Journal of Experimental Botany, Vol. 54, No. 384, pp. 1085-1091,
March 1, 2003
© 2003 Oxford University Press
Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules
Received 23 August 2002; Accepted 8 November 2002
1 Agronomy and Range Science, University of California, 1 Shields Ave, Davis, CA 95616, USA
2 Land, Air and Water Resources, University of California, 1 Shields Ave, Davis, CA 95616, USA
3 To whom correspondence shoud be sent. Fax: +1 530 752 4361. E-mail: rfdenison{at}ucdavis.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Respiration in support of N2 fixation by rhizobia in legume root nodules depends on an adequate supply of O2, but excessive O2 can damage nitrogenase, the key enzyme. The movement of O2 into and within the nodule is driven by gradients in the concentration of O2 or in the oxygenation of the O2-carrier, leghaemoglobin. Steeper gradients may increase flux to the sites of respiration, but gradients also raise the possibility of inadequate O2 in some nodule zones and excessive O2 in others. No detailed study of O2 gradients in the interior of nodules has been published previously. Spectral changes in leghaemoglobin with oxygenation, previously used to measure the average O2 status of the nodule interior, were used to map longitudinal gradients in O2 and in respiratory capacity in the elongated nodules of alfalfa (Medicago sativa L.) and sweetclover (Melilotus officinalis L.). Variability among nodules under air in the magnitude and direction of internal O2 gradients was seen in both species. Despite consistently higher respiratory capacity near the meristematic tip, a majority of nodules had higher O2 towards the tip than towards the base. These results contrast with a previous report, apparently based on limited data, but they are consistent with anatomical and tracer studies showing higher gas permeability near the tip.
Key words: Diffusion, legumes, lucerne, oxygen, oximetry, rhizobia.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Under ideal conditions, N2-fixation by bacteroids, a differ entiated form of rhizobia (Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, or Azorhizobium spp.) inside legume root nodules, can supply a large fraction of plant N needs. N2-fixation is powered by bacteroid respiration, so an adequate O2 supply is essential. However, excessive O2 concentrations can cause irreversible damage to nitrogenase. Even O2 concentrations well below atmospheric levels can cause reversible inhibition of nitrogenase activity (Denison et al., 1992b). Legumes have evolved various sophisticated mechanisms that maintain a low O2 concentration (mol m–3) around the bacteroids, while providing a high O2 flux (mol s–1) to support high respiration rates by the bacteroids. These adaptations include a physical barrier to gas diffusion, surrounding the nodule interior, and leghaemoglobin, which facilitates O2 diffusion within rhizobium-infected cells.
Steep gradients in O2 across the diffusion barrier have been measured with microelectrodes (Tjepkema and Yocum, 1974; Soupene et al., 1995), but detailed data on O2 gradients within the nodule interior have not been published. Diffusion of O2 through gas spaces between cells is driven by gradients in O2 concentration, whereas diffusion within infected cells also depends on gradients in Lb oxygenation. Steeper O2 gradients would increase O2 flux, but could also result in O2 concentrations high enough to inhibit nitrogenase (or low enough to limit rhizobial respiration) in some parts of a nodule, even if average O2 concentration were within the optimum range for N2 fixation.
Longitudinal gradients in the elongated nodules that result from indeterminate nodule growth in legumes like alfalfa (Medicago sativa L.) or sweetclover (Melilotus officinalis L.) are of particular interest. These nodules typically contain several developmental zones (Vasse et al., 1990). Near the growing tip, rhizobia, similar in appearance to the free-living form of these bacteria, are mostly contained within infection threads, which are tubular structures of plant origin. Closer to the point of attachment to the root, membrane-bound compartments containing rhizobia are released into plant cells where the rhizobia differentiate into bacteroids, the form capable of fixing N2. Older nodules typically have a senescent zone, where little or no N2 is fixed, near the root. Longitudinal O2 gradients, if they exist, could have a role in the development or maintenance of these zones.
It has been proposed that decreases in nodule O2 permeability, which conserve photosynthate and protect nitrogenase from O2-inactivation after defoliation (Hartwig et al., 1987; Denison et al., 1992a), may also limit losses to ‘ineffective’ rhizobia, which fix little or no N2 (Udvardi and Kahn, 1993; Denison, 2000). Longitu dinal O2 gradients could enhance or reduce the effects of such ‘legume sanctions’ on reproductive rhizobia near the tip, relative to the effects on terminally differentiated bacteroids.
