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Brief Communication |
Crop and Soil Systems Research Group, SAC, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, UK
Received for publication April 20, 2007. Accepted for publication October 4, 2007.
ABSTRACT
Cortical senescence is an important feature of the roots of a number of graminaceous species because it may contribute to rhizodeposition of carbon and influence the efficiency of nutrient uptake. A major limitation to understanding the physiological control of senescence and its impact on rhizosphere processes has been the lack of reliable techniques for characterizing the progress of senescence along the root. The use of a single-cell pressure probe was evaluated for quantifying cell integrity in different regions of wheat roots. The percentage of locations with turgid cells declined with increasing distance behind the root apex. The decline preceded visible collapse of the cortex but after the loss of nuclear staining in the outer cortex. The percentage of locations with turgid cells was closely associated with root diameter, which suggests that measurements of changes in diameter, made using minirhizotrons under well-watered conditions in the field, could provide estimates of in situ rates of cortical senescence.
Key Words: cell turgor • cortex • Poaceae • rhizodeposition • root • senescence • Triticum aestivum • viability • wheat
Concerns over global climate change have resulted in a major drive to develop methods to sequester carbon (C) in soils and to improve the efficiency of fertilizer use in crop production. The manufacture and use of inorganic nitrogen (N) fertilizer has a significant negative impact on the energy balance of crop production, and thus reductions in fertilizer application and improvements in the efficiency of N uptake from soil are desirable. However, the need to sequester C may not be compatible with attempts to improve fertilizer recovery. For example, approximately 60% of fertilizer N is captured by cereal crops in the UK, and the majority of that not recovered is thought to be temporarily immobilized by the microbial biomass following rhizodeposition of C (King et al., 2001
). To identify opportunities to increase C storage and improve nutrient use efficiency, researchers need to be able to accurately measure and model inputs and losses of C from soils and its interactions with N cycling (Rees et al., 2005
).
A feature of the main root axes of some graminaceous species, including wheat, barley, and oats, is the senescence and eventual sloughing of the cortex in mature regions. This process is distinct from the formation of aerenchyma in response to flooding in a number of species and from the loss of the cortex resulting from secondary thickening in many dicotyledonous plants. Cortical senescence has potential implications for the uptake of nutrients and water because contact between the root surface and soil aggregates may be reduced and the radial transport pathway across the remaining cortex to the stele impeded (Clarkson et al., 1968). Cortical senescence must also contribute to the rhizodeposition of C (with likely consequences for the mineralization/immobilization of N), although few attempts have been made to quantify it (Robinson et al., 1989
; Rees et al., 2005
). In general, our understanding of the processes leading to the death and collapse of the cortex is poor, hindering investigations into processes such as nutrient uptake, nutrient remobilization, and rhizodeposition of C. The major limitation has been the lack of reliable techniques to characterize the progress of senescence along the root and to identify the location and age at which cortical cells die. Observations of a collapsed or sloughed cortex provide no direct information on when and at what location the cells died. Cell death probably precedes the collapse of the tissue, but it is by no means certain.
Staining techniques, pioneered by Holden (1975)
and developed by Deacon and coworkers (Henry and Deacon, 1981
; Lascaris and Deacon, 1991a
), suggested that an early event in senescence was the loss of nuclei from otherwise intact, healthy-looking tissue. When whole root pieces are stained with acridine orange, a steady decline in the number of cells with visible nuclei is observed with increasing distance from the root apex (Henry and Deacon, 1981
). There is also a radial decline in the number of visible nuclei from the outer cortical cells inward (Henry and Deacon, 1981
). Because this longitudinal and radial decline occurs under both sterile and nonsterile conditions and the rate differs between species, cultivars, and chromosome substitution lines, researchers have concluded that cortical senescence is a programmed developmental event, under some genetic control (Henry and Deacon, 1981
; Lewis and Deacon, 1982
; Lascaris and Deacon, 1991b
; Liljeroth, 1995
). The loss of visible nuclei appears to be relatively insensitive to environmental conditions (Lascaris and Deacon, 1991b
). Low nutrient supplies can enhance the rate of loss but at supply levels that limit plant growth (Lascaris and Deacon, 1991b
). Defoliation and infection by foliar pathogens has little effect on the rate of loss, but shading can reduce it (Deacon and Mitchell, 1985
; Lascaris and Deacon, 1991b
). More recently, the loss of stainable nuclei in wheat and barley has been associated with an increase in DNA fragmentation as shown by an in situ TUNEL-assay (Liljeroth and Bryngelsson, 2001
).
