JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 399, pp. 1071-1078, May 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
Why hypocotyl extension mutants need to be characterized at the cell level: a case study of axr3-1
Received 21 July 2003; Accepted 26 January 2004
Department of Biology, Plant Laboratory, University of York, Heslington, York YO10 5YW, UK
* E-mail: drf1{at}york.ac.uk
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Abstract |
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Since the discovery of auxin, a debate has taken place as to whether the auxin distribution in elongating organs can account for the distinctive cell elongation profiles that have been found. In an attempt to address this important issue, the elongation profiles of cells have been compared in the hypocotyls of wild-type and auxin-hypersensitive axr3-1 Arabidopsis Columbia ecotype seedlings. Clear differences in cell elongation profiles were found in the two types of seedling, whether they were light- or dark-grown. However, it was not possible unambiguously to ascribe the cell elongation profile differences to the proposition that the axr3-1 mutation causes the hypocotyl to be hypersensitive to auxin. The possibility that the abnormal hypocotyl elongation profile of the mutant was a secondary effect, consequent on a more fundamental effect of the axr3-1 mutation, is considered. It is clear from this study that cell elongation and its control needs to be studied at the cell, and not the organ, level. To characterize a mutant as having a short, or long, hypocotyl is inadequate. To determine which factors control the timing and the magnitude of cell elongation requires the demonstration of correlations between the growth rate of cells and their content of regulating substances or their sensitivity to that substance. Studies of the cell elongation profiles of the many hypocotyl length mutants could also be a very effective means of probing the co-ordination of root and shoot elongation.
Key words: Arabidopsis, dark, epidermal cell elongation, growth rate, hypocotyl, light, nutrition, spatial, temporal.
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Introduction |
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The simplest model for the regulation of cell elongation by an individual substance was that proposed over 50 years ago when it was postulated that cell elongation rate of coleoptiles, epicotyls, or hypocotyls were proportional to the auxin content of those organs. However, that simple model has been difficult to challenge due to the fact that the manipulation of the endogenous supposed regulator, indolyl-3-acetic acid (IAA), is hard to achieve. Mutants of Arabidopsis which have altered responses to exogenous auxin offer new approaches to exploring the role of auxin in controlling organ extension. While the hypocotyl length of such mutants is easy to measure, such measurements fail to address the fact that the overall organ extension is the sum of many different cell elongation profiles. The auxin-response mutant axr3-1 was selected for the present study. The AXR3 gene was isolated from two mutagenized Arabidopsis populations which were selected by their resistance to sub-lethal concentrations of auxin and the axr3-1 mutant is shorter in the light and dark (Leyser et al., 1996; Rouse et al., 1998). The axr3-1 phenotype has been postulated to be the result of cells showing an increased sensitivity to auxin. However, it is already known that the response to exogenous auxin of excised bean hypocotyl segments is dependent on the position which those segments occupied in the intact organ which suggests that the hypocotyl is a heterogeneous system (Gotô and Esashi, 1974). By examining how the AXR3 gene influences the cell elongation profiles, it would be possible to learn more about the way in which auxin controls hypocotyl elongation.
Because of the small size of Arabidopsis hypocotyls, most previous investigators interested in the control of hypocotyl elongation have not felt that it was possible to examine its cell growth distribution profiles. A methodology is reported in this paper which provides a means of measuring the elongation profiles of cells in very young Arabidopsis seedling. This methodology has been used to characterize the elongation profiles of seedlings grown in light and darkness (Barley, 2003) and it has also enabled the elongation profiles to be compared in wild-type and axr3 seedlings during the first few days of growth. Since this work was initiated, another method of measuring Arabidopsis hypocotyl cell elongation profiles has been reported (Gendreau et al., 1997) and that has enabled further conclusions to be drawn about the effect of the growing conditions on the cell elongation profiles.