Unfortunately, O2 concentration in the nodule interior is typically too low for accurate measurements using microelectrodes. Quantitative estimates of dissolved O2 concentration in the nodule interior (Oi) have therefore been based on non-invasive optical measurements of the oxygenation of Lb, whose spectral properties change when it binds O2 (Klucas et al., 1985; King et al., 1988). Previous applications of this approach to the problem of nodule O2 gradients have been quite limited, however.
Lee et al. (1995) reported that the older half of yellow sweetclover nodules ‘always showed more oxygenation’. Their result is surprising because studies of nodule anatomy and tracer experiments using iodine vapour (Jacobsen et al., 1998) suggested that gases enter mainly near the meristematic tip, at least in alfalfa nodules. A suberized endodermis apparently limits gas influx from the atmosphere into older parts of the nodule. Although the direction of O2 gradients reported by Lee et al. for sweetclover nodules is opposite from what might be predicted from the point of O2 entry in alfalfa nodules, O2 gradients would also depend on longitudinal gradients in respiratory O2 consumption. If respiration rates near the root were low enough, then even the limited O2 flux through the endodermis might maintain Oi as high or higher than it is near the tip.
Given the possible importance of nodule O2 gradients in the nodule interior, and the lack of statistical information on these gradients in previous work (Lee et al., 1995), O2 gradients were examined in representative numbers of both alfalfa and sweetclover nodules. Spectral changes in leg haemoglobin with oxygenation were used as an intrinsic indicator of internal O2 in intact, attached nodules.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Growth of plants and measurement of fractional oxygenation of leghaemoglobin (FOL) followed the methods of Shimada et al. (1997), except that plants were grown in growth pouches rather than slide chambers, and a 10-bit rather than an 8-bit frame grabber was used to improve precision in measuring light transmittance through nodules. A small hole was cut through the plastic and the paper wick of the growth pouch to expose a nodule, and the pouch was placed on the stage of an inverted microscope. Video images of the selected nodule were captured using 580 nm filters on both illumination system and camera.
Transmittance of 580 nm light through a nodule decreases with increasing FOL, but transmittance also depends on Lb concentration and on absorbance by materials other than Lb. Therefore, FOL was calculated for each pixel in an image, using reference values for that same pixel with FOL=0 and 1, corresponding to fully deoxygenated and fully oxygenated Lb, respectively (Shimada et al., 1997). To obtain these reference images, a nodule initially under humidified air (20% O2 in N2) was exposed successively to humidified N2, 70% O2, and N2 until a steady-state was achieved in overall FOL (typically within 30 s) under each gas, before returning to air. Overall FOL (a single value representing the entire nodule) was simultaneously monitored during this process using a modulated 660 nm LED connected to a lock-in amplifier (Layzell et al., 1990), to confirm that exposure to elevated O2 and pure N2 resulted in fully oxygenated and deoxygenated Lb, respectively, by the time the reference images were captured. Differences between these reference images and sample images captured at other points during the gas exposure sequence were used to map initial FOL gradients in air and to visualize patterns of changing FOL in response to external O2.
For each pixel, FOL at time t was calculated using the equation:
FOL(t)x,y=[I(t)x,y–I(N2)x,y]/[I(O2)x,y–I(N2)x,y](1)
where FOL(t)x,y is the FOL at time t and pixel coordinates x,y; I(t)x,y is the intensity at 580 nm of the same pixel at time t; and I(O2)x,y and I(N2)x,y are the corresponding intensities under fully oxygenated and fully deoxygenated conditions, respectively (Shimada et al., 1997).
Image analysis software was used to measure longitudinal profiles in FOL for each nodule. Because Lb is contained inside infected cells, O2 fluxes at scales larger than an individual cell are assumed to occur mainly as O2 in intercellular airspaces, in equilibrium with dissolved free O2 in the infected cells. Therefore, dissolved free O2 was also estimated, using the equation of Layzell et al. (1990)
Oi=(FOLx37 nM)/(1–FOL)(2)
The coefficient, 37 nM, equivalent to the O2 concentration at which Lb is 50% oxygenated, may vary among and within species. Legumes often have more than one type of Lb, and these may differ in O2 affinity and in distribution within the nodule (Kawashima et al., 2001). Longitudinal variation in the O2 affinity of Lb could affect the quantitative accuracy of dissolved O2 estimates derived from FOL, but probably not our qualitative conclusions about the direction of O2 gradients in nodules.