Although the above evidence suggests that changes are taking place within the root cortex as the tissue ages, the acridine orange staining technique used has been criticized and the conclusion that nuclei are lost before cell death has been challenged (Wenzel and McCully, 1991
). Using Nomarski optics, these authors were able to find intact nuclei and cytoplasmic streaming in cells of whole tissues and tissue macerates that failed to stain with fluorescent dyes. The lack of staining in older tissues and certain cell layers was suggested to result from poor penetration of the stain (Wenzel and McCully, 1991
).
There is a need, therefore, for additional nonstaining techniques to determine cell viability in the root cortex and in particular to characterize the loss of membrane integrity during the progress of senescence, as this is likely to significantly influence rhizodeposition of organic compounds. The objective of experiments reported here was to investigate the potential for using measurements of the presence and absence of turgor to characterize the spatial distribution of cortical cell death in cereal roots. The premise on which the technique is based is that when cell membrane integrity is impaired, turgor pressure will collapse and cell viability will be lost. The potential of this technique was evaluated by testing the hypothesis that the proportion of locations with turgid cells decreases with increasing distance (and hence tissue age) behind the root apex. The presence or absence of turgor was measured along the length of seminal roots of wheat using a single-cell pressure probe. Pressure probes have been used to examine water relations in the growth zone of roots (e.g., Pritchard et al., 1991), but measurements in mature regions have rarely been made. A second objective was to determine the relationship between the proportion of locations with turgid cells and root diameter. If a strong relationship is found, then relatively simple measurements of changes in root diameter made using minirhizotron techniques could provide valuable information on the temporal dynamics of cortical senescence under field conditions. The pattern of cortical senescence derived from the pressure probe measurements was compared with that obtained from the acridine orange staining technique.
MATERIALS AND METHODS
Plant growth
All experiments were conducted on a winter wheat variety (Triticum aestivum L. cv. Savannah). Caryopses were imbibed in aerated, purified (reverse osmosis) water for 24 h and placed on moist paper towel for 4 d to germinate. One seedling was then transplanted into each culture vessel. The culture vessels were designed to isolate a single seminal root from each plant, enabling the root to be recovered from the growing medium with minimal physical damage to the cortex (Fig. 1).
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. Seedlings were transplanted with one seminal root of median length in the front (narrow) compartment and the remainder in the main compartment. A piece of polythene sheet was placed over the surface of the growth medium in the narrow compartment, with one edge over the dividing partition and buried under the surface in the main compartment. The single root was trained through a cut in the polythene sheet. The sheet then acted as a physical barrier to prevent later formed adventitious roots entering the narrow compartment and becoming tangled with the seminal root. Small open-ended plastic tubes were placed through the sheet, and sealed against it, to permit gas exchange but not entry of roots.
The culture vessels were placed in a controlled environment room at 19 ± 1°C. Light was supplied by compact-type fluorescent lamps providing a photon fluence of 325 µmol·m–2·s–l photosynthetically active radiation (PAR) at initial plant height, over a 16-h photoperiod. Relative humidity was not controlled but ranged from 85–90%. Plants were kept supplied with water and nutrients by pumping nutrient solution through the irrigation ports for 5 min four times a day. Volumes were not determined, but the regime was more than enough to return the growth medium to field capacity, as shown by the daily drainage of excess solution.
Plants were harvested 31–34 d after germination. The front cover was removed from the narrow compartment, and the sand/calcined clay gently washed from the sole seminal root with reverse osmosis water. The length of the root was measured to the nearest millimeter.
