Physiological Responses of Soybean Plants Grown in a ... - NCBI

Plant Physiol. (1991) 96, 305-309 0032-0889/91/96/0305/05/$01 .00/0

Received for publication September 18, 1990 Accepted January 15, 1991

Communication

Physiological Responses of Soybean Plants Grown in Nitrogen-Free or Energy Limited Environment1

a

Yu-xian Zhu, Karel R. Schubert, and Daniel H. Kohl* Biology Department, Washington University, St. Louis, Missouri 63130 ABSTRACT

the cowpea seedlings. Based on N isotopic fractionation data, Shearer et al. ( 18) also concluded that part of recently fixed N is incorporated into nodule tissue. Parsons and Day (14) reported that nodules of 25- to 30-d-old soybean plants grown in Ar:O2 (80:20) still had significant ARA (over 100 ,mol g-' nodule h-'). On a somewhat shorter (9-10 d) exposure of nodule-bearing white lupin plants to Ar:02 environment, Pate et al. (15) found no significant decrease in final nodule weight of treated plants compared with those of plants grown in ambient atmosphere. Nitrogenase activity in these nodules on Ar:O2 treated plants, as measured by H2 evolution, was not statistically different from nodules of plants grown in ambient atmosphere. Similarly, from studies of the effects of glutamate synthase mutants on the nutritional phenotype and symbiotic properties of Rhizobium meliloti, Lewis et al. ( 11) concluded that ammonium assimilation may not be required for the establishment of a functional nodule. In treatments such as stem-girdling, which decreases phloem sap supply to nodules, the mechanism of nitrogenase inhibition has been attributed either to a decrease in the availability of carbohydrate (20), or to an increase in the resistance to 02 diffusion exerted by the inner cortex of the nodule (6, 20). Upon measuring both the concentration of free, dissolved 02 within infected nodule cells and nitrogenase activity, Layzell et al. (10) concluded that inhibition of N2 fixation in soybean nodules after stem-girdling is caused by a decrease in free, dissolved O2 concentration in the infected cells of the nodule and limitation of nodule respiration. Heckmann et al. (7) reported that both the ATP/ADP ratio and energy charge increased when external 02 partial pressure increased from 20 to 30 kPa. The increased energy status was correlated with a twofold increase in nodule ARA. Our motivation in designing experiments in which nodules from soybean plants grown in an N2-free environment and from stem-girdled plants was to measure the activity of proline metabolizing enzymes under these conditions (to be reported subsequently). In the process, we made the unexpected collateral observations reported in this communication.

Soybean (Glycine max [L.] Merr.) seedlings grown in the absence of combined N and in an Ar:02 (79:21, volume/volume) atmosphere had greater seedling and nodule mass, threefold higher acetylene reducing activity per gram fresh weight nodules, no observable increase in nitrogenase Fe-protein, and a higher energy charge than did control plants. A sharp fall in acetylene reducing activity and energy charge accompanying stem-girdling was prevented by exogenous succinate, a result consistent with a path from the roots to the nodule other than via the phloem.

Studies of N metabolism in N2-fixing legumes are often conducted using plants grown hydroponically in the absence of combined N to separate events connected with reduction of nitrate from the fixation of atmospheric N2 and the assimilation of the resultant NH3. To dissociate further functions that are normally coordinated, plants are sometimes grown hydroponically in the absence of N2, typically in an Ar:02 atmosphere. In this circumstance, nitrogenase function carries on while assimilation does not, since, in the absence of N2, nitrogenase reduces protons to H2. Atkins et al. (1, 2) studied the responses of nodules from air versus Ar:02 (80:20, v/v) grown cowpea seedlings. They found that nitrogenase activity in the latter, as measured by H2 evolution, was consistently higher throughout the 22-d time course of their experiment. As measured by C2H2 reduction, the Ar:02-grown plants had about 50% of the activity ofthe controls on a per gfw2 nodule basis. They also found that nodule initiation, infected and noninfected nodule tissue differentiation, as well as the ultrastructure of bacteroid-containing cells were similar in experimental and control plants up to 16 d after sowing. In longer experiments (>16 d), the Ar:02 treatment caused cessation of growth of the nodules, a reduction in protein levels in bacteroid and nodule plant cells, and progressive degeneration of nodule structure leading to premature senescence of these organs. They concluded that direct incorporation of fixed Ncompounds into nodules may be a critical feature in the establishment and continued growth of effective nodules in

MATERIALS AND METHODS Plant Material Seeds of soybean (Glycine max L. Merr. cv Williams 82) were obtained from Manglesdorf Seed Co. (St. Louis, MO) and were inoculated with Bradyrhizobium japonicum strain 6 1A89, also known as USDA 1 10, obtained from the Nitragin

'This research was supported by grant GM38786 from the National Institutes of Health to D.H.K. 2Abbreviations: gfw, g fresh weight; ARA, acetylene reducing

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Table I. Some Properties of 21-d-old, Ar:02Grown and Control Soybean Plants Plant Weight

Nodule Weight

ARA

Treatment

Fresh

Dry

Fresh

98

Dry

mga 22.2 ± 2.0

nmol/min/gfw nodule

404.5 ± 33.4b Ar:02 14.1 ± 1.2 136.4 ± 10.6b Control 96±6 13.7 ± 1.4 132.1 ±10.1d 3.14±0.1 0.44±0.01 GHC b a Error terms are ± 1 SEM of six measurements. Error terms are ± 1 SEM of two experiments. 4.07 ± 0.24 3.25 ± 0.38

0.58 ± 0.03 0.45 ± 0.02

136 ± 12 97 ± 10

In each experiment, three sets of replicate plants were used and ARA was measured three times on d Error term is ± 1 SEM of three each set. c Greenhouse-grown seedlings at the same age. replicate measurements on plants grown in the greenhouse at the same time as the first set of plants grown in Ar:02.