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Materials and methods |
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Surface-sterilized (10 min in 10% Chlorox, rinsed in 70% ethanol p.a. and three rinsings in sterile distilled water) and imbibed (4 °C for 2 d) Arabidopsis ecotype Columbia wild-type and axr3-1 mutant seeds were surface planted at approximately ten seeds per plate and 8 mm apart in a sterile tissue culture room on 90 mm Petri dishes prepared with an autoclaved minimal Arabidopsis tissue salt (ATS) medium containing 8 g l–1 agar, 10 g l–1 sucrose, and the Arabidopsis tissue salt supplement (ATS) of 5 mM KNO3, 2.5 mM KH2PO4 (pH 5.5), 2 mM MgSO4, 2 mM Ca(NO3)2, and 50 µM Fe-EDTA, with micronutrients 70 µm H3BO3, 14 µM MnCl2, 0.5 µM CuSO4, 1 µM ZnSO4, 0.2 µM NaMoO4, 10 µM NaCl, and 0.01 µM CoCl2 (Wilson et al., 1990). In addition, in an effort to mimic the markedly different epidermal cell growth distribution patterns of the axr3-1 dark-grown hypocotyl, wild-type seedlings were grown in the dark for 5 d on media supplemented with aliquots of the auxin transport inhibitor NPA (naphthylphthalamic acid) or on auxin (IAA, indole-3-acetic acid). The sealed dishes (Micropore tape: 3M Company, Minneapolis, Mn.) were grown for 1–5 d in a vertical position at 20–21 °C in a sterile growth room, either triple-wrapped in foil or in 16 h photoperiods of 45–60 photons µmol m–2 s–1 irradiance.
Light-grown and dark-grown seedlings harvested daily over 5 d, although dramatically varying in thickness and length, were successfully preserved, cleared, stored, and mounted in 70% ethanol p.a. at room temperature. Seed specimens (T0) were stored in 70% ethanol p.a., and after seed coat removal, mounted in water and/or ethanol miscible High Viscosity Mountant CMCP-10 (Polysciences, Inc., Warrington, Pa).
The growth distribution profiles of columns comprising non-stomatal hypocotyl epidermal cells, defined as cell files, were measured by open field Nikon image analysis photomicroscopy at 200x or 400x magnification. One cell file per seedling was chosen from cell files falling within two standard deviations of mean overall hypocotyl length. Cell width changes were considered minimal (Gendreau et al., 1997), and at least three independent sets of measurements were taken from at least five specimens at each growth point.
Growth distributions could not be defined by statistical methods such as analysis of variance due to cell length dependence on both spatial and temporal histories, however, because cell count number of non-stomatal files remained unchanged throughout the first 5 d of growth (Nagatani et al., 1991; Scheres et al., 1995; Smalle et al., 1997), direct comparisons were made between wild-type and axr3-1 light- and dark-grown seedlings with cell files selected for 16±2 cells per file. Cumulative growth was calculated by the sequential summing of the cell length measurements from the base to the apex of the hypocotyl with rates of elongation derived from these values.
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Results |
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Comparison of the overall hypocotyl lengths of wild-type and mutant axr3-1 seedlings
Many earlier studies that have measured hypocotyl elongation have done so by monitoring the elongation of an entire hypocotyl. In order to compare the individual cell length measurements of this study with these previous works, the overall hypocotyl length was calculated by summing together the individual lengths of the cells in non-stomatal epidermal cell files.
The overall hypocotyl length profiles of the wild-type light- and dark-grown seedlings markedly differed throughout the first 5 d of early seedling growth. For example, the wild-type dark-grown hypocotyl began to elongate rapidly at day 3 and was over five times longer than the wild-type light-grown hypocotyl (15.5±0.9 mm versus 3.0±0.2 mm) by day 5. Within standard error, however, the wild-type light-grown hypocotyl did not elongate past day 4.
By contrast with the wild-type overall hypocotyl lengths in the dark and light, the overall lengths of the light- and dark-grown axr3-1 hypocotyls were nearly equal at day 1 and again at day 2. By day 3, the rate of elongation in the axr3-1 dark-grown hypocotyl increased, with the hypocotyl reaching an overall length of 4.2±0.3 mm by day 5, or about a quarter of the length of the wild-type dark-grown hypocotyl at this time. The axr3-1 light-grown hypocotyl did not elongate past day 2, but notably remained longer than the wild-type light-grown hypocotyl until day 4. (Fig. 1).
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Comparison of the growth distribution profiles of wild-type and mutant axr3-1 seedlings
Cell files were measured in dark- and light-grown seedlings that contained 16–18 non-stomatal cells per file (Fig. 2). Cell file profiles of light- and dark-grown seedlings differed markedly and could not be explained by any single factor such as by a shift of the auxin dose-response curve. For example, if the axr3-1 mutant was singularly hypersensitive to auxin then its growth distribution profiles would reflect this hypersensitivity as a uniform change in the auxin dose-response curve. However, at day 3, whilst the basal cells of the wild-type dark-grown hypocotyl had stopped elongating, in the axr3-1 dark-grown hypocotyl, cells continued to elongate beyond the mid-hypocotyl. By comparison with these two dark-grown patterns of elongation, all of the cells of the axr3-1 light-grown hypocotyl had stopped elongating by day 2, but the mid-hypocotyl cells of the wild-type light-grown hypocotyl continued to elongate with noticeable mid-hypocotyl elongation occurring from day 3 to day 5.
