When these nodules are exposed to the atmos- phere, they experience a large change in p02 that may cause an oxygen shock. During an oxygen shock, nitrogenase activity is depressed arid then recovers gradually (Skeffington and Stewart 1976; Silvester et al. 1988; Silvester et al. 1990). The extent of the depression and the time needed for the recovery depend on the severity of the shock. Consistent with the oxygen shock hypothesis, these nodules had only about 35 % as much specific nitrogenase activity as the nodules with typical time courses (Table 1).
Effects of disturbance Caution must be used in using the results of nitrogenase activities measured in field nodules because the measure-
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Schwintzer and Tjepkerna 1421
I Fig. 4. Relative rates of acetylene reduction as a function of time after addition of acetylene in nodules of intact speckled alder seedlings grown in a growth chamber in hydroponics or vermiculite. See Table 2 for more information. Values are mean f SE, n = 6. : rwi---$ Hydroponical ly grown
I I I I I I I
-
- x x x P
-
-
- Vermicul i te grown
I I I I I I I
T i m e a f t e r a d d i t i o n o f a c e t y l e n e ( rn in)
Table 2. Dry mass and specific activity of nodules on growth chamber grown seedlings of speckled alder, red alder, and sweet gale used to determine the time course of acetylene reduction in seedlings and the effect of disturbance on the peak rate of nitrogenase activity.
Plant Nodule dry masso Specific activityb
N (8) (pmol C2H, . min-' . g-I) Run 21Run 1'
Speckled alder, hydroponic 6 Speckled alder, vermiculite 6
Red alder Untreated control 5 Disturbed 4
Sweet gale Untreated control 5 Disturbed 5
Time course 0.033 f 0 . 0 0 5 ~ 2.03f 0 . 5 1 ~ 0.053f0.007b 4.85 +0.53b
Effect of disturbance
0.052 f 0 . 0 0 7 ~ 1 .48f0 .28~ 0 . 9 9 f 0 . 0 1 ~ 0.044f0.006~ 1.67f 0 . 2 9 ~ 0 .96 f0 .02~
0.061 +0.008a 1.64f 0 . 2 8 ~ 1 .O1 f 0 . 0 2 ~ 0.062 +0.006a 1.72+0.39a 0 .96f0 .04~
Note: Values are mean + SE. Within species and within columns, values followed by the same letter are not significantly different (P 5 0.05).
"Nodule biomass for the entire root system. "Specific activity values are calculated, using nodule dry mass, for the peak rate of acetylene reduction. 'Ratio of the peak rate for the second exposure to acetylene to the peak rate for the first exposure.
O 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Can. J. Bot. Vol. 75, 1997
ments inevitably disturb the nodules, and it is difficult to obtain random samples of field nodules. Disturbances to nodules could result from changes in the composition of the atmosphere around the nodules, detachment from the plant, and mechanical damage. The atmospheric concentration of both oxygen (Silvester et al. 1990) and carbon dioxide (Winship and Tjepkema 1982; Brioua and Wheeler 1994) affect the rate of nitrogenase activity in actinorhizal plants. Well-aerated soils have p02 close to atmospheric levels, and nodules from such soils would be little affected by measure- ment at atmospheric pOz. However, nodules from poorly aerated soils such as wet or compacted soils may be affected strongly. All field nodules are likely to experience a change in C 0 2 concentration when exposed to atmospheric CO,, but the direction and magnitude of the resulting effect on nitro- genase activity cannot be predicted without further research.
Nodules may also suffer disturbance effects caused by removal of the shoot, detachment from the plant, or mechan- ical damage. A wide range of such effects has been reported in actinorhizal plants, and the evidence is contradictory, in part. Some of the differences are probably due to differences in species, growing conditions, length of time between the disturbance and the measurement, and measurement proce- dures. On the one hand, in black alder (Alnus glutinosa (L.) Gaertn.) seedlings, detopping the plant or transferring it to a new incubation vessel, which involved some abrasion and compression of the root system, reduced nitrogenase activity to approximately 85% of its pretreatment value, but com- plete detachment of nodules reduced rates to 9 % of the pretreatment value (Wheeler et al. 1978). In gray alder seedlings, detached, nodulated roots that had all gravel shaken off retained 83 % of their pretreatment activity (Huss- Dane11 and Ahlqvist 1984), and seedlings that were detopped retained about 85% of their original activity 45 min after detopping (Sundstrom and Huss-Dane11 1987). In field nodules of black alder, complete excision from the root resulted in marked reduction in nitrogenase activity, sometimes by as much as 50% (Akkermans and van Dijk 1976). On the other hand, we observed no significant disturbance effect in red alder and sweet gale seedlings in this study when we detached the shoots and the lower part of the root system and then shook them to remove much of the vermiculite in which they had grown (Table 2). Moreover, in sweet gale, removal of the shoot and roots beneath the nodulated zone had little effect on the rate of nitrogenase activity of hydroponically grown seedlings (Tjepkema et al. 1988). Further, in Casuarina, excising the nodules from the plant but leaving them attached to a 2 - 10 mm root segment reduced the rate of nitrogenase activity by only 10% (Tjepkema and Murry 1989). Finally, in field nodules of black alder, severing the subtending root at a distance of 20-50 mm from the nodule did not affect nitrogenase activity in the subsequent 3 h (Akkermans and van Dijk 1976). In summary, it appears that nodules can often be detached from actinorhizal plants with little loss of activity as long as a short segment of the subtending root remains attached to the nodule, and care is taken to avoid damage to the nodule. This is in contrast to legume nodules, which are much more susceptible to disturbance (Minchin et al. 1986; Vessey 1994).