Quantifying the presence and absence of turgor
The presence or absence of turgor in the root cortex was determined using a single-cell pressure probe (Tomos and Leigh, 1999
). The lateral roots were excised a few millimeters from their base to facilitate ease of handling of the main axis. The root was held under water against a transparent acrylic back-plate using modelling clay and, at defined points along the root, a glass microcapillary filled with silicon oil was inserted into the cortex. The microcapillary was controlled with a micromanipulator and viewed under a dissection microscope operated with transmitted light. The tip of the microcapillary was opened by rubbing it gently against the surface of a glass slide. Opened tips were approximately 20 µm in diameter. Prior to insertion, oil in the microcapillary was placed under a slight positive pressure to prevent entry of water or cell sap by capillary action. An attempt was made to keep the depth of insertion the same at each region along the root. The depth was measured on a set of young test root tissues using a calibrated vernier scale on the micromanipulator. The average depth was 42 µm. After the root was impaled, any entry of cell sap into the capillary was noted. If no sap was seen, the microcapillary was inserted another increment, i.e., approximately 42 µm deeper. If sap entered at either the first or second increment, then the location was recorded as being turgid. If no sap entered at this second increment, the absence of turgor was recorded. An inevitable consequence of probing roots in regions where the cortex begins to collapse is that measurements will not be confined to the outer cell layers; following their collapse, the central and ultimately inner cortical layers might be measured. At each region along the root, the cortex was probed 20 times within a distance of 2–3 mm, and the number of probed locations with turgor was expressed as a percentage of the total number of locations probed (20) within the region. Areas close to a lateral root (e.g., <0.8 mm) were avoided because these may have delayed senescence compared to other regions (Henry and Deacon, 1981
). The diameter of the same region of root was measured using an eyepiece graticule fitted to the microscope.
Young, fresh root tissue and roots killed by immersion in boiling water served as positive and negative controls, respectively, for the reliability of microcapillary function. The same microcapillary was used for multiple impalements but was replaced on the rare occasions when the tip broke or became plugged with tissue debris or viscous material. Before each insertion into the root, freedom from plugging was determined by visually assessing the rate of flow of oil droplets from the tip after pressurizing the oil reservoir. If some plugging was suspected, the operation of the microcapillary was further checked using the young turgid and killed tissue controls. For avoiding possible systematic bias in the results, the order in which specific regions of the root were probed was varied randomly between replicates.
Histology
A separate set of plants was used for staining of nuclei and measurements of the cortex and stele. Every 50 mm away from the root apex, a 10-mm piece was excised and fixed in 70% aqueous methanol. Staining was as described by Henry and Deacon (1981)
. Root pieces were hydrolyzed in 3% HCl in 95% aqueous ethanol for 5 min at room temperature and rinsed twice in phosphate-citrate buffer, pH 3.8. They were then stained with acridine orange in phosphate–citrate buffer for 15 min, before being rinsed twice in fresh buffer. Serial transverse sections were cut by hand, mounted on a glass slide in buffer, and viewed under a fluorescence microscope. Sections were taken midway between lateral roots. The cortex was subdivided into three groups: the inner group (the three cell layers adjacent to the endodermis), the outer group (the first two cell layers underlying the epidermis), and the middle or central group (the two cell layers between the inner and outer group). The absolute point of reference for identifying cell layers was the endodermis.
A scoring system was devised to record the proportion of cell layers with visible nuclei. The presence or absence of visible nuclei in each group was scored for up to five serial sections per location along the root axis, and the percentage of sections with at least one visible nucleus in a particular cell layer group recorded. In addition, on one section per location, the radial width of the cortex plus epidermis, and the diameter of the stele, were determined using a calibrated eyepiece graticule. Two measurements were made perpendicular to each other and averaged to give the radial width of the cortex (plus epidermis) and diameter of the stele. A single seminal root from six replicate plants was measured.