Linear regression was used to quantify the longitudinal gradient in free O2 along each nodule. Equation 1 is quite sensitive to the difference in the intensity of a pixel between fully oxygenated and fully deoxygenated conditions. As I(O2)x,y–I(N2)x,y approaches zero, estimated FOL will approach infinity, for any value of I(t)x,y. Therefore, pixels for which this difference was less than 30 (frame-grabber units) were excluded from the regression analysis. Typically, this meant excluding the nodule tip, where there was little change in intensity with oxygen status, due to lack of Lb. In addition, nodules were excluded whose overall change in intensity with the gas treatments was below this threshold, presumably due either to low Lb concentration or to excessive overall thickness or opacity. A frame grabber with more resolution (12 bits or more) would be needed to map FOL in such nodules. The conclusions about O2 gradients for nodules in air are based on 24 alfalfa and 28 yellow sweetclover nodules. Nodule age ranged from 19 d to about 30 d.
It is possible that the conditions under which FOL was measured in these experiments could stress nodules in ways that might affect nodule O2 gradients. To test this hypothesis, representative data for control and stressed nodules from a field experiment on grazing effects on white clover (Trifolium repens L.) are included for comparison with these laboratory data. The effects of stress on O2 gradients were also examined directly by mapping FOL as above in nodules attached to plants that had been detopped 4–6 h previously. Previous work with alfalfa showed that this is enough time to affect overall FOL (Denison et al., 1992a).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Calculated FOL images for representative alfalfa nodules under steady- and non-steady-state conditions are shown in Fig. 1A–I. Under air, a few alfalfa nodules had much higher FOL towards the meristematic tip than towards the base (Fig. 1A), many had slightly higher FOL towards the tip (Fig. 1B), and a few had slightly lower FOL towards the tip (Fig. 1C). Quantitative data on the frequency of each are given below.
|
Calculations of FOL assumed that any change in the brightness of a given pixel is due only to changes in FOL near that pixel. Movement of a bright or dark feature in or out of a given pixel therefore resulted in an underestimate of FOL at one point and a corresponding overestimate at an adjacent point. Such movements do not affect large-scale patterns, but they can create small-scale variation in FOL images unrelated to actual FOL. The adjacent black and red spots near the base of the nodule in Fig. 1B illustrate the effects of movement, perhaps of a small necrotic region. Noise due to movement was especially apparent near the nodule tip, where there is little or no Lb. With little Lb, there is little change in brightness with FOL, so the denominator in equation 1 is small, magnifying the effects of even small changes in brightness due to movement. As explained in the Materials and methods, these data were excluded from the regression used to quantify nodule O2 gradients.
Calculated Oi decreased markedly from tip to base in some nodules under air, as exemplified by Fig. 2A. The other two representative nodules showed slight overall trends in opposite directions from each other (Fig. 2B, C), although a short segment in Fig. 2C was more similar to the corresponding part of the nodule in Fig. 2A. Nodule tips showed great variability in apparent Oi over short distances. Usually, this zone was excluded when the O2 gradient was calculated by regression, as explained in the Materials and methods, because the small difference between images acquired under N2 and under elevated O2 indicated a lack of Lb in this zone, limiting the accuracy of FOL estimates. However, differences between the O2 and N2 images for the nodule in Fig. 1A were large enough, even near the tip, that no data were excluded.
|
Figure 3 shows changes in overall (whole-nodule) FOL, monitored at 660 nm, over the course of one of these assays. Whole-nodule FOL decreased to zero within 30 s in each of the two exposures to an O2-free atmosphere (N2). Exposure to 70% O2 resulted in complete oxygenation of Lb in about 4 s. Maps of FOL from 580 nm images acquired during increases and decreases in FOL (Fig. 1D–I) provide information on spatial patterns in O2 diffusion (influx from the atmosphere or longitudinal diffusion within the nodule) and O2 consumption by respiring bacteroids and mitochondria. The arrows in Fig. 3 indicate the approximate times at which the images used to calculate FOL maps in Fig. 1B, E, and H were acquired. During the rapid transition from the fully deoxygenated to the fully oxygenated state, alfalfa nodules often showed transiently higher O2 towards the tip, as seen in Fig. 1D and E. Only three out of 39 alfalfa nodules had the opposite pattern, i.e. noticeably higher FOL towards the root, during this transition. During the transition (under N2) from fully oxygenated to deoxygenated Lb, FOL was almost always lower towards the tip than towards the base, as seen in Fig. 1G–I. Only one nodule out of 39 had higher FOL towards the tip during this transition.
