Evidence for protection of nitrogenase from O(2) by colony structure in the aerobic diazotroph Gluconacetobacter diazotrophicus.

Gluconacetobacter diazotrophicus is an endophytic diazotroph of sugarcane which exhibits nitrogenase activity when growing in colonies on solid media. Nitrogenase activity of G. diazotrophicus colonies can adapt to changes in atmospheric partial pressure of oxygen (pO(2)). This paper investigates whether colony structure and the position of G. diazotrophicus cells in the colonies are components of the bacterium's ability to maintain nitrogenase activity at a variety of atmospheric pO(2) values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO(2) of 2 and 20 kPa. Imaging of live, intact colonies by confocal laser scanning microscopy and of fixed, sectioned colonies by light microscopy revealed that at 2 kPa O(2) the uppermost bacteria in the colony were very near the upper surface of the colony, while the uppermost bacteria of colonies cultured at 20 kPa O(2) were positioned deeper in the mucilaginous matrix of the colony. Disruption of colony structure by physical manipulation or due to 'slumping' associated with colony development resulted in significant declines in nitrogenase activity. These results support the hypothesis that G. diazotrophicus utilizes the path-length of colony mucilage between the atmosphere and the bacteria to achieve a flux of O(2) that maintains aerobic respiration while not inhibiting nitrogenase activity.

Response of the endophytic diazotroph Gluconacetobacter diazotrophicus on solid media to changes in atmospheric partial O(2) pressure.

Gluconacetobacter diazotrophicus is an N(2)-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O(2) pressures (pO(2)) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO(2) (5 to 60 kPa). Nitrogenase activity was measured by H(2) evolution in N(2)-O(2) and in Ar-O(2), and respiration rate was measured by CO(2) evolution in N(2)-O(2). To validate the use of H(2) production as an assay for nitrogenase activity, a non-N(2)-fixing (Nif(-)) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup(+)) activity (0.016 +/- 0.009 micromol of H(2) 10(10) cells(-1) h(-1)) when incubated in an atmosphere enriched in H(2). However, Hup(+) activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO(2) tested. However, when the assay atmospheric pO(2) was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO(2) for nitrogenase activity was 0 to 20 kPa above the pO(2) at which the bacteria had been grown. As atmospheric pO(2) was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO(2) was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO(2) from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O(2), 80% of nitrogenase activity was recovered within 10 min, indicating a "switch-off/switch-on" O(2) protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N(2) at a wide range of atmospheric pO(2) and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO(2).

Stimulation of nodulation in field peas (Pisum sativum) by low concentrations of ammonium in hydroponic culture.

Although the inhibitory effects of high concentrations of mineral N (> 1.0 mM) on nodule development and function have often been studied, the effects of low, static concentrations of NH4+ (< 1.0 mM) on nodulation are unknown. In the present experiments we examine the effects of static concentrations of NH4+ at 0, 0.1 and 0.5 mM in flowing, hydroponic culture on nodule establishment and nitrogenase activity in field peas [Pisum sativum L. cv. Express (Svalöf AB)] for the initial 28 days after planting (DAP). Peas grown in the presence of low concentrations of NH4+ had significantly greater nodule numbers (up to 4-fold) than plants grown without NH4+. Nodule dry weight per plant was significantly higher at 14, 21 and 28 DAP in plants grown in the presence of NH4+, but individual nodule mass was lower than in plants grown without NH4+. The nodulation pattern of the plants supplied with NH4+ was similar to that often reported for supernodulating mutants, however the plants did not express other growth habits associated with supernodulation. Estimates of N2 fixation indicate that the plus-NH4+ peas fixed as much or more N2 than the plants supplied with minus-NH4+ nutrient solution. There were no significant differences in nodule numbers, nodule mass or NH4+ uptake between the plants grown at the two concentrations of NH4+. Nodulation appeared to autoregulate by 14 DAP in the minus-NH4+ treatment. Plant growth and N accumulation in the minus-NH4+ plants lagged behind those of the plus-NH4+ treatments prior to N2 fixation becoming well established in the final week of the experiment. The plus-NH4+ treatments appeared not to elicit autoregulation and plants continued to initiate nodules throughout the experiment.

Expression of characteristics of ammonium nutrition as affected by pH of the root medium.