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Profiles of supplemented growth seedlings
In a brief additional experiment, to estimate the disparity of the wild-type and axr3-1 profiles, wild-type seedlings were grown on sterile plates in media supplemented with aliquots of either NPA (naphthylphthalamic acid) or IAA (indole-3-acetic acid). Although the predominantly radial uptake of either supplement and constant levels of hormone supplementation are atypical of normal plant-growth processes, if the axr3-1 mutant does exhibit a uniform growth pattern then it should be possible to mimic the phenotype and the growth distribution profile of the axr3-1 mutant by a controlled mediation of auxin level. The supplements used were chosen because the exogenous application of NPA is believed to act by blocking a component of the auxin polar transport system and hence might elevate levels of auxin within the cell. Additional auxin is the simplest way of elevating auxin levels.
The supplement concentrations used were determined first by the viability of the seedlings, and then by the match of overall hypocotyl length of day 5 dark-grown axr3-1 seedlings. Dark-grown seedlings were used because of the marked difference in overall hypocotyl length between wild-type and axr3-1 dark-grown seedlings. At the highest viable concentration of NPA (10–6 M), the overall hypocotyl length of the NPA growth-supplemented seedlings were about 30% shorter than the wild-type seedlings (13 mm versus 16 mm). The overall hypocotyl length of the IAA (10–5 M) growth-supplemented seedlings (about 5.6 mm) more closely approximated that of axr3-1 dark-grown seedlings (about 4 mm), but the cell length growth distribution patterns were distinctly different (Fig. 3). The basal cells of the supplemented growth wild-type seedlings remained unaffected by either treatment, suggesting that the early growth patterns of wild-type dark-grown seedlings were unaffected by external auxin mediation. From the mid-hypocotyl the cell length growth profiles had altered, but only the mid-hypocotyl tenth cell of the IAA supplemented seedling directly corresponded to any cell length in the axr3-1 seedling, and the lengths of their neighbouring cells differed widely. The IAA supplemented seedlings were also shorter than the axr3-1 seedlings, and distorted by numerous small cells constricting the root formation and the sub-apical structure of the hypocotyl.
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Comparison of seed size
It was noticed that the seed size from axr3-1 plants was noticeably larger than wild-type seeds at T0. From this observation, it could be assumed that a large seed size would produce a large hypocotyl. When measured, the larger seeds of axr3-1 already had a 7% increase in hypocotyl length and individual cell lengths at the start of the experiment (data not shown).
Comparison of cumulative growth curves and normalized cumulative growth curves
Altogether the methodology of using the daily sampling of seedlings from populations of individuals has been a convenient way of defining a growth continuum, but in relying on daily cell length growth distribution profiles there is no temporal link of cell length patterns between previous and ensuing days of growth. Cumulative growth curves (Fig. 4), derived from the sequential summing of the cell length measurements from the base to the apex of the hypocotyl, and compared on consecutive days, were used as a means of describing regional rates of hypocotyl elongation. When these growth curves were normalized with respect to their overall hypocotyl lengths and compared between the sets of hypocotyls (Fig. 5), the growth curves of the day 3 axr3-1 hypocotyls closely matched those of the day 2 wild-type hypocotyls in both the light and dark. The rate of growth of each seedling set remained broadly similar between day 2 and day 5 (Fig. 6), thus the accelerated pattern of axr3-1 extension continued throughout the remainder of the 5 d growth frame. As one of the pleiotropic effects of the axr3-1 mutant is an earlier senescence, it may therefore be more appropriate to describe its growth processes by means of a developmental age, independently of the daily harvesting of sample sets of seedlings. Similarly, other mutants may have altered senescences or other temporal characteristics that are consequently reflected in altered rates of growth processes.
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To summarize the results, growth distribution profiles measured from intact files of Arabidopsis hypocotyl epidermal cells have demonstrated that the elongation of these cells in the dark or light is dependent on numerous factors including a cell’s temporal and spatial position along the hypocotyl as well as the prevailing light environment and nutrient conditions. When these analyses were applied to the characterization of the auxin-response mutant axr3-1 the results suggest that factors other than a global hypersensitivity to auxin must be considered to explain the hypocotyl cell length growth distribution profiles of this mutant.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The patterns of cell length differences that were measured in both light and dark-grown wild-type and mutant axr3-1 hypocotyls have demonstrated that no single factor can readily describe their distinct profiles of elongation. Although the pleiotropic phenotype of the axr3-1 mutant has in the past been attributed to a global hypersensitivity to auxin, the results of this study suggest that other factors within its phenotype may be more directly affecting the development process of the axr3-1 plant system.