RESULTS
Presence and absence of turgor
In the apical 200 mm of the root, an average of 94% of the locations probed per root were turgid (Fig. 2). This percentage began to decline between 200 and 300 mm and continued to decline until 650 mm, beyond which it was rare to find turgid cells. Roots were approximately 90 mm long when transplanted and had an average length of 809 mm when harvested, giving an extension rate of approximately 25 mm·d–1 over their modal 32-d lifespan. With these values, the approximate age of any region along the root axis can be calculated. Thus, the percentage of locations with turgid cells began to decrease when the tissue was between 8 and 12 d old and reached a minimum by 26 d. The diameter of the root also decreased with distance from the apex. The average diameter was just under 0.9 mm at 100 mm from the apex and 0.23 mm at the base of the root. Regression analysis of root diameter against distance behind the root apex indicated an average reduction in diameter of 9 µm·mm–1 increase in distance behind the apex (r2 = 0.986, P < 0.001). There was a significant, positive linear relationship between the percentage of turgid cells in any particular region of the root and the root diameter (r2 = 0.65, P < 0.001; Fig. 3).
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; Liljeroth, 1995
). The percentage of sections with visible nuclei correlated well with the percentage of locations with turgid cells. Correlation coefficients were 0.87, 0.96, and 0.76 for the outer, middle, and inner groups of cell layers, respectively (based on the mean values presented in Figs. 2 and 4). The width of the cortex was calculated as two times the radial width measured on transverse sections. There was a steady decline in cortical width basipetally along the root axis, which commenced relatively close (50–150 mm) to the root apex (Fig. 5). The width in the oldest regions (32 d old) was only half that of young regions near the root apex (2 d old). Linear regression analysis indicated an average reduction in cortical width of 4.85 µm·mm–1 length of root axis (P < 0.001, r2 = 0.90) and a small (0.89 µm·mm–1) but significant (P < 0.001, r2 = 0.77) reduction in the diameter of the stele. The sum of the cortical width and stele diameter gives the total root diameter, as measured on sectioned tissue. The average decline in diameter was 5.9 µm·mm–1 length of root axis (P < 0.001, r2 = 0.94). These values agree reasonably well with those derived from measurements on intact roots (Fig. 2), although direct comparisons cannot be made because a different set of plants was used in each case.
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With a pressure probe, the actual turgor pressure of a cell, rather than a simple presence or absence, can be recorded (Tomos and Leigh, 1999
), but the accuracy of the measurement depends on how well the membrane seals around the microcapillary after the cell is impaled. In mature regions of the wheat roots in the present study, the seal was poor, and the system leaked when pressure was applied to the oil reservoir to force the sap back into the cell. Thus, presence and absence of turgor was considered to be the most reliable measurement of cell integrity. Close to the root apex, the percentage of probed locations that were turgid averaged 94%. It is to be expected that the occasional cell would lack turgor, even in young tissue because minor damage may occur on removing the root from the sand/clay growth substrate, even when great care is taken. The progressive decline in the percentage of locations with turgid cells beyond 200 mm from the root apex supported the initial hypothesis.
Measurements of the depth of insertion of the probe suggest that in regions with no obvious collapse of cell layers, the outer two cortical layers were being probed. Once these cells began to collapse, inserting the probe to the same depth would have meant that the presence or absence of turgor was being determined on the central and, eventually, the inner cell layers. Observations on hand-cut sections indicated that visible degeneration and collapse of the outer cortex, in general, commenced about 600 mm behind the apex. In only one of the roots examined did it occur earlier (450 mm from the apex). Thus, reductions in the percentage of locations with turgid cells were observed before (i.e., closer to the root apex) there was any visible collapse of specific cortical layers. Measurements of the width of the cortex, on the other hand, revealed some "shrinkage" of the cortex commencing much closer to the apex (100–200 mm from the apex). A similar pattern of basipetal decline in root diameter was observed in fresh tissues. The similarity between fresh and preserved tissues suggests that most of the variation in diameter along the length of fresh roots was associated with structural changes in the cortex rather than with changes in tissue hydration.