To study the effect of root-zone pH on characteristic responses of NH4+ -fed plants, soybeans (Glycine max¿L.¿ Merr. cv. Ransom) were grown in flowing solution culture for 21 d on four sources of N (1.0 mol m-3 NO3-, 0.67 mol m-3 NO3- plus 0.33 mol m-3 NH4+, 0.33 mol m-3 NO3- plus 0.67 mol m-3 NH4+, and 1.0 mol m-3 NH4+) with nutrient solutions maintained at pH 6.0, 5.5, 5.0, and 4.5. Amino acid concentration increased in plants grown with NH4+ as the sole source of N at all pH levels. Total amino acid concentration in the roots of NH4+ -fed plants was 8 to 10 times higher than in NO3(-)-fed plants, with asparagine accounting for more than 70% of the total in the roots of these plants. The concentration of soluble carbohydrates in the leaves of NH4+ -fed plants was greater than that of NO3(-)-fed plants, but was lower in roots of NH4+ -fed plants, regardless of pH. Starch concentration was only slightly affected by N source or root-zone pH. At all levels of pH tested, organic acid concentration in leaves was much lower when NH4+ was the sole N source than when all or part of the N was supplied as NO3-. Plants grown with mixed NO3- plus NH4+ N sources were generally intermediate between NO3(-)- and NH4+ -fed plants. Thus, changes in tissue composition characteristic of NH4+ nutrition when root-zone pH was maintained at 4.5 and growth was reduced, still occurred when pH was maintained at 5.0 or above, where growth was not affected. The changes were slightly greater at pH 4.5 than at higher pH levels.

Increase in nitrate uptake by soybean plants during interruption of the dark period with low intensity light.

Diurnal patterns of net NO3- uptake by nonnodulated soybean [Glycine max (L.) Merr. cv. Ransom] plants growing in flowing hydroponic culture at 26 and 16 degrees C root temperatures were measured at hourly intervals during alternate days of a 12-day growth period. Ion chromatography was used to determine removal of NO3- from the culture solution. Day and night periods of 9 and 15 h were used during growth. The night period included two 6-h dark periods and an intervening 3-h period of night interruption by incandescent lamps to effect a long-day photoperiod and repress floral initiation. At both root temperatures, the average specific rates of NO3- uptake were twice as great during the night interruption period as during the day period; they were greater during the day period than during the dark periods; and they were greater during the dark period immediately following the day period than during the later dark period that followed the night interruption. While these average patterns were repetitious among days, measured rates of uptake varied hourly and included intervals of net efflux scattered through the day period and more frequently through the 2 dark periods. Root temperature did not affect the average daily specific rates of uptake or the qualitative relationships among day, dark and night interruption periods of the diurnal cycle.

Cyclic variations in nitrogen uptake rate of soybean plants: effects of pH and mixed nitrogen sources.

To determine if the daily pattern of NO3- and NH4+ uptake is affected by acidity or NO3- : NH4+ ratio of the nutrient solution, non-nodulated soybean plants (Glycine max) were exposed for 21 days to replenished, complete nutrient solutions at pH 6.0, 5.5, 5.0, and 4.5 which contained either 1.0 mM NH4+, 1.0 mM NO3- [correction of NO3+], 0.67 mM NH4+ plus 0.33 mM NO3- (2:1 NH4+ : NO3-) [correction of (2:1 NH3+ : NO4-)], or 0.33 mM NH4+ plus 0.67 mM NO3- (1:2 NH4+ : NO3-). Net uptake rates of NH4+ and NO3- were measured daily by ion chromatography as depletion from the replenished solutions. When NH4+ and NO3- were supplied together, cumulative uptake of total nitrogen was not affected by pH or solution NH4+ : NO3- ratio. The cumulative proportion of nitrogen absorbed as NH4+ decreased with increasing acidity; however, the proportional uptake of NH4+ and NO3- was not constant, but varied day-to-day. This day-to-day variation in relative proportions of NH4+ and NO3- absorbed when NH4+ : NO3- ratio and pH of solution were constant indicates that the regulatory mechanism is not directly competitive. Regardless of the effect of pH on cumulative uptake of NH4+, the specific nitrogen uptake rates from mixed and from individual NH4+ and NO3- sources oscillated between maxima and minima at each pH with average periodicities similar to the expected interval of leaf emergence.