The accuracy of estimates of nitrogenase activity based on measurements of field nodules can also be reduced by diffi-
culties in obtaining random samples. This has been reported for legume nodules (Minchin et al. 1994) and was noted under methods in this study as well.
Recommendations for field measurements Overall, it is clear that nitrogenase measurements of detached field nodules are subject to a wide variety of errors. These errors apply equally to measurements made with I5N2 incor- poration and acetylene reduction. However, use of careful methodology can minimize these errors and allow estimates of nitrogen fixation via acetylene reduction in natural vegeta- tion at sites that are unsuitable for use of I5N dilution and I5N natural abundance techniques. Such methodology would include use of an open flow-through system, preliminary testing to assess the importance of differences in pC02 and p 0 2 between the soil atmosphere and the measurement sys- tem, use of nodules subtended by a root segment at least 20 mm long, preliminary testing to determine how long detached nodules retain full nitrogenase activity, and peri- odic measurements during the growing season to assess the seasonal curve of nitrogenase activity.
Conclusions
Overwintering field nodules of speckled alder and sweet gale nodules with typical time courses have pronounced acetylene- induced declines in nitrogenase activity similar to those of growth chamber grown seedlings. In speckled alder, there is little or no seasonal change in the time course of acetylene reduction even though there is a large seasonal change in specific nitrogenase activity. Because these nodules have pronounced acetylene-induced declines, the initial peak rate is the only reliable measure of nitrogenase activity, and this can only be measured accurately with an open flow-though system with a flow rate (mL . min-I) at least three times the volume of the measurement cuvette (mL). A simple, inex- pensive flow-through system for field use can be assembled using a lightweight battery-operated pump and a motorcycle battery. Caution must be used in interpreting nitrogenase activities measured in field nodules because the measure- ments invariably disturb the nodules. Nodules can probably be detached from the plant with little loss of activity as long as a short segment of the subtending root remains attached to the nodule and care is taken to avoid damage to the nodule.
Acknowledgements
We thank Kimberly J. Petersen for helping with the field experiments and the experiment with hydroponically grown seedlings and Daniel M. Moore for growing the vermiculite- grown seedlings and helping with the experiments on dis- turbance effects. This work was supported in part by U.S. Department of Agriculture NRICGP grant No. 92-37305-7758 to J.D.T.
References
Akkermans, A.D.L., and Van Dijk, C. 1976. The formation and nitrogen-fixing activity of the root nodules of Alrzus glutirzosa under field conditions. In Symbiotic nitrogen fixation in plants. Edited by P.S. Nutman. Cambridge University Press, London, U.K. pp. 511-520.
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
Schw~ntzer and Tjepkerna
Berry, A.M., and Sunell, L.A. 1990. The infection process and nodule development. In The biology of Frankia and actinorhizal plants. Edited by C.R. Schwintzer and J.D. Tjepkema. Academic Press, San Diego, Calif. pp. 61 -81.
Brioua, A.-H., and Wheeler, C.T. 1994. Growth and nitrogen fixation in Alnus glutinosa (L.) Gaertn. under carbon dioxide enrichment of the root atmosphere. Plant Soil, 162: 183- 191.
Harris, S., and Silvester, W. 1994. Acetylene- and argon-induced declines in nitrogenase activity in Coriaria arborea. Soil Biol. Biochem. 26: 641 -648.
HUSS-Danell, K., and Alqvist, A.-S. 1984. Nitrogenase activity in root nodule homogenates of Alnus incana. Plant Soil, 78: 159- 170.