In most studies in which acridine orange has been used to quantify the apparent loss of nuclei during cortical senescence, intact root pieces were viewed and scored for the number of cell layers with anucleate cells (Henry and Deacon, 1981
; Lewis and Deacon, 1982
; Lascaris and Deacon, 1991b
). In my laboratory, the technique has proved difficult to use. The main problems are associated with focusing the microscope on cell layers deep in the cortex and the need to account for the curvature of the root surface. Thus, confidently identifying the cell layer under observation was difficult. Liljeroth (1995)
overcame some of these problems by freezing the root, cutting it in half longitudinally, and examining the cut surface. In the present study, a different approach was adopted. Transverse sections were cut, which enabled the easy identification of the different cortical layers. The disadvantage of transverse sections is that the nucleus in a cell may not be visible if the section is cut above or below it. The risk of mistakenly designating a particular cell layer as anucleate by simply missing the nucleus during sectioning was minimized by (1) examining serial sections from the same piece of root and (2) adopting a conservative method of scoring the nucleated status. Thus, for a cell group in a given section to be classed as nucleate, only one nucleus per group needed to be visible. Because different techniques and forms of expressing the data have been used, the measurements in this study are not directly comparable with those reported in the literature. Nevertheless, the general loss of nuclear staining basipetally along the root and radially inward (Fig. 4) is consistent with previous reports (Henry and Deacon, 1981
; Liljeroth, 1995
). In the current experiments, the loss of cell integrity commenced further from the apex than did the decline in nuclear staining in the outer cortical cell layers, which suggests that nuclei fail to stain earlier in senescence than the cell membranes lose integrity. The earlier decline in visible nuclei may result from the genuine degeneration of nuclei, or a failure of nuclei to stain, prior to the impairment of membrane integrity. The latter explanation is most consistent with the observations of intact nuclei and cytoplasmic streaming in cells whose nuclei failed to stain (Wenzel and McCully, 1991
).
Although the absence of turgor has been interpreted to result from the loss of membrane integrity in the present work, an alternative but less likely explanation is conceivable. The absence of turgor could result from the active reduction in the concentration of solutes, with the cell membrane remaining intact. However, the question over interpretation of the loss of turgor relates to its underlying mechanism rather than its functional consequence. In either case, the loss of turgor would seem to be associated with the final stages of senescence and cell death because loss of turgor is followed by the collapse of the outer cortex.
In conclusion, these experiments have demonstrated that measurements of the presence and absence of turgor can be used to map cortical cell viability along wheat root axes. The distribution of senescence broadly follows that revealed by nuclear staining, but the pressure probe technique has the advantage that it provides a more direct measure of the loss of turgor, and probably membrane integrity, which is likely to have important consequences for rhizodeposition of organic compounds. The other major advantage of the pressure probe technique is that it can be adapted for simultaneous sampling of individual cells (Tomos and Sharrock, 2001
), thereby facilitating investigation of the relationships between cell solute concentrations, cell senescence, and rhizodeposition. Currently, the contribution of cortical senescence to C deposition is unknown. A major uncertainty is the extent to which soluble organic compounds are depleted by respiration, biosynthetic processes, or re-translocation prior to cell death and hence how much organic C is lost to the rhizosphere.
The close linear association between the percentage of locations probed with turgid cells and root diameter in these well-irrigated roots suggests that nondestructive measurements of changes in root diameter made using minirhizotrons could provide an indirect estimate of the temporal dynamics of cortical senescence in the field under conditions of ample water supply. Care will be needed in interpreting such measurements because other factors, such as the transpiration rate, could alter the root diameter. However, with suitable controls and after appropriate calibration, more realistic estimates of the contribution of cortical senescence to rhizodeposition of C in the field might then be possible (Rees et al., 2005
). Only with a better understanding of the quantity and nature of C compounds deposited in the rhizosphere as a result of cortical senescence can we determine whether manipulating cortical senescence will substantially increase C deposition or improve nutrient use efficiency of cereals.
FOOTNOTES
1 The author is indebted to E. Stevenson for skilled technical assistance, K. Oparka for the loan of a pressure probe, and J. Pritchard for additional advice on its use. SAC receives financial assistance from the Scottish Government Rural and Environment Research and Analysis Directorate. 
2 E-mail: ian.bingham{at}sac.ac.uk
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