Cyclic variations in nitrogen uptake rate in soybean plants: uptake during reproductive growth.

Net uptake of NO3- by non-nodulated soybean plants [Glycine max (L.) Merr. cv. Ransom] growing in flowing hydroponic culture was measured daily during a 63 d period of reproductive development between the first florally inductive photoperiod and [unknown word] seed growth. Removal of NO3- from a replenished solution containing 1.0 mol m-3 NO3- was determined by ion chromatography. Uptake of NO3- continued throughout reproductive development. The net uptake rate of NO3- cycled between maxima and minima with a periodicity of oscillation of 3 to 7 d during the floral stage and about 6 d during the fruiting stage. Coupled with increasing concentrations of carbon and C : N ratios in tissues, the oscillations in net uptake rates of NO3- are evidence that the demand for carbohydrate by reproductive organs is contingent on the availability of nitrogen in the shoot pool rather than that the demand for nitrogen follows the flux of carbohydrate into reproductive tissues.

Nitrogen nutrition and temporal effects of enhanced carbon dioxide on soybean growth.

Plants grown on porous media at elevated CO2 levels generally have low concentrations of tissue N and often appear to require increased levels of external N to maximize growth response. This study determines if soybean [Glycine max (L.) Merr. Ransom'] grown hydroponically at elevated CO2 requires increases in external NO3- concentrations beyond levels that are optimal at ambient CO2 to maintain tissue N concentrations and maximize the growth response. This study also investigates temporal influences of elevated CO2 on growth responses by soybean. Plants were grown vegetatively for 34 d in hydroponic culture at atmospheric CO2 concentrations of 400, 650, and 900 microliters L-1 and during the final 18 d at NO3- concentrations of 0.5, 1.0, 5.0 and 10.0 mM in the culture solution. At 650 and 900 microliters L-1 CO2, plants had maximum increases of 31 and 45% in dry weight during the experimental period. Plant growth at 900 microliters L-1 CO2 was stimulated earlier than at 650 microliters L-1. During the final 18 d of the experiment, the relative growth rates (RGR) of plants grown at elevated CO2 declined. Elevated CO2 caused increases in total N and total NO3(-)-N content and leaf area but not leaf number. Enhancing CO2 levels also caused a decrease in root:shoot ratios. Stomatal resistance increased by 2.1- and 2.8-fold for plants at the 650 and 900 microliters L-1 CO2, respectively. Nitrate level in the culture solutions had no effect on growth or on C:N ratios of tissues, nor did increases in CO2 levels cause a decrease in N concentration of plant tissues. Hence, increases in NO3- concentration of the hydroponic solution were not necessary to maintain the N status of the plants or to maximize the growth response to elevated CO2.

Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources.

Soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 21 days on 4 sources of N (1.0 mM NO3-, 0.67 mM NO3- plus 0.33 mM NH4+, 0.33 mM NO3- plus 0.67 mM NH4+, and 1.0 mM NH4+) in hydroponic culture with the acidity of the nutrient solution controlled at pH 6.0, 5.5, 5.0, and 4.5. Dry matter and total N accumulation of the plants was not significantly affected by N-source at any of the pH levels except for decreases in these parameters in plants supplied solely with NH4+ at pH 4.5. Shoot-to-root ratios increased in plants which had an increased proportion [correction of proporiton] of NH4(+)-N in their nutrient solutions at all levels of root-zone pH. Uptake of NO3- and NH4+ was monitored daily by ion chromatography as depletion of these ions from the replenished hydroponic solutions. At all pH levels the proportion of either ion that was absorbed increased as the ratio of that ion increased in the nutrient solution. In plants which were supplied with sources of NO3- plus NH4+, NH4+ was absorbed at a ratio of 2:1 over NO3- at pH 6.0. As the pH of the root-zone declined, however, NH4+ uptake decreased and NO3- uptake increased. Thus, the NH4+ to NO3- uptake ratio declined with decreases in root-zone pH. The data indicate a negative effect of declining root-zone pH on NH4+ uptake and supports a hypothesis that the inhibition of growth of plants dependent on NH4(+)-N at low pH is due to a decline in NH4+ uptake and a consequential limitation of growth by N stress.