Minchin, F.R., Witty, J.F., Sheehy, J.E., and Muller, M. 1983. A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. J. Exp. Bot. 34: 641 -649.
Minchin, F.R., Sheehy, J.E., and Witty, J.F. 1986. Further errors in the acetylene reduction assay: effects of plant disturbance. J. Exp. Bot. 37: 1581-1591.
Minchin, F.R., Witty, J.F., and Mytton, L.R. 1994. Reply to 'Measurement of nitrogenase activity in legume root nodules: in defense of the acetylene reduction assay' by J.K. Vessey. Plant Soil, 158: 163-167.
Monz, C.A., and Schwintzer, C.R. 1989. The physiology of spore-negative and spore-positive nodules of Myrica gale. Plant Soil, 118: 75-87.
Rosendahl, L., and Huss-Danell, K. 1988. Effects of elevated oxygen tensions on acetylene reduction in Alnus incatla - Frarzkia symbioses. Physiol. Plant. 74: 89-94.
Schwintzer, C.R. 1979. Nitrogen fixation by Myrica gale root nodules in a Massachusetts wetland. Oecologia, 43: 283-294.
Schwintzer, C.R., and Tjepkema, J.D. 1994. Factors affecting the acetylene to I5Nz conversion ratio in root nodules of Myrica gale L. Plant Physiol. 106: 1041 - 1047.
Schwintzer, C.R., Berry, A.M., and Disney, L.D. 1982. Seasonal patterns of root nodule growth, endophyte morphology, nitro- genase activity, and shoot development in Myrica gale. Can. J . Bot. 60: 746-757.
Silvester, W.B., and Harris, S.L. 1989. Nodule structure and nitrogenase activity of Coriaria arborea in response to varying PO,. Plant Soil, 118: 97-109.
Silvester, W.B., and Winship, L.J. 1990. Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured Frankia. Plant Physiol. 92: 480-486.
Silvester, W.B., Whitbeck, J., Silvester, J.K., and Torrey, J.G. 1988. Growth, nodule morphology, and nitrogenase activity of Myrica gale with roots grown at various oxygen levels. Can. J. Bot. 66: 1762- 1771.
Silvester, W.B., Harris, S.L., and Tjepkema, J.D. 1990. Oxygen regulation and hemoglobin. In The biology of Frankia and actinorhizal plants. Edited by C.R. Schwintzer and J.D. Tjepkema. Academic Press, San Diego, Calif. pp. 157- 176.
Skeffington, R.A., and Stewart, W.D.P. 1976. Evidence from inhibitor studies that the endophyte synthesises nitrogenase in the root nodules of Alnus glutinosa L. Gaertn. Planta (Berlin), 129: 1-6.
Sundstrom, K.-R., and Huss-Danell, K. 1987. Effects of water stress on nitrogenase activity in Alnus incana. Physiol. Plant. 70: 342-348.
Tjepkema, J.D., and Murry, M.A. 1989. Respiration and nitro- genase activity in nodules of Casuarina cunninghatniana and cultures of Frankia sp. HFP020203: effects of temperature and partial pressure of 0,. Plant Soil, 118: 11 1 - 11 8.
Tjepkema, J.D., and Schwintzer, C.R. 1992. Factors affecting the acetylene-induced decline during nitrogenase assays in root nodules of Myrica gale L. Plant Physiol. 98: 145 1 - 1459.
Tjepkema, J.D., Schwintzer, C.R., and Monz, C.A. 1988. Time course of acetylene reduction in nodules of five actinorhizal genera. Plant Physiol. 86: 581-583.
Vessey, J.K. 1994. Measurement of nitrogenase activity in legume root nodules in defense of the acetylene reduction assay. Plant Soil, 158: 151-162,
Wheeler, C.T., Cameron, E.M., and Gordon, J.C. 1978. Effects of handling and surgical treatments on nitrogenase activity in root nodules of A l t ~ ~ t s glutinosa, with special reference to the application of indole-acetic acid. New Phytol. 80: 175- 178.
Winship, L., and Tjepkema, J.D. 1982. Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules. Plant Physiol. 70: 361-365.
Witty, J.F., Minchin, F.R., Skot, L., and Sheehy, J.E. 1986. Nitrogen fixation and oxygen in legume root nodules. Oxf. SUN. Plant Mol. Cell Biol. 3: 275-314.
@ 1997 NRC Canada
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y U
NIV
ER
SIT
Y O
F N
OR
TH
TE
XA
S L
IBR
AR
Y o
n 11
/25/
14Fo
r pe
rson
al u
se o
nly.