For example, the axr3-1 mutant is described by single inflorescences, reduced seed set numbers, and an earlier senescence than wild-type seedlings (Leyser et al., 1996). Possibly as a result of these factors which may be affecting nutrient stores and partitioning, axr3-1 seeds are noticeably larger (around 7% longer) than wild-type seeds, and an earlier senescence suggests that the total energy stores of axr3-1 seedlings are conserved. In addition, in earlier sequential transport studies, albeit using wheat seedlings, auxin levels were not detected before the second day of growth (Wright, 1961). Exogenous application of NPA and IAA did not affect early basal cell growth in the dark-grown wild-type seedlings. Thus in both light and dark-grown axr3-1 seedlings, the longer basal cell lengths measured on day 1 and day 2 may be due solely to nutritional strategies.
When examined in a spatial and temporal framework throughout the life cycle of a plant system, nutrient partitioning theories imply that a hierarchical structure is operating within a plant system. A plant can be partially characterized by the contributions of any single organ, but a plant is, in actuality, a set of competing organs and there is no set developmental plan (Kutschera, 2000). If the balance of one organ is disturbed then the growth patterns of the other organs are also likely to be affected (MacIntyre, 2001). Furthermore, the role of long-distance hierarchical signalling is known in apple tree grafting procedures, and recent Arabidopsis mutant studies of max (more axillary growth) mutant roots grafted to wild-type hypocotyls similarly illustrate the root-transmitted effects of altered hypocotyl elongation (Turnbull et al., 2002).
In another consideration of chosen analytical frameworks, it became clear when analysed that the length of a seedling was independent of the age of the seedling and that the progress of a seedling might be better described by its developmental age. For example, all mid-hypocotyl rates of growth remained broadly constant between day 2 and day 5 of growth. When growth rate curves were normalized, however, by day 2 the relative cell length changes in both light- and dark-grown axr3-1 seedlings had overtaken those of the wild-type seedlings by approximately one 24 h period (16 h photoperiod). If other plant organs are examined by similar criteria, it may be, for example, that the compacted structure of the axr3-1 root organ system is due to the axr3-1 roots developing within a more accelerated growth frame than that of axr3-1 hypocotyl development.
In practice, a hormone mutant’s behaviour has generally been described by the presumed global contributions of a discrete one hormone system, but this set of assumptions has been shown here not to provide a sufficient explanation of all developmental ramifications. If the more pleiotropic aspects of a mutant remain unexplored, then the opportunity has been lost to use these singularities as a possible means of revealing the full nature and behaviour of this mutant. Whilst in a process such as elongation a hormone such as auxin may be acting on different organs at different times or in different ways, for example, a mutant’s behaviour might be better explained by the effects of multiple or interacting hormones, or by the effects of environmental conditions. Within these possibilities also lie the prospects of end-results from whole series of both long-term and short-term processes, and patterns of development that may describe entire families of mutants.
Ideally the temporal, spatial, and phenotypic aspects of physiology would be characterized as standard procedure for every mutant. Clearly, a methodology sampling from populations of individuals is restricted by the frequency of sampling, and any short-term effects within a daily timeframe may be impossible to discern by such methods. There will have to be development in the technology to turn genes on and off for short periods before the primary and secondary effects of mutation can be considered.
The methodologies described in this paper have used an image analysis system to maintain a sharp focus along the length of the hypocotyl, and a simple procedure of preserving, clearing, storing, and mounting harvested seedling samples in 70% ethanol p.a. The detail that has emerged from studying the individual cell length profiles within the overall length of the hypocotyl cannot readily be substituted by assumptions of uniform cell length or growth distribution within the hypocotyl, yet overall hypocotyl length remains a frequent choice as the means of characterizing mutant growth physiology. The characterizations of root growth patterns in numerous mutants (Evans et al., 1994) and growth models of leaves (Chandler and Robertson, 1999; Granier and Tardieu, 1999) support the role of growth distribution analyses for any extending organ. The results of the present study show how valuable it is to profile the hypocotyl growth distributions in what is believed to be a well-characterized mutant. It could be interesting to monitor the changes and developmental patterns of other mutants with altered statures when studied in a similar way.
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Acknowledgements |
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Richard Firn, Ottoline Leyser, the University of York Plant Laboratory.
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References |
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