|
Results for sweetclover nodules were similar to those for alfalfa, as seen in FOL maps of representative sweetclover nodules in air (Fig. 1J–L). Steady-state longitudinal gradients in FOL for alfalfa and sweetclover nodules under air are summarized in Fig. 4. There were more nodules having higher O2 towards the base in sweetclover (Fig. 4B) than in alfalfa nodules (Fig. 4A). However, there were still more sweetclover nodules with higher O2 towards the tip than the other way around. The median values for O2 gradients in alfalfa and sweetclover nodules were –5.1 and –4.2 nM mm–1, respectively, where negative values indicate that Oi decreases from the tip to the base. As discussed below, these gradients are in the opposite direction from those reported by Lee et al. (1995).
|
Could this discrepancy be the result of some form of stress, either in these experiments or in those of Lee et al.? In this study’s assays, O2 influx was typically rapid, as shown in Fig. 3. This pattern is characteristic of healthy nodules in the field, for example, nodules attached to healthy white clover plants (Fig. 5A), and quite different from that seen in severely stressed plants, for example, after grazing (Fig. 5B). Similar patterns have been seen in soybean (Glycine max) in the field (Denison et al., 1991).
|
In a direct test of the hypothesis that stress may reverse the direction of O2 gradients in nodules, only three out of 39 alfalfa nodules and two out of 27 sweetclover nodules attached to detopped plants showed higher FOL towards the base (proximal end) under air. Of the remainder, a majority of nodules of each species had gradients in the opposite direction, i.e. higher Oi near the tip, while others showed little or no gradient. The direction of nodule FOL gradients during O2 influx was similar to that under air, and opposite that when FOL was dominated by respiration under N2. These results are qualitatively similar to those for non-detopped control plants.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A majority of nodules in this study had O2 gradients in the opposite direction from those reported by Lee et al. (1995). Some of the nodules from this study showed results consistent with their report (Fig. 4B), and the number of nodules on which their measurements were made was not stated, so it is possible that this discrepancy simply reflects a limited number of nodules in their study. Both studies used spectral measurements of Lb to infer O2 gradients. There were a number of differences in methodological details, but any obvious problems with either method have not been identified. The consistency between Fig. 3 and Fig. 5A (and additional field and laboratory data not shown) suggests that stress was not a problem in these experiments. Lee et al. (1995) did not publish data showing that the physiological status of their nodules was also representative of field conditions, but the current study’s detopping experiment shows that stress will not necessarily reverse the direction of O2 gradients in nodules. O2 gradients could change quantitatively or qualitatively with other factors, such as nodule age.
There are two possible explanations for the transient FOL gradients often seen during increasing FOL (e.g. Fig. 1D, E). First, nodule permeability to O2 could be higher near the tip, allowing faster O2 influx into this zone. This hypothesis is consistent with nodule anatomy and with iodine vapour tracer experiments (Jacobsen et al., 1998). An alternative hypothesis is that nodule respiratory capacity is lower in or near the meristematic tip, so that respiration is unable to keep pace with O2 influx into this zone from an external atmosphere of 70% O2. The reverse FOL gradients in Fig. 1G–I are inconsistent with this second hypothesis, however. These figures illustrate the result, consistently obtained, that FOL in a nodule whose Lb had been O2-saturated decreased (when exposed to an O2-free atmosphere) more quickly nearer the tip than near the base. This indicates higher, not lower, respiratory capacity near the tip. A higher rate of respiration in the meristematic zone or in zone II (Vasse et al., 1990), where there is active cell expansion and growth of infection threads, also seems more physiologically plausible. The consistently lower FOL near the tip of nodules in which FOL was decreasing due to respiration also gives additional credence to the observations of gradients in the opposite direction under air.
Because there is little Lb in the meristematic zone, O2 availability to undifferentiated rhizobia in infection threads cannot be assessed directly. The FOL in Lb-containing cells nearest the tip suggests that O2 supply is unlikely to limit rhizobial respiration there in unperturbed nodules, but any limitations on short-range diffusion could alter this conclusion. A lack of functional Lb in would probably preclude application of this method to senescent nodules or senescent zones in older nodules.