Effect of ammonium sulfate, ammonium chloride and root-zone acidity on inorganic ion content of tobacco.

Tobacco plants (Nicotiana tabacum L. cv NC82) were supplied with (NH4)2SO4 or NH4Cl at root-zone pH of 6.0 and 4.5 in hydroponic culture for 28 days. Dry matter accumulation, total N and C content, and leaf area and number were not affected by the NH4+ source or root-zone pH. Plants supplied with NH4Cl accumulated up to 1.2 mM Cl g DW-1, but accumulated 37% less inorganic H2PO4- and 47% less SO4(2-) than plants supplied with (NH4)2SO4. The large Cl- accumulation resulted in NH4Cl- supplied plants having a 31% higher inorganic anion (NO3-, H2, PO4-, SO4(2-), and Cl-) charge. This higher inorganic anion charge in the NH4Cl-supplied plants was balanced by a similar increase in K+ charge. Plants supplied with NH4Cl accumulated greater concentrations of Cl- in leaves (up to 5.1% of DW) than plants supplied with (NH4)2SO4 (less than -% DW). Despite the high Cl- concentration of leaves in NH4Cl supplied plants, these plants showed no symptoms of Cl- toxicity. This demonstrates that toxicity symptoms are not due solely to an interaction between high Cl- concentration in tissue and NH4+ nutrition. The increase in root-zone acidity to pH 4.5 from 6.0 did not induce toxicity symptoms.

Uniformity of environmental conditions and plant growth in a hydroponic culture system for use in a growth room with aerial CO2 control.

A portable system of hydroponic culture was developed that maintained temperature, pH, and nutrient concentrations of circulating nutrient solutions. The hydroponic system is used within a controlled-environment room (CER) for control of aerial environment. The CER was equipped with an auto-calibrating system for atmospheric CO2 control. The control systems for the hydroponic chambers were able to maintain acidity within +/- 0.2 pH units and the temperature with +/- 0.5 degree C. Mixing time for the 200-liter volume of solution within a hydroponic chamber was less than 12 min. The CO2 control system was able to maintain aerial concentrations within +/- 10 ppm CO2 during the light period. The only gradient found to occur within the hydroponic chambers or CER was a slight gradient in aerial temperature along the length of hydroponic chambers. Growth of soybeans [Glycine max (L.) Merr.] was characterized during a 3-week period of vegetative development by leaf number and area, plant dry weight, total N content of plants, and N depletion from the nutrient solution. The growth characteristics among populations for three hydroponic chambers within the CER were not significantly different, and the percent standard errors of means of the measurements within populations from each chamber were nearly all less than 10%. Thus, the uniformity of plant growth reflected the uniformity of environmental conditions.

Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling.

When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO(2) and H(2) evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO(2) and H(2) evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.

Regulation of Assimilate Partitioning in Soybean : Initial Effects following Change in Nitrate Supply.

Increased concentrations of nitrate in a nutrient solution (2, 5, and 10 millimolar KNO(3)) were correlated with increased shoot:root ratios of non-nodulated soybeans (Glycine max [L.] Merr.) grown in sand culture. While altering the pattern of C and N partitioning, the N treatments did not affect whole plant photosynthesis over the study period. To determine the mechanism responsible for the observed changes in assimilate partitioning, detailed C and N budgets were worked out with plants from each N treatment over three consecutive 4-day periods of midvegetative growth. The information for the C and N budgets from the 2 and 10 millimolar NO(3) (-) treatments was combined with data on the composition of xylem and phloem exudates to construct a series of models of C and N transport and partitioning. These models were used to outine a ;chain-reaction' of cause-and-effect relationships that may account for the observed changes in assimilate partitioning in these plants. The proposed mechanism identifies two features which may be important in regulating the partitioning of N and other nutrients within the whole plant. (a) The concentration of N in the phloem is highly correlated with the N concentration in the xylem. (b) The amount of N which cycles through the root-from phloem imported from the shoot to xylem exported by the root-is regulated by the root's requirement for N: only that N in excess of the root's N requirements is returned to the shoot in the xylem. Therefore, roots seem to have the highest priority for N in times of N stress.