Division of Lipha Chemical (Milwaukee, WI). After inoculation, seeds were sown in perlite in two adjacent cabinets (50 x 50 x 70 cm) at the density of 60 seeds/cabinet. The cabinets were fabricated in two pieces and placed inside a temperature controlled growth chamber (Conviron; Controlled Environments Inc., Pembina, ND). Illumination was provided by a mixture of fluorescent and incandescent lamps for an average irradiance of 580 Amol photons/m2/s at "soil" level and 650 ,umol/m2/s at maximum plant height. The plants were subjected to a 12 h light/dark cycle during which temperatures were maintained at 26 to 30C in the light and 22 to 23°C in the dark. The plants were supplied with N-free nutrients (4) twice a day. Excess solution drained from the bottom of the cabinets. Nutrient solution pumped into the Ar:02-cabinet was outgased with pure Ar and kept under Ar pressure

throughout the whole experimental period. Air from outside the building was pumped in (1 L/min) and exhausted from the control cabinet and Ar:02 (79:21, v/v) was pumped in and exhausted from the other cabinet. The cabinets, which were under a slight positive pressure, were fitted with a relief valve to prevent excessive pressure buildup inside. (This is an essential safety feature.) The level of CO2 in the Ar:02-cabinet was maintained at 330 to 400 ,uL/L by a feedback system, which included an IR gas analyzer (model 202; MSA, Pittsburgh, PA), a digital indicating controller (model UT30, style B; Yokogawa Electric Co., Musashino-shi, Tokyo, Japan), and a mass flow controller (model 5850C; Brooks, Hartfield, PA). The cabinets were assembled from their parts immediately after the seeds were sown. A high flow rate of Ar:02 (800 mL/min) was maintained for the first 5 d to flush out the atmospheric N2, which was in the cabinet when it was assembled. This resulted in the Ar:02 environment being established prior to seed germination and nodule formation. The Ar:02 flow rate was reduced by one-half after 5 d. At the time of harvest (21 d after planting), either both Ar:02-grown and control plants were used immediately for ARA assays or their roots were immersed in liquid N2 and stored at -80°C until further use. Thus, the results of Western blots, quantitation of adenine nucleotides, and ARA were not

from the same plant, but the same batch. The frozen nodules picked at a later time, placed on ice, rinsed with tap water, blotted dry, and weighed before being gently crushed with grinding buffer (2 volumes/gfw nodules). The grinding buffer consisted of 100 mM Tricine buffer (pH 8.0), 400 mM sucrose, and 2.5 mM MgCl2. Bacteroids were prepared as described previously (8). The supernatant fraction taken from above pelleted bacteroids will be referred to as cytosol. The results presented here are the average of two replications for were

A

B:.. C D

Figure 1. Western immunoblot of R. rubrum nitrogenase Fe-protein antiserum against bacteroidal extracts of root nodules from Ar:02grown (lanes A and C), ambient air grown (lanes B and D) 21-d-old soybean plants. Of the solubilized extracts (2 mL/gfw nodule), 40 1sL (lanes A and B) or 10 1sL (lanes C and D) each were loaded (see "Materials and Methods" for details). The nitrogenase Fe-protein that migrated at 33 kD is indicated by an arrow.

Table I. Effect of Nitrogen-free Environment on Nodular ATP, ADP, AMP Content, and Energy Charge of 21-d-old Soybean Plants AMP ATP ADP Energy Charge Treatment nmol/gfw nodule

64.2 ± 6.2a 51.4 ± 6.6 42.5 ± 5.7 Control 25.4 ± 2.7 63.4 ± 4.8 57.0 ± 3.1 a Error terms are ± 1 SEM of three replicates.

Ar:02

ratio

0.57 ± 0.05 0.40 ± 0.03

Ar/02 ATMOSPHERE AND STEM-GIRDLING EFFECTS ON SOYBEAN PLANTS

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Table Ill. ARA and Energy Status of Soybean Nodules from 50-d-old Stem-girdled Plants Treatment

ARA nmol/min/gfw nodules

ADP AMP nmol/gfw nodules 42.0 ± 3.6 65.2 ± 2.6 54.7 ± 3.6 26.2 ± 1.8 79.1 ± 2.3 65.5 ± 4.1 44.8 ± 1.7 61.9 ± 5.9 54.0 ± 3.7 ATP

Energy Charge ratio

0.46 ± 0.03 206.0 ± 14.5 Control 0.39 ± 0.02 93.4 ± 5.0 Girdled for 2 h 0.47 ± 0.03 203.9 ± 9.1 Girdled + succinateb b a 500 mL of 50 mm sodium succinate solution was Error terms are ± 1 SEM of three replicates. supplied to the roots at the time of stem-girdling. Measurements were done on nodules harvested 2 h later.

plants grown under an Ar:02 atmosphere. Other soybean plants were grown in the greenhouse as described (8). Stemgirdling was performed as described by Vessey et al. (20). Chemicals

All chemicals were purchased from Sigma Chemical Co. CO2 (certified 289 gL/L), and regular grade C02, C2H2, and Ar gas were purchased from Acetylene Gas Co. (St. Louis, MO); 79%:21% Ar:02 (certified,