It is concluded that alfalfa and sweetclover nodules usually have higher O2 permeability, but also higher respiratory capacity, towards the meristematic tip. The balance between permeability and respiratory capacity differs among nodules, resulting in variability among nodules with respect to FOL gradients (Fig. 4). There is only a slight FOL gradient along the nodule in many cases, but O2 is more likely to be higher near the tip than near the root. The detopping experiments suggest that this may be the case even in nodules with reduced O2 permeability. If so, then a decrease in permeability that reduced O2 supply to reproductive rhizobia near the nodule meristematic tip would also limit O2 supply to bacteroids, as well as to any saprophytic rhizobia that may occur nearer the root (Timmers et al., 2000).
|
Acknowledgements |
|---|
Supported by the National Science Foundation grant 0077903 and USDA grant 99-35305-8646. The grazing study was conducted at USDA’s Appalachian Soil and Water Conservation Research Laboratory. We thank Bob Rousseau for assistance with the experiments and Karin Jacobsen for comments on the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Denison RF. 2000. Legume sanctions and the evolution of symbiotic cooperation by rhizobia. American Naturalist 156, 567–576.[CrossRef][Web of Science]
Denison RF, Hunt S, Layzell DB. 1992a. Nitrogenase activity, nodule respiration and O2 permeability following detopping of alfalfa and birdsfoot trefoil. Plant Physiology 98, 894–900.
Denison RF, Smith DL, Legros T, Layzell DB. 1991. Non-invasive measurement of internal oxygen concentration of field-grown soybean nodules. Agronomy Journal 83, 166–169.
Denison RF, Witty JF, Minchin FR. 1992b. Reversible O2 inhibition of nitrogenase activity in attached soybean nodules. Plant Physiology 100, 1863–1868.
Hartwig U, Boller B, Nösberger J. 1987. Oxygen supply limits nitrogenase activity of clover nodules after defoliation. Annals of Botany 59, 285–291.
Jacobsen KR, Rousseau RA, Denison RF. 1998. Tracing the path of oxygen into birdsfoot trefoil and alfalfa nodules using iodine vapour. Botanica Acta 111, 193–203.
Kawashima K, Suganuma N, Tamaoki M, Kouchi H. 2001. Two types of pea leghaemoglobin genes showing different O2-binding affinities and distinct patterns of spatial expression in nodules. Plant Physiology 125, 641–651.
King BJ, Hunt S, Weagle GE, Walsh KB, Pottier RH, Canvin DT, Layzell DB. 1988. Regulation of O2 concentration in soybean nodules observed by in situ spectroscopic measurement of leghaemoglobin oxygenation. Plant Physiology 87, 296–299.
Klucas RV, Lee K, Saari L, Erickson BK. 1985. Factors affecting functional leghaemoglobin in legume nodules. In: Ludden PW, Burris JE, eds. Nitrogen fixation and CO2 metabolism. New York: Elsevier Science Publishing Co. Inc., 13–20.
Layzell DB, Hunt S, Palmer GR. 1990. Mechanism of nitrogenase inhibition in soybean nodules. Pulse-modulated spectroscopy indicates that nitrogenase activity is limited by O2. Plant Physiology 92, 1101–1107.
Lee K, Shearman LL, Erickson BK, Klucas RV. 1995. Ferric leghaemoglobin in plant-attached leguminous nodules. Plant Physiology 109, 261–267.[Abstract]
Shimada S, Rousseau R, Denison RF. 1997. Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules. Journal of Experimental Botany 48, 1251–1258.
Soupene E, Foussard M, Boistard P, Truchet G, Batut J. 1995. Oxygen as a key developmental regulator of Rhizobium meliloti N2 -fixation gene expression within the alfalfa nodule. Proceedings of the National Academy of Sciences, USA 92, 3759–3763.
Timmers ACJ, Soupene E, Auriac MC, de Billy F, Vasse J, Boistard P, Truchet G. 2000. Saprophytic intracellular rhizobia in alfalfa nodules. Molecular Plant–Microbe Interactions 13, 1204–1213.
Tjepkema JD, Yocum CS. 1974. Measurement of oxygen partial pressure within soybean nodules by oxygen microelectrodes. Planta 119, 351–360.[CrossRef][Web of Science]
Udvardi MK, Kahn ML. 1993. Evolution of the (Brady) Rhizobium–legume symbiosis: why do bacteroids fix nitrogen? Symbiosis 14, 87–101.
Vasse J, de Billy F, Camut S, Truchet G. 1990. Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. Journal of Bacteriology 172, 4295–4